Herbal Drugs and Fingerprints

Devi Datt Joshi

Herbal Drugs
and Fingerprints
Evidence Based Herbal Drugs
Devi Datt Joshi
Amity Institute of Phytochemistry
& Phytomedicine
Amity University, Uttar Pradesh
Noida, UP, India

ISBN 978-81-322-0803-7 ISBN 978-81-322-0804-4 (eBook)

DOI 10.1007/978-81-322-0804-4
Springer New Delhi Heidelberg New York Dordrecht London

Library of Congress Control Number: 2012952527

© Springer India 2012

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or
part of the material is concerned, specifically the rights of translation, reprinting, reuse of
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way,
and transmission or information storage and retrieval, electronic adaptation, computer software,
or by similar or dissimilar methodology now known or hereafter developed. Exempted from this
legal reservation are brief excerpts in connection with reviews or scholarly analysis or material
supplied specifically for the purpose of being entered and executed on a computer system, for
exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is
permitted only under the provisions of the Copyright Law of the Publisher’s location, in its
current version, and permission for use must always be obtained from Springer. Permissions for
use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable
to prosecution under the respective Copyright Law.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are
exempt from the relevant protective laws and regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of
publication, neither the authors nor the editors nor the publisher can accept any legal responsibility
for any errors or omissions that may be made. The publisher makes no warranty, express or
implied, with respect to the material contained herein.

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Foreword

Globally the demand is increasing for medicines, pharmaceuticals, tonics,

cosmetics and other plant based products. India is a major player in this
area along with China, which is the world leader in the production, con-
sumption and export of herbal products as well as raw materials. The cur-
rent interest in the commercial production of these preparations and products
has put tremendous pressure on the supply of raw materials. The burgeoning
gap between availability of bioresources and demand on one hand and
between commercial demand and supply on the other has led to use of both
inappropriate (e.g., unsustainable collection from the wild, well beyond
natural regeneration) and spurious practices (e.g., collection of raw material
without any consideration of quality, deterioration of quality due to poor
storage, adulteration or even substitution by a different part of the same
plant or from an altogether different plant source and/or even material being
spiked with synthetics). Such a situation calls for proper guiding material
and techniques for fast and easy determination of genuineness of crude
materials and for quality assurance in respect of herbal products. Fortunately
literature is now becoming available, albeit slowly, that provides the much
needed know how for determining the reliability of the raw material used
along with the plant source and information on the availability, production
and quality requirements.
Herbals are derived from whole plants or plant parts and used to prevent,
relieve and treat illness. Since antiquity they are valued for medicinal, aro-
matic, nutritional and even rejuvenating qualities, and have played crucial
role in maintaining the well being and vitality of human body, and for allevi-
ating human suffering across civilizations.

v
vi Foreword

The complex metabolic pathway of the human body can be adversely

affected by the use of non-standardized herbals and herbal drugs. Good man-
ufacturing practices, among others, insist on use of standardized protocols
during the processing of raw materials (collection, transport, storage) and
production of the final products or drugs. Standardized herbals and herbal
drugs contain active ingredients, present in the naturally occurring plant
source, in certain quantity and the proportion between different constituents/
active principles is a key quality parameter for the efficacy of the product. It
is in this regard that the modern tools and techniques of analysis provide vital
support and required evidence. The present compilation is a unique and wel-
come attempt by the author to bring together current tools of finger printing
and analyses for various classes of phytochemicals and natural compounds.
The book Herbal Drugs and Fingerprints should be able to bridge an impor-
tant gap, and would be useful for herbal healthcare professionals, scientists
and researchers as well as to industries based on the herbals.

G.B. Pant Institute of Himalayan Lok Man S. Palni

Environment and Development
Kosi-Katarmal, Almora, Uttarakhand,
INDIA
(An Autonomous Institute of Ministry
of Environment and Forests, Government of India)
Preface

Herbal drugs are time tested and valuable resource for healing, even today,
globally. As the demand and commercial value of these drugs is increasing
tremendously, assurance of safety, quality, and efficacy of medicinal plants
and produces is becoming a crucial issue. The need of the hour is to develop
an evidence-based market of herbal drug raw materials and herbal drugs.
Herbal drugs are composed of many constituents and are therefore very capa-
ble of variation; hence, it is very important to obtain reliable fingerprints that
bear pharmacologically active and chemically characteristic components of
the herbal drug. The information generated based on fingerprints pattern has
a potential application in the identification of an authentic drug, in excluding
the adulterants, and in maintaining the quality and consistency of the drug.
Several analytical techniques have been developed for obtaining fingerprinting
profiles of the herbal drugs and have assured to be a valuable tool for proving
constant composition of herbal preparations by establishing relevant criteria
for uniformity. These fingerprints deal with the advanced extraction as well
as analytical techniques with the help of which qualitative and quantitative
evaluation of herbal drugs and formulations can be carried out and serve as a
rapid and unambiguous tool in the herbal research thereby allowing the man-
ufacturers to set quality standards, specifications, and seek marketing approval
from regulatory authorities. Quality control of herbal drugs is a tedious and
difficult job as herbs differ from that of the conventional drugs, so some inno-
vative methods are into practice for the sake of quality assessment. Fingerprint
analysis using chromatography has become the most potent tool for quality
control of herbal drugs because of its simplicity and reliability, for
identification, authentication, and adulteration. These fingerprints have global
acceptance by regulatory authorities to determine authenticity and reliability
of chemical constituents of herbal drugs and formulations.
The herbal raw material is prone to a lot of variation due to several factors,
the important ones being the identity of the plants and seasonal variation
(which has a bearing on the time of collection); the ecotypic, genotypic and
chemotypic variations; drying and storage conditions; and the presence of
xenobiotics. World Health Organization (WHO) stresses the importance of
the qualitative and quantitative methods for characterizing the samples,
quantification of the biomarkers and/or chemical markers, and the fingerprint
profiles. In case a principle active component is known, it is most logical to
develop fingerprints for the same as main evidence, on the whole fingerprint

vii
viii Preface

profile, whereas the active ingredients contributing to therapeutic efficacy are

not yet known as marker substance should be specific for the medicinal herb
for evidence-based fingerprints. The advancements in modern methods of
analysis and the development of their application have made it possible to
solve many of these problems. Extremely valuable are techniques like TLC,
HPTLC, HPLC, GC, MS, LC–MS, GC–MS, NMR, LC–MS–NMR, and
GC–MS–NMR. Starting from raw material, processing, and formulation into
suitable dosage form, claims are deciphered by fingerprints at every step. At
each and every step, fingerprints have to be generated and a multiple-marker-
based fingerprints strategy needs to be adopted to minimize batch-to-batch
variation and to maintain quality and ensure safety and efficacy.
In order to have a good coordination between the quality of raw materials,
in-process materials and the final products, it has become essential to develop
reliable, specific, and sensitive fingerprints using a combination of classical
and modern instrumental method of analysis. It also encompasses the entire
field of study from birth of a plant to its clinical application, including herbal
drug preparation of a defined content of a constituent or a group of substances
with known therapeutic activity, respectively, by adding excipients or by
mixing herbal drugs or herbal drug preparations.
Sometimes, same species of medicinal herb has different pharmacological
activities depending on its area of origin, the methods used to extract it, and
the part of the herb used, among other factors. After establishing differences
and similarities in fingerprints among different samples of the same type of
herb, we can attempt to have an answer as to why these different samples give
different pharmacological activities, ultimately explaining the “cooperative
effects” of components and effectively controlling the quality of these drugs
during production, using hyphenated techniques. Fingerprints by hyphenated
techniques lead to high robustness with all information along structural
characterization.
The sole aim of this book is to collect all the fingerprint techniques for
herbal drugs at one place, by various case studies globally for different rea-
sons, using latest innovations, so that it may be helpful in the development of
evidence-based herbal drugs and new herbal drug development.

D.D. Joshi
Acknowledgements

The author wishes to record his sincere thanks to Dr. Ashok K. Chauhan,
Founder President, Ritnand Balved Education Foundation for providing
logistic support to promote research on utilization of herbals as evidence-
based drugs. Sincere thanks to Mr. Atul Chauhan, Chancellor, Amity
University Uttar Pradesh, for providing opportunities with different intellec-
tual forum via workshop/seminar and exhibitions of national and interna-
tional standards from time to time.
Many other individuals are responsible for text. Author would like to
thank Dr. L.M.S. Palani (Director, GBPIHED), Dr. Rajeev Kr. Sharma
(Director, PLIM, Govt. of India, Ghaziabad), Dr. D.K. Uprati (Scientist,
NBRI-Lucknow), Dr. A.B.S. Rawat (Scientist, NBRI-Lucknow), Professor
(Dr.) B.S. Kaphalia (University of Texas, USA), Dr. Harendra Kharkwal
(Dy. Director, MoEF, Govt. of India), Col. (Retd.) Dr. Y.P. Singh (N. Delhi),
Dr. P. Joshi (HPL, Govt. of India), Dr. Vidhu Aeri (Associate Professor,
Jamia Hardard, New Delhi), and Dr. Hismi Jamil Husain (Sr. Environment
Advisor, Rio Tinto) for their sincere advices and time to time guidance.
Due permission for discussion from Prof. (Dr.) P. Pushpangadan,
Prof. (Dr.) S.N. Raina, Dr. Amit C. Kharkwal, Prof. (Dr.) Deepsikha Pande
Katare, Dr. Harsha Kharkwal, Prof. (Dr.) Rajni singh, Dr. Kirti Rani Sharma,
Mr. N.C. Nainwal, and Dr. Ram Prasad (from Amity Group) is thankfully
acknowledged.
Finally, the author would like to thank family members for allowing taking
time away from their precious company. To, wife Kummu and sons Gaurav
and Harshit, for your inspiration, motivation, and computer guidance; your
untiring love has given support and enthusiasm to chase the dream.
A special appreciation is due to Dr. Mamta Kapila (Editor, Life Sciences,
Springer India, N. Delhi) who has helped from the beginning, especially to
design the excellent chapters found in this volume; without her inputs, the
publication would not have been in the present face.

D.D. Joshi

ix
Contents

Part I Herbal Drugs: A Review on Practices

1 Herbal Drugs: A Review on Practices ........................................ 3

Introduction .................................................................................... 3
Asian Continent and Traditional Herbal Drugs ............................. 3
China and Japan ........................................................................ 3
Indian Subcontinent .................................................................. 5
Traditional Herbal Drugs in Europe............................................... 6
Traditional Herbal Drugs in America ............................................ 7
Traditional Herbal Drugs in Australia ........................................... 8
Traditional Herbal Drugs in Africa ................................................ 8
Industrialization of Herbal Drugs and Legislation......................... 8
Phytochemical Standardization ...................................................... 9
Extraction of Therapeutics ........................................................ 10
Analysis for Marker and Chromatographic Fingerprint............ 11
Preparative HPLC to Isolate Therapeutic.................................. 11
Biochemical Approach .............................................................. 12
Reverse Pharmacology ................................................................... 12
New Drug Development ................................................................ 13
Synthetic Drug Development .................................................... 13
Modern Approach for Drug Development ................................ 14
Fingerprints of Drugs: Needs and Values ...................................... 14
Multidisciplinary Strategy to Develop Fingerprints ...................... 15
Modern Chemistry and Pharmacology...................................... 15
Standardization of Fingerprints ................................................. 16
Pharmacokinetics of Standardized Form ....................................... 16
Case Study................................................................................. 17
Commercial Manufacturing and Quality Control .......................... 18
Regulatory Norms for Herbal Drugs.............................................. 19
The Therapeutic Goods Act ...................................................... 19
Drug Administration Law ......................................................... 19
Ayush ......................................................................................... 20
Ministry of Health and Welfare, Japan...................................... 21
Ministry of Health, Saudi Arabia .............................................. 22
WHO Guidelines for Assessment of Herbal Drugs .................. 22
References ...................................................................................... 24
Bibliography .................................................................................. 24

xi
xii Contents

Part II Herbal Drugs and Chromatographic Fingerprints

2 TLC: Herbal Drugs and Fingerprints........................................ 29

Thin-Layer Chromatogram ............................................................ 30
Preparation of TLC Plate in Laboratory.................................... 30
Ready-Made TLC Plate............................................................. 30
Selection of Suitable Plate Size................................................. 30
Spotting ..................................................................................... 30
Preparation of Developing Chamber ......................................... 32
Chromoplate Generation ........................................................... 33
Evaluation of TLC Plate............................................................ 33
Retention Factor ........................................................................ 34
Presentation of TLC Results ..................................................... 34
Procedure to Determine Rf Value of Unknown ......................... 34
Spot Development by Iodine Vapors ......................................... 35
Documentation of Fingerprints ................................................. 35
Two-Dimensional TLC ............................................................. 35
TLC Bioautography .................................................................. 37
Bioluminescence and TLC Analysis ......................................... 37
Detection of Colorless Compounds .......................................... 38
Combination of TLC with Other Techniques............................ 38
Criteria for Selectivity, Reproducibility and Robustness .......... 39
Special Hazards ......................................................................... 39
Troubleshooting in TLC Analysis ............................................. 39
Identification of Marker Compounds in Herbal Drugs .................. 40
TLC Analysis for Alkaloids ...................................................... 42
TLC Analysis for Phenols ......................................................... 43
TLC Analysis for Saponins ....................................................... 43
TLC Analysis for Terpenoids .................................................... 44
TLC with DART–MS ................................................................ 45
TLC Fingerprints for Batch to Batch Consistency ........................ 46
References ...................................................................................... 47
Bibliography .................................................................................. 48
3 HPTLC: Herbal Drugs and Fingerprints .................................. 49
Operational Summary of HPTLC .................................................. 49
HPTLC Pre-Coated Plates ............................................................. 50
Detection and Visualization ........................................................... 50
Nondestructive Techniques ....................................................... 50
Nonreversible Reactions ........................................................... 52
Destructive Techniques ............................................................. 52
Common Visualizing Reagents ................................................. 53
Group-Specific Reaction ........................................................... 55
Coupling of HPTLC with Spectrometry ........................................ 57
References ...................................................................................... 59
Bibliography .................................................................................. 59
Contents xiii

4 HPLC: Herbal Drugs and Fingerprints ..................................... 61

A Glance on HPLC Column .......................................................... 62
Analytical Mobile Phases and HPLC ............................................ 63
Solvent Changeover in HPLC ........................................................ 64
Buffers as Mobile Phase ................................................................ 64
Selection of Buffer and Analytical Method Development ........ 65
Buffer Concentration ................................................................. 65
Buffer Solubility........................................................................ 65
Buffer and Its Impact on Detection ........................................... 65
Buffers as Mobile Phase in HPLC ............................................ 66
Detectors in HPLC: Detection, Quantification, and Fingerprints ..... 66
HPLC–UV ................................................................................. 66
HPLC–FD ................................................................................. 68
HPLC–CL ................................................................................. 68
HPLC–ECD............................................................................... 69
HPLC Detection in Hyphenated System........................................ 69
HPLC–RID................................................................................ 69
HPLC–ELSD............................................................................. 70
HPLC–CAD .............................................................................. 70
HPLC–FID ................................................................................ 71
HPLC–MS ................................................................................. 71
HPLC–NMR ............................................................................. 72
LC–Hyphenation for Online Structural Identification ................... 72
HPLC–DAD (Diode-Array Detector) ............................................ 74
HPLC–MS, MS–MS, and MSn ................................................. 74
HPLC–NMR (Nuclear Magnetic Resonance)........................... 74
HPLC Biochemical Detection ................................................... 75
HPTLC–HPLC for Quality Assurance .......................................... 75
Case Study................................................................................. 75
Different Chemical Classes from Natural Products
and LC Fingerprints ....................................................................... 76
Fingerprints for Flavonoids ....................................................... 77
Fingerprints for Terpenoids ....................................................... 77
Fingerprints for Alkaloids ......................................................... 78
Fingerprints for Coumarins ....................................................... 78
Fingerprints for Alkamides ....................................................... 78
References ...................................................................................... 78
Bibliography .................................................................................. 81
5 GC: Herbal Drugs and Fingerprints .......................................... 83
Instrumentation .............................................................................. 83
GLC Columns ........................................................................... 84
Detectors in GLC ...................................................................... 85
Development of GC Fingerprints and Data Interpretation............. 86
Sample Preparation ................................................................... 86
Separation and Detection .......................................................... 87
Analytical Results ..................................................................... 87
Utility of GC Fingerprints in Herbal Drugs ................................... 88
Contamination/Adulteration...................................................... 88
xiv Contents

Traceability................................................................................ 88
Qualification and Quantification of Therapeutics ..................... 88
Nutraceutical Discovery ............................................................ 91
New Cosmeceuticals ................................................................. 93
In Aromatherapy ....................................................................... 93
To Decipher Traditional Formulations ...................................... 94
References ...................................................................................... 96
Bibliography .................................................................................. 96

Part III Herbal Drugs and Spectral Fingerprints

6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints .......... 101

Instrumentation .............................................................................. 102
UV–Vis. Spectrometry and Herbal Drugs ..................................... 103
To Study the Phenolics and Derivatives .................................... 103
UV Spectrometry for Different Derivatives .............................. 105
UV–Vis. Coupled Methods for Analysis .................................. 106
Preparation of Sunscreens ......................................................... 106
Evaluation of Vitamin C Content ................................................... 109
Preparation of Stock Solutions .................................................. 109
Green Synthesis of Nanoparticles .................................................. 110
SNPs from C. tamala Twigs...................................................... 110
Purification of Geometrical Isomers .............................................. 113
Analysis of Ultra-Diluted Drugs .................................................... 114
Identification of Powdered Medicinal Plants ................................. 116
Biological Assay Using Hyphenated Techniques .......................... 116
To Differentiate Various Species of a Genus ................................. 116
References ...................................................................................... 119
Bibliography .................................................................................. 120
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints............... 121
Interpretation of IR Spectra ........................................................... 123
Authentication of Herbal and Herbal Drugs .................................. 125
Interpretation of Data ................................................................ 131
Identification and Comparison of Biomolecules............................ 133
Authenticity of Herbal and Herbal Drugs ...................................... 137
To Distinguish Herb from Its Morphological Fakes ...................... 138
Standardization of Metal-Based Herbal Drugs .............................. 141
References ...................................................................................... 145
Bibliography .................................................................................. 145
8 Mass Spectroscopy: Herbal Drugs and Fingerprints ............... 147
Interpretation of Mass Spectra ....................................................... 149
Techniques for Analysis of Ions in MS.......................................... 149
Detection and Recording of Fingerprints ....................................... 149
Electrospray MS for Herbal Fingerprints.................................. 150
Matrix-Assisted Laser Desorption Ionization ........................... 156
Hyphenated Techniques ................................................................. 160
References ...................................................................................... 161
Bibliography .................................................................................. 161
Contents xv

9 NMR Spectroscopy: Herbal Drugs and Fingerprints............... 163

Metabolic Fingerprints ................................................................... 165
Case Study 1.............................................................................. 165
1
H-NMR Detection .................................................................... 172
1
H-NMR Spectra to Recognize Patterns and Find
Discriminating Signals .............................................................. 173
2D-NMR Spectroscopy to Compare Metabolites ..................... 174
Evaluation of Quality of High Therapeutic-Value Herbals ............ 176
Case Study 2.............................................................................. 176
Materials and Chemicals ........................................................... 176
Molecular Modeling....................................................................... 178
Case Study 3.............................................................................. 178
To Decipher the Herbal Constituents ............................................. 179
Case Study 4.............................................................................. 179
Metabolic Fingerprints to Distinguish Plant Species ..................... 182
Case Study 5.............................................................................. 182
Quality Assurance of Traditional Medicines ................................. 183
Case Study 6.............................................................................. 183
Detection of Contaminations ......................................................... 184
References ...................................................................................... 185
Bibliography .................................................................................. 186

Part IV Pollutants in Herbal Drugs and Fingerprints

10 Metals in Herbal Drugs and Fingerprints ................................. 189

Atomic Absorption Spectroscopy .................................................. 189
Instrumentation in AAS ............................................................ 190
Case Study 1.............................................................................. 191
Case Study 2.............................................................................. 192
The Graphite Furnace Atomizer .................................................... 193
Inductively Coupled Plasma .......................................................... 193
WDXRF Spectroscopy................................................................... 194
Heavy Metals ................................................................................. 195
Removal of Heavy Metals from Herbal Drugs .............................. 195
Case Study 3.............................................................................. 196
Bhasmas as Nanoherbal Drugs ...................................................... 198
References ...................................................................................... 199
Bibliography .................................................................................. 200
11 Pesticide Residues in Herbals and Detection ............................. 201
Regulatory Norms .......................................................................... 202
Analysis of Pesticide Residues ...................................................... 202
Isolation and Purification Methods ........................................... 203
Hyphenated Techniques for Isolation and Purification ............. 204
Contemporary Trends in Pesticide Residue Analysis ............... 205
Degree of Uncertainty of Analysis ............................................ 207
Pesticides Residues in Natural Products ........................................ 208
Current Scenario of Pesticides Residues in Herbals ...................... 209
xvi Contents

Pesticide Residues in Dry Fruits .................................................... 210

TLC Fingerprints....................................................................... 211
GC Fingerprints......................................................................... 211
References ...................................................................................... 212
Bibliography .................................................................................. 212
12 Radiation Detection in Herbals................................................... 213
Microorganisms in Herbals ............................................................ 214
Safety and Effectiveness of Irradiation .......................................... 216
Irradiation of Spices and Dried Vegetable Seasonings .................. 216
Irradiation Dose for Essential Oils and Organoleptic Characters .. 217
Radiation Quantity and Antioxidant Activity ................................ 218
Detection of Irradiated Foods and Food Supplements ................... 218
Thermoluminescence ................................................................ 220
Pulse Photo-Stimulated Luminescence ..................................... 221
Electron Paramagnetic Spin Resonance Spectroscopy ............. 221
References ...................................................................................... 226
Bibliography .................................................................................. 226

Part V DNA Techniques for Evidence-Based Herbals

13 Herbal Drugs and DNA Fingerprints......................................... 231

Authentication of Traditional Formulations................................... 232
Experimental Details ................................................................. 232
RAPD Reaction ......................................................................... 232
Identification of Chemotypes, Ecotypes, and Substitutes .............. 233
Case Study................................................................................. 233
Identification of Adulterants .......................................................... 234
DNA Fingerprint Techniques for Identification of Ingredients...... 234
Polymerase Chain Reaction ...................................................... 235
Simple Sequence Repeats ......................................................... 235
Restriction Fragment Length Polymorphisms .......................... 235
Amplified Fragment Length Polymorphism ............................. 236
Random Amplified Polymorphism ........................................... 237
Single-Nucleotide Polymorphism ............................................. 238
Short Tandem Repeat ................................................................ 238
DNA Barcode ............................................................................ 238
Genetic Markers ........................................................................ 239
Trouble Shooting in DNA Fingerprinting ...................................... 239
Commercial Utility of Genetic Markers in Herbal Technology ....... 240
Comparative Genetic Analysis....................................................... 242
Future Scope of Genetic Markers in Herbal Drug Research ......... 244
References ...................................................................................... 244
Bibliography .................................................................................. 245

About the Author ................................................................................. 247

Index ...................................................................................................... 249

 Part I
Herbal Drugs: A Review on Practices

The use of herbals as drug is older than recorded history, as mute witness to
this fact are marshmallow (Althaea officinalis) root, hyacinth (Hyacinthus
orientalis), and yarrow (Achillea millefolium), found carefully tucked around
the bones of a Stone Age man in Iraq. These herbs are used as demulcent,
diuretic, and common cold remedy, respectively, even today. Ayurveda and
TCM are two great living traditions of the world which are related to herbal
healthcare. Every continent has its folk system for healing and caring, and a
few are still recognized by the state healthcare agencies, in different coun-
tries. King Hammurabi of Babylon (1800 bc) had prescribed the use of mint
for digestive disorders; now, it has been established by modern science that
peppermint indeed relieves nausea and vomiting by mildly anesthetizing the
lining of the stomach. The knowledge of herbal drugs was widely dissemi-
nated throughout Europe by the seventeenth century. Nicholas Culpeper had
written “A Physical Directory” and “The English Physician,” the first manu-
als that a layperson could use for healthcare, and it is still widely referred and
quoted. The first publication of the US Pharmacopoeia (1820) included an
authoritative listing of herbal drugs, with descriptions of their properties,
uses, dosages, and tests of purity. It was periodically revised and became the
legal standard for medical compounds in 1906, but with development of
synthetic chemistry, synthetic therapeutic ingredients gained preference, and
herbal drugs became the secondary choice.
The synthetic therapeutic products could not be accepted more due to
severe side effects, and there was relook for herbal therapy, and natural prod-
ucts were found as storehouse of different molecular designs, even beyond
the human imagination. A few traditional remedies directly passed the modern
molecular approach for treatment, for example, ephedrine, an active ingredi-
ent of Ephedra, is used in the commercial pharmaceutical preparations for the
relief of asthma symptoms and other respiratory problems, as it helps the
patient to breathe more easily, while Ephedra in TCM was in use since 2,000
years back to treat the same ailments. Foxglove (Digitalis lanata) leaves are
known since 1775, till the date, as cardiac stimulant and keeps alive the
millions of heart patients worldwide. The active ingredient has been identified
as digoxin, a medicine of all pharmacopoeias.
2 I Herbal Drugs: A Review on Practices

There are over 750,000 plants on earth, but relatively, only a very few
have been studied for healing. Modern pharmacology looks for one active
ingredient and seeks to isolate it, with the exclusion of all others. Most of
the research done on herbals is focused on identifying and isolating active
ingredients, rather than studying the medicinal properties of whole herb.
Herbalists, however, consider that the power of herbal lies in the interaction
of all its ingredients. Used as a drug, herbals offer synergistic interactions
between ingredients both known and unknown. FDA has categorized
most of the herbals as food supplements or nutraceuticals, knowingly
acknowledging the wisdom of centuries-old practices. The sole aim of this
section is a review on the different approaches adopted globally, by different
regimes, at different times, based on sociopolitical scenario to ensure
for correct quantity and quality of herbal drugs to induce the desired ther-
apeutic effects.
 Part II
Herbal Drugs and Chromatographic
Fingerprints

Chromatography is a technique for the separation of mixture of solutes

brought about by the dynamic partition or distribution of dissolved or dis-
persed material between two immiscible phases, one of which is moving on
the other. Every type of chromatography contains a mobile phase and a
stationary phase. The moving phase may be liquid or gas. Chromatography,
in its various forms, is used for concentrating/identification of component
which is in dilution but has high commercial value (e.g., Taxol from T. baccata,
digoxin from D. lanta, and forskolin from C. forskohlii). This is an extremely
valuable technique for the separation, isolation, purification, and identification
of components from the mixture. Chromatographic fingerprints of herbals
and herbal drugs may be defined as the chromatographic pattern of pharma-
cologically active and or chemically characteristic constituents present in the
extract, which is featured by the fundamental attributions of integrity and
fuzziness with the reference or reference standard. Due to geo-climatic fac-
tors, there is high variability of chemical components, even in the same
species, collected from different zones. As the therapeutic effect of herbals is
based on interaction of numerous ingredients present on it, so different kinds
of chromatographic fingerprint techniques for quality control of herbal drugs
have gradually come into practice, such as TLC, HPTLC, HPLC, and CLC,
for the purpose of species authentication, evaluation of quality and ensuring
the consistency, and stability of herbal drugs and their related products.
In herbals, a large number of chemical components are involved, and many
of them are in low concentration. Chromatographic instruments and experi-
mental conditions are difficult to reproduce during real analysis resulting in
baseline and retention time shifts from one chromatogram to another, and a
few associated complications such as abnormal chromatograms and ghost
peaks are additional difficulties during fingerprint development, so chemo-
metric approaches such as variance analysis, peak alignment, correlation
analysis, and pattern have been employed to deal with the chromatographic
fingerprint. Many mathematical algorithms are used for data processing in
chemometric approaches. The basic principles for this approach are variation
determination of common peaks/regions and similarity comparison with sim-
ilarity index and linear correlation coefficient. Similarity index and linear
correlation coefficient can be used to compare common pattern of the
28 II Herbal Drugs and Chromatographic Fingerprints

chromatographic fingerprints obtained. In general, the mean or median of the

chromatographic fingerprints under study is taken as the target, and both are
considered to be reliable. To facilitate the data processing, a “computer aided
similarity evaluation” (CASE) software has been developed.
Currently, many hyphenated techniques with chromatography are in prac-
tices for quality assessment, authentication, and content quantification, and
there is still lookout for further innovations. A brief on the utility of a few
chromatographic techniques with latest update to develop fingerprints of
herbals and herbal drugs is described in this section.
 Part III
Herbal Drugs and Spectral Fingerprints

The relook on herbal healthcare has developed a challenge and demand to

regulatory authorities and scientists to authenticate that products/produces
have not been adulterated with contaminants or cheaper ingredients. Analytical
authentication is an important quality criterion for efficacy and safety, as it is
focused on specific molecular markers or on recording compositional profiles
of the ingredients. Spectroscopic techniques, which are based on interaction
between electromagnetic radiation and atoms/molecules of the sample, are
very attractive tools due to simplicity, fast and easy mode of operation.
Complex mixtures are analyzed with hyphenated chromatographic devices to
cross validate the analysis. Electromagnetic spectrum is a classification of
photons with various energies into different spectral regions. UV–Vis. spec-
troscopy is used when high-energy photons (wave length 200–400 nm) are
absorbed by atoms/molecules of sample which causes electronic excitation.
Visible wavelengths cover a range from 400 to 800 nm.
IR spectroscopy is an analytical technique used to identify organic, as well
as some inorganic, materials in the herbals and products, in solid as well as
liquid forms. Mid-infrared spectroscopy (400–40,000 cm−1) is useful to iden-
tify a variety of adulteration problems using experimental and statistical
methods. NIR (4,000–14,000 cm−1) is another rapid, reliable, and nondestruc-
tive technique that is widely used for quality and processing control for the
qualitative characterization of various products in exploring bulk material
with little or no sample preparation. FTIR, an advanced form of IR, looks at
the mid-infrared spectrum. The region between 1,500 and 400 cm−1 is referred
to as the fingerprint region. Absorption bands in this region are generally due
to intramolecular phenomena and specify molecular composition and
structure.
The m/z value of molecule and its elucidation is characteristic of mass
spectrometry fingerprints while NMR for the environment of protons around
it. LC–MS has become method of choice in many stages of drug development.
LC–NMR improves speed and sensitivity of detection and found useful in the
areas of pharmacokinetics, toxicity studies, drug metabolism, and drug dis-
covery process. The identification of adulterants in a Chinese herbal medi-
cine was done by LC–NMR technique. GC–MS instruments have been used
for identification of large number of components present in natural and biological
100 III Herbal Drugs and Spectral Fingerprints

systems. The identification and quantification of chemical constituents present

in polyherbal oil formulations is carried out by GC–MS method.
For trace-level analysis, a combination of column liquid chromatography
or capillary gas chromatography with a UV–Vis. or an MS has become the
preferred approach to develop reliable fingerprints. Various hyphenated pro-
cedures used for the analysis of herbal drugs are HPLC–DAD, CE–DAD,
GC–MS, LC–MS, HPLC–MS, HPLC–DAD–MS, and LC–DAD–MS. The
data obtained from such hyphenated instruments are the so-called two-way
data; say one way for chromatogram and the other way for spectrum, which
could provide much more information than the classic one-way chromatography.
A “total analysis device” has been recently demonstrated in the case of online
HPLC–UV (DAD)–FTIR–NMR–MS analyses. In this section, efforts have
been focused on to gather all related potential information based on important
case studies, in summarized form, as a clue for any new problem, to develop
validated fingerprints.
 Part IV
Pollutants in Herbal Drugs
and Fingerprints

Environmental pollution especially with heavy metals, pesticides, radioactive

particles, mycotoxins, and microbes including pathogens poses serious prob-
lem on quality of herbal and herbal drugs. It is a common misperception that
natural herbs, used as drugs since centuries, cannot be toxic, though at once
it was true, but due to environmental pollution, a few toxic compounds are
being accumulated at alarming level on plants. Heavy metals such as Cu, Zn,
Cr, Fe, and Co are required in very trace quantities for the proper functioning
of enzyme systems, hemoglobin formation, and vitamin synthesis in human
and for the growth and photosynthesis in plants. Metabolic disturbances are
encountered in case of both deficiency and excess of these essential metals.
On the other hand, Pb, Cd, As, and Hg (toxic metals) are not required by the
body, and they produce deleterious effects upon exposure even at very low
concentrations. Along heavy metals, pesticides used for protection from
certain insects also have presence in the herbals and transported finally to
human body with different routes. Aflatoxins (AF) and Aflatoxin B1 (AFB1),
the most biologically active form of AF produced by the fungi Aspergillus
parasiticus and Aspergillus flavus, are major contaminants in herbals, respon-
sible for poor performance, liver lesions, and immunosuppression. Unregulated
irradiations of herbals for longer durability are the major cause of presence of
radiations. The contamination of pollutants in herbals remains and continues
during their transportation and storage. Such contaminated herbs are one of
the major potential sources of pollutants in the human organs and systems,
because these are not only utilized as herbal drugs and food supplements, but
many of them are consumed as condiments in daily routine.
To get the desired therapeutic outcome, quality of the finished products
and plant raw materials must be ensured. Many reports have shown that one
of the major quality problems frequently encountered is high concentration of
these pollutants. The development of phyto-radioprotective agents has been
the subject of intense research in view of a radiation environment, such as
space exploration, radiotherapy, and even nuclear war. However, no ideal,
safe synthetic radio-protectors are available to date. In Ayurveda, several
plants have been used to treat free radical-mediated ailments that may be
relevant to the mitigation of ionizing radiation-induced damage in mammalian
systems (e.g., Allium sativum, Aloe vera, Tinospora cordifolia, Hippophae
188 IV Pollutants in Herbal Drugs and Fingerprints

rhamnoides, Curcuma longa, Centella asiatica, Stephania tetrantra, Spirulina

platensis, Syzygium cumini, Ocimum sanctum, Moringa oleifera, and Zingiber
officinale).
The use of herbal drugs undetected for presence of these pollutants
as antioxidants, cyto-protective, ACE-inhibitors, immunomodulators, metal-
loelements, prostaglandins, radioprotective, etc., will have different results,
so WHO has made it mandatory to estimate the pollutants in every batch.
This section is composed with objectives to familiarize from the technique
used for developing fingerprints of these pollutants citing different case stud-
ies to have evidence-based herbal drugs.
 Part V
DNA Techniques for Evidence-Based
Herbals

Herbal authentication in itself involves many parameters including gross

morphology microscopy, chemical analysis, and DNA fingerprinting. DNA
technology became more important in case of folk healthcare system where
different plants species are used under the generic name, so reliable authenti-
cation and quality control is necessary for the protection of consumers, sus-
tainable development of the industry, and integration of folk herbal drugs into
mainstream. Each herb contains large number of compounds, so it is not
possible to analyze for presence or absence for all compounds. The DNA
fingerprints remain the same irrespective of the plant part used, while the
phytochemical content varies with the plant part used, physiology, and envi-
ronment. Interspecies variation has been reported using DNA marker in
different genera such as Glycyrrhiza, Echinacea, and Curcuma. DNA markers
are helpful to identify cells, individuals, or species as they can be used to
produce normal, functioning proteins to replace defective ones. Moreover,
these markers help in treatment of various diseases and distinguishing the
genuine herb from adulterated. DNA fingerprinting provides an objective
evaluation of genetic identity of plants based on species, cultivars, or geographic
origin and ensure genetic uniformity of raw herbal materials. Chromatographic
techniques such as TLC, HPTLC, and HPLC provide chemical fingerprinting
(i.e., profiling of various chemical constituents). The combination of DNA
and chemical fingerprint techniques is an effective tool in authentication and
quality control.
Britain is using DNA fingerprinting of residues of orange to establish the
substitution of premium citrus fruits with those from lower quality variety by
an orange juice manufacturer. France has adopted DNA fingerprint tech-
niques to ascertain the fraudulent adulteration of Chianti wines with inferior
quality grapes. The method also facilitates the management of biodiversity, as
several international plant resource germplasm collection centers are exploiting
DNA fingerprinting on maintaining and propagating those unique collections.
DNA-based molecular markers have been found useful in differentiating
different accessions of Taxus wallichiana, Azarchdichta indica, Juniperus
communis, Codonopsis pilosula, Allium schoenoprasum, and Andrographis
paniculata collected from different geographical regions.
230 V DNA Techniques for Evidence-Based Herbals

Presently, there is no legal control model for trade of raw herbals, globally.
Different countries define herbals or produce in different ways and have
adopted different approaches to licensing, dispensing, manufacturing, and
trading to ensure their safety, quality, and efficacy. Only fingerprinting of
herbal drugs is utilized for the authenticity and quality control. With the
advancement of DNA fingerprints techniques, it is in practice as a rapid and
specific tool in the herbal research, thereby, allowing the manufacturers to set
quality standards and specifications and to seek marketing approval from
regulatory authorities.
 Herbal Drugs: A Review on Practices
Introduction
Herbal drugs have been used throughout history
similar to the way modern pharmaceuticals are
utilized today (i.e., to improve human health). Fossil
evidence suggests that plants were used medici-
nally in prehistoric times. Although the first use
of plant-derived pharmaceuticals may be difficult
to pinpoint, but use of herbals for human health is
the basis for the modern pharmaceutical industry.
History makes a man wise, so we start from the
point to the present to have prosperous future on
the subject to be discussed. Traditional herbal
drugs are very popular in different systems of
medicines like Indian system of medicine (ISM),
traditional Chinese system (TCM), Unani natur-
opathy, oestropathy, and homeopath. All over the
world, plants play an important role in healthcare
of majority of population. In India alone, nearly
two million traditional health practitioners use
plants for treatment of various ailments [1]. In
traditional system, only a few markers of phar-
macologically active constituents were employed
to assess the quality and authenticity of complex
herbal medicines; however, the therapeutic effects
of herbal drugs are based on the complex interac-
tion of numerous ingredients in combination,
which are totally different from those of synthetic
drugs. Thus, many kinds of chemical fingerprint
methods to control the quality of herbal drugs
have gradually come into practices, such as thin-
layer chromatography, gas chromatography, and
high-performance liquid chromatography. The
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_1, © Springer India 2012
1
chromatographic fingerprint analysis represents a
comprehensive qualitative approach for the
purpose of species authentication, evaluation of
quality, and ensuring the consistency and stability
of herbal drugs and their related products. The
entire composition of compounds is evaluated to
determine not only the presence or absence of
desired markers or active constituents but the
complete set of ratios of all detectable analytes.
The chemical fingerprints obtained by chromato-
graphic and electrophoretic techniques, espe-
cially by hyphenated chromatographies, are
strongly recommended for the purpose of quality
control of herbal drugs, to decipher ingredients,
and therefore be used for authentication and
identification of the herbal products. Current
global regulations recommend a drug, which is
assessed to toxicity, and other clinical data.
Asian Continent and Traditional
Herbal Drugs
The two famous ancient human civilizations of
world, the Chinese civilization and the Vedic
civilization, were from Asia, had well-flourished
healthcare system, mainly based on herbals, and
still have acceptance as time tasted system.
China and Japan
Traditional Chinese medicines (TCM) have been
used by Chinese people from ancient times,
3
4 1 Herbal Drugs: A Review on Practices
although animal and mineral materials have been
used, but primary source of remedies is botanical.
In TCM, about 12,000 herbals are used by tradi-
tional healers, of which 500 are common. Herbals
are generally used only after processing, for
example, stir-frying or soaking in vinegar or
wine. In clinical practice, traditional diagnosis
may be followed by the prescription of a complex
and often individualized remedy. TCM are still
common use in China. More than half of the
population regularly uses traditional remedies,
with the highest prevalence in rural areas. About
5,000 traditional remedies are available in
China, accounting for approximately one fifth of
the entire Chinese pharmaceutical market [2].
Chinese herbalists usually do not prescribe single
herb for their patients, but recommend in combi-
nations of 8–15 herbs. There are three major rea-
sons to support the combination practice. Mutual
reinforcement involves combining two or more
very similar herbs together to create a stronger
effect. Mutual assistance is the way to use one
herb to help another work better. Mutual restraint
is meant to use one herb to reduce or eliminate
side effects of another herb in the combination.
Historically, Chinese medicines were practiced
largely within a family-based lineage system,
from generation to generation. The specific tech-
niques and knowledge required to practice were
transmitted from teacher to student in the context
of an apprenticeship relationship; because of this
way of transmitting the art and science of Chinese
medicine, there emerged many different styles of
practice, each associated with a particular family
lineage. At the time of the Chinese Cultural
Revolution, a decision was made to standardize
and secularize the practice of Chinese medicine.
This was carried out by examining the various
family lineages, extracting what they seemed to
have in common, eliminating anything that the
communist government considered to be too
overtly spiritual, and naming the resulting collec-
tion of knowledge and techniques as traditional
Chinese medicine (TCM), officially approved
version of Chinese medicine, which subsequently
is being taught largely in government-sponsored
schools instead of within a family-based appren-
ticeship system, and has three main objectives:
(a) to treat acute diseases by killing pathogen;
(b) to heal chronic illness (e.g., gastrointestinal
disorder, respiratory disorder, allergies, immune
system deficiency) by strengthening the body,
helping to recover it; and (c) maintaining daily
life healthy by keeping the balance of human
body.
In general, under TCM, herbs can treat a wide
variety of diseases and conditions, as compared
to synthetic drugs, and have much gentler and
safer impact. Most of Chinese herbs do not cause
side effects; even a few can be easily counter-
acted with other herbs. For these reasons, people
turned to TCM. More and more people rely on
TCM as alternative after synthetic medicine
failed and is a very good alternative for those who
are looking for a natural alternative for the con-
ventional western medicine. Presently, there are
many highly efficient Chinese herbal patented
medicines, for pain syndromes, gastrointestinal
disorders, neurological disorders, stress-related
syndromes, respiratory disorders, heart problems,
sexual dysfunction, allergies, and immune system
deficiencies, as well as replacements for antibiotics
and anti-inflammatory drugs [3]. A major disad-
vantage of the standardization of TCM is lack of
its spiritual roots. Contemporary practitioners
wishing to revive this spiritual rooting, and the
knowledge/techniques associated with it, often
name themselves as practitioners of “Classical
Chinese Medicine” (i.e., the form of the medicine
prior to the Cultural Revolution) or five-element
practitioners. Generally speaking, a TCM practi-
tioner will rely primarily upon the “eight-principle”
diagnostic framework. A TCM practitioner is
likely to pay more attention to physical symp-
toms and design treatment to eliminate the symp-
toms. The five-element practitioners, on the other
hand, tend to be more focused on the emotional
and spiritual aspects of the imbalance and aim
their treatments at the root cause of the dishar-
mony. Great variety exists among the Chinese
practitioners, but both the “eight principles”
and “five-element” frameworks are important
aspects of theoretical foundation of the medicine.
The uniqueness of Chinese medicines is the
insight look that physical, emotional, mental, and
spiritual aspects of an individual are always
Asian Continent and Traditional Herbal Drugs
interconnected and exist within a larger web
which ultimately includes the entire cosmos.
Some herbs are too strong for pregnant women
and may cause miscarriage. Certain foods can
have adverse effects on the herbal therapy. It is a
thumb rule that during use of TCM, one should
avoid the raw food (e.g., vegetables should be
cooked, but fruits are okay), greasy, strong-tasting
or -smelling, difficult to digest (such as beef), or
irritating to the digestive system (like spicy
foods). So it is recommended to consult to the
health expert before using TCM.
In Japanese traditional healing system, many
herbal remedies found their way from China
into the Japan, through Korea. Herbs native to
Japan have been classified in the first pharmaco-
poeia of Japanese traditional medicine in the
ninth century [4].
Indian Subcontinent
Ayurveda, a medical system primarily practiced
in India known for nearly 5,000 years, includes
diet and herbal remedies, while emphasizing the
body, mind, and spirit in disease prevention and
treatment. During Vedic civilization, Ayurvedic
scholars were very particular for quality control
and site for collection of suitable raw herbs for
medicine (e.g., Himalaya is the best place for
collection of medicinal plants of hill origin [5]).
There is description that scholars were not afraid
to diseases even to death, as they had expertise to
keep it away up to certain period by use of their
unique formulations [6]. In Vedic civilization,
Ayurveda was a system of healing cognized by
sages of the Vedanta philosophy, which describe
the relation of doctor (Vaidhya) and patient as
friends. The doctor had prime duty to protect the
life of his patient without any discrimination, as
the royal doctor of Ravan treated Laxaman, the
younger brother of Lord Ram during Ram–Ravan
battle. He disclosed the habitat of the unique
healing herb and instructed to Hanuman to collect
it [7]. The collection practices were of high moral
value as Lord Hanuman had difficulty in
identification of the herb, and to avoid adultera-
tion, he pulled the whole hill and went to Lanka
5
from Himalaya; finally, Sushen selected proper
herb and treated Laxaman [8]. Ayurveda, generally
mistaken with exotic herbs, oils, and therapies,
this is not what it is. It is finding of harmony with
the whole of life and ultimately realizing our true
universal nature as pure cosmic consciousness
beyond koshas (bodies) or ahamkara (ego). The
word Ayur + Veda = Ayurveda [(life) + (knowledge
or wisdom)] describes how the ancient sages
cognize Ayurveda, where first we have to go
beyond our own limited perspective of our current
experience of mind and try to see with more clarity
what mind really is and how all of our experience
is directly affected by it. The eternity of Ayurveda
is also described in the Charak Samhita (one of
the scriptures in Sanskrit language). Acharya
Charak (600 bc) was the father of Ayurvedic
medicine. His renowned work “Charak Samhita”
which is considered the encyclopedia of Ayurveda
today goes in depth about his principals, diagnoses,
and cures that still retain their potency and truth
even after a couple of millennia. His research led
to the facts of the human anatomy, embryology,
pharmacology, blood circulation, and diseases
like diabetes, tuberculosis, and heart disease.
Charak Samhita describes medicinal qualities
and functions of 100,000 medicinal plants on
which present scientists are still doing research to
find the fact as per their language. Therefore,
even Ayurveda, popularly known as the fifth
Veda, is originated in the divine mind and
descended from the divine sources to the ancient
physicians.
This ancient Indian science of healing seeks to
reestablish the harmony between the body and its
habitat by creating the optimum health environ-
ment. Over centuries, Ayurveda has had a nurturing
influence on ancient TCM, Unani, and the
humoral medicine practiced by Hippocrates in
Greece. The entire science of Ayurveda is based
on the “five great elements” (Panchabhuta) theory.
These five elements are earth (prithvi), water
(jal), fire (agni or tej), air (vayu), and ether (space
or akash). Ayurveda strongly advocate for ele-
mental structure of the body. There are also five
mahabhutas (elements) in the human body, and
these five mahabhutas are represented in the form
of doshas, dhatus, and malas. Outside the body,
6 1 Herbal Drugs: A Review on Practices
they form the basic ingredients of the drugs and
food ingredients. In a normal body of a living
being, these substances remain in a particular
proportion. However, because of enzymatic
action inside the human body, this ratio of five
mahabhutas or their equilibrium inside the body
gets disturbed. The body has, however, a natural
tendency to maintain equilibrium. It eliminates
some of the mahabhutas which are in excess and
takes some of the mahabhutas which are in short-
age. This shortage of mahabhutas is replenished
through the ingredients of herbs, food, drinks, etc.
Traditional Herbal Drugs in Europe
The “doctrine of signatures,” adopted and pro-
moted by Dioscorides, a Greek, working as a
Roman military physician, and wrote his De
Materia Medica, whereby plants have been used
for medicinal purposes according to their resem-
blance to parts of the human anatomy, for exam-
ple, shape or color. Theophrastus (372–286 bc)
introduced the word “orchids” for the under-
ground tubers, which resemble to testicles
(Fig. 1.1). The Greeks referred to testicles as
“orchis.” In Enquiry into Plants, Theophrastus
has reported that the orchids had medicinal
properties. Naturally, this led to orchid tubers
Fig. 1.1 Tubers of early purple orchid [9]
being used to heal diseases of the testicles, and
to stimulate lust (Table 1.1), and supposed to
produce a male progeny if given to men as whole
new tubers; and if the shriveled old tubers were
given to women, this should produce female
children [9].
William Turner was the first English herbalist
(1568) to describe the four main uses, including
the treatment of alcoholic gastritis [8], and after
11 years, Williams Langham reported antipyretic,
anti-consumption, and anti-diarrheal effects [9].
John Parkinson in 1640 described the tubers to
increase fertility in men, and the Ottomans
extracted the Salep of the dried tubers [10]. In the
East, Salep was (and is) mainly made from Orchis
morio, but it could be made in the UK from
Orchis mascula, the early purple orchid, or from
Orchis maculata or Orchis latifolia. Orchids, pre-
sumably as Salep, were dispensed in London in
Oliver Cromwell’s time, and before the introduc-
tion of coffee, hot drinks of Salep were sold at
stalls in the streets of London. The tubers were
mainly imported from the East but also came
from Oxfordshire. In Hamlet, Ophelia’s fantastic
garlands included “long purples” that were gen-
erally known either by a rude name or by the
name “dead man’s fingers” in rude scenes, a refer-
ence to testicles, then the orchids must have been
of the genera Orchis or Ophrys, second from the
Traditional Herbal Drugs in America 7

Table 1.1 Summarized detail of orchid products in European healing system [9]
Proposed use of orchid
Author Year Preparation Indication
Turner W 1568 Tuber with goats milk Aphrodisiac
Dry Antiaphrodisiac
Topical Antiseptic
Tuber Gastrointestinal due to wine
Langham W 1579 Tuber Antipyretic
Tuber Anti-consumption
Tuber Anti-diarrhea
Parkinson J, Ottomans 1640 Tuber Increase fertility in men
Tuber (Salep) Aphrodisiac
Make ice cream (Turkey)
Coffee substitute (Albania)

Table 1.2 Medical uses of vanilla [9]

Author Year Proposed use of vanilla derivatives
Aztec herbal 1552 Flavoring and perfume prevent fatigue in those holding
public office, bestow the bodily strength of a gladiator
Drive weariness far away
Drive out fear and fortify the human heart
Menashian et al. 1992 Improve food intake and reduce nausea and vomiting in
patients given chemotherapy
Fladby et al. 2004 Diagnostic of Alzheimer’s disease (patients cannot smell
vanilla)
Fitzgerald et al. 2004 Antimicrobial against Escherichia coli, Lactobacillus
plantarum, and Listeria innocua

implies genus Dactylorhiza where the tubers are
palmate and resemble fingers. Today, Salep is
largely collected from Asia. Turkey uses the
greatest bulk in making ice cream and beverages,
but not allowed to export the tubers any more.
Turkey still uses vast quantities, as takes 2,600
tubers to obtain 1 kg of dried tubers [9].
Traditional Herbal Drugs in America
Vanilla, an aromatic oil, is exuded from the seed
pods of vanilla, a well-known example of tradi-
tional herbal healing knowledge of America
(Table 1.2) [9]. Apparently the word “vanilla” is
derived from the Spanish word “vainilla” which
in turn came from the Latin “vagina” or pod or
sheath. The most important vanilla species is
Vanilla planifolia, introduced into Europe by the
Spanish in 1510 and brought to popularity in the
UK when the Marquess of Blandford introduced
it here in 1800. Unlike Salep, vanilla can be
farmed, but the flavor and aroma molecule, vanil-
lin (4-hydroxy-3-methoxy-benzaldehyde), is now
produced synthetically. The Aztecs had several
uses for vanilla, but today its medicinal uses are
confined to relieving nausea and improving food
intake in patients receiving chemotherapy and as a
diagnostic aroma for Alzheimer’s disease, with
loss of the sense of smell being an early manifes-
tation of this condition. It has been described as
an antimicrobial agent and acts as preservative to
prolong the life of food products. Vanilla pom-
pona was also used to flavor tobacco in Cuba [9].
8 1 Herbal Drugs: A Review on Practices
Table 1.3 Use of medicinal plants by Australian aborigi-
nes and early settlers [9]
Name of medicinal plants Uses
Cymbidium canaliculatum Cure for dysentery
Food
Cymbidium madidum Oral contraceptive
Cure for dysentery
Dendrobium teretifolium (bruised Rub to relieve pain
leaves)
Dendrobium discolor Poultice
Young canes
Mature canes (bruise and extract Cures ringworm
with spirit)
Traditional Herbal Drugs in Australia
The historical records describe the use of orchids
by Australian aborigines and early settlers
(Table 1.3). In addition, many orchid bulbs were
employed as emergency bush food, for example,
Gastrodia sesamoides (roasted), Dendrobium
speciosum, and Caladenia species. Diuris
maculata has sweet-tasting tubers, but Lawler
and Slaytor warn that some Australian herbal
bulbs have toxic alkaloids, for example, Liparis
reflexa [9].
Traditional Herbal Drugs in Africa
The traditional healing systems of Africa are still
a matter of study. Brian Morris has described 12
medicinal plants currently used as medicine in
Malawi. Nine of these are used for stomach com-
plaints and two for fertility problems. Interestingly,
two species, Cyrtorchis arcuata and Eulophia
cucullata, are employed to promote friendship,
the former being dried and pounded into a powder
and the latter prepared as an infusion of the roots.
Cyrtorchis arcuata is also employed to treat
diabetes or skin infections and Eulophia cucul-
lata to prevent epilepsy. An infusion of the leaves
and pseudobulbs of Bulbophyllum maximum is
used to protect against sorcery and Tridactyle
tricuspis to treat madness [9].
In Zambia, medicinal plants as boiled root
tubers of terrestrial orchids are used to make a
food dish, chikanda or kinaka. The orchids
involved are from three genera Disa, Habenaria,
and Satyrium. The orchids have become scarce in
Zambia and are now illegally imported from
Tanzania. Four million Tanzanian herbals are
currently sent from Tanzania to Zambia each
year. In Africa, an amulet of leaves of Ansellia
africana impregnated with a paste made from the
pseudobulbs is considered as a contraceptive but,
most conveniently, only in the short term for
unmarried women. In the Molucca islands, the
seeds of Grammatophyllum scriptum have been
added to a woman’s food to ensnare her for life.
Berliocchi also pointed out that Bourbon tea,
popular in the nineteenth century, was made from
an infusion of orchids from Mauritius and
Reunion that included Angraecum fragrans. The
tea was thought to be a sedative. A tincture was
also made to apply to the fingertips and improve
the sense of touch. Both vanilla and Salep are
widely in use for a delicious flavoring and won-
derful perfume, respectively, and used in making
ice cream and beverages, although many are not
enthusiastic about the aroma of Salep [9].
Industrialization of Herbal Drugs
and Legislation
The safety of some herbal ingredients have been
recently called into question because of the
identification of adverse events associated with
their use and, increasingly, because of the dem-
onstration of clinically relevant interactions
between herbs and prescribed drugs, for example,
the adverse events (stroke, heart attacks, heart-rate
irregularities, liver toxicity, seizures, psychoses,
and death) associated with use of ephedra in
formulations for weight loss. The bodybuilding
effects and increased energy due to kava-kava
(also known as kawa), widely used in Europe and
increasingly in Canada to treat anxiety, nervous-
ness, insomnia, pain, and muscle tension, have
raised issues to some countries to enforce regula-
tions restricting or banning these products. A few
herbs in common use have been suspected of
causing cancer. These include Aristolochia, Rubia
tinctorum, Morinda officinalis, and Senecio riddellii.
Phytochemical Standardization
Although prolonged and apparently uneventful
use of a substance usually offers testimony of
its safety, investigation of the potential toxicity
of naturally occurring substances may reveal
previously unsuspected problems.
The recent global resurgence of interest in
herbal drugs has led to an increase in the demand
for them. The need of the hour is to evolve a sys-
tematic approach and to develop well-designed
methodologies for the standardization of herbal
raw materials and herbal formulations. Traditional
systems of medicine are in use since centuries all
over the world. According to one estimate, 80%
of the world population still depends on herbal
products for their primary healthcare needs.
The toxic side effect of drugs of modern medi-
cine and the lack of medicines for many chronic
ailments have led to the reemergence of the herbal
drugs, with possible treatments for many health
problems. They have stood the test of time for
their safety, efficacy, cultural acceptability, and
lesser side effects. The chemical constituents
present in them are a part of the physiological
functions of living flora, and hence they are
believed to have better compatibility with the
human body. Most diseases, like diabetes, heart
diseases, cancer, and psychiatric disorders, are
multifactorial and hence need therapeutic inter-
vention at more than one level. Plants with com-
plex phytochemical mixtures have advantage
over single molecules in treating such diseases,
with an added advantage of being devoid of toxic
side effects [10].
With commercialization of the herbal drugs
assurance of safety, quality and efficacy has
become an important issue. The herbal raw mate-
rial is prone to a lot of variations due to several
factors, the important ones being the identity of
the plants and seasonal variation (which depend
on the time of collection), the ecotypic, genotypic,
and chemotypic variations, drying and storage
conditions, and the presence of xenobiotics.
WHO stresses the importance of the qualitative
and quantitative methods for characterizing the
samples and quantification of the biomarkers
and chemical markers with fingerprint profiles.
A known ingredient of the herbal leads to logical
therapeutic efficacy, whereas the active ingredients
9
are not yet known; the marker ingredient specific
for that particular botanical is chosen for analytical
purpose. The advancements in modern methods
of analysis and the development of their applica-
tion have made it possible to solve many of these
problems. Techniques like HPTLC, GC, MS,
HPLC, LC–MS, and GC–MS are extremely
valuable to establish the markers for unknown
herbals. Starting from sourcing of the raw mate-
rial, standardization, and preparation of the
extracts to formulation of the extracts into suitable
dosage form, the problems vary with each plant
species and part of the plant that is being used.
At each and every step, phytochemical profiles
have to be generated and a multiple-marker-based
standardization strategy needs to be adopted to
minimize batch-to-batch variation and to maintain
quality and ensure safety and efficacy [10].
Phytochemical Standardization
In herbals and herbal drugs, standardization starts
with correct identity of the sample, organoleptic
evaluation, pharmacognostic evaluation, volatile
matter, quantitative evaluation (ash values, extrac-
tive values), phytochemical evaluation, test for
the presence of xenobiotics, microbial load test-
ing, toxicity testing, and biological activity. The
phytochemical profile has a special significance
as it is directly linked with the activity of the
herbal drugs. The fingerprint profiles serve as
guideline to the phytochemical profile of the drug
in ensuring the quality, while quantification of
the marker compound(s) serves as an additional
parameter in assessing the quality of the sample.
Phytochemical standardization encompasses
all possible information generated with regard
to the chemical constituents present in an herbal
drug. Hence, the phytochemical evaluation for
standardization purpose includes (1) preliminary
testing for the presence of different chemical
groups, (2) quantification of chemical groups of
interest (e.g., total alkaloids, total phenolics, total
triterpenic acids, total tannins), (3) establishment
of fingerprint profiles, (4) multiple-marker-based
fingerprint profiles, and (5) quantification of
important chemical constituents.
10
Extraction of Therapeutics
The phytochemical evaluation is done by
scientifically designed work plan and finally vali-
dated before the chemical characterization of the
herb. Based on the physical and chemical proper-
ties, the therapeutics may be extracted by:
Supercritical Fluid Extraction
This is the most technologically advanced extrac-
tion system. Supercritical fluid extraction (SFE)
involves use of gases, usually CO2, and com-
pressing them into a dense liquid. This liquid is
then pumped through a cylinder containing the
material to be extracted. From there, the extract-
loaded liquid is pumped into a separation chamber
where the extract is separated from the gas and
the gas is recovered for reuse. Solvent properties
of CO2 can be manipulated and adjusted by
varying the pressure and temperature. The advan-
tages of SFE are the versatility it offers in pin-
pointing the desired constituents to extract from
a given material and the fact that end product has
no solvent residues left in it (CO2 evaporates
completely). The downside is that this technology
is quite expensive. There are many other gases
and liquids that are highly efficient as extraction
solvents when put under pressure. Coupled
SFE-SFC System in which a sample is extracted
with a supercritical fluid which then places the
extracted material in the inlet part of a super-
critical fluid chromatographic system. The extract
is than chromatographed directly using super-
critical fluid. Coupled SFE–GC and SFE–LC
system in which a sample is extracted using a
supercritical fluid which is then depressurized to
deposit the extracted material in the inlet part or a
column of gas or liquid chromatographic system,
respectively. SFE is characterized by robustness
of sample preparation, reliability, less time-
consuming, high yield, and also has potential
for coupling with a number of chromatographic
methods [10].
Microwave-Assisted Extraction
An innovative, microwave-assisted solvent
extraction technology known as microwave-
assisted processing (MAP) offers many advantages
1 Herbal Drugs: A Review on Practices
over conventional methods. Applications include
the extraction of high-value compounds from
natural sources, including nutraceuticals and
functional food ingredients, and pharmaceutical
actives from biomass. MAE technology offers a
few advantages as (a) improved products,
increased purity of crude extracts, and improved
stability of marker compounds, possibility to use
less toxic solvents and (b) reduced processing
costs, increased recovery and purity of marker
compounds, very fast extraction rates and reduced
energy and solvent usage. With microwaves drive
extraction as opposed to diffusion, very fast
extraction rates and greater solvent flexibility are
possible. Many variables, including the micro-
wave power and energy density, can be tuned to
deliver desired product attributes and optimize
process economics. The process can be custom-
ized to optimize for commercial/cost reasons,
and excellent extracts are produced from widely
varying substrates. Examples include, but are not
limited to, antioxidants from dried herbs, carote-
noids from single cells and plant sources, taxanes
from taxus biomass, essential fatty acids from
microalgae and oilseeds, phytosterols from
medicinal plants, polyphenols from green tea,
flavor constituents from vanilla and black pepper,
essential oils from various sources, and many
more [10].
Solid-Phase Extraction
This involves sorption of solutes from a liquid
medium into a solid adsorbent by the same mech-
anisms by which molecules are retained on chro-
matographic stationary phases. These adsorbents,
like chromatographic media, come in the form of
beads or resins that can be used in column or in
batch form. They are often used in the commer-
cially available form of syringes packed with
medium (typically a few hundred milligrams to a
few grams) through which the sample can be
gently forced with the plunger or by vacuum.
Solid-phase extraction media include reverse
phase, normal phase, and ion-exchange media.
This is method for sample purification that sepa-
rates and concentrates the analyte from solution
of crude extracts by adsorption onto a disposable
solid-phase cartridge. The analyte is normally
Phytochemical Standardization
retained on the stationary phase, washed and then
evaluated with different mobile phases, for exam-
ple, when an aqueous extract is passed down a
column containing reverse-phase packing mate-
rial, everything that is fairly nonpolar binds,
whereas everything polar passes through [10].
Analysis for Marker and
Chromatographic Fingerprint
A chromatographic fingerprint of an herbal drug
is a chromatographic pattern of the extract of
pharmacologically active ingredients. This chro-
matographic profile is featured by the fundamental
attributions of integrity and fuzziness or same-
ness and differences so as to chemically represent
the herbal drug investigated. The chromatographic
fingerprints are used for authentication and
identification of herbal drugs accurately. Herbal
drug and its extract have hundreds of unknown
components, and many of them are in low amount,
sometimes in various concentrations, so it is very
important to obtain reliable chromatographic
fingerprints that represent pharmacologically
active and chemically characteristic components
of the herbal drug. In the phytochemical evalua-
tion of herbal drugs, TLC is being employed
extensively for the following reasons: (i) It
enables rapid analysis of herbal extracts with
minimum sample cleanup requirement, (ii) it
provides qualitative and semiquantitative infor-
mation of the resolved compounds, and (iii) it
enables the quantification of chemical constituents.
Fingerprinting using HPLC and GLC is also carried
out in specific cases. In TLC fingerprinting, the
data that can be recorded using a high-performance
TLC (HPTLC) scanner includes the chromato-
gram, Rf values, the color of the separated bands,
their absorption spectra, l max, and shoulder
inflection(s) of all the resolved bands. All of
these, together with the profiles on derivatization
with different reagents, represent the TLC
fingerprint profile of the sample. The information
so generated has a potential application in the
identification of an authentic drug in excluding
the adulterants and in maintaining the quality and
consistency of the drug. HPLC fingerprinting
11
includes recording of the chromatograms, retention
time of individual peaks, and the absorption
spectra (recorded with a photodiode array
detector) with different mobile phases. Similarly,
GLC is used for generating the fingerprint profiles
of volatile oils and fixed oils of herbal drugs.
Furthermore, the recent approaches of applying
hyphenated chromatography and spectrometry such
as high-performance liquid chromatography–
diode array detection (HPLC–DAD), gas
chromatography–mass spectroscopy (GC–MS),
capillary electrophoresis–diode array detection
(CE–DAD), high-performance liquid chromatog-
raphy–mass spectroscopy (HPLC–MS), and
high-performance liquid chromatography–nuclear
magnetic resonance (HPLC–NMR) spectroscopy
are in use to generate additional spectral informa-
tion, which is very helpful for the qualitative
analysis and even for the online structural eluci-
dation [10, 11].
Preparative HPLC to Isolate Therapeutic
There are basically two types of preparative
HPLC. One is low-pressure (typically under
5 bar) traditional PLC (pressure liquid chroma-
tography), based on the use of glass or plastic
columns filled with low-efficiency packing mate-
rials of large particles and large size distribution.
A more recent form PLC, preparative high-
performance liquid chromatography (Prep HPLC)
has been gaining popularity in pharmaceutical
industry. In preparative HPLC (pressure >20 bar),
larger stainless steel columns and packing materi-
als (particle size 10–30 mm) are needed. The
examples of normal-phase silica columns are
Kromasil 10 mm, Kromasil 16 mm, and Chiralcel
AS 20 mm, whereas for reverse phase are
Chromasil C18, Chromasil C8, and YMC C18.
The aim is to isolate or purify compounds,
whereas in analytical work, the goal is to get
information about the sample. Preparative HPLC
is closer to analytical HPLC than traditional PLC
because its higher column efficiencies and faster
solvent velocities permit more difficult separa-
tion to be conducted more quickly. In analytical
HPLC, the important parameters are resolution,
12
sensitivity, and fast analysis time, whereas in
preparative HPLC, both the degree of solute
purity as well as the amount of compound that
can be produced per unit time, that is, throughput
or recovery, are important. This is very important
in pharmaceutical industry of today because new
products (natural, synthetic) have to be intro-
duced to the market as quickly as possible; using
such a powerful purification technique makes it
possible to spend less time on the synthesis
conditions [10, 12].
Most traditional drugs are administered as
mixtures of many components, and with today’s
knowledge of the many possible interactions
between drugs, and between food and drugs,
ethnopharmacological research deals with this
aspect too. Additive, synergistic, or antagonis-
tic effects are all possible. Various admixtures
have also been shown to affect the bioavailabi-
lity of pharmacologically active principles.
Pharmacological studies of traditional herbal
drugs provides clue to the isolation of active
principles [10].
Biochemical Approach
The traditional medicines, which are generally
prepared by means of aqueous extracts, have
hundreds of chemical compounds. Modern clinical
trial proved that a complex formulation com-
posed of up to 20 herbs had greater efficacy than
single herb used. Obviously, there exists certain
relation between biological activity and chemical
composition of herbal medicine, and it is called
as quantitative composition–activity relationship
(QCAR). Experimental studies, such as random
controlled trials (RCT), often provide the most
trustworthy methods for establishing causal rela-
tionships from data, in which one or more vari-
ables is changed (typically random) to measure
its effect on other variables. In recent years, the
relation between active ingredients of herbal drugs
and biological activity is one type of causal rela-
tionship which has been attempted. When the
amount of active components in certain formula-
tion varied, its therapeutic effect correspondingly
changed. Thus, causal analytical methods are
1 Herbal Drugs: A Review on Practices
employed to study the relation of chemical
composition and bioactivity of herbs, which is
helpful to discover active components. There are
few methods available for discovering causal
relationships, in which the actual process of con-
trolled experiment can be stimulated through
series of conditional dependence tests. A recent
approach called “stepwise causal adjacent rela-
tionship discovery” (STEPCARD) method has
been developed to overcome the disadvantage of
existing causal discovery algorithm, as well as
the unreliability of traditional statistical methods,
for example, stepwise regression. The main idea
of STEPCARD is using conditional dependency
test to determine causal adjacent relationships
between explanatory variables and predictor. For
a given data set containing chemical composition
matrix (parameter X) and bioactivity information
matrix (parameter Y), STEPCARD algorithm
can be applied to choose the components or
component combinations most correlative to
the biological activity of original formulation
(comparison drug). The computational results of
STEPCARD algorithm dealing with chemical and
biological data represents the minimal significant
level used in conditional independent test to pick
out at least one variable. But there is lack of
scientific approach to study correlations of their
chemical constitution and pharmacological mech-
anism. However, this work affords a new strategy
to identify active component or component com-
binations of ethnic medicine and is helpful to
accelerate the speed of new drug discovery [13].
Reverse Pharmacology
In normal drug discovery course, “laboratories to
clinic” approach is followed; while for herbal medi-
cine research, “clinics to laboratories” approach –
a true reverse pharmacology – is followed (Table 1.4).
The ethnomedicine is based on its use for many
years, and its clinical existence is presumed. For
bringing more objectivity and to confirm ethnic
claims, systematic clinical trials are necessary.
In latter, clinical experiences, observations, or
available data becomes a starting point, whereas
with conventional drug research, it is at the end.
New Drug Development 13

Table 1.4 Search for new pharmacological agents; general pathways [14]
Forward pharmacology Reverse pharmacology
Compound discovered Isolate a therapeutic target

Assay for biological activity Identify a compound that affects target

Determine mechanism Modify drug to maximize effects

Demonstrate the desired biological function in vivo

Process
1. Cell- or physiologically directed
2. Unbiased as to the compound’s mechanism of action
3. Must determine the mechanism of action often using
in vitro methods
Process
1. Molecular target-directed
2. Compound has demonstrated in vitro activity
3. Must demonstrate in vitro activity
4. Must demonstrate the compound acts by the proposed
mechanism of action

Expensive, time consuming, numerous bottlenecks

TARGET LEAD LEAD PRECLINICAL CLINICAL

IDENTIFICATION IDENTIFICATION OPTMIZATION STUDIES TRIALS

Drug to market 10-15 years

Fig. 1.2 Synthetic route for drug discovery [14, 15]

Reverse pharmacology is the science of integrating New Drug Development

documented clinical/experiential hits into leads
by transdisciplinary exploratory studies and fur-
ther developing these into drug candidates by
experimental and clinical research. In reverse
pharmacology approach process, safety remains
the most important starting point, and efficacy
becomes a matter of validation. The scope of
reverse pharmacology is to understand the mech-
anisms of action at multiple levels of biological
organization and to optimize safety, efficacy, and
acceptability of the leads in natural products
based on relevant science [14].
Synthetic Drug Development
In synthetic drug discovery, we have to recognize
an event at first and the molecule as possible drug
is applied, which can provide valuable insights
for drug development. Historically, several such
clinical hits do not often pursued quickly and
rigorously by the drug discovery teams. The
potential molecule, if any, as desired has to cross
the long journey of about 10–15 years, to be
called drug (Fig. 1.2) [14].
14
Economic, time spring, least bottlenecks
Reverse Pharmacology
RELEVANT
LARGE SCALE TRIALS SCIENCE
Drug to market 4-5 Years
Fig. 1.3 Modern approach for herbal drugs development [14, 15]
Modern Approach for Drug
Development
Reverse pharmacology (RP) is designed to dis-
cover new herbal drugs as an academic discipline
to reduce three major bottlenecks of costs, time,
and toxicity, major challenges associated with
synthetic route. RP can be perceived to comprise
of three phases: first, the experiential phase that
includes robust documentation of clinical obser-
vations of the biodynamic effects of standardized
herbal drugs by meticulous record keeping.
Secondly, it includes exploratory studies for
tolerance, drug interactions, dose-range finding
in ambulant patients of defined subsets of the
disease, and para-clinical studies in relevant
in vitro and in vivo models to evaluate the target
activity. Third phase includes experimental studies,
basic and clinical, at several levels of biological
organization, to identify and validate the reverse
pharmacological correlates of herbal drug safety
and efficacy. The scope of reverse pharmacology
is to understand the mechanisms of action at
multiple levels of biology and to optimize safety,
efficacy, and acceptability. In this approach,
scientist travels a reverse path from “clinics to
laboratory” rather than classical “laboratory to
clinics” (Fig. 1.3), with journey of 4–5 years, as a
new drug [14, 15].
Fingerprints of Drugs: Needs
and Values
In recent years, interest on plant-based drugs has
increased considerably, with annual growth rate
between 5 and 15 %, but the quality control and
1 Herbal Drugs: A Review on Practices
CLINICAL
SAFETY PARACLINICAL
TRIALS
STUDIES STUDIES
PHASE II & I
quality assurance still remains a challenge
because of the high variability of chemical com-
ponents involved, which has generated a need to
developed methods for fingerprinting. Herbal
drugs, singularly and in combinations, contain a
myriad of compounds in complex matrices in
which no single active constituent is responsible
for the overall efficacy. This creates a challenge
in establishing quality control standards for raw
materials and standardization of finished herbal
drugs. The main cause of confusion is language
as during translating from Chinese pin-yin termi-
nology into western languages or when the same
name is used in different regions for different
parts of the plant, or even different species or
genera, for example, a problem and confusion
resulting in the mistaken use of an herb in the
beginning of the 1990s in Belgium. Stephania
tetrandra, which is used in herbal treatment
against obesity, was exchanged with Aristolochia
fangchi, a herb resulting in a severe nephropathy
because of the presence of aristolochic acid.
Confusion probably occurred because of the sim-
ilarity in the pin-yin terminology of both plants:
feng fang ji vs. guang fang ji, respectively [11].
To ease out such problems, the identity and
quality can be derived from chromatographic or
spectral fingerprints. These fingerprints can be
defined as “a chromatographic pattern of an
herbal extract showing some common pharmaco-
logically active and/chemical characteristic com-
pounds.” The entire fingerprints are used as a
source of information because by assaying only a
number of compounds from the extract, the total
intrinsic quality of the herb is not necessarily
assessed. The fingerprint chromatograms and
spectra are also accepted by the WHO as an
Multidisciplinary Strategy to Develop Fingerprints
identification and qualification technique for
medicinal herbs and herbal drugs. Analysis and
handling of the fingerprint data is an important
aspect for stricter quality control to check the
conscious adulterations, where another plant is
sold, or to the unconscious mistaken use of “look-
alikes” [11].
The concept of phytoequivalence is in practice
in order to ensure consistency of herbal products.
According to this concept, a chemical profile,
such as chromatographic fingerprint, for herbal
product should be constructed and compared with
the profile of a clinically proven reference prod-
uct. Chinese State Food and Drug Administration
have framed a regulation for the compositions of
liquid injections with herbal ingredients using
stringent quality procedures for chemical assay
and standardization. Fingerprints of herbal medic-
inal liquid injections are compulsorily to follow
the above procedure of standardization and finger-
prints. In addition, among the various experi-
mental techniques, chromatographic methods are
highly recommended for finding out fingerprints
of herbal products because of the high separation
ability.
Multidisciplinary Strategy to Develop
Fingerprints
Modern Chemistry and Pharmacology
Herbal drugs are just considered as encapsulated
dried plant material, without the quality check
and/of the plant material, but it is not at all. Most
of the ingredients are extracts produced from a
defined part of a plant and represent a large col-
lection of compounds, and these compounds are
responsible for the overall activity of the drug.
Extracts are standardized on the basis of the con-
tent of these compounds, for example, the purified
fraction of opium poppy (Papaver somniferum),
highly effective for pain and insomnia, has addic-
tive nature. When it was realized, there was
tension ignited between China and England in
the mid-nineteenth century. Similar example may
be cited that hypericin, the active compound in
extracts of St John’s wort (Hypericum perforatum),
15
is used to prevent and treat some forms of depression.
Standard operating procedures and raw material
specification were developed to have St John’s
wort extracts of defined hypericin content, and
consistent yield, from different batches of plant
material. Despite the apparent simplicity of this
botanical, a study comparing 10 products showed
variations from the label claim for hypericin
ranging from 22 to 140 %. Another example may
be cited using ginseng products, standardized for
two types of chemical moieties of active com-
pounds, (1) ginsenosides and (2) eleutherosides,
and are generally standardized for the total con-
tent of each class of compound. Although this
form of standardization provides a means of
comparing products, it does not ensure that large
variations do not exist within each class of com-
pound that could affect the effectiveness of a
preparation. Similarly, a widely advertised diet
supplements from the South African plant Hoodia
gordonii were found to contain no putative active
ingredients and were likely derived from different
plants [13].
Standardization of herbals and herbal products
refers to the production of a plant preparation that
is consistent in terms of composition and efficacy.
The standardization processes begin with the
source of the raw material and continue till the
characterization of finished product with the
same quality as drugs. The environmental condi-
tions significantly affect phytochemical profiles
and finally the efficacy of the end product. Herbal
extracts prepared by the herb of certain location
can vary from year to year, as the secondary
metabolite production is regulated by tempera-
ture, drought, or flood, as well as by geographical
location. Therefore, biochemical profiling for
each batch is used to ensure that a consistent
material is being used to produce a quality
material and rejection of a particular crop, also.
A variety of molecular techniques including
restriction fragment length polymorphism, random
amplification of polymorphic DNA, and DNA
sequencing are used to authenticate plant mate-
rial and detect adulterant plant species. Each
technique has advantages and disadvantages in
terms of cost, accuracy, reproducibility, time, and
taxonomic level of identification. New technologies
16
and advances in molecular biochemistry have
strong scientific logic to prove that these tech-
niques are correct for standard regulatory norms
[13].
Standardization of Fingerprints
With the awareness of side effects of synthetic
drugs and rise of strains resistant to antibiotics,
pharmaceutical industries are turning to plant-
based drug. There is a common trend to look the
chromatographic fingerprints for unknown herb,
at the first. A consistent general profile is moni-
tored without regard for spot/peak identity that
may not be related to activity. The American
Herbal Pharmacopoeia describes the use of
marker compounds for characterizing products
with LC and/HPTLC fingerprint, which is useful
for establishing a baseline for specificity and
sensitivity. Other chromatographic techniques
with advantages and limitations are also in
practice [13].
Identifying active compounds in herbals is
often a challenge even though the process is
based on simple principles. Usually, active
compounds are isolated from a complex matrix
by activity-guided fractionation, which uses
chromatographic techniques such as HPLC for
separating compounds, followed by activity mea-
surements made with an in vitro assay related to
in vivo activity (e.g., measuring the inhibitory
activity of aldose reductase, associated with dia-
betes). The process is complicated when herbals
have activity related to complex metabolic disor-
ders that involve multiple metabolic pathways.
Sensitive in vitro assays are essential to the pro-
cess because fractionation can produce hundreds
of fractions requiring testing in sub-milligram
quantities. The active components may be major
components of the botanical or minor compo-
nents with high activity.
A major hypothetical advantage of herbals
over conventional single-component drugs is the
presence of multiple active compounds that
together provide a potentiating effect that may
not be achievable by any single compound. This
advantage presents a unique challenge for the
1 Herbal Drugs: A Review on Practices
process of activity-guided fractionation, however,
because the relative activity of fractions may
decrease with greater purity and may even be lost
entirely. The potentiating activity of the individu-
ally active components of herbals can be assessed
by recombining the fractions after separation
followed by confirmation of biological activity.
The complexity of the process increases when
multiple in vitro assays are used for activity-
guided fractionation, each yielding a different set
of active compounds. Alternatively, the interac-
tions of herbal components could be negative, as
in the case of the diminished bioavailability of
caffeine resulting from the flavonoids in tea [1].
Compounds identified by activity-guided
fractionation are tested in appropriate animal
models to confirm in vivo activity. The isolation
steps may involve chromatographic procedures
by solvent partitioning, medium-pressure liquid
chromatography, and countercurrent chromatog-
raphy. Depending on the compound and informa-
tion about its chemistry, it is possible to synthesize
the compound also. Synthesis of the compounds
may be difficult and expensive yet more economical
and efficient than isolating a comparable amount
from a plant. Pure active compounds are gener-
ally obtained for testing and quality control.
Pharmacokinetics of Standardized
Form
The extraction and purification process, often
necessary to concentrate therapeutic ingredients
to a sufficient level, may alter the properties,
mainly as their solubility and bioavailability.
The active compounds within herbal preparations
are very often hydrophobic and tend to precipi-
tate at high concentrations, so attempting to
improve efficacy by increasing concentration can
be counterproductive. The use of solubilizers and
bioenhancers is a logical consideration for many
standardized herbals just as for drugs. A wide
range of enhancers is available, each with specific
solubility properties. One widely used enhancer
is Capmul MCM C10, a glyceryl monocaprate
produced from edible fats and oils and commonly
used in lip products. In a rat study examining the
Pharmacokinetics of Standardized Form
a
0.2
AU
0.0
b
1.0
AU
0.0
10 20
Time (min.)
Fig. 1.4 Quality control assessment of Artemisia dra-
cunculus plants. Shown are HPLC chromatograms mea-
sured by a photodiode array detector at 254 nm from
identical ethanolic extractions of plants of Artemisia dra-
cunculus that were vegetative at age 6 weeks (a) and were
enteric bioavailability of the antibiotic ceftriax-
one, Capmul increased bioavailability by as much
as 80 %. In a similar study, coenzyme Q10 was
formulated with self-emulsifying drug delivery
systems, which are mixtures of an oil, a surfac-
tant, a co-surfactant, and the active substance
used for improving the bioavailability of lipo-
philic compounds. The optimized formulation
doubled the bioavailability of coenzyme Q10 in
dogs. A similar formulation of a lipid-based self-
micro-emulsifying drug delivery system was
used to enhance the bioavailability of a silymarin
preparation from Silybum marianum (milk thistle)
for liver disease in rabbits. Bioenhancers are
effectively used for pharmaceuticals, dietary
supplements, and botanicals [13, 16].
Case Study
In a study for therapeutic herb and value addition
on it using standardized extract, the ethanolic
extract of Artemisia dracunculus was evaluated.
Artemisia dracunculus is a potential herb for pre-
venting and treating type 2 diabetes. The extract
is active in both chemically induced diabetic mice
17
30 40
mature flowering plants at age 17 weeks (b). The profile
of B is consistent with the profile of the reference extract,
whereas the profile of A contains both quantitative and
qualitative differences, making it unacceptable as material
for production [13]
and a genetic model that develops severe type 2
diabetes. The plants used for the production of
the extract were grown hydroponically. The
extract was initially characterized by LC–MS
with a photodiode array detector and an electron
impact mass detector using spectral database
matching. The chromatograms of the extract,
measured at 254 nm, and the total ion current
electron impact mass spectrometry (EI–MS) were
determined. The crude extract was fractionated
into ten simple fractions based on HPLC reten-
tion. The active window was defined by using
activity-guided fractionation and in vitro assays
such as glucose uptake into muscle cells. Because
the identity of the compounds from spectral
matching of chromatographic peaks was only
tentative, initial standardization of the extract for
scientific quality control was based on the area of
the most abundant peaks in the retention time
period thought to contain the active compounds.
Preparatory HPLC was subsequently used to
isolate subfractions from the active fractions and
pure compounds from the subfractions. Activity-
guided fractionation and compound purification
led to the identification of active compounds
within the peaks (Fig. 1.4b). Compound
18
identification was confirmed with the use of com-
mercial standards, mass fragmentation pattern
libraries, or a combination of LC–MS, LC–MS/
MS, and NMR. The abundance of these com-
pounds is used for direct standardization of the
extract in addition to ultraviolet spectra, thereby
decreasing the likelihood that unknown compounds
can interfere with the analysis. Electrospray
ionization, a sensitive detection method, was
used to enhance the level of standardization by
confirming the molecular weights of the active
compounds and providing additional chemical
information about active compounds. Assays
were then used to validate the activity component
of the standardization process. Hence, this is an
example of a standardization technique that has
evolved together with the development of the
herbal drug [13].
Development of standardized method to prepare
Artemisia dracunculus extract. (A) Photodiode
array detection at 254 nm from HPLC separation
provides a sensitive profile of the extract that was
used for general fingerprinting. (B) LC-electron-
impact (EI)–MS analysis of the extract provides
both qualitative and quantitative data for the com-
ponents and provides partial or complete com-
pound identification, especially when confirmed
by comparison with chemical standards and
other spectroscopy techniques such as NMR.
Compounds identified in B are 1-6-demethoxy-
capillarisin, 2-davidigenin, 3-sakuranetin, 4-2,
4-dihydroxy-4-methoxydihydrochalcone, and
5-2, 4-dihydroxy-4-methoxy-di-hydrochalcone.
Further, the LC–MS-electrospray ionization
(ESI) analysis of the extract provides sensitive
and quantitative selected ion chromatograms
specific for the molecular weights of the com-
pounds of interest (M-H for negative ESI),
validating the other chromatographic techniques
as well as the ability to detect compounds that
may not be detectable by other means (e.g.,
compound with m/z 515)
Multi-herbal preparations are a rich resource
with a potential as future ingredients in herbal
dietary supplements. Scientific validation of herbal
therapeutics is necessary to ensure consistent
results between research studies and herbal prod-
ucts. Although many challenges exist at every
1 Herbal Drugs: A Review on Practices
level of development, the identity of the active
compounds and the validation of their efficacy
will be a requisite for the quality control and
standardization of herbal drugs. Patenting of
drugs derived from indigenous systems of
medicine has started to take epidemic proportions.
The current value of the world market for medic-
inal plants from leads given by indigenous and
local communities is estimated to be $43 billion.
Using traditional knowledge increased the
efficiency of screening plants for medical proper-
ties by more than 400%. The failures and non-
sustainability of the chemical route to agriculture
and health care provide an opportunity to reeval-
uate traditional knowledge systems and move
from the false hierarchy of these systems to a
plurality. Such a pluralistic view of knowledge
systems will imply respect for the different sys-
tems in their own logic and in their own episte-
mological foundations. It will also mean that one
system does not have to serve as the measure of
scientific adequacy for all systems and diverse
systems do not need to be reduced to the language
and logic of dominating knowledge systems.
Evidence-based herbal drugs have a universal
acceptability [13].
Commercial Manufacturing
and Quality Control
The term “standardized herbal extract” deciphers
the extract for free from any potentially hazard-
ous chemical manufactured by raw plant materi-
als obtained from plants that are cultivated
specifically for producing the extract or are
obtained as a by-product from the production of
another product, or are collected from the wild,
for example, ginseng is grown specifically for the
production of related extract, whereas grape seed
extract is a by-product of the commercial wine-
making industry. Herbals have been collected as
they grow in their native environments, but this
practice raises concerns about the consistency of
end products and is considered an irresponsible
collection technique that can lead to environmental
destruction and the endangerment of species
when not restricted to research purposes.
Regulatory Norms for Herbal Drugs
Plants grown specifically for the production
of specific extract for basic research are ideally
sourced from a characterized and uniform
genetic material with a taxonomic record of the
genus, species, and cultivar or other additional
identifiers. Records are maintained for the
source of the seed, locations and conditions of
cultivation, and exposure to possible chemical
treatments such as pesticides. Ideally, herbals
are cultivated under controlled conditions such
as hydroponics within climate-controlled green-
houses yielding consistent plant material. Only
with tight control over the entire process of
herbals production can seed-to-pill standardiza-
tion be achieved. When source plants are pro-
cured in various regions of the world, records of
plant identification, maintenance of voucher
specimens, and biochemical profiling may
suffice. Raw materials from international sources
also be carefully monitored for contamination
from a variety of sources including co-harvested
weed plants, toxic phytochemicals, and heavy
metals. Plants that were not directly treated with
pesticides can become contaminated by chemi-
cal drift. Depending on the formulation, humid-
ity, and temperature, pesticides can drift from
the site of intended application by as much as
4.8 km (3 miles) [12, 17–19].
Regulatory Norms for Herbal Drugs
The increasing acceptance of herbals as drug has
alarmed the regulating authorities globally. The
speculators and opportunist are under scan using
the different norms by each country; a few are as
below:
The Therapeutic Goods Act
In Australia, the “Therapeutic Goods Act 1989,”
sets out the legal requirements for the import,
export, manufacture, and supply. It has details of
the requirements for listing or registering all ther-
apeutic goods in the Australian Register of
Therapeutic Goods (ARTG), as well as many
other aspects of the law including advertising,
19
labeling, and product appearance. Australian
manufacturers of therapeutic goods must be
licensed, and their manufacturing processes must
comply with the principles of GMP. All medicines
manufactured for supply in Australia must be
listed or registered in the ARTG, unless they are
specifically exempt or excluded. Listed medicines
are considered to be of lower risk than registered
medicines. Most complementary medicines (e.g.,
herbal, vitamin, and mineral products) are exam-
ples of listed products. Medicines assessed as
having a higher level of risk must be registered
(not listed). Registered medicines include non-
prescription (low-risk, OTC) medicines and pre-
scription (high-risk) medicines. Complementary
medicines (also known as “traditional” or “alter-
native” medicines) include vitamin, mineral,
herbal, aromatherapy, and homeopathic products.
Complementary medicines may be either listed
or registered, depending on their ingredients
and the claims made. Most complementary
medicines are listed in the ARTG, and some are
registered (Therapeutics Good Administration,
1999). In New Zealand, supplements in the
market are largely manufactured in the USA,
follow the TGA, but regulations are not restric-
tive, and there are no limits on ingredients or
potencies, and structure/function claims are
allowed [20].
Drug Administration Law
In China, many herbal drugs have been used for
hundreds of years, and it is assumed in many
cases that they must work, for example, about
7,000 species of plants are used in China as herbal
drugs, but only 230 are most common, with in-
depth pharmacological, analytical, and clinical
studies. The 2000 edition of the Chinese
Pharmacopoeia included 784 items on TCM and
509 on Chinese patent medicines. Herbal medi-
cines in China are normally considered as medic-
inal products with special requirements for
marketing. New drugs have to be investigated
and approved according to the drug administra-
tion law. New traditional Chinese medicines are
classified under five categories based on the
20
amendment and supplement regulation of
approval of new traditional medicines [20]:
Class 1
1. Artificial alternatives of Chinese crude drugs
2. Newly discovered Chinese crude drugs and
their preparations
3. Active constituents extracted from Chinese
crude drugs and their preparations
4. Active constituents extracted from a composite
formulation of traditional Chinese medicines
Class 2
1. Injection of traditional Chinese medicines
2. Use of new medicinal parts of Chinese crude
drugs and their preparations
3. Effective fractions extracted from Chinese
crude drugs or natural drugs and their
preparations
4. Chinese crude drugs artificially developed in
an animal body and their preparations
5. Effective fractions extracted from a composite
formulation
Class 3
1. New composite formulations of traditional
Chinese medicines.
2. Composite preparations of traditional Chinese
medicines and chemical drugs with the main
efficacy due to the traditional Chinese
medicine.
3. Domestically cultivated or bred crude drugs
originally imported and commonly used in
China, and their preparations.
Class 4
1. Preparation with a change of dosage form or
route of administration.
2. Botanical crude drugs acclimatized from their
origin or crude drugs from a domesticated
wild animal in China.
Class 5
In 1995, the preparatory committee on Chinese
medicines was formed to manage the imple-
mentation of these recommendations; as a
result, 31 potent Chinese medicines that may
potentially cause adverse effects have been
1 Herbal Drugs: A Review on Practices
identified. Proprietary preparations containing
a combination of herbal ingredients and
conventional drugs are regulated in the same
manner as other conventional drugs. The
majority of suppliers are state-owned or state-
connected. The pharmacopoeial TCM allows
the parallel manufacturing and sale of both
pharmaceutical drugs and traditional herbal at
a point also.
Ayush
For traditional medicines in India, an authorized
body Ayush has adopted strict guidelines for all
herbal drugs (related to Ayurveda, Yoga, Unani,
Siddha, and Homeopathy) to be exported from
India [14]. With respect to this, Department of
AYUSH Govt. of India gave some parameters for
Drug Development, Standardization & Quality of
Ayurveda, Siddha, and Unani drugs, which
include five protocols as [21]:
Protocol-I: Standardization of single plant
material
Protocol-II: SOP of preparation of extracts
Protocol-III: Standardization of plant extract
Protocol-IV: SOP of finished product
Protocol-V: Standardization of formulations
These protocols based on most common
parameters such as morphological evaluation,
microscopic evaluation, physicochemical evalua-
tion, particle size, bulk density and tap density
(in case of powder crude drugs or powder formu-
lations), and assay for constituents (marker %,
major compounds like alkaloids, glycosides,
flavonoids/saponin). With respect to above
parameters are test for heavy/toxic metals (lead,
cadmium, mercury, and arsenic), microbial con-
tamination (total viable aerobic count, total
Enterobacteriaceae, and total fungal count), test
for specific pathogen (E. coli, Salmonella spp.,
S. Aureus, Pseudomonas aeruginosa), pesticide
residue (DDT, HCH, endosulfan, aldrin, mala-
thion, and parathion), test for aflatoxine (B1, B2,
G1, G2), and chelating agent (for Bhasma, Lepa,
Aswarista, etc.). Further stability assessment and
self life, safety assessment, documentation of
safety based on experience or toxicological studies,
Regulatory Norms for Herbal Drugs
Table 1.5 Status of various medical systems in India [14]
Medical system
Characteristics Ayurveda Siddha
Medicinal plants known 2,000 1,121
Licensed pharmacies 8,533 384
Hospitals 753 276
Dispensaries 15,193 444
Registered practitioners 438,721 17,560
Undergraduate college 219 6 37
Postgraduate college 57 3 8
assessment of efficacy by ethnomedicinal infor-
mation, and biological activity evaluation are
essential [18, 21]. Ayush has made it mandatory
for all ISM to be exported to meet the interna-
tional standards for contamination including
heavy metals in 2005. These guidelines can be
accessed on the Ayush website (http://www.indi-
anmedicine.org) [14]. The popularity of ISM is in
rise day by day and widely accepted (Table 1.5).
Ministry of Health and Welfare, Japan
Japanese traditional medicine is in use since more
than a thousand years: may be divided into folk
medicine and Chinese-concept-based Japanese
medicine (i.e., Kampo medicine). Kampo medi-
cine is so popular that the per capita consumption
of herbal drugs in Japan seems to be the highest
in the world. One hundred and forty-six Kampo
drugs are registered as drugs by the Ministry of
Health and Welfare (MHW) and are included in
coverage under the national health insurance
(NHI). Acceptance of Kampo drugs took place
without clinical validation studies. In 1989, about
80% of physicians reported prescribing Chinese
medicine. Physicians generally recognize the raw
herbs which have long been used as folk medicine
and which have also been used for a considerable
period as components of an industrial product.
These products are freely usable for the purposes
indicated in the monograph. Local traditional
usage is not sufficient for approval as a drug; the
claims and rules of combinations of herbal ingre-
dients are determined on the basis of the pharma-
cological actions of the ingredients. In absence of
21
Unani Tibetan Homeopathy
751 337 482
462 – 613
74 – 223
1,193 – 5,634
43,578 – 217,460
– 178
– 31
monograph, the claims reported in the Japanese
Pharmacopoeia are used as a guide [20].
In Japan, empirical facts or experience, such
as reference data and clinical test reports, is used
for the evaluation of a Chinese medicine, but
importance is given to the pharmacological action
of each ingredient. Safety and efficacy have been
estimated based on general methods employed
by modern medical science. In 1972, the MHW
designated 210 formulae as OTC drugs; this
selection was based primarily on the experience
of doctors actually practicing traditional Chinese
medicine. In 1976, the MHW specified 146 for-
mulations as NHI applicable prescription drugs.
In the case of an application for approval of a pre-
scription drug other than those previously listed,
specified data on safety, stability, comparison
with other drugs, clinical test results, etc., must
be submitted.
New Kampo drugs are regulated in essentially
the same way as western drugs in Japan. The
same data required for new western drugs are
required for new Kampo drugs, including data
from three-phase clinical trials. Since 1971, the
MHW has been running a program for reevalua-
tion of all drugs marketed before 1967; a new
system to reevaluate the efficacy and safety for
all drugs every 5 years was launched in 1988. An
advisory committee for Kampo drugs was estab-
lished in 1982 in close association with the MHW
in order to improve quality control of Kampo
drugs. Since the 1986 good manufacturing prac-
tice Llw, the standard applied to all pharmaceuti-
cal drugs has also applied to Kampo drugs. In
addition, in 1985, guidelines for ethical extract
products in oriental medicine formulations were
22 1 Herbal Drugs: A Review on Practices

Table 1.6 Different national regulatory authorities and guidelines [19]

S. no. Country Regulatory authority Guidelines
1 Australia Australian Department of Health Guidelines for preparation and presentation of
applications for investigational drug and drug
products
2 Canada Health Protection Board Preclinical toxicology guidelines
3 European Union Committee for Proprietary Recommendation for the development of
Medicinal Products nonclinical testing strategies
4 France Ministry of Public Health and Guidelines for analytical, pharmacological, and
Social Security toxicological testing of pharmaceuticals
5 India Directorate General of Health Drug and cosmetic rules
Services
6 Japan Ministry of Health and Welfare Guidelines for toxicity studies of drugs
7 Nordic countries Nordic Council on Medicines Guidelines for registration of new drugs
8 UK Department of Health and Social Guidance notes on application for product licenses
Security
9 USA Food and Drug Administration Guidelines for assessment of drugs and medical
device, safety in animals (issued by PMA, prepare
in conjugation with FDA)

developed. The MHW has three major systems
for collection of adverse reaction data. The first is
a voluntary system involving 2,915 monitoring
hospitals. The second system, the pharmacy
monitoring system, which includes 2,733 phar-
macies, collects data on cases [20].
Ministry of Health, Saudi Arabia
In Saudi Arabia, registration of medicinal prod-
ucts by the ministry of health is obligatory for
any ingredient having medicinal effects such as
herbal preparations, health and supplementary
food, medicated cosmetics, antiseptics, or medical
devices [20].
A few important organizations known for
strict regulations related to herbal drugs are as
per Table 1.6 [19] have guidelines for safe use of
herbal drugs.
WHO Guidelines for Assessment
of Herbal Drugs
The WHO has recently defined traditional medi-
cine as comprising therapeutic practices that have
been in existence, often for hundreds of years,
before the development and spread of modern
medicine and are still in use today. As per WHO,
every herbal formulation must be standardized.
WHO collaborates and assists health ministries
in establishing mechanisms for the introduction
of traditional plant drugs into primary healthcare
programs, in assessing safety and efficacy, and in
ensuring adequate supplies and the quality control
of raw and processed materials. According to
WHO guidelines, less stringent selection proce-
dures could be applied for the screening, chemical
analyses, clinical trials, and regulatory measures,
but the procedure for pure phytochemicals for
quality control should be identical to that for syn-
thetic drugs according to WHO guidelines. The
traditional preparations comprise medicinal
plants, minerals, organic matter, etc. Some of the
important parameters are stability testing, safety
assessment, specific therapeutic activity analysis,
and estimation of the active constituents in plant
raw material and finished products. The objective
of WHO guidelines is to define basic criteria for
the evaluation of quality, safety, and efficacy of
herbal drugs and therefore to assist national regu-
latory authorities, scientific organizations, and
manufacturers to undertake an assessment of the
documentation/submission/dossiers in respect of
such products. The manufacturing procedure and
Regulatory Norms for Herbal Drugs
formula including the amount of excipients
should be described in detail. A method of
identification, and quantification, if applicable, of
the plant material in the finished product should
be defined. If the identification of an active prin-
ciple is not possible, it should be sufficient to
identify a characteristic substance or mixture of
substances (e.g., chromatographic fingerprint) to
ensure consistent quality of the product.
According to WHO, “herbal drugs” should be
regarded as finished, labeled medicinal products
that contain as active ingredients aerial or under-
ground parts of plants or other plant material, or
combinations thereof, whether in the crude state
or as plant preparations. Plant material includes
juices, gums, fatty oils, essential oils, and any
other substance of this nature. Herbal medicines
may contain excipients in addition to the active
ingredients. Drugs containing plant material
combined with chemically defined active sub-
stances, including chemically defined, isolated
constituents of plants, are not considered to be
herbal medicines. Exceptionally, in some coun-
tries, herbal drugs may also contain, by tradition,
natural organic or inorganic active ingredients
which are not of plant origin. Multicomponent
herbal formulations can be standardized with
newer techniques such as DNA fingerprinting,
HPTLC, liquid chromatography, and mass spec-
troscopy. The value of animal testing to establish
safety and toxicity is not so critical if the herbs
are used in traditional forms. Nevertheless, all the
critical pharmacopoeial tests such as dissolution
time, microbial, pesticide, and heavy metals con-
tamination must be in accordance with global
standards, and all the Ayurvedic medicine manu-
facture must be in accordance with current good
manufacturing procedures for herbs [20].
Analysis of Raw Herb
Raw material can be defined as starting material
or any intermediate which will be utilized for
further processing. Before finished pharmaceutical
dosage forms are produced, the identity, purity,
and quality of raw materials as per specifications
for impurities and other related substances present
must be established with use of suitable test
methods. Pharmacopoeias and formularies of
23
various countries provide standardized test methods
for the most common and widely used materials
in their monographs. Stored drug samples are
prone to attack by harmful mycotoxin-producing
fungi. Detection of mycotoxins (aflatoxin B,
acliratoxin, citrinin, and zearalenone) is certainly
a matter of great concern in stored drugs of
important medicinal plants, for example, fruits of
Emblica officinalis (1.51 mg/g); Terminalia
chebula (1.19 mg/g) have developed an HPTLC
method for the detection of aflatoxins B1, B2, G1,
and G2 from herbal raw materials and estimated
the production of aflatoxins in caffeinated and
decaffeinated tea samples. Studies revealed that
caffeine acts as a good inhibitor for the growth of
aflatoxins in stored drugs [20].
Quality Control Techniques as per WHO
A lot of analytical techniques have been devel-
oped for renewed for quality control of drugs
from plant origin. The quality control step is
for selecting targets to assess authenticity and
inherent quality, but many traditional drugs are
claimed to exert their effects because each type
of chemical compound present many have a
different activity and then seem of all of these
may modify the action of the major active
component. Chromatographic fi ngerprinting
emphasizes an integral formulation of pharmaco-
logically active and phytopharmaceutically char-
acteristic components of samples with similar or
different attributions. This technique can be used
for the assessment of quality consistency and sta-
bility of herbal extracts or products by visible
observation and comparison of the standardized
fingerprint pattern. Fingerprinting of the herbals
is done by HPLC, HPTLC, MS, LC–MS [1],
H–NMR, etc. Apart from this, there are some
methods to evaluate the fingerprint quality of
herbal materials or pharmaceutical products,
such as correlative chromatography, comparative
analysis, wavelet analysis, and artificial neural
networks (ANN) [20].
Evaluation of plant materials and their derived
products has always been an important part of the
professional expertise of a pharmacognosist.
However, over the years the nature and degree of
this evaluation have changed. Initially, it was
24
considered sufficient to authenticate the plant
material by comparison with a standard botanical
description or monograph. Later, it was realized
that, for detection of adulterants, this practice
must be supplemented with other important pro-
cedures like microscopy, chemical tests, and
advanced analytical techniques. The main goal of
pharmacognosy is to assess the value of raw
materials and to ensure that the final product is of
the required standard. Strict standardization
procedures and pharmacognostical studies of
medicinal plants reduce drastically much of the
accidents in wrong prescriptions of traditional
herbal drugs, where a plant can be differentiated.
The use of herbal drugs is growing steadily,
particularly for the treatment of asthma and aller-
gic diseases, as health supplements and tonics.
Numerous reports on the presence of heavy metals
in herbal drugs are being presented regularly.
Federal agencies have put in a lot of efforts to
regulate the use of herbal drugs; nevertheless, the
situation is worse in developing and underdevel-
oped countries. Some of the crucial discoveries
in the safe use of herbal drugs were brought out
through intensive pharmacognostical research, so
need of hi-tech fingerprints is stressed by regula-
tory authorities.
References
1. Venkatesh V. Medicinal plant scenario in India.
Bangalore: Foundation for Revitalization of Local
Health Traditions; 2002.
2. Li L. Opportunity and challenge of traditional Chinese
medicine in face of the entrance to WTO (World Trade
Organization). Chin Inform Tradit Chin Med.
2000;7:7–8 (in Chinese).
3. Reninger E. TCM (Traditional Chinese Medicine) and
five element styles of practice. 2011. http://taoism.
about.com/od/qigongchinesemedicine/a/TCM.htm .
Accessed 7 Sept 2011.
4. Saito H. Regulation of herbal medicines in Japan.
Pharmacol Regul. 2000;41:515–9.
5. Shastri P K. Charak Samhita. 2nd ed., (Chaukhamba
Sanskrit Sansthan, Varanasi, Edited by Ganga Sahay
Pande). 1983;Sloka number 39, p. 7.
6. Shastri P K. Charak Samhita. 2nd ed., (Chaukhamba
Sanskrit Sansthan, Varanasi, Edited by Ganga Sahay
Pande). 1983;Sloka number 39, p. 14.
7. Tulshidas. Ramcharitmanas, Lanka Kand. (Published
by Gita Press Gorakhpur, India) p. 809.
1 Herbal Drugs: A Review on Practices
8. Tulshidas. Ramcharitmanas, Lanka Kand. (Published
by Gita Press Gorakhpur, India) p. 811.
9. Bulpitt CJ. The uses and misuses of orchids in medi-
cine. Q J Med. 2005;98:625–31.
10. Patil PS, Shettigar R. An advancement of analytical
techniques in herbal research. J Adv Sci Res.
2010;1(1):08–14.
11. Heyden YV. Extracting information from chromato-
graphic herbal fingerprints. LCGC Europe. 2008;
21(9): also available at http://chromatographyonline.
findanalytichem.com/lcgc/Column%3A+Practical+
Data+Handling/Extracting-Information-from-
Chromatographic-Herbal/ArticleStandard/Article/det
ail/565826?contextCategoryId=47195. Accessed 8
Aug 2011.
12. Mukherjee PK, Ponnusankar S, Venkatesh M. Ethno
medicine in complementary therapeutics. In:
Ethnomedicine: a source of complementary therapeu-
tics. Trivandrum: Research Signpost; 2010. p. 29–52.
13. Ribnicky DM, Poulev A, Schmidt B, Cefalu WT,
Raskin I. Evaluation of botanicals for improving
human health. Am J Clin Nutr. 2008;87(2):472S–5S.
14. Joshi DD, Sharma RK. Herbal drugs in patent regime.
Saarbrücken: VDM Publisher; 2011. p. 23–40. p.
69–70. ISBN ISBN-13: 978-3639321821.
15. Patwardhan B, Vaidya ADB, Chorghade M, Joshi SP.
Reverse pharmacology and systems: approaches for
drug discovery and development. Curr Bioact Compd.
2008;4:201–12.
16. Lazo JS. Rear-view mirror and crystal balls: a brief
reflection on drug discovery. Mol Interv. 2008;8(2):60–
3. Speaking of Pharmacology. Editorial.
17. Giri L, Andola HC, Purohit VK, Rawat MSM, Rawal RS,
Bhatt ID. Chromatographic and spectral fingerprinting
standardization of traditional medicines: an overview
as modern tools. Res J Phytochem. 2010;4:234–41.
18. Morgan K. Medicine of the gods: basic principles of
Ayurvedic medicine. 2002. [http://www.compulink.
co.uk/~mandrake/ayurveda.htm].
19. Rath SK, Sharma S, Srivastava S. Regulatory toxicology
for pharmaceuticals. CRIPS. 2001;2(2):2–9.
20. IARC Monograph. Some traditional herbal medicines.
Vol. 82. p. 43–67.
21. Patra KC, Parta SK, Harwansh RK, Kumar KJ.
Traditional approaches towards standardization of
herbal medicines: a review. J Pharm Sci Technol.
2010;2(11):372–9.
Bibliography
Andola HC, Gaira KS, Rawal RS, Rawat MSM, Bhatt ID.
Habitat dependent variations in berberine content of
Berberis asiatica Roxb. Ex DC in Kumaun, West
Himalaya. Chem Biodivers. 2010;7:415–20.
Andola HC, Rawal RS, Rawat MSM, Bhatt ID, Purohit
VK. Analysis of berberine content using HPTLC
fingerprinting of root and bark of three Himalayan
Berberis species. Asian J Biotechnol. 2010;2:239–45.
Bibliography
Gong F, Wang BT, Chau FT, Liang YZ. Data preprocessing
for chromatographic fingerprint of herbal medicine with
chemometric approaches. Anal Lett. 2005;38:2475–92.
Guo FQ, Liang YZ, Xu CJ, Li XN, Huang LF. Analyzing of
the volatile chemical constituents in Artemisia capillaris
herba by GC-MS and correlative chemometric resolu-
tion methods. J Pharm Biomed Anal. 2004;35:469–78.
Joshi DD, Uniyal RC. Different chemo-types of Gokhru
(Tribulus terrestris): a herb used for improving phy-
sique and physical performance. Int J Green Pharm.
2008;2(3):138–41.
Joshi DD, Kharkwal H. Commercial manufacturing of
herbal medicines: recent trends to select suitable chemo-
types as raw herb. Indo Italian workshop on bacteria and
fungi for environmental sustainability. 2010. p. 79–81.
Kala CP. Health traditions of Buddhist community and
role of Amchis in trans-Himalayan region of India.
Curr Sci. 2005;89(8):1331–8.
Li L. Opportunity and challenge of traditional Chinese
medicine in face of the entrance to WTO (World Trade
Organization). Chin Inform Tradit Chin Med.
2000;7:7–8 (in Chinese).
25
Meester FD, Waston RR. Wild type food in health promo-
tion and disease prevention. Totowa: Humana Press
Inc; 2009.
Ran XR, Yang HH, Liang QL, Chen J, Wang YM, Luo
GA, Li P, Li KM, Chen YW. Difference analysis of
mass spectra and its application to research of Chinese
multiherb remedy. Gaodeng Xuexiao Huaxue Yanjiu.
2007;2:250–3.
Sagar Bhanu PS, Zafar R, Panwar R. Herbal drug stan-
dardization. Indian Pharm. 2005;4(35):19–22.
Wohlmuth H, Penman KG, Pearson T, Lehmann RP.
Pharmacognosy and chemo-types of passionflower
(Passiflora incarnata). Biol Pharma Bull.
2010;33(6):1015–8.
Xie PS. Discussion of the present situations, development
and problems of fingerprint of Chinese medicines.
Zhongyaocai. 2007;3:257–9.
Ye M, Han J, Chen HB, Zheng JH, Guo DA. Analysis of
phenolic compounds in rhubarbs using liquid chroma-
tography coupled with electrospray ionization mass
spectrometry. J Am Soc Mass Spectrom.
2007;18:82–91.
 TLC: Herbal Drugs and Fingerprints
In an attempt to develop primary fingerprints of
herbals and herbal drugs, thin-layer chromatogra-
phy (TLC) is used to decipher the active principals,
identification of claimed herb, and possible con-
tamination/adulterants. The TLC method is efficient
and rapid and combines the sensitivity and sim-
plicity with low cost for the determination of
main active principles of medicinal plants (alka-
loids, anthraquinones, coumarins, essential oils,
flavonoids, glycosides, saponins, tannins, etc.),
which are the main ingredient responsible for
potential pharmacological effects. The technique
is so efficient that it is being used as primacy ana-
lytical tool in the new drug discovery for herbal
drugs. The technique is based on simple principle
that as the solvent moves over the spot that was
applied on the plate (stationary phase), equilibrium
is established for each component of the mixture
between the molecules of that component which
are adsorbed on the solid and the molecules which
are in solution. In principle, the components will
differ in solubility and strength of their adsorption
to the adsorbent (i.e., stationary phase), resulted a
few components carries faster up in the plate than
others. When the solvent has reached the desired
distance on the TLC plate (i.e., solvent front), it is
removed from the developing chamber, dried, and
evaluated. If the compounds are colored, visual-
ization is straightforward. Usually the compounds
are not colored, so UV lamp is used to visualize
the spots on the plate (sometimes plate itself
contains flour which fluoresces everywhere except
where an organic compound is on the plate).
Different compounds in the sample mixture travel
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_2, © Springer India 2012
2
different distances according to how strongly they
interact with the adsorbent. This property gener-
ates the concept of Rf (retention factor) value and
can be compared to standard compounds to aid in
the identification of an unknown substance in the
chromoplate. This technique is typically the start-
ing place for any sample of unknown composi-
tion. Confirmation of identity is achieved either
co-spotting (i.e., sample is spotted with stan-
dard) or using multiple mobile phases/two-
dimensional TLC in which it will exhibit a
change in Rf, if compound is different. TLC eval-
uation and identification depends on the Rf value
and upon color generated with a visualization
reagent. The mobile phase used for TLC may have
many factors together as applicable, when neces-
sary as: (1) mover in the solvent of high eluting
power, (2) restrainer, that is, of low eluting power,
(3) homogenizer is used in case the mixture of the
mover and restrainer is immiscible, (4) pH con-
troller is used when the mixture to be separated
has both acidic and basic functional groups, for
example, amino acids, (5) sharpener (i.e., the sol-
vent used to produce more compact spot), and (6)
viscosity reducer, the solvent used to reduce devel-
opment time of plate. In preparing the mixed sol-
vent, it is essential to measure the component
carefully because the small variation in composi-
tion can change the reproducibility/repeatability
and reuse of mobile phase for more than one devel-
opment is generally not recommended as the com-
position can change due to evaporation and the
optimum angle of 45° should be there between
adsorbent layer and solvent surface.
29
30
Thin-Layer Chromatogram
The quick screening of herbals/herbal products
for fingerprints and quality is possible, using
thin-layer chromatogram, even in least available
facilities. The entire process may be summarized
as below:
Preparation of TLC Plate in Laboratory
In TLC, an adsorbent is applied to a supporting
plate in a thin layer; generally, a binding agent is
used to adhere the adsorbent to the support,
although some work is done without a binder
using very finely divided adsorbent which clings
to the support and forms a rather soft layer. This
is to be distinguished from the loose-layer chro-
matograms in which the adsorbent does not
adhere to the supporting plate and must therefore
be developed in a horizontal or near-horizontal
position. Mostly, a mixture of adsorbent and
binder is applied as thin slurry, and the excess
moisture is removed under varying conditions
depending on the adsorbent, the binder, and the
desired degree of activity. TLC plates are made
by mixing the adsorbent, such as silica gel, with
a small amount of inert binder like calcium sulfate
(gypsum) and water. This mixture is spread as
thin slurry on an unreactive carrier sheet, usually
glass, thick aluminum foil, or plastic, and the
resultant plate is dried and activated by heating in
an oven for 30 min at 110°C. The thickness of the
adsorbent layer is typically around 0.1–0.25 mm
for analytical purposes and around 1–2 mm for
preparative TLC. After the starting point is
marked about 1.0 cm from the bottom of the
plate, the finish line is marked a convenient
distance from the starting point. This is done with
a very soft lead pencil, and care is taken not to
disturb the adsorbent layer at the point of sample
application since this leads to deformed spots.
The solution of the compound is deposited at the
starting line by means of a micropipette, and the
plates are then placed in a closed container con-
taining a layer of solvent about 0.5 cm deep. The
solvent ascends the plate by capillary attraction
2 TLC: Herbal Drugs and Fingerprints
until it reaches the finish line, at which time the
plate is removed and solvent allowed to evapo-
rate. The locations of the various substances are
then determined by developing spots to the color-
less compounds making visible.
Ready-Made TLC Plate
Presently, ready-made TLC plates with station-
ary phase either of silica gel (SiO2) or alumina
(Al2O3) are easily available in market. A TLC
plate is a sheet of aluminum foil, glass, metal, or
plastic which is coated with a thin layer of a solid
adsorbent (usually silica, modified silica, or alu-
mina) and may easily reduce to the desired size.
The stationary phase on the plates is of uniform
thickness and consists of fine particle size. The
aluminum sheets are preferred, as can be cut
without much more labor.
Selection of Suitable Plate Size
The ready-made TLC plates generally used are of
20 × 20-cm sheets. Each large sheet is cut hori-
zontally into small pieces which are 10 cm in
length and of desired widths; the more samples
we can plan to run on a plate, the wider it needs
to be. Skillful handling is required as coating of
adsorbent may disturb and/dirty (Fig. 2.1) during
reduction of size, of the TLC plate.
Spotting
Sample to be analyzed is spotted by either capil-
lary or syringe on the TLC plate; on the baseline
(Fig. 2.2) the process is called “spotting.” If the
sample is not already in solution, it is dissolve in
solvent of volatile nature such as hexanes, ethyl
acetate, or methylene chloride to have 1% solu-
tion. For concentrated sample, it is necessary to
dilute it to avoid a smear or streak; however, some-
times only we have to go by trial and error to have
well-sized spot for easy reading. Microcaps with
capillary (Fig. 2.3) or syringe are used for volu-
metric spotting with care that it does not disturb
Thin-Layer Chromatogram 31

Fig. 2.1 Cutting suitable

size of TLC plate

Fig. 2.2 Baseline marks for

spotting

Fig. 2.3 Microcaps with

capillary
32
Fig. 2.4 Sample application
on the plate
Fig. 2.5 Mobile phase in
developing chamber
the coating of adsorbent. A small spot of solution
containing the sample is applied on the plate,
about one centimeter above from the base (Fig. 2.4).
The plate is then dipped in to a suitable solvent
(i.e., mobile phase) and placed in a sealed con-
tainer. As solvent moves up in the plate by capil-
lary action and meets the sample mixture, which is
dissolved and is carried up the plate by the solvent.
Different compounds in the sample mixture move
at different rates due to differences in solubility in
the solvent and due to differences in their polarity
to the stationary phase. Results also vary depend-
ing on the solvent used, for example, if the solvent
is 90:10 mixture of hexane to ethyl acetate, then
2 TLC: Herbal Drugs and Fingerprints
the analyte would be mostly nonpolar. This means
that during separation on TLC plate, the nonpolar
parts will have move further up the plate.
Preparation of Developing Chamber
The developing container for TLC may be a
specially designed chamber, a jar with a lid, or a
beaker with a watch glass on the top/aluminum
foil/butter paper with rubber band; pour mobile
phase into the beaker to a depth of just less than
0.5 cm, cover the beaker, swirl it gently, and allow
it to stand till dip TLC plate on it (Figs. 2.5 and 2.6).
Thin-Layer Chromatogram
Fig. 2.6 TLC plate in
developing chamber
Nowadays, there are applicators aided with
software for mounting purpose, where in spite of
spots, narrow bands are produced. Such type of
devices is recommended when manual contact,
with samples due to safety reasons, is strictly
prohibited as extremely toxic solutions, microbi-
ologically contaminated samples, and radioactive
compounds.
Chromoplate Generation
When we place the spotted TLC plate in the
developing chamber, cover it and allow undis-
turbed on bench top until the solvent is about
half a centimeter below the top of the plate.
During the span when TLC plate is inside chamber,
a partially competing process occurs as (1) in
between the components of developing solvents
and their vapor, an equilibrium is established,
known as chamber saturation; (2) stationary
phase adsorb molecules from gas phase (adsorp-
tive saturation) and loaded into the surface of
stationary phase; (3) the wet part of the layer with
mobile phase also interacts with gas phase; and (4)
during migration, the components of the mobile
33
phase can be separated by the stationary phase
under certain conditions, causing the formation
of secondary fronts.
Evaluation of TLC Plate
In TLC plates, often a small amount of a
fluorescent compound, usually manganese-acti-
vated zinc silicate, is added to the adsorbent
that allows the visualization of spots under UV
254-nm wavelength. The adsorbent layer itself
has fluorescence, but spots of analyte quench it.
Compounds separated may not be UV detectable,
so several methods exist to visualize the spots by
spraying reagents [1]. Once visible, the Rf value
of each spot is determined by dividing the dis-
tance traveled by the product by the total distance
traveled by the solvent (the solvent front). These
values depend on the solvent used and the type
of TLC plate and are not physical constants.
Evaluation can be either visible or scanner is used
to measure the spot density, enabling the analyst
for quantitative results by the density at a specific Rf
value and then calculated the marker compound
in the sample with reference to area [2]:
34
Area (sample) × Volume (standard)
% ingredient =
Area (standard) × Volume (sample)
Cone. of standard
× × 100
Cone. of sample
Retention Factor
The retention factor (Rf) is defined as the distance
traveled by the compound divided by the distance
traveled by the solvent, from the baseline, for
example, if a compound travels 2.1 cm and the
solvent front 2.8 cm, the Rf is 0.75:
Solvent front
new position
of compound
Rf = distance travelled by the compound
distance travelled by the solvent
Presentation of TLC Results
The position of substance zone (spot) in the
TLC can be described with the aid of the reten-
tion factor Rf, which is defined as quotient
obtained by dividing distance between the sub-
stance zone and the starting line by the distance
between solvent front and starting line. Rf value
does not give any information about the meth-
odology used and other boundary parameters.
When Rf value is multiplied by 100, it is
referred as hRf value. Before presenting the
results, selectivity reproducibility and robust-
ness of the analytical method are necessary.
The Rf for a compound is a constant from one
experiment to the next, only if the chromato-
graphic conditions are similar (i.e., solvent
system, adsorbent, thickness of the adsorbent,
amount of analyte mounted, and temperature).
If the identity of a compound is suspected but
not yet proven, an authentic sample of the com-
pound, or standard, is spotted and run on a TLC
2 TLC: Herbal Drugs and Fingerprints
plate side by side (or on top of each other, i.e.,
co-spotting) with the compound in question. If
two substances have the same Rf value, they are
likely (but not necessarily) the same com-
pound. If they have different Rf values, they are
definitely different compounds. Notable point
is that this identity check should be performed
on a single plate because it is difficult to dupli-
cate all the factors which influence Rf exactly
from experiment to experiment.
2.1 cm 2.8 cm
origin
Rf = 2.1 = 0.75
2.8
Procedure to Determine Rf Value
of Unknown
On a TLC plate, mark a spot with pencil approxi-
mately 1 cm from the end of the slide, far enough
so that it will stay above the solvent in the devel-
oping jar. Pens should never be used on TLC
plate because the ink will also develop as spots
in the plate. Care is to be taken not to disturb
the surface of the silica while marking. On top of
the plate, label the spots with pencil according
to choice (i.e., A = anthracene, C = cholesterol,
T = test sample). Notable point, never to touch a
TLC plate on the face, only on the sides and back
are used; otherwise, extra spots may appear. The
plate now looks like as below (Fig. 2.7).
Now using volumetric capillary/clean cutoff-
flat syringe needle, dip it in the solution to be
spotted. The liquid rises in the needle by capil-
lary action. Now very briefly touch the needle to
the TLC slide on the pencil mark to spot the
material. The spot should be as small in diameter
Thin-Layer Chromatogram
Fig. 2.7 Pre-spotting plan Bottom of slide
on the TLC plate
(Sideways view of plate) •
as possible to keep the spots sharp and must not
run into another spot. Let the first spot dry, then
touch the needle down on top of it two or three
more times to ensure adequate sample. Between
spotting new samples, the needle should be
cleaned with suitable solvent. Now let the spot
dry, then carefully place the TLC plate in a devel-
oping chamber/jar containing about ½ cm of the
appropriate mobile phase on the bottom. Cover
the chamber/jar to ensure saturation of the air in
the chamber with solvent. Two or three plates
may be developed at the same time, if desired.
When the mobile phase has reached about three
fourths of the way up the plate, take it out of the
chamber and quickly mark the solvent front with
pencil before it evaporates. Let the plate dry and
visualize the spots in the following way. First,
hold the plate under an ultraviolet light and mark
the spots that fluoresce (never look directly at a
UV light). Next step is to place the plate in iodine
chamber and mark new spots, if any that become
visible. Third, after removing the plate from the
iodine chamber, dip it quickly into a 2% solution
of phosphomolybdic acid in 95% ethanol, wipe
off the back, and set it on a warm hot plate. The
hot plate should be on a setting low enough not to
melt the plastic plate but high enough to cause the
spots to appear. If the plate curls, it may be neces-
sary to hold it down with tongs or wire gauze.
When the dark green, orange, or brown spots
have appeared, remove the plate from the hot
plate. Measure the distance from the original
position of spotting to the spot and to the solvent
front. Calculate the Rf value and record it [3].
Spot Development by Iodine Vapors
Iodine vapor is used for quantitative estimation of
lipids on TLC plates is based on the fact that most
lipids can be stained by iodine vapor, in con-
trolled conditions, and the intensity of staining is
35
Top of slide
• A
C
• T
proportional to the actual amount of lipid in the
spot. The method consists of (1) exposing the
developed plate to iodine vapor, (2) spraying it
with a suitable solvent to prevent halogen evapo-
ration, (3) collecting the stained lipids by scrap-
ing the spots off the plate, and (4) determining by
a rate-sensing method the absorbed iodine. The
method has been successfully applied to analysis
of several common phospholipids, long chain
fatty acids, cholesterol, etc. [3].
Documentation of Fingerprints
The choice for fingerprints depends on the nature
of the constituents that is present in plant material
or on customer’s specification. TLC is widely
employed in herbal authentication, and the major-
ity of pharmacopoeial monographs for herbs
include a TLC identification test. TLC separates
mixtures of compounds to leave a “fingerprint”
of separated compounds on a plate coated with
silica gel. This fingerprint can be compared with
that of an authentic sample or pure reference
compounds. The chemical profile (fingerprint) of
raw material or of intermediate product (specific
extracts) or of the finished products against refer-
ence material defines claims made on certificate
of analysis as routine methodology for quality
control (Fig. 2.8) [3].
Two-Dimensional TLC
When the analyte to be studied is unknown, there
may be many components of very close polarity,
and clear-cut separation of the components may
not be achieved. In such cases, two-dimensional
TLC (2D-TLC) separation has advantage
(Fig. 2.9). In this case, a single spot of a mixture
is applied near to one of the corner of a 20 × 20-cm
plate and developed in one direction as usual.
36 2 TLC: Herbal Drugs and Fingerprints

Fig. 2.8 TLC fingerprints (where 1 = reference, 2 = sample). saponins during extract (20% asiaticoside) preparation.
TLC No. 1: Fingerprint of forskolin during purification TLC No. 4: Fingerprints of withanolides in Withania
from Coleus forskohlii. TLC No. 2: Fingerprint of baco- somnifera for herb selection
sides during herb selection. TLC No. 3: Fingerprint of

Fig. 2.9 2D-TLC of C. forskohlii root extract [3]

Thin-Layer Chromatogram
Fig. 2.10 Toxicity profiles for 15 wastewater samples; decreased luminescence indicates toxic substance zones
(Source: ChromaDex)
The plate is then removed, dried, and redeveloped
in a second mobile-phase system so that direction
of solvent flow is at right angle with respect to the
first run. The spot location is detected as previous
case. Each spot will with new Rf values [3].
TLC Bioautography
Use of TLC for chemical and biological screening
together is known as “TLC bioautography,” a
multidisciplinary approach, which provides a
ground for efficient collaboration of different
disciplines for a new drug discovery as well as
analysis of food and feed. It is based on the fact
that toxins or other negatively acting agents
reduce the metabolic activity of the microbes,
which is proportional to the luminescence and
the stability of compounds on the TLC plate
which can be verified easily. For a new drug dis-
covery, isolation of single component, followed
by assay for its biological activity, is a tedious
and expensive process. Under such stress, TLC
bioautography has a good compatibility with
large number of samples which can be studied
for various activities (e.g., acetylcholinesterase
inhibitor, antibacterial, antifungal, free radical
scavenger), whose biological properties have not
been documented earlier. This technique is used
for selection of herbals to study therapeutic
37
properties, combines TLC with in situ bioassay,
and allows localization of active constituents in
complex mixture. Agar diffusion, direct TLC
bioautography, and agar overlay bioautography
are in general practices. For discovering new
antioxidants in herbals, TLC plate is sprayed with
2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical.
Antioxidant reduces the radical, producing white
spots on the purple ground. The TLC bioautog-
raphy is a future tool for the study of herbal for-
mulation to understand/explain the synergistic
phenomena in new drug discovery [3].
Bioluminescence and TLC Analysis
Bioluminescence is the production and emission
of light by a living organism as the result of a
chemical reaction during which chemical energy
is converted to light energy. The bioluminex
assay is a unique biosensor method that directly
couples natural bioluminescence to TLC (Fig. 2.10).
This rapid assay can be used to support material
identity, detect toxins and chemical adultera-
tions, identify potential bioactive compounds, and
monitor manufacturing processes. TLC has tradi-
tionally been used as a reliable and economical
analytical technique for the simultaneous separa-
tion of multiple samples using a minimum of harsh
chemicals or solvents. A developed TLC plate is
38
coated with the nonpathogenic, bioluminescent
marine bacteria Vibrio fischeri. The compound
which interferes with the metabolic process of the
bacteria inhibits bioluminescence and is, there-
fore, detected as contrasting dark spots on the
luminescent background of the TLC plate. Activity
is measured by a reduction in light emission of
the bacteria producing a toxicity pattern charac-
teristic of each analyzed sample that can be
viewed and quantified directly on the TLC plate.
Results occur within minutes and can be docu-
mented photographically (CCD camera, X-ray,
Polaroid, or 35-mm film). This technology has
been developed into a kit system that provides an
effective means of prescreening a variety of
complex mixtures in order to minimize the use of
environmentally costly conventional methods [3].
Bioluminex has been tested with a wide variety
of toxins, from naturally occurring biotoxins
such as biocides to heavy metals. Bioluminex
sets the standard for consistent regulatory use
and helps ensure the quality of products, from
biomass to bottle. Standard toxicity tests only
establish the overall toxicity (biological activity)
for complex mixtures like wastewater or crude
natural product extracts. Identification of the
active compounds requires the tedious isolation of
single components followed by assays of their
biological effects. In addition, there is the risk of
false results due to interference or interaction
by any number of compounds in complex sub-
stances. Bioluminex overcomes the limitations of
this conventional approach by specifically assign-
ing biological activity to single components of
mixtures. Because of parallel sample processing,
the TLC bioluminescence technique represents a
versatile and rugged method with high sample
throughput. Bioluminex has been successfully
validated in environmental applications for
food samples, natural products, and a multi-
tude of toxicity-related problems. Furthermore,
ChromaDex has customized this advanced TLC–
bioassay technique for specific research needs by
employing genetically engineered microorgan-
isms, as BioluminexTS Kit, available in market.
The technique has wide scope in the analysis
of drinking water and wastewater/effluent for
screening biocide, leaching, in food, beverage,
2 TLC: Herbal Drugs and Fingerprints
dietary supplements screening (raw materials and
product), in veterinary applications (testing feed,
supplements, and urine), in testing for toxic
residues in soils, in testing for residues in manu-
facturing equipment, in validation of organic
products or certify soil, and in equipment and
water used in organic farms as well as in determi-
nation of an unknown compound’s toxicity.
Detection of Colorless Compounds
In general, the detection of colored compounds
causes no problem, and the same is true for com-
pounds which exhibit fluorescence or phospho-
rescence under ultraviolet light. In some cases
where the color is not very intense, a spray
reagent may be used to increase the sensitivity
by spraying the chromatogram. There are numer-
ous spray reagents which can be used to make
the various colorless compounds visible on the
chromatogram. These can be divided into two
classes: (1) those which are general reagents and
will detect a large number of different types of
compounds (e.g., 10% H2SO4, iodine vapor) and
(2) those which are more specific in nature, indi-
cating the type of compound or functional group
that is present (e.g., Dragendorff’s reagent for
alkaloids, potassium hydroxide for coumarins
and anthraquinones, ferric chloride for tannins
and phenolic compounds, ninhydrin for alpha
amino acids) [3].
Combination of TLC with Other
Techniques
TLC has been directly coupled with column chro-
matography by using various splitters with a vari-
able drive to control the application of a portion of
the column elute to the TLC plate. Similarly, TLC
is used for the selection of proper mobile phase to
HPLC and optimizes the analytical conditions.
The spots obtained from TLC may be eluted, con-
centrated, and then subjected to either HPLC or
GLC analysis. The high boiling substances are
preferred for TLC–GLC analysis. Mass spec-
trometry (MS) and TLC are combined together to
Thin-Layer Chromatogram
know the molecular weight of the compound and
fragmentation patterns. Desired spot from TLC
with defined Rf value can be scraped, dissolved in
suitable solvent, filtered, and may be studied by
NMR or IR spectrometry for structure elucida-
tion, for authentication of the target component.
Criteria for Selectivity, Reproducibility,
and Robustness
TLC analysis mainly concerned with determi-
nation of identity, purity, and assay or together
all, for which two- or multidimensional TLC
are the options. The selection of stationery
phase with analyte is a part of experience and
theoretical knowledge to the chemical compo-
sition of stationary phase. For satisfactory sep-
aration efficiency, the mean particle size,
particle size distribution, and morphology of
particle are to be considered. It is advisable to
use prepared TLC plate to secure better repro-
ducibility of good fame for quality. Solvent
system up to six components is used, provided
that this must have the appearance of single-
phase system with no sign of cloudiness. The
analogy between solvent system and mobile
phase becomes logical when we use mixture of
solvents, as the solvent system placed in the
development chamber releases some of its
components into the pores of stationery phase,
where it forms a liquid stationary phase. In this
equilibrium, the mobile phase and solvent have
different meaning [3].
Special Hazards
Looking directly at an ultraviolet (UV) light tends
to cause eyes cataracts, and shining it on the skin
promotes skin cancer. For these reasons, the UV
lamp should be kept in the hood and only held
over the TLC slides for a few seconds to visualize
them. The lamp should be pointed downward at
all times and turned off immediately after use.
UV rays are absorbed by glass, so a transparent
glass is used in UV cabinet to observe the TLC
plate. Cyclohexane and toluene are flammable
39
and are narcotic in high concentrations. Chloroform
is an anesthetic and a possible carcinogen under
conditions of daily exposure for several years.
All organic solvents and plate developing cham-
bers are kept in the fume hood to avoid solvent
exposure [3].
Troubleshooting in TLC Analysis
The aforesaid steps seem that TLC is quite an
easy procedure, but what about the first time we
run a TLC, and see spots everywhere and blurred,
streaked spots, etc., as with any technique, with
practice we get better. A few notable points during
process development for unknown compounds
may be helpful as [3]:
1. When compound runs as a streak rather than a
spot indicates that sample is overloaded. To
have desired results, run the TLC again after
diluting sample, or sample might just contain
many components, creating many spots which
run together and appear as a streak. Perhaps,
the experiment did not go as well as expected,
change mobile phase as per own analytical
judgment.
2. When sample runs as a smear or an upward
crescent, compounds which possess strongly
acidic or basic groups (amines or carboxylic
acids) sometimes show up on a TLC plate
with this behavior. Addition of a few drops of
ammonium hydroxide (amines) or acetic acid
(carboxylic acids) to the eluting solvent helps
to obtain a clear plate.
3. If sample runs as a downward crescent, likely
the adsorbent may be disturbed during the
spotting, causing the crescent shape.
4. If plate solvent front runs crookedly, either the
adsorbent has flaked off the sides of the plate
or the sides of the plate are touching the sides
of the container (or the paper used to saturate
the container) as the plate develops. Crookedly
run plates make it harder to measure Rf value
accurately.
5. Many random spots are seen on the plate,
indicating that during operation analyst have
accidentally dropped any organic compound
on the plate.
40
Fig. 2.11 TLC of Equisetum species, where: 1 = rutoside, 2 = hyperoside, 3 = caffeic acid, 4 = E. arvense, 5 = E. arvense
(China), 6 = E. palustre, 7 = E. arvense [4]
Identification of Marker Compounds
in Herbal Drugs
TLC is used for the analysis of aliphatic mono-
carboxylic acids, keto acids, hydroxy acids,
dicarboxylic acids, aromatic carboxylic acids,
phenolcarboxylic acids, alcohols (except methanol
and ethanol) and glycols, their derivatives, alkaloids
(purine, pyridine, phenyl alkylamine, dipyridine,
pyridine–pyrrolidine, quinoline, isoquinoline,
indole, and pyrrolizidine), using Dragendorff
reagent, amino acids, proteins, peptides, antibiotics,
carbohydrates, dyes (both oil soluble as well as
water soluble), hydrocarbons (using hexane as
mobile phase in silica TLC plate), lipids, nucleic
acids and nucleosides, pesticides (chlorinated,
phosphorus, carbamates, pyrethrins, etc.), phenolic
compounds, screening of pharmaceuticals (samples
for drug of abuse, sulfa drugs, contraceptives,
laxatives, etc.), flavonoids, steroids, vitamins,
antioxidants, inorganic ions, etc. The herbal raw
material as well as herbal drugs may have adul-
teration; hence, the proper identification of
material is essential before use. There is simple
quantitative test by TLC against the reference
standard, as the fingerprints and TLC profile
clearly indicate about the adulteration. In this
way, substitution by exhausted drugs can be
avoided as the dried exhausted cloves and umbel-
liferous fruits after extraction of their volatile oil
2 TLC: Herbal Drugs and Fingerprints
closely resemble to the genuine drug. Similarly,
addition of synthetic to fortify inferior products,
such as adding citral to the oil of lemon or benzyl
benzoate to balsam Peru, can be checked.
In an attempt to establish a certified quality of
marketed herbal drugs, crude, powdered, or in
combination, TLC is used to decipher the active
principals, identification of claimed herb, and
possible contamination/adulterants. The TLC
method is efficient, rapid, and combines the sen-
sitivity and simplicity with low cost for the deter-
mination of main active principles of medicinal
plants (alkaloids, anthraquinones, coumarins,
essential oils, flavonoids, glycosides, saponins,
tannins, etc.), which are the main ingredients
responsible for potential pharmacological effects.
The technique is so efficient that it is being used
as primacy analytical tool in the new drug discov-
ery for herbal drugs. Herbal drugs are obtained
from cultivated or wild plants. They vary more or
less in composition and properties depending on
the habitat, climate zone, and annual variations.
This causes problems in the identification and
purity tests of herbal drugs.
Case Study 1: There are many Equisetum sub-
species and hybrids species described in litera-
ture. These Equisetum species along with having
alkaloid (species like Equisetum palustre) have to be
detected by TLC analysis (Figs. 2.11 and 2.12) [4].
Identification of Marker Compounds in Herbal Drugs
Fig. 2.12 E. arvense and E. palustre in TLC analysis [4]
Case Study 2: Passionflower (Passiflora incar-
nata) is used in phytotherapy as a mild sedative
and anxiolytic agent and has considerable quali-
tative and quantitative variability with respect to
its content of C-glycosyl flavones, some of which
are used as marker compounds for extracts.
Analysis of plant material cultivated in Australia
revealed two chemically distinct groups; hence,
an investigation was carried out to determine
whether distinct intraspecific chemotypes exist in
this species. Eleven P. incarnata samples were
analyzed by HPLC, LC–MS, and two different
TLC methods. The samples fell into two distinct
groups with respect to their C-glycosyl flavone
profile, with little within-group variation. One
chemotype was dominated by isovitexin and
schaftoside/isoschaftoside. The other chemotype
was characterized by a high level of swertisin,
with low levels of schaftoside/isoschaftoside.
The two chemotypes were readily identified by
both HPLC and TLC [5].
TLC Method 1: This was the method for
passionflower in the British Pharmacopoeia
(2007). Mobile phase was composed of water-
anhydrous formic acid methyl ethyl ketone ethyl
acetate (10:10:30:50, v/v), and the plates were
41
developed to a distance of 150 mm at room tem-
perature. Detection was by way of two spray
reagents, natural products reagent (2-aminoethyl
diphenylborinate, sigma, 1% in methanol) fol-
lowed by Macrogol 400 (polyethylene glycol,
sigma, 5% in methanol) [5].
TLC Method 2: This method was adopted from
Widmer, Meier, and Schaffner and was published
by CAMAG, Switzerland, on their website (http://
www.camag.com/index.php; n.d.). Mobile phase
was composed of tetrahydrofuran-toluene-formic
acid-water (16:8:2:1, v/v), and the plates were
developed to a distance of 52 mm at room tem-
perature. The heated plates were sprayed with
natural products reagent (0.5% in ethyl acetate)
followed by polyethylene glycol 4000 (Fluka,
5% in dichloromethane) [5].
TLC Method 2 was superior to the Method 1(i.e.,
the British Pharmacopoeial method) in terms of
clarity and resolution, and Method 2 allowed for
the differentiation between the two P. incarnate
chemotypes (Fig. 2.13) [5].
Similarly, qualitative analysis for different
chemical classes of therapeutics, using different
mobile phases (Table 2.1), can be performed by
TLC as:
42 2 TLC: Herbal Drugs and Fingerprints

Fig. 2.13 TLC plate showing P. incarnata swertisin chemotype (1–6) and isovitexin chemotype (7, 8), where C = chlo-
rogenic acid, H = hyperoside, R = rutin [5]

Table 2.1 Brief list of common solvent systems for TLC [3]
Chemical group Absorbent Solvent system frequently used Detection
Alkaloids Silica gel 1. Methanol–chloroform (85:15) 1. UV
2. Toluene–ethyl acetate– 2. Dragendorff
diethylamine (70:20:10)
Anthocyanins Silica gel, cellulose n-butanol–acetic acid–water 1. UV
(40:10:20) 2. Anisaldehyde- sulfuric acid
Cardiac glycosides Silica gel 1. Ethyl acetate–methanol–water 1. Kedde reagent
(81:11:8)
2. Chloroform–methanol–water 2. Antimony chloride
(65:35:10)
Flavonoids Silica gel Chloroform–acetone–formic acid UV
(75:16.5:8.5)
Indoles Silica gel Chloroform–ethyl acetate–formic p-Dimethylamino
acid (5:4:1) cinnamaldehyde
Monosaccharides Silica gel n-butanol–acetic acid–ether–water 1. Aniline hydrogen phthalate
(9:6:3:1) 2. UV
Phenols Silica gel Acetic acid–chloroform (1:9) Folin reagent
Polyacetylenes Silica gel Chloroform–methanol (1:9) 10% H2SO4
Saponins Silica gel 1. Chloroform–methanol–water 1. Vanillin/ sulfuric acid
(60:35:5)
2. n-butanol–water (1:1) (upper 2. Anisaldehyde sulfuric acid
phase)
Terpenes Silica gel 1. Chloroform–methanol (95:5) 1. 10% Sulfuric acid
2. Ethyl acetate–cyclohexane (60:40) 2. Anisaldehyde- sulfuric acid

TLC Analysis for Alkaloids in analysis of new unknown herbo-combination

The systematic procedure for the analysis of
alkaloids by means of thin-layer chromatography,
with the mixture of cyclohexane-chloroform-
diethylamine (5:4:1) as screening the alkaloids
on the basis of polarity and further analysis using
chloroform–acetone–diethylamine (5:4:1) and
chloroform–diethylamine (9:1), is still a guideline
for alkaloids by TLC, subsequently developing
spots with Dragendorff’s reagent. The most
preferred adsorbent TLC plate for alkaloids has
been silica gel 60 F254. Once detecting the alka-
loid, the mobile phase is determined based on
satisfactory and validable parameters. The authors
were benefited by the above concept during
developing in-house protocol for Gloriosa glabra
Identification of Marker Compounds in Herbal Drugs
methanolic extract, evaluating for both colchicines
and colchicoside in a single TLC plate using
mobile-phase chloroform–methanol (8.5:1.5) and
evaluating at 254 nm, previously as it was been
done in two steps: chloroform–methanol (9.5:0.5)
for colchicines and chloroform–methanol (8:2)
for colchicoside. The best industrial application
of TLC is the determination of selected substance in
pharmacopoeial products, when it is being measured
at the level of less than 0.1% (e.g., related sub-
stances in yohimbine, berberine hydrochloride,
thiocolchicoside, reserpine as pharmacopoeial
product especially in USP). Test solution: A
50-ml separating funnel was charged with 10 ml
of the preparation to be tested added 1.0 ml of
concentrated aqueous ammonia and 10 ml of
chloroform, after which the mixture is shaken for
3 min. Then the chloroform extract is separated
and, in the case of emulsification, centrifuged for
3 min at 2,500 rpm to complete phase separation.
Reference solution: The reference solution is
prepared by dissolving 0.01 mg of sanguiritrine
(working sample) solution in 19.5 ml of methyl
alcohol and 0.5 ml of ammonia solution. Spotting
on TLC plate: Sample of the test solution (30 ml)
and of the reference solution (20 ml) was applied
on the start line of a TLC plate (silica gel 60 F254).
The plate is dried in air for 3 min, placed into a
vertical cell with the diethyl ether–petroleum
ether–methanol mixture (35:15:1) and chromato-
graphed in the ascending mode. When the solvent
front reaches the end of the path, the plate is
extracted from the cell, dried in air at room
temperature until the solvent mixture is removed,
and examined on exposure to UV radiation with
a wavelength of 360 nm. The chromatogram of
sanguiritrine shows two spots: orange (sangui-
narine) and yellow (chelerythrine). The chro-
matogram of the test solution must display band
of the same color and mobility (i.e., Rf value) as
those on the reference pattern [3].
TLC Analysis for Phenols
Volumes have been written about the TLC anal-
ysis of phenolic compounds (derivatives of benzoic
acid, cinnamic acid, coumarins, flavonoids,
43
tannins, and lignins). For simple phenols, the
best solvents are petroleum ether (60–80°C)-carbon
tetrachloride-acetic acid (4:6:1) and chloroform–
acetone–diethylamine (4:2:0.2). Polyhydric
phenols are best separated by using chloroform-
acetic acid (5:1), chloroform–acetone–acetic
acid (10:2:1) and benzene–acetic acid (5:1).
These spots are made visible by spraying with an
acetone solution of p-nitrobenzene diazonium
fluoroborate. Phenolic compounds are ubiquitous
in the plant kingdom, being the most abundant
secondary metabolites. Though bio-phenols are
found in all plants, their quantitative distribution
varies between different tissues of plant and
within different populations of the same plant
species. Bio-phenols of fruits, vegetables, herbs,
spices, and cereals have high therapeutic poten-
tial and are in use as herbal drug. TLC is the
primary one and best screening technique for
bio-phenols analysis. Lignin, the second most
abundant compound in the nature, on hydrolysis,
gives bio-phenols. Methanol has been reported as
a suitable solvent for short-time extraction as
phenolic glycosides degraded with longer span.
The primary characterization of phenolics is
usually done by TLC [3].
TLC Analysis for Saponins
Saponins are glycosides which produce stable
foams when their aqueous solution shaken. On
acid hydrolysis, saponins split into sugars and
the corresponding sapogenins. Saponins based
on the carbon skeleton of sapogenin are termed
as terpenoid saponins (e.g., Centella asiatica,
Bacopa monnieri, Stevia rubidiana) and steroidal
saponins (e.g., Tribulus terrestris, Withania
somnifera, Gymnema sylvestre) were analyzed
by TLC, in authors lab, using mobile-phase chlo-
roform – methanol and water in different compo-
sitions and subsequently developing spots with
anisaldehyde–sulfuric acid and drying plate at
110°C for 10 min. The spots were evaluated
against reference preparation, either as qualitative
or quantitative determination (withanolides in
Withania somnifera, have steroidal skeleton, but
these are steroidal lactones). Chromoplates were
44
examined before and after spraying under UV
and day light. The individual optimization of
TLC techniques was helpful in the process devel-
opment of purification at higher purity levels of
phytochemicals (>98% purity of the ingredient)
to detect the impurity profile of the ingredient,
using organic solvents with water (sometimes in
different pH values, as buffer, etc.), as well as to
develop HPLC analysis methods, selection of
analytical column, and to determine combination
of mobile phase and gradient parameters, as well
as regeneration of column prior to the next
analysis.
As plant saponins are sensitive to the struc-
tural variation (e.g., saponins mostly are hydro-
philic, but digoxin is also lipophilic), so these are
standardized with saponin mixtures isolated from
the plant species in which the concentration is
measured (gravimetric analysis). However, one
plant species may contain some saponins which
can be determined with a biological test and
others cannot be. That is why biological and
colorimetric determinations do not provide accu-
rate data and have to be recognized as approximate
(hence gravimetric analysis is preferred). TLC (on
normal and reversed phases) and 2D TLC provide
excellent qualitative information and in combina-
tion with online coupling of a computer with
dual-wavelength flying-spot scanner and two-
dimensional analytical software which can be
used for routine determination of saponins in
plant material. The densitometry of saponins has
been very sensitive, however, to plate quality,
spraying technique, and the heating time, and
therefore appropriate saponin standards have to
be run in parallel with the sample. Gas–liquid
chromatography has limited application for deter-
mination since saponins are quite big molecules
and are not volatile compounds. Thus, there are
only few applications of GC for determination
of intact saponins. The method has been used
for determination of TMS, acetyl or methyl
derivatives of an aglycones released during
saponin hydrolysis. However, structurally differ-
ent saponins show different rates of hydrolysis,
and precise optimization of hydrolysis conditions
is essential. Besides, during hydrolysis, a number
of artifacts can be formed which can influence
2 TLC: Herbal Drugs and Fingerprints
the final results. High-performance liquid
chromatography on reversed-phase columns
remains the best technique for saponin determi-
nation and is the most widely used method for
this group of compounds. However, the lack of
chromophores allowing detection in UV limits
the choice of gradient and detection method. The
pre-column derivatization with benzoyl chloride,
coumarin, or 4-bromophenacyl bromide has been
used successfully in some cases, allowing UV
detection of separation. Standardization and
identification of the peaks in HPLC chromato-
grams have been based on comparison of the
retention times with those observed for authentic
standards. But new hyphenated techniques,
combining HPLC with mass spectrometry and
nuclear magnetic resonance, are developing
rapidly and allow online identification of sepa-
rated saponins. Capillary electrophoresis has
been applied for saponin determination only in a
limited number of cases, and this method is still
being developed [3].
TLC Analysis for Terpenoids
The low polar nature of terpene and its deriva-
tives, solvents of low polarity, are used for TLC
separation of these components; spots are mea-
sured either UV-visible or developed using iodine
vapors or 5% sulfuric acid after iodinization and
heating at 110°C for 10 min in oven. Author in
his laboratory is evaluating forskolin from Coleus
forskohlii, using cyclohexane-ethyl acetate (6:4)
and developing spots with 7.5% H2SO4 after
iodinization and subsequently heating it at 110°C
for 10 min. The technique is too valuable to assay
purity of forskolin which matches with HPLC
assay. The TLC fingerprints for essential oils
Ocimum sanctum (tulsi), Centella asiatica (gotu
kola), and Convolvulus pluricaulis (shankh-
pushpi), in a combo-herbal drugs, were developed
using mobile-phase toluene–ethyl acetate (85:15),
which was evaluated at 365 nm after drying in
hot air, and finally spots were developed using
7.5% H2SO4 and heating plate at 110°C for
10 min in oven, by the authors, where separate
marker appears for individual ingredient, using
Identification of Marker Compounds in Herbal Drugs 45

b c
N O
100 N
H N

Relative intensity
288.11301[MH]+
rutaecarpine
[MH]+;288.1107
ritaecarpine
a m/z
0
200 250 300 350 400 450 500

N O
100 N
[MH]+ H
Relative intensity
304.14758 N
H2C
evodiamine
[MH]+;304.1450
evodamine

m/z
0
200 250 300 350 400 450 500

Fig. 2.14 Evodiae fructus extract in TLC DART–MS [8]. (a) Evodiae fructus, (b) TLC chromatogram of the extract of
Evodiae fructus, under 365 nm, (c) DART–MS spectra of rutaecarpine and evodiamine

against reference botanical material (RBM).
While in another combo-herbal formulation, the
TLC fingerprints for O. sanctum were developed
by ether-hexane (7:3), and spots were developed
with 2% H2SO4 and drying it at 110°C for 10 min,
reddish fluorescence marker for O. sanctum, vis-
ible at 365 nm. The polarity of compounds
increases with increasing number of –OH groups,
while the alkyl derivative of hydroxyl group
decreases the polarity, and further increase in size
of alkyl group again decreases the polarity.
Hydrogenation of side chain had only slight effect
on polarity; similarly, hydrogen band formation
also reduces the polarity in terpenoids. In order to
select the optimum mobile phase for TLC, mobile
phase was toluene–ethyl acetate (4:1), n-hexane–
ethyl acetate (9:1), hexane–chloroform (5:1), and
benzene–ethyl acetate (5:1). The best separation
of components was achieved with the last solvent
mixture. The bands of terpenoids in the chro-
moplate were revealed by treating the TLC plates
with an anisaldehyde–sulfuric solution, followed
by heating to 100°C for 10 min.
shaken for 3 min, and hexane phase was transferred
into a 50-ml round-bottom flask. The extraction
was repeated with the same amount of n-hexane.
Then extracts are combined, and the solvent is
distilled off to dryness in a rotary evaporator at a
temperature not exceeding 50°C. The residue was
dissolved in 1 ml of ethyl acetate. Reference solu-
tion: The reference solution is prepared by dis-
solving 10 mg of menthol in 2 ml of ethyl acetate.
Spotting on TLC plate: Sample of the test solution
(20 ml) and of the reference solution (5 ml) were
applied, on the start line of a TLC plate [silica gel
60 F254, (mobile-phase benzene: ethyl acetate
mixture) (5:1)] and chromatographed in the
ascending mode until the solvent reaches a level of
10 cm in the plate and dried the plate in air at room
temperature until the solvent disappears. Then the
plate is treated with anisaldehyde solution and
heated at 105°C for 10 min. The developed chro-
matogram is examined in daylight [3].
TLC with DART–MS

Test solution: A 50-ml separating funnel was Using TLC, to generate more versatile and
charged with 10 ml of the preparation to be tested specific information on extract of herbal drugs,
10 ml of n-hexane; after which the mixture is “direct analysis in real time (DART) ion source”
46
is being in use. This hyphenation system of TLC
and DART–MS provides unique and specific
information on the major constituents of crude
plant drug on TLC through uncovering high-
resolution mass number of each band on the TLC
plate directly in real time.
Case Study-1: The three well-known Korean
Pharmacopoeial herbal drugs were extracted
and developed on a silica-coated TLC plate
with preestablished conditions, as per Korean
Pharmacopoeia IX, and developed TLC plate
were placed between the DART ion source and
TOF-MS analyzer to get real-time mass spectra
from the bands on the plate directly. The marker
coumarin compounds, decursin and decursinol,
were successfully identified from the TLC plate
developed with Angelicae gigantis radix, along
with alkaloid compounds of rutaecarpine and
evodiamine from Evodiae fructus and lignan
molecules of gomisin A, N, and schisandrin from
Schisandrae fructus (Fig. 2.14) [6].
Case Study-2: The curcuminoids (mixture of
curcumin, demethoxy curcumin, and bis-
demethoxy curcumin) were successfully detected
directly from the raw rhizome of Curcuma longa
when a turmeric extract was separated on a TLC
plate, studied with DART–MS, each band pro-
duced molecular ion peaks corresponding to cur-
cumin, demethoxy-curcumin, and bis-demethoxy
curcumin. Molecular ions of curcuminoids in
turmeric-containing beverages and curry powder
were also efficiently detected at the range of
5–100 mg/ml. To establish the validity of analytical
methods for curcuminoids (curcumin and its
derivatives) from various types of samples with
DART–MS, data were compared with the results
of HPLC and were strong proofs for specificity of
the method. As DART–MS produces [M + H]+
molecular ions of most compounds, so relatively
simple and clear mass spectra are obtained even
of multicomponent samples [7]. In order to take
advantage of the capacity of DART–MS for the
real-time analysis of individual compounds in
natural raw materials, the technology has a huge
scope in herbal drug industries [8].
2 TLC: Herbal Drugs and Fingerprints
TLC Fingerprints for Batch to Batch
Consistency
A study for TLC fingerprints of an Unani formu-
lation, named “Majoon-e-Sandal,” which is
widely used in the ailments of stomachic, antibil-
ious, psychoneurosis, vomiting, and nausea, and
prepared by mixing the powders of the Santalum
album (110 g), Bambusa bambos (15 g), and
Styrax benzoin (15 g) for the formulation compo-
sition and kept separately. Tamarindus indica
was soaked in water for 2 h, crushed with hand,
and filtered through muslin cloth and kept sepa-
rately. Punica granatum seeds were crushed with
hand and filtered it through muslin cloth and kept
separately. Crocus sativus was grinded by adding
rose water and kept separately. Dissolve 750 g of
sugar in 500 ml of water; at the boiling stage,
0.1% citric acid was added, mixed thoroughly,
and filtered through muslin cloth. Then, boil the
filtrate on slow heat and add the mixed extract
of Tamarindus indica and Punica granatum
followed by Crocus sativus. Then, with the content
mixed thoroughly, prepare the 79% consistency
of quiwam. Remove the vessel from the fire,
while hot condition is added to the mixed powders
of the Santalum album, Bambusa bambos, and
Styrax benzoin, followed by 0.1% of sodium ben-
zoate; mix thoroughly to prepare the homogenous
product. Allow to cool to room temperature, and
packed it in tightly closed container to protect
from light and moisture. Majoon-e-Sandal is a
semisolid, brown-colored characteristic of its
own odor and in sweet taste [9].
Preparation of extracts: From three different
batches, 2 g of sample, each separately, was
soaked in chloroform and alcohol, respectively,
for 18 h, refluxed for 10 min on water bath, and
filtered. The filtrates were concentrated on water
bath and made up to 5 ml in a standard flask sepa-
rately. Development of TLC fingerprints: The
chloroform and alcohol extracts were applied on
precoated silica gel 60 F254 TLC plate (E. Merck)
as absorbent and developed the plate using sol-
vent systems, toluene : ethyl acetate 9:1 and 1: 1,
References
Fig. 2.15 TLC for chloroform extracts [9]
respectively. After developing, the plates were
dried and observed the color spots at UV-254,
UV-366 nm, and spots were developed using van-
illin–sulfuric acid spraying reagent. TLC studies
of chloroform and alcoholic extract of all the three
batch samples had identical spots in UV (254,
366 nm), similar Rf values, even on development
with vanillin sulfuric acid reagent, after heating at
105º for 10 min (Figs. 2.15 and 2.16) [9].
TLC is a widely applied technique in herbal
authentication and used in majority of pharmaco-
poeia’s monograph for correct identification. Using
Co-TLC, unknown compounds can be easily
identified with an authentic compound, so it is
widely adopted because of less time-consum-
ing, semiquantitative, and a cheap technique.
Furthermore, it also provides fingerprints of the
material under consideration; if the marker com-
pound is known, then it becomes more precise, so it
is an important tool for monitoring the identity and
47
Fig. 2.16 TLC for alcohol extracts [9]
purity of the plant material under consideration; in
addition, it also provides information about substi-
tution and adulteration.
References
1. Abu-Hamdah S, Afifi FU, Shehadeh M, Khalid S. Simple
quality control procedures for selected medicinal plants
commonly used in Jordan. Pharm Biol. 2005;43(1):1–7.
2. Sachan AK, Sachan NK, Kumar S, Sachan A, Gangwar
SS. Evaluation and standardization of essential oils for
development of alternative dosage forms. Eur J Sci
Res. 2010;46(2):194–203.
3. Joshi DD. Thin layer chromatography and biotechnol-
ogy at molecular level. In: Textbook of molecular bio-
technology. New Delhi: I.K. International Pub. House;
2009. p. 1181–96. Sample Chapter 53.
4. Klier B. Current problems with identification of
herbal drugs. PhytoLab GmbH & Co. KG 91487
Vestenbergsgreuth Germany. http://www.ga-online.
org/files/Graz/WS-4_Klier.pdf. Accessed 29 Mar 2012.
5. Wohlmuth H, Penman KG, Pearson T, Lehmann RP.
Pharmacognosy and chemotypes of passionflower
48
(Passiflora incarnata L.). Biol Pharma Bull.
2010;33(6):1015–8.
6. Kim HJ, Jee EH, Ahn KS, Choi HS, Jang YP. Identification
of marker compounds in herbal drugs on TLC with
DART-MS. Arch Pharm Res. 2010;33(9):1355–13559.
7. Kim HJ, Jang YP. Direct analysis of curcumin in turmeric
by DART-MS. Phytochem Anal. 2009;20(5):372–7.
8. Alsbou E. Ambient mass spectrometry desorption
electrospray ionization (DESI) & direct analysis in real
time (DART). http://www.ecplaza.net/tradeleads/
seller/5638482/evodiamine_98_99_hplc.html# none
(2010). Accessed 28 Mar 2012.
9. Meena R, Meena AK, Khan SA, Mangeswari S.
Evaluation of an Unani compound formulation-Majoon-
e-Sandal. Int J Pharm Sci Res. 2010;1(5):238–42.
Bibliography
Di X, Kelvin KC, Hei WL, Carmen WH. Fingerprint
profiling of acid hydrolyzates of polysaccharides
extracted from the fruiting bodies and spores of
Lingzhi by high-performance thin-layer chromatogra-
phy. J Chromatogr A. 2003;1018:85–95.
Hardman R, Abu-Al-Futuh IM. The detection of isomers
of 4-hydroxyisoleucine by the Jeol amino acid analyser
and TLC. Planta Med. 1979;36:79–84.
Joshi DD. Utilisation of medicinal plants for health and
wealth. Proceedings of national workshop on role of
2 TLC: Herbal Drugs and Fingerprints
forestry in employment generation and rural development.
IFRI Dehradun, India; 2006. p. 59–68.
Kirchner JG. Thin layer chromatography. In: Techniques
of chemistry. 2nd ed. New York: Wiley; 2000. XIV.
Kopylova E, et al. Standardization of a complex prepara-
tion for the treatment of periodontal diseases. Pharm
Chem J. 2002;36:504–6 [Translated from Khimiko-
Farmatservticheskii Zhurnal. 36 (9): 44–46].
Li BY, Hu Y, Liang YZ, Xie PS, Du YP. Quality evalua-
tion of fingerprints of herbal medicine with chromato-
graphic data. Anal Chim Acta. 2004;514:69–77.
Pascual ME, et al. Simplified screening by TLC of plant
drugs. Pharm Biol. 2002;40(2):139–43.
Sharma J, Fried B. Handbook of thin layer chromatogra-
phy, Chromatographic Science Series, vol. 55. New
York: Marcel Dekker; 2003.
Stationary Office. British Pharmacopoeia 2007. London:
The Stationary Office; 2006.
USP. United States pharmacopeial convention. USP
29–NF 24. Rockville: United States Pharmacopeial
Convention, Inc. electronic version; 2006.
Verbitski SM, et al. Rapid screening for complex mixtures by
thin layer chromatography-bioluminescence. Technical
article. Am Biotechnol Lab. 2006;24(9):40–1.
WHO Monographs on Selected Medicinal Plants –
Volume 2. http://apps.who.int/medicinedocs/en/d/
Js4927e/. Accessed 28 Mar 2012.
Zhao LH, Huang CY, Shan Z, Xiang BR, Mei LH.
Fingerprint analysis of psoralea corylifolia L. By HPLC
and LC-MS. J Chromatogr B. 2005;821:67–74.
 HPTLC: Herbal Drugs
and Fingerprints
Plants contain thousands of constituents and are
valuable source of new therapeutic molecules.
For new and effective herbal drug development,
it is important to have a validated process to pre-
pare plant extract and to isolate ingredients for
full structure elucidation and biological testing.
The combination of biological and chemical
screening leads to the important information
about plant constituents. The chemical screen-
ing by TLC analysis is illustrated in the form of
hi-tech art using high-performance thin-layer
chromatography (HPTLC) for better separation,
eliminating manual errors, and better repeatability
as well as reproducibility of the test results. It
provides a great deal of preliminary information
about the content and nature of constituents
found in the active fraction. Once the chemical
nature of a constituent is established via HPTLC
analysis, it is easier to develop validated process
to prepare standardized extract and isolate
ingredient in pure form, structure elucidation,
and biological testing with synergistic explanation
[1]. HPTLC is a very simple and economical
analytical method, useful for high-potential
qualitative characterization and quantitative
determination of herbals and products. Its field
of application covers virtually all classes of
substance with the exception of readily volatile
and gaseous substances and can be extended
easily to the preparative scale by using thicker
layers [preparative layer chromatography
(PLC)]. The separated substances, depending
on their optical properties, can be detected,
identified, and quantified in visible, infrared, or
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_3, © Springer India 2012
3
UV light, sometimes only after derivatization
with a suitable reagent.
Currently, quality evaluation is a main concern
in herbal formulations due to variation in the con-
tent of markers/active ingredients in the raw
materials, due to different geo-climatic factors
and business reasons. A computerized densitom-
eter is used for the fingerprinting, of concern
spot, on its area and intensity, for true authentica-
tion of test samples, against standard. Such
chemical fingerprinting is helpful for industries,
research institutions, and regulatory authorities
for quality evaluation and to decipher the claims
made for the products [2].
Operational Summary of HPTLC
The whole analytical process for HPTLC may be
summarized in the following steps [3]:
1. Selection of stationary phase for HPTLC
analysis
2. Sample preparation, clean up, and pre-chro-
matographic derivatization, if any
3. Application of sample on stationary phase
4. Development of chromoplate
5. Detection of spots including post-chromato-
graphic derivatization
6. Quantification
7. Documentation
Stationary phase selection for a new product is
based on the subject knowledge of the analyst
which is supported by the gained knowledge dur-
ing experiments and TLC analysis for the same.
49
50
Table 3.1 Comparison between silica gel pre-coated HPTLC and TLC plates [3]
Property HPTLC layer
Particle size 5–6 mm
Pore diameter 60 Å
Plate dimensions 10 × 10 cm, 20 × 20 cm,
10 × 20 cm
Layer thickness 0.20–0.25 mm
Analysis per plate Up to 75
Spot size recommended ~1 mm
Spot loading 50–200 nl
Band size recommended 5–10 mm
Band loading 1–4 ml
Sensitivity limit Upper pg (fluorescence)
Normal development time 2–30 min
The steps as spotting, evaluation, and documen-
tation have been connected with computers and
cameras respectively, which make the technique
more hi-tech. HPTLC leads to difficulty in auto-
mation, and because of its open character, it is
highly influenced by environmental factors. It is
therefore essential that each step which may
require specific approach must be carefully vali-
dated, much more than TLC analysis.
HPTLC Pre-Coated Plates
The uniformity and homogeneity of the station-
ary phase during HPTLC analysis is directly
linked with reproducibility and versatility of the
analytical results. HPTLC uses the same type of
silica gel 60 layers, as in traditional TLC, with a
thickness of 0.20–0.25 mm. However, the particle
size is much smaller, typically ranging from 4 to
8 mm, with an optimum of 5–6 mm (Table 3.1).
The commercial pre-coated HPTLC plates with
polymeric binders are sufficiently hard so as not
to be easily damaged by the capillary tubes used
for sample application. Use of smaller particles
of stationary phase, similar in size and quality to
HPLC packing materials, gives a lower theoreti-
cal plate height (H) and hence higher efficiency
but can be fully utilized if the plates are not over-
loaded with too much sample, the spot size is
kept small (about 1.0 mm), and the plate is devel-
oped only to the extent necessary for complete
3 HPTLC: Herbal Drugs and Fingerprints
TLC layer
10–12 mm
40, 60, 80, 100 Å
5 × 10 cm, 5 × 20 cm,
10 × 20 cm, 20 × 20 cm
0.20–0.25 mm
Up to 16
2–5 mm
1–5 ml
10–15 mm
5–10 ml
ng
15–20 min
resolution (often only 5 cm and rarely more than
8 cm). A direct comparison of theoretical plates
in HPTLC with HPLC serves little purpose as the
number found is only valid for the spot used for
calculation. The basic problem is that all analytes
do not travel the same distance and are not
measured in retention time as in column
chromatography.
As HPTLC have higher performance than
TLC, so it is possible to carry out separations on
HPTLC that were not possible on TLC plates
and, for those where it was possible, to shorten
the time of separation dramatically. HPTLC is
therefore a more rapid, efficient, and sensitive
technique than conventional TLC. For in situ
quantitative analysis using spectro-densitome-
ters, it is essential that HPTLC layers are used for
the most reliable results.
Detection and Visualization
Like TLC, HPTLC requires the visualization and
detection, and similar practices are used for that
but at more precise level. These practices may be
categorized as [3]:
Nondestructive Techniques
In this practice, the chromoplate remains intact,
may be evaluated by:
Detection and Visualization 51

Table 3.2 Some fluorescence intensifier and their application areas [3]
Intensifier Compounds detected Enhancement Stabilization
Triton X-100 (1% v/v solution in Fatty acids asdansyl amides At least tenfold Yes
hexane or heptane)
Polyethylene glycol 400 or 4,000 Compounds with alcoholic 20- to 25-fold Unknown
(10% w/v in methanol) (-OH) functional groups.
Paraffin liquid (33% v/v in hexane) Aflatoxins threefold to fourfold Unknown
Paraffin liquid (33% v/v in hexane) Ketosteroids, cholesterol, tenfold Unknown
cortisol
Paraffin liquid (33% v/v in hexane) Dansyl amides tenfold Yes
Paraffin liquid (33% v/v in hexane) Gentamicins Yes, but level unknown Yes

Visible Detection at shorter wavelengths in the UV. Depending

There are compounds that have color, for example,
natural and synthetic dyes, chlorophyll, and nitro-
phenols, to give an absorption in the visible part
of the electromagnetic spectrum. These are
clearly seen in visible light and do not require
any further treatment for visualization.
Ultraviolet Detection
There are many compounds that appear color-
less in normal light but can absorb electromag-
netic radiation at shorter wavelengths. These are
often detected in the UV range, normally at
200–400 nm. Often exposure to UV light at
short-wave radiation (254 nm) or long-wave
radiation (365 nm), with commercial UV lamps
and cabinets, which function at either or both of
these wavelengths. To aid visualization, many
commercial pre-coated HPTLC layers contain
an inorganic phosphorescent or an organic
fluorescent indicator (Table 3.2). Detection by
absorbance in these cases relies on the phospho-
rescence or fluorescence being quenched by the
sample components. This process is commonly
called “fluorescence quenching” in both cases,
although more accurately for most indicators
designated F254 it is described as phosphores-
cence quenching.
Reversible Reactions
Many compounds do not absorb visible or UV
light, quench fluorescence, or fluoresce when
excited by visible or UV light. In these cases,
suitable detection reagents are used to give
colored chromatographic zones in visible light or
upon the nature of analyte and developing reagent,
it may be reversible reactions (i.e., nondestruc-
tive techniques), for example, iodine vapor and
ammonia.
Iodine is a universal reagent detecting the
presence of many organic species on thin layers,
but some reactions with iodine are irreversible.
The use of iodine as a vapor enables the detection
of separated substances rapidly and economically
before final characterization with a group-specific
reagent. Where lipophilic zones are present on a
chromatographic layer, the iodine molecules con-
centrate in the substance zones giving yellow–
brown chromatographic zones on lighter yellow
background. The preparation of the reagent sim-
ply involves putting a few iodine crystals in a dry
chromatography tank, replacing the lid, and
allowing the iodine vapor to fill the air space for
a few hours. The developed chromatogram is
then introduced into the chamber, and as soon as
the chromatographic zones are recognized, the
layer is removed and the results recorded. The
adsorbed iodine is allowed to slowly evaporate
from the layer surface under a dry stream of air at
room temperature; a fume cupboard facility is an
ideal location for this. These chromatograms can
be subjected to further treatment with other uni-
versal or with more specific functional group
reagents. If more permanent results of the iodine
impregnation are required, then the chromato-
graphic zones are sprayed or dipped in a starch
solution (0.5–1% w/v) to give blue starch–iodine
inclusion complexes. However, it is important to
carry out this procedure after partial evaporation
52
of iodine from the layer. Starch treatment has
the best results when iodine is still retained in the
separated chromatographic zones but has gone
from the background layer. Otherwise, it will be
difficult to distinguish the zones from a back-
ground that will also be stained blue.
Iodine detection works well on silica gel 60
and aluminum oxide layers. However, results are
usually poor on reversed-phase layers as the lipo-
philicity of the layer does not differ appreciably
from the chromatographic zones. Iodine vapor
reversible reactions occur with a wide range of
organic lipophilic molecules, for example, fats,
waxes, some fatty acids and esters, steroids, anti-
oxidants, detergents, emulsifiers, and many mis-
cellaneous pharmaceuticals.
Ammonia vapor is often used in conjunction
with other reagents to improve the contrast
between the separated chromatographic zones
and the layer background. The most common
usage is in the visualization of organic acids with
pH indicators. Although indicators, such as bro-
mocresol green and bromophenol blue, detect the
presence of a variety of organic acids, further
treatment with ammonia vapor sharpens the con-
trast between analytes and background layer
resulting in greater sensitivity. On segregation of
ammonia source, ammonia gradually evaporates
away from the chromoplate, and the sensitivity of
detection reverts to that prior to treatment.
Exposure to ammonia vapor can be achieved by
simply holding the chromatographic plate face-
down over a beaker of strong ammonia solution.
However, more elegantly, it can be performed by
pouring ammonia solution into one compartment
of a twin-trough developing tank and placing the
TLC plate in the dry compartment. With the lid in
place, the TLC plate is exposed to an almost even
concentration of vapor. The process is reversible
with time as the ammonia soon evaporates from
the sorbent surface.
Nonreversible Reactions
A few techniques and practices used to visualize
the spots for HPTLC have chemical reactions
that cannot be in original stage; such practices are
known as nonreversible reactions. Fluorescent
3 HPTLC: Herbal Drugs and Fingerprints
dyes are commonly used for the nondestructive
detection of lipophilic substances, for example,
fluorescein, dichlorofluorescein, eosin, rhodamine
B and 6 G, berberine, and pinacryptol yellow.
Reagents for dipping chromatograms are prepared
as dye (10–100 mg) in methanol or ethanol (100 ml).
After air drying, the detected chromatographic
zones appear brightly fluorescent on a lighter
fluorescent background under UV light (254 nm).
Although very effective on silica gel, cellulose, and
kieselguhr layers (sensitivity from low micro-
gram to low nanogram range), these dyes do not
respond on reversed-phase silica gels; sometimes
exposure to ammonia vapor after dye treatment
improves sensitivity.
Destructive Techniques
Oxidation and/or derivatization due to chemical
reactions occurring on the chromatographic layer
between a reagent and separated analytes is a
destructive technique. In this case, the visualized
compounds are no longer the original one. The
major techniques as destructive are charring and
thermal activation. Charring techniques involve
treatment of the developed chromatogram with a
suitable reagent, followed by heating the layer at
relatively high temperatures to degrade any
organic species to carbon. As can be appreciated,
the reaction is somewhat nonspecific, and hence,
charring has been included in what is termed uni-
versal reagents. The most popular charring
reagent is sulfuric acid, applied to the chromato-
graphic layer as a dilute solution (10–20% v/v in
methanol/water); however, orthophosphoric acid
and chromosulfuric acid have proved successful
in more of the specific circumstances. The tem-
perature and heating time depends on the nature
of the compounds to be charred. This can vary
from 5 to 20 min at 100–180°C. Dilute solution
of sulfuric acid in water/methanol ensures ade-
quate wetting of the TLC/HPTLC layers. On
heating, the solvents evaporate steadily and acid
concentrates and finally chars the organic material
present. Although it is a very simple detection
technique, but sulfuric acid charring does have
limitations especially where commercially man-
ufactured chromatography plates are concerned.
Detection and Visualization
Most binder whether present in homemade or
commercial plates affected to a greater or lesser
extent depending on the temperature and time of
heating. Overheating of plates with organic bind-
ers may have a gray or even black background,
rendering it useless.
It has been observed that some developed
zones on a TLC/HPTLC layer when heated at
high temperatures have fluoresced on exposure to
UV light, for example, lysergol and lumilysergol
(using mobile phase chloroform–methanol–
ammonium hydroxide, 85:14.5:0.5, and heating
at 100°C). This process has been given the title
thermochemical activation. Separations on mod-
erately polar aminopropyl-bonded silica gel lay-
ers have been observed to give the most consistent
and sensitive results for this process of detection.
The reaction mechanism by which thermochemi-
cal activation takes place is not fully elucidated,
but the following has been suggested as a proba-
ble sequence. The surface of the silica gel-bonded
layer acts as a catalyst. Under the influence of the
catalytic adsorbent surface, substances rich in
p-electrons are formed that conjugate to form
products having fluorescent at excited state. It has
been observed that compounds with possible het-
eroatoms, such as nitrogen, oxygen, sulfur, or
phosphorus, will more readily respond to thermal
activation than pure hydrocarbons. Changes in
pH often alter the excitation and emission wave-
lengths. The fluorescent compounds formed are
quite stable. The fluorescence can frequently be
intensified and stabilized by coating the chro-
matogram with liquid paraffin or a polyethylene
glycol. The fluorescent enhancer is dissolved in
hexane or heptane (5% w/v). If the aminopropyl-
bonded layer contains a fluorescent indicator
(F254), then appreciable fluorescence quenching
can occur under UV light at 254 nm. A few com-
pounds that have weak fluoresce, like vanillic
acid and homovanillic acid, can exhibit strong
fluorescent absorption after thermal activation
and fluorescence enhancement. Thermal activa-
tion is also effective for the detection of cate-
cholamines, fruit acids, and some carbohydrates.
Spots are also detected by derivatization
reactions either before or after development;
however, the popularity of detection of the
chromatographic zones after development with
53
chemical reagents compared with chemical
derivatization before development is reflected in
the number of methods available in the scientific
literature. Many hundreds of reagents and reagent
procedures are available for the post-chromato-
graphic visualization, whereas relatively few
describe pre-chromatographic detection. In case
where visualization before chromatographic
development has been recommended, the results
are quite unique and specific.
The post-chromatographic visualization is
similar to the TLC detection, which is achieved
by spraying or dipping. Some reactions occur
immediately, and colored chromatographic zones
appear on contact with the reagent or more usu-
ally after drying or heating at a defined tempera-
ture (Table 3.3). The choice of whether the
reagent is applied as a spray or by dipping
depends on a number of factors. Spraying uses
less solvent, can be accomplished with simple
atomizer devices, and is completed in a short
period. However, spraying exposes the surround-
ing atmosphere; uneven spray, etc. are drawbacks
of the techniques. The salient features of spray-
ing reagent are below:
1. Sensitivity for detection
2. Specificity of the reagent for the analyte of
interest
3. Background effects, more specific when plates
are to be scanned spectrophotometrically
4. Stability of detection reagent
5. Stability of the chromatogram after chemical
or thermal treatment
6. Ease of preparation of the spraying or dipping
reagent
7. Hazards associated with the preparation and
use of a particular detection reagent
Common Visualizing Reagents
A few common reagents for detection of non-
UV–Vis. compounds in HPTLC are as follows.
Iodine Vapor/Solution
It is also called “iodine reaction” possibly results
in an oxidative product. The reaction pathway is
normally irreversible (but sometimes reversible
also, as previously discussed); in most instances,
54 3 HPTLC: Herbal Drugs and Fingerprints

Table 3.3 Some popular visualization reagents for TLC/ HPTLC [3]
Visualization reagent
Ehrlich’s reagent
Folin and Ciocalteu’s
reagent
Gibb’s reagent
Blue tetrazolium reagent
Tillman’s reagent
Iron (III) chloride reagent
EP reagent
Jensen’s reagent
N-Bromosuccinimide
reagent
O-Phthalaldehyse-
sulfuric acid reagent
Reagent conditions Groups detected
4-Dimethylaminobenzaldehyde (2%, w/v) in Amines, indoles
25% (w/w) hydrochloric acid/ethanol (50:50,
v/v). After treatment, heat at 110°C for 2 min
As per literature Phenols
2, 6-Dibromoquinone-4-chloroimide Phenols, indoles, thiols,
(0.5%, w/v) in methanol. After treatment, barbiturates
heat at 110°C for 5 min
Blue tetrazolium (0.25%, w/v) in sodium Corticosteroids,
hydroxide solution (6%, w/v in water)/ carbohydrates
methanol (25:75, v/v)
2, 6-Dichlorophenolindophenol sodium salt Organic acids including
(0.1%, w/v) in ethanol. After treatment, heat vitamin C
at 100°C for 5 min
Iron (III) chloride (1%, w/v) in ethanol/water Phenols, ergot alkaloids,
(95:5, v/v). After treatment, heat at 100°C inorganic anions, enols,
for5 min hydroxamic acids,
cholesteryl esters
4-Dimethylaminobenzaldehyde (0.2%, w/v) Terpenes, sesquiterpene
and orthophosphoric acid (3%, v/v) in acetic esters
acid/water (50:50, v/v). After treatment, heat
at 80°C for 10 min
Chloramine T (10%, w/v) and trichloroacetic Digitalis glycosides
acid (0.4%, w/v) in chloroform–methanol–
water (80:18:2, v/v). After treatment, heat
at 120°C for 10 min
0.5%, w/v solution in acetone. After Amino acids, Z-protected
treatment, heat at 120°C for 20 min amino acids, hydroxyl
flavones, hydroxyl
quinones
O-Phthalaldehyde (1%, w/v) in methanol/ Ergot alkaloids,
sulfuric acid (90:10, v /v). After treatment, b-blockers, indole
heat at 80°C for 3 min derivatives, histidyl
peptides

it is observed with organic unsaturated compounds
present in the separated chromatographic zones.
Electrophilic substitutions, addition reactions,
and the formation of charge-transfer complexes
occur with iodine. An added feature is that iodine
also possesses fluorescence-quenching proper-
ties; the chromatographic zones that have iodine
appear as dark zones on a TLC layer containing
fluorescent indicators (Table 3.4) [3].
Nitric Acid Vapor
Many compounds such as ephedrine, sugars, tes-
tosterone, and xanthine derivatives have yellow
or blue fluoresce after nitration, at 365 nm. Most
aromatic compounds can be nitrated with the
fumes from concentrated fuming nitric acid. The
developed chromatogram is heated to about
160°C for 10 min and kept while still hot into a
chamber containing the nitric acid vapor. Nitration
proceeds at a reasonable rate, and generally the
chromatographic zones are rendered yellow or
brown.
Redox Reaction
Oxidation and reduction reactions are frequently
used for visualization techniques as reactions are
group specific, depending on the particular
reagent used. The main redox reactions for
Detection and Visualization
Table 3.4 Iodine reactions on the TLC layer [3]
Compounds
Polycyclic aromatic hydrocarbons, indole,
and quinoline derivatives
Quinine alkaloids, barbiturates, unsaturated
lipids, capsaicins, and calciferol
Opiates, brucine, ketazone, and trimethazone
Thiols and thioethers
Alkaloids, phenothiazines, and sulfonamides
developing visible spots are as follows: Emerson’s
reagent [4-aminoantipyrine-potassium hexacy-
anoferrate (III)] for detection of arylamines and
phenols; chlorine-o-toluidine reagent for vita-
mins B1, B2, and B6 and triazines; chloramine T
for steroids and purine derivatives; and chlorine–
potassium iodide–starch reagent for amino,
imino, and amido groups and triazine herbicides.
By contrast, reduction reactions include phosph-
omolybdic acid for lipids, phospholipids, and
some steroids; tin(II) chloride-4-dimethylamin-
obenzaldehyde reagent for the detection of aro-
matic nitrophenols; blue tetrazolium reagent for
corticosteroids; Tillman’s reagent (2,6-dichloro-
phenolindophenol) for organic acids, including
vitamin C; and silver nitrate–sodium hydroxide
reagent for reducing sugars and sugar alcohols.
Iodoplatinate Reagent
This is an effective reagent for a wide range
of nitrogen containing compounds, including
alkaloids, ketosteroids, quaternary ammonium
compounds, thiols, thioethers, opiates, sulfoxides,
tricyclic antidepressants, and vitamins D3, K1,
and B1. A range of colors are produced on the
chromatogram depending on the analyte. The limit
of sensitivity for detection is often in the low
nanogram range. Iodoplatinate reagent, a typical
dipping reagent, consists of the following: 10%
(w/v) hexachloroplatinic acid aqueous solution
(3 ml), 6% (w/v) potassium iodide aqueous solu-
tion (100 ml), and 10% (v/v) methanol aqueous
solution (97 ml). After dipping, the TLC plates
are dried at 80°C for 5 min. Further heating at
55
Reaction
Formation of oxidation products
Addition of iodine to the double bonds
Iodine addition to the tertiary nitrogen for the opiates. Addition
reaction withthe -OCH3 group of the brucine. Ring-opening
reaction for the ketazone and trimethazone
Oxidation of sulfur and addition across the double bond
in the thiazole ring
Complex formation
115°C for 5 min can improve sensitivity for some
analyte.
Group-Specific Reaction
Many reagents are functional group specific
meaning that they give specific reactions with
certain organic and inorganic chemical groups. In
most cases, the reaction mechanism has been
fully elucidated. As general rule, these reagents
are very sensitive with detection limits usually in
middle to low nanogram range, for example [3].
Hydrazone Formation
A hydrazone is a class of organic compounds with
the structure R1R2C = NNH2. They are related to
ketones and aldehydes by the replacement of the
oxygen with the = NNH2 functional group. They
are formed usually by the action of hydrazine on
ketones or aldehydes. The reagent employed for
hydrazone formation is2,4-dinitrophenylhydra-
zine in acidic solution [100 mg in 100-ml ethanol/
phosphoric acid (50:50)]. After dipping or spray-
ing the chromoplate with the reagent, the reaction
is completed by heating at 110°C for 10 min.
This is a specific reagent for aldehydes, ketones,
and carbohydrates. Yellow or orange-yellow
hydrazones, or osazones in the case of carbohy-
drates, are formed on the chromoplate. Ascorbic
acid and dehydroascorbic acid are also detected
by this reagent giving yellow zones on a white
background. The sensitivity limit is in the order of
10 ng per chromatographic zone.
56
Dansylation
Dansyl [5-(dimethylamino)-1-naphthalenesulfo-
nyl] chloride and other derivatives are used to
produce fluorescent dansyl derivatives of amino
acid, primary and secondary amines, fatty acid, and
phenols. The dansylation of carboxylic acid is indi-
rect as the acid amides must first be formed. This
conversion is readily achieved with the reagent.
The detection limit is 1–2 ng for fatty acids; how-
ever, one of the problems with post-chromato-
graphic dansylation is the background fluorescence
it produces. Unfortunately, the fluorescent contrast
between the chromatographic zones and back-
ground results in reduced sensitivity.
Diazotization
Azo dyes are strongly colored and can be pro-
duced readily from aromatic nitro- and primary
amines and phenols present in the separated chro-
matographic zones. This can be achieved in two
basic ways. Nitro compounds are reduced to pri-
mary arylamines. These are diazotized with
sodium nitrite and then coupled with phenols to
form the azo dyes. Conversely, phenols can be
detected by reaction with sulfanilic acid in the
presence of sodium nitrite. The resulting azo dyes
are often stable for a period of months. A novel
approach to the detection of phenols is to impreg-
nate the layer with sulfanilic acid hydrochloride
(2.5% w/v in water) before chromatography and
application of the sample. After drying the plate
120°C for 30 min, the phenolic samples are
applied in the usual way. Following development
and drying, the layer is sprayed with fresh sodium
nitrite solution (5% w/v). The azo dyes formed
have a high stability, immediately appearing as
colored zones that maintain their color for weeks
after first visualization.
Metal Complexes
A number of transition metals act as electron
acceptors to form complexes with organic com-
pounds that are rich in electrons. Colored metal
complexes are formed by electron movement to
different energy states in the transition metal ion.
Copper (Cu2+) readily forms such complexes or
chelates with carboxylic acids including thiogly-
colic and dithioglycolic acids. A suitable detection
3 HPTLC: Herbal Drugs and Fingerprints
reagent is copper(II) sulfate 5-hydrate (1.5% w/v,
water/methanol). Most acids appear as blue zones
on a pale blue background. The limit of sensitiv-
ity is 5 mg/zone. Copper is also used in the biuret
reaction with proteins, resulting in the formation
of a reddish-violet complex, and with aromatic
ethanolamines to form blue-colored chelates.
Iron (Fe3+) and cobalt (Co2+) can also be used in a
similar way with the formation of reddish-violet
zones for phenolic compounds and blue zones in
the presence of ammonia vapor for barbiturates,
respectively.
Ninhydrin Test
Ninhydrin is a well-known detection reagent for
the visualization of amino acids, peptides, amines,
and amino sugars. The limit of sensitivity ranges
from 0.2 to 2 mg per chromatographic zone
depending on the amino acid. The colored zones
can vary from yellow and brown to pink and vio-
let, depending on the sorbent layer and pH. The
colors fade quickly unless stabilized by the addi-
tion of metal salts of tin, copper, or cobalt.
Copper(II) nitrate or acetate is the usual salts
chosen as additives. A typical formulation for
such a ninhydrin dipping reagent is 0.3% (w/v) in
propan-2-ol with the addition of 6 ml/100 ml of
aqueous copper(II) acetate (1% w/v). After dip-
ping, the TLC layer is heated at 105°C for 5 min.
For better resolution between glycine and serine,
collidine is added to the ninhydrin at conc. of
5-ml/100-ml reagent.
Natural Product Reagent
Natural product reagent (NPR), as diphenyl boric
acid-2-aminoethyl ester, readily forms complexes
with 3-hydroxyflavones via a condensation reac-
tion and is used extensively for visualization of
components in herbal preparations in TLC/HPTLC
analysis. A suitable dipping reagent consists of
diphenyl boric acid-2-aminoethyl ester (1 g) dis-
solved in methanol (100 ml). This solution should
be freshly prepared when needed, especially where
quantitative results are required. The chromoplate
is thoroughly dried, dipped in the reagent for a few
seconds, dried again in a stream of warm air, and
then dipped in a polyethylene glycol (PEG) 4000
(5% w/v) solution in ethanol. The reagent is
Coupling of HPTLC with Spectrometry
especially good for the detection of rutin, chloro-
genic acids, hypericum, and other flavonoids. It
can also be used on most sorbent layers including
both the normal and reversed-phase silica gels.
The limit of sensitivity is about 1–5 ng/chromato-
graphic zone. The purpose of the PEG 4000 is to
enhance the fluorescence and to stabilize the emis-
sion of light.
Case Study
The root, stem bark, and fruits of various Berberis
species in the Himalayan region are well recog-
nized for their alkaloid contents. Due to global
demand for berberine alkaloids and their deriva-
tives, various analytical tools such as HPLC, GC,
and GC-MS have been used for berberine estima-
tion. HPTLC, a technique for quality control and
standardization of traditional herbs like Berberis
for berberine content in root and stem bark of
three Berberis (i e., B. asiatica, B. aristata, B.
lycium), was used, and comparative analytical
assessment revealed that the berberine content
varied both in root and stem bark samples. More
berberine content observed in root samples as
compared to bark of all the investigated species.
Among the species, Berberis asiatica contains
more berberine as compared B. lycium and
B. aristata (Figs. 3.1 and 3.2) [4].
B asiatica B aristata B lycium Berberine Berberine Berberine B. asiatica B. aristata B. lycium
(root) (root) (root) 2 µL
Fig. 3.1 HPTLC at 254-nm root and stem bark samples of various Berberis species [4]
57
Coupling of HPTLC with Spectrometry
HPTLC is coupled with ultraviolet–visible, infra-
red spectrometry, Raman spectrometry, photoa-
coustic spectrometry, and mass spectrometry.
FTIR has a high potential for the elucidation of
molecular structures, and the characteristic
absorption bands are the clue for specific detec-
tion, as it indicates the presence/absence of
specific functional group. Almost all chemical
compounds yield good FTIR spectra that are
more useful for identification of unknown sub-
stances and discrimination between closely
related substances. The HPTLC–FTIR spectra
make possible the detection and quantification of
even non-UV-absorbing substances on HPTLC
plates. These reasons make this hyphenated tech-
nique more universally applicable. The HPTLC
and FTIR coupling can be divided into two
groups, that is, indirect and direct methods [5].
For indirect coupling there is transfer of the
substance from a TLC spot to a non-absorbing IR
material (KBr or KCl) or in situ measurement of
excised HPTLC spots when the spectra are
recorded directly from the plate. The direct
online-coupled HPTLC–FTIR offers some advan-
tages relative to other hyphenated techniques
4 µL 6 µL (bark) (bark) (bark)
58
B asiatica B aristata B lycium Berberine
(root) (root) (root) 2 µL
Fig. 3.2 HPTLC at 366-nm root and stem bark samples of various Berberis species [4]
(HPTLC–Raman spectroscopy, HPTLC–PA, and
HPTLC–MS), such as the ease of operation and
the optimized operational aspects of online cou-
pling. In direct-coupling HPTLC–FTIR method,
a major difficulty is the absorption by conven-
tional stationary phases, for example, silica gel,
which absorb strongly in the IR range. It is very
difficult to obtain reliable spectra in the regions
where the layer shows strong IR absorption. The
silica gel, the most widely used adsorbent in
HPTLC, presents absorption bands between
1,350 and 1,000 cm−1 and above 3,550 cm−1 which
are superimposed on the spectra of compounds,
and only the region between 3,550 and 1,350 cm−1
can be evaluated. Therefore, measurements in this
region are not possible, but it is possible to make
measurements up to 1,000 cm−1 on cellulose. The
best results are obtained when the mixture of silica
gel 60 and magnesium tungstate (1:1) is used as
stationary phase. This adsorbent improves signal-
to-noise ratios and enhances the performance of
the diffuse reflectance of the matrix. Another
problem is due to the particle size, particle-size
distribution, and the layer thickness, which affect
the scattering, remitting, and absorbing of the
radiation by the matrix. A stationary phase with a
particle diameter of 10 mm, a narrow particle-size
3 HPTLC: Herbal Drugs and Fingerprints
Berberine Berberine B. asiatica B. aristata B. lycium
4 µL 6 µL (bark) (bark) (bark)
distribution, and a layer thickness of 200 mm on
glass is found to be ideal in the mid-IR range.
Finally, the binder or the fluorescence indicator
added to the adsorbent and the mobile phase could
lead to altered HPTLC–FTIR spectra [5].
The identification can be realized by fitting the
reference spectra to sample spectra and visual com-
parison. The compounds separated by HPTLC can
be also identified using an HPTLC–FTIR library.
The band position, width, and intensity are automati-
cally compared, and the reliability of the results is
described in terms of hit quality. Quantitative analy-
sis with the HPTLC–FTIR technique is generally
applied for the substances that do not absorb in the
UV–Vis. range and when the precision required is
not too high. The lack of precision is due to the
increase of sample spot broadening with increased
migration distance and to the measurement not being
exactly at the peak maximum. These problems are
due to the circular infrared beam with small diame-
ter. The determination of compounds is made on the
basis of evaluation of the peak areas in the Gram-
Schmidt trace or in the window diagram, or by the
evaluation of Kubelka-Munk spectra with integra-
tion of their strongest bands. The method using the
Gram-Schmidt traces indicates the changes in absor-
bance over the whole spectral region, and therefore,
Bibliography
it is suitable and practical for rapid determinations.
The evaluation of the peak areas in the window chro-
matogram is appropriate for the quantification of
individual substances. An advantage of this method
is a better signal-to-noise ratio, but the disadvantage
is the poorer precision. More precise results are
obtained using the evaluation of Kubelka-Munk
spectra. The limit of identification and determination
is 10 times higher than those obtained by densitom-
etry. This method has the disadvantages of the mea-
surement only of the fraction of the substance in the
peak maxima and the additional processing step. In
conclusion, none of these methods is perfect and
appropriate for all samples. The choice of a method
depends on the goals of the analysis [5].
TLC and HPTLC are valuable tools for quali-
tative determination of small amounts of impuri-
ties. Lack of chemical markers is a major problem
for the quality control of herbal medicines. In
many cases, we do not have sufficient chemical
and pharmacological data of chemical markers.
Furthermore, there are many technical challenges
in the production of chemical markers, for exam-
ple, temperature, light, and solvents often cause
degradation and/or transformation of purified
components; isomers and conformations may
also cause confusions of chemical. Under such
conditions, HPTLC fingerprints have its values,
using reference botanical standard for compari-
sons and quality management policies [ISO 9000
certification, good laboratory practices (GLP),
good manufacturing practices (GMP), total qual-
ity management (TQM) and validated instruments
and services, etc.] in pharmaceuticals to have a
better quality of drugs.
References
1. Giri L, Andola HC, Purohit VK, Rawat MSM, Rawal RS,
Bhatta ID. Chromatographic and spectral fingerprinting
standardization of traditional medicines: an overview as
modern tools. Res J Phytochem. 2010;4:234–41.
59
2. Rajkumar T, Sinha BN. Chromatographic fingerprint
analysis of budmunchiamines in Albizia amara by HPTLC
technique. Int J Res Pharm Sci. 2010;1(3):313–6.
3. Wall PE. Thin layer chromatography: a modern
practical approach, RCS chromatography monograph.
Cambridge: Royal Society of Chemistry; 2005. ISBN
0-85404-535-X.
4. Andola HC, Rawal RS, Rawat MSM, Bhatta ID, Purohit
VK. Analysis of berberine content using HPTLC
fingerprinting of root and bark of three Himalayan
berberis species. Asian J Biotechnol. 2010;2(4):239–45.
5. Cimpoiu C. Qualitative and quantitative analysis by
hyphenated (HP)TLC-FTIR technique. J Liq Chromatogr
Relat Technol. 2005;28:1203–13.
Bibliography
Ahmad I, Aqil F, Owais M. Turning medicinal plants into
drugs. Modern phytomedicine, vol. 384. Weinheim:
Wiley; 2006. p. 67–72.
Bhutani KK. Fingerprinting of Ayurvedic drugs. East
Pharm. 2000;507:21–6.
Bobby N, Wesely EG, Johnson M. HPTLC profile studies
on the alkaloids of Albizia lebbeck. Asian Pac J Trop
Biomed. 2012;2:1–3.
Dhandapani A, Kadarkarai M. HPTLC quantification of
flavonoids, larvicidal and smoke repellent activities of
Cassia occidentalis L. (Caesalpiniaceae) against
malarial vectore Anopheles Stephensi Lis (Diptera:
Culicidae). J Phytol. 2011;3(2):60–71.
Liang YZ, Xie P, Chan K. Quality control of herbal medi-
cines. J Chromatogr B. 2004;812:53–70.
Long F. Bio-pharmaceutical characterization of herbal
medicinal products. Drugs. 2001;44(4):102–8.
Sagar BPS, Zafar R, Panwar R. Herbal drug standardiza-
tion. Indian Pharm. 2005;4(35):19–22.
Shahare MD, Mello PM. Standardization of Bacopa mon-
nieri and its formulations with reference to Bacoside
A, by high performance thin layer chromatography.
Int J Pharmacogn Phytochem Res. 2010;2(4):8–12.
Shanbhag DA, Khandagale NA. Application of HPTLC in
the standardization of a homoeopathic mother tincture
of Syzygium jambolanum. J Chem Pharm Res.
2011;3(1):395–401.
Soni K, Naved T. HPTLC–its applications in herbal drug
industry. Pharma Rev. 2010 (July-August);112–7.
WHO. Quality control methods for medicinal plant mate-
rials. Geneva: WHO; 1998.
Yadav D, Tiwari N, Gupta MM. Simultaneous quantification
of diterpenoids in Premna integrifolia using a validated
HPTLC method. J Sep Sci. 2011;34(3):286–91.
 HPLC: Herbal Drugs and Fingerprints
High performance liquid chromatography
(HPLC) has been the most reliable tool for the
separation of complex mixtures. The complex
crude extracts from medicinal plants have differ-
ent closely related compounds along with
metabolites and require efficient analytical sep-
aration methods for HPLC fingerprints. HPLC
has been recognized since the early 1980s as
the most versatile technique for the efficient
separation, and since that time, there are regular
innovations through the years in terms of con-
venience, speed, choice of column stationary
phases, high sensitivity and applicability to a
broad variety of sample matrices, and ability to
hyphenate the chromatographic method to spec-
troscopic detectors. From the chromatography
viewpoint, the development of columns with
different phase chemistry (especially reversed
phase) enabled the separation of almost any
type of plant product. HPLC allows an analyst
to identify unknown compounds by comparison
of their HPLC retention times with known and
UV spectra. The HPLC analytical methods are
validated for specificity, linearity, limit of
detection (LOD), precision and accuracy, rug-
gedness, and robustness. The lack of a sensitive
and universal detection, suitable for both quali-
tative and quantitative analysis, under a wide
range of conditions is one of the reasons to look
for universal analytical technique. A literature
search on journals focused on natural product
chemistry revealed that more than 70% of
HPLC applications are performed by HPLC–UV
and HPLC–DAD, with HPLC–MS applications
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_4, © Springer India 2012
4
accounting for about 20% (one third being
related to MS–MS), HPLC–ECD for 3%,
HPLC–NMR for 2%, and ELSD for 1.5%. The
other detection techniques are reported in less
than 1% of all HPLC applications. The large
majority of analyses are still performed by
HPLC–UV, which is widespread in many labo-
ratories. For the detection of non-UV-active
constituents, aerosol-based detectors, such as
ELSD or CAD, are in use with HPLC–MS as a
powerful tool for detection ability. More
recently, the advent of electrospray ionization
(ESI) and atmospheric pressure chemical ion-
ization (APCI) interfaces has provided mass
spectrometry (MS) interfaces which are appli-
cable to the analysis of a wide range of
molecules and are compatible with liquid
chromatography [1]. For the separation of crude
extracts, either raw mixtures or samples
enriched by extraction via simple solid-phase
extraction (SPE) or liquid–liquid extraction
(LLE) are injected into HPLC after passing
through 0.45-m filter. The separations are per-
formed mostly in reversed-phase chromatogra-
phy on C18 material with the MeCN-H2O or
MeOH-H2O solvent system in the gradient elu-
tion mode. In order to improve the separation
efficiency, various modifiers are added to the
mobile phase that might strongly influence the
sensitivity of detection. In multi-hyphenated
systems, online presence of several different
detectors (hyphenated systems) leads to the need
for an eluent composition that is compatible
with all detectors [2].
61
62 4 HPLC: Herbal Drugs and Fingerprints

Table 4.1 Properties of solvents commonly used in HPLC [3]

Miscible
S. no. Solvent Polarity with water UV cutoff
1 Hexane Nonpolar No 200
2 Isooctane Nonpolar No 200
3 Carbon Nonpolar No 263
tetrachloride
4 Chloroform Nonpolar No 245
5 Methylene chloride Nonpolar No 235
6 Tetrahydrofuran Nonpolar Yes 215
7 Diethyl ether Nonpolar No 215
8 Acetone Nonpolar Yes 330
9 Ethyl acetate Nonpolar Poorly 260
10 Dioxane Nonpolar Yes 215
11 Acetonitrile Nonpolar Yes 190
12 Isopropanol Nonpolar Yes 210
13 Methanol Nonpolar Yes 205
14 Water Polar Yes —

A Glance on HPLC Column possible while retaining adequate resolution.

HPLC column, the heart of the instrument, con-
sists of a metal cylinder packed with tiny silica or
modified silica particles to separate compounds
in a mixture. The mobile phase passes through
the column using high pressure, resulted each
component in the mixture coming out at different
times, and elution is based on polarity (Table 4.1)
[3]. Selection of suitable column is entirely based
on the chemical nature of ingredient to be ana-
lyzed. The column specification (length, diame-
ter, and particle size) has impact on the resolution
and analysis time (Table 4.2) [4]. In case analyte
is strongly retained by the stationary phase, a
short column length significantly decreases anal-
ysis time. Column selectivity is also controlled
by changing the column temperature and the pH
of the mobile phase and addition of organic
modifiers such as acetonitrile into the mobile
phase to reduce stationary phase–analyte interac-
tion and elute more rapidly.
The length of column is a factor to decide the
nature and number of compounds to be analyzed.
Analytical HPLC columns are at the range of
5–25 cm in length; long columns provide better
resolution and efficiency but require longer reten-
tion times and higher pressure. Due to the reason,
analysts prefer to choose the shortest columns as
The internal diameter of the column has a vital
role for adequate resolution, which is inversely
proportional (i.e., decrease in internal diameter,
the resolution is more fine), as smaller diameters
increase efficiency and resolution, but it requires a
lower flow rate of solvent and, thus, increases the
time of analysis. At low flow rate, impurities, if
any, such as dirt quickly clog up the column with
small internal diameters. Most columns have a
particle size of 5 mm. The smaller particle size,
such as 3 mm, will offer greater separation and will
decrease the time of analysis. However, smaller
particles require a higher amount of pressure to
push the mixture through the column. Every strat-
egy in column selection has an advantage and dis-
advantage. It is up to the analyst to choose each
parameter skillfully to achieve an optimal balance
in separation, efficiency, and pressure in the system.
HPLC is a sophisticated form of column chro-
matography, especially designed to separate and
classify components of a sample based on its
interactions with a specific column. HPLC is dis-
tinct from column chromatography because it
utilizes a more pressurized environment to propel
the liquid samples through the densely packed
column more efficiently. Based on the nature of
silica particles used, columns are termed as nor-
mal phase or reverse phase.
Analytical Mobile Phases and HPLC 63

Table 4.2 Different stationary phases in HPLC column and utility-USP categories [4]
S. no. USP code Particle dia. (mm) Bonding Description
1 L1 3–10 C-18 Octadecylsilane chemically bonded to porous silica or
ceramic particles, used in analysis of wide range of
compounds, neutral to weakly basic
2 L2 30–50 C-18 Octadecylsilane chemically bonded to silica gel of a
controlled surface
3 L3 5–10 None Porous silica microparticles, used for normal phase
separation
4 L4 30–50 Silica Silica gel of controlled surface porosity bonded to a solid
spherical core
5 L5 30–50 Alumina Alumina of controlled surface porosity bonded to a solid
spherical core
6 L6 30–50 Cation Strong cation-exchange packing-sulfonated fluorocarbon
exchange polymer coated on a solid spherical core
7 L7 5–10 C-8 Octyl silane chemically bonded to totally porous microsilica
particles, provides shorter retention times for hydrophobic
compounds than C-18
8 L8 10 Amino An essentially monomolecular layer of amino-propylsilane
(−NH2) chemically bonded to totally porous silica gel support
9 L9 10 Cation Irregular, totally porous silica gel having a chemically
exchange bonded, strongly acidic cation-exchange coating
10 L 23 10 Anion Anion-exchange resin made of porous polymethacrylate or
exchange polyacrylate gel with quaternary ammonium groups

Analytical Mobile Phases and HPLC
HPLC is a high-value instrument, between the
commercial utility and capital investment; it is
difficult to dedicate instrument for a single ana-
lytical mode, so there is a frequent need for shift
in the operation from normal phase (NP) to
reverse phase (RP) and vice versa. When aqueous
mobile phase is replaced with nonaqueous condi-
tions, immiscibility situations arise during this
changeover. We can have a smooth analytical
operation by following the certain practices at the
instant of switchover from one mode to another.
The instrument has different types of seals, which
may suffer by expansion, contraction, and extra
friction due to change in solvent, so:
1. On regular practice, there is a trend to replace
the column with tubing or a union; the system
is flushed extensively with isopropanol (IPA)
before going over to water or hydrocarbon.
2. Before beginning of the changeover process,
remove the HPLC column, cap, and store it at
the appropriate storage solvent, unless the
same column is to be used in the new mode.
3. Columns such as cyano- and fluorophenyl (F 5)
can work in either RP or NP mode and can
remain installed, if desired.
4. After IPA flush, the column can be removed
and capped to avoid excessive wear on the
valuable component. In the flushing steps,
we have to be sure to include the entire fluid
path (pump, auto-sampler, valves. detector,
including the sample loop and any other fluid
paths) that is encountered for the normal
operation of making injections. As part of all
the washes, the injection needle (syringe) is
to be washed well with IPA, where manual
injections are carried out.
5. For loop-type injector, it is desired to flashed-out
by several IPA injections (as IPA is miscible
with both high-aqueous and high organic
mobile phases); the total volume of IPA
required may vary with instrument design, but
the waste volume can be monitored for future
data and observed for uniform appearance of
absorbance at the detectors, indicating the job
done, that is, the system has returned to a
stable baseline.
64
6. The second step after the IPA wash is to be
followed with alcohol (methanol/ethanol).
The trend with alcohol is similar as with IPA
wash before going to water. Methanol will
help flush the IPA out faster than going directly
from IPA to water. If excessive baseline noise
or drift is observed with a UV detector, repeat
the procedures and allow more time to flush
out the poorly swept flow regions.
Solvent Changeover in HPLC
The first best practice is to dedicate the instru-
ment for specific mode of analysis. Another best
desirable point is that at least columns should be
dedicated to have trouble-free operation. Whenever
changeover becomes necessary, the analyst uses
his professional skills, and a few notable points
are shown below.
1. Remove all the additives and start with 100%
isopropyl alcohol in all reservoirs.
2. Isopropyl alcohol is fully miscible with all
common solvents and is the safest changeover
solvent.
3. Flow rate of isopropyl alcohol should at low,
about half of normal, to avoid excessive seal
wear and damage due to over-pressuring.
4. The use of acetonitrile routinely as the change-
over solvent should be avoided as it is better
than methanol but is not fully miscible with
pure hydrocarbons.
5. The use of methanol routinely also should be
avoided as it is not fully miscible with many
normal-phase conditions, for example,
n-hexane and heptanes.
6. If isopropyl alcohol is not available, first check
miscibility (using small external vessel) with tar-
get mobile phase before starting the operation.
7. In a gradient system, isopropyl alcohol should
be in all lines of instrument to make certain
that water or hydrocarbon is removed from all
fluid areas, including the injector valve and
any other selector valves.
8. Analyst should have a focus on the pressure
mark and detector signals during changeover
as these are excellent indicators to confirm
that the system has been fully in equilibrium,
4 HPLC: Herbal Drugs and Fingerprints
but evaporative detectors such as MS and
ELSD cannot be used for this purpose.
9. The severe detector baseline noise or pres-
sure fluctuations are the indication of incom-
plete mixing, resulting to the globules of
immiscible solvent that may resemble as
bubbles or particles.
10. The detectors and all other components
should be flushed if baseline is not stable.
11. Total time for changeover can vary, depending
on the chemical composition of mobile phase
used, but normal span should be of about an
hour. The reduction in changeover time may
result into slowdown of the process.
12. The changeover and baseline equilibration
with refractive index (RI) detectors, are
extremely slow and achieve equilibrium only
after the complete changeover.
13. For excessive baseline noise and drift, in gra-
dient system, degassing should be properly
checked as it may have pockets of immiscible
solvent.
14. There should be always a blank run before real
analysis. For first time HPLC analysis of a new
compound specificity of the analytical method
should be established, along with reproduc-
ibility, repeatability, linearity, limit of detection,
precision, and accuracy of the method used.
15. To start HPLC analysis on routine analysis,
first have a blank run, than standard/reference
standard, standard mixed with sample, and
finally the sample.
16. For successful changeover into a different
chromatography, where columns and instru-
ments are frequently used in different modes,
labeling can be adopted to alert new user
about possible solvent compatibility issues,
indicating previous use also.
Buffers as Mobile Phase
Reversed-phase HPLC analysis is affected by
pH, type of buffer in use and its concentration,
and presence of organic modifier and its volume.
An improper choice of buffer, in terms of buffer-
ing species, ionic strength, and pH, can result in
poor or irreproducible retention and tailing in
Buffers as Mobile Phase 65

Table 4.3 HPLC buffers, pKa values, and useful pH bases gain a proton and become ionized when pH
range [6] decreases. For mixtures containing acids and/or
Buffer pKa (25°C) Useful pH range bases, it is necessary to control the pH of the mobile
TFA 0.5 <1.5 phase using an appropriate buffer in order to achieve
Sulfonate 1.8 <1–2.8 reproducible results. The separation of ionic com-
Phosphate 2.1 1.1–3.1 pounds at satisfactory level can only be achieved
Chloroacetate 2.9 1.9–3.9 with a mobile phase of certain pH where retention
Formate 3.8 2.8–4.8 and resolution are sensitive to it. Generally, a buffer
Acetate 4.8 3.8–5.8
concentration of 10–50 millimolar (mM) is ade-
Sulfonate 6.9 5.9–7.9
quate for small molecules [7, 8].
Phosphate 7.2 6.2–8.2
Ammonia 9.2 8.2–10.2
Phosphate 12.3 11.3–13.3
Buffer Solubility

reverse-phase separation of polar and ionizable
compounds due to partial ionization of the ana-
lyte and strong interaction between analytes and
residual silanols or other active sites on the sta-
tionary phase. It can be overcome by proper
mobile-phase buffering (maintaining the pH
within a narrow range) and choosing the right
ionic species and its concentration (ionic strength)
in the mobile phase [5]. In LC–MS, separations
depend heavily on the correct choice of acid–base
buffering species and other additives. A suitable
buffer always has ability to maintain and not sup-
press analyte ionization in the MS interface.
Selection of Buffer and Analytical
Method Development
Although buffer solubility is determined readily by
precipitation, but this method is tedious and time-
consuming. The relationship between the buffer’s
solubility limit and volume fraction of organic
cosolvent is useful in reversed-phase LC as a
general rule is that less than 50% organic should be
used with buffer, which also depends on the specific
buffer as well as its concentration. The most com-
mon buffer systems, including ammonium acetate
at pH 5.0, ammonium phosphate at pH 3.0 and pH
7.0, and potassium phosphate at pH 3.0 and pH 7.0.
The inorganic buffer salts are less soluble in typical
organic cosolvents compared with organic buffer
salts. The sodium salts are avoided because they
are expected to have lower solubility, which makes
them less useful for liquid chromatography [8].

The typical pH range for reversed phase on silica-
based packing is pH 2–8; choice of buffer is typi-
cally governed by the desired pH. It is important
that the buffer has a pKa. A rule of thumb is to
choose a buffer with a pKa value <2 units of the
desired mobile-phase pH (Table 4.3) [6].
Buffer Concentration
In reversed-phase HPLC, the retention of analytes
is related to their hydrophobicity. The more hydro-
phobic the analyte, the longer it is retained. When
an analyte is ionized, it becomes less hydrophobic,
and therefore, its retention decreases. Acids lose a
proton and become ionized when pH increases, and
Buffer and Its Impact on Detection
Slight variations in mobile-phase preparation can
result in pH changes that can have dramatic
effects on selectivity, capacity factor (retention
factor), peak shape, resolution, and reproducibility.
Good laboratory practices in preparing mobile
phases are to be followed to ensure that results
can be reproduced within and between laboratories.
While instrument solvent blending has become
very convenient for fast method development, it
is best to evaluate premixed solvent whenever
possible to ensure accuracy and equilibration
before completing and publishing HPLC method.
The choice of buffer is also dependent upon
means of detection. For traditional UV detection,
66
the buffer needs to be effectively transparent in
this region, especially, critical for gradient separa-
tions. Buffers listed in Table 4.3 have low enough
absorption below 220 nm [7].
Buffers as Mobile Phase in HPLC
Always, buffers should be prepared freshly on the
day requirement to ensure that the buffer pH is
unaffected by prolonged storage and that there is
no microbial growth present. Changes in pH and
microbial growth affect chromatography up to a
drastic extent. Buffer reagents can contain a stabi-
lizing agent, for example, sodium metabisulphite,
which affects the optical and chromatographic
behavior of buffer solutions, so it is often worth
buying reagents that contain no stabilizer. The
buffer is to be filtered through a 0.45-mm filter
before use to remove any particulate matter that
may cause blockages. After filtration, the solvents
are stored in a covered reservoir to prevent con-
tamination with dust, fumes, etc. The freshly pre-
pared mobile buffer phase is thoroughly to be
degassed to remove all dissolved gasses, by either
bubbling with helium or sonication and/or vac-
uum filtration. Ammonium acetate, ammonium
formate, ammonium hydroxide solution in water,
ammonium phosphate monobasic, ammonium
trifluoroacetate, potassium phosphate dibasic
anhydrous, sodium formate, sodium phosphate
dibasic dehydrate, sodium phosphate monobasic
anhydrous, sodium trifluoroacetate, trifluoroacetic
acid, triethylamine, etc. are a few reagents used as
buffer during HPLC analysis. Phosphoric acid
and its sodium or potassium salts are the most
common buffer systems for reversed-phase HPLC.
Phosphonate buffers can be replaced with sul-
fonate buffers when analyzing organophosphate
compounds. In LC–MS, volatile buffer systems,
such as trifluoroacetic acid and trifluoroacetate
(sodium or potassium), are ideal choices for posi-
tive-ion mode detection. TFA can negatively impact
detector response even in positive-ion mode, while
it strongly suppresses ionization with negative-ion
mode. Acetic acid is good for negative-ion mode.
LC–MS applications further limit buffer selection
and buffer concentration [7, 8].
4 HPLC: Herbal Drugs and Fingerprints
Detectors in HPLC: Detection,
Quantification, and Fingerprints
Different detectors are used for medicinal plant’s
profiling and fingerprinting, for quantitative analyses,
and quality control purposes in HPLC. In each
case, the potential range of detection, their sensitiv-
ity and selectivity and their potential to provide
online structure information, is unique and specific.
A few common HPLC detectors are as follows:
UV, DAD, FU, EC, RI, ELSD, CAD, MS, MS–MS,
NMR, etc. HPLC detectors can be categorized into
two groups: (a) Simple detectors used for the
recording of chromatographic traces for profiling
or quantification purposes (e. g., UV, ELSD, ECD)
and (b) detectors in hyphenation are used to gener-
ate multidimensional data for online identification
and dereplication (e.g., UV–DAD, MS, NMR).
A few detectors in HPLC are selective for compounds
having specific spectroscopic or physicochemical
properties; they may be summarized as follows:
HPLC–UV
Ultraviolet (UV) detector is most widely used
among all HPLC detectors, although it suffers
from some limitations, particularly for natural
product compounds that do not possess UV chro-
mophores. It has the best combination of sensitiv-
ity, linearity, versatility, and reliability of all the
LC detectors developed so far. Most natural prod-
ucts absorb UV light in the range of 200–550 nm,
including all substances having one or more dou-
ble bonds and all substances that have unshared
electrons. Thus, even compounds having weak
chromophores, such as triterpene glycosides (e.g.,
asiaticoside from Centella asiatica), can be suc-
cessfully detected by UV at short wavelengths
(203 nm) [9]; in such cases, mobile phase exhibit-
ing high UV cutoffs is avoided because they might
blind the detection with weak chromophores [10].
In UV detection, the relationship between the
intensity of light transmitted through the detector
cell and the solute concentration is governed by
Beer’s law [11]. The two factors that control the
detector sensitivity are the magnitude of the
Detectors in HPLC: Detection, Quantification, and Fingerprints
extinction coefficient of the analyte of interest at
a given wavelength and the path length of the
light passing through the UV cell. The sensitivity
increase with increasing path length but cell vol-
ume is designed in such a way to help avoid peak
dispersion [12].
Presently, three types of UV detectors are
available: fixed wavelength, multiple wave-
length, and photodiode array (PDA) [also known
as diode-array detector (DAD)]. The fixed-
wavelength detector is the least expensive and
has higher intrinsic sensitivity because the light
is emitted at specific wavelengths with given
lamps and a single wavelength for the detection
of nearly all natural products having a chro-
mophore for both profiling and quantification
purpose. It has been in practice for flavonoids,
terpenes, alkaloids, coumarins, alkamides, and
polyacetylene. It is used in several pharmacope-
ias, as it is simple and inexpensive, for the
quantification of individual natural product in
the quality control of herbal drugs or phyto-
preparations. In the United States Pharmacopeia
(USP) and European Pharmacopeias, HPLC–UV
(203 nm) methods are used for the determina-
tion of the total ginsenoside content of ginseng
roots and the measurement of the ratio of the
ginsenosides [the ginseng saponins, structurally
grouped into two groups: the panaxadiol (PD)
fraction (e.g., ginsenoside-Rb1, ginsenoside-
Rb2, ginsenoside-Rc, ginsenoside-Rd) and the
panaxatriol (PT) fraction (e.g., ginsenoside-
Rg1, ginsenoside-Rg2, ginsenoside-Re, ginse-
noside-Rf, depending on their glycons). The
ginsenoside-Rb1 and ginsenoside-Rg1 are
believed to be the most pharmacologically effec-
tive compounds] Rg1 and Rb1. This analysis is
important to assess pharmacological effects, as
a low Rg1/Rb1 ratio is linked to the calming
properties of American ginseng, while the high
Rg1/Rb1 ratio to be for the stimulating charac-
teristics of Asian ginseng [13].
HPLC–UV detector for the quantification of
flavonoid, naphthodianthrone, and phloroglucinol
constituents of Hypericum perforatum by a ter-
nary solvent system in the gradient mode (50 min)
had separated 16 main constituents, which were
quantified based on their relative response factors
67
at 270 nm and compared with rutin as an external
standard. A more rapid method (5 min) using
fast-gradient elution for a specific determination
of naphthodianthrones (590 nm) and phlorogluci-
nols (270 nm) also has been reported [14].
HPLC–UV analysis also applied for Ginkgo
biloba; a complete profiling of most of the
flavonoids (350 nm) (33 compounds detected by
comparison with standards) was reported by
Hasler et al. [15]. The same authors also proposed
an HPLC–UV method, which includes the hydro-
lysis of all glycosides and the subsequent
quantification of three main flavonoid aglycones
(quercetin, kaempferol, and isorhamnetin) for the
determination of the total flavonoid content.
Recently, another HPLC–UV method (350 nm)
has been reported, which includes the
quantification of two glycosides (rutin and quer-
citrin) as markers, in addition to the three agly-
cones [16]. An analysis of ten commercial
samples revealed that problems of adulteration
with rutin exist to fulfill the total flavonoid
requirement (24%), and thus, the method is
highly helpful in quality control and total
fingerprint profiling [17].
A multivariate data analysis (MVDA) of
Ginkgo products, based on their HPLC–UV
profiles, and comparison with the traditional
flavonol quantification method [18] revealed that
there are significant variations in the relative con-
centrations of individual flavonols in commercial
products. The other active ingredients in Ginkgo
are the terpene trilactones, which are standard-
ized at 6% in the extracts. These terpenes have
weak chromophores (at wavelength 220 nm) and
are difficult to detect in the presence of the
strongly UV-absorbing flavonoids, so quantified
by other methods such as ELSD [19]. In Ginkgo,
the level of highly allergenic compound alky-
lphenols (ginkgolic acids, cardanols, and cardols)
are part of specification, and maximum limit is
5 ppm, as per EU and US Pharmacopeias. The
total amount of alkylphenols is determined by a
simple HPLC–UV (210 nm) [20].
The separation of crude extracts using gradi-
ent elution causes a shift of the baseline at low
wavelengths that can be avoided when working
with modifiers having low UV cutoffs such as
68
phosphate buffers or trifluoroacetic acid (TFA).
These modifiers are not optimal in multi-hyphen-
ated systems with HPLC–MS (due to nonvolatile
solvents, which have impact for ion suppression)
and are avoided [21]. In other cases, for the same
plant extract, quantification may require both
HPLC–UV and HPLC–MS. A simple HPLC–UV
method for the standardization of herbal supple-
ments as Pueraria lobata [known as Kudzu
(Japan)], a fully validated as per the ICH guide-
lines for the quantification of the isoflavone, is in
practice [22, 23].
HPLC–UV detector is used to compare the
fingerprint profiles of closely related species or
the same species from different locations. Herba
Oldenlandiae, a plant of the Chinese Pharmacopeia,
which can be adulterated by different Oldenlandia
species. A comparative analysis of chemical
components was obtained by software calcula-
tion of correlation coefficients of the entire UV
chromatograms for appraising similarity [24].
Fingerprint-based similarity also can be
employed for chemotaxonomic study by apply-
ing hierarchical clustering analysis (HCA) and
principal component analysis (PCA) to a set of
HPLC–UV profiles, as was used for different
Taxus species [25].
HPLC–FD
In the fluorescence detector, the detection is
based on the molecular absorption of a photon,
which triggers the emission of another photon
with a longer wavelength. This difference in
wavelengths (absorption vs. emission) provides
more selectivity, and the fluorescent light is
measured against a very low-light background,
thus improving the signal/noise (S/N) ratio.
There are very few natural product compounds
having fluorescent in a practical range of wave-
lengths, but compounds can be made to fluoresce
by forming appropriate derivatives [12]. It has
higher sensitivity and selectivity compared with
HPLC–UV. The technique is used for detection of
aflatoxins in food because aflatoxins have natural
fluorescence [26]. An FD method for the
4 HPLC: Herbal Drugs and Fingerprints
quantification of aflatoxins in herbs also has
been recently reported [27]. It combines sample
clean-up with immuno-affinity columns fol-
lowed by HPLC–FD, and it enabled the detec-
tion of aflatoxin content ranging from 7 to 20 mg/
kg in plant material. HPLC–FD also has been
used for the screening of major benzo-phenan-
thridine alkaloids produced by cell cultures of
Eschscholzia californica [28]. The detection of
natural products that do not have natural
fluoresce has been successfully achieved after
the addition of fluorescent tags. This was the
case for the specific and sensitive detection of
the plant hormone jasmonic acid which was
tagged with zink cyanide [Zn(CN)2] (Adam’s
reagent) [29].
HPLC–CL
Chemiluminescence (CL) may be defined as the
emission of light from a molecule or atom at an
electronically excited state due to chemical reac-
tion at an ordinary temperature without any addi-
tional generation of heat. Chemiluminescent (CL)
nitrogen detection is a new technology for the
detection of nitrogen-containing molecules, includ-
ing a broad range of pharmaceuticals with very
high sensitivity up to the femtogram range [30]. As
many compounds are not chemiluminescent, they
can only be detected chemiluminescently after
derivatization. There are many chemiluminogenic
reagents, which helps in chemiluminescence detec-
tion. The chemiluminogenic reagents are applied at
post-column stage; for example, addition of potas-
sium hexacyanoferrate (III) enables the detection
of various carboxylic acids in the form of N-(4-
aminobutyl)-N-ethylisoluminol derivatives [31].
Though it is a very sensitive method, but till the
time, it has not been much applied to the analysis of
crude plant extracts. A study reported the sensitive
HPLC–CL detection of flavonoids (LOD 3 ng/ml)
in phytopharmaceuticals containing Hippophae
rhamnoides. The procedure was based on chemilu-
minescent enhancement by flavonols of the cerium
(IV)–rhodamine 6 G system in a sulfuric acid
medium [32].
HPLC Detection in Hyphenated System
HPLC–ECD
Electrochemical detector (ECD) detection is
usually performed by maintaining the potential
of the working electrode at a fixed value relative
to the potential of the electrolyte, which is mea-
sured by the reference electrode. The fixed
potential difference applied between the work-
ing and the reference electrodes drives the elec-
trochemical reaction, and the resulting current is
measured as a function of the elution time. This
allows for detection limits at the picomole level.
The selectivity of ECD depends on the accessi-
ble potential range, the number of compounds
that are active in this range, and the half-widths
of the individual signals [10]. Compounds hav-
ing electrolytic characters are measured and
detected by HPLC with electrochemical detector
(ECD). This detection method is inexpensive,
sensitive, selective, and widely accepted [10].
Numerous functional groups are sensitive to oxi-
dation, including phenols, aromatics, amines,
thiols, and quinolines, and many are compatible
with reduction, such as olefins, esters, ketones,
aldehydes, ethers, and quinones. ECD differs
from other methods of detection because it alters
the sample. Cells for HPLC–ECD usually con-
sist of three electrodes, the working, counter,
and reference electrodes, which can be aligned
in several different geometries and coulometric
systems; the eluent is directed through the elec-
trode, while in amperometric systems, the eluent
passes over it. Pulsed techniques, which use
multistep potential-time waveforms to realize
amperometric/coulometric detection while main-
taining uniform and reproducible electrode activ-
ity, are also available for detection [33].
HPLC–ECD has special recognition for the
study of the metabolism of aromatic compounds
as it is associated with electrochemistry. The
proper use of LC–ECD requires knowledge of
redox reactions and their dependence on mobile-
phase composition. In this respect, the study of
electrode reaction mechanisms is helpful in the
elucidation of mechanism of their interaction
with living cells, such as antioxidant character of
natural products [34].
69
HPLC–ECD is a common technology used in
many pharmaceutical, clinical, and biochemical
laboratories to evaluate the antioxidant properties
as it is most suitable for their accurate detection
and quantification with high accuracy in food
ingredients [35]; for example, ECD was found to
be more sensitive than UV for polyphenols esti-
mated by HPLC with coulometric detection
because they are reducing agents, and their elec-
trochemical responses result from the donation of
electrons. Some degree of correlation between
the electrochemical profiles of 17 flavonoids and
3 cinnamic acid derivatives and 3 different anti-
oxidant assays has been established. Artemisinin
was selectively detected and quantified in plasma
using a glassy carbon electrode by reductive ECD
[36]. Recently, different amino acid derivatives
characteristic for Korean ginseng have been ana-
lyzed by pulsed amperometric detection in both
ginseng and plasma samples [37].
HPLC Detection in Hyphenated
System
HPLC in hyphenation is used to generate multidi-
mensional data (i.e., both chromatographic as well
as spectrometric) using the following practices:
HPLC–RID
Refractive index detector (RID) detection is
based on the simple principle of refraction, in
which we have zero response to mobile phase,
but, as soon as an analyte enters in detector with
mobile phase, after selective segregation by col-
umn, is detected by detector using refractive
properties and recorded by the recorder. Though
it is a most simple and least expensive detector,
the refractive index (RI) detector, which was
reported by Tiselius and Claesson in 1942 [38],
lacks sensitivity; is very susceptible to changes in
ambient temperature, pressure, and flow rate; and
cannot be used for gradient elution. Despite these
disadvantages, this detector has been useful for
detecting compounds such as carbohydrates and
70
polymers. Because of its inherent limitations,
HPLC–RID has been favorably replaced by other
detection methods such as HPLC–ELSD, HPLC–
CAD, and HPLC–FID.
HPLC–ELSD
Evaporative light-scattering detector (ELSD),
introduced in 1966 by Ford and Kennard [39], is
a quasi-universal detector for liquid chromatog-
raphy, as it can detect any analyte which is less
volatile than mobile phase, regardless of the opti-
cal, electrochemical, or other analyte properties
[40]. The operating principle of the ELSD is that
the eluent is nebulized using a flow of nitrogen,
and the resulting aerosol is transported through a
heated drift tube where the volatile components
and solvents are evaporated. The remaining solid
fraction is subsequently introduced into a cell,
where a light beam is directed into the particles
that cause scattering of the incident light and is
detected by a photodiode or a photomultiplier.
The most important parameters affecting the
ELSD signal response are the nebulizer gas flow
rate and the drift tube temperature [41]. The gas
flow rate influences the droplet size of the col-
umn effluent before evaporation occurs. Higher
flow rates result in the formation of smaller aero-
sol droplets and less scattering of light, with sub-
sequent lower sensitivity, but they allow for a
more stable baseline. The drift tube temperature
facilitates the evaporation of the nebulized aero-
sol so that the light-scattering response of the
nonvolatile solute can exclusively be determined.
Higher temperatures are needed for the mobile
phase of high-aqueous-content or nonvolatile sol-
utes vs. semi-volatile ones. However, the new
generation of ELSD is able to vaporize the eluent
at low temperature, facilitating the detection of
semi-volatile analytes. It is pertinent to optimize
these parameters to ensure that the optimal signal-
to-noise ratio (S/N) is achieved [42].
ELSD is a mass-dependent detector; that is, it
generates a similar response for moieties of equal
mass. However, the ELSD response also depends
on the volatility of the compound and the mobile-
4 HPLC: Herbal Drugs and Fingerprints
phase composition (in the case of gradient elution),
which makes such quantification procedures
experimentally inaccessible. Thus, this interplay
of several factors leads to a nonlinear response,
which is one disadvantage of this type of detector
and ultimately renders direct linear regression
for making calibration curves inaccurate [43].
In order to avoid the variation generated by the
gradient elution, the post-column addition of
mobile-phase flow having an opposite composi-
tion is an interesting alternative [44].
The mass-dependent detection property of
ELSD is entirely in contrast to UV, which is a
concentration-based detector, and response gen-
erated does not depend on the spectral or physic-
ochemical properties of the analyte.
The increased interest in ELSD as a universal
detector is that it is more compatible with gradi-
ent elution than RID, and it is much less costly
and easier to maintain than an HPLC–MS plat-
form. It is an additional detector rather than a
substitute in HPLC.
The applications related to natural products,
ESLD has been used mainly for the detection of
compounds with weak chromophores such as
terpenes [45], in both aglycone [42] and glyco-
sidic forms [46], saponins, and some alkaloids
[47]. The method also has been applied in com-
plement to HPLC–MS for surveying natural
product libraries [48].
HPLC–CAD
In charged aerosol detector (CAD), the dried particle
stream is charged with a corona discharge needle,
and the resulting electrical charge is measured by
an electrometer. CAD operates by detecting
charge particles with a selected mobility range
rather than measuring individual gas-phase ions
as in MS. Generally, CAD is more sensitive than
ELSD, and the operating principle of CAD is
roughly comparable to that of ELSD. Contrary to
ELSD, the detection method provides a uniform
response to nonvolatile analytes, but detection is
based on mass, whereas CAD is a charge based
detection.
HPLC Detection in Hyphenated System
Charged aerosol detection (CAD) is a new
technology for universal HPLC detection, intro-
duced by Dixon and Peterson in 2002 [49], and
is more dedicated to the analysis of non-UV-
absorbing compounds [43]. The operating prin-
ciples of CAD are roughly comparable to that of
ELSD. Because it is a new detection method, so
it has not yet been popularized for natural prod-
uct’s detection to the large extent, although it has
been used for a wide range of pharmaceutical
products. Both CAD and ELSD were evaluated
for a library of more than 700 compounds in the
150- to 700-Da range and failed only in the
detection of two compounds having small MWs
[50]. The method is advantageous in that no nor-
malization of the peak area is needed, as with
HPLC–MS.
HPLC–FID
Flame ionization detector, a most common ana-
lytical detection for organic solvents in gas–liq-
uid chromatography (GLC) applied on the same
principle in HPLC, detects all carbon-containing
molecules, segregated by column at the same
instant, in both normal- and reversed-phase
modes. HPLC–FID coupling implies working
with 100% water as mobile phases. Water is a
very weak mobile phase at room temperature, but
with superheated water under pressure to tem-
peratures between 80 and 250°C, the polarity of
liquid water decreases markedly to the extent that
it can replace the organic modifier and act as the
sole eluent [51]. The successful applications of
HTLC–FID have been described [52], mainly for
the separations of alcohols, carbohydrates, and
amino acids. As per literature search, this detec-
tion technology is rare in practice.
HPLC–MS
The mass spectrometry with HPLC as a detector
has excellent sensitivity and selectivity for the
analysis of natural products including important
structural information such as molecular weight,
71
molecular formula, and diagnostic fragments,
which are crucial for dereplication and rapid
online characterization.
The high volume of mobile-phase flow in
HPLC analysis is a major problem to compete
with the high vacuum required for the mass
spectrometer; it has been overcome through the
use of atmospheric pressure ionization (API)
interfaces including electrospray ionization (ESI)
and atmospheric-pressure chemical ionization
(APCI). For both ESI and APCI, a combination
of high voltage and heat is used to produce the
ions that are assayed by the MS system. In ESI,
the high-voltage field (3–5 kV) produces nebuli-
zation of the column effluent, resulting in charged
droplets that are directed toward the mass ana-
lyzer. These droplets get smaller as they approach
the entrance to the mass analyzer. As the droplets
get smaller, individual ions emerge in a process
referred to as “ion evaporation”; these ions are
then separated by the MS system. In APCI, heat
is used to vaporize the column eluent, and then a
corona discharge is used to ionize solvent mole-
cules, which then produce the analyte ions via
chemical ionization mechanisms. More recently,
a third type of ionization source, termed atmo-
spheric pressure photoionization (APPI), has
been introduced. In APPI, heat is used to vapor-
ize the column eluent (similar to APCI), but the
ionization is produced by way of an ultraviolet
(UV) lamp that produces 10-eV photons.
Depending upon the solvent system used, the
10-eV photons ionizes either the mobile-phase
solvent or a dopant (e.g., toluene) added to the
column effluent; these ions then produce the ana-
lyte ions through various ionization mechanisms
including charge or proton transfer [53].
These API methods generally provide a soft
ionization and mainly molecular ion species in
the form of either protonated molecules [M + H]+
[positive-ion mode, (PI)] or deprotonated mole-
cules [M − H]− [negative-ion mode, (NI)].
Different adducts (e.g., [M + Na]+ (PI) or
[M + HCOO]− (NI)) are also produced, depending
on the solutes and the modifiers used. The notable
point is to understand that analyte ionization is
largely dependent on analyte and is governed
72
mainly by proton affinity. As a rule of thumb
for a good approximation, acidic molecules
(e.g., carboxylic acids or phenolics) will produce
mainly [M − H]− in NI, while bases (e.g., alka-
loids, amines) will generate [M + H]+ in PI.
Compounds such as glycosides have a high
affinity for salts and tend to form sodium adducts
in PI. Molecule fragmentation or partial de-
clustering of the adducts can be induced in one of
the high-pressure regions of the ion beam from
the source to the mass analyzer by collision-
induced dissociation (CID) either at the API
source (in-source CID) or in conjunction with
tandem MS (MS–MS) experiments [54].
Presently, there are many types of mass spec-
trometers that can be used as detector with HPLC.
Those that are low resolution (LR), such as the
single-quadrupole (Q) mass spectrometers, are
the most commonly used and least expensive,
whereas in high resolution (HR) and exact mass
capabilities, such as time-of-flight (TOF) instru-
ments, they are becoming increasingly popular
because they provide molecular formula infor-
mation online and very precise ion trace extrac-
tion (+/− 0.01 DA) for a specific detection. For
measuring structurally relevant fragments or for
very specific detection, triple-quadrupole (QQQ)
MS–MS systems are the most commonly used,
for particularly bio-analytical assays and for
specific quantification purposes in complex
matrices through multiple reaction monitoring
(MRM) [55]. Ion-trap mass spectrometers have
the unique capability of producing multistage
MS–MS (MSn) data that may be essential for
structural elucidation purposes. In addition to
these four kinds of mass spectrometers, there are
a growing number of additional types, including
hybrid systems that combine LR and HR analyz-
ers for specific applications [53].
Besides their widespread use for qualitative
analysis [dereplication studies and crude extract
profiling], HPLC–MS and HPLC–MS–MS have
been successfully used for the specific detection and
quantification of a large range of natural products.
While mainly simple quadrupole or triple-quadru-
pole analyzers are considered for quantitative stud-
ies, TOF instruments have recently increasingly
been used as quantification tools, though histori-
cally it suffered from a narrow dynamic range [56].
4 HPLC: Herbal Drugs and Fingerprints
HPLC–MS–ELSD for natural product’s
profiling including phenolics, alkaloids, amides,
terpenoids, steroids, and polyketides, with gradient
elution, using ESI (in both PI and NI mode) were
well detected in PI and NI mode [48]. It was con-
cluded in an investigation that ESI (PI) is used
mainly for alkaloids, APCI (PI) for pigments and
carotenoids, ESI (PI/NI) for triterpene glyco-
sides, and APCI or ESI (PI/NI) for phenolics and
flavonoids. APCI is generally more suitable for
nonpolar constituents, while ESI provides a softer
ionization and gives a good response for polar
metabolites. On the other hand, APCI is gener-
ally recognized to be less prone to matrix effects
than ESI [57]. Only a few studies have referred to
the use of APPI for natural product’s detection
[58]. As ionization of analyte is dependant (in
HPLC–MS) on the selection of the interface, the
ion mode polarity and the eluent composition
might significantly affect the sensitivity. HPLC–MS
is the method of choice, while HPLC–UV and
HPLC–ELSD provide sensitive and selective
detection at shorter analysis time, but MS pro-
vides useful structural information for a con-
firmation of the identity for the detection of
natural products in biological fluids, related sam-
ples, and quantification of flavonoids, alkaloids,
saponins, sesquiterpenoids, etc.
HPLC–NMR
It is a high-cost technique, basically used for online
structural information [1]. H–NMR can detect
all analyte-bearing protons. HPLC–19F–NMR
has been used for the specific detection of
fluorinated derivatives, for example, for studying
the metabolic fate of 3-chloro-4-fluoroaniline in
rat body fluids [59].
LC–Hyphenation for Online Structural
Identification
The hyphenated HPLC with other techniques is
generally termed as LC–hyphenation, in which
both chromatographic and spectroscopic dimen-
sions to generate structurally relevant data for
the online identification and dereplication of a
LC–Hyphenation for Online Structural Identification
compound by the validated method are used,
for example, DAD, MS, NMR, CD, and IR used
in hyphenation with HPLC for their online
identification and dereplication purposes.
The ability to rapidly identify known or
undesirable compounds using natural product
extract libraries (for natural product discovery
program) is called dereplication. It is important
to prevent the unnecessary use of resources on
the isolation of compounds of little or no value
for development from extracts used in the screen-
ing process and best utilization of resources to
focus on samples containing the most promising
leads. Dereplication strategies typically employ
a combination of separation sciences, spectro-
scopic methods, and database searching. HPLC
has been the most reliable tool for the separation
of complex mixtures of small molecules.
Reversed-phase HPLC on octadecylsilane (ODS
or C-18) is recognized as the most broadly appli-
cable of bonded phases for this purpose. The
interfaced with a diode-array detector (DAD),
HPLC allows an analyst to identify known com-
pounds by comparison of their HPLC retention
times and UV spectra. At present days, the use of
electrospray ionization (ESI) and atmospheric-
pressure chemical ionization (APCI) interfaces
has provided mass spectrometry (MS) interfaces
which are in use of the analysis of a wide range
of molecules and are compatible with liquid
chromatography. LC–MS has become a widely
used tool for the dereplication of natural prod-
ucts because the nominal molecular weight of a
compound can be used as a search query in
nearly all databases. Dereplication of the active
component(s) in crude natural product extracts
requires some form of feedback from the bioas-
say, which was initially used to detect the bio-
logical activity. This is necessary regardless of
the separation technique and analytical method
(DAD or MS) used. This approach requires des-
iccation of fractions to remove the HPLC sol-
vents, which are usually incompatible with the
bioassay, re-suspending the fractions in a com-
patible solvent (water or DMSO), and then indi-
vidual assaying of each fraction. This process is
not cost effective, being both time and labor
intensive. Consequently, as a result of the
increasing emphasis on the generation of new
73
lead compounds, faster cycle times, and high
efficiency, many pharmaceutical companies have
moved away from the natural product’s area.
Currently, almost every large pharmaceutical
company has established HTS infrastructures
and possesses large combinatorial compound
libraries, which cover a wide range of chemical
diversity. However, the ability to detect the
desired biological activity directly in the HPLC
effluent stream and chemically characterize the
bioactive compounds online, eliminates much of
the time and labor taken in the fraction collection
strategy. This way, cycle times, expenses, and
the isolation of known or undesirable compounds
would be reduced dramatically, allowing natural
products to be screened in an efficient and cost-
effective manner [60].
Dereplication of natural product analysis
using hyphenated technique is strategic for guid-
ing an efficient lead-finding process, but this task
is not easy to perform, as no exhaustive LC spec-
troscopic databases are available. Furthermore,
even if libraries of natural product spectra could
be efficiently generated on a given LC-hyphenated
system, their widespread use is complicated by
the fact that, contrary to GC–MS, most of the
data, especially HPLC–MS and HPLC–MS–MS,
are instrument dependant. Thus, for each new
compound, both the order of the atoms and the
stereochemical orientations have to be elucidated
de novo. Consequently, a single LC-hyphenated
technique does not exist that is capable of identi-
fying online all secondary metabolites of a plant.
As each technique presents its limitations, but
coupling of various hyphenated methods together
has the advantage that all structural information
is obtained within one single analysis [61]. For
natural product analysis, the dereplication proce-
dures rely mainly on HPLC–DAD–MS and
chemotaxonomic information provided by natu-
ral product databases. When unknown natural
products cannot be dereplicated by these means,
complementary information can be obtained by
NMR either directly hyphenated with LC
(HPLC–NMR) [62] or used at line with pre-con-
centration methods such as HPLC–SPE–NMR
[63] or after micro-fractionation with micro-flow
HPLC–NMR methods or even with a combina-
tion of both techniques by CAP–SPE–NMR
74
[64]. Other hyphenated methods, such as HPLC–IR
or HPLC–CD, can generate useful additional
structural information. A few commonly used
techniques are as follows:
HPLC–DAD (Diode-Array Detector)
It is also known as photodiode-array detection
(PDA) for online structural determination.
HPLC–DAD–MS is a key strategic tool, and it is
used for dereplication purposes in laboratories
involved in drug discovery. It is a manifold tech-
nique in the analysis of natural products, with
characteristic chromophores (e.g., polyphenols,
phenolics, polyketides, alkaloids, and terpe-
noids), commonly used as multi-hyphenated with
MS or ELSD detectors. For such compounds,
DAD–UV spectral libraries are consulted for
dereplication, but the compound has to be ana-
lyzed under the same HPLC conditions, as the
composition of the mobile phase might affect the
UV bands slightly. Furthermore, the choice of
modifiers affects UV detection and might blind
the bands in the low-wavelength range. The pos-
sibility to acquire several UV spectra across a
given LC peak allows the assessment of peak
purity [65]. Comparison of the genuine and
shifted online DAD spectra enables a precise
localization of the hydroxy groups on the poly-
phenols online. Another interesting point regard-
ing DAD–UV detection is that all wavelengths
are stored during analysis, and thus, multiple
wavelengths can be monitored at the same time
for detection of different classes of compounds.
This has been found to be very useful for simul-
taneous detection of the naphthodianthrones
(590 nm) and phloroglucinols (270 nm) of
Hypericum perforatum [14].
HPLC–MS, MS–MS, and MSn
“HPLC–MS” detection is a key technique for the
online identification of natural products by gener-
ating nominal mass of molecular ions or accurate
mass measurements for the determination of
empirical formulas. Tandem or hybrid MS instru-
ment is used for structural information through
4 HPLC: Herbal Drugs and Fingerprints
fragmentation of the molecular species by CID
reactions. For online dereplication purposes, the
determination of molecular weight is high priority
and necessitates the comparison of MS data
obtained under different detection conditions
(protonated or deprotonated molecules from
adducts or fragments). The use of high-resolution
instruments, such as a TOF–MS or FT–MS sys-
tem, enables direct determination of the molecu-
lar formula of crude mixtures. This strategic
information allows for more precisely targeting
the search in natural product libraries for derepli-
cation purposes [66].
In natural product chemistry, MS–MS spectra
are mainly useful for the partial determination of
sugar sequences of various glycosides, detection
of characteristic losses, such as those of preny-
lated compounds, classic fragmentation of
flavonoids, and the determination of substituted
positions on A or B rings as well as the differen-
tiation of isomers. For the analysis of fully
unknown constituents, MS–MS usually cannot
provide enough information to ascertain structure
determination, and thus, the combination with
other online information is mandatory.
HPLC–NMR (Nuclear Magnetic
Resonance)
It is a powerful tool to those compounds which
have been partially identified with MS–MS data-
bases, as additional spectroscopic information to
ascertain the identity of, in some cases, a complete
structural assignment I.D. obtained by HPLC–
NMR. The limiting factor of online HPLC–NMR
is the need for solvent suppression because HPLC
separation is performed with MeCN-D2O and
MeOH-D2O. In practice, the very large signal of
MeCN or MeOH and the residual HOD signal are
completely eliminated by powerful solvent-sup-
pression sequences. The main drawback of this
procedure is that analyte signals localized under
the solvent resonances also will be suppressed,
and thus, some information can potentially be lost.
Another problem occurring in HPLC–NMR is that
the chemical shifts recorded in a typical reversed-
phase solvent differ from those reported in stan-
dard deuterated NMR solvents [67].
HPTLC–HPLC for Quality Assurance
HPLC Biochemical Detection
The online HPLC–biochemical detection
(LC–BCD) system describes a range of targets,
such as the human estrogen receptor (hER), the
urokinase receptor, and acetylcholinesterase and
phosphodiesterase. After injection, the complex
mixture is chromatographically separated, and
subsequently, separated compounds are intro-
duced in a biochemical assay (microtiter-type
bioassays, where interactions between extracts
and the biochemical reagents proceed at high
speed in a closed continuous-flow reaction detec-
tion system. When sufficient resolution is
achieved, the individual contribution of the bio-
active compounds to the total bioactivity is obtained
within a single run). The chemical information
using aforesaid type online biochemical detection
with complementary chemical analysis techniques
[as mass spectrometry, (i.e., LC–BCD–MS)] is
obtained for the characterization and identification
of bioactive molecules, in real time, compared to
traditional screening approaches of complex mix-
tures, which are often characterized by a repeat-
ing cycle of HPLC fractionation and biological
screening and LC–BCD–MS analysis [60].
HPTLC–HPLC for Quality Assurance
Quality assessment has been established by using
HPTLC–HPLC, where color and rRf (relative
retention factor) of bands are compared with
those of reference markers in case of HPTLC,
while rRt (relative retention time) and applied
information content (j) are used for HPLC data.
These are not only for controlling the quality of
herbal raw materials which directly affects their
efficacy and safety but also controlling the con-
sistency of different batches and offer integral
characterization of a complex system with a
quantitative reliability. These two techniques,
in combination or alone, can be successfully
used to determine most of the phytochemical con-
stituents of herbal products in order to (a) ensure
the reliability and reproducibility of pharmaco-
logical and clinical researches, (b) to understand
bioactivities and possible side effects, and (c) to
enhance quality control of herbal products,
75
especially in traditional formulation/recipes where
basic scientific information is still lacking [68].
Case Study
A Thai traditional medicine, “Ayurved Siriraj
Prasachandaeng,” from 12 raw herbs is used
extensively as an antipyretic for both children
and adults. The formula, prepared in powder,
requires the combination of 12 medicinal herbs
based on historical references and several lines
of empirical evidence in Thai traditional medi-
cine practice. Ayurved Siriraj Prasachandaeng
belongs to the Center of Applied Thai Traditional
Medicine (CATTM), Faculty of Medicine Siriraj
Hospital, and Mahidol University, Thailand,
which formerly was the Clinic of Thai Traditional
Medicine established by Prof. Ouay Ketusingh
in 1982. CATTM has many Thai traditional rec-
ipes which have been produced and used with
satisfaction for a long time. In a study using
HPTLC and HPLC, 12 medicinal herbal compo-
nents and three batches of Ayurved Siriraj
Prasachandaeng produced from different times
were analyzed. The chromatograms from three
batches were regarded as the original fingerprints
of Ayurved Siriraj Prasachandaeng. Phenolic
composition, probably found in some compo-
nents such as gallic acid, caffeic acid, p-coumaric
acid, ferulic acid, vanillic acid, and kojic acid
was also determined. For HPLC analysis, all
solvents were HPLC grade. Water was purified
by a Milli-Q system (Millipore Corp., USA).
Other chemicals and reagents were analytical
grade, purchased from Merck, Germany. Gallic
acid, caffeic acid, p-coumaric acid, ferulic
acid, vanillic acid, and kojic acid were from
Sigma, USA. Ayurved Siriraj Prasachandaeng
and its 12 raw herbs were obtained from an
authentic source. All of them were indepen-
dently identified by at least two Thai traditional
pharmacists of CATTM according to morpho-
logical characteristics and produced following
the standard operating procedures (SOPs) of
good manufacturing. Twelve plants were
ordered for component no. 1–12 which were
Citrus aurantifolia Swing., Bouea macrophylla
Griff., Knema globularia Warb., Dracaena loureiri
76
Gagnep., Myristica fragrans Linn., Caesalpinia
sappan Linn., Conioselinum univitatum Trucz.,
Kaempferia galanga Linn., Mesua ferrea Linn.,
Jasminum sambac (Linn.) Ait., Mammea sia-
mensis Kosterm., and Nelumbo nucifera Gaertn.,
respectively [68].
The HPTLC system (Camag, Switzerland)
consisted of a Linomat 5 sample applicator
equipped with a 100-ml syringe and a Scanner 3.
Aluminum HPTLC plates (20 cm × 10 cm) pre-
coated with silica gel 60. HPLC system (Waters,
Milford, MA, USA) consisted of a Waters 2695
Separation Module system equipped with a pho-
todiode-array detector. An X-Terra RP18 column
100 mm × 4.6 mm ID., with 1.7-mm particle size
was used [68].
Three batches of Ayurved Siriraj Prasachan-
daeng and 12 herbal components were extracted
separately in 80% ethanol and lyophilized to
dry powder. Each powder was dissolved sepa-
rately in 80% ethanol for HPTLC and 50%
methanol for HPLC and then filtered through a
0.2-mm membrane filter before used. For the
HPTLC qualification of retention factor (Rf)
value, gallic acid, caffeic acid, p-coumaric acid,
ferulic acid, vanillic acid, and kojic acid (1 mg
each) were dissolved separately in 80% etha-
nol. Gallic acid and caffeic acid solutions were
prepared likewise for the HPTLC qualification
of relative retention factor (rRf) value. For
HPLC, gallic acid, caffeic acid, and vanillic
acid (1 mg each) were dissolved separately in
50% methanol. Each solution was vortexed for
2 min and filtered through a 0.2-mm membrane
filter to obtain the filtrate as the reference
marker [68].
The mobile phase for HPTLC was of hexane–
ethylene acetate–acetic acid (31:14:5). Each ref-
erence standard and each sample were spotted on
the HPTLC plate and developed in a saturated
mobile phase to a distance of 90 mm. After plate
drying in a stream of cold air for 3 min, each plate
was visualized under 254- and 366-nm UV and
then sprayed with 0.5% Fast Blue B Salt (FBS)
heated at 110°C until the bands were clearly vis-
ible under visible light. Finally, Rf and rRf values
were calculated [68].
4 HPLC: Herbal Drugs and Fingerprints
The mobile phase for HPLC was composed
of (A) O-phosphoric acid (0.1%, v/v) and
(B) acetonitrile using an isocratic condition
of 95% A and 5% B at 0–4 min; a gradient
elution of 95–85% A, at 4–8 min; 85–0%
A, at 8–13 min; and 0–95% A, at 13–16 min.
The flow rate was 1.0 ml/min; UV spectra
were recorded over the range of 200–
400 nm. The mathematic methods for
HPLC analysis, relative retention time
(rRt) value, and applied information content
(j) value were used to validate similarities
among batches [68]. The calculated factors,
including Rf and rRf from HPTLC and rRt
and j from HPLC, were applied for the
identification and quality control of each
batch of the recipe, and each component
was compared with the reference markers.
Like bioequivalent (BE) assessment,
coding a standard BE range for basic
pharmacokinetic parameters 80–125% of
mean has been generally accepted.
Moreover, the similarity among different
batches of the recipe was indicated by
the relative standard deviation (RSD) of
the rRt and j values less than 15%, and the
HPTLC and HPLC procedures used in this
study exhibited a satisfactory repeatability
which potentially provides a reliable
measure for quality control of the herbal
products.
Different Chemical Classes from
Natural Products and LC Fingerprints
Presently, scientists are looking for lead com-
pounds with specific structures and pharmacologi-
cal effects from natural sources. The wide
experiences and successes of Chinese scientists in
this specialized area have resulted in a number of
widely used drugs. The tremendous progress made
in life sciences has not only revealed many patho-
logical processes of diseases but also led to the
establishment of various molecular and cellular
Different Chemical Classes from Natural Products and LC Fingerprints
bioassays in conjunction with high-throughput
technologies. This is advantageous and permits
certain natural compounds that are difficult to iso-
late and purify, and compounds that are difficult to
synthesize, to be assayed. This technical report
compiles and analyzes the current scientific knowl-
edge on herbal drugs and highlights the practical
ways for ensuring the safety of herbal preparations
and evaluating their claimed efficacy, supported by
fingerprints. Examples for a few chemical classes
from natural products may be cited as follows:
Fingerprints for Flavonoids
Flavonoids, the natural polyphenols, are benzo-
pyrane derivatives with a phenyl group in the sec-
ond position, widely spread among different plant
species. Flavonoids can be O-glycosides, usually
in the positions three or seven. According to the
degree of oxidation, the flavonoids can be classified
as calcones, flavanones, flavones, flavonols, cate-
chins, terpenylflavonols, isoflavonols, etc. Many
flavonoids have an antioxidant activity and are
involved in the oxidation processes which take
place into the cell. Their biological activity is
very important, and many allopathic, ayurvedic,
or homeopathic drugs rich in flavonoids are on
the market, with high consumer demands, so it is
necessary to have more reliable analytical meth-
ods, with fingerprints for the same.
Flavonoids can be extracted from plants by
various methods, most of them involving an
extraction in ethanol, followed by a precipitation,
at room temperature, and purification. Flavonoids
are generally stable compounds and may be
extracted from the dried, ground medicinal plant
material and pharmaceutical preparation with
cold or hot solvents (aqueous mixtures with etha-
nol, methanol, acetone, and dimethylformamide,
etc.); further purification is usually done by sol-
vent–solvent exchange. The use of a large num-
ber of methods, individually or hyphenated, has
been published (TLC, HPLC, GC, electrophore-
sis, gravimetry, spectrophotometry, coulometry,
IR, NMR, MS) for the quantification of flavonoids
from plants [69].
77
Fingerprints for Terpenoids
The large class of terpenoids includes not only
the compounds with a triterpene and steroidal
structure (saponins, sterols, cardiotonic glyco-
sides) but also the compounds with an isoprenic
structure, such as volatile oils, terpenes (bitter
principles), and carotenoids. The volatile oils are
aliphatic or aromatic hydrocarbons, aldehydes,
alcohols, acids, esters, etc. and are widely distrib-
uted in nature. Structurally, the volatile oils are
monoterpenes, sesquiterpenes, and azulenes.
Volatile oils are obtained by distillation with
water or with nonpolar solvents. The most used
method for their analysis is GC, but some HPLC
methods are reported in the literature on a silica
stationary phase and a binary mixture, n-pentane/
water, as mobile phase. LC–MS methods have
also been applied. Standardized extracts of
Ginkgo biloba leaves contain large quantities of
terpene-like compounds and are mainly used in
the treatment of peripheral and cerebral circula-
tion disorders or as a remedy against asthma,
coughs, bladder inflammation, blennorrhagia,
and alcohol abuse. The leaf extracts contain
biflavones, flavonol glycosides, and terpene lac-
tones. A method based on liquid chromatogra-
phy, coupled with electrospray mass spectrometry,
has been reported for the analysis of terpenoids in
G. biloba extracts. This method allows the rapid
isocratic separation of underivatized ginkgolides
(GA, GB, GC, and GJ) and bilobalide at very low
levels (10 pg on the column) and their quantita-
tive detection by external standardization with
relative standard deviations of 3 and 5% for intra-
and inter-day analyses, respectively [70].
Carotenoids are compounds with a terpenoid
structure with 30–50 carbon atoms in the mole-
cule. Carotenoids are widely spread in nature,
being the yellow/red pigments which can be
found together with chlorophylls. Carotenoids
have a provitamin A structure and have an impor-
tant biological activity in the visual process and
regarding the epithelial tissue. These compounds
can be extracted in nonpolar solvents and can
be analyzed by liquid chromatography (TLC,
HPLC, etc.) [71].
78
Fingerprints for Alkaloids
Alkaloids have been in use for a long time for
antiarrhythmic, hypotensive, platelet-aggregation-
inhibiting, histamine-antagonizing, and anti-
flagellated protozoa properties. Lysergol, an
alkaloid, is used in the treatment of schizophrenia
and motor disorders. It is also used in the synthe-
sis of lumilysergol and nicergoline. The drug
consisting of seeds is locally used for its aperient
action in MP of India, whereas the powder is used
as an antipyretic. The purity of the product is
defined by TLC (mobile phase; chloroform–
methanol–ammonia solution: 95:5:0.5, using sil-
ica gel 60 F254, at 254 and 365 nm) as well as
HPLC [(silica column 250 × 4.6 mm, I.D., 5 m,
mobile phase (n-heptane–THF–MeOH–diethyl
amine: 55:35:6:0.02), wavelength 230 nm, flow
rate 1.5 ml/min)]. Elymoclavin is a geometrical
isomer of lysergol, which is considered as an
associated impurity. According to global market
specification, it should be less than 0.5% (wt/wt)
by HPLC analysis, as a high-value product [69].
Fingerprints for Coumarins
Coumarins (a-benzopyrones) are natural deriva-
tives of benzopyrane with a lactonic structure.
The name comes from “coumara” the name of
tonka seeds which contain large quantities of
coumarins. Coumarins can be found in numerous
species such as algae, mushrooms, and lichens
and also in superior plants (Umbelliferae,
Rutaceae, Labiatae, Orchidaceae, etc.). Bio-
chemically, coumarins are formed by photosyn-
thesis from phenylalanine and cinnamic acid. As
crystals, coumarins have blue, green, or violet
fluorescence and show a good absorbance of the
UV light, being an effective UV screen. Many of
them are thermally labile. Coumarin derivatives
are biologically active on the nervous system
level, and some of them have an anticoagulant
activity. Coumarin glycosides are soluble in water
and polar organic solvents, while terpenylcou-
marins and furanocoumarins are soluble in non-
polar organic solvents. The coumarins have been
analyzed by TLC or HPLC and easily detected
4 HPLC: Herbal Drugs and Fingerprints
due to their natural fluorescence. HPLC has also
been extensively used, mainly with reversed-phase
stationary phases [69].
Fingerprints for Alkamides
Alkamides are a distinct class of natural products,
containing an aliphatic acid (mostly unsaturated)
residue linked with various amine moieties.
Approximately 200 alkamides have been isolated
from nature. It is well known that alkamides from
Echinacea species have immunostimulating prop-
erties, and 15 new isobutyl- and 2-methyl-butyl-
amides have been identified in Echinacea
angustifolia and purpurea roots [72]. Extraction
with chloroform is widely used for the dried, pow-
dered plant material, and solvent is distilled out
completely; residue redissolved in a small volume
of alcohol and filtered using 0.45-m filter [72].
Among chromatographic techniques, HPLC is
rapid and does not lead to decomposition, material
loss, or artifact formation. Due to these reasons,
the isolation, even at small quantities (milligrams
or less) for structure determination, comprehen-
sive biological testing, semisynthetic work, or
even for production of therapeutic agents, HPLC
is being used. The increasing requirements for
quality control, profiling and fingerprinting,
dereplication, and metabolomics and the detec-
tion issue by HPLC are still challenging. New
detectors still have to be developed to fulfill the
needs of sensitivity, linearity, speed, robustness,
and universal response.
References
1. David F, Vanhoenacker G, Tienpont B, Francois I,
Sandra P. Coupling columns and multidimensional
configurations to increase peak capacity in liquid
chromatography. Lc Gc Eur. 2007;20:154–8.
2. Wilson ID, Brinkman UAT. Hyphenation and hyper-
nation – the practice and prospects of multiple
hyphenation. J Chromatogr A. 2003;1000:325–56.
3. Henry R, Santasania CT. Reporter. Separation of closely
related compounds. Sigma-Aldrich. 2010;28.5: 6–7.
4. The United States Pharmacopoeia. XXIIIth revision,
Natural formulas 18, Rockville: United States
Pharmacopoeial Convention; 1995.
References
5. Mcmaster MC. HPLC a practical user’s guide. New
York: VCH; 1994. p. 85.
6. Verma S. Reporter. Separation of closely related com-
pounds. Sigma-Aldrich. 2010;28.5: 9.
7. Schobel U, Frenay M, van Elswijk DA, McAndrews
JM, et al. High resolution screening of plant natural
product extracts for estrogen receptor alpha and beta
binding activity using an online HPLC-MS biochemical
detection system. J Biomol Screen. 2001;6:291–303.
8. Oosterkamp AJ, van der Hoeven R, Glassgen W, Konig
B, et al. Gradient reversed-phase liquid chromatography
coupled on-line to receptor-affinity detection based on the
urokinase receptor. J Chromatogr B. 1998;715(1):331–8.
9. Gao XF, Dan M, Zhao AH, Xie GX, Jia W. Simultaneous
determination of saponins in flower buds of Panax
notoginseng using high performance liquid chromatog-
raphy. Biomed Chromatogr. 2008;22:244–9.
10. Ozkan SA. LC with electrochemical detection, recent
application to pharmaceuticals and biological fluids.
Chromatographia. 2007;66:S3–13.
11. Vial J, Jardy A. Study of the linear range in HPLC anal-
yses with UV detection: methodology and experimental
application to the influence of the analyte UV spectrum.
J High Resolut Chromatogr. 1999;22:217–21.
12. Brantner AH, Males Z. Quality assessment of Paliurus
spina-christi extracts. J Etnopharmacol. 1999;66:175–9.
13. Fuzzati N. Analysis methods of ginsenosides. J
Chromatogr B. 2004;812:119–33.
14. Tolonen A, Hohtola A, Jalonen J. Fast high-perfor-
mance liquid chromatographic analysis of naphthodi-
anthrones and phloroglucinols from Hypericum
perforatum extracts. Phytochem Anal. 2003;14:306–9.
15. Hasler A, Sticher O, Meier B. Identification and deter-
mination of the flavonoid from Ginkgo biloba by high
performance liquid chromatography. J Chromatogr.
1992;605:41–8.
16. Dubber MJ, Kanfer I. High performance liquid chro-
matographic determination of selected flavonols in
Ginkgo biloba solid oral dosage forms. J Pharm Pharm
Sci. 2004;7:303–9.
17. Sloley BD, Tawfik SR, Scherban KA, Tam YK.
Quality control analyses for ginkgo extracts require
analysis of intact flavonol glycosides. J Food Drug
Anal. 2003;11:102–7.
18. Chen P, Ozcan M, Harnly J. Chromatographic
fingerprint analysis for evaluation of Ginkgo biloba
products. Anal Bioanal Chem. 2007;389:251–61.
19. Li WK, Fitzloff JF. HPLC determination of flavonoids
and terpene lactones in commercial Ginkgo biloba
products. J Liq Chromatogr Relat Technol. 2002;
25:2501–14.
20. Fuzzati N, Pace R, Villa E. A simple HPLC-UV
method for the assay of ginkgolic acids in Ginkgo
biloba extracts. Fitoterapia. 2003;74:247–56.
21. Leitner A, Emmert J, Boerner K, Lindner W. Influence
of solvent additive composition on chromatographic
separation and sodium adduct formation of peptides
in HPLC-ESI MS. Chromatographia. 2007;65:649–53.
22. Mohn T, Potterat O, Hamburger M. Quantification of
active principles and pigments in leaf extracts of Isatis
tinctoria by HPLC/UV/MS. Planta Med. 2007;73:151–6.
79
23. Bebrevska L, Bravo L, Vandervoort J, Pieters L,
Vlietinck A, Apers S. Development and validation of
an HPLC method for quality control of Pueraria
lobata flower. Planta Med. 2007;73:1606–13.
24. Liang Z, Jiang Z, Ho H, Zhao Z. Comparative analy-
sis of Oldenlandia diffusa and its substitutes by high
performance liquid chromatographic fingerprint
and mass spectrometric analysis. Planta Med.
2007;73:1502–8.
25. Ge GB, Zhang YY, Hao DC, Hu Y, Luan HW, Liu XB,
et al. Chemotaxonomic study of medicinal Taxus spe-
cies with fingerprint and multivariate analysis. Planta
Med. 2008;74:773–9.
26. Jaimez J, Fente CA, Vazquez BI, Franco CM, Cepeda
A, Mahuzier G, et al. Application of the assay of
aflatoxins by liquid chromatography with fluorescence
detection in food analysis. J Chromatogr A.
2000;882:1–10.
27. Braga S, de Medeiros FD, Oliveira ED, Macedo RO.
Development and validation of a method for the quan-
titative determination of aflatoxin contaminants in
Maytenus ilicifolia by HPLC with fluorescence detec-
tion. Phytochem Anal. 2005;16:267–71.
28. Klvana M, Chen JK, Lepine F, Legros R, Jolicoeur M.
Analysis of secondary metabolites from Eschscholtzia
californica by high-performance liquid chromatogra-
phy. Phytochem Anal. 2006;17:236–42.
29. Kristl J, Veber M, Krajnicic B, Oresnik K, Slekovec
M. Determination of jasmonic acid in Lemna minor
(L.) by liquid chromatography with fluorescence
detection. Anal Bioanal Chem. 2005;383:886–93.
30. Li FM, Zhang CH, Guo XJ, Feng WY.
Chemiluminescence detection in HPLC and CE for
pharmaceutical and biomedical analysis. Biomed
Chromatogr. 2003;17:96–105.
31. Ohba Y, Kuroda N, Nakashima K. Liquid chromatog-
raphy of fatty acids with chemiluminescence detec-
tion. Anal Chim Acta. 2002;465:101–9.
32. Zhang QL, Cui H. Simultaneous determination of quer-
cetin, kaempferol, and isorhamnetin in phytopharma-
ceuticals of Hippophae rhamnoides L. by
high-performance liquid chromatography with chemi-
luminescence detection. J Sep Sci. 2005;28:1171–8.
33. LaCourse WR, Modi SJ. Microelectrode applications
of pulsed electrochemical detection. Electroanalysis.
2005;17:1141–52.
34. Jean-Luc W. HPLC in natural product analysis: the
detection issue. Planta Med. 2009;75(7):719–34.
35. Skrinjar M, Kolar MH, Jelsek N, Hras AR, Bezjak M,
Knez Z. Application of HPLC with electrochemical
detection for the determination of low levels of anti-
oxidants. J Food Compos Anal. 2007;20:539–45.
36. Chan KL, Yuen KH, Jinadasa S, Peh KK, Toh WT. A
high-performance liquid chromatography analysis of
plasma artemisinin using a glassy carbon electrode for
reductive electrochemical detection. Planta Med.
1997;63:66–9.
37. Joo KM, Park CW, Jeong HJ, Lee SJ, Chang IS.
Simultaneous determination of two amadori com-
pounds in Korean red ginseng (Panax ginseng) extracts
and rat plasma by high-performance anion-exchange
80
chromatography with pulsed amperometric detection.
J Chromatogr B. 2008;865:159–66.
38. Tiselius A, Claesson S. Adsorption analysis by means
of interferometric study. Arkiv Kemi Minearl Geol.
1942;15:1–6.
39. Ford DL, Kennard W. Vaporization analyzer. J Oil
Colour Chem Assoc. 1966;49:299–313.
40. Megoulas NC, Koupparis MA. Twenty years of evap-
orative light scattering detection. Crit Rev Anal Chem.
2005;35:301–16.
41. Guillarme D, Rudaz S, Schelling C, Dreux M, Veuthey
JL. Micro liquid chromatography coupled with evapo-
rative light scattering detector at ambient and high
temperature: optimization of the nebulization cell
geometry. J Chromatogr A. 2008;1192:103–12.
42. Dubber MJ, Kanfer I. Determination of terpene trilac-
tones in Ginkgo biloba solid oral dosage forms using
HPLC with evaporative light scattering detection. J
Pharm Biomed Anal. 2006;41:135–40.
43. Vervoort N, Daemen D, Torok G. Performance evalua-
tion of evaporative light scattering detection and
charged aerosol detection in reversed phase liquid chro-
matography. J Chromatogr A. 2008;1189:92–100.
44. Gorecki T, Lynen F, Szucs R, Sandra P. Universal
response in liquid chromatography using charged
aerosol detection. Anal Chem. 2006;78:3186–92.
45. Li P, Zeng LJ, Lin G. The extraction of imperialine
and imperialine-3 beta-glucoside from Fritillaria
pallidiflora Schrenk and quantitative determination
by HPLC-evaporative light scattering detection.
Phytochem Anal. 2002;13:158–61.
46. Kim SN, Ha YW, Shin H, Son SH, Wu SJ, Kim YS.
Simultaneous quantification of 14 ginsenosides in
Panax ginseng C.A. Meyer (Korean red ginseng) by
HPLC-ELSD and its application to quality control. J
Pharm Biomed Anal. 2007;45:164–70.
47. Schaneberg BT, Molyneux RJ, Khan IA. Evaporative
light scattering detection of pyrrolizidine alkaloids.
Phytochem Anal. 2004;15:36–9.
48. Cremin PA, Zeng L. High-throughput analysis of nat-
ural product compound libraries by parallel LC-MS
evaporative light scattering detection. Anal Chem.
2002;74:5492–500.
49. Dixon RW, Peterson DS. Development and testing of
a detection method for liquid chromatography based
on aerosol charging. Anal Chem. 2002;74:2930–7.
50. Gamache PH, McCarthy RS, Freeto SM, Asa DJ,
Woodcock MJ, Laws K, et al. HPLC analysis of non-
volatile analytes using charged aerosol detection. Lc
Gc Eur. 2005;18:345–9.
51. Smith RM. Superheated water chromatography –
a green technology for the future. J Chromatogr A.
2008;1184:441–55.
52. Guillarme D, Heinisch S. Detection modes with high
temperature liquid chromatography – a review. Sep
Purif Rev. 2005;34:181–216.
53. Korfmacher WA. Principles and applications of
LC-MS in new drug discovery. Drug Discov Today.
2005;10:1357–67.
4 HPLC: Herbal Drugs and Fingerprints
54. He XG. On-line identification of phytochemical
constituents in botanical extracts by combined high-
performance liquid chromatographic-diode array
detection-mass spectrometric techniques. J
Chromatogr A. 2000;880:203–32.
55. Pisitkun T, Hoffert JD, Yu MJ, Knepper MA. Tandem
mass spectrometry in physiology. Physiology.
2007;22:390–400.
56. Williamson LN, Bartlett MG. Quantitative liquid
chromatography/time-of-flight mass spectrometry.
Biomed Chromatogr. 2007;21:567–76.
57. Jessome LL, Volmer DA. Ion suppression: a major
concern in mass spectrometry. Lc Gc N Am.
2006;24:83–9.
58. Syage JA, Short LC, Cai SS. Atmospheric pressure
photoionization – the second source for LC-MS? Lc
Gc N Am. 2008;26:286–300.
59. Duckett CJ, Lindon JC, Walker H, Abou-Shakra F,
Wilson ID, Nicholson JK. Metabolism of 3-chloro-4-
fluoroaniline in rat using [C-14]-radiolabelling, F-19-
NMR spectroscopy, HPLC-MS/MS, HPLC-ICPMS
and HPLC-NMR. Xenobiotica. 2006;36:59–77.
60. Elswijk DAV, Irth H. Analytical tools for the detection
and characterization of biologically active compounds
from nature. Phytochem Rev. 2003;1:427–39.
61. Wilson ID, Brinkman UAT. Hype and hypernation:
multiple hyphenation of column liquid chromatogra-
phy and spectroscopy. Trends Anal Chem.
2007;26:847–54.
62. Jaroszewski JW. Hyphenated NMR methods in natu-
ral products research, part 1: direct hyphenation.
Planta Med. 2005;71:691–700.
63. Clarkson C, Madikane EV, Hansen SH, Smith PJ,
Jaroszewski JW. HPLC-SPE-NMR characterization of
sesquiterpenes in an antimycobacterial fraction from
Warburgia salutaris. Planta Med. 2007;73:578–84.
64. Lambert M, Wolfender JL, Staerk D, Christensen B,
Hostettmann K, Jaroszewski JW. Identification of
natural products using HPLC-SPE combined with
CapNMR. Anal Chem. 2007;79:727–35.
65. Larsen TO, Hansen MAE. Dereplication and discov-
ery of natural products by UV spectroscopy. In:
Colegate SM, Molyneux RJ, editors. Bio-active natu-
ral products: detection, isolation, and structural deter-
mination. 2nd ed. London: CRC Press; 2008. p.
221–44.
66. Romani A, Vignolini P, Isolani L, Ieri F, Heimler D.
HPLC-DAD/MS characterization of flavonoids and
hydroxycinnamic derivatives in turnip tops (Brassica
rapa L. subsp. sylvestris L.). J Agric Food Chem.
2006;54(4):1342–6.
67. Wolfender JL, Queiroz EF, Hostettmann K.
Phytochemistry in the microgram domain – a LC-NMR
perspective. Magn Reson Chem. 2005;43:697–709.
68. Akarasereenont P, Thitilertdecha P, Chotewuttakorn
S, Palo T, Seubnooch P, Wattanarangsan J, et al.
Chromatographic fingerprint development for quality
assessment of “Ayurved Siriraj Prasachandaeng” anti-
pyretic drug. Siriraj Med. 2010;62(1):4–8.
Bibliography
69. Cimpan G, Gocan S. Analysis of medicinal plants by
HPLC: recent approaches. J Liq Chromatogr Relat
Technol. 2002;25(13):2225–92.
70. Mauri P, Migliazza B, Pietta P. Liquid chromatography/
electrospray mass spectrometry of bioactive terpe-
noids in Ginkgo biloba L. J Mass Spectrom.
1999;34(12):1361–7.
71. Lacey ME, Tan ZJ, Webb AG, Sweedler JV. Union
of capillary high-performance liquid chromatogra-
phy and microcoil nuclear magnetic resonance spec-
troscopy applied to the separation and identification
of terpenoids. J Chromatogr A. 2001;922:139–49.
72. Bauer R, Remiger P. TLC and HPLC analysis of alk-
amides in Echinacea drug. Planta Med. 1989;
55:367–71.
Bibliography
Bindseil KU, Jakupovic J, Wolf D, Lavayre J, Leboul J,
Van der Pyl D. Pure compound libraries; a new per-
spective for natural product based drug discovery.
Drug Discov Today. 2001;6(16):840–7.
Deli J, Matus Z, Toth G. Comparative study on the carote-
noid composition in the buds and flowers of different
Aesculus species. Chromatographia. 2000;51(Suppl):
S179–82.
Dillard CJ, German JB. Phytochemicals: nutraceuticals and
human health. J Sci Food Agric. 2000;80:1744–56.
Glasl S, Gunbilig D, Narantuya S, Werner I, Jurenitsc J.
Combination of chromatographic and spectroscopic
methods for the isolation and characterization of polar
guaianolides from Achillea asiatica. J Chromatogr A.
2001;936:193–200.
Grabley S, Thiericke R. Bioactive agents from natural
sources: trends in discovery and application. Adv
Biochem Eng Biotechnol. 1999;64:101–54.
Gu L, Gu W. Characterisation of soy isoflavones and
screening of novel malonyl glycosides using high-
81
performance liquid chromatography- electrospray
ionisation-mass spectrometry. Phytochem Anal.
2001;12:377–82.
Herrmann K. Occurrence and content of hydroxycinnamic
and hydroxybenzoic acid compounds in foods. Crit
Rev Food Sci Nutr. 1989;28:315–47.
Kim ND, Mehta R, Yu W, Neeman I, Livney T, Amichay
A, Poirier D, Nicholls P, Kirby A, Jiang W, Mansel
R, Ramachandran C, Rabi T, Kaplan B, Lansky E.
Chemopreventive and adjuvant therapeutic potential
of pomegranate (Punica granatum) for human
breast cancer. Breast Cancer Res Treat. 2002;
71(3):203–17.
Lutz ESM, Irth H, Tjaden UR, Van der Greef J. Applying
hollow fi bres for separating free and bound label
in continuous flow immunochemical detection. J
Chromatogr A. 1996;755(2):179–87.
Mueller-Harvey I. Analysis of hydrolysable tannins. Anim
Feed Sci Technol. 2001;91:3–20.
Oosterkamp AJ, Irth H, Tjaden UR, Vander-Greef J.
On-line coupling of liquid chromatography to bio-
chemical assays based on fluorescent-labeled ligands.
Anal Chem. 1994;66(23):4295–301.
Rashid MA, Gustafson KR, Crouch RC, Groweiss A,
Pannell LK, Van ON, Boyd NR. Application of high-
field NMR and cryogenic probe technologies in the
structural elucidation of poecillastrin a, a new antitu-
mor macrolide lactam from the sponge Poecillastra
species. Org Lett. 2002;4(19):3293–6.
Schofield P, Mbugua DM, Pell AN. Analysis of condensed
tannins: a review. Anim Feed Sci Technol. 2001;
91:21–40.
Swatsitang P, Tucker G, Robards K, Jardine D. Isolation
and identification of phenolics compounds in Citrus
sinensis. Anal Chim Acta. 2000;417:231–40.
Weber L. High-diversity combinatorial libraries. Curr
Opin Chem Biol. 2000;4(3):295–302.
Zani CL, Alves TMA, Queiroz R, Fontes ES, Shin YG,
Cordell GA. A cytotoxic diterpene from Alomia
myriadenia. Phytochemistry. 2000;53:877–80.
 GC: Herbal Drugs and Fingerprints
Gas–liquid chromatography (GLC) was introduced
by James and Martin in 1952, to separate volatile
substances by percolation of a gas stream over a
stationary phase [1]. It has gained widespread
acceptance for different applications such as
process control in chemical plants, quality con-
trol in the food and pharma sectors, monitoring
sample composition in the oil industries, environ-
mental and biomedical sciences, and some time
the term GLC is also abbreviated as GC (though
in GC the stationary phase is solid, while, in
GLC, the solid stationary phase is coated with an
inert liquid of high boiling point). The basis of
separation in GLC is partitioning of the sample in
and out of the film of liquid spread over an inert
solid. There exists a wide range of liquid phases
usable up to 450 °C. In herbal drug industry,
GLC is used mainly for (a) assay of raw materials
and finished goods for process and quality
control, (b) quantification of ingredients in
formulations, and (c) assay for impurities and
residual solvents and volatiles. It is a key tech-
nique to obtain pure compound for structure
elucidation, pharmacological testing, and
development of new therapeutics. It has a very
wide field of application, but its first and main
area of use is in the separation and analysis of
multicomponent mixtures such as essential oils,
hydrocarbons and solvents, their identification,
and structural elucidations. GLC (commonly
termed as GC), fingerprints, especially by hyphen-
ated techniques, are strongly recommended for
quality control of herbal drugs, to authenticate
the herbal part used for (a) volatile oils, plant acids,
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_5, © Springer India 2012
5
alkaloids, steroids, glycosides and aglycons, amino
acids, etc.; (b) separation of complex mixtures
into constituents; and (c) sample in parts of
milligram that is sufficient for analysis.
Instrumentation
The GC separation is carried out in a tubular
column made of glass, metal, or Teflon. Gas
chromatography, specifically gas–liquid chro-
matography, involves the vaporization of sample
injected into head of the chromatographic
column, a specially manufactured device with
rubber septum, that is termed as injector. The
sample is transported through the column by
the fl ow of inert gaseous mobile phase, the
carrier gas (generally, helium, argon, nitrogen).
The stationary phase of column is an inert solid
which is adsorbed into the surface by inert and
thermally stable liquid. The choice of carrier
gas is often dependent upon the type of detector
used. The carrier gas system also contains a
molecular sieve to remove water and other impu-
rities. Microsyringe is generally used to inject
sample through a rubber septum into a flash
vaporizer port at the head of the column. The
carrier gas enters into the port, and sample
vaporizes to form a mixture (vaporized solvent
and vaporized solutes) with carrier gas. Each
chemical species tends to migrate at its own
characteristic rate, hence separated from other
species by the time they emerge from the col-
umn and reach at detector. The detector senses
83
84
Fig. 5.1 GLC schematic diagram for FID system
the emergence of each sample component and
provides an electrical signal to the recorder,
proportional to the concentration of each com-
ponent in the emergent stream. The temperature
of the sample port is usually about 50 °C higher
than the boiling point of the least volatile compo-
nent of the sample.
GC has a very wide field of applications, but
its first and main area of use is in the separation
and analysis of multicomponent mixtures
such as essential oils, hydrocarbons, and
solvents. Intrinsically, with the use of the flame
ionization detector (FID) (Fig. 5.1) and the
electron capture detector (ECD), have very high
sensitivity to, determines materials present at
very low concentrations in the matrix, due to the
reason it is the second most important technology
for pollution studies, forensic work, and general
trace analysis. Because of its simplicity, sensi-
tivity, and effectiveness in separating components
of mixtures, it is one of the most important
tools in chemistry. It is widely used for quantita-
tive and qualitative analysis of mixtures, for the
purification of compounds, and for the determi-
nation of thermochemical constants as heats of
solution and vaporization, vapor pressure, and
activity coefficient.
In summarized way, we can say that GLC is a
six components system, as of:
1. Pneumatic system to gas supply and flow
control and measurement.
2. Injector, a specially designed port for sample
injection.
3. Column, the device for separation of ingredi-
ents present in matrix.
5 GC: Herbal Drugs and Fingerprints
4. Oven to provide thermostatically controlled
atmosphere to the column.
5. Detector detects the ingredient.
6. Recorder to record the response of detector as
chromatogram.
The selection of column and detector for every
analysis is matter of subject expertise, a summa-
rized detail on these is as follows:
GLC Columns
There are two general types of column, packed
and capillary (open tubular), used with GLC.
Packed columns contain a finely divided, inert,
solid support material (commonly based on dia-
tomaceous earth) coated with liquid stationary
phase. Most packed columns are 1.5–10 m in
length and have an internal diameter of 2–4 mm.
Capillary columns have an internal diameter of a
few tenths of a millimeter. They can be one of
two types: wall-coated open tubular (WCOT) and
support-coated open tubular (SCOT). Wall-coated
columns consist of a capillary tube whose walls
are coated with liquid stationary phase. In
support-coated columns, the inner wall of the
capillary is lined with a thin layer of support
material such as diatomaceous earth, into which
the stationary phase has been adsorbed. SCOT
columns are generally less efficient than WCOT
columns, but both types of capillary columns are
more efficient than packed columns. A new type
of WCOT column with fused silica known as
fused silica open tubular (FSOT) column is a new
innovation, which has much thinner walls than
Instrumentation
the glass capillary columns and is given strength
by the polyimide coating. These columns are
flexible and can be round into coils. They have
the advantages of physical strength, flexibility,
and low reactivity.
Detectors in GLC
There are many detectors which can be used in
gas chromatography. Different detectors have
different types of selectivity. A nonselective
detector responds to all compounds except the
carrier gas, while a selective detector responds to
a range of compounds with a common physical
or chemical property, and a specific detector
responds to a single chemical compound. The
most popular detector in gas chromatography is
the FID. Its high sensitivity, fast response, wide
dynamic range, and simplicity of construction
make it the most widely used detector. Several
other detectors that are available which offer high
selectivity are ECD for halogen-containing com-
pounds, TID (thermo-ionic detector) for nitrogen
and phosphorous-containing compounds, and the
SCD (sulfur chemiluminescence detector) for
sulfur-containing compounds. Another detector
with a large popularity in gas chromatography is
the mass spectrometer. GC–MS is very adequate
for the identification of unknown compounds but
has a more limited dynamic range than the FID.
FID
FID (flame ionization detector), a general detec-
tor, is a useful for the analysis of organic com-
pounds, specially solvents, essential oils, etc. It
has high sensitivity, a large linear response range,
and low noise. In FID, the effluent from the col-
umn is mixed with hydrogen and air and ignited
at detector. The resulted flame produces ions and
electrons which conduct electricity through the
flame. A large electrical potential is applied at the
burner tip, and a collector electrode is located
above the flame. The current resulting from the
pyrolysis of organic compounds is measured,
which is recorded by recorder. FID, a nonselec-
tive detector, has a potential possibility of inter-
fere by many nontarget compounds present in
85
samples. The FID works by directing the gas
phase output from the column into a hydrogen
flame. A voltage of 100–200 V is applied between
the flame and an electrode located away from the
flame. The increased current due to electrons
emitted by burning carbon particles is then mea-
sured. Although the signal current is very small
(the ionization efficiency is only 0.0015%), the
noise level is also very small (<10–13 amp) and
with a well-optimized system, sensitivities of
5 × 10−12 g/ml for n-heptane at a signal/noise ratio
of 2 can be easily realized. Except for a very few
organic compounds (e.g., carbon monoxide), the
FID detects all carbon-containing compounds.
The detector also has an extremely wide linear
dynamic range that extends over, at least five
orders of magnitude with a response index
between 0.98 and 1.02. In case of FID, the
response is proportional to the carbon content of
the compound, and this is useful for general anal-
ysis of organic compounds.
ECD
The electron capture detector (ECD) is extremely
sensitive for those compounds having strong
electron capturing properties such as halogenated
molecules. Although ECD is simple in design
and operation, it is actually a complex chemical
reactor, and many processes are involved in the
production of a signal. ECD is based on forma-
tion of plasma of thermalized electrons. The elec-
trons are collected by applying a small voltage to
produce standing current. The compound enter-
ing into the cell captures the thermalized elec-
trons, resulting decrease in standing current,
which is recorded by recorder, proportional to the
content of eluting moiety.
FPD
The flame photometric detector (FPD) is a most
reliable detector for the analysis of phosphorus
and sulfur compounds. It is based on specific
chemiluminescence produced by burning in
hydrocarbon-rich flame. The sulfur and phospho-
rus emission bands are derived from excited S2
and HPO species, respectively, and are formed by
reactions with hydrogen atoms in the upper part
of the flame. Carbon gives the flame emission
86
bands, which are formed in the lower region of
the flame. In original FPD design, the column
effluent was mixed with air and combusted in
burner of similar design to an FID. The flame is
burned in the stream of hydrogen gas, and only
the upper part of the flame is monitored by hori-
zontal optical system, which focuses the emis-
sion into a photomultiplier. Filters are used to
improve selectivity with transmission maxima at
394 nm for sulfur and 526 nm for phosphorus
detection. The high selectivity and reproducibil-
ity of FPD have made it a preferred detector for
the analysis of P- or S-containing compounds,
provided the nature of the sulfur response and the
of quenching carbon radicals (the direct interfer-
ence from hydrocarbons due to CH and C2 emis-
sions is not a major problem in the P mode due to
high selectivity, although hydrocarbon fragment
can directly quench HPO and S2 emission) are
kept in the mind, it gives excellent results.
AFID
Alkali flame ionization detector (AFID) is widely
used for the detection of organophosphorus com-
pounds. The basic feature of all AFIDs is the
introduction of an alkali salt into a relatively cool
hydrogen flame or plasma. The long-term stabil-
ity of AFIDs is not as of FID, and certain precau-
tions are taken for best results. Gas flow to the
detector is carefully controlled with individual
pressure regulation for each instrument. A flame
stabilization period of about an hour is sufficient
as the alkali source is in good health during this
period; overnight operation is not recommended
as it shortens the life of alkali source and increases
detection contamination.
GC–MS
Mass spectrometry is the most sensitive and
selective method for molecular analysis and can
yield information on the molecular weight as
well as the structure of the molecule. Combining
chromatography with mass spectrometry (GC–
MS) provides the advantage of both gas chro-
matography for separation method and mass
spectrometry as an identification method. In mass
spectrometry, there is a range of methods to ion-
ize compounds and then separate the ions.
Common methods of ionization used in conjunction
5 GC: Herbal Drugs and Fingerprints
with gas chromatography are electron impact
(EI) and electron capture ionization (ECI). EI is
primarily configured to select positive ions,
whereas ECI is usually configured for negative
ions (ECNI). EI is particularly useful for routine
analysis and provides reproducible mass spectra
with structural information which allows library
searching [2]. The GC–MS, in herbal drug stud-
ies with capillary column, have very good separa-
tion, necessary to generate fingerprint of high
quality, and the spectral database is useful for the
further research to elucidating the relationship
between chemical constituents and its pharma-
cology. Thus, GC–MS is the most preferable
tool for the analysis of the volatile chemical
compounds in herbal drugs especially in
aromatherapy.
Development of GC Fingerprints
and Data Interpretation
GC analysis is highly informative and unique, as
is used where TLC, HPTLC, and HPLC techniques
do not work satisfactorily. The entire operation
may be discussed in a generalized way as:
Sample Preparation
The collection and identification of plant part is
carried out as per established procedure and a
voucher samples deposited to herbarium with reg-
istration number. The collected plant part is dried
and reduced to suitable size. A few steps common
in the sample preparation for GC analysis are to
select the desired plant part and extract it with
suitable solvents, and, partitioned between solvents,
desired fraction is charcoalized, if pigmented and
filtered, using filter aid, passed through Na2SO4
column to dehydrate it, and finally make up in
suitable solvent (either n-hexane, n-heptane, or
in alcohol of chromatography/spectral grade),
based on the nature of solubility of the desired
ingredient(s). During extraction and purification,
TLC, HPTLC, and HPLC analysis may be guiding
for process chemistry and suitable solvent selec-
tion, for a new moiety. Extraction steps are recom-
mended to carried out in dim light to avoid the
Development of GC Fingerprints and Data Interpretation
Fig. 5.2 GLC chromatogram of walnut oil [5]
photochemical reactions. In case of volatile and
thermosensitive compounds, solvent extraction is
preferred than to steam distillation, to avoid the
evaporation and decomposition, respectively [3].
The moisture content on the sample is also consid-
ered, to calculate the assay on dry basis. The sample
preparation procedure for different classes of
chemical compounds may be summarized as:
Alkaloids
The air-dried plant part is ground altogether as a
fine powder, extracted with alcohol, defatted
using n-hexane or petroleum ether (60–80°C),
alcohol distilled out under vacuum, the soft mass
dissolved in acidic water (10% HCl), filtered, and
the filtrate basified with NH4OH. The basified
fraction (i.e., the alkaloidal fraction) is extracted
with chlorinated solvent, for example, CHCl3 and
MeCl2, which may be directly injected into GC
after passing through Na2SO4 and subsequent
filter through 0.45 mfilter.
Flavonoids
Flavonoids are isolated by dissolving the alcoholic
soft mass of the plant with hot water, followed
by extraction with chloroform or ethyl acetate,
and assayed mostly after derivatization.
Lipids
Air-dried and powder plant parts are extracted
with petroleum ether (40–60°C) for lipid fraction.
The marc is macerated with ethanol (80%) at
room temperature till exhaustion. The resulting
alcoholic extract is concentrated to obtain a crude
residue. The obtained lipid fraction is treated with
hot acetone. Acetone is distilled out, and residue
87
is saponified. On dissolving in water and further
extraction with ether both saponified and non-
saponified matter separates. The non-saponified
matter may be analyzed by GC either as such or
after methylation.
Separation and Detection
In injection port, the sample vaporized and
transported through the column by the flow of
carrier gas. The column temperature, stationary
phase, and flow rate of carrier gas play a vital
role in the separation of different ingredients.
Each chemical species tends to migrate at its
own characteristic rate, hence separated from
other species by the time they emerge from the
column and reach at detector. The separation is
also dependent upon the boiling point of the
sample, which is controlled to within tenths of
a degree for accuracy of results. There is a rule
of thumb that temperature slightly above the
average boiling point of the sample results in an
elution time of 2–30 min. Minimal tempera-
tures give good resolution but increase elution
times. For a sample of a wide boiling range,
temperature programming is useful, in term of
temperature programmed retention indices
(IPTGC) [4].
Analytical Results
The chromatogram for standard and sample are
compared against RT of ingredients (Fig. 5.2)
and calculated for assay.
88
Utility of GC Fingerprints in Herbal
Drugs
The GC fingerprints for herbal drugs are unique
as used in determination of:
Contamination/Adulteration
The raw material to be used for herbal drugs and
finished goods should be free from pesticides,
herbicides, and residual solvents, as the presence
of these has high health risk. There are extensive
legislations controlling the quality of all human
foods, drinks, and drugs derived from plant-based
products and offensives carry very serious penal-
ties. In addition, the condition of the herbal-based
food is also of great concern to the food scientist
who looks for those trace materials that have been
established to indicate the onset of microbial
action with time, storage conditions, rancidity, or
decomposition and are analyzed by GC technique.
Traceability
The features of GC fingerprint are effective in the
identification of spurious herbal products. The
source of many plants (herbs and spices) can
often be identified from the peak pattern of the
chromatograms obtained directly from GC analy-
sis. The unique qualitative and quantitative pat-
tern from a GC analysis helps in identification of
source of many alcoholic beverages and raw
material of specific geographical origin. The peak
pattern in chromatographic fingerprints, for eval-
uating the quality of herbal drugs, is an intuitive
evaluation method, to compare the percentage,
concentration, and distribution of components.
Qualification and Quantification
of Therapeutics
Plants contain minerals, vitamins, volatile oils,
glycosides, alkaloids, bioflavanoids, tannins, and
other substances that are important in supporting
5 GC: Herbal Drugs and Fingerprints
a particular herb for its medicinal properties, and
these elements also provide an important natural
safeguard for identity and authenticity of the
plant used for medicine purposes. A brief discus-
sion on the analysis of these using GC is as:
Alkaloids
Capillary gas chromatography often coupled with
a mass spectrometer as a detector (GC–MS) is a
well-established technique for analyzing com-
plex mixtures of alkaloids. There are a large
number of publications dealing with GC or
GC–MS of underivatized alkaloids. Mirko et al.
[6]. have reported that GC–MS of alkaloids pro-
vides much structural information in a short time.
The characteristic fragmentation pattern of the
different structural types and the high sensitivity
of GC allow a fast identification even of minor
compounds of a mixture without laborious isola-
tion procedures. However, even under optimal
conditions, some decomposition reactions may
occur. A properly deactivated column, as well as
mild conditions (use of on-column injection), is
recommended for optimal results. Twenty-nine
alkaloids from the extracts of D. stramonium
(Bulgarian origin) have been detected by GC-MS,
which have all the characteristics for the tropane
nucleus ions at m/z 124, 113, 96, 95, 94, 83, 82,
and 81 [7].
Flavonoids and Flavones
Due to biological and physiological importance,
flavonoids have considerable attention to be
analyzed by gas chromatography coupled to mass
spectrometry (GC–MS) technique for the analy-
sis of flavonoid and aglycones. Because of the
increasing interest in structure elucidation of
flavonoids, use of hyphenated technique of gas
chromatography with mass spectrometer is espe-
cially in practice. Monitoring the presence
of flavonoids with GC–MS provides a fast and
efficient separation and requires minimal sample
preparation to monitor the herbal product quality.
Tannins and Other Components
Vegetable tannins influence the characteristics
of many plant products, their taste, palatability,
Utility of GC Fingerprints in Herbal Drugs
Fig. 5.3 GLC fingerprints of Cinnamomum verum bark for essential oils [9]
nutritional value, pharmacological and toxic effects,
and their microbial decomposition because of
their ability to form complex with proteins,
carbohydrates, nucleic acids, alkaloids, and
minerals, and gas chromatography is unfeasible
due to their large molecular weight, polarity, and
thermal liability. However, molecules with these
chemical features are in principle suitable to
be degraded by pyrolysis (PY), an effective
technique for the study of complex, nonvolatile
samples, which can be integrated with a gas chro-
matograph mass spectrometer (PY–GC–MS) to
provide a rapid analysis of the degradation products
and hence the characterization of the original
sample. Headspace sampling coupled with gas
chromatography (HSGC) is a widely used
technique for the analysis of beer throughout
the world. HS–GC is typically used for quality
control to identify problems or changes occurring
in the fermentation process that affect the taste or
quality of the final product. It has been observed
that a large number of companies, and a few
government agencies, stimulating their standard-
ization and the development of reliable quality
control analytical methodologies to support their
safety for such products. Furthermore, for herbal
preparations, some tests include quantification of
the active compounds and uniformity of dosage,
disintegration, and dissolution among others.
Concerning the quantification assay and consid-
ering the high complexity of this type of matrix,
89
special attention has been given to chromatographic
techniques, mainly gas chromatography, due to
its advantages such as good separation, efficiency,
and speed properties [8].
Terpenes
Various herbs and spices (including orange,
lemon, lime, grapefruit rosemary, sage, spear-
mint, nutmeg, black peppercorns, cloves, cara-
way seeds, and vanilla beans) are analyzed
through GC and GC–MS. [8]
Essential Oils
The analysis of the volatile oils has a number of
advantages as volatile oil gives a reasonable
fingerprint which can be used to identify the
plant. The composition and relative concentra-
tion of the organic compounds in the volatile oil
are characteristic of the particular plant, and the
presence of impurities in the volatile oil can be
readily detected (Fig. 5.3) [9].
The extraction of the volatile oil is relatively
straightforward and can be standardized, and
components can be readily identified using
GC–MS analysis (Figs. 5.4 and 5.5) [9].
The relative quantities of components used in
the herbal drugs. Changes in composition of the
volatile oil may also be used as indicators of oxi-
dation, enzymatic changes, or microbial fermen-
tation, so GC and GC–MS are unanimously
accepted methods for the analysis of volatile
90
Fig. 5.4 GLS fingerprint of essential oils from Murraya koenigii leaflet [9]
CH3
O
N CH3
H CH2CH2CH C Examples
Mahanimbine CH3
Fig. 5.5 Structure of mahanimbine [9]
constituents of herbal drugs, due to their sensitiv-
ity, stability, and high efficiency. Especially, the
hyphenation with MS provides reliable informa-
tion for the qualitative analysis of the complex
constituents [10]. GC–MS was the first success-
ful online combination of chromatography with
mass spectrometry and is widely used in the anal-
ysis of essential oil in herbal medicines [11, 12].
With the GC–MS, scientists can produce not only
a chromatographic fingerprint of the essential oil
of the herbal drugs but also the information
related to its qualitative and quantitative compo-
sition. The chemical structure of the individual
components of many of the oils, elucidated by the
GC–MS tandem systems, provided the knowl-
edge necessary to synthesize a number of com-
mercially important synthetic flavors. For
example, the synthetic flavors that closely imitate
those of the peach, melon, and other fruits that
are presently available to the contemporary food
5 GC: Herbal Drugs and Fingerprints
chemist are a direct result of the separating
capabilities of GC techniques [11].
GC–MS Analysis of Garlic
Analysis of the two garlic (Allium sativum) sam-
ples was carried out using a GC-17A gas
chromatographic instrument hyphenated with a
QP-5000 mass spectrometer from Shimadzu. The
chromatographic column was a DB-5 quartz cap-
illary column (30 m, 0.25 mm I.D.). The injec-
tion temperature was 50 °C initially (maintained
for 3 min), which was increased to 230 °C (main-
tained at 10 min) at rate of 10 °C/min. The inlet
temperature was constant at 250 °C. In the exper-
iment, the sampling volume was 0.4 ml. Electron
impacts (EI) were recorded in the MS at 70 eV
ionization energy in the full-scan mode. The
analytical range of m/z was 30–350 amu, with
0.2 s/scan, and ionization source temperature was
set to 150°C [10].
GC–MS Analysis of Thiophene Derivatives
Natural thiophenes are biologically active com-
pounds, and the activity is enhanced by irradiation
with long wavelength ultraviolet light, exhibit
substantial antiviral, antibacterial, antifungal,
nematocidal, and insecticidal properties under
Utility of GC Fingerprints in Herbal Drugs
irradiation, whereas their toxicity is much reduced
in darkness. In presence of UV light, these sulfur-
containing polyacetylenes react as type II photo-
sensitizers producing singlet oxygen that causes
a wide spectrum of microbial and animal toxicity.
The separation of thiophenes by capillary GLC
and the group-specific MS fragmentation with
the typical sulfur isotope peaks allowed the
unequivocal assignment of individual thiophenes
in complex mixtures, even when occurring in
traces and in the presence of different geometri-
cal isomers. GLC and GC–MS are suitable
methods for the analysis of complex mixtures of
thiophenes which contain several stereoisomers
or differ only in the number of unsaturated bonds
and methyl groups, for example, Tagetes patula.
IPTGC were calculated according to Van den
Dool and Kratz (1963) [4]. The purified extracts
were diluted in hexane. 1 ml hexanic solution of
23 mg/ml n-hexadecane was added as an internal
standard at 4 °C to each gram of plant material of
the diluted extract source. Thiophenes were
quantified by comparing the peak areas of the
n-hexadecane and thiophenes (response factor
of 0.2440). The analysis was carried out with
a Carlo Erba Mega 5360 GC equipped with
fused silica capillary column (15 m × 0.25 mm
I.D.) and coated with PERMABOND OV-1 DF.
GLC conditions: injector = 250°C, temp. program
T0 = 120°C, t0 = 2 min, rate of temperature
increase = 10°C/min, T1 = 300°C, t1 = 5, pressure =
1 atm, split ratio = 1:20, injection volume = 2 ml,
and detector = FID. Data evaluation was carried
out using a Hewlett Packard 3393 Integrator.
GC–MS was carried out as above, and the only
differences were a 30-m capillary column, the
heating rate was reduced to 6°C/min, the detec-
tor was a Finnigan MAT 4500 quadrupole mass
spectrometer, and mass spectra were recorded
at 45 eV [3].
Nutraceutical Discovery
Plant sterols have chemical and biological func-
tions as like cholesterol in human, a critical role
in stabilizing the phospholipid bilayer in plant
cell membrane, just as cholesterol does in animal
91
cell membrane. Plant sterols fall into one of three
categories, namely, 4-desmethylsterol (cholestane
series), 4-monomethylsterols (4-methylcholestane
series), and 4,4-dimethylsterols (lanostane series,
i.e., triterpene alcohols). In plants, more than 40
sterols have been identified of which campes-
terol, ß-sitosterol, and stigmasterol are the most
abundant. These 3 sterols are structurally similar
to cholesterol (they are all 4-desmethyl sterols)
and differ only in a side-chain substitution of a
methyl group (campesterol), an ethyl group
(sitosterol), or an ethylidene group (5-avenasterol)
and a diene at C-22 (stigmasterol). These are
completely saturated forms of sterols and lack
the carbon–carbon double bonds found in choles-
terol and sterols. Plant sterols (and stanols) exist
in 4 different forms: as free alcohol, fatty acid
esters, and conjugates with sugar moieties or
phenolic acids. The sterol pattern of vegetable oil
is used to characterize and detect adulteration
(sitosterol). However, nutrition research has
focused mainly on five types – campesterol,
ß-sitosterol, stigmasterol, campestanol, and
ß-sitostanol [13].
The cholesterol esters, dietary plant sterol,
and stanol esters are hydrolyzed to free sterols
and stanols, respectively, and solubilized in mixed
micelles. In general, only 0.1–5% of the ingested
plant sterols (depending on the type) are absorbed;
[14] consequently, blood levels of plant sterols in
humans are only 0.1–0.14% of cholesterol levels
[15]. The poor efficiency of absorption of plant
sterols and stanols is attributed to the ABCG5
and ABCG8 transporter in enterocytes which
selectively pump plant sterols and stanols from
the enterocytes into the intestinal lumen, poor
solubility of the sterols in mixed micelles, and the
poor efficiency of intestinal cells to re-esterify
the absorbed sterols [15]. The human body does
not synthesize plant sterols endogenously. Plant
sterols block both biliary and dietary cholesterol
absorption in the intestine by different mecha-
nisms, by decreasing the absorption of choles-
terol. The net result reduced intestinal absorption
and less cholesterol reaching the liver via chylo-
micron remnants. In response to the decreased
supply of exogenous cholesterol, receptor-mediated
uptake of lipoprotein cholesterol is increased,
92
resulting in reduction of serum LDL cholesterol
levels [13].
Plant sterols and stanols occur naturally in
almost all vegetable foods, but the content and
composition vary widely. Nuts and oilseeds
have higher amounts of plant sterols than other
foods. Corn and sorghum have higher plant sterol
content than rice and wheat. Whole wheat and
brown rice are better sources of plant sterols than
refined flour and polished rice, respectively.
Legumes contain moderate levels. The plant ste-
rol content of fresh vegetables and fruits is
2–20 mg/100 g and 10–32 mg/100 g, respec-
tively. Mustard, fenugreek, and cloves contain
higher amounts of plant sterols than other
spices. Plant sterols get concentrated in the
unsaponifiable fraction of vegetable oils. In
most vegetable oils, the total sterol content is
200–370 mg/100 g (olive, groundnut, safflower,
sunflower, soya bean, cottonseed). Rice bran,
corn, mustard/rapeseed, and sesame oils contain
high levels (740–1,156 mg/100 g), whereas
palm oil and coconut oil contain low levels
(88–110 mg/100 g) [13 ]. The major sterols
are ß-sitosterol, campesterol, and stigmasterol.
Brassicasterol, characteristic of the Cruciferae
family, is present in rapeseed and mustard seed
oils but is in trace amounts in other oils. Plant
stanols are present in smaller quantities in many
of the above foods. Sterols are heat-stable mole-
cules. Cooking does not affect the plant sterol
content of cereals, legumes, and vegetables, but
industrial processing of vegetable oils results in
20–70% loss [13, 16]. The early human diet was
probably rich in plant sterols, providing up to 1 g
per day. However, most current western diets pro-
vide 200–400 mg/day of plant sterols [15].
Studies in humans have documented an inverse
relationship between intake of plant sterols and
both serum total and LDL cholesterol (corrected
for age, body mass index and energy intake,
dietary fiber, and saturated fat) [17]. Consumption
of wheat germ (328 mg plant sterols) reduced
cholesterol absorption by 42.8% compared with
plant sterol-free wheat germ in human subjects.
A study comparing purified corn oil triglycerides
with or without corn oil plant sterols reported that
in a single test meal, cholesterol absorption
5 GC: Herbal Drugs and Fingerprints
increased 38% with corn oil devoid of plant
sterols. The effect was attributed to natural corn
oil plant sterols because adding back 150 or
300 mg plant sterol to a test meal reduced choles-
terol absorption by ~12 and 27.9%, respectively
[18]. The sterols in rice bran oil are present as a
component of gamma-oryzanol (group of esters
of phenolic acids with sterols, ferulate esters
of triterpene alcohols, and ferulate esters of
ß-sitosterol and campesterol), free sterols, and
4,4¢-dimethylsterols (cycloartenol and 24-methylene
cycloartanol). Animal and human studies have
shown that rice bran oil reduces serum total and
LDL cholesterol and triglycerides and increases
serum HDL cholesterol concentrations [13, 19].
In human studies, 300 mg of gamma-oryzanol/
day reduced LDL cholesterol and consumption
of margarine containing 2.1g/day of plant sterols
from rice bran oil for 3 weeks reduced serum
LDL cholesterol by 9% [20]. These findings
suggest that sterols in foods and vegetable oils
are important dietary components for lowering
serum LDL cholesterol.
Phytostanols are a fully saturated subgroup
of phytosterols (contain no double bonds).
Phytostanols occur in trace levels in many plant
species, and they occur in high levels in tissues of
only in a few cereal species. Phytosterols can
be converted to phytostanols by chemical hydro-
genation. More than 200 different types of
phytosterols have been reported in plant species.
In addition to the free form, phytosterols occur as
four types of conjugates, in which the 3b-OH
group is esterified to a fatty acid or a hydroxycin-
namic acid or glycosylated with a hexose (usually
glucose) or a 6-fatty-acyl hexose. The most popular
methods for phytosterol analysis involve hydroly-
sis of the esters (and sometimes the glycosides)
and capillary GLC of the total phytosterols,
either in the free form or as trimethylsilyl (TMS)
or acetylated derivatives. Several alternative
methods have been reported for analysis of free
phytosterols and intact phytosteryl conjugates.
Recently, phytosterols and phytostanols have
received much attention for their cholesterol-
lowering properties. In the last several years, two
spreads, one containing phytostanyl fatty acid
esters and the other phytosteryl fatty acid esters,
Utility of GC Fingerprints in Herbal Drugs 93

−2.76

ARCHID: −21.82
STEARI. −17.89

LINCLEI.−19.72
CLEICA. −18.50

CIS.11,1. −23.72
MYRIST− −8.97

PALMITI. −15.17
Response [mV]

TRICOS. −27.55
BEHENI −25.25
ERUCICi −26.59
150

DHA. −30.02
EPA −28.74
100

50

0
Retention time (min.)

Fig. 5.6 GLC fingerprints of P. armeniaca seeds [23]

have been commercialized and were shown to immune system; oleic acid is able to enhance the
significantly lower serum cholesterol at dosages insulin in INS-1, also can be used for the preser-
of 1–3 g/day. The popularity of these products vation of confectionary items; stearic acid is
has caused the medical and biochemical commu- widely used in cosmetic formulations in antiper-
nity to focus much attention on phytosterols, and spirants, emollient creams and lotions, shampoos,
consequently, research activity on phytosterols and shaving creams; and linolenic and linoleic
has increased dramatically [21, 22]. The afore- acid, an essential fatty acids, required in normal
said data have been generated with the GC and human growth, development, and other biologi-
GC–MS techniques. cal process [23].
Prunus armeniaca seeds were collected from
Pithoragarh, Uttarakhand, India, during crop
New Cosmeceuticals ripening season and kept at −20 °C until ana-
lyzed. Oil was extracted with n-hexane in the
Apricot (Prunus armeniaca) cultivated at higher Soxhlet at 70 °C. The n-hexane fraction was
altitudes (1,000–2,700 m) and is native to China distilled out at 70 °C, and temperature was raised
and Indian Himalaya and in Japan but also found to 80°C, just after the distillation stopped, to
in Turkey, Southern Europe, South Africa, and remove the traces of n-hexane. Pale-yellow oil
Australia. Its pits yield 22–38% kernels, nor- was stored at 4°C until testing, and fatty acids
mally, which are considered as waste by farmers were analyzed by using GC–FID (Clarus 500, a
and have no commercial value, but these seed PE Auto-System, GC with inbuilt auto-sampler),
kernels are rich source of oil with great medicinal after saponification using 2 N-KOH in methanol,
value due to presence of different unsaturated subsequent esterification using 5% HCl, and
fatty acids. Fourteen different fatty acids have extracting the methyl ester phase into petroleum
been identified by GC–FID (Fig. 5.6), mainly, ether for GC analysis. The fatty acids methyl
myristic acid (25.63%), oleic acid (24.75%), esters (FAMEs) were identified by comparing
stearic acid 1(2.91%), linolenic acid (16.41%), their relative retention times with reference
and linoleic acid (9.50%) and in traces of archidic standards (FAMEs) [23].
acid, archidonoic acid, behenic acid (often used
as hair conditioners and moisturizers their
smoothing properties), erucic acid (has high tol- In Aromatherapy
erance to temperature that makes it suitable for
transmission oil), and tricosanoic acid. All these Aromatherapy uses natural oils (especially
fatty acids are source of fat in diet which increases essential oils) refined to 100% from flowers,
the immunity and also known by their utility in leaves, seeds, trunks, pericarp, resin, etc.
cosmetic and pharma industry. Myristic acid, an Essential oils that are applied to the skin can be
important saturated fatty acid, stabilizes many absorbed into the blood stream. The constitu-
different proteins, including proteins used in the ents of essential oils aid in health, beauty, and
94
hygiene conditions. Since essential oils are so
powerful and concentrated, they should never
be applied to the skin in their undiluted form.
Before application to the skin, essential oils
are diluted into a carrier such as a cold-pressed
vegetable oil. The common carrier oils include
sweet almond oil, apricot kernel oil, and grape
seed oil. In addition to therapeutic benefit at
the emotional and physical level, essential oils
are helpful in other applications as in house-
hold and laundry cleaners. Some oils act as a
natural insect repellent and pesticide, for exam-
ple, use of citronella candles during the sum-
mer to keep away mosquitoes. Essential oils
are blended together to create appealing and
complex aromas, for a specific therapeutic
application, and are referred as an essential oil
synergy. A synergistic essential oil blend is
considered to be greater in total action than
individual oil working independently. Lavender
oil promotes the recovery of skin. Tea tree is
effective in the treatment of atopic dermatitis
and suppresses the growth of Staphylococcus
aureus on the skin surface and decreases itch.
It enhances the immune system and helps in
the correct functioning of the defensive sys-
tem. Aromatic bathing reduces the stress.
Fragrance changes the body temperature, blood
pressure, and glucose level, feeling positive
and reduces fatigues. The essential oil from
Citrus is photosensitive, and degradation prod-
uct is toxic to skin, so it is necessary to avoid
direct sun light for 4–5 h after use, hence is
recommended at nighttime only [24]. Similarly,
the list may be matters of thousand examples,
and there is the need to establish evidence-
based medical aromatherapy system through
clinical studies and scientific research of essen-
tial oils, where GC, GC–MS fingerprints, has a
major role, to predict for stability, composi-
tion, and ingredients.
To Decipher Traditional Formulations
The resurgence of old herbal remedies that
arouse sexual activity and the use of new exotic
synthetic preparations have coincided with an
5 GC: Herbal Drugs and Fingerprints
increase in poisonings associated with the use of
these aphrodisiacs. References to such sub-
stances have crept into holy texts from the Kama
Sutra and the Bible to the Koran and the litera-
ture from Shakespeare and Ovid to Gilbert and
Sullivan plays in the twentieth century [25]. The
common aspects of different cultures and ethni-
cal groups have often emerged for the use of
plants as aphrodisiacs. The seventh part of the
Kama Sutra is dedicated to the occult practices
(Aupanishadika). A mixture of powdered thorn-
apple (Datura stramonium) seeds, black pepper
(Piper nigrum), long pepper (Piper longum) and
honey, applied on the penis before coitus, is
reputed to make a woman subject to the man’s
will. The thorn-apple seeds have atropine and
scopolamine, responsible for inducing an initial
excitement followed by sedation and hallucina-
tions. These two constituents are adsorbed
through the mucous membranes of both the
penis and the vagina and have a central nervous
system action producing behavioral effects in
both partners [26]. The peppers have a counter-
irritant or rubefacient action, increasing the
blood flow around the area of application. In the
man, this local inflammation of the clitoris
increases the sexual desire. Ginger (Zingiber
officinale) is described in the “Kama Sutra” and
employed by man to increase potency. The
honey is used as skin friendly base of the formu-
lation as well as useful lubricant.
Another example may be cited from the tradi-
tional formulation by the people of, Uttarakhand,
India, from hill lemon (Citrus pseudolimon)
fruits, (in Hindi, known as galgal nimbu and
chukh in local dialect) which is squeezed and
juice is concentrated over the wood fire in iron
pot till evaporation stops and is considered for
long stability (more than 10 years); with many
medical utilities, when analyzed by GC for vit. B
complex, it confirms the presence of ingredients
(Figs. 5.7 and 5.8) [27].
GC and GC hyphenized with MS are important
tools in the elucidation of the volatiles compo-
nents in complex matrix, as of herbal products.
Although only volatiles components can be analyzed,
this methodology is important to the creation
of fingerprint and monitoring of the herbals for
Utility of GC Fingerprints in Herbal Drugs 95

Fig. 5.7 GLC fingerprints of C. pseudolimon [27]

Fig. 5.8 GLC fingerprints for various ingredients of vit. B complex [27]

seasonal and geographic chemical variations non-identified components, along with verifying
and stability and to compare the composition changes in compositions and degradations, for
of selected extracts based on identified and quality and regulatory purposes.
96
References
1. James AT, Martin AJP. Gas-liquid partition chroma-
tography; the separation and micro-estimation of
volatile fatty acids from formic acid to dodecanoic
acid. Biochem J. 1952;50(5):679–90.
2. Liang YZ, Xie P, Chan K. Quality control of herbal
medicines. J Chromatogr B. 2004;812:53–70.
3. Margl L, Tei A, Gyurja´n I, Wink M. GLC and
GLC-MS analysis of thiophene derivatives in plants and
in in vitro cultures of Tagetes patula L. (Asteraceae).
Z Naturforsch. 2002;57C:63–71.
4. van den Dool H, Kratz PD. A generalization of the
retention index system including linear temperature
programmed gas liquid partition chromatography.
J Chromatogr. 1963;11:463–71.
5. Saxena R, Joshi DD, Singh R. Chemical composition
and antimicrobial activity of walnut oil. Int J Essent
Oil Ther. 2009;3(2–3):115–8.
6. Kreh M, Matusch R, Witte L. Capillary gas chroma-
tography-mass spectrometry of Amaryllidaceae
alkaloids. Phytochemistry. 1995;38(3):773–6.
7. Philipov S, Berkov S. GC-MS investigation of tropane
alkaloids in Datura stramonium. Z Naturforsch.
2002;57C:559–61.
8. Sathya A, Ramasubramaniaraja R, Brindha P. Gas
chromatography in phytochemistry – an overview.
J Pharm Res. 2010;3(8):1823–6.
9. George V. Plant metabolites in human welfare. Lead
Lecture. International conference on current trends in
medicinal plants research. Department of Botany,
University of Pune, 12 Jan 2012.
10. Teo CC, Tan SN, Yong JWH, Hewb CS, Ong ES.
Evaluation of the extraction efficiency of thermally
labile bioactive compounds in Gastrodia elata blume by
pressurized hot water extraction and microwave assisted
extraction. J Chromatogr A. 2008;1182:34–40.
11. Patra KC, Pareta SK, Harwansh R, Kumar KJ. Traditional
approaches towards standardization of herbal medicines
– a review. J Pharm Sci Technol. 2010;2(11):372–9.
12. Zeng ZD, Liang YZ, Xu CJ. Comparing chemical
fingerprints of herbal medicines using modified
window target-testing factor analysis. Anal Bioanal
Chem. 2005;381:913–24.
13. Ghafoorunissa. Impact of quality of dietary fat on
serum cholesterol and coronary heart disease: focus
on plant sterols and other non-glyceride components.
Natl Med J India. 2009;22(3):126–32.
14. Heinemann T, Axtmann G, Von-Bergmann K.
Comparison of intestinal absorption of cholesterol
with different plant sterols in man. Eur J Clin Investig.
1993;23:827–31.
15. Ostlund Jr RE. Phytosterols in human nutrition. Annu
Rev Nutr. 2002;22:533–49.
16. Verleyen T, Forcades M, Verhe R, Dewettinck K,
Huyghebaert A, De Greyt W. Analysis of free and
5 GC: Herbal Drugs and Fingerprints
esterified sterols in vegetable oils. J Am Oil Chem Soc
(JAOCS). 2002;79:117–22.
17. Ellegard L, Bosaeus I, Andersson H. Will recommended
changes in fat and fibre intake affect cholesterol
absorption and sterol excretion? An ileostomy study.
Eur J Clin Nutr. 2000;54:306–13.
18. Ostlund Jr RE, Racette SB, Okeke A, Stenson
WF. Phytosterols that are naturally present in
commercial corn oil significantly reduce cholesterol
absorption in humans. Am J Clin Nutr. 2002;75:
1000–4.
19. Cicero AF, Gaddi A. Rice bran oil and gamma-
oryzanol in the treatment of hyperlipoproteinaemias
and other conditions. Phytother Res. 2001;15:
277–89.
20. Vissers MN, Zock PL, Meijer GW, Katan MB.
Effect of plant sterols from rice bran oil and triter-
pene alcohols from Shea nut oil on serum lipoprotein
concentrations in humans. Am J Clin Nutr. 2002;72:
1510–5.
21. Moreau RA, Whitaker BD, Hicks KB. Phytosterols,
phytostanols, and their conjugates in foods: structural
diversity, quantitative analysis, and health-promoting
uses. Prog Lipid Res. 2002;41(6):457–500.
22. John S, Sorokin AV, Thompson PD. Phytosterols and
vascular disease. Curr Opin Lipidol. 2007;18(1):
35–40.
23. Singh R, Gupta S, Joshi DD, Nainwal N. Wild apricot
(Prunus armeniaca) kernel oil: a strategic alternative
to value added fatty acids. Int J Essent Oil Ther.
2010;4:1–5.
24. Shibamoto K, Mochizuki M, Kusuhara M. Aroma
therapy in anti-aging medicines. Jpn Soc Anti-Aging
Med. 2010;7(6):55–9.
25. Wedeck HE. Love potions through the ages, a study of
amatory devices and mores. New York: Philosophical
Library; 1963.
26. Hostettmann K. Tout Savoir sur les aphrodisiaques
naturels. Lausanne: Favre; 2000.
27. Joshi DD, Singh R. Amity Institute of Phytochemistry
& Phytomedicine, AUUP, Noida, (Un-published data);
2011.
Bibliography
Belec L, Georges AJ, Steenman G, Martin PM. Antibodies
to human immunodeficiency virus in the semen of
heterosexual men. J Infect Dis. 1989;159:324–7.
Bilia R, Flamini G, Taglioli V, Morelli I, Vincieri FF.
GC–MS analysis of essential oil of some commercial
fennel teas. Food Chem. 2002;76(3):307–10.
Gamble W, Vaughan M, Kruth HS, Avigan J. Procedure
for determination of free and total cholesterol in
micro- or nanogram amounts suitable for studies with
cultured cells. J Lipid Res. 1978;19:1068–70.
Bibliography
Gattuso G, Barreca D, Gargiulli C, Leuzzi U, Caristi C.
Flavonoid composition of Citrus juices. Molecules.
2007;12:1641–73.
Kroll U, Cordes C. Pharmaceutical prerequisites for a
multi-target therapy. Phytomedicines. 2006;13(1):12–9.
Lin H, Lee MC, Chaung WC. Application of LC/MS and
ICP/MS for establishing the fingerprint spectrum of
the traditional Chinese medicinal preparation Gan-Lu-
Yin. J Sep Sci. 2006;29(1):172–9.
97
Sato K. Aromatherapy and free radicals. J Jpn Soc
Aromather. 2007;6:28–34 (in Japanese).
Troszynsk A, Ciska E. Phenolic compounds of seed
coats of white and coloured varieties of pea (Pisum
sativum L.) and their total antioxidant activity. Czech
J Food Sci. 2002;20(1):15–22.
Xiao YS, Zhang TZ, Hou JD. Determination of flavonoids
of Pleioblastus amarus by HPLC. J Ningbo Coll.
2001;133:89–91.
 UV–Vis. Spectroscopy: Herbal Drugs
and Fingerprints
The absorption, transmission, and emission of
ultraviolet–visible (UV-Vis.) light wavelengths by
herbal and herbal products are primary indication
for purity and the amount present. Generally, it is
used to study molecules and inorganic ions in
solution. The ultraviolet and visible light are both
distinct regions of the electromagnetic spectrum,
but there are similarities between the two regions
for analytical procedures, due to the reason the
same research techniques and tools are used
for both regions together. Ultraviolet and visible
light comprise only a small portion of the wide-
ranging electromagnetic radiation spectrum, which
is lower in frequency and therefore lower in
energy than cosmic, gamma, and X-rays. Both
UV and Vis. light are of a higher frequency and
therefore of higher energy than infrared, micro-
wave, and radio waves, so on exposure, the
absorption, emission, or scattering of electro-
magnetic radiation by atoms or molecules are
expressed. Measurements of these properties
expressed by the atoms or molecules present
in a sample and decipher their concentration or
abundance, with high degree of accuracy.
The ultraviolet band of the electromagnetic
spectrum (200–400 nm) is grouped into three
regions termed UV-A, UV-B, and UV-C. All
scientists do not agree on the exact division of
these wavelengths. UV-A (320–400 nm), UV-B
(290–320 nm), and UV-C (200–290 nm) are based
on wavelength. Absorption of ultraviolet or visible
light electromagnetic radiation causes electron to
move from lower energy levels to a higher energy
levels. As the spectrum of an atom or molecule
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_6, © Springer India 2012
6
depends on its electron energy levels, UV–Vis.
absorption spectra are useful for identifying
unknown substances. Emission of electromag-
netic radiation (e.g., ultraviolet and visible light)
occurs when electrons move from higher
energy levels to lower energy levels. Scattering
of electromagnetic radiation occurs when light
is deflected or scattered in other directions.
Rayleigh scattering describes light that is redi-
rected at the same wavelength. Some interactions,
however, may occur that result in a partial transfer
of energy. As a result, the scattered electro-
magnetic radiation is of a different wavelength.
Brillouin scattering and Raman scattering are
examples of this type of scattering.
The way electromagnetic radiation affects
atoms and molecules depends on the energy of
the light. The energy is dependent upon the
frequency of the light. Both ultraviolet and visible
light promote electrons into higher energy
orbitals. Infrared light excites atomic and molec-
ular vibrations. Microwaves excite atomic and
molecular rotation. Special instrumentation is
used in UV–Vis spectroscopy. Hydrogen or
deuterium lights provide the source of light
for ultraviolet measurements. Tungsten lamps
provide the light for visible measurements. These
light sources generate light at specific wavelengths.
Deuterium lamps generate light in the UV range
(190–380 nm). Tungsten–halogen lamps generate
light in the visible spectrum (380–800 nm).
Xenon lamps which can produce light in the UV
and visible portions of the spectrum are used to
measure both UV and visible spectra.
101
102
UV–Vis. methods were pioneered by American
chemist Arnold Beckman, 1940. He invented
a spectrophotometer that had the ability to
measure the transmission of ultraviolet and visi-
ble light through a target. In UV spectroscopy, a
beam of light is split into a sample and reference
beams. As its name implies, the sample beam is
allowed to pass through the target sample.
Alternately, the reference beam passes through
the control solvent or a portion of the solvent
that does not contain the actual target. Once the
light passes through the target sample of interest,
it is measured by a special meter termed a
spectrometer designed to compare the difference
in the transmissions of the sample and reference
beams. Double beam UV spectroscopy instru-
ments allow scientists to simultaneously measure
transmissions through the target sample and
solvent. The absorption of ultraviolet or visible
light by a substance result in a unique ultravio-
let–visible spectroscopic signature is known as
UV–Vis. spectrum.
The concentration of a substance present in
sample has relationship with absorbance as per
Beer–Lambert law. In general, there is a linear
relationship between increasing amount of sub-
stance and a decreasing percentage of light
transmitted (increase in absorbance) through
the target sample. When ultraviolet or visible
light strikes atoms or molecules, they can either
bounce off or cause electrons to jump between
energy levels. Due to absorption of ultraviolet or
visible light, electromagnetic radiation causes
electron to move from lower energy levels to a
higher energy levels. Depending on the nature of
analyte, there may be either emission (i.e., the
jump of electrons from higher energy levels
to lower energy levels) or scattering (i.e., light
is deflected or scattered in other directions.
Rayleigh scattering describes light that is redi-
rected at the same wavelength) of electromag-
netic radiations. Some interactions, however,
may occur that result in a partial transfer of
energy. As a result, the scattered electromagnetic
radiation is of a different wavelength. Brillouin
scattering and Raman scattering are examples
of this type of scattering.
6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints
Instrumentation
A specially designed instrument is used in
UV–Vis spectroscopy, with hydrogen or deute-
rium lamps as source of ultraviolet light, while
the tungsten lamp is used for visible measure-
ments. These light sources generate light at
specific wavelengths. Deuterium lamps generate
light in the UV range (190–380 nm). Tungsten–
halogen lamps generate light in the visible
spectrum (380 to about 800 nm). The xenon
lamps which can produce light in the UV and
visible portions of the spectrum are used to
measure both UV and visible spectra. Visible
wavelengths cover a range from 400 to 800 nm
and the near ultraviolet region out to 200 nm.
Electromagnetic spectrum is a classification of
photons with various energies into different
spectral regions. UV–Vis. spectroscopy is used
when involving the absorption of these high-
energy lights by atoms or molecules, which
causes electronic excitation. This phenomenon
only occurs if conjugated pi-electron systems
are present. Energies of 36–72 kcal/mol and the
extensions of energies to 143 kcal/mol are
sufficient to promote a molecular electron to a
higher energy orbital in the visible region and at
the near UV region, respectively. Molecule
absorbs light energy when exposed to light and
excites electron from the highest occupied
molecular orbital (HOMO) to the lowest unoc-
cupied molecular orbital (LUMO). Then, an
optical spectrometer records wavelengths where
the absorption happens and also the intensity
of the absorption. Result is usually presented by
a graph of absorbance (A) versus wavelength.
The absorbance of a sample is proportional to
the quantity of absorbing molecules in a spec-
trometer light beam. Chromophores are molecular
moieties such as pi-electron functions and het-
eroatoms (having nonbonding valence-shell
electron pairs) which tend to absorb light of
200–800 nm wavelengths. Most instruments are
not able to detect the absorption of wavelength
below 200 nm, but the characteristic of conjuga-
tion which moves the absorption maxima to
UV–Vis. Spectrometry and Herbal Drugs
longer wavelengths solved the problem.
Conjugated pi-electron system shifts the absorp-
tion maxima in the same direction to the right in
a graph with every additional of double or triple
bond. It is called the bathochromic shifts because
the increased conjugation narrows the gap
between the HUMO and LUMO orbitals, and
therefore, the energy required for the promotion
of electrons is then less, meaning the wavelengths
increased correspondingly. UV–Vis. spectrome-
ter may be grouped into five sections: (1) radia-
tion source, hydrogen discharge tube is commonly
used but can be replaced by a tungsten incandes-
cent lamp for energy by emitting light; (2) mono-
chromator, disperses light from the source into its
separate wavelengths; (3) photometer, splits the
monochromatic light into sample and reference
beams and then reflects these beams into the sam-
ple area; (4) sample area, consists of small cells
with samples; and (5) detection area, generates
voltage which proportional to energy when radi-
ation beams are focused on separate photomulti-
plier tubes.
In UV spectroscopy, a beam of light is split
into a sample beams and reference beams. As its
name implies, the sample beam is allowed to
pass through the target sample. Alternately, the
reference beam passes through the control sol-
vent or a portion of the solvent that does not con-
tain the actual target. Once the light passes
through the target sample of interest, it is mea-
sured by a special meter termed a spectrometer
designed to compare the difference in the trans-
missions of the sample and reference beams.
Double beam UV spectroscopy instruments
allow scientists to simultaneously measure trans-
missions through the target sample and solvent.
The amount or percentage of a substance present
is often of great importance. In general, there is
a linear relationship between increasing amount
of substance and a decreasing percentage of light
transmitted through the target sample. If there is
more of a substance to absorb the UV light, then
less light will pass through to the detector.
Correspondingly, less absorption by the target
sample allows increased transmission light
through the sample.
103
UV–Vis. Spectrometry and Herbal
Drugs
The knowledge of optical properties of atoms or
molecules in UV–Vis. range has revolutionized
the chemical world as it reveals the many hidden
truth and deciphers the material. The utilization
of UV–Vis. spectral knowledge in case of herbal
drugs may be cited as:
To Study the Phenolics and Derivatives
All phenolic moieties have common features
of deriving from the shikimate pathway. The
flavonoids, phenolic derivatives, are aromatic
secondary plant metabolites, which have potential
medicinal important due to their physiological
and pharmacological role and health benefits,
having strong antioxidant and radical-scavenging
activity, and appear to be associated with reduced
risk for certain chronic diseases, prevention of
some cardiovascular disorders, and certain kinds
of cancerous processes. Flavonoids exhibit anti-
viral, antimicrobial, and anti-inflammatory activ-
ities, beneficial effects on capillary fragility, and
an ability to inhibit human platelet aggregation
properties. The actual in vivo mechanism of
action of flavonoids is largely unknown. One of
the reasons is that most studies have focused on
in vitro tests at doses or concentrations much
higher than those documented in humans,
whereas few clinical investigations have been
carried out on biomarkers of some of diseases
mentioned above, but results have been contra-
dictory. Epidemiological studies have shown an
inverse association between risk and intake level
of some particular flavonoids. Flavonoids are
frequently found in fruits, vegetables, and cere-
als. Food preparation and processing of fresh
fruits and vegetables may decrease flavonoids
content by 50% owing to their leaching into water
or by removal of the richest parts of the plant. In
order to overcome this problem, and even more
to make processed foodstuffs a better vehicle
for flavonoid intake, a considerable amount of
104 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Fig. 6.1 (a) Flavone a b

skeleton. (b) Flavanone
skeleton B
O B
O
A C
A C

O
O

284
100

90
Band II
80

70

60

50
A

40 Band I

30

20 326

10

0
240 260 280 300 320 340 360 380
nm

Fig. 6.2 UV spectrum of flavanone hesperidine [2]

work has been done on the modification of their
biosynthesis in plants.
Fresh fruits and their hand-squeezed or indus-
trially processed juices contain mostly flavanones
and flavones. Although more than 5,000 flavonoid
derivatives have been characterized, only a limited
number of representative derivatives have been
found and identified. Many others are often only
present in very low concentrations and have
yet to be identified. Their significance, however,
may outweigh their simple concentration levels.
A flavonoid skeleton is composed of two aromatic
rings (commonly designated as A and B), which
are connected through a pyrone ring (C) in the
case of flavones or a dihydropyrone ring in the
case of flavanones (Fig. 6.1) [1].
Flavonoids are mainly present as their glyco-
syl derivatives (hydrophilic nature), while the
aglycones (i.e., the forms lacking the sugar
moieties) owing to their lipophilic nature and
hence their low solubility in water. The presence
of large number of flavonoids with different
combinations that are possible between polyhy-
droxylated aglycones and a limited number of
mono- and disaccharides are studied by UV–Vis.
spectroscopy. As the absorbed wavelengths by a
substance, results in unique ultraviolet–visible
spectra for each substance, for example, the UV
spectra of flavones and related glycosides,
show two strong absorption peaks commonly
referred to as band I (300–380 nm) and band II
(240–280 nm) (Fig. 6.2) [2].
UV–Vis. Spectrometry and Herbal Drugs
a R3
R4
R2
HO O HO
R1
OH O OH
Fig. 6.3 (a) Flavone aglycones. (b) Flavanone aglycones
Band I is associated with the presence of a
B-ring cinnamoyl system. Band II absorption is
due to an A-ring benzoyl system. Substitutions
on the A or B ring may produce hypsochromic or
bathochromic shifts of the absorptions, which are
useful for clarifying structures (Fig. 6.3) [1].
Similar type of studies for other horticulture
product may have new methods of quality assur-
ance and relook to the process adopted for the
preparation of the same.
A group of compounds classified as polyme-
thoxyflavones (PMFs) usually found as compo-
nents of the essential oils fraction of Citrus peels
[3]. Hand-squeezed juices contain no detectable
traces of this class of compounds [4]. Commercial
juices, on the other hand, are rich in PMFs
because the industrial processing of fruits
leads to juices being contaminated with the
peel constituents (vide infra). The flavanone
O-glycosides have a glycosyl substitution exclu-
sively at the C-7 position (on ring A), although a
3-O-rutinoside has also been reported (viz., rutin),
and di-C-glycosides, along with smaller amounts
of mono-C-glycosides. For these compounds,
substitution is generally on either the C-6 or the
C-8, or on both positions [1].
UV Spectrometry for Different
Derivatives
UV spectra of different derivatives of a compound
can be easily distinguished and characterized,
for example, the quercetin, 3-O-glucosyl quercetin,
and 3,4¢-di-O-glucosyl quercetin in UV–Vis.
spectrometry have spectrum as Fig. 6.4 [5].
105
b R2
R3
O
R1
O
Flavonoids contribute to fruit and juice quality
in many ways, influencing the appearance, the
taste, and the nutritional value of the product from
the plant. In lemon and orange juices, for instance,
hesperidin can contribute to the formation of
sediments which result in undesirable cloudiness,
whereas naringin markedly influences the bitter-
ness of the juice of grapefruits and bergamots.
Flavonoids, most common in our diet, may be
cited from Citrus fruits as their glycosyl deriva-
tives. Aglycones occur less frequently in juices,
owing to their lipophilic nature and hence their
low solubility in water. The presence of a rela-
tively large number of flavonoids in Citrus
juices is a result of the many different combina-
tions that are possible between polyhydroxylated
aglycones and a limited number of mono- and
disaccharides. The most common sugar moieties
include d-glucose and l-rhamnose. The glyco-
sides are usually O-glycosides, with the sugar
moiety bound generally to the aglycone hydroxyl
group at C-7 or at the C-3 in some cases. In addi-
tion to these, C-glycosides have also been detected
in various citrus fruits or juices [1].
Many analytical procedures require no pre-
liminary separation of the flavonoid fraction,
and analysis is performed directly on the crude
juices. Extraction with solvents of graded polarity
leads to the separation of flavonoids from the
other components. Methanol, ethanol, acetone,
water, ethyl acetate, and, to a lesser extent, propa-
nol, dimethylformamide, and combinations of
these are frequently used for their extraction.
Hydrolysis of glycosides is a useful method to
obtain structural elucidation and characterization
information. The rate of acid or basic hydrolysis
106
100
90
80
70
60
A
50
40
30
20
10
0
200 250 300
Fig. 6.4 UV spectrum of quercetin and derivatives [5]
of glycosides depends on the acid or base strength,
the nature of the sugar, and the position of attach-
ment to the flavonoid nucleus. In fact, acid hydro-
lysis, which does not affect C-glycosides, is a
good method to distinguish these derivatives
from their O-glycosidic counterparts as the latter
are quickly hydrolyzed [1].
UV–Vis. Coupled Methods for Analysis
A number and variety of methods for the detection
and quantification of natural product compounds
have been developed, and several analytical
procedures allow the simultaneous determination
of the various kinds of flavonoid glycosides as
flavanone O-glycosides, flavone O-glycosides,
flavone C-glucosides, and polymethoxyflavones,
for example, liquid chromatography (LC) and
more recent coupled methods as LC–MS and
LC–MS–MS [6]. HPLC–PDA chromatographic
fingerprint was developed for ethanolic extract
of Dioscorea membranacea, which have the
dioscorealide A, dioscorealide B, and dioscore-
anone, the three main markers of the species.
The samples were separated with reversed-phase
6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints
3,4’-di-O-glucosyl quercetin
3-O-glucosyl quercetin
quercetin
350 400 450
nm
HPLC by linear gradient elution using water
and acetonitrile as mobile phase at a flow rate of
1.0 ml/min and detector wavelength at 270 nm
for dioscorealide A and dioscorealide B and at
245 nm for dioscoreanone. This HPLC fingerprint
along UV spectra has proven the identification
and assessment for quality D. membranacea
(Fig. 6.5) [7].
Preparation of Sunscreens
Sunscreens are chemicals that provide protection
against the adverse effects of solar and in particu-
lar UV radiation [8]. Sunscreens has wide variety
of chemicals that have specific absorbance in
some parts of the UV spectrum. There are very
few single chemical substances that have absor-
bance over the full range of UV. This property
(having absorbance over the full range of UV) is
needed for a product to be considered as a suitable
wide-spectrum sunscreen. Plant extracts, due to
wide range of natural compounds, usually cover
this full range of UV wavelengths. The best
approach to protecting the body from the harmful
effects of UV irradiation is to use these active
UV–Vis. Spectrometry and Herbal Drugs 107

Dioscorealide B

Dioscorealide A
a b

Dioscorealide B

Dioscorealide A
40 60
50
30 40
mAU

mAU
20 30
20
10
10
0 0
0 10 20 30 0 10 20 30
RT (min.) RT (min.)
c d
100 Dioscorealide A 175

150
Dioscorealide B
270 nm
80 270 nm
125
60
mAU

100

mAU
40 75

50
20
25

0 0

200 250 300 350 200 250 300 350

nm nm

Dioscoreanone
e f
Dioscoreanone

10 30
25
8
20
mAU
mAU

6
15
4
10
2 5
0 0
0 10 20 30 0 10 20 30
RT (min.) RT (min.)

g
245 nm
150

125 Dioscoreanone

100
mAU

75

50

25

200 250 300 350

nm

Fig. 6.5 (a) HPLC fingerprints of extract, (b) HPLC fingerprints of standard, (c) UV fingerprints, (d) UV fingerprints,
(e) HPLC fingerprint of extract, (f) HPLC fingerprint of standard, (g) UV fingerprints

photoprotectives. In recent years, naturally occur- radicals. There are also reports of flavonoids
ring compounds have gained considerable atten- inhibiting the activities of many enzymes, including
tion as protective agents [9]. There are reviews lipoxygenase, cyclooxygenase, monooxygenase,
about the photoprotective effects of some naturally xanthine oxidase, mitochondrial succinate dehy-
occurring herbal polyphenols and phenolic-rich drogenase and NADH-oxidase, phospholipase
extracts in skin damage induced by UV irradia- A2, protein kinases, and nuclear transcription
tion. Several studies have shown the flavonoids factor. Phenolics are believed to be capable of
to act as scavengers of superoxide anions, singlet acting in redox-sensitive signaling cascades to
oxygen, hydroxyl radicals, and lipid peroxyl inhibit DNA damage. Many flavonoids such as
108
quercetin, luteolin, and catechins are better
antioxidants than the nutrients vitamin C, vitamin
E, and b-carotene. Therefore, the phenolics may
be beneficial in preventing UV-induced oxygen
free radical generation and lipid peroxidation,
that is, events involved in pathological states such
as photoaging and skin cancer, and to prepare
topical sunscreen formulations from these two
extracts and measure their SPFs.
Since time immemorial human in different
civilizations protected themselves against the
environment by paintings, clothes covering the
body, veils, and large brim hats were used by
ancient Greeks and umbrellas by Mesopotamia,
Chinese, and Indian civilization. Natural sub-
stances extracted from plants have been recently
considered as potential sunscreen resources
because of their ultraviolet ray absorption in the
UV region and their antioxidant activity. Green
tea polyphenols, Aloe vera extract, aromatic
compounds isolated from lichens, and several
glycosides of aesculin are examples of natural
substances evaluated for their sunscreen proper-
ties. There is strong evidence that DNA-damaging
radiations induce the accumulation of UV light
absorbing flavonoids and other phenolics in der-
mal tissue of the plant body, which may be a
potential source of sunscreen [10].
To evaluate the UV protective activities of
plants, the air-dried herbal materials are ground
into fine powders and extracted by maceration
method two times with 90 and 50% aqueous
methanol, respectively [11]. After filtration of
total extracts, the solvent is removed under
reduced pressure to give the gummy mass. The
gummy mass is dissolved in 20% hydro-metha-
nol solution and kept in refrigerator for 48 h until
the chlorophyll was precipitated. The extracts are
filtrated and extracted with ethyl acetate, and this
fraction is rich in flavonoids and other phenolics,
the most important chemicals for sunscreen. The
sun protection factor (SPF) is calculated by dif-
fuse transmittance method.
In a study, the ethyl acetate extracts of plants
are analyzed for SPF in vitro. The extract is dis-
solved in methanol, and spectra of the samples
are obtained by running from 337.5 to 292.5 nm
(at 5 nm intervals). The SPF determination model
6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints
used was based on the equation proposed by
Gharavi et al. [12]:
∑
337.5
E (λ )ε (λ )
SPF = 292.5
∑
337.5
E (λ )ε (λ )T (λ )
292.5
where
T(l) is the measured sunscreen transmittance
at l,
E(l) is the spectral irradiance of terrestrial
sunlight at l,
and e(l) is the erythemal action spectrum at l.
The values of the E(l) and e(l) were calcu-
lated according to the report by Gharavi et al.
[12]. T(l) was mean value of four experimental
observations. Three different concentrations of
each extract (according to Beer’s law) were used
for obtaining the standard curve and calculating
SPF in 2 mg/ml solution according to Gharavi
et al. method [12].
A few phenolics have also been found in vitro
to be mutagenic. These harmful effects are sus-
pected to result from the prooxidant rather than
antioxidant action of these compounds. Therefore,
it is necessary to investigate their toxic effects
before human use. In the past decade, the antioxi-
dant activity of herbal phenolics, namely, pheno-
lic acids and flavonoids, has been given much
attention. Many flavonoids such as quercetin,
luteolin, and catechins are better antioxidants
than the vitamin C, vitamin E, and b-carotene.
Therefore, the phenolics may be beneficial in
preventing UV-induced oxygen free radical gen-
eration and lipid peroxidation (i.e., events
involved in pathological states such as photoaging
and skin cancer) and to prepare topical sunscreen
formulations from plant extracts having phenolic
acids and flavonoids and measure their SPFs.
Presently, physicians have increasing stress
that public and professionals should protect
themselves from the potential harmful effects of
ultraviolet radiation when they go out in the sun
for extended periods [9]. Studies have shown that
UV radiation can drive the chemical reactions
that produce highly reactive radicals that have
the potential to damage DNA and other cell regu-
lating chemicals and the cell structures also.
Evaluation of Vitamin C Content 109

Thus, UV–Vis. spectroscopy allows us in discovery molybdate solution 2 ml in each volumetric

and authenticity of testing of potential sun blocking flask and make up to 25 ml with distilled water.
substances [9]. 7. Preparation of sample solutions: Accurately
weigh 1 g of each sample in a 25-ml conical
flask and add 10 ml of oxalic acid (0.05 M)
Evaluation of Vitamin C Content solution, and the sample was placed under
shade for 24 h for extraction of vitamin C
l-Ascorbic acid (vitamin C), an important precur- content. After 24 h, the samples were filtered
sor of redox reaction, is an integral part of drugs,
to enhance the quality and efficacy of product. Table 6.1 Concentration of vitamin C in medicinal
Presently, UV–Vis. analysis is in practice to esti- plants (mg/100 g) [13]
mate the concentration of vitamin C by measuring Medicinal plant Absorbance Concentration
spectrum at 760 nm, after sample preparation Jasmine nudiflorum 1.864 202.74
against standard preparation (Tables 6.1 and 6.2) Ricinus communis 1.322 145.74
[13]. The whole process may be summarized as: Ficus bengumera 0.362 45.169
Ficus pumila 0.338 42.634
Policaria crispa 0.188 26.988
Preparation of Stock Solutions Narcissus tazetta 0.553 65.199
Fagophyrum esculentum 1.216 134.80
Cassia fistula 0.747 85.548
The following stock solutions were prepared:
Cherry chrysthanum 0.255 33.913
1. Ammonium molybdate solution (5%, m/v):
Ruscus hypophyllum 0.524 62.202
Weigh 5 g of ammonium molybdate and
Taraxicum officinale 0.266 35.052
dissolve it in 100 ml of distilled water.
Verbascum thapsus 0.221 30.339
2. Oxalic acid solution (0.05 M): Weigh required Rumax aluculatus 0.365 45.489
quantity of oxalic acid, add on it 1 ml of Fumaria parviflora 0.592 69.297
freshly prepared solution containing 0.02 M Bombax ceiba 0.143 69.297
EDTA, and then make up the volume 100 ml Stellaria media 0.395 48.653
with distilled water. Pectus forums 0.307 39.432
3. Sulfuric acid solution (5%, v/v): Measure 5 ml Olea europaea 0.549 64.839
of concentrated sulfuric acid and make up the Rosa indica 0.714 82.116
volume 100 ml with distilled water. Euphorbia helioscopia 0.622 72.473
4. Metaphosphoric acid with acetic acid solu- Oxalis latifolia 0.644 74.714
tion: Dissolve with shaking required quantity Cestrum nocturnum 0.800 91.144
of metaphosphoric acid pellets in required Pridium guajava 1.321 145.75
quantity of acetic acid and then make the Cupressus sempervirens 1.280 141.43
volume up to 100 ml with distilled water. Withania somnifera 0.361 45.029
Galium tricorne 0.381 47.154
5. Standard l-ascorbic acid solution (0.1%,
Tecoma stonz 618 71.999
w/w): Weigh 0.1 g of l-ascorbic acid and dis-
Lactuca sativa 0.142 22.067
solve in oxalic acid (0.05 M) solution freshly
Zizyphus jajuba 0.115 19.211
prepared and make up the volume 100 ml.
6. Preparation of different standard solution: The
0.5, 1, 2, 3, 4, and 4.5 ml, respectively, of stan- Table 6.2 Different concentration of standard solutions
and absorbance (mg/100 g) [13]
dard l-ascorbic acid (0.1%, w/v) solution taken
in separate 25-ml volumetric flasks, add 4.5, S. no. Absorbance Concentration
4, 3, 2, 1 and 0.5 ml of oxalic acid (0.05 M) 1 0.3 40
solution in each, and then add separately meta- 2 0.62 80
phosphoric acid with acetic acid 0.5 ml, sulfuric 3 1.03 120
acid (5%, v/v) solution 1 ml, and ammonium 4 1.52 160
110
Fig. 6.6 (a) Change in color with AgNO3 [15], (b) UV–Vis. spectrum of SNPs [15]
through a 0.45-mm filter. Add separately
metaphosphoric acid with acetic acid 0.5 ml,
sulfuric acid (5%, v/v) solution 1 ml, and
ammonium molybdate solution 2 ml, in each
volumetric flask and make up the volume
25 ml with distilled water.
To avoid photochemical reactions, amber-colored
volumetric flasks are used in the aforesaid whole
analysis. By this method, 29 different medicinal
plants were studied (Table 6.1), and each sample
was analyzed for vitamin C at 760 nm using
UV–Vis. spectrophotometer and compared with
the standard.
The temperature, solvents, pH, light, and metal
ions (Cu+2 and Fe+3) degrade the ascorbic acid [14].
The mean concentration of Cu+2 in drinking water
is 60 mg/l (0.06 ppm). Water concentration is com-
paratively more in fruits and vegetables than other
plants parts, so vitamin C degrades comparatively
fast on these items. Human body cannot produce
vitamin C and acquires from exogenous sources
only. The daily intake, as per American standard of
vitamin C, is 60 mg/250 ml, so to monitor different
herbal drugs and preparations as food supplements,
nutraceuticals, cosmaceuticals, etc. [13].
Green Synthesis of Nanoparticles
The biologically synthesized silver nanoparticles
(SNPs) are widely in use in the field of medicine,
for antimicrobial and antifungal properties, as an
alternate for conventional antibiotics. Extracellular
6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints
biosynthesis of silver nanoparticles is carried out
by using medicinal plant extracts for the reduction
of aqueous silver ions in short period. The silver
nanoparticles formation is confirmed by the color
change of plant extracts and further confirmed with
the help of UV–Vis spectroscopy. These photosyn-
thesized silver nanoparticles are tested for antibac-
terial and antifungal activities using disk diffusion
method. The test cultures are Proteus, Pseudomonas,
Klebsiella, Bacillus, and E. coli species of bacte-
ria, and Aspergillus, Fusarium, Curvularia, and
Rhizopus species of fungal have been studied.
The microbial property of silver nanoparticles is
analyzed by measuring the inhibition zone. The
silver nanoparticles synthesized from stem bark
extracts of Boswellia (Fig. 6.6) [15] and twigs of
Cinnamomum tamala (Figs. 6.7 and 6.8) [16]
extract have been studied, and the SNPs synthe-
sized, and studied for antimicrobial properties, and
results indicate that the silver nanoparticles have an
important advantage over conventional antibiotics.
The silver nanoparticles exhibit yellowish-brown
color in aqueous solution due to excitation of sur-
face plasmon vibrations in silver nanoparticles [17].
During experiment, the appearances of yellowish-
brown color in the reaction vessels indicate the
formation of silver nanoparticles (SNPs) [18].
SNPs from C. tamala Twigs
Cinnamomum tamala twigs were collected
from Thalkedar Forest, District Pithoragarh,
Green Synthesis of Nanoparticles
Fig. 6.7 Color change with AgNO3 and conc.biomass [16] where 1 and 2 are controlled and 3, 4, 5, and 6 are experi-
mental. The digit deciphers: 1 AgNO3 only, 2 twig only, 3 0.01% twig, 4 0.05% twig, 5 0.1% twig, and 6 0.5% twig
Uttarakhand, India. The freshly harvested C. tamala
twigs were exposed to vacuum until they were
completely dried, at 25°C. The biomass used for
the reduction was prepared by crushing the dried
twigs to the 60 mesh size, weighted, and added to
in the range of 40–60 ml of 1–10 mM aqueous
AgNO3 solution to obtain 0.01–05% final con-
centrations, in conical flasks of 100 ml, at room
temperature. The flasks are thereafter shaken at a
rotation rate of 100–200 rpm in the dark at
20–50°C for 24 h. Change in appearance, from
colorless to yellowish-brown, indicates the
presence of silver nanoparticles in the reaction
mixture, and the intensity of color increases with
the concentration of biomass (Fig. 6.6). The
formation of silver nanoparticles is confirmed
by UV–Vis. spectroscopy. UV spectra showed
the peak at 282 nm indicating the formation
of spherical nanoparticles (Figs. 6.8–6.10). The
size of the silver nanoparticle is in the range of
80–150 nm and is spherical in shape [16].
Silver nitrate is used as reducing agent as silver
has distinctive properties such as good conductiv-
ity, catalytic, and chemical stability. The aqueous
111
silver ions when exposed to herbal extracts
were reduced in solution, thereby leading to the
formation of silver hydrosol. The time duration
of change in color varies from plant to plant.
The synthesis of SNPs had been confirmed by
measuring the UV–Vis spectrum of the reaction
media. The weak absorption peak at shorter
wavelengths is due to the presence of several
organic compounds which are known to inter-
act with silver ions [17]. Three different routes
have been described for the reduction of silver
in plant extracts. The secondary metabolites
present in plant systems may be responsible for
the reduction of silver and synthesis of nano-
particles, as the first route. The second biogenic
route is the energy (or) electron released during
glycolysis (photosynthesis) for conversion of
nicotinamide adenine dinucleotide (NAD) to
nicotinamide adenine dinucleotide dehydroge-
nase. (NADH) led to transformation of Ag(NO3)2
to form nanoparticles, and the third postulate is
release of an electron during formation of
ascorbate radicals, from ascorbate, and reduces
the silver ions.
112 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Fig. 6.8–6.10 UV spectra indicating the formation of SNPs using C. tamala twigs [16]
Purification of Geometrical Isomers
The silver nanoparticles synthesized biologi-
cally using medicinal plants have been found of
high toxicity against different pathogenic bacte-
ria and fungi of selected species. The ionic silver
strongly interacts with thiol group(–SH) of vital
enzymes and inactivates the enzyme activity.
Experimental evidence indicates that DNA loses
its replication ability once the bacteria have been
treated with silver ions. The pathogenic effect of
nanoparticles can be attributed to their stability
in the medium as a colloid, which modulates the
phosphotyrosine profile of the pathogen proteins
and arrests its growth. The growth of microor-
ganisms was inhibited by the green synthesized
SNPs showed variation in the inhibition of
growth of microorganisms may be due to the
presence of peptidoglycan, which is a complex
structure and after contains teichoic acids or
lipoteichoic acids which have a strong negative
charge. This charge may contribute to the seques-
tration of free silver ions. Thus, gram-positive
bacteria may allow less silver to reach the cyto-
plasmic membrane than the gram-negative bac-
teria [19]. The SNPs synthesized from plant
species are toxic to multidrug resistant microor-
ganisms. It shows that they have great potential
in biomedical applications. Similar observation
was found in Allium cepa, Argemone mexicana,
and Artocarpus heterophyllus and concluded
that silver nanoparticles have an ability to inter-
fere with metabolic pathways. The data and
results by Ahmad N et al. indicate that the inhi-
bition of oxidation based biological process by
penetration of metallic nano-sized particles
across the microsomal membrane [20]. The use
of silver ions as preventing agents in cosmetics
has been tested by a challenged list in a set of
cosmetic dispersions with the addition of known
preservative inhibitors or microorganism’s
growth promoters. The silver nanoparticles syn-
thesized from leaf extract showed higher toxicity
than that of bark extracts. The reason could be
that the leaf extract synthesized higher concen-
tration of silver nanoparticles than the bark sam-
ples. Moreover, green leaves are the site of
photosynthesis and availability of more H+ ions
to reduce the silver nitrate into silver nanoparti-
cles. The molecular basis for the biosynthesis of
113
these silver crystals is speculated that the organic
matrix contain silver binding proteins that pro-
vide amino acid moieties that serve as the nucle-
ation sites [21].
Purification of Geometrical Isomers
Commiphora wightii has been widely used in
Ayurvedic medicine for more than 2,000 years,
mainly to treat arthritis and inflammation. The
active ingredients in gugulipid are the ketoster-
oids cis- and trans-4, 17 (20)-pregnadiene-3,16-
dione, also known as E- and Z-guggulsterone
[22], and are extracted from the resin, that is safer
and more effective than many cholesterol-lower-
ing drugs [23]. After extracting the ole-gum resin
with ethyl acetate, it separates into two parts,
gum and resin. The ethyl acetate insoluble part
of gum chemically characterized as a carbohydrate
gum (belonging to the class of sugar), and the
resin in ethyl acetate fraction contains bioactive
components, especially E- and Z-guggulsterones.
After filtration and evaporation of the solvent
under reduced pressure, compounds are sepa-
rated. Fractionated in petroleum ether, the steroi-
dal compounds were isolated by dissolving in
petroleum ether (40–60°C). For isolation of E-
and Z-guggulsterones from the mixture of steroi-
dal compounds, after being evaporated of the
solvent, of this was dissolved in small amount of
ethyl acetate and transferred on to a silica gel
60 F254 column chromatography as stationary
phase and eluted with the mixtures of solvents,
toluene–acetone (9:1), which resulted in 15 frac-
tions of 20 ml, based on TLC identification and
quantitative analysis of these isomers in herbal
extracts, the HPLC technique with photodiode-
array detector and gradient solvent was applied.
At first, the calibration curve for E- and
Z-guggulsterones was developed (Fig. 6.11) [23].
The guggulsterones peaks were identified at
242 nm using C-18 column (125 × 4.0 mm,
5.0 mm). The mobile phase was acetonitrile–
water (46:54, v/v), at ambient temperature
(~28°C) with flow rate of 1.0 ml/min.
In a study for authenticity of Commiphora
wightii resin, the presence of mangiferolic acid, a
114
Fig. 6.11 (a) E-Guggulesterone, (b) Z-guggulesterone
metabolite of Mangifera indica, raised the sus-
pension on adulteration, so genuine samples of
C. wightii and M. indica were collected from
India and Pakistan, and their ethyl acetate extract
was subjected to the TLC analysis, together with
the suspicious sample. The four compounds pres-
ent in commercial sample were not detected in
any authentic sample of C. wightii resin; how-
ever, they were present in M. indica specimen.
Finally, it was concluded that commercial sam-
ple, C. wightii sample, was adulterated with M.
indica resin, and TLC fingerprints had detected
the same, as morphologically it is not easy to dis-
tinguish between the closely related resins [24].
Analysis of Ultra-Diluted Drugs
The ultra-diluted drugs (lower than micro-
volumes) have billions dollar market, globally.
The UV-sensitive organic substances/compounds
of a drug, which has been ultra diluted, are char-
acterized by its UV–Vis. fingerprints, for evi-
dence-based status, significantly to claim that the
formulation is more than a placebo, for example,
the ultra-diluted Digitalis purpurea has wide
acceptance in treating ailments (heart diseases,
dropsy) in a medical system, known as homeopa-
thy. It is in use since long as narrow poison (1500
bc). The purified and active principles of D. pur-
purea have been studied for their identification
by UV spectroscopy. The saturated foxglove (D.
purpurea) extract in aqueous ethanol (91.4% eth-
anol and glass double-distilled water mixture) is
presented as q or Q (mother tincture) in term of
ultra-diluted medicine (i.e., in homeopathy).
Dilution or potency 1 means that one part of
6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints
O
O
OH
CH3
H
CH3
H OH
CH3
O
O H
OH
OH
3
Fig. 6.12 Structure of digoxin: calculation of theoretical
lmax [25]
medicine is mixed with 99 parts of aqueous
ethanol. When the same process is repeated at 30
times by adding one part of previous potency in
99 parts of aqueous ethanol, as diluents and suc-
cussed, after each stage starting from first dilu-
tion, next 29 times, 30 potency is obtained and so
on. The active ingredient of D. purpurea is
digoxin (Fig. 6.12), has medicinal preparations
Q, 6, 30, 200 in Indian market (manufactured by
M/S Hahnemann Publishing Company Pvt. Ltd.,
Kolkota) have been analyzed for digoxin content
by UV–Vis. spectroscopy (Figs. 6.13 and 6.14),
for potency of the digoxin [25].
1. A five member cyclic , b-unsaturated ketone:
202 nm
2. Acyloxy substitution (–OCOR): 25 nm
3. b-Unsaturated alkyl (including part of cyclic
ring): 24 nm (i.e., 2 × 12 nm)
Analysis of Ultra-Diluted Drugs 115

2.5

Digitalis P Q
2.0

1.5
A

1.0 Digitalis P 6

0.5
Digitalis P 30

0.0

Digitalis P 200
−0.5

100 200 300 400 500 600 700 800 900 1000
nm

Fig. 6.13 UV–Vis. spectra of Q, P 6, P 30, and P 200

Fig. 6.14 Absorption

maxima with dilution [25] 2.5

2.0

1.5
A

1.0

0.5

0.0

1.0 1.5 2.0 2.5 3.0 3.5 4.0

conc.

The above calculation predicts that the compound
in ethanol medium absorbs at wavelength 251 in
UV–Vis. spectrum. The UV–Vis. spectra of D.
purpurea Q, P 6, P 30, and P 200 (Fig. 6.13), and
the relation with absorption intensity (peak
heights) on concentration (Fig. 6.14) have been
studied for the authentication of the claims made
on it [25].
Ingredients other than , b-unsaturated ketone
of D. purpurea do not show any other specific
UV–Vis. absorbance, so any change brought due
to dilution could not be deciphered, but the
method is useful to generate the fingerprints of
large number of ultra-diluted drugs using medici-
nal plants having UV–Vis. active ingredients, for
evidence-based drugs.
116 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Table 6.3 Ultraviolet analysis of leaf powder of H. rosa-sinensis [26]

Treatment Visible light UV (254 nm) UV (365 nm)
Drug powder Light green Dark green Blackish brown
Sulfuric acid (conc.) Dark green Black Black
Sulfuric acid (dilute) Light brown Greenish black Black
Hydrochloric acid (conc.) Green Black Black
Hydrochloric acid (dilute) Brown Greenish black Black
Acetic acid Brown Greenish black Black
Nitric acid (dilute) Brown Greenish black Black
Methanol Green Dark green Black
Ethanol Brown Brownish black Black
Chloroform Brown Dark green Black
Petroleum ether Green Greenish black Black
Distilled water Brown Dark green Black
10% NaOH Brown Green Black
5% Iodine Greenish brown Dark green Black
Picric acid Green Greenish red Black
FeCl3 solution Brown Dark green Black
Ammonia solution Brown Green Greenish black

Identification of Powdered Medicinal bioautography of the dichloromethane extract of

Plants
The powdered part of plant tissues has specific
fluorescence in UV range, a tool to detect adul-
teration, contamination, and stability and prove
for claims made on products, for example, the
leave powder of Hibiscus rosa-sinensis, known
as China rose, examined under UV–Vis. light
treated with different reagent emits various
color radiations (Table 6.3), which helps in the
identification of the drug in fragmentary condi-
tion as well as in whole form. The results are
sufficient for preliminary phytochemical screen-
ing and act as biomarkers for identification and
authentification of raw drug samples and play an
important role in quality control and prevention
of adulteration [26].
S .calycina showed the presence of a compound
which strongly inhibit the growth of Cladosporium
cucumerinum. HPLC–UV and HPLC–MS analy-
ses of the extract revealed the presence of three
main compounds: a bitter principle, a xanthone,
and a naphthoquinone derivative with a molecu-
lar weight of 188. Comparison of online UV and
MS data with a data bank allowed identification
of the bitter principle as sweroside and the xan-
thone as decussatin. As these have no antifungal
properties, the strong activity of the dichlo-
romethane extract was attributed to the naphtho-
quinone, a class of compounds which is known to
have strong antimicrobial properties. Targeted
isolation afforded the active compound, identified
as 2-methoxy-1,4-naphthoquinone. Interestingly,
quinines were previously not known to occur in
the Gentianaceae [27].

Biological Assay Using Hyphenated

Techniques
Swertia calycina, a small plant found in Rwanda,
family Gentianaceae, was studied with the com-
bined use of TLC and HPLC in the search for
new antifungal metabolites (Fig. 6.15). TLC
To Differentiate Various Species
of a Genus
The basic aim to develop fingerprint is the estab-
lishment of a characteristic chromatogram/spec-
trum for the substance of interest, in the medicinal
To Differentiate Various Species of a Genus 117

Fig. 6.15 Structure and UV–Vis fingerprint of 2-methoxy-1,4-naphthoquinone [27]. (a) 2-Methoxy-1,4-
naphthoquinone, (b) UV–Vis. spectrum

Fig. 6.16 Fingerprint chromatograms of (a) Euphorbia ebracteolata Hayata, (b) Euphorbia fischeriana Steud, and (c)
Stellera chamaejasme L, where P1 = ebracteolata compound B, P2 = jolkinolide B, and P3 = neochamaejasmin [28]

herb, and at least one marker compound is
identified in the fingerprint, as characteristic,
capable to explain the uniqueness (as large peaks
with good resolution). Example may be cited
from the Chinese herb Langdu, which have
three different species (Euphorbia ebracteolata
Hayata, Euphorbia fischeriana Steud, and Stellera
chamaejasme L.). The experiment was with strat-
egy to develop optimum experimental conditions
for one of the species first and then apply these
optimum conditions to the other two species and
other related herbs. The species chosen for the
optimization was Euphorbia ebracteolata Hayata.
In this process, different experimental conditions
were adopted for establishing the optimum exper-
imental conditions for Euphorbia ebracteolata
Hayata HPLC fingerprints, with repeatability and
reproducibility. Using the optimized experimen-
tal conditions, fingerprints of other two species of
Langdu Euphorbia fischeriana Steud and Stellera
chamaejasme L. were obtained (Fig. 6.16) [28].
The characteristic HPLC pattern, as fingerprint,
approved as authentic by the identification of
marker compounds of the herb in the fingerprint
118
Fig. 6.17 Chemical structure and UV spectrum of the marker compounds [ 28 ] ( a ) ebracteolata compound B,
( b ) neochamaejasmin, and (c) jolkinolide B
chromatogram. This is the second reason to
optimize the experimental conditions under
which the number of intense peaks and their reso-
lution in the chromatogram are maximized. To
meet this requirement, peak-tracking method
using diode-array detector was employed to
find out if the marker compounds of the herbal
medicine could be identified in the fingerprint
chromatograms. As per literature, ebracteolata
compound b, jolkinolide B, and neochamae-
jasmin were recognized as the characteristic
marker compounds for Euphorbia ebracteolata
Hayata, Euphorbia fischeriana Steud, and Stellera
6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints
chamaejasme L. respectively. These marker
compounds were identified according to their
distinctive UV spectra in their characteristic
fingerprint chromatogram (Fig. 6.17) [28].
UV–Vis. spectroscopy is a reliable technique
for the assessment of herbals and herbal drugs, as
the absorption, transmission, and emission of
wavelengths are unique fingerprints for each.
The technique has effective potential when used
in conjunction with chemical screening methods
so that ubiquitous and unimportant compounds
can be excluded. For chemical screening, HPLC
coupled with different detection methods, for
References
example, UV, MS provides a great deal of
preliminary information about the content and
nature of constituents found in the active extracts,
as well as distinguished the adulteration of M.
indica and C. wightii resins. In certain cases,
combination with a spectral library and pre- or
post-column derivatization allows structure deter-
mination online.
References
1. Gattuso G, Barreca D, Gargiulli C, Leuzzi U, Caristi
C. Flavonoid composition of Citrus juices. Molecules.
2007;12:1641–73.
2. Polydera AC, Stoforos NG, Taoukis PS. Effect of high
hydrostatic pressure treatment on post processing
antioxidant activity of fresh Navel orange juice. Food
Chem. 2005;91:495–503.
3. Miyake Y, Yamamoto K, Morimitsu Y, Osawa T.
Isolation of C-glucosylflavone from lemon peel and
antioxidative activity of flavonoid compounds in
lemon fruit. J Agric Food Chem. 1997;45:4619–23.
4. Leuzzi U, Caristi C, Panzera V, Licandro G. Flavonoids
in pigmented orange juice and second-pressure
extracts. J Agric Food Chem. 2000;48:5501–6.
5. Mabry TJ, Markham KR, Thomas MB. The ultraviolet
spectra of flavones and flavonols. In: The systematic
identification of flavonoids. Berlin: Spinger; 1970.
6. De-Rijke E, Out P, Niessen WMA, Ariese F, Gooijer
C, Brinkman UAT. Analytical separation and detec-
tion methods for flavonoids. J Chromatogr A. 2006;
1112:31–63.
7. Sirikatitham A, Chuchom T, Itharat A. Development
of the chromatographic fingerprint analysis of diosco-
realides and dioscoreanone from Dioscorea mem-
branacea Pierre. Songklanakarin J Sci Technol.
2007;29 Suppl 1:101–7.
8. Elmets CA, Young C. Sunscreen and photo carcino-
genesis: an objective assessment. Phytochem
Photobiol. 1996;63:435–9.
9. Svobodova A, Psotova J, Walterova D. Natural pheno-
lics in the prevention of UV-induced skin damage, a
review. Biomed Pap. 2003;147:137–45.
10. Khazaeli P, Mehrabanti M. Screening of sun protec-
tive activity of the ethyl acetate extracts of some
medicinal plants. J Pharm Res. 2008;7(1):5–9.
11. Markham KR. The techniques of flavonoid
identification. London: Academic; 1982. p. 16.
12. Gharavi SM, Tavokoli N, Pardakhti A, Baghaei ZN.
Determination of sun protection factor of sunscreens
by two different in vitro methods. J Res Med Sci.
1999;2:53–4.
13. Hussain I, Khan L, Khan MA, Khan FU, Ayaz S,
Khan FU. UV spectrophotometric analysis profile of
ascorbic acid in medicinal plants of Pakistan. World
Appl Sci J. 2010;9(7):800–3.
119
14. Anna RB, Ada SV. Evaluation of sampling and extrac-
tion procedures for the analysis of ascorbic acid from
pear fruit tissue. Food Chem. 2002;77:257.
15. Savithramma N, Rao ML, Rukmini K, devi
Suvarnalatha P. Antimicrobial activity of silver nano-
particles synthesized by using medicinal plants. Int J
ChemTech Res. 2011;3(3):1394–402.
16. Singh R, Goel A, Joshi DD. Biosynthesis of silver nano-
particles by Cinnamomum tamala twigs. Indian Patent,
Pending. Application no. 2026/DEL/2011; 2011.
17. Thirumurgan A, Tomy NA, Jai Ganesh R, Gobikrishnan
S. Biological reduction of silver nanoparticles using
plant leaf extracts and its against clinically isolated
organism. De Phar Chem. 2010;2:279–84.
18. Shankar SS, Rai A, Ahmad A, Sastry MJ. Rapid synthe-
sis of Au, Ag and bimetallic Au shell nanoparticles using
neem. J Colloid Interface Sci. 2004;275:496–502.
19. Uthaman S, Annapoorna SKS, Ravindranath M,
Shanti KC, Lakshmanan VK. Novel boswellic acids
nanoparticles induces cell death in prostate cancer
cells. J Nat Prod. 2012;5:100–8.
20. Ahmad N, Sharma S, Singh VN, Shamsi SF, Fatma A,
Mehta BR. Biosynthesis of silver nanoparticles from
Desmodium triflorum: a novel approach towards weed
utilization. Biotechnol Res Int. 2011;1:1–8.
21. Prabhu N, Divya TR, Yamuna G. Synthesis of silver
phyto nanoparticles and their antibacterial efficacy.
Dig J Nanomater Biostruct. 2011;5:185–9.
22. Ding X, Staudingerm JL. The ratio of constitutive
androstane receptor to pregnane x receptor determines
the activity of guggulsterone against the Cyp2b10 pro-
moter. J Pharmacol Exp Therap. 2005;314(1):120–7.
23. Szapary PO, Wolfe ML, Bloedon LAT, Cucchiara AJ,
Dermarderosian AH, Cirigliano MD, Rader DJ.
Guggulipid for the treatment of hypercholesterolemia:
a randomized controlled trial. J Am Med Assoc.
2003;290:765–72.
24. Ahmad R, Ali Z, Wa Y, Kulkarni S, Avery R,
Chaudhary MQ, Rahman A, Khan IA. Chemical char-
acterization of commercial Commiphora wightii resin
sample and chemical profiling to assess for authentic-
ity. Planta Med. 2011;77:945–50.
25. Sharma A, Purkait B. Energy of commercially avail-
able ultra diluted natural cardiotropic drug Digitalis
purpurea: an UV spectroscopic study. Res J Pharmacol.
2009;3(4):58–62.
26. Gupta V, Bansal P, Garg A, Meena AK. Pharmacopoeial
standardization of Hibiscus rosa sinensis Linn. Int J
Pharm Clin Res. 2009;1(3):124–6.
27. Hostettmann K. Strategy for the biological and chemi-
cal evaluation of plant extracts. Invited lecture pre-
sented at the International Conference on Biodiversity
and Bioresources: Conservation and Utilization, 23–37
November 1997, Phuket, Thailand. Available at http://
www.iupac.org/symposia/proceedings/phuket97/
hostettmann.html. As on 20 Sept 2011.
28. Siu-kay W, Siu-kay T, Sik-yiu k, Kwan-li S, et al.
Establishment of characteristic fingerprint chromato-
gram for the identification of Chinese herbal medi-
cines. J Food Drug Anal. 2004;12(2):110–4.
120
Bibliography
Arya V. Living system: eco-friendly nano factories. Digest
J Nanomater Biostruct. 2010;5:9–21.
Ferreres F, Llorach R, Gil-Izquierdo A. Characterization
of the inter glycosidic linkage in di-, tri-, tetra- and
pentaglycosylated flavonoids and differentiation of
positional isomers by liquid chromatography/electro-
spray ionization tandem mass spectrometry. J Mass
Spectrom. 2004;39:312–21.
Gardea-Torresdey JL, Gomez E, Peralta Videa J, Parsons
JG, Troiani HE, Jose-Yacaman M. Synthesis of gold
nanotriangles and silver nanoparticles using Aloe vera
plant extract. Langmuir. 2003;13:1357.
Gauthaman KK, Saleem MT, Thanislas PT, Prabhu VV,
Krishnamoorthy KK, Devaraj NS, et al. Cardioprotective
effect of the Hibiscus rosa-sinensis flowers in an oxidative
stress model of myocardial ischemic reperfusion injury in
rat. BMC Complement Altern Med. 2006;6:32–9.
Ip M, Lui SL, Poon VKM, Lung I, Burd A. Antimicrobial
activities of silver dressings: an in vitro comparison.
J Med Microbiol. 2006;55:59–63.
Huie CW. A review of modern sample-preparation tech-
niques for the extraction and analysis of medicinal
plants. Anal Bioanal Chem. 2002;373:23–30.
6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints
Kimmatkar N, Thawani V, Hingorani L, Khiyani R.
Efficacy and tolerability of Boswellia serrata extract
in treatment of osteoarthritis of knee – a randomized
double blind placebo controlled trial. Int J Phytother
Phytopharm. 2003;10(1):3–7.
Nagarajan M, Waszkuc TW, Sun J. Simultaneous determi-
nation of E- and Z-guggulsterones in dietary supple-
ments containing Commiphora mukul extract
(guggulipid) by liquid chromatography. J AOAC Int.
2001;84:24–8.
Pal S, Tak YK, Song JM. Does the antibacterial activity
of silver nanoparticles depend on the shape of the
nanoparticle? A study of the gram-negative bacterium
Escherichia coli. Appl Environ Microbiol. 2007;73:
1712–20.
Robards K. Strategies for the determination of bioactive
phenols in plants, fruit and vegetables. J Chromatogr
A. 2003;1000:657–91.
Sharma A, Ajay GN, Mahadik KR. Review article molec-
ular markers: new prospects in plant genome analysis.
Pharmacogn Rev. 2008;2(3):21–3.
Urizar NL, Moore DD. Gugulipid: a natural cholesterol
lowering agent. Ann Rev Nutr. 2003;23:303–13.
Zhang J, Yang CJ, Wu DG. Studies on the diterpenes
of Euphorbia prolifera. Chin Trad Herb Drug.
1998;29:73–6.
 FTIR Spectroscopy: Herbal Drugs
and Fingerprints
In infrared (IR) spectroscopy, IR radiations are
passed through a sample, where some of the
infrared radiations are absorbed by the sample.
When the radiant energy matches to the energy of
specific molecular vibration, absorption occurs.
In IR spectrum, wave number (sometimes called
as frequency) is plotted on the x-axis and is pro-
portional to the energy. The band intensity can be
expressed as absorbance (A) or percentage trans-
mittance (%T) as plotted on y-axis (zero trans-
mittance corresponds to 100% absorption of light
at that wave number) (Fig. 7.1). Absorbance
(A) is logarithms, to the base 10, of the reciprocal
to the transmittance (T).
A = log10 (1 / T ) (7.1)
The absorbance or transmittance with cor-
responding wave numbers as a peak in spectra
gives information about the molecule to the
analyst, by matching it with spectrum of known
compound, peak-by-peak correlation. The spec-
trum represents a fingerprint of a sample, unique
characteristic, as no two different molecular
structures can produce the same spectra. This
makes infrared spectroscopy useful for several
types of analysis as each different material
is a unique combination of atoms. Therefore,
infrared spectroscopy results are used in molecu-
lar identification (qualitative analysis) of every
different kind of material, and the size of
peaks in the spectrum is a direct indication of
the amount of material present (i.e., purity) in
the sample.
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_7, © Springer India 2012
7
The original infrared instruments were of the
dispersive type (i.e., the separation of individual
frequencies of energy emitted from the infrared
source, by a prism or grating). Grating is a more
modern dispersive element, used to separate the
frequencies of infrared energy. The detector
measures the amount of energy at each frequency
which has passed through the sample. This results
in a spectrum which is a plot of intensity versus
frequency. The slow scanning process is a main
difficulty in this technique. In order to overcome
this limitation, the Fourier transform infrared
(FTIR) spectrometry is developed on the disper-
sive instruments.
To measure all infrared frequencies simultane-
ously, rather than individually, a very simple
optical device known as interferometer is added
with the instrument. The interferometer produces
a unique type of signal which has all of the
infrared frequencies encoded into it. The signal,
known as interferogram, is measured very quickly,
usually on the order of 1 s. The interferogram is
decoded to individual frequencies, via a well-
known mathematical technique called the Fourier
transformation, to have a frequency spectrum
(a plot of the intensity at each individual wave
number/frequency). This transformation is per-
formed by the computer with modern algorithms
software as the spectrum. All the spectral features
in the spectrum are only strictly due to the
sample. A single background measurement is
sufficient for many sample measurements because
the background spectrum is characteristic of the
instrument itself. Thus, the time span per sample
121
122
Fig. 7.1 IR spectrum plotted using transmittance (left) and absorbance (right)
Source Interferometer
Interferogram Computer
Fig. 7.2 A schematic presentation of IR spectroscopy
is reduced to a few seconds rather than several
minutes, as in the case of IR. The normal instru-
mental process (Fig. 7.2) may be described as a
black body source of radiations which passes
through an aperture, into the interferometer
(where spectral encoding results into the inter-
ferogram), to the surface of the sample, finally to
the detector, specially designed to measure the
special interferogram signals. The measured
signals are digitized and sent to the computer
where the Fourier transformation takes place, as
infrared spectrum.
The uniqueness of IR spectroscopy is that
any sample virtually at any state may be studied
[1, 2]. Infrared region starts immediately after
the visible region at 700 nm. The classical infra-
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints
Sample Detector
IR Spectrum
red region extends from 2,500 to 50,000 nm.
This spectral region encompasses three subdivi-
sions: the far-infrared (FIR: 400–33 cm−1 or
30–300 mm), mid-infrared (MIR: 4,000–400 cm−1
or 3–30 mm), and near-infrared (NIR: 12,820–
4,000 cm−1 or 0.78–3 mm), named in relation to
the visible region. Infrared analysts often use
wave numbers to describe the infrared spectral
region. The energies of infrared radiations range
from 48 kJ/mol at 2,500 nm to 2.4 kJ/mol at
50,000 nm. These low energies are not sufficient
to cause electron transitions, but they are sufficient
to cause vibrational changes within molecules,
so IR spectroscopy is also known as vibrational
spectroscopy. As spectrum generates through
the incident radiation that is absorbed at a particular
Interpretation of IR Spectra
frequency (i.e., wave number), so it is based on
the absorption of electromagnetic radiation by a
molecular system. In other words, IR spectra
provide images of vibrations of the atoms of a
compound. FTIR spectroscopy has developed
quickly due to its low noise, rapid speed, high
repeatability, easy operation, low expense, and
so on. It is also associated with other sciences
just like mathematics or computers, or with
other techniques such as two-dimensional cor-
relation analysis (2D-IR); FTIR has become
increasingly useful in the field of evaluating
herbal qualities. The vast majority of molecules
exhibit infrared peaks in the mid-infrared region
(4,000–400 cm−1). The position and intensity of
a vibrational peaks are characteristics of the
underlying molecular motion and consequently
of the atoms participating in the chemical bond,
their conformation, and their immediate environ-
ment. Thus, a certain submolecular group pro-
duces peaks in a characteristic spectral region.
These characteristic peaks form the empirical
basis for the interpretation of vibrational spectra.
Moreover, characteristic absorption peaks are
used for a specific compound detection. FTIR
spectrometers have almost entirely replaced dis-
persive instruments because of their better per-
formance in nearly all respects. The application
of this technique has improved the acquisition of
IR spectra dramatically. The major advantage
of IR over other spectroscopic techniques is
that practically all compounds show absorption
and can thus be analyzed both qualitatively
and quantitatively. FTIR spectroscopy is nonde-
structive and allows in situ and remote measure-
ments of almost any sample, irrespective of the
physical state and without elaborate sample
preparation [3, 4], applied to identify the func-
tional groups of chemical constituents but has
been widely used and applied in recent years
for the identification, quality control, and manu-
facturing process supervision of pharmaceutical
drugs.
Two units are used in vibrational spectroscopy:
cm−1 (wave numbers) or nm. The choice of one of
the units depends either on the type of spectrom-
eter [dispersive vs. Fourier transform (FT)] or to
avoid too large numbers in the NIR range where
123
nm is more often used. The relationship between
the two units is as follows [5]:
1
[cm −1 ] = (7.2)
[nm] × 10 −7
The basic principle of IR spectroscopy is the
measurement of the amount of IR radiations
absorbed by a sample has a high potential for the
elucidation of molecular structures [6]. The
regions of an IR spectrum where bond-stretching
vibrations are seen depend primarily on whether
the bonds are single, double, or triple or bonds to
hydrogen, etc., as [7]:
Bond Absorption region, cm−1
C–C, C–O, C–N 800–1,300
C=C, C=O, C=N, N=O 1,500–1,900
C=C, C≡N 2,000–2,300
C–H, N–H, O–H 2,700–3,800
Interpretation of IR Spectra
IR spectroscopy is an extremely effective method
for determining the presence or absence of a wide
variety of functional groups in a molecule. One of
the most preferred methods to decipher IR spec-
trum is to start at the high wave number end of the
spectrum (typically 4,000 cm−1) and look for the
presence and absence of characteristic absorptions
(Table 7.1) as move toward lower wave numbers.
The intensity of an absorption peak in the IR
spectrum is related to the change in dipole that
occurs during the vibration. Consequently,
vibrations that produce a large change in dipole
(e.g., C=O stretch) result in a more intense absorp-
tion than those that result in a relatively modest
change in dipole (e.g., C=C). Vibrations that do
not result in a change in dipole moment (e.g., a
symmetrical alkyne C≡C bond stretch) have little
or no absorption. The application of IR spectros-
copy in herbal analysis is still very limited com-
pared to its applications in other areas (food and
beverage industry, microbiology, pharmaceutical,
etc.), but infrared spectroscopy is certainly one of
the most important analytical techniques available
to scientists in herbal sciences [8]. The utility of
FTIR fingerprints may be highlighted as follows.
124 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Table 7.1 Important regions of the IR spectrum

Wave number (cm−1) Functional group Characteristic features of the peak
3,600–2,700 cm−1 (X–H stretch region)
3,600–3,300 Alcohol O–H The alcohol OH stretch is usually a broad and strong absorption
Amine or Amide N–H near 3,400. The NH stretch is typically not as broad or strong as
Alkyne C–H the OH, and in the case of an NH2, it may appear as two peaks.
The terminal alkyne C–H may be confirmed by a weak C≡C bond
stretch near 2,150 cm−1
3,300–2,500 Acid O–H This is normally a very broad signal centered near 3,000 cm−1
3,200–3,000 Aromatic (sp2) =C–H The aromatic CH character usually appears as a number of weak
Alkene (sp2) =C–H absorptions, while the alkene C–H is one or a couple stronger
absorptions
3,000–2,800 Alkyl (sp3) C–H Almost all organic compounds have alkyl CH, so this is not
usually too informative, but the intensity of these peaks relative to
other peaks gives indication as to the size of the alkyl group
2,850 and 2,750 Aldehyde C–H Two medium-intensity peaks on the right-hand shoulder of the
alkyl C–H. The response for the presence of carbonyl C=O peak
in spectrum to confirm
2,300–2,100 cm−1 (C≡X stretched region)
2,260–2,210 Nitrile C≡N A sharp, medium intensity peak. Carbon dioxide in the atmo-
sphere may also result in an absorption in this area if not
subtracted/pursed-out
2,260–2,100 Alkyne C≡C The peak intensity varies from medium to nothing; since the
intensity is related to the change in dipole moment, symmetrical
alkynes will show little or no absorption here
1,850–1,500 cm−1 (C=X stretch region)
1,850–1,750 Anhydride C=O Anhydrides have two absorptions, one near 1,830–1,800 and one
3–4 member ring C=O near 1,775–1,740. The absorption frequency increases as the ring
size decreases, e.g., cyclohexanone = 1,715, cyclopen-
tanone = 1,745, cyclobutanone = 1,780, cyclopropanone = 1,850
1,750–1,700 Aldehyde C=O It is usually the most intense absorption in the entire spectrum
Ketone C=O
Ester C=O
Acid C=O
1,700–1,640 Amide C=O Due to resonance, amides, and conjugated carbonyl, there comes
Conjugated C=O slightly lower response than normal C=O. In general, conjugation
lowers the absorption by 20–50 cm−1
1,680–1,620 Alkene C=C Absorption is not as intense as for C=O. It is variable and may be
fairly small in symmetrical or nearly symmetrical cases. For
confirming alkene response of C–H peaks above 3,000 cm−1
1,600–1,400 Aromatic C=C Multiple sharp, medium peaks appear. The pattern of peaks varies
depending upon the substitution pattern. Usually there is one peak
around 1,600 cm−1 and several others at lower wave numbers
1,500–400 cm−1(fingerprint region)
1,300–1,000 C–O A strong peak
1,500–400 Various functional Interpretation of peaks in the fingerprint region is complicated
groups due to the large number of different vibrations that occur here.
These include single bond stretches and a wide variety of bending
vibrations. This region gets its name since nearly all molecules
(even very similar ones) have a unique pattern of absorptions in
this region
Authentication of Herbal and Herbal Drugs
Authentication of Herbal
and Herbal Drugs
During the previous two decades, there have been
much more emphasis on the use of FTIR for
herbal authentication, as data from literature
search indicate that most of the papers are related
to qualitative assays of an active compound, but
there are also many papers dedicated to quantita-
tive methods. Authors have priority to proposing
new methods for qualitative and quantitative
determinations, but a few are also concern with
establishing new methods for analytical estima-
tion of geographic origin. Examples may be cited
as follows:
Radix Aconiti kusnezoffii has been analyzed
from various processed products and from their
ether extracts by using FTIR spectroscopy and
2D-IR [9]. There are distinctive differences in the
absorption peaks in the range of 1,800–1,500 cm−1
in the second derivative spectra of different pro-
cessed products. The authors used the second-
derivative spectra because of their better resolution.
With the use of high resolution, high speed, and
convenience FTIR, quickly and precisely distin-
guish various processed products such as Radix
A. kusnezoffii can be applied to predict the ten-
dency of transformation of the complicated
chemical mixture systems under heat perturbation.
The root of Angelica sinensis (olive) is well
known in traditional Chinese medicine for common
use. Modern pharmacological research indicates
that it could be used to treat anemia, apoplexy,
hypertension, coronary heart disease, thromboangii-
tis obliterans, superficial thrombosed phlebitis, etc.
FTIR associated with second-derivative infrared
spectroscopy and 2D-IR has been used to study the
main constituents in traditional Chinese formula-
tion made from Angelica and its different extracts
(extracted by petroleum ether, ethanol, and water in
turn). This method to use macroscopic fingerprint
characters of FTIR and 2D-IR spectrum to identify
the main chemical constituents of plant in their
different extracts and compare the components
for differences among the similar samples is
highly rapid, effective, visual, and accurate for
pharmaceutical research [9].
125
The quality of buchu oil obtained from two
South African species, Agathosma betulina and
Agathosma crenulata (family Rutaceae, both),
has been studied using FTIR spectroscopy.
Samples of A. betulina and A. crenulata are col-
lected from different natural localities and culti-
vation sites in South Africa. The essential oil was
extracted by hydro-distillation and scanned using
three spectroscopy techniques, such as near-
infrared (NIR), mid-infrared (MIR), and Raman.
A comparison of the three spectroscopy tech-
niques indicates that MIR together with PLS
(partial least squares) algorithms produces the
best model for the quantification of six of the
seven major oil constituents [9].
FTIR has been used to simultaneously analyze
the main chemical constituents in different sol-
vent extracts of several kinds of Chrysanthemum
samples of different regions [9]. The findings
indicate that different Chrysanthemum samples
have dissimilar fingerprint characters in FTIR
spectra. These spectral fingerprints provide struc-
tural information for complicated test samples.
Liu et al. have reported that they are able to iden-
tify the main components of different extracts
and distinguish the origins of the Chrysanthemum
samples from different regions easily [10].
Multistep infrared spectroscopic methods, including
conventional FTIR, second-derivative spectros-
copy, and 2D-IR correlation spectroscopy, have
been proved to be effective methods to examine
complicated mixture system such as the inves-
tigation on the effect of flowering on the phar-
maceutical components of Cistanche tubulosa.
The results provide a scientific explanation for
the traditional experience that flowering con-
sumes the pharmaceutical components in stem
and the seeds absorb some nutrients of stem after
flowering. A new method using single-reflection
Fourier transform infrared spectroscopy was
proposed for direct and fast determination of
Corydalis yanhusuo W. T. Wang and of tradi-
tional Chinese herbal medicines and its confus-
able varieties. FTIR spectroscopy and 2D-IR
have been employed to propose a new method for
analysis of Cordyceps. Their second-derivative
spectra can amplify the differences and confirm
the potential characteristic IR absorption bands.
126
The different fingerprints display different chem-
ical constitutes. Chestnut (Castanea sativa) shell
and eucalyptus (Eucalyptus globulus) bark, waste
products of the food and wood industries, respec-
tively, have been analyzed as potential sources of
antioxidant compounds. The antioxidant activity
and the total phenols content of the extracts had
positive linear correlations. FTIR spectroscopy
confirmed the higher content of phenolic com-
pounds in chestnut shell extracts compared to
eucalyptus bark extracts [9].
The determination of sweet cherry anthocya-
nins in crude material of three varieties using dif-
fuse reflectance infrared Fourier transform
spectroscopy (DRIFTS) and the curve-fitting
deconvolution method has been described. A lin-
ear relationship between the sweet anthocyanins
content and the peak area at 1,640–1,630 cm−1
was established with a high correlation coefficient
(0.990). The deconvolution analysis using the
curve-fitting method allowed the elimination of
spectral interferences from other cell wall com-
ponents. The proposed method is simple, rapid,
and nondestructive and could be applicable to
any cherry varieties [9].
A systematic study for the effect of the com-
mercially available, ultra-diluted drug from
Digitalis purpurea (extract of foxglove leaves)
with aqueous ethanol has been conducted to mon-
itor changes in its chemical structure/functional
group arrangements using vibrational (FTIR and
Raman) spectroscopy. These changes suggest a
significant effect of ultra-dilution (micro-vol-
umes) in the spectrum profile of Digitalis bands in
the fingerprint region. The technique is useful in
the detection and identification of compounds/
chemical groups present at levels lower than
micro-volume in drugs used in alternative/com-
plementary medicine [9].
The composition of fenugreek seeds in the
form of powder, ash, and oil has been investigated
through FTIR and Raman spectra measurements.
The results indicate that fenugreek seeds (in pow-
der form) are rich in proteins, but lipids and starch
are in small amounts. The fenugreek oil FTIR
absorbance ratios A3009/A2924, A3009/A2854, and
A3009/A1740 have been studied for iodine values, and
data reveals that the iodine value of fenugreek oil
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints
is higher than that of other oils. On the other
hand, the ash of fenugreek is very rich in phos-
phate compounds. The spectra showed some
absorption bands that are due to phosphate
compounds. It could be concluded that the inor-
ganic part of fenugreek consists mainly of
phosphate compounds [9].
Ginseng, an expensive herb of high therapeu-
tic value, is mostly adulterated with other cheaper
products, so quality assurance is required as its
commercial products such as capsules, powder,
soft gels, and teas are available globally with high
demand. Yap et al. have discussed a rapid means
of distinguishing American and Asian ginsengs
from two morphological fakes, namely, sawdust
and Platycodon grandiflorum, via pattern differ-
ences and principal component analysis of their
infrared spectra [8]. The results showed that
ginseng can be distinguished from both sawdust
and Platycodon grandiflorum; hence, there is a
potential for the use of infrared spectroscopy as a
novel analytical technique in the authentication
of ginseng. A simple and rapid protocol based on
infrared wavelengths and principal component
analysis for identification and categorization of
ginseng has been elaborated. The advantage of
this protocol is that it is able to provide rapid
identification of natural products because it
avoids tedious extraction or purification proce-
dures. The results showed that this protocol was
able to discriminate not only raw ginseng roots
but also different types of ginseng in three com-
mercial ginseng products [9].
The misidentification of ginseng using tradi-
tional methods of authentication via morphology
has been overcome by infrared spectroscopy.
Molecular spectroscopy (including near-infrared
diffuse reflection spectroscopy, Raman spectros-
copy, and infrared spectroscopy) with OPUS/Ident
software (Thermo Scientific-Nicolet, Waltham,
MA) has been applied to clustering ginseng
according to species and processing methods.
The results demonstrate that molecular spectro-
scopic analysis provides a rapid, nondestructive,
and reliable method for identification of this
Chinese traditional herbal drug. The traditional
methods, which are laborious and time consuming,
have been replaced by molecular spectroscopic
Authentication of Herbal and Herbal Drugs
Fig. 7.3 FTIR spectra of (a) Asian ginseng, (b) American ginseng, (c) notoginseng [9]
analysis, as more effective method. The herbal
materials of Asian ginseng (the root of Panax gin-
seng), American ginseng (the root of Panax
quinquefolius), and notoginseng (the root of Panax
notoginseng) have been differentiated by conven-
tional Fourier transform infrared spectroscopy
(1D-FTIR) (Fig. 7.3) and 2D correlation FTIR
applying a thermal perturbation. These species
of herbs were further identified based on the
positions and intensities of relatively strong
auto-peaks and positive or negative cross-peaks
in their 2D-FTIR spectra. The findings provide
a rapid and new operational procedure for the
differentiation of these notable herbs.
Huanglongbing (HLB), also known as citrus
greening, has greatly affected citrus orchards in
Florida and has been studied for the detection
using FTIR. Leaf samples of healthy, nutrient-
deficient, and HLB-infected trees were processed
and analyzed using a rugged, portable mid-
infrared spectrometer. The spectral peak in the
region of 952–1,112 cm−1 was found to be dis-
tinctly different between the healthy and HLB-
infected leaf samples. This carbohydrate peak
could be attributed to the starch accumulation in
the HLB-infected citrus leaves. Thus, this study
demonstrated the applicability of mid-infrared
spectroscopy for HLB detection in citrus [9].
127
FTIR spectroscopy has been emphasized as a
widespread technique in the quick assess of food
components. In one such work, procyanidins
have been extracted with methanol and acetone/
water from the seeds of white and red grape vari-
eties. FTIR spectroscopy allowed the creation of
a partial least squares (PLS1) regression model
with eight latent variables (LVs) for the estima-
tion of the degree of polymerization (DPn), giv-
ing a root mean square error of cross-validation
(RMSECV) of 11.7%. The application of orthog-
onal projection to latent structures (O-PLS1)
clarifies the interpretation of the regression model
vectors. Moreover, the O-PLS procedure removed
88% of non-correlated variations with the DPn
[9]. Kava has been used as a folk medicine and
well-known traditional beverage. A study focused
on quantitative analyses of kava lactones in kava
samples using FTIR data results is similar to
values with GC (R2 = 0.75–0.98), indicating that
the FTIR method is suitable for determination
of the contents of the six kava lactones in kava
samples and thereby the chemotypes [9].
Studies for relative abundance of active ingre-
dients in many deciduous perennial fruit crops
reveal requirement of winter chilling for adequate
bud break and flowering. Recent research has shown
that changes in sugar and amino acid profiles are
128
3354 1652 1515
1036
2929 1319
782
A
A
B
C 1692
D 1516 1468
wave number cm−1
Fig. 7.4 Conventional IR spectra (panel I) and second
derivative IR spectra (panel II) of four patchouli samples
of different geographical origins at room temperature [9].
associated with the release of buds from dormancy.
Judd et al. applied FTIR spectrometry to provide
an alternative mechanism for tracking metabolic
changes in the meristems of kiwi fruit buds during
winter dormancy [11]; results suggested that the
application of multivariate analysis to FTIR spec-
tra has the potential to be a reliable and fast
method for detecting structural and compositional
changes in fruit crops. These wave numbers
appear to be associated with carbohydrate, pectin,
and cellulose levels in the meristems. It is
expected that this FTIR signature can be used to
advance our understanding of the influence of the
various environmental and physiological factors
on the breaking of bud dormancy and shoot out-
growth, including the optimum timing and con-
centrations of applications of bud break regulators,
such as hydrogen cyanamide. FTIR for herbal
analysis is used to decipher the geographical ori-
gin of the raw herb. In addition to the applications
already mentioned related to Agathosma betulina
and Agathosma crenulata and ginseng analysis,
there are other reports where FTIR was used
as discrimination technique for Epimedium
Korean Nakai from Jilin province, China, and
patchoulis (Pogostemon cablin Blanco) from
different Chinese provinces (Nanxiang, Paixiang,
Zhanxiang, and Zhaoxiang) (Fig. 7.4).
Studies on the identification of four species
of Mongolian herbal medicine by ultraviolet
(UV)/fluorescence/IR spectroscopy have been
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints
A
B
C
A
D
1500 1455
1737 1659 1606
wave number cm−1
(A) Nanxiang, (B) Paixiang, (C) Zhanxiang, and (D)
Zhaoxiang
carried out. The methanolic extracts of Mongolian
herbal drug Rubus sachalinensis Leveille, its
substitute materials Sambucus williamsii Hance
and Uncaria rhynchophylla (Miq.) Jacks and its
adulterant Cinnamomum cassia Presl showed
clear differences in the IR spectra. IR spectros-
copy provided more information through the
fingerprints region of herbal medicines, render-
ing the technique direct and simple. Lakshimi
Prasuna et al. studied the FTIR spectra of Basella
rubra and Moringa oleifera leaves and showed
evidence for the protein matrix bands and those
corresponding to carboxylic C–O bonds (Fig. 7.5)
[12]. Phyllagathis praetermissa collected from
Pasoh Forest Reserve (Negeri Sembilan), Ampang
Forest Reserve (Selangor), and Bukit Lagong
(Selangor) in Peninsular Malaysia were differenti-
ated based on their chemical constituents by using
multistep infrared macro-fingerprinting.
These data provided useful information for the
best choice of soil, geographical location, and
transplantation of herb culture. According to
these spectral fingerprint features, we cannot only
identify the main components of different herbal
plants and extracts but can also distinguish the
origins of samples from different regions easily,
which is troublesome using existing analytical
methods. Compared with traditional methods,
which are laborious and time consuming, the
molecular spectroscopic analysis is more effec-
tive and efficient.
Authentication of Herbal and Herbal Drugs
Fig. 7.5 FTIR spectrum of Basella rubra leaf (at room temperature) [9, 12]
Interpretation of Herbal Drug FTIR Spectra
by Chemometrics: The data interpretation with
the help of personal computers, generated by
modern analytical instruments, for the acquisi-
tion and processing needs the education of chem-
istry, mathematics, and statistics with numerical
software. This computer-based chemical disci-
pline that uses mathematical and statistical meth-
ods, (a) to design or select optimal measurement
procedures and experiments and (b) to provide
maximum chemical information by analyzing
chemical data, is known as chemometrics.
The FTIR spectral fingerprint provides the
information about the source of pure ingredient
either of natural or synthetic in origin. Due to the
inherent complexity of the IR spectrum, the
actual interpretation may be difficult, and opera-
tion requires much experience. Indeed, slight dif-
ferences in the spectra within the same plant
species may not be obvious and generally not vis-
ible to the naked eye. The quality of plant sam-
ples from different localities and growing
conditions based on their geographical origin
may vary. Thus, the identification of origin of
crude herbs based on geographical origins is cru-
cial in order to ensure authenticity, quality, safety,
and efficacy of the raw material before it is con-
verted to the final product. Herbal product manu-
facturers are always looking for such faster and
cost-effective verification method for traceability,
129
as wet chemistry analysis is too laborious and
time consuming.
A case study was conducted by incorporating
appropriate chemometric methods [principal
component analysis (PCA) and soft independent
modeling of class analogy (SIMCA)] as tools for
extracting relevant chemical information from
the infrared spectra. The team of scientists under-
took this effort to introduce and to develop a rapid
quality verification method with the integration
of statistical and mathematical modeling for
extracting relevant information base on the infra-
red spectroscopy data and extending the used of
FTIR transmission spectroscopy, selecting
Orthosiphon stamineus Benth (Java tea), used for
treating infection of the urinary tract, kidney, and
bladder stones disease based on its geographical
origin and varieties. The dried leaves of O. sta-
mineus from ten different geographical origins of
two varieties (white and purple flowers) were col-
lected from Malaysia and Indonesia and coded
(Table 7.2) [13].
The dried leave samples were milled until fine
powder and were filtrated with sieves 0.071 and
0.500 mm mesh size. KBr of spectroscopy grade
was also filtrated with sieves 0.071 mm mesh
size. 2-mg samples were mixed uniformly with
100 mg KBr (2% w/w) and homogenized by
using stir vortex CENCO 34 (Breda, Netherlands).
The FTIR spectra were recorded in the mid-IR
130 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Table 7.2 List of O. stamineus samples as per its geographical origin and varieties [13]
S. no. Code Location State Group ID
1 BLKBPM Bumbung Lima Penang A
2 SRKBPM Kepala Batas Penang B
3 SZBKAM Bota Kanan Perak C
4 MABTRM Bohor Temak Perlis D
5 ZARWBM Rawang Selangor E
6 STJGCM Jengka Pahang F
7 NNPPDM Pasir Puteh Kelantan G
8 MSSMSM Semonggok Sarawak H
9 USKGSM Kuching Sarawak I
10 NHPJI Pulau Jawa Jakarta J
11 BPPM_P Balik Pulau Penang K
12 NNPPDM_P Pasir Puteh Kelantan L
13 SABAH_P -b Sabah M
14 USKGSM_P Kuching Sarawak N
a
Example BLKBPM: BL: distributor; KB: location; P: state; M: country (M: Malaysia;
I: Indonesia); [13] P: purple flowers (white flowers not indicated)
b
Information not available

region 4,000–400 cm−1 at resolution 4 cm−1 with
16 scans using Thermo Nicolet FTIR Nexus
spectrometer coupled with DTGS (deuterated
triglycine sulfate) detector. The interferometer
and the detector chamber were purged with dry
nitrogen to remove spectral interference due to
atmospheric carbon dioxide and water vapor. Air
background spectrum was recorded before each
sample, and all experiment were performed in six
triplicates (six pellets KBr with three scan each),
and baseline corrected for each spectrum; absor-
bance was normalized so that peak absorbance of
the most intense band is set to unity. The spectra
were transferred via a JCAMP.DX format into
the statistical software program. As per Beer’s
law, the absorption of light at a given wavelength
is due entirely to the absorptivity of the constitu-
ents in the sample. Therefore, any variations in
the spectral due to spectrometer and sampling
error should be eliminated prior to data analysis.
So in this study, two chemometric preprocessing
steps, namely, baseline correction and normaliza-
tion, were used. Baseline effects introduced by
the spectrometer (e.g., detector drift, changing
environmental condition such as temperature,
spectrometer purge, and sampling accessories)
were removed by auto-correcting the spectrum
baseline, and normalization of the absorbance
spectra to the most intense band (hydroxyl group,
3,406 cm−1) deletes the differences between spec-
tra due to different amounts of sample or path
length variation [13].
Chemometric data analysis was done using
the PCA algorithms in the study for reducing the
high-dimensional spectroscopic data by con-
structing a linear combination of the original
variable into a few orthogonal principle compo-
nents which contain most of the variability of the
data set. This projection method allows (a) visu-
alization of the natural clustering in the data,
(b) primary evaluation of the between-class
similarity, and finally (c) finding the reasons behind
the observed pattern by making correlation
with the chemical or physicochemical properties
of the studied samples. SIMCA (soft independent
modeling of class analogy), a popular classification,
method was used to assign unknown samples in
order to test the robustness of this study. The five
steps followed were as follows: (1) constructing
separate PCA models for each class; (2) deter-
mining the optimal number of PCs by validation;
(3) fitting the unknown samples to each predefine
model, provided that the class are distinct enough;
(4) deciding whether the samples belong to the
corresponding class by referring to the object-to-
model distance and leverage (distance of the
Authentication of Herbal and Herbal Drugs
NHPJI
SZBKAM
MSSMSM
ZARWBM
MABTRM
A
SRKBPM
NNPPDM
STJGCM
USKGSM
BLKBPM
4000.0 3000 2000 1500 1000 400.0
wave number cm−1
Fig. 7.6 The characteristic FTIR spectra of O. stamineus samples [13]. Where white flowers (left), purple flowers
(right) from different origin
sample to the model center); and finally (5) vali-
dating the classification results with statistical
test called significance test [13].
Interpretation of Data
As per Malaysian Herbal Monograph, the main
chemical constituents of O. stamineus are
flavonoids, caffeic acid derivatives, diterpene ester,
triterpene saponins, and other minor compounds.
Each of these compounds plays an indispensable
role in the complicated system of mixture con-
tributing to the efficacy and potency due to the
synergistic effect contributed from a number of
constituents present in the herb. In FTIR spectra
(4,000–400 cm−1) of ten different sample’s origin
of (a) white flowers and (b) purple flowers variet-
ies (Fig. 7.6) [13], the sharp absorption peak at
1,600–1,760 cm−1 is assigned to C=O stretching
vibration in carbonyl compounds which may be
characterized by the presence of high content of
terpenoids and flavonoids in the complex mixture
of O. stamineus. The presence of a narrow and
sharp peak at ~2,925 and ~2,853 cm−1 was assigned
to C–H and C–H (methoxy compounds) stretch-
ing vibration, respectively. The presence of diter-
penes was further proven with the absorption band
131
USKGSM_P
SABAH-P
A
NHPJI
NNPPDM-P
USKGSM
4000 3000 2000 1500 1000 400
wave number cm−1
of hydroxyl (3,500–3,480 cm−1), ester carbonyl
(1,270–1,150 cm−1), and phenyl (1,600,
1,420 cm−1) [13].
By visual recognition, there is no significant
difference in the characteristic absorption bands,
but the intensity of certain wavelength does differ
from each other especially at the fingerprint
region (1,800–800 cm−1). This interprets the sim-
ilarity of certain main chemical component
observed among different O. stamineus sample
origin. The presentation of spectrum in 3-D
(Fig. 7.7) [13] further enhances the visualization
of the variability in the intensity absorption bands
between the respective origins. Differences
between spectra are generally not visible to the
naked eyes; it is more practical to incorporate sta-
tistical method for the aid of interpreting the
obtained measurement results from spectroscopic
analysis. Since the discrimination of different
geographical origin of herb based on the slight
differences among particular absorption bands is
too subjective, the results may vary between ana-
lysts as reported [13].
The PCA was carried out using 520 points
(normalized absorbance) between the ranges of
the selected spectral region 1,800–800 cm−1.
A total of 18 data sets from six triplicate measure-
ments of each sample, 12 data sets were randomly
132
Fig. 7.7 3-D absorbance matrix spectra (1,800–800 cm−1) of different sample’s origin [13]
selected to represent the respective sample’s
origin and varieties (as shown in Table 7.1).
Natural grouping of O. stamineus from ten differ-
ent geographical origins, the first three principal
components (PCs) represent only 61 % of the
total variance (PC1 = 29 %, PC2 = 22 %, and
PC3 = 10 %). This indicates that these three com-
ponents are not sufficient to provide effective
clustering of the sample’s origin with clear sepa-
ration between the groups.
The rule of thumb to make a good classification
is to make sure that the variation within different
sample must be greater than the variance between
individual samples. Thus, spectral reproducibil-
ity is important for creating a robust classification
model. The systematic variation between repli-
cate spectra due to baseline effect is highlighted
and removed after derivatization. Generally, the
use of spectra derivatives with Savitzky–Golay
algorithm as a chemometric preprocessing tech-
niques is widely reported in most classification
based on FTIR spectroscopy. The potential rela-
tionship and pattern associate between different
O. stamineus samples source with regard to its
complex mixtures were further investigated by
computing a PCA based on the first-derivative
spectral data. The first three PCs represent almost
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints
90 % of the total variance in the data set. The first
PCs consist 48 % of the total variability followed
by the second PCs with 34 % variance and only
8 % variance carried by the third PCs. Overall,
each sample was able to form distinct cluster in
the two-dimensional plot. Examining the space
defined by the first and second PCs, (a) samples
from the same origin (state), for example, Kelantan
(group G and L) and Sarawak (group H, I and N),
tends to groups itself together in the first PCs.
Samples from Penang origin only observed this
relationship when it is projected onto the third
PCs, which contain only 8 % of the total data
variability. From this inherent structure, we can
make a sound assumption that the composition of
complex chemical constituent does vary accord-
ing to its origin. But there are no significant dif-
ferences in the complex mixture observed
between the different plant varieties as sample
with purple flower (rectangular shape) forms a
close cluster with the white flower samples (oval
shape). The first and second PCs which contain
almost 60 % of the data variances separate the
four respective sample’s origin into four well-
defined spaces.
The overall result from aforesaid studies pro-
vides evidence that O. stamineus samples from
Identification and Comparison of Biomolecules
different geographical origin and varieties have
varied complex chemical mixture. Sample ori-
gin seems to have more dominant effect to the
chemical constituent of the plant compared to
plant varieties. The good classification model
obtained from both PCA and SIMCA further
proved that classification of samples from vari-
ous sourcing and varieties is possible with the
incorporation of chemometric techniques and
FTIR spectroscopy. Therefore, the obtained
classification model may be of great use for
quality inspection of raw herbal material on a
continuous basis as new batches are produced.
Chemometrics analysis of spectra data is rapid
and simple since no chemical treatment of sam-
ples is required [13].
Identification and Comparison
of Biomolecules
During the decade of 1940, IR spectroscopy has
become an accepted tool for the characterization
of biomolecules, and with technological advance-
ment, the FTIR has been proven to be useful in
studying compositional changes in plant cell
walls. This property was utilized to determine
changes in cell wall, if any, due to biotic/abiotic
factors. In the beginning, use of IR spectroscopy
was restricted only for structural elucidation of
isolated compounds from the herbal matrices.
Drug discovery from the plants continued to pro-
vide new and important leads against various
pharmacological targets including cancer, HIV/
AIDS, Alzheimer’s, malaria, infections, and pain.
A number of events for new drug discovery and
interaction of a drug with biological target and
pharmacological effects have to be considered,
and these involve absorption, distribution, metab-
olism, and elimination. In order to assess the
importance of each of these factors on drug
action, both structural and physicochemical prop-
erties of the drug are taken into account. In bio-
logical systems, properties such as electrostatic
bonds, hydrogen bonds, van der Waals bonds,
as well as effects related to electron transfer
and hydrophobic effects, are of major importance.
Although the hydrogen bond is fairly weak
133
compared to other interactions, it is of paramount
importance in biological systems to investigate
the drug metabolism, its biotransformation
pathways, and toxicological, pharmacological,
and biomedical interest. FTIR spectroscopy has
been used for identification and comparison of
biomolecules in two different species of medicinal
plants, for example, Atylosia albicans and Tephrosia
tinctoria [7].
Plants Atylosia albicans and Tephrosia tincto-
ria were collected from the western Ghats region
of Hassan District, Karnataka, India. The leaf,
flower, fruit, and stem were carefully excised
from the plant, cleaned, shade dried, and placed
in polythene bags (to avoid contamination, and
reference sample was kept in the herbarium), and
powdered. The plant powders were kept in a lyo-
philizer to remove water. The samples were again
ground in an agate mortar and pestle in order to
obtain fine powder. Each powdered plant mate-
rial was mixed with completely dried potassium
bromide (99:1 ratio), and the mixture was sub-
jected to a pressure of 5 × 106 pa in an evacuated
die to produce a KBr pellet for use in an FTIR
spectrometer. FTIR spectra were recorded with
an FTIR 460 plus Jasco. The powdered samples
of both Atylosia albicans and Tephrosia tinctoria
were mixed with dried potassium bromide and
prepared as pellets and scanned at room tempera-
ture (25 ± 2 °C) at 4,000–400 cm−1 spectral range.
To improve the signal to noise ratio for each spec-
trum, 100 interferograms with a spectral resolu-
tion of ±4 cm−1 were averaged. Background
spectra, which were collected under identical
conditions, were subtracted from the sample
spectra. Each sample was scanned under the same
conditions with six different pellets. Special care
was taken to prepare the pellets at the same thick-
ness by taking the same amount of sample and
applying the same pressure. Therefore, in the
present study, it was possible to directly relate the
intensities of the absorption bands to the concen-
tration of the corresponding functional groups.
The results of functional group analysis
(Table 7.3) using FTIR revealed the existence of
various characteristic functional groups in leaves,
stem, flower, and fruit of A. albicans and T. tinctoria
(Fig. 7.8, 7.9, 7.10, 7.11 and 7.12) [7].
134 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Table 7.3 General band assignments of the FTIR spectra of biological tissue [7]
S. no. Peak Assignment
1 521 cm−1 Torsion and ring torsion of phenyl
2 600–900 cm−1 CH out of plain bending vibrations
3 892 cm−1 C–C, C–O, deoxyribose
4 940 cm−1 Carotenoid
5 1,000–14 cm−1 Protein amide I absorption
6 1,000–200 cm−1 C–OH bond in oligosaccharides such as mannose and galactose
7 1,000–350 cm−1 Region of phosphate vibration, carbohydrate residues attached
to collagen and amide III vibration (in collagen)
8 1,020–50 cm−1 Glycogen
9 1,030 cm−1 Collagen
10 1,105 cm−1 Carbohydrates
11 1,145 cm−1 Phosphate and oligosaccharides
12 1,180–300 cm−1 Amide III band region
13 1,206 cm−1 Amide III collagen
14 1,244/5 cm−1 PO−2 asymmetric (phosphate –I)
15 1,255 cm−1 Amide III
16 1,312–131 cm−1 Amide III band components of protein collagen
17 1,456 cm−1 CH3 bending vibration (lipid and protein)
18 1,482 cm−1 Benzene
19 1,504 cm−1 In plane CH bending vibration from the phenyl rings
20 2,800–3,000 cm−1 C–H lipid region
21 3,500–600 cm−1 OH bond
22 3,000–700 cm−1 O–H stretching (water)

Fig. 7.8 FTIR spectra of

T. tinctoria leaves [7]

The dominant bands at 1,655 and 1,546 cm−1
were attributed to protein amide І and ІІ bands.
The shoulder at about 1,750 cm−1 was attributed
to lipid C=O stretching vibration. The band at
1,465 cm−1 was assigned to the =CH2 bending
mode of the cell lipids. The band at 1,460 cm−1
represents asymmetric –CH3 bending modes
of end ethyl group proteins. The band at 1,402 cm−1
represents C=O symmetric stretching of COO–
and assigned to lipids. Band at 1,377 cm−1 rep-
resents C–H bending mode of =CH 2. The
very strong absorption band observed around
Identification and Comparison of Biomolecules 135

Fig. 7.9 FTIR spectra of

A. albicans leaves [7]

Fig. 7.10 FTIR spectra of

T. tinctoria flower [7]

Fig. 7.11 FTIR spectra of

A. albicans fruit [7]
136
Fig. 7.12 FTIR spectra of A. albicans stem [7]
3,373–3,422 cm−1 may be due to the presence of
bonded N–H/C–H/O–H stretching of amines and
amides. A very strong absorption at 3,400 cm−1
shows the presence of amino acids, and the very
strong absorption band appearing in the region
2,933–2,922 cm−1 is due to N–H stretching. The
lone C=O stretching vibration band correspond-
ing to saturated aliphatic ester 1,743 cm−1 is
present in all parts of the plants. The bands at
900–1,350 cm−1, 1,020 cm−1, 1,024 cm−1, and
1,050–100 cm−1 are attributed to phosphordiester
stretching bands region (for absorbance due to
collagen and glycogen), DNA, glycogen (C–O
stretch associated with glycogen, phosphate, and
oligosaccharides PO−2 stretching modes), P–O–C
antisymmetric stretching mode of phosphate
ester, and C–OH stretching of oligosaccharides,
respectively. A band at 1,051 cm−1 is attributed
to C–O–C stretching of DNA and RNA. The
more intense bands occurring at 3,419, 2,927,
2,853, 1,633,1,421, 1,260, 1,073, 816, and 635cm−1
corresponding to O–H/N–H, C–H, C–O, and
C–Cl/C–S stretching/bending vibrations, respec-
tively, indicate the presence of amino acids,
alkenes, nitrates, ethers, organic halogen com-
pounds, and carbohydrates in A. albicans and
T. tinctoria [7].
A symmetrical stretching of NO2− group
results into strong absorption in the region
1,660–1,625 cm−1. The observed absorption
band at 1,630 cm−1 indicates the presence of
amines (protein). This gives the evidence that
the plants A. albicans and T. tinctoria are rich
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints
in proteins. The weak absorption band observed
between 1,421 and 1,415 cm−1 in the plant parts
of A. albicans and T. tinctoria may be due to the
presence of bonded C–O/O–H bending. The
medium absorption band of 620 cm−1 indicates
the presence of sulfate. A strong absorption
band occurs at 597 and 580 cm−1 in the stem,
leaves, fruits, and flower of two species which
is possibly due to aliphatic C–Cl absorption and
brominated compounds. The brominate com-
pounds show an infrared band region at 600–
500 cm−1. The weak absorption band at 539 cm−1
indicates the presence of phosphates in the
leaves, fruits, flowers, and stem in the examined
plants. The very weak band occurring at
780 cm−1 in the flowers, leaves, stem, and fruits
of these plants can be attributed to out-of-plane
N–H wagging, primary and secondary amide
and nitrite group. Five major peaks 1,590,
1,348, 1,051, 3,385, 1,063, and 456 cm−1 were
observed in the FTIR spectra. This is a
significant observation made in A. albicans as it
is not reported in any legumes so far. A weak
absorption band at 940 cm−1 is attributed to car-
otenoid being present only in the fruit of A.
albicans. A weak absorption band at 965 cm−1
is attributed to C–O stretching of the phospho-
diester and the ribose and is present only in A.
albicans. A very weak peak at 892 cm−1 is
attributed to C–C and C–O deoxyribose and
seen only in A. albicans. A medium peak at
1,340 cm−1 is due to CH2 wagging collagen
present in A. albicans. A very strong peak at
Authenticity of Herbal and Herbal Drugs
1,581, 1,358, and 520 cm−1 is attributed to ring
C–C stretch of phenyl, stretching C–O, defor-
mation C–H, deformation N–H, and phenyl
group respectively, are present in fruits, leaves,
and stem of A. albicans and leaves and flowers
of T. tinctoria. A medium peak at 3,300 and
1,020–1,050 cm−1 is attributed to amide І bands
stemming from N–H stretching modes in pro-
teins, nucleic acids, and glycogen [7].
From above investigations, it was concluded
that A. albicans and T. tinctoria are rich in pheno-
lic compounds and also show the presence of
oligosaccharides, phosphates, proteins, carbohy-
drates, and carotenoid. This work also indicates
the presence of biomolecule concentration which
is different in different parts of the plants. This
work offers scope for further research in phy-
tochemical analysis and biological activity of
medicinal plants. Fourier transform infrared
spectroscopy is proved to be a reliable and sensi-
tive method for detection of biomolecular com-
position of cells. From this study, we examined
the potential of FTIR spectroscopy for easy and
rapid discrimination and identification of various
functional groups responsible for medicinal prop-
erties. Spectral area ranged between 4,000 and
400 cm−1 is important for an easy and reliable dis-
crimination between different plant species based
on biomolecules due to unique fingerprint for a
particular moiety.
Authenticity of Herbal and Herbal
Drugs
The true and false identification of herbal drugs
can be performed by the characteristic bend of
their IR fingerprints using the common and
variant peak ratio sequent analytical method to
evaluate the quality of the same medicinal plant
species. For this purpose, three methods are in
practice for collecting IR fingerprints for quality
control of herbal drugs. In method one, solvents
of different polarities are used to extract compo-
nents. After evaporating the solvent, the compo-
nents are blended with KBr powder and pressured
into a pellet, and then the IR fingerprints are gen-
erated. The IR spectrum of samples enables us to
137
distinguish different herbal drugs effectively.
In method two, powder of herbal drug is blended
with KBr powder and pressured into a pellet;
then, the IR spectra of the samples are collected,
while in method three, the reflectance IR spectra
are obtained directly from herbal materials.
Although methods two and three are convenient
methods, they provide lower resolution ability in
identification of herbal drugs as compared to
method one [14].
The IR fingerprint spectra are an effective
and convenient for the primary determination
of true and false identification of herbals and
herbal drugs. Under the same experimental
conditions, the IR fingerprints of different
herbal drugs have differences in their specific
bends. Herbal drugs can be identified clearly by
specific bends and their relative intensities.
Since early 1987, the fingerprint spectra are in
use for the identification of herbal drugs, by the
extraction of components from the herb with
certain solvents of different polarities on the
order of petroleum ether (or cyclohexane),
chloroform, ethanol, and water, and their UV
fingerprint spectra are also measured [15].
Chinese herbal drugs, which are complex mix-
tures of many herbals, can be easily identified
for purity using their IR spectra. The specific
bends and the relative intensities of the IR spec-
tra are the main criteria for authenticity.
In a study by Zou et al., the IR spectra of nine
kinds of roots of Glycyrrhiza uralensis Fisch and
Glycyrrhiza inflata Batal, including planted and
wild samples from different regions, looked very
similar (Fig. 7.13) [16]. The IR spectra of their
water extract showed significant differences.
The common peak ratios of the different species
were minimum, while their variant peak ratios were
maximum. The common peak ratio of the same
species of the wild and the planted from near
producing regions was of slight difference, but
their variant peak ratios were obviously larger
than that of planted samples. The common and
variant peak ratios of the same species samples
from the same region had nonsignificant difference.
On further investigation, they find IR spectra
having great differences once extracted with
chloroform and with water [17].
138
Fig. 7.13 The IR spectra of roots of planted G. uralensis Fisch, wild G. uralensis Fisch, and wild G. inflata Batal [16]
To Distinguish Herb from Its
Morphological Fakes
The quality control of herbals and herbal prod-
ucts is important for consumer’s safety and
efficacy, as traditional herbal drugs are not regu-
lated, as considered dietary supplements and not
drugs. At present, these commercial products
are available in various formulations such as
capsules, powder, softgels, and tea. The tradi-
tional means of authentication via smell, taste,
or physical appearance are hardly reliable for
these high-value products, and adulteration with
other cheaper products may occur. Example
may be cited from ginseng, the famous Chinese
herb, as it exhibits an adaptogenic effect and
improves physical and mental performance.
Different parts and species of ginseng are
believed to have different medicinal properties.
There are many species of ginseng, of which the
two most common and widely used are Panax
ginseng C.A. Meyer (Asian ginseng) and Panax
quinquefolius L. (American ginseng). Panax
ginseng was previously known as “Panax schin-
seng Nees” and is cultivated in China, Germany,
Japan, Korea, and Russia, while Panax quinque-
folius, previously known as Aralia canadensis,
is found in the United States of America and
Canada. Besides these two major varieties, there
are 11 other ginseng species: Panax japonicus
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints
C.A. Meyer (Japanese ginseng), Panax major
Ting, Panax notoginseng (Burk.) F.H. Chen
(Sanqi or Tianqi ginseng), Panax omeiensis
J. Wen, Panax pseudoginseng Wallich (Himalayan
ginseng), Panax sinensis J. Wen, Panax stipule-
anatus H.T. Tsai and K.M. Feng, Panax trifolius
L. (dwarf ginseng), Panax vietnamensis Ha et
Grushv (Vietnamese ginseng), Panax wangianus
Sun, and Panax zingiberensis C.Y. Wu and K.M.
Feng [8].
Asian ginseng is not a generic term and is used
to refer to ginsengs which originate from Asian
countries. Ginseng with other species such as
Panax japonicus and Panax notoginseng, Panax
ginseng, is also classified under Asian ginseng.
The chemical composition of ginseng includes
the saponins, naphtha class, carbohydrates, and
starch. Its pharmacological activity is due to the
mixture of its constituents and not the presence of
a single compound. The triterpene saponins,
called ginsenosides, are the major active ingredi-
ents in ginseng, and there are more than 30 differ-
ent ginsenosides. These triterpene glycosides
are characterized by a four trans-ring steroid
aglycone skeletons with attached sugar moieties,
and they can be grouped into the 20-(S)-
protopanaxadiols and 20-(S)-protopanaxatriols,
except for the oleanolic acid-derived ginsenoside
Ro. The major ginsenosides Rb1, Rb2, Rc, Rd,
Re, and Rg1 are used as markers of ginseng
To Distinguish Herb from Its Morphological Fakes
quality because they account for 90 % of the total
ginsenosides [8].
American and Asian ginsengs are distin-
guished by their chemical profiles. The glucogin-
senoside Rf is detectable in Asian ginseng but not
in American ginseng, and the ocotillol-type trit-
erpene 24-(R)-pseudoginsenoside F11 is present
in American ginseng but absent in Asian ginseng.
The relative abundances of ginsenosides can
also be used to distinguish between the Asian
and American species, as a higher amount of
protopanaxadiol ginsenosides exists in American
ginseng, in contrast to a higher amount of pro-
topanaxatriol ginsenosides in Asian ginseng. In
addition, ginsenoside ratios are also indicative of
the types of ginseng. A higher Rb1/Rg1 value
usually indicates P. quinquefolius, and a high Rf/
F11 ratio of more than 700:1 distinguishes the
Asian from the American varieties [8].
Herbs like ginseng have traditionally been
authenticated by morphological and histological
means. In Asia, ginseng quality is based on the
age, origin, as well as the physical characteristics
of the root. The potency of the ginseng roots is
determined by its shape and can be classified into
three kinds based on the numbers and sizes of the
lateral branches on the main root: “pencil” roots,
“chunky” roots, and “complex” roots. These
methods are hardly reliable at present hi-tech and
commercial scenario, as many commercial prod-
ucts are in the form of various formulations.
Additional commercial aspect is that ginseng is
expensive, and cultivation of ginseng is slow and
difficult, and it takes more than 4 years before it
can be harvested. Under these circumstances, the
possibility of adulterating it with other cheaper
products is more expected [8].
FTIR technique is based on the fact that when
low-energy transitions occur in molecules of the
sample material, sample absorbs the IR radiation,
resulting as in absorbance spectra represent the
quality of the major structural features/basic
functional groups of the molecules, present in the
sample. No two chemical structures can have iden-
tical IR spectra, as each type of bond in different
compounds has different vibrational frequencies.
The IR spectra, which are usually plot of percent
transmittance or absorbance versus frequency
139
expressed in wave numbers (cm−1), are used as a
fingerprint for compound identification [8].
In a case study, American ginseng, Asian gin-
seng, and Platycodon grandiflorum (jiegeng) root
samples were cut into small pieces and ground
into fine powder. Each sample was mixed uni-
formly with spectroscopic grade potassium bro-
mide (KBr) powder (1 % w/w) in an agate pestle
and mortar and then pressed into a pellet. The
spectra were recorded in the region of 4,000–
400 cm−1 on a Shimadzu IRPrestige-21 FTIR
spectrometer (Shimadzu Corporation Pte. Ltd.,
Asia Pacific) equipped with a KBr beam splitter
and a deuterated L-alanine triglycine sulfate
(DLATGS) detector. Each spectrum was an aver-
age of 40 scans co-added at 4 cm−1 resolution,
and pure KBr background spectra were recorded
before analysis of the samples. Base line correc-
tion was done for each spectrum, and their absor-
bance normalized with the Shimadzu
IRsolution 1.10® software program, prior to
data analysis, so that the peak absorbance of the
most intense band was set to unity. The cor-
rected spectra were then analyzed for pattern
similarity and functional group identification
(Fig. 7.14a). With complex mathematical pro-
cessing techniques, tiny differences in the IR
spectrum were amplified via their derivative
spectra, for the separation of overlapping bands
and determination of exact peak locations
(Fig. 7.14b). The spectra were converted to their
corresponding second derivatives with the same
Shimadzu IR solution 1.10® software program
(Creon Lab Control AG, Shimadzu Corporation
Pte. Ltd., Asia Pacific) using the 23-point
Savitzky and Golay algorithm. Principal com-
ponent analysis (PCA) is a method of chemo-
metric analysis which is widely used for
reorganizing information and explaining causes
of variance in a set of data, in this case IR spec-
tra (Figs. 7.15 and 7.16) [8].
In aforesaid studies, it is concluded that FTIR
spectroscopy is a novel and interesting innova-
tion for detecting adulteration in very short time
before the utilization of raw herb for commercial
production to distinguish between morphological
and chemical fakes, specifically cases similar to
ginseng.
140 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 7.14 Comparison of (a), general; (b), second-derivative MIR spectra of sawdust with American and Asian gin-
sengs [8]

Fig. 7.15 Comparison of the (a) general MIR spectra, (b) magnified fingerprint region (2,000–600 cm−1), and (c)
second-derivative MIR spectra of American and Asian ginsengs with jiegeng [8]
Standardization of Metal-Based Herbal Drugs
Fig. 7.15 (continued)
Standardization of Metal-Based
Herbal Drugs
Metal-based herbal drugs have its own repute
since ancient as gold and silver has been
described beneficial in healing and anti-disease
drugs. In olden days, people used silver bottles
for storing water, wine, and milk to prevent from
spoiling. In “Siddha” medicine, a form of south
Indian medicine, apart from gold and silver,
mercury, sulfur, mica, arsenic, zinc, and several
other minerals are treated with indigenous herbs,
and given as “Bhasma” and “Chendurams.”
“Bhasma” means a fine ash obtained through
141
incineration. “Chendurams” are prepared by the
process of sublimation, and they are much more
potent. In “Siddha,” there is a faith for prolonga-
tion of life through rejuvenation using mercurial
drugs. In Indian market, Siddha drugs like
Poorna Chandrodaya Chenduram (PCC), Kshya
kulanthanga Chenduram (KsKc), Velli Parpam
(SP), Naga Chenduram (NC), Naga Parpam
(NP), and three different popular brands of Linga
Chenduram (LC1, LC2, and LC3) are prepared
from purified metal, triturated with decoction of
herbal juices, generally prescribed in the dose of
100–200 mg/day, and recommended to be taken
with suitable adjuvant [18].
142
Fig. 7.16 2D plots of jiegeng, sawdust, and American
and Asian ginsengs. (a) General spectra of sawdust and
ginsengs, PC1 = 82 % versus PC2 = 10 %; (b) second-
derivative spectra of sawdust and ginsengs, PC1 = 74 %
versus PC2 = 11 %; (c) general spectra of jiegeng and gin-
sengs, PC1 = 76 % versus PC2 = 14 %; (d) second-derivative
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints
spectra of jiegeng and ginsengs, PC1 = 51% versus
PC2 = 29%; (e) general spectra of jiegeng, sawdust, and
ginsengs, PC1 = 80 % versus PC2 = 7 %; and (f) second-
derivative spectra of jiegeng, sawdust, and ginsengs,
PC1 = 57 % versus PC2 = 17 % [8]
Standardization of Metal-Based Herbal Drugs 143

Fig. 7.16 (continued)

144 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

LC3 SP

LC2

KsKc
LC1

592
PCC

4000 3500 3000 2500 2000 1500 1000 500

4000 3500 3000 2500 2000 1500 1000 500
Fig. 7.17 FTIR of LC (LC1, LC2, LC3)
Fig. 7.19 FTIR of PCC, KsKc, SP (Au- and Ag-based
formulation)

The spectra clearly indicate that drug sample

NC
NP
981
884
4000 3500 3000 2500 2000 1500 1000
Fig. 7.18 FTIR of NC and NP (zinc-based formulation)
In a study for quality and quantity determina-
tion for these formulations, about 0.1–0.2 g of the
drug was digested with nitric acid: perchloric
acid (2:1) and allowed to cool and transferred
into beaker after complete digestion. The beaker
was heated to remove acids; the resulting solu-
tion was diluted to 50 ml, with deionized water
(perchloric acid was used as it does not form
complexes with metals and metalloids). IR spec-
tra (4,000–450 cm−1) were recorded (Figs. 7.17,
7.18, 7.19), using Perkin Elmer FTIR spectro-
photometer in KBr pellets [18].
does not have any organic compound, strong
evidence of being “Bhasma.” The three different
brands of LC showed a similar pattern (Fig. 7.16).
Spectrum of PCC is similar to LC. The peak at
592 cm−1 in KsKc is due to Fe3O4, but the exact
form of silver in SP could not be concluded using
IR spectrum. LC is used to treat fevers, skin dis-
eases, and venereal diseases. PCC is used for
treating tuberculosis, jaundice, fever, and bron-
chitis. KsKc is given for all respiratory diseases
including tuberculosis. SP is used to treat cough,
tuberculosis, piles, leucorrhoea, gonorrhea and
500 spermatorrhea. NC is used to treat piles, skin dis-
eases, and white discharge. Thus, the fingerprints
for purity of metal-based herbal drugs using FTIR
are quite helpful to decipher the quality [18].
Herbal drugs play an important role in modern
human life and have significant effects on treat-
ing diseases, but the quality and safety of these
herbal products have now become a serious issue
due to increasing pollution in air, water, soil, and
modern life style. Using FTIR spectroscopy, the
technique for liquid and solid samples, along
with the statistical method of principal compo-
nent analysis (PCA) to identify and discriminate
herbal drugs, and traditional formulation may
be scrutinized for quality purpose. Traditional
drugs are widely accepted in China, India, Japan,
Bibliography
Pakistan, Sri Lanka, and Thailand. In China,
about 40 % of the total medicine consumption is
attributed to traditional tribal medicines. In Japan,
herbal medicinal preparations are more in demand
than mainstream pharmaceutical products. Due
to globalization, ethics are being substituted by
commercial opportunities, so there are more
chances of intentional adulteration and contami-
nation in herbal drugs. The FTIR spectra are
helpful in developing methods to test raw materi-
als and finished products, identification and iso-
lating active components, as provides clue for the
functional groups of ingredients.
References
1. Stuart BH. Infrared spectroscopy: fundamentals and
applications, analytical techniques in the sciences.
London: Wiley; 2004.
2. Holler FJ, Skoog DA, Crouch SR. Principles of instru-
mental analysis. 6th ed. New York: Brooks/Cole
Publishing; 2007.
3. Somsen GW, Visser T, Meyers RA. Liquid chroma-
tography/infrared spectroscopy. In: Encyclopedia of
analytical chemistry. Chichester: Wiley; 2000.
4. Visser T, Meyers RA. Infrared spectroscopy in envi-
ronmental analysis. In: Encyclopedia of analytical
chemistry. Chichester: Wiley; 2000.
5. Bunaciu AA, Aboul-Enein HY, Fleschin S. Application
of Fourier transform infrared spectrophotometry in
pharmaceutical drugs analysis. Appl Spectrosc Rev.
2010;45(3):206–19.
6. Steele D, Chalmers JM, Griffiths PR. Instrumentation
for near infrared spectroscopy. In: Handbook of vibra-
tional spectroscopy. London: Wiley; 2002. p. 44–70.
7. Kumar JK, Devi Prasad AG. Identification and com-
parison of bio-molecules in medicinal plants of
Tephrosia tinctoria and Atylosia albicans by using
FTIR. Rom J Biophys. 2011;21(1):63–71.
8. Yap KYL, Chan SY, Lim CS. Authentication of tradi-
tional Chinese medicine using infrared spectroscopy:
distinguishing between ginseng and its morphological
fakes. J Biomed Sci. 2007;14:265–73.
9. Bunaciu AA, Aboul-enein H, Fleschin S. Recent
applications of Fourier transform infrared spectropho-
tometry in herbal medicine analysis. Appl Spectrosc
Rev. 2011;46:251–60.
10. Liu HX, Zhou Q, Sun S-Q, Bao H-J. Discrimination of
different Chrysanthemums with Fourier transform infra-
red spectroscopy. J Mol Struct. 2008;883–884:38–47.
11. Judd MJ, Meyer DH, Meekings JS, Richardson AC,
Walton EF. An FTIR study of the induction and
release of kiwifruit buds from dormancy. J Sci Food
Agric. 2010;90:1071–80.
145
12. Lakshmi Prasuna CP, Chakradhar RPS, Rao JL, Gopal
NO. EPR and IR spectral investigations on some leafy
vegetables of Indian origin. Spectrochim Acta A Mol
Biomol Spectros. 2009;74:140–7.
13. Sim CO, Hamdani MR, Ismail Z, Ahmad MN.
Assessment of herbal medicines by chemometrics-
assisted interpretation of FTIR spectra. Send to J Anal
Chim Acta. January 2004. Available at, http://www.
camo.com/downloads/resources/application_notes/
Assessment%20of%20Herbal%20Medicines%20
by%20Chemometrics%20-%20Assisted%20
Interpretation%20of%20FTIR%20Spectra.pdf. As on
28 Apr 2012.
14. Zou H-B, Yang G-S, Qin Z-R, Jiang W-Q, Du A-Q,
Aboul--Enein HY. Progress in quality control of
herbal medicine with IR fingerprint spectra. Anal
Lett. 2005;38(9): 1457–75. Available at, http://dx.doi.
org/10.1081/AL-200062153. As on 15 Nov 2011.
15. Yuan JR, Li JL. The study on identification of Chinese
herbal medicine with ultraviolet spectra group. J
Shandong Univ Tradit Chin Med. 1987;11(1):58–63.
16. Zou H-B, Yuan J-R, Du A-Q, Sun L-L, Qin Z-R. The
dual-index sequence method of common and variant
peak ratio for IR fingerprint spectra of the components
extracted from Radix Glycyrrhizae with water. Chin
Tradit Pat Med Pat Med. 2004;26(10):779–83.
17. Zou H-B, Yuan J-R, Du A-Q, Sun L-L. The dual-index
sequence analytical method of common peak ratio and
variant peak ratio for IR fingerprint spectra of the com-
ponents extracted from Radix Glycyrrhizae with chlo-
roform. China J Chin Med Mater. 2005;30(1):16–20.
18. Sudha A, Murty VS, Chandra TS. Standardization of
metal based herbal drugs. Am J Infect Dis.
2009;5(3):200–6.
Bibliography
Bugay DE, Brittain HG. Infrared absorption spectroscopy.
In: Brittain HG, editor. Spectroscopy of pharmaceutical
solids. New York: Taylor & Francis; 2006. p. 235–65.
Bunaciu AA, Aboul-Enein HY, Fleschin S. FTIR spectro-
photometric analysis of co-enzyme Q10 (CoQ10) and
its pharmaceutical formulations. Prep Biochem
Biotechnol. 2007;37:59–65.
Chan KL, Elkhider N, Kazarian SG. Spectroscopic imag-
ing of compacted pharmaceutical tablets. Chem Eng
Res Des. 2005;83:1303–10.
Dubois J, Wolff J-C, Warrack JK, Schoppelrei J, Lewis
EN. NIR chemical imaging for counterfeit pharmaceu-
tical products analysis. Spectroscopy. 2007;22:40–50.
Jespersen N, Ahuja S. General principles of spectroscopy and
spectroscopic analysis. In: Modern instrumental analysis,
Wilson and Wilson’s comprehensive analytical chemistry,
vol. 47. Amsterdam: Elsevier; 2006. p. 111–37.
Movasaghi MS, Rehman IU. Fourier transform infrared
(FTIR) spectroscopy of biological tissues. Appl
Spectrosc Rev. 2008;43:134–79.
146
Silva FEB, Ferrão MF, Parisotto G, Müller EI, Flores
EMM. Simultaneous determination of sulpha-methox-
azole and tri-methoprim in powder mixtures by attenu-
ated total reflection-Fourier transform infrared and
multivariate calibration. J Pharm Biomed Anal.
2009;49:800–5.
Sankaran S, Ehsani R, Etxeberria E. Mid-infrared spec-
troscopy for detection of Huanglongbing (greening) in
citrus leaves. Talanta. 2010;83:574–81.
Sun S-Q, Tang J-M, Yuan Z-M, Bai Y. FTIR and
classification study on trueborn tuber Dioscoreae sam-
ples. Spectrosc Spectr Anal. 2003;23(2):258–61.
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints
Wang J, Qu W, Jun S, Bittenbender HC, Li QX. Rapid
determination of six kava lactones in kava root and rhi-
zome samples using Fourier transform infrared spec-
troscopy and multivariate analysis in comparison with
gas chromatography. Anal Method. 2010;2:492–8.
Wu Z-F, Deng D-H, Qin B-C, Li H-Y. Identifying the
quality of Semmecta by FTIR. Chin J Spectrosc Lab.
2003;20(2):312–4.
Yu M, Lin J, Fang J. Silica spheres coated with YVO4:
Eu3+ layers via sol-gel process: a simple method to
obtain spherical core-shell phosphors. Chem Mater.
2005;17:1783–91.
 Mass Spectroscopy: Herbal Drugs
and Fingerprints
In order to measure the characteristics of indi-
vidual molecules, in herbals and herbal drugs, a
mass spectrometer (MS) is used to convert them
into ions so that they can be moved and manipu-
lated by external electric and magnetic fields for
individual characterization and define them at the
level of molecular structure. The mass spectrum
is a two-dimensional representation where x-axis
represents the mass number and y-axis represents
the relative intensity (relative abundance) of the
ions produced. The mass spectrum is always
recorded over a range of mass number, and the
mass range is ideally more than the expected
molecular weight of the compound. Once a mass
spectrum is recorded, one has to look at the spec-
trum in totality. The first step toward interpreting
a mass spectrum is to look for the molecular ion,
which provides the molecular mass of the ana-
lyte. In many mass spectra, the molecular ion is
easily identified as the ion with the highest mass
number (a careful assessment is necessary as the
highest mass number ion may be due to an isoto-
pic peak or an impurity). Many times under elec-
trospray ionization mode, molecule undergoes
near total fragmentation, and hence, no molecu-
lar ion is visible. It is very important to note that
the ion with the maximum abundance is the base
peak and is not necessarily the molecular ion.
Although the molecular ion gives information
about the molecular mass of the compound, it
does not provide much information about the
molecular structure. The structural information is
obtained from the fragmentation pattern of the
mass spectrum. After a molecule is ionized, the
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_8, © Springer India 2012
8
molecular ion retains the excess ionization
energy. If this excess energy is greater than the
energy required to break a chemical bond, the
molecule can fragment. The fragmentation pro-
cesses are typically categorized as direct cleav-
age or rearrangement. Cleavage reactions are
simply the breaking of a chemical bond to pro-
duce two fragments. Rearrangements are more
complex reactions that involve both making and
breaking bonds. These rearrangement fragments
often provide important clues about the location
and identity of some functional groups. Functional
group can have a significant effect on the frag-
mentation patterns observed in mass spectros-
copy. The McLafferty rearrangement is a classic
example of a rearrangement reaction. The
McLafferty rearrangement is often observed for
carbonyl compounds that contain a liner alkyl
chain.
The MS is a four-component system and
(Fig. 8.1) may be summarized for essential func-
tions of it as (1) ionization chamber to generate
ions, usually to cations by loss of an electron;
(2) mass analyzer to separate the different ions
according to their mass and charge ratio (m/z)
basis; (3) detector to detect and characterize the
separated ions; and (4) recorder to record the data
of detector and displayed as spectrum. Due to high
reactivity and short life of ions, their formation
and manipulation are conducted in reduced
pressure. The pressure under which ions may be
handled is around 10−5 to 10−8 Torr (mmHg)
(i.e., less than a billionth of an atmosphere). Each
of the aforesaid four tasks may be accomplished
147
148
Mass
Ionization
Analyzer
Chamber (m/z)
Fig. 8.1 Graphic presentation of mass spectrometer
Fig. 8.2 Simplified schematic of MS
in different ways. In one common procedure,
ionization is effected by a high-energy beam of
electrons, and ion separation is achieved by accel-
erating and focusing as a beam, which is then bent
by an external magnetic field. The ions are then
detected electronically, and the resulting informa-
tion is stored and analyzed in a computer and pre-
sented in the form of peaks.
During operation when a high-energy electron
collides with a molecule, it often ionizes by
knocking away one of the electrons (either bond-
ing or nonbonding), producing a molecular ion.
The residual energy from the collision may cause
the molecular ion to fragment into neutral pieces
and smaller fragment ions. The molecular ion is a
radical cation, but the fragment ions may either be
radical cations or carbocations, depending on the
nature of the neutral fragment. The ion source is
8 Mass Spectroscopy: Herbal Drugs and Fingerprints
Detector
Recorder
the heart of the spectrometer, where the molecules
of sample are bombarded by electrons issuing
from a heated filament (process is known as
electron impact). Gases and volatile liquid sam-
ples are allowed to leak into the ion source from
a reservoir. Cations formed by the electron bom-
bardment are pushed away by a charged repeller
plate and accelerated toward other electrodes,
having slits through which the ions pass as a
beam. Some of these ions fragment into smaller
cations and neutral fragments. A perpendicular
magnetic field deflects the ion beam in an arc
whose radius is inversely proportional to the
mass of each ion. Lighter ions are deflected more
than heavier ions. By varying the strength of the
magnetic field, ions of different mass are focused
progressively on a detector fixed at the end of a
curved tube (Fig. 8.2).
Detection and Recording of Fingerprints
Interpretation of Mass Spectra
A mass spectrum is usually a vertical bar graph,
in which each bar represents an ion having a
specific mass-to-charge ratio (m/z) and the length
of the bar indicates the relative abundance of
the ion. The most intense ion is assigned an
abundance of 100, and it is referred to as the
base peak. Most of the ions formed in a mass
spectrometer have a single charge, so the m/z
value is equivalent to mass itself. Modern mass
spectrometers easily resolve ions differing by
only a single atomic mass unit (amu), and thus
provide completely accurate values for the
molecular mass of a compound. The highest mass
ion in a spectrum is normally considered to be
the molecular ion, and lower mass ions are
fragments from the molecular ion, assuming the
sample is a single pure compound.
Most stable organic compounds have an even
number of total electrons, reflecting the fact that
electrons occupy atomic and molecular orbitals in
pairs. When a single electron is removed from a
molecule to give an ion, the total electron count
becomes an odd number, and we refer to such ions
as radical cations. The molecular ion in a mass
spectrum is always a radical cation, but the frag-
ment ions may either be even-electron cations or
odd-electron radical cations, depending on the
neutral fragment lost. The simplest and most
common fragmentations are bond cleavages pro-
ducing a neutral radical (odd number of electrons)
and a cation having an even number of electrons.
A less common fragmentation, in which an even-
electron neutral fragment is lost, produces an odd-
electron radical cation fragment ion. Fragment
ions themselves may fragment further. As a rule,
odd-electron ions may fragment either to odd- or
even-electron ions, but even-electron ions frag-
ment only to other even-electron ions. The masses
of molecular and fragment ions also reflect the
electron count, depending on the number of hetero
atoms in the species. The molecular ion peak is the
signal of the parent ion corresponding to the
molecular weight of the compound. Allowing for
isotopes, it is the peak of highest mass in the
spectrum. The nature of the fragments often
149
provides a clue to the molecular structure, but if
the molecular ion has a lifetime of less than a
few microseconds, it will not survive long enough
to be observed, and without a molecular ion peak
as a reference, it is difficult to decipher a mass
spectrum. Fortunately, most organic compounds
give mass spectra that include a molecular ion.
The most stable molecular ions are those from
aromatic rings, other conjugated pi-electron
systems, and cycloalkanes. Alcohols, ethers, and
highly branched alkanes generally show the
greatest tendency toward fragmentation [1].
Techniques for Analysis of Ions in MS
The main function of the mass analyzer is to
separate (resolve) the ions formed at ionization
source of the mass spectrometer according to
their m/z ratios. There are a number of mass
analyzers but frequently in use are quadrupoles,
time-of-flight (TOF) analyzers, magnetic sectors,
and both Fourier transform and quadrupole ion
traps. These mass analyzers have different features,
including the m/z range that can be covered for
the mass accuracy and achievable resolution.
The compatibility of different analyzers with
different ionization methods varies as tandem
mass spectrometers (MS–MS) are instruments
that have more than one analyzer and so can be
used for structural and sequencing studies. In
tandem mass spectrometers, two, three, and four
analyzers have all been incorporated into avail-
able instruments, and the analyzers do not neces-
sarily have to be of the same type, in which case
the instrument is a hybrid one. More popular
tandem mass spectrometers include those of the
quadrupole–quadrupole, magnetic-sector qua-
drupole, and, more recently, the quadrupole time-
of-flight geometries [1].
Detection and Recording
of Fingerprints
The detector detects the ion current and amplifies
it into signals; signals are then transmitted to the
data system where it is recorded in the form of
150
mass spectra. The m/z values of the ions are
plotted against their intensities to show the number
of components in the sample, the molecular mass
of each component, and the relative abundance of
the various components in the sample. The more
common detectors are the photomultiplier, the
electron multiplier, and the micro-channel plate
detectors [1].
Fast atom bombardment (FAB) and ther-
mospray techniques have restriction for highly
volatile and polar moieties, so electrospray
ionization (ESI) and nanospray ionization tech-
niques are currently in practice. Matrix-assisted
laser desorption ionization (MALDI) deals well
with thermolabile, nonvolatile organic com-
pounds especially those of high molecular mass
and are used successfully in biochemical areas
for the analysis of proteins, peptides, glycopro-
teins, oligosaccharides, and oligonucleotides.
Electrospray ionization is the technique used at
an atmospheric pressure for ionization, so fre-
quently known as “atmospheric pressure ioniza-
tion” (API) techniques and is well suited to the
analysis of polar molecules ranging from less
than 100 Da to more than 1,000,000 Da in
molecular mass. During standard electrospray
ionization, the sample is dissolved in a polar,
volatile solvent and pumped through a narrow,
stainless steel capillary (75–150 mm i.d.) at a
flow rate of 1 ml/min. A high voltage of 3 or
4 kV is applied to the tip of the capillary, which
is situated within the ionization source of the
mass spectrometer, and as a consequence of this
strong electric field, the sample emerging from
the tip is dispersed into an aerosol of highly
charged droplets, a process that is aided by a
coaxially introduced nebulizing gas flowing
around the outside of the capillary. This gas, usu-
ally nitrogen, helps to direct the spray emerging
from the capillary tip toward the mass spectrom-
eter. The charged droplets diminish in size by
solvent evaporation, assisted by a warm flow of
nitrogen (acts as a drying gas) which passes
across the front of the ionization source.
Eventually charged sample ions, free from sol-
vent, are released from the droplets, some of
which pass through a sampling cone or orifice
into an intermediate vacuum region, and from
8 Mass Spectroscopy: Herbal Drugs and Fingerprints
there through a small aperture into the analyzer
of the mass spectrometer, which is held under
high vacuum. The lens voltages are optimized
individually for each sample. In nanospray, ion-
ization technique is a low flow rate version of
electrospray ionization, where a small volume
(1–4 ml) of the sample dissolved in a suitable
volatile solvent, at a concentration of 1–10 pmol/
microl, is transferred into a miniature sample
vial. A reasonably high voltage (700–2,000 V) is
applied to the specially manufactured gold-
plated vial resulting in sample ionization and
spraying. The flow rate of solute and solvent
using this procedure is very low, 30–1,000 nl/
min. This technique has advantage over the ESI
that it needs less sample size as well as has scope
for multi- analysis. A common application of
this technique is for a protein digest mixture to
be analyzed to generate a list of molecular
masses for the components present, and then
each component to be analyzed further by tan-
dem mass spectrometric amino acid sequencing
techniques. ESI and nanospray ionization are
highly sensitive analytical techniques, but the
sensitivity deteriorates with the presence of non-
volatile buffers and other additives, which should
be avoided as far as possible. With reference to
natural product chemistry, the term “electrospray
ionization” is replaced by “electrospray” as
(except in a few cases) electrospray does not
create ions but transfers the analyte into gases
phase from condensed phase [1].
Electrospray MS for Herbal Fingerprints
In this technique, test solution is passed through
a capillary which is held at high potential. The
effect of the high electric field as the solution
emerges is to generate a mist of highly charged
droplets which pass down a potential and pres-
sure gradient toward the analyzer portion of
the mass spectrometer. During that transition, the
droplets reduce in size by evaporation of the
solvent or by “Coulomb explosion” (droplet sub-
division resulting from the high charge density).
Ultimately, fully de-solvated ions result from
complete evaporation of the solvent or by field
Detection and Recording of Fingerprints
Fig. 8.3 Essential features of the electrospray interface [1]
desorption from the charged droplets. Nebulization
of the solution emerging from the capillary may
be facilitated by a sheath flow of nebulizer gas, a
technique for which the term “ion spray” was
originally coined by its developers. In practice,
the facility to use a nebulizer gas is commonly
incorporated on commercial instruments; the
need for its use or not is determined by the flow
rate employed, the composition of the solvent,
and the sign of the potential applied to the capil-
lary tip (since a high negative potential, in par-
ticular, may lead to a corona discharge unless
suppressed by the use of an appropriate sheath
gas). In addition, a flow of bath gas is usually
applied to the interface to promote droplet evapo-
ration; controlled heating of the interface pro-
vides an alternative approach. Sampling of the
fully or partially de-solvated ions is made using
a capillary or a skimmer device (Fig. 8.3).
Numerous elaborations have been reported, but
majority of the electrospray literature involves
implementation on quadrupole mass spectrome-
ters, but this position is rapidly changing with the
increasing use of Paul ion traps and Fourier trans-
form ion cyclotron resonance instruments to
enhance resolution. Implementation of electro-
spray on magnetic sector instruments is compli-
cated and needs to avoid collisional activation
during ion acceleration; enhanced resolution is
151
achieved, however, in comparison with quadrupole
instruments. Installation of an electrospray source
on a time-of-flight instrument also presents some
advantages.
Electrospray technique is a convenient tech-
nique may be studied into three stages as droplet
formation, droplet shrinkage, and gaseous ion
formation. In this process, the solution delivered
to the tip of the electrospray capillary experiences
the electric field associated with the maintenance
of the tip at high potential. Due to a positive
potential, positive ions in solution accumulate
at the surface and get drawn in a downfield
direction to establish a “Taylor cone” (Fig. 8.4).
A high enough imposed field, the cone is drawn
to a filament which produces positively charged
droplets via a budding process when the surface
tension is exceeded by the applied electrostatic
force. The diameter of the droplets formed is
influenced by a number of parameters, including
the applied potential, the solution flow rate, and
solvent properties. Evaporation of solvent from
the initially formed droplets, as they traverse a
pressure gradient toward the analyzer of the mass
spectrometer, leads to a reduction in diameter,
with collisional warming preventing freezing.
Fission (Coulomb explosion) will occur at the
point (the Rayleigh limit) at which the magnitude
of the charge is sufficient to overcome the surface
152
Fig. 8.4 Droplet production in the electrospray interface [1]
tension holding the droplet together. Continuous
and continual depletion of the droplet size (by
solvent evaporation and fission, respectively)
may be envisaged to result eventually in the
formation of droplets containing a single ion,
from which the non-solvated analogue may be
derived by further evaporation of solvent (aided
by activating collisions in the interface). During
the whole process, electrochemical oxidation can
also generate radical cations from neutral analytes,
thereby permitting this technique in some selected
instances, inspite of as a true ionization method
rather than a procedure for phase transfer of pre-
formed ions.
Currently electrospray at reduced flow rates is
in use, where sample solution flows at electro-
spray interface with 3–20 ml/min. In the interests
of direct compatibility with conventional scale
analytical HPLC, some effort has been devoted to
the accommodation of much higher flow rates.
The technique is best utilized for the recording of
8 Mass Spectroscopy: Herbal Drugs and Fingerprints
full mass spectra at the nanogram level. The
mechanisms underlying the electrospray process
and the manner of its implementation have
significant implications for the properties of the
gas-phase ions produced. The followings are
notable points [1].
(a) The charge states of the gaseous ions reflect
the charge states in the condensed phase;
although somewhat modified following ion–
molecule collisions in the interface, resulting
multiply charged species are commonly
observed.
(b) The transfer of ions to the gas phase is not an
energetic process, yet the de-solvation pro-
cess effectively cools the ions. Under appro-
priate conditions, therefore, low internal
energy ions are introduced into the mass
spectrometer for conventional analysis or for
selection as precursor ions in a tandem MS
analysis. Application of a suitable potential
difference between focusing components in a
Detection and Recording of Fingerprints
region of intermediate pressure allows ions
to achieve kinetic energies consistent with
the applied potential and therefore undergo
activating collisions.
(c) The electrospray process involves the step-
wise disruption of non-covalent interactions
(principally the removal of molecules of sol-
vation); interception of this process allows the
preservation of relatively strong non-covalent
interactions of analytical importance.
Case Study 1
LXD (Longdan Xiegan decoction) is a herbal
decoction used in traditional Chinese medicine
and consists of ten medicinal herbs, Gentianae
Radix, Scutellariae Radix, Gardeniae Fructus,
Rehmanniae Radix, Alismatis Rhizoma, Plantaginis
Semen, Angelicae Sinensis Radix, Clematidis
Armandii Caulis, Glycyrrhizae Radix et Rhizoma,
and Bupleuri Radix. The decoction has attracted
a great deal of attention for its wide range of
pharmaceutical and therapeutic uses. It is used
to treat damp heat in the liver, gall bladder,
congested eyes, swelling, and pain in the ear.
Flavonoids (from Scutellariae Radix, Glycyrrhizae
Radix et Rhizoma), iridoid glycosides (from
Gentianae Radix, Gardeniae Fructus, Rehmanniae
Radix), and triterpenoids (from Glycyrrhizae Radix
et Rhizoma, Bupleuri Radix, and Gentianae Radix)
were the three main classes of active compounds
reported according to chemical research on
each of the individual herbs, but there was no
report on the interpretation of the chemical
constitution of the decoction, so LXD was stud-
ied by high-performance liquid chromatography
coupled to electrospray ionization tandem mass
spectrometry and photodiode array detection
method [2].
HPLC coupled to electrospray ionization (ESI)
tandem mass spectrometry and photodiode array
detection (HPLC–DAD–ESI–MSn) was used to
identify and characterize the flavonoids in LXD.
In total, 51 flavonoids (27 flavones, 10 flavanones,
7 chalcones, 5 flavonols, and 2 isoflavones) were
characterized. Eighteen compounds among them
including a newly detected flavonoid, naringin,
from the ingredient herbs, were unambiguously
determined by comparing the retention times, UV
153
spectral data, and mass fragmentation behaviors
with those of the reference compounds. Another 33
compounds were tentatively identified by refer-
encing to the reported data of their UV and MS
spectra. The ESI–MS/MS fragmentation behav-
ior of flavones (OMe-substituted, O-glycosides,
C-glycosides), chalcones, flavonols, and their appro-
priate characteristic pathways were proposed.
In negative ion ESI-MS, all the flavonoids yielded
prominent [M–H]− ions in the first-order mass
spectra. Fragmentation with a loss of mass of
15 Da (CH3), 18 Da (H2O), 28 Da (CO), 44 Da
(CO2), and 56 Da (2CO) and the residues of glu-
cose and glucuronic acid observed in the MS/MS
spectra were useful for aiding the structural
identification of flavonoids investigated [2].
The researcher team has emphasized in their
research paper that it was a matter of hard work
and high professional skill to optimize the HPLC
and MS conditions for satisfactory separation of
the chemical constituents of LXD, as it was not
an easy task because of the vast number of chem-
ical components present in the decoction with
many of them having similar physicochemical
properties [2]. Eighteen flavonoid compounds
were used as standards for optimization of the
separation of flavonoids using HPLC and the
ionization and fragmentation using ESI-MS. In
order to optimize the separation and detection
of flavonoids in LXD, variables such as column
types, column temperatures, mobile phase, elu-
tion conditions, flow rates, and detection wave-
length have been investigated. A number of C18
RP-ODS (250 mm × 4.6 mm, 5 mm) columns,
from different manufacturers, were tested for
their suitability in this analytical design. The
Waters ODS column was finally selected due to
the best peak separation capacity. Two column
temperatures, 30 and 40°C, were tested, and
results were better at 30°C. Acetonitrile with
water (different pH) was used as mobile phase,
and no enhancement was obtained by changing
the pH in the aqueous mobile phase, so the best
mobile phase was a mixture of acetonitrile and
water. Five wavelengths (210, 254, 270, 280, and
330 nm) were tested for the best detection sensi-
tivity, and best detection result was at 270 nm.
Three different flow rates (0.6, 0.8, and 1.0 ml/min)
154
were tested, and 0.8 ml/min had good separation.
In MS analysis, the negative ion mode of ESI was
selected for its better sensitivity and separation
than the positive ion mode. For the peaks of the
18 isolated standards in the negative ion LC/
ESI-MS total ion current chromatogram, separa-
tion conditions were used to record the LC/MS
chromatograms of the LXD. In addition, colli-
sion-induced dissociation (CID) fragmentation
of the [M–H]− ion provided extensive informa-
tion for the characterization of flavonoids in the
decoction [2].
All the solvents for used for analysis were of
high purity HPLC grade, and herbs for the prepa-
ration of the LXD were purchased from authentic
supplier. Eighteen pure compounds were used as
reference standards, and their structures were
fully characterized by nuclear magnetic reso-
nance (NMR) spectroscopic methods, and their
purities were >98% as determined by HPLC.
Stock solutions (1.0 mg/ml) of all the above
compounds were prepared by dissolving them in
methanol [2].
The decoction was analyzed by online HPLC/
ESI using an HPLC system coupled to a LCQ
ion trap mass spectrometer. LC separation was
conducted using a ThermoFinnigan HPLC and
a C-18 column (250 mm × 4.6 mm, 5 mm),
Symmetry from Waters, at a flow rate of 0.8 ml/
min. The mobile phase consisted of (A) acetonitrile
and (B) water (v/v), using an iso-gradient elution
of 22% A at 0–35 min, a gradient elution of
22–30% A at 35–40 min, 30–50% A at 40–80 min,
and 50–90% A at 80–90 min. The temperature of
the column oven was 30°C, and detection wave-
length was 270 nm (Fig. 8.5). The UV spectra
from 190 to 400 nm were also recorded for peak
characterization. Mass spectra were acquired using
an LCQ ion trap mass spectrometer (Thermo-
Finnigan, San Jose, CA, USA) equipped with an
ESI source. Nitrogen was used as the sheath and
auxiliary gas, and helium was used as the damp-
ing and collision gas. All mass spectra were
acquired in the negative ion mode with an ion
spray voltage at 4.5 kV, a capillary temperature
at 275°C, a capillary voltage at 19 V, a sheath
gas flow rate at 20 (arbitrary units), an auxiliary gas
flow rate at 45 (arbitrary units), and a tube lens
8 Mass Spectroscopy: Herbal Drugs and Fingerprints
offset at 40 V. The first-order mass spectra were
recorded in the range m/z 80–1,000. The relative
collision energy was varied from 35 to 45% of
the maximum to produce optimum yields of frag-
ment ions [2].
The powdered samples of Gentianae Radix
(0.6 g), Scutellariae Radix (0.3 g), Gardeniae
Fructus (0.3 g), Rehmanniae Radix (0.6 g),
Alismatis Rhizoma (0.6 g), Plantaginis Semen
(0.3 g), Angelicae Sinensis Radix (0.3 g),
Clematidis Armandii Caulis (0.3 g), Glycyrrhizae
Radix et Rhizoma (0.3 g), and Bupleuri Radix
(0.6 g) were immersed in 168 ml of deionized
water for 1 h and then were decocted to boil for
30 min, filtered and cooled. Acetic acid was
added to adjust the pH to 3.0. It was then left to
stand for 30 min and extracted with ethyl acetate
(1:1, v/v) twice. The ethyl acetate extracts were
combined and evaporated to dryness. The residue
was dissolved in 2-ml methanol and was filtered
through a 0.45-mm syringe filter before injecting
into the HPLC instrument for analysis [2].
The aforesaid study is an array for identification
and characterization of the flavonoids present in
LXD-type formulations. The knowledge of the
profile of the flavonoids (flavones, isoflavones,
flavonols, flavanones, and chalcones) in the LXD
is important for the understanding of its biologi-
cal and medical significance, and such a profile
may also prove useful to evaluate the quality of
the decoction, as flavonoids, polyphenols, and
its derivatives are responsible for potent
biological properties (antibacterial, antiviral,
anti-inflammatory, antiallergic, neoplastic, anti-
mutagenic, antithrombotic, and vasodilatory),
which have a very complex profile of chemical
components. Hence, it is very difficult to identify
all the compounds in the decoction. High-
performance liquid chromatography/electrospray
ionization tandem mass spectrometry (HPLC/
ESI–MS/MS) is a most powerful analytical tech-
nique for the analysis of natural products. It is
widely used in the analysis of complex mixtures
such as plant metabolites. ESI is considered to
provide soft ionization since it can lead to only
protonated or deprotonated molecules. MS/MS,
particularly multistage MS/MS with an ion trap
(IT), provides further information about the struc-
Detection and Recording of Fingerprints
Fig. 8.5 LC/MS chromatograms of the flavonoid standards. (a) Negative ion LC/ESI-MS total ion current (TIC) and
(b) LC-UV chromatogram monitored at 270 nm [2]
tures of the compounds under analysis. By frag-
menting the precursor ion, it is possible to obtain
structurally important fragment ions. Thus, online
HPLC/ESI-ITMS provides a fast method to sepa-
rate and determine the molecular mass and
resolve the subtle differences in their molecular
structures. The tandem mass spectrometric frag-
mentation pattern of flavonoids (except chal-
cones) has been investigated extensively, and thus
allows the characterization of unknown com-
pounds even when reference standards are
unavailable [2].
Case Study 2
A study on characterization of polyphenols by
HPLC–PADESI/MS of Equisetum telmateia
(family Equisetaceae, is a species of the subgenus
Equisetum, widely distributed in Europe; aerial
155
parts are used for the treatment of prostatitis,
stomachaches, and cystitis in traditional medicine)
was performed using retention characteristics in
RP-HPLC, through analysis of UV–vis. and MS
spectra, and by HPLC–PAD–ESI/MS analyses.
Flavan-3-ols (i.e., catechins and proanthocyani-
dins) were also analyzed by HPLC using detec-
tion following post-column derivatization with
p-dimethylaminocinnamaldehyde (DMACA). The
chromatographic profile of the ethyl acetate frac-
tion from the infusion of E. telmateia is recorded
at 280 nm. The UV spectra of the same suggested
that the major flavonoids present in this fraction
were kaempferol derivatives and flavan-3-ols.
Retention characteristics and UV spectra also
allowed the identification of protocatechuic and
p-hydroxybenzoic acids, confirmed by compari-
son with standards, as well as the presence of vari-
156
ous caffeic acid derivatives. Further identification
of compounds was made from their MS. The
retention time and spectral characteristics of
components detected in the HPLC–PAD–MS/
MS analysis are clues for the tentative identities
of these compounds.
Analysis of polyphenols was performed
using HPLC with an auto-injector, spectrometer
equipped with an API/ES ionization chamber.
Separation was performed on a Waters Spherisorb
ODS2 column (150 mm × 4.6 mm, 3.0 mm) main-
tained at 25°C. The mobile phase consisted of
2.5% acetic acid (solvent A), a 90:10 mixture of
2.5% acetic acid, and acetonitrile (solvent B) and
acetonitrile (solvent C). The gradient profile used
was from 100:0:0 (A: B: C) to 0:100:0 between 0
and 5 min, changing to 0:85:15 between 5 and
30 min, changing to 0:50:50 between 30 and
35 min, and followed finally by isocratic elution
with 0:50:50 between 35 and 40 min; the flow
rate was 0.5 ml/min. The first detection was by
photodiode array detector (PAD) with the
spectrophotometer set at 280 and 360 nm, and
the second detection using ESI-MS. The capillary
temperature was 225°C, and the capillary voltage
was 45 V. Spectra were obtained in the positive
ion mode, and the MS was programmed to
perform two scans, a full mass scan and an MS2
scan of the most abundant ion using a collision
energy of 45 V.
Reference standards as kaempferol 3-O-
rutinoside and kaempferol 3-O-glucoside were
from Extrasynthese; standards of flavan-3-ols
were obtained from grape seeds by repeated
extraction with methanol and fractionation
of the combined extracts on a Sephadex LH-20
Fluka column (45 × 5 cm) using ethanol as mobile
phase. Catechins and proanthocyanidins were
further separated from the Sephadex fractions by
semi-preparative HPLC.
Dried aerial parts of E. telmateia Ehrh. were
obtained from authentic supplier, and plant
material was identified by Dr. J. Paiva, Botany
Department, University of Coimbra, Portugal,
and a voucher was deposited at the Department of
Pharmacognosy, Faculty of Pharmacy, University
of Coimbra. Extracts were prepared from the
pulverized plant material according to their
8 Mass Spectroscopy: Herbal Drugs and Fingerprints
uses in traditional medicine. An infusion was
prepared by adding 100 ml of hot water to 5 g of
the plant material. The mixture was kept hot
and left to stand for 10 min, following which the
mixture was filtered under vacuum and the filtrate
cooled and its volume made up to 100 ml with
water. An aliquot of the infusion was washed
with n-hexane and further extracted with ethyl
acetate. The organic phase was collected, dried
over anhydrous sodium sulfate, and concentrated
under vacuum at 30°C until all of the ethyl ace-
tate had been removed.
The infusion and the ethyl acetate fraction
were frozen with liquid nitrogen and then lyo-
philized. A decoction was prepared by adding
100 ml of cold water to 5 g of the plant material
and heating the mixture to boiling for 20 min.
After cooling, the mixture was filtered under vac-
uum and its volume made up to 75 ml with water.
In order to prepare a tincture, 40 ml of 45% aque-
ous ethanol was added to 2.5 g of the plant mate-
rial and the mixture allowed standing at room
temperature for 13 days in the dark. After this
time, the hydroalcoholic extract was filtered and
concentrated under vacuum and its volume made
up to 10 ml with 45% aqueous ethanol. From the
aforesaid studies, it is clear that it is possible to
decipher the traditional anti-inflammatory uses of
E. telmateia at molecular level [3].
Matrix-Assisted Laser Desorption
Ionization
This technique is relatively straightforward to
use and reasonably tolerant to buffers and other
additives. The mass accuracy depends on the
type and performance of the analyzer of the mass
spectrometer, as most modern instruments are
capable of measuring masses to within 0.01% of
the molecular mass of the sample, at least up to
ca. 40,000 Da. Matrix-assisted laser desorption
ionization (MALDI) is based on the bombard-
ment of sample molecules with a laser light to
bring about sample ionization. The sample is
premixed with a highly absorbing matrix com-
pound for the most consistent and reliable result,
and a low concentration of sample to matrix is
Detection and Recording of Fingerprints
Fig. 8.6 Simplified laser
schematic of MALDI-TOF
MS (linear mode) [4]
ions
high
voltage
= matrix and analyte
the best option. The matrix transforms the laser
energy into excitation energy for the sample,
which leads to sputtering of analyte and matrix
ions from the surface of the mixture. In this way,
energy transfer is efficient, and also the analyte
molecules spared excessive direct energy that
may otherwise cause decomposition. Most com-
mercially available MALDI mass spectrometers
now have a pulsed nitrogen laser of wavelength
337 nm [4].
The sample to be analyzed is dissolved in an
appropriate volatile solvent, usually with a trace
of trifluoroacetic acid if positive ionization is
being used. Sinapinic acid is a common one for
protein analysis, while alpha-cyano-4-hydroxy-
cinnamic acid is often used for peptide analysis.
An aliquot of the final solution is applied to the
sample target which is allowed to dry prior to
insertion into the high vacuum of the mass spec-
trometer. The laser is fired, the energy arriving at
the sample/matrix surface optimized, and data
accumulated until an m/z spectrum of reasonable
intensity has been amassed (Fig. 8.6). The time-
of-flight analyzer separates ions according to
their mass (m)-to-charge (z) (m/z) ratios by mea-
suring the time it takes for ions to travel through
a field-free region known as the flight, or drift,
tube. The heavier ions are slower than the lighter
ones. The m/z scale of the mass spectrometer is
calibrated with a known sample that can either be
157
detector
field free region
analyzed independently (external calibration) or
premixed with the sample and matrix (internal
calibration) [4].
MALDI is also a soft ionization method and
so results predominantly in the generation of sin-
gly charged molecular-related ions regardless of
the molecular mass; hence, the spectra are rela-
tively easy to interpret. Fragmentation of the
sample ions does not usually occur. In positive
ionization mode, the protonated molecular ions
are usually the dominant species, although they
can be accompanied by salt adducts, a trace of the
doubly charged molecular ion at approximately
half the m/z value and/or a trace of a dimeric spe-
cies at approximately twice the m/z value. Positive
ionization is used in general for protein and pep-
tide analyses. In negative ionization mode, the
deprotonated molecular ions are usually the most
abundant species, accompanied by some salt
adducts and possibly traces of dimeric or doubly
charged materials. Negative ionization can be
used for the analysis of oligonucleotides and oli-
gosaccharides [4].
Case Study
The problem of adulteration is a key concern
for the herbal-based industries, where botanical
mis-identification and intentional addition of
cheap substitutes are common practices. Final
product quality is entirely based on the quality and
158
consistency of the starting material, subsequent
handling, extraction, and further processing. The
introduction of other botanical and non-botanical
contaminants can occur throughout the process.
Therefore, rapid and cost-effective methods, like
MALDI-TOF MS, can be used to monitor these
materials for complex product purity and consis-
tency, for example, species in the genus Echinacea
(primarily E. purpurea and to a lesser extent E.
angustifolia) are used in literally thousands of
dietary supplements. Product quality and species
identification are generally determined using
HPLC and TLC/HPTLC. Less utilized assays
such as near-infrared spectroscopy (NIR) have
also been developed for Echinacea species
identification and correlation to phytochemical
concentration. All of these techniques have par-
ticular strengths, although in general these meth-
ods are low throughput and require sampling and
drying of relatively large portions of leaf or root.
Automated, high-throughput processing meth-
ods, teamed with matrix-assisted laser desorp-
tion/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) analysis, to differentiate
Echinacea species by their mass profiles, have
been developed. Analysis of these samples high-
lighted key MS signal patterns from both small
molecules and proteins that characterized the
individual Echinacea materials analyzed. Based
on analysis of pure Echinacea samples, off-the-
shelf products containing Echinacea could then
be evaluated in a streamlined process.
Corresponding analysis of dietary supplements
was used to monitor for product composition,
including Echinacea species, and plant materials
used. These results highlight the potential for
streamlined, automated approaches for agricul-
tural species differentiation and botanical prod-
uct evaluation.
Based on the growing maturity and success
of matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrome-
try (MS) for determination of biomarkers in
numerous plant and animal species, research
has been conducted to evaluate this technique as
a higher-throughput assay using relatively small
amounts of fresh and dry plant tissue. For botan-
ical product quality control, MALDI-TOF MS
8 Mass Spectroscopy: Herbal Drugs and Fingerprints
has the potential to address the most challenging
issues of species and adulterant identification
and product consistency, while also having the
potential to support breeding programs and
rapid plant screening for phytochemical quan-
tity and quality [5].
A vital component of higher-throughput
method development is sample preparation prior
to MALDI-TOF MS analysis. There is always
thrust for a user-friendly robotic system that
allows for method development and method
deployment flexibility and the objective to
develop an automated, high-throughput method
to process samples and differentiate Echinacea
species by their MALDI-TOF mass profiles. MS
analysis of plant materials for a variety of
Echinacea species demonstrates the power of
MALDI-TOF MS to generate complex, sample-
specific profiles. Comparison of MS profiles col-
lected from the different plant tissues for a single
Echinacea species or similar plant materials for
the three species characterized to the low-molec-
ular-weight (expanded view, 240–560 Da) and
high-molecular-weight (expanded view, 2,000–
15,000 Da) profiles, respectively, for seed extracts
from the three Echinacea species is analyzed.
While MS fingerprints characteristic of the gen-
eral Echinacea genus can be discerned, species-
specific signal markers can be easily distinguished
within the two mass ranges. The unique
fingerprints generated from the individual plant
materials and Echinacea species analyzed can be
used to evaluate off-the-shelf Echinacea prod-
ucts. The MS patterns were compared between
samples to potentially further characterize or
confirm sample composition. As an example, the
mass spectra for leaf materials from E. angustifo-
lia and E. purpurea, and the Rexall Sundown
herbal supplements, of which E. purpurea is a
component. Specific signal characteristic of E.
purpurea was identified by visual comparison in
the corresponding spectrum from the Sundown
product, along with general Echinacea leaf mate-
rial MS signals (for comparing the E. angustifo-
lia, E. purpurea, and Rexall Sundown profiles).
Comparison of these MS patterns shows that the
composition of Rexall Sundown includes E. pur-
purea aerial parts. In addition to these, a few
Detection and Recording of Fingerprints
low-intensity signal (not characteristic of either
the E. angustifolia or E. purpurea) profiles were
also present within the Rexall Sundown profile
and indicative of an additional compounds present
in the supplement [5].
Dried Echinacea materials including E. pur-
purea, E. pallida, and E. angustifolia seeds, roots,
and aerial parts used in this study were from
American Herbal Pharmacopoeia. Off-the-shelf
products included Echinacea supplements from
Nature’s Resource Products (Mission Hills, CA)
and Rexall Sundown, Inc. (Boca Raton, FL).
MALDI matrixes a-cyano-4-hydroxycinnamic
acid (CHCA) and 3, 5-dimethoxy-4-hydroxycin-
namic acid (sinapinic acid, SA) were purchased
from Fluka (Sigma-Aldrich Switzerland). Solvents
included HPLC grade acetonitrile obtained from
Honeywell Burdick & Jackson and sequencing
grade triflouroacetic acid (TFA) from Fluka.
A Milli-Q gradient water system was used for
molecular-grade water. This buffer and phenyl-
methanesulfonyl fl ouride (PMSF) were from
Sigma-Aldrich [5].
Three types of plant tissues – seed, root, and
aerial parts (leaf/stem/flower) – and two commer-
cial Echinacea supplements were analyzed in
this study. The commercial supplements were of
composition of the various Echinacea tissues
analyzed. Samples were prepared in triplicate,
approximately 100 mg of each, in 2.0-ml conical
screw cap micro tubes. Roots were shaved prior
to weighing. Samples of Echinacea supplements
from Nature’s Resource Products and Rexall
Sundown, Inc. were extracted from capsules [5].
MALDI-TOF-MS analysis was performed on
a Voyager-DE STR MALDI-TOF mass spec-
trometer. Analysis of CHCA prepared samples
was performed in reflector mode, over the mass
ranges 50–600 (300 shots/sample) and 600–
5,000 Da (500 shots/sample). Samples prepared
with SA were analyzed in linear mode, over the
mass range 2,000–60,000 Da (500 shots/sample).
External mass standards were used to calibrate
the data [5].
Small molecules, peptides, and proteins from
aerial parts, seeds, and roots from three Echinacea
species (E. purpurea, E. pallida, and E. angusti-
folia) and two off-the-shelf supplements com-
159
posed of specific Echinacea tissues were extracted
using streamlined, automated instrumentation for
downstream sample profiling by MALDI-TOF
MS. Samples were homogenized using a mini
Bead-Beater, and the supernatant was collected,
diluted, and deposited onto a MALDI target plate
using a ProPrep liquid-handling system.
Automated sample analysis was then performed
on the mass spectrometer, resulting in profile-rich
spectra for the individual samples analyzed. This
approach allowed for a systematic sample-prepa-
ration method that significantly improved sample
throughput, preparation reproducibility, and data
reliability [5].
Using these procedures, sample-preparation
time was reduced by half. The ProPrep performed
sample preparation and deposition tasks for 32
plant extracts and 2 blanks in approximately
30 min, compared to a manual preparation time
of over 60 min. The ProPrep liquid-handling
steps included the following: (1) transferring
supernatant from the homogenized samples
(Extract-1) to a clean microtiter plate (Collection
1), (2) transferring MALDI matrix (CHCA or
SA) from the solution vial to a second microtiter
plate (Collection 2), (3) pipetting an extract ali-
quot from Collection 1 to an individual well in
Collection 2, (4) mixing the extract/matrix solu-
tion in Collection 2, and (5) depositing an aliquot
of the extract/matrix solution onto a clean MALDI
target plate. Robust and reproducible sample
preparation and MS data were obtained using
these procedures. Final MALDI sample deposi-
tion performed by this instrument produced
reproducible, well-formed droplets, properly
located and without overlap. MALDI-TOF MS
analysis over the 50–600 m/z range resulted in
spectra with over 200 small-molecule compounds
detected. Sample analysis performed in triplicate
demonstrates the reproducibility of the sample-
preparation and MS analysis techniques used.
Triplicate analysis of Echinacea samples indi-
cates high reproducibility. Similar mass spectra
are observed for the individual preparation, and
MS analyses performed for the E. pallida root
replicate. Small signal-intensity differences are
identified between the triplicate analyses; how-
ever, the similarity in molecular profile patterns
160
was enough to be easily distinguished. Similar
results were seen for analysis of the various plant
extracts over the mass ranges 50–600, 600–5,000,
and 2,000–60,000 Da.
MS analysis of plant materials for a variety
of Echinacea species demonstrates the power
of MALDI-TOF MS to generate complex,
sample-specific profiles. Comparison of MS
profiles collected from the different plant tis-
sues for a single Echinacea species or similar
plant materials for the three species is charac-
terized. The highlights of the species specificity
of the profiles generated show the low-molecu-
lar-weight (expanded view, 240–560 Da) and
high-molecular-weight (expanded view, 2,000–
15,000 Da) profiles, respectively, for seed
extracts from the three Echinacea species ana-
lyzed. While, MS fingerprints characteristic of
the general Echinacea genus can be discerned,
species-specific signal markers can be easily
distinguished between two mass ranges [5].
Using the aforesaid techniques, larger sam-
ple database can be prepared for other plants
and plant materials sampled for their possible
by-product due to degradation and plant materi-
als used during supplement composition, and
materials and products following stability test-
ing would enhance capabilities to monitor and
characterize botanical products. The ability to
analyze large numbers of samples quickly using
these technologies makes them ideal tools for
botanical product analysis and biomarker dis-
covery. Fingerprint comparisons, based on a
library of analyzed materials and by-product,
yield the opportunity to characterize raw mate-
rials and products. The data gathered from
large sample sets can be subjected to multi-
variant statistical analysis methods for pattern
identification. Further analysis can also be per-
formed to identify, characterize, and validate
potential biomarkers. The techniques presented
show promise for characterizing botanical
materials and products for important aspects
such as product quality control, including
species and adulterant identification; product
consistency and stability, as well as the ability
to support plant-breeding programs; and early
8 Mass Spectroscopy: Herbal Drugs and Fingerprints
rapid screening for phytochemical quantity and
quality [5].
In pharmaceutical industry, LC–MS has
become method of choice in many stages of drug
development. Recent advances include electro-
spray, thermospray, and ion spray ionization
techniques which offer unique advantages of high
detection sensitivity and specificity, liquid sec-
ondary ion mass spectroscopy; later, laser mass
spectroscopy with 600 MHz offers accurate
determination of molecular weight proteins, pep-
tides. Isotopes pattern can be detected by this
technique.
Hyphenated Techniques
Hyphenated chromatography-MS systems emp-
loyed for metabolomic fingerprinting consist of
three parts. The first part is the chromatographic
separation section, used to separate complex
mixtures obtained from the plant material. The
most commonly used chromatographic systems
are HPLC, GC, or CE. The second part consists
of the ionization chamber where the molecules
are being ionized by different ionization sources
such as electron ionization (EI), electrospray
ionization (ESI), atmospheric pressure chemical
ionization (APCI), and atmospheric pressure
photoionization (APPI). Another ionization source
being used, but not in combination with flow-
based systems, is matrix-assisted laser desorp-
tion/ionization (MALDI). The third part consists
of the MS where the ionized molecules are being
detected as their mass-to-charge ratio. The most
commonly used MS systems include single
quadrupole, triple quadrupole, ion trap, and time-
of-flight spectrometers. More advanced high-
resolution spectrometers include the orbitrap or
the Fourier transform cyclotron. For a targeted
approach, MS is usually employed as the detec-
tor, while nuclear magnetic resonance (NMR)
and MS are used without the chromatography
step as an untargeted analytical tool. There is little
difference in the sample preparation and data
handling between MS-based and NMR-based
technologies, although the solvents being used
Bibliography
differ between the two analytical systems. NMR
makes use of deuterated solvents, while MS uses
non-deuterated solvents [6].
In MS, ionized components are identified and
quantified at very low quantities making the MS a
very sensitive analytical tool for metabolic
fingerprinting. The lack of ionization and there-
fore the inability to quantify or identify compo-
nents that do not ionize limits the use of MS to the
more targeted metabolic profiling techniques.
Various probes are being employed to overcome
this inherent ionization problem in order to
increase the dynamic range of components that
can be detected with the use of MS. Other limita-
tions of MS-based technologies are that the
equipment needs to be calibrated regularly and
fragmentation of molecules occurs which compli-
cates the mass spectra. Fragmentation does how-
ever lead to structural information which in turn
leads to identification of unknown components
with the use of MS–MS-based technologies [6].
GC equipment can be directly interfaced with
rapid scan mass spectrometer of various types.
In GC, the flow rate from capillary column is
generally low enough that the column output can
be fed directly into ionization chamber of MS.
The simplest mass detector in GC is the ion trap
detector (ITD). In this instrument, ions are cre-
ated from the eluted sample by electron impact or
chemical ionization and stored in a radio fre-
quency field. The trapped ions are then ejected
from the storage area to an electron multiplier
detector. The ejection is controlled so that scan-
ning on the basis of mass-to-charge ratio is
possible. The ion trap detector is remarkably
compact and less expensive than quadrupole
instruments. GC–MS instruments have been used
for identification of hundreds of components that
are present in natural and biological system [7].
MS is an instrument which can provide struc-
tural information and molecular weight of the
compound under study. With a clear understand-
ing of mass spectrometry and the various factors
governing the fragmentation process, a meaning-
ful interpretation of the mass spectrum can be
achieved. High-resolution instruments, including
double focusing and FT-ICR mass spectrometers,
are capable of determining the exact mass of ion.
161
High-resolution mass spectrometry can distin-
guish compounds with the same nominal mass
but different exact mass caused by different ele-
mental composition. The exact structural eluci-
dation of a compound calls for hyphenated
techniques, and each one provides a vital piece of
information. It is a collective pooling of all the
information that are collected from various equip-
ments that ultimately results in the total under-
standing of an unknown molecule.
References
1. Gaskell SJ. Electrospray: principles and practice. J
Mass Spectrom. 1997;32:677–88.
2. Wang Y, Yang L, He Y, Wang H, Welbeck EW, Annie
Bligh SW, Wang Z. Characterization of fifty-one
flavonoids in a Chinese herbal prescription longdan
xiegan decoction by high-performance liquid chroma-
tography coupled to electrospray ionization tandem
mass spectrometry and photodiode array detection.
Rapid Commun Mass Spectrom. 2008;22:1767–78.
3. Correia H, González-Paramás A, Amaral MT, Santos-
Buelga C, Batista MT. Characterisation of polyphenols
by HPLC-PADESI/MS and antioxidant activity in
Equisetum telmateia. Phytochem Anal. 2005;16:380–7.
4. Hillenkamp F, Karas M, Beavis RC, Chait BT. Matrix-
assisted laser desorption/ionization mass spectrometry
of biopolymers. Anal Chem. 1991;63:1193.
5. Greene LA, Issa I, Gray DE, Schwartz SA. Streamlining
plant sample preparation: the use of high-throughput
robotics to process Echinacea samples for biomarker
profiling by MALDI-TOF mass spectrometry. J Biomol
Tech. 2007;18(4):238–44.
6. Kooy FV, Maltese C, Cho YH, Kim HK, Verpoorte R.
Quality control of herbal material and phytopharma-
ceuticals with MS and NMR based metabolic
fingerprinting. Planta Med. 2009;75:763–75.
7. Patil PS, Shettigar R. An advancement of analytical tech-
niques in herbal research. J Adv Sci Res. 2010;1(1):8–14.
Bibliography
Chen XJ, Ji H, Zhang QW, Tu PF, Wang YT, Guo BL, Li
SP. A rapid method for simultaneous determination of
15 flavonoids in epimedium using pressurized liquid
extraction and ultra-performance liquid chromatogra-
phy. J Pharm Biomed Anal. 2008;46:226–35.
Dunn BW. Current trends and future requirements for the
mass spectrometric investigation of microbial, mamma-
lian and plant metabolomes. Phys Biol. 2008;5:1–24.
Grata E, Boccard J, Guillarme D, Glauser G, Carrupt PA,
Farmer EE, Wolfender J-L, Rudaz S. UPLC-TOF-MS
162
for plant metabolomics: a sequential approach for
wound marker analysis in Arabidopsis thaliana. J
Chromatogr B. 2008;871:261–70.
Ho HM, Chen R, Huang Y, Chen ZY. Vascular effects of a
soy leaves (Glycine max) extract and kaempferol gly-
cosides in isolated rat carotid arteries. Planta Med.
2002;68:487–91.
Hope JL, Prazen BJ, Nilsson EJ, Jack RM, Synovec RE.
Comprehensive two-dimensional gas chromatography
with time-of-flight mass spectrometry detection: anal-
ysis of amino acid and organic acid trimethylsilyl
derivatives, with application to the analysis of metabo-
lites in rye grass samples. Talanta. 2005;65:380–8.
Kang J, Zhou L, Sun J, Han J, Guo DA. Chromatographic
fingerprint analysis and characterization of furocou-
marins in the roots of angelica dahurica by HPLC/
DAD/ESI-MSn technique. J Pharm Biomed Anal.
2008;47:778–85.
8 Mass Spectroscopy: Herbal Drugs and Fingerprints
Seger C, Sturm S. Analytical aspects of plant metabolic
profiling platforms: current standings and future aims.
J Proteome Res. 2007;6:480–97.
Shin MH, Hong MK, Kim WS, Lee YJ, Jeoung YC.
Identification of a new analogue of sildenafil added
illegally to a functional food marketed for penile
erectile dysfunction. Food Addit Contam.
2003;20:793–6.
Thiocone A, Farmer EE, Wolfender JL. Screening for
wound-induced oxylipins in Arabidopsis thaliana
by differential HPLC-APCI/MS profiling of crude
leaf extracts and subsequent characterisation by
capillary- scale NMR. Phytochem Anal. 2008;19:
198–205.
Zhong DF, Xing J, Zhang SQ, Sun L. Study of the electro-
spray ionization tandem mass spectrometry of
sildenafil derivatives. Rapid Commun Mass Spectrom.
2002;16:1836–43.
 NMR Spectroscopy: Herbal Drugs
and Fingerprints
Nuclear magnetic resonance (NMR) spectroscopy
was first developed in 1946 by research groups
at Stanford and MIT, in the USA, since that day
it has developed into the premier organic spec-
troscopy available to scientists to determine the
detailed chemical structure and new drug dis-
covery. Another well-known product of NMR
technology has been the magnetic resonance
imaging (MRI), extensively utilized in the medical
radiology to obtain image slices of soft tissues.
In recent years, NMR has moved out from
research laboratories to the on-line process anal-
ysis market. The NMR spectroscopy is based on
the fact that nucleus of atom has magnetic prop-
erty that can be utilized to yield chemical infor-
mation. As electrons have spin around nucleus,
these spins are paired in some atoms (e.g., 12C,
16
O, 32S) and cancel the effect of each other, so
that the nucleus of the atom has no overall spin.
However, in many atoms (1H, 13C, 31P, 15N, 19F,
etc.) nucleus possesses an overall spin. If an atom
has even number of neutrons and protons, the
nucleus has no spin, but if the number of neutrons
plus the number of protons is odd, then the
nucleus has a half-integer spin (i.e., 1/2, 3/2, 5/2).
But when the number of neutrons and the number
of protons are both odd, then the nucleus has an
integer spin (i.e., 1, 2, 3).
When we apply an external magnetic field to
the atom, the nucleus may align with it or in
opposite direction. Each nucleus has a character-
istic value of magnetogyric ratio (g), which is
defined as a constant of proportionality between the
nuclear angular momentum and magnetic moment.
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_9, © Springer India 2012
9
For a proton magnetogyric ratio, (g ) = 2.674 × 104
gauss−1 sec−1. This precession process generates
an electric field with frequency precession rate
(wo) (also known as Larmor frequency). When
we irradiate the sample with radio waves (in the
MHz frequency range), the proton absorbs the
energy and is promoted to the less favorable higher
energy state. This stage is called resonance because
the frequency of the applied radiation and the
precession coincide or resonate. The field strength
of a magnet is usually reported at the resonance
frequency for a proton. Therefore, for different
nuclei with different gyromagnetic ratios, differ-
ent frequencies are needed in order to achieve
resonance. The orientations at which a nucleus
magnetic moment can take against an external
magnetic field are not of equal energy. Spin states
which are oriented parallel to the external field
are lower in energy than in the absence of an
external field. In contrast, spin states whose ori-
entations oppose the external field are higher in
energy than in the absence of an external field.
Due to resonance, there is a possibility to
induce a transition between the various spin states.
By irradiating the nucleus with electromagnetic
radiation of the correct energy (as determined
by its frequency), a nucleus with a low-energy
orientation can be induced to transition to an
orientation with a higher energy. The absorption
of energy during this transition forms the basis of
the NMR method, as other spectroscopic methods
(as IR and UV–Vis.). The amount of signal inten-
sity is proportional to the population difference
between the two energy levels involved. Energy
163
164
levels have a large energy difference in case of
UV–Vis. spectroscopy, but in NMR, the energy
separation of the spin states is comparatively very
small, even it is difficult to distinguish from noise
level. This property leads to difficulties in using
NMR for trace analysis, so NMR is not consid-
ered accurate below a quantitation of 0.05 wt.%
(500 ppm). There are two general types of NMR
instrument: continuous wave (CW) and Fourier
transform (FT). Early experiments were con-
ducted with CW instruments, and in 1970 the first
FT instruments became available and now domi-
nates the market.
In a molecule different protons have different
electronic environments and experience slightly
different applied magnetic fields owing to the
shielding /de-shielding effect of the induced elec-
tromagnetic fields. In order to standardize the
NMR scale, it is necessary to set a “zero” refer-
ence point to which all protons can be compared.
The standard reference selected is tetramethylsi-
lane (TMS). This compound has four methyl
(−CH3) groups bonded (i.e., single bonded) to a
silicon atom. All of the protons on the methyl
groups are in the same electronic environment.
Therefore, only one NMR signal generates. The
electronegativity of the carbon atoms is actually
higher than the silicon atom to which they are
bonded, resulting sigma-electrons shifted toward
the carbon atoms in the methyl groups and, conse-
quently, the heavily shielded protons causing one
signal at a very high magnetic field strength. So
this NMR signals works as reference. Any differ-
ence against the reference signal is called the
chemical shift. This shift is measured in parts per
million (ppm). NMR signals occurring near the
TMS resonance are said to be in an upfield
position, while those shifted away by de-shielding
are said to be downfield. All NMR signals are
downfield from the TMS signal because of the
heavily shielded nature of the methyl protons in
TMS molecule. The proton NMR chemical shift
range is 0–12 ppm. The ppm scale is another form
of standardization that allows one to compare
directly the 1H spectra obtained on NMR instruments
with different magnetic fields. With referenced to
the TMS, resonance is 0 ppm; the actual NMR
peak position in Hz is divided by the resonance
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
frequency of the spectrometer, which is in MHz.
Thus, one is dividing Hz by MHz which is a part
per million (ppm). One ppm on a 58-MHz NMR
instrument is actually 58 Hz from the resonance
position of TMS, while on a 300-MHz NMR
instrument 1 ppm is 300 Hz from the TMS reso-
nance position. With this standardization/normal-
ization in place, one can always unequivocally say
that all benzene protons resonate at 7.16 ppm no
matter what NMR instrument is being used in
the analysis. A few considerable points are as
follows: (1) Electronegative groups are de-shielding
and tend to move NMR signals from neighboring
protons further downfield (to higher ppm values).
(2) Protons on oxygen or nitrogen have highly
variable chemical shifts, which are sensitive to
concentration, solvent, temperature, etc. (3) The
system of alkenes, aromatic compounds, and car-
bonyls strongly de-shields attached protons and
moves them downfield to higher ppm values. In
most spectroscopic techniques the energy absorp-
tion by the sample is not a primary concern, but in
NMR, the energy absorption is a matter of pri-
mary concern, and the NMR process is so-called
an absorption process, where nuclei in the excited
state must be able to relax and return to the ground
state. The timescale for this relaxation is crucial to
the NMR studies as relaxation of electrons to the
ground state in UV–Vis. spectroscopy is a very
fast process, on the order of picoseconds, but in
NMR, the excited state of the nucleus can persist
for minutes. Because the transition energy between
spin levels is so small, attaining equilibrium has
much longer timescale (i.e., the relaxation period).
Such stereochemical data have high potential to
decipher the active ingredients of traditional
herbal drugs and lined with the modern medical
practices. Traditional herbal drugs are mainly in
raw and semi-standardized form and often based
on empirical evidence, so certainly NMR spec-
troscopy has an important role, for selecting herb-
als and products of potential therapeutic interest,
and offers tremendous opportunities to discover
new drugs of herbal origin [1]. Herbals and their
products in traditional practices (such as Ayurveda,
Unani, Kampo, TCM) are chemically complex
mixture and contain one or many structurally related
active compounds that produce a combined effect.
Metabolic Fingerprints
Studies at molecular level help in standardizing
the herbal preparations to an optimal concen-
tration of active constituents and preserving
their activities. The marker system used in chro-
matography is not sufficient alone to decipher the
stereochemistry of the molecule. NMR-based
fingerprints are additional supporting documents
for the chemical profile of the product. The
fingerprint pattern approach is quite often used
by the pharmaceutical industry to examine the
source of the drug substance and the method of
chemical preparation, as a “phytochemical logo.”
Example may be cited from an important medici-
nal plant of Indian system of medicine (ISM),
Withania somnifera (ashwagandha, Indian gin-
seng, winter cherry), studied by using NMR and
chromatographic (HPLC and GC–MS) tech-
niques. A total of 62 major and minor, primary
and secondary metabolites from leaves and 48
from roots were unambiguously identified. Twenty-
nine of these were common in the tissues of
both parts. These included fatty acids, organic
acids, amino acids, sugars, and sterol-based
compounds. Between leaves and roots, highly
significant, qualitative and quantitative differ-
ences were noticed, particularly with respect to
the secondary metabolites. W. somnifera is used
in Ayurveda for over 3,000 years, in various
forms (decoctions, infusions, ointments, powder,
and syrup), and presently used in different parts
of the world for all age groups of patients without
any side effects even during pregnancy. The
extracts as well as different isolated bioactive
constituents of W. somnifera have been reported
to possess adaptogenic, anticancer, anticon-
vulsant, immunomodulatory, anti-oxidative, and
neurological effects. The plant is also considered
efficacious in the treatment of arthritis, geriatric,
behavioral, and stress-related problems. Several
bioactive alkaloids and sterol lactone-based
phytochemicals (e.g., ashwagandhine, cuscohy-
grine, isopelletierine, anaferine, anhygrine,
tropine, sitoindosides), as well as saponins (with-
anolides, withanamides, and glycowithanolides),
have been isolated from different parts of this
plant [1]. The application of NMR spectral
fingerprints in herbal-based industry may be cited
as below.
165
Metabolic Fingerprints
Technological advances in analytical systems
like NMR, GC–MS, and HPLC have opened up
new avenues to understand the complex bio-
chemical processes within a plant and total anal-
ysis of metabolome of a plant. This has become
an important area of investigations in pharmacol-
ogy and functional genomics of medicinal plants.
Comprehensive chemical analysis establishes the
correlation between complex chemical mixtures
and molecular pharmacology, along with com-
plex cellular processes and biochemical pathways
via metabolite-to-gene network [2].
Case Study 1
In view of the aforesaid objectives, leaves and
root tissues of W. somnifera were extracted with
n-hexane followed by warm (~35 °C) methanol–
water (90–70% methanol, stepwise successively).
After liquid–liquid partition of methanolic water
portion with chloroform followed by n-butanol,
metabolite repertoire of the plant was distributed
into four fractions of different polarities
(n-hexane, aqueous-methanol, chloroform, and
n-butanol). Each fraction was then subjected to
NMR and GC–MS analysis and sometimes to
HPLC–PDA analysis. Metabolic content of leaf
and root tissues is extracted by n-hexane, chloro-
form, n-butanol, and methanolic water; the quantity
of metabolite in leaves was much higher than that
in roots, particularly in the aqueous–methanolic
fraction. The metabolomic analysis and its inter-
pretation, for different fractions, were carried out
as below [3].
n-Hexane Fraction
The metabolomic analysis of n-hexane extract of
W. somnifera leaves and roots was performed by
NMR spectroscopy and GC–MS. 1H-NMR spec-
tra of both leaf (Fig. 9.1) and root extracts predom-
inantly have different saturated and unsaturated
fatty acids. Signals at d 1.6 and at d 2.3 represented
the b-CH2 and a-CH2 of the fatty acids [4]. The
signals of all other protons of hydrocarbon chain
166 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

Pheophytins a
Pheophytins b

Pheophytins a
Pheophytins a

signals of saturated alkyl chain of fatty acids

Methyl (terminal) signal of Linolenic acid (Specific)

11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 ppm

Carotenoids

Porphyrins
Me signals of fatty acids
6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 ppm

Pheophytins a

Pheophytins a
TAG FAME

Porphyrins
β−CH2 of fatty acids
4.30 4.25 4.20 4.15 ppm 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 ppm −1.5 −1.4 ppm
allylic CH2
Olefinic protons of fatty acids
α−CH2 of fatty acids
di allylic CH2 of PUFA

sterol
11 10 9 8 7 6 5 4 3 2 1 0 −1 ppm

Fig. 9.1 1H-NMR spectra of n-hexane extract of W. somnifera leaves [3]

of fatty acids appeared at d 1.3. The appearance of double doublet (dd) signals (d 4.1–4.3) of sn1
olefinic protons at d 5.35 indicated the presence of and sn3 protons of triacylglycerol (TAG). Minor
unsaturated fatty acids, whereas signals at d 2.07 amounts of methyl esters of fatty acids were
indicated the allylic protons of unsaturated fatty indicated by singlet (for O–Me group) signals at d
acid. In addition, the characteristic bis-allylic sig- 3.6. The characteristic signal of 18-CH3 group of
nals (triplet) of di- and tri-unsaturated fatty acids sterol appeared distinctly at d 0.7. Several signals
appeared at d 2.8. Homodecoupling experiment of at d 6.0–6.5 might be attributed to the carotenoids.
the olefinic protons (d 5.35) altered the triplet sig- Integration ratio with respect to trimethylsilyl
nals of bis-allylic protons into two distinguished propionate (TSP) signals indicated that the caro-
singlets at d 2.77 and d 2.83, indicating the pres- tenoid content of leaf extract was higher than that
ence of di- and tri-unsaturated fatty acids. The of root. This is expected, as major role of carote-
18:3 fatty acids were identified by their character- noids in leaves is to protect leaf from excessive
istic triplet methyl signals at d 0.99 particularly in light stress. It was further observed that percent-
the spectrum of leaf n-hexane extract. The age of TAG was much higher in root extract than
downfield shift of methyl signals for 18:3 (lino- in leaves. The presence of two signals in the
lenic acid) fatty acids occurred due to proximity of upfield region (d 1.43 and d 1.6) of the spectrum
the unsaturated double bond. of leaves was a characteristic for N–H group of
Terminal methyl signals of other fatty acids the porphyrins. Pheophytins are the degraded
appeared collectively at d 0.90. In case of root products of chlorophylls. During metabolite
samples, the signals of the terminal methyl were extraction, the chlorophylls lose their magnesium
not very distinct due to overlap. The 1H-NMR ions and become pheophytins. The signals of part
spectrum of n-hexane extract showed characteristic of phytal fragments [−O–CH2–CH=C(CH3)–] of
Metabolic Fingerprints 167

Overlaping Signal of β sitosterrol and C-21 Me of

WF-A and WN
C-19 Me (WF-A)

C-19 Me (WF-A)
C-19 Me (WN)

C-18 Me (WN)
C-28Me (WF-A)
C-28Me (WN)
C-27 Me (WN)
C-22H (WF-A)
C2-H (WF-A)

C-7H (WN)
C-4H (WF-A)

C-6H (WF-A)
C-6H (WN)
C-3H (WF-A)

C2-H (WN)

C-22H (WN)
C-3H (WN)

Un assigned
Un assigned
Un assigned
Un assigned
Un assigned

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

Fig. 9.2 1H-NMR spectrum of chloroform fraction of the W. somnifera leaves. WF A = withaferin A, WN = withanone [3]

chlorophylls and other parts of pheophytins also respective molecular ion peak in the mass spec-
appeared at d 9.52, 9.35, 8.5, 8.0, 3.4, and 3.24. trum together with the characteristic peak at m/z
The ratio of chlorophylls a and chlorophyll b was 74 (base peak) that appeared due to McLafferty
determined as 3:1 by integration of singlet sig- rearrangement and the peak at m/z 87 that
nals at d 9.37 and d 9.55. Other minor signals in appeared due to loss of (CH2)2COOCH3+. Other
the range of d 11–7 may have appeared due to respective logically defined mass fragments also
oxidized products of chlorophylls. The observed appeared in the spectrum. Presence of campes-
signals of all the protons and the corresponding terol and stigmasterol at tR 53.09 and 53.35 was
13
C signals were identified by 1H−13C heteronu- indicated in the GC–MS analysis, particularly in
clear single-quantum (HSQC) mapping. To deter- the n-hexane extract of root [3].
mine the composition of individual fatty acids
and sterols, the n-hexane extract was subjected to Chloroform and n-Butanol Fractions
1
GC–MS analysis after esterification (methyl H-NMR spectrum of chloroform fraction of the
ester). Palmitic acid (16:0), oleic acid (18:1), W. somnifera leaf extract (Fig. 9.2) has presence
linoleic acid (18:2), and linolenic acid (18:3) of double doublet at d 6.8 and d 6.5 together with
were major fatty acids present in the leaf and root distinguished doublet signals at d 6.3 and d 5.8
samples. These fatty acids belong to membrane that indicated the presence of withanolide skele-
lipids of plant tissues. Percentage peak area of ton. Characteristic singlet signals of methyl series
the GC chromatograms revealed that palmitic of withanolide framework were observed in the
acid and linolenic acid were the predominant range of d 0.6–2.2. Comparison of the spectrum
fatty acids present in the leaves, whereas roots with the purified withanolide standards estab-
were richer in palmitic acid and linoleic acid. The lished the presence of withaferin A and witha-
GC–MS analysis further suggests the presence of none in the mixture. The 13C signals of each
many other minor but very long-chain fatty acids, metabolite were recognized by 1H-13C HSQC
particularly in the root samples, indicating higher spectrum and compared with the literature data
activity in the stearoyl-CoA elongation activity in of the pure compounds. Both 1H and 13C signals
the root tissue as compared to the leaf tissue. clearly indicated the presence of withaferin A
Presence of each fatty acid was indicated by and withanone as the major metabolites in the
168
chloroform fraction of leaves. Branch signals
at d 0.9 of the 1H spectrum appeared due to ali-
phatic chain of b-sitosterol. GC–MS analysis of
the fraction indicated the presence of b-sitosterol.
There were some unassigned signals appearing in
the range of d 5.2–5.7 of the 1H-NMR spectrum
which may be due to the presence of minor
amounts of withanolides in the mixture. The cor-
responding 1H-NMR spectrum of the root was
not distinct like leaf, but it was clear enough to
indicate the presence of withanolide skeleton.
The 1H signals at d 6.6 (m), 5.85 (d), 4.2 (m),
1.95 (s), l.85 (s), 1.2 (s), and 0.95 (s) and corre-
sponding 13C signals at d 12.0, 13.0, 14.0, 22.0,
57.0, 129.5, and 140.0 were identified by HSQC
spectrum, providing sufficient evidence for the
presence of withanolide A. Broadness of the
spectrum indicated the presence of significant
amounts of similar type of compounds. HPLC
analysis of the chloroform partition of both
leaf and root samples was performed for further
identification of other NMR-nonidentifiable witha-
nolides. Assignments of resultant chromatograms
were performed by matching with the chromato-
gram of purified withanolides and, subsequently,
by cochromatography [3].
The analysis suggested that withaferin A and
withanone are the major metabolites present in
the leaf as shown by NMR, and withanolide A
and withanone are major metabolites in the root.
Other metabolites detected are 27-hydroxy with-
anone, 17-hydroxy-27-deoxy withaferin A,
27-hydroxy withanolide B, 27-deoxy withaferin
A, and 12-deoxy withastramonolide. Each of
the withanolides was quantified by HPLC using
the calibration curve of the standard samples. The
qualitative and quantitative data on the metabo-
lites were established by NMR and HPLC. The
NMR analysis was not enough to provide any
useful information about chemical constituents
of the n-butanol fraction. The HPLC–PDA analy-
sis of the n-butanol fraction of leaf is indicating
the presence of physagulin, withanoside IV,
and withanoside VI. However, chromatogram of
same fraction of root is cumbersome but reason-
able enough to indicate the presence of withano-
side IV and withanoside VI. Presence of these
compounds was further confirmed by cochro-
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
matography with standard. To explore further the
metabolic composition of chloroform and
n-butanol fractions, GC–MS analysis was carried
out, which indicates the presence of 1-octanol,
different aromatic alcohols, and aromatic acids.
Presences of these compounds are logically sup-
ported by their respective mass fragmentation
patterns obtained from GC–MS analysis [3].
Aqueous Fraction
The metabolic profiling of aqueous fraction of W.
somnifera was analyzed mainly by 1H-NMR
(Fig. 9.3), though in case of root samples, GC–MS
was also applied. Assignment of the compounds
was thoroughly done comparing the 1H spectra of
reference compounds together with Biological
Magnetic Resonance Data Bank (http://www.
bmrb.wisc.edu/metabolomics/) and wherever
necessary, by spiking with appropriate internal
standards. Two-dimensional (2D) correlation
spectroscopy (COSY) and HSQC spectra were
also extensively used to resolve the complexity of
the overlapping/interfering spectral regions to
identify the exact molecule in the extract [3].
The entire 1H spectrum of aqueous fraction
may be divided into three major regions. d 0.0–
3.5 region is rich with amino acids. d 3.5–5.5
contains sugars, and rest of the spectrum is domi-
nated by aromatic compounds. In the 1H-NMR
spectrum of the leaves, the region of amino acids
started with distinct triplet signals of isoleucine,
followed by two sharp doublets of valine. The
doublet signals of two d-CH3 (0.95, 0.96) leucines
were detected by enlarging the spectral segment
using Bruker X-WIN–NMR software. Multiplet
signals of g-CH2 of isoleucine appeared at d 1.35,
and it also shows distinct correlation spectros-
copy (COSY) interactions with d -CH3 of isoleu-
cine. Complex multiplet signal at d 1.48 was
observed due to overlapping signals of b-CH3 of
alanine (doublet) and multiplet signals of g-CH2
of isoleucine. Branch broad multiplet signals at d
1.6–1.8 may be due to the presence of ornithine
in the extract. The presence of g-aminobutyric
acid (GABA) in the mixture was indicated by
characteristic signals at d 1.90 (m, b-CH2), 2.29
(t, a-CH2), and 3.01 (t, g-CH2) and then distinct
correlation in COSY spectrum. Characteristic dd
Metabolic Fingerprints 169

Phenyl alanine

Fumanic acid
Trigonelline

Trigonelline

Trigonelline

Tyrosine
Tyrosine
Uracil

Uracil
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 ppm

Choline
Glycine
Malic acid
Tartaric acid
β−glucose

Threonine
α−glucose

Lactate
5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 ppm

Isoleucine
succinate

Isoleucine
Threonine
Lactate
GABA

Isoleucine
Malic acid

Alanine

Lactate
GABA

GABA

Valine
Glutamate
Glutamine
Aspargin

Valine
Glutamate
Asparatate

Orinithine

3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 ppm

Fig. 9.3 1H-NMR spectrum of the aqueous fraction of W. somnifera leaves [3]

signals of malic acid appeared at d 2.47–2.68 J = 7.9 Hz; a, d 5.2 and J = 3.8 Hz). GC–MS
(geminal protons), and it showed the expected analysis further indicated presence of other sugars,
correlation with dd signals of neighboring pro- that is, galactose, N-acetylglucosamine, and myo-
tons at d 4.34 (H attached with C–OH). Respective inositol in the extract. The singlet signals at d 4.4
carbon signals appeared at d 43.5 and d 71.5. The appeared due to the presence of tartaric acid. 1H
dd signal at d 2.68–2.79 is indicative of b-CH2 of signals of threonine and lactate generally appeared
aspartate, whereas similar signals at d 2.85–2.90 side by side at d1.33 and d 4.2 due to their low
suggested the presence of asparagine. Strong sin- abundance in the extract. The characteristic signals
glet signal at d 2.40 was the signature of succinic were not clearly observed in the 1D spectrum,
acid. The presence of glutamine and glutamate in but in the 2D-COSY spectrum they appeared dis-
the water part was identified mainly by COSY tinctly. The respective carbon peaks appeared at d
cross peak. Overlapping signals of associated 22.5. Strong singlet signals at d 5.8 indicated one
protons appeared in regions at d 2.0–2.44. Strong of the olefinic protons of uracil and showed clear
singlet signal at d 3.2 is indicative of N(Me3)3 of cross peak at d 7.85 corresponding to olefinic pro-
choline in the extract. Related carbon signals tons signals. Sharp singlet around d 6.5 represented
appeared at d 55.1 and d 75.3. The 1D spectral fumaric acid in the extract. Signals at d 6.88 and d
range at d 3.2–4.2 of the carbohydrate region is 7.18 were assigned to tyrosine, which was sup-
highly congested. It was very difficult to identify ported by COSY experiments. The branch spectral
any particular signals, but all the carbon signals band from d 7.3 to d 7.45 regions and correspond-
related to carbohydrate skeleton were relatively ing COSY analysis suggested the occurrence of
distinct (d 63.0, 73.0, 74.2, 76.6, 78.2, and 96.9) phenylalanine. The signatures of trigonelline were
in the HSQC spectrum. Overall nature of the observed at d 8.1, 8.8, and 9.1 ppm [3].
1
spectrum suggested the presence of high amount H spectral complexity (overlapping signals)
of sugars in the extract. However, the presence of did not allow quantification of all the metabolites.
a- and b-anomers of glucose was clearly identified However, a number of them were quantified by
by their respective doublet signals (b, d 4.61 and integrating the distinct characteristic signals of
170
each metabolite with respect to signal intensity of
quantified amount of TSP (trimethylsilyl propi-
onate). NMR spectroscopy of the aqueous ali-
quots of root samples was not distinctly
informative as very high concentration of sugar
in this fraction masked other minor signals.
However, GC–MS analysis of the extract of root
samples indicated the presence of higher amounts
of fructose, galactose, glucose, and glycerol
besides some minor amino acids [3].
The metabolomic fingerprinting of herbal
extracts is desirable to standardize drugs and to
establish the scientific basis of their pharmaco-
logical action. This study recruited 1D- and
2D-NMR, HPLC–PDA, and GC–MS techniques
for rapid metabolome analysis of Withania leaf
and root extracts. Such analysis is desirable for
developing herbal drugs and establishing associa-
tion with their action through functional genom-
ics and molecular pharmacology. Such knowledge
is essential to evolve directions for genetic
improvement of medicinal plants for the enhance-
ment of the biosynthesis of bioactive molecules.
High-resolution NMR like 800/1,000 MHz or
high-resolution instruments like Fourier trans-
form ion cyclotron resonance mass spectrometer
(FTICR MS) can resolve even higher number of
molecules and establish those quantitatively in
different plant parts. Such a wide metabolomic
analyses is helpful to generate discrete parame-
ters for better defining and quality control of
herbal extract, as well as to serve as diagnostics
for their true identity and adulteration with other
plants or non-usual parts of the same plant [3].
Plants are one of the most important sources
for the development of new drugs. Presently,
plant extracts and plant-derived components are
being used in a multitude of herbal remedies and
phytopharmaceuticals. In the literature three
terms are in practice to describe the analysis of
metabolites produced by plants are as follows:
the metabolic profiling, metabolic fingerprinting,
and metabolomics. A very simplistic view is that
metabolomics can be seen in two different ways.
Firstly, there is the microscopic view (targeted
approach) which looks at a specific set of com-
pounds (e.g., phenolics or terpenoids). This
approach is also called metabolic fingerprinting
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
or metabolic profiling. The second view is the
macroscopic view (untargeted approach) which
aims at identifying and quantifying all the metab-
olites present in a specific organism. Metabolomics
is therefore a “systematic study of the unique
chemical fingerprints that specific cellular pro-
cesses leave behind,” and more specifically, the
study of small molecules produced during the
metabolism and their profiles is known as metab-
olomics. The metabolome represents the collec-
tion of all metabolites in a biological organism,
which are the end products of its gene expression.
The metabolic fingerprinting can give an instan-
taneous snapshot of the physiology of that cell.
The analytical tools used to study the metabo-
lome include mainly chromatography and spec-
troscopy. Chromatography is used to separate
the metabolites which are then being quantified
and identified with the use of various detectors.
The two main chromatography systems employed
in metabolic fingerprinting are GC and HPLC.
The detectors used in these chromatography sys-
tems depend largely on the type of components
to be detected and quantified. For a targeted
approach, MS is usually employed as the detec-
tor, while NMR and MS are used without the
chromatography step as an untargeted analytical
tool. Presently with 1H-NMR and 13C-NMR
techniques, metabolomics is rapidly developing
for studying plants, quality control of plant-
based products, and drugs [5].
For a proton(s)-bearing molecule, the inten-
sity of all proton signals is absolutely propor-
tional to the molar concentration of the molecule.
Using a proper internal standard, the real concen-
tration of the molecule is easily calculated [6] as
NMR spectroscopy has a very high reproducibil-
ity. NMR, as a tool for metabolomics, has unique
advantages over MS-based methods as it provides
a detailed analysis on the biomolecular composi-
tion very quickly with relatively simple sample
preparation [7]. It is a universal detector for all
molecules containing NMR-active nuclei, unlike
MS where detection of analytes is influenced by
selective ionization or ultraviolet spectrometers
where only chromophore-bearing compounds are
detected. In metabolomics all data obtained from
the analytical methods are further analyzed by
Metabolic Fingerprints
Sample preparation
(harvesting, drying and extraction)
1H-NMR analysis
Data bucketing
Multivariate data analysis
Differentiating signal sorting
2D-NMR analysis of sorted signals
(Semi) Purification using chromatography
(or on-line LC-NMR)
Structure elucidation
Fig. 9.4 Schematic flow chart for NMR-based metabolomic analysis [5]
statistical methods in order to extract all possible
information.
For the accuracy and correctness of the data,
further analysis is done by the statistical methods
for inevitably reliant on the robustness of the raw
analytical data set. Thus, NMR has a unique
advantage, the highest reliability in metabolom-
ics. Unlike the retention time in chromatography-
based techniques, with a few exceptions, the
chemical shift, coupling constant, and integral of
each signal in an NMR spectrum do not change
as long as they are measured under the same con-
ditions: the field strength applied, the solvent, the
pH, and the temperature. Despite the low intrin-
sic sensitivity, the robustness of data and ability
to cover a broad range of metabolites has enabled
NMR to be a favored tool for overall macroscopic
metabolomics and fingerprinting. In addition to
the advantages of data robustness, the utility of
NMR techniques for structure elucidation of
metabolites is also matchless [5].
Sample Preparation, Extraction,
and Purification
NMR-based metabolomics includes sample prep-
aration, extraction, multivariate data analysis,
and identification of metabolites (Fig. 9.4) (like
other analytical methods). The sample prepara-
tion steps need much caution in order to obtain
reproducible and reliable results. Because in the
metabolomic analysis, the age, type of tissues,
developmental stage, environmental conditions
171
1H-1H-J-resolved
COSY, TOCSY
Long range-COSY
HMQC (HSQC)
HSQC –TOCSY
HMBC
(e.g., water drought, sunlight, temperature, and
pH of soil), and harvesting time greatly affect the
metabolome obtained even from the same geno-
type. For these aspects, sampling of the target
materials is carefully planned to avoid increased
biological variation interfering with the final data
interpretation. After harvesting, the sample is fro-
zen immediately to avoid any (bio) chemical
change in the material. The next step is grinding
and extraction of the material to liberate the
metabolites from the cells. During the extraction
(bio) chemical reactions may occur in the mate-
rial, reflected in changes in the metabolome. This
is avoided by drying the material before extrac-
tion either by heat or freeze drying, keeping the
material at a low temperature, or grinding at low
temperatures and/or in the presence of a solvent
that denatures enzymes involved in metabolite
alteration. Denaturation can also be achieved by a
brief microwave treatment. For the development
of an optimum extraction method, deep freezing
in liquid nitrogen followed by mechanical grind-
ing, sonication with a probe head or cup horn, and
bead beating prior to solvent extraction is evalu-
ated for the efficient breakage of the cell using a
mycobacteria, Mycobacterium bovis. From these
evaluated methods, sonication has been found to
be superior to others [8], as there is no single
extraction method to extract all possible metabo-
lites in an organism. The polarity of the solvent
limits the range of metabolites that can be
extracted. pH is a factor that affects the profile of
172
metabolites extracted; for example, alkaloids are
soluble in nonpolar solvents at basic pH and in
aqueous solvents at acidic pH. To overcome these
problems in part, a two-phase solvent system con-
sisting of chloroform–methanol–water (2:1:1) has
been applied to extract both polar and nonpolar
compounds in a single extraction [5]. This method
was also applied to metabolic fingerprinting of
Ephedra with as light modification, by addition of
NH4OH, for benzylamine alkaloids extraction [9].
However, this elaborate two-phase extraction
method was found to be problematic when deal-
ing with a large number of samples because of the
long processing time and the possibility of degra-
dation or losses during processing. Recently, a
simple direct extraction method with deuterated
NMR solvents has been developed for sample
preparation, which in general gave better quality
NMR spectra. Single solvent systems have
been used for some studies. Methanol with
trifluoroacetic acid (TFA) has been found to be a
suitable solvent for the extraction of alkaloids,
while perchloric acid was routinely used for the
more polar metabolites, which prevents enzymatic
degradation of metabolites. However, a combina-
tion of CD3OD and D2O was found to be a more
preferable solvent, since it can extract more
diverse metabolites. The combination of these
two solvents was used in different ratios, for
example, 30–70% of CD3OD depending on the
study. In most of our work, a mixture of metha-
nol-d4 and KH2PO4 buffer in D2O (pH = 6.0) has
been used, which extracts a wide range of metab-
olites including amino acids, carbohydrates,
fatty acids, organic acids, phenolics, and terpe-
noids in a single step. The direct extraction
method with deuterated NMR solvents saves
time and makes it possible to deal with a large
number of samples. Moreover, the buffer in the
solvent can avoid possible fluctuation of chemi-
cal shifts of signals in the NMR spectra. However,
when commercial herbal preparations are ana-
lyzed, a more targeted approach is used, focus-
ing on the known major bioactive compounds in
the plant material. For instance, in the case of
St. John’s wort (Hypericum perforatum), both
tablets and capsules were analyzed using a mixture
of methanol–pyridine (6:4, v/v) [5].
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
1
H-NMR Detection
In NMR-based metabolomics, the 1H-NMR spec-
trum provides a wealth of chemical information.
With the aid of chemical shifts and coupling con-
stants, some metabolites (amino acids, phenolics,
sugars, and TCA-related organic acids) can be
easily identified. The observed chemical shift
positions and spin–spin coupling pattern for
each proton provide information as to what
kinds of protons are found in the molecules
and, subsequently, how the protons are arranged.
Furthermore, the concentration of each metabo-
lite in the sample can be easily calculated from
the integration of the signals in the spectra, and
there is no requirement for calibration curves to
convert signal intensity into concentration as
used in other methods. For obtaining reproduc-
ible results on certain concentration, the relax-
ation delay, the pulse width, and the acquisition
time are the main parameters. The optimum range
of these parameters in 1H-NMR has been excel-
lently reviewed by Pauli et al [10]. One of the
problems in measuring 1H-NMR spectra is the
water signal, a huge signal caused by residual
water, which overlaps with the anomeric protons
of sugars or glycosides (d = 4.8–5.2). To suppress
this undesired water signal, several methods have
been applied, for example, addition of paramag-
netic ions like Mg2+ followed by Meiboom–Gill
modification of the Carr–Purcell (CPMG) spin
echo pulse program and pre-saturation using an
additional pulse. When the pre-saturation tech-
nique is applied during relaxation delay, the most
common method, unwanted reduction of the sig-
nal intensity close to the suppressed water might
occur. For correct suppression, the temperature
and pH of samples are carefully adjusted since
the water signal is highly affected by these fac-
tors. Depending on the molecular size of metabo-
lites, specific pulse sequences have been applied.
To filter out the signals from small molecules
from those of large ones, spin diffusion differ-
ences are utilized. It has been established in an
NMR-based study on toxin-induced changes in
lipoprotein profiles. In the case of a matrix con-
taining macromolecules (e.g., proteins or lipid
vesicles), the application of a spin echo sequence
Metabolic Fingerprints
like the Carr–Purcell–Meiboom–Gill (CPMG)
pulse sequence allows the attenuation of unwanted
resonances from macromolecules. However, in
any 1H-NMR spectra of plant extracts using
diverse pulse sequences, there are congested sig-
nals of metabolites. The multivariate data analy-
sis is a key step for sorting out the discriminating
signals from the complex spectra. Prior to the
multivariate data analysis, all signals in the
obtained 1H-NMR spectra should be digitalized.
This is achieved by the so-called bucketing pro-
cedure, which divides the spectrum into small
bins and sums all intensities in each bin. Ideally,
a smaller bucket size is better to examine subtle
perturbations in the metabolome. However,
because of unfavorable fluctuations of chemical
shifts, even with a fixed temperature and pH of
samples, one can only use 8–16 Hz (0.02–
0.04 ppm in 400-MHz NMR) for the bucket size.
It is also possible to use full-resolution NMR
data for the multivariate data analysis, and full-
resolution NMR data sets (0.15 Hz per data point
resulting in 30,000 variables) gave more precise
information about constituents responsible for
data clustering, compared to the use of integrated
NMR data sets (0.04 ppm bucketing resulting in
200 variables). Some aligning methods were
found to improve the separation of NMR data for
further statistical analysis [5, 11].
1
H-NMR Spectra to Recognize Patterns
and Find Discriminating Signals
Currently in metabolomics studies, many hun-
dreds or sometimes thousands of samples are
routinely analyzed, and a minimum of several
hundreds of signals is detected in each sample. It
is crucial to extract the relevant information from
such a huge data set. The samples are grouped
based on the analysis of the metabolome (pattern
recognition), and a discriminating metabolite
(biomarker) can be found by multivariate data
analysis. The pattern of a class represents the
information about the relations between the
observations within the class, discerning, which
are similar, which are different, or which are
atypical outliers. Through the pattern recognition,
173
some metabolites can be found that are character-
istic of a class. Of the multivariate data analysis,
principal component analysis (PCA) and partial
least squares projections to latent structures
(PLS) are on the verge of becoming routine pro-
cessing steps for raw analytical data. PCA is
designed to extract and display the systematic
variation in a data matrix, and PLS is a regression
extension of PCA, which is used to reveal the
correlation between two kinds of data sets [12].
Both PCA and PLS are projection methods by
reducing original data to a few principal compo-
nents in order to describe maximum variation
within the data. All raw data (e.g., the integral
values of all chemical shifts in a 1H-NMR spec-
tra) of samples are plotted in a K-dimensional
space (K = number of variables, for example,
chemical shifts in 1H-NMR spectra), and the plot-
ted samples are projected onto the line generated
by least square sense (principal component line).
The score of each sample is obtained along the
PC line. The next principal components can also
be calculated by projection onto the line which is
orthogonal to the previous PC line or space. In
general, the first three PCs are used for the analy-
sis. For the data analysis, score and loading plots
are used. The score plot is useful for observation
of any groupings in the data set and in addition
will highlight outliers that may be due to errors in
sample preparation, experimental conditions, or
instrumentation parameters. The coefficients by
which the original variables should be multiplied
to obtain the PC are called loadings. The numeri-
cal value of a loading of each variable on a PC
shows how much the variable has in common
with that component. Prior to the PCA and PLS
analyses, raw data are scaled in different ways.
There are several ways to scale the data. For unit-
variance scaling, the standard deviation and the
scaling weight as the inverse standard deviation
are calculated. Subsequently, each variable is
multiplied by the inverse standard deviation.
Each scaled variable then has equal variance.
This method is useful when the classification
relies more on differences between minor com-
pounds, since it increases the influence of weaker
signals. However, applying the unit-variance scal-
ing to an NMR data set, it is difficult to interpret
174
the loading plot obtained from the unit-variance
scaling method because all signals have the same
variance and consequently are different from the
original NMR spectra. With mean centering,
the average value of each variable is calculated
and then subtracted from the data. This improves
the interpretability of the loading plot since it
resembles the original NMR spectra. Also, the
spectral noise is removed for the analysis as long
as intensities of noise are lower than real signals.
However, the influence of minor signals might
be excluded since the mean-centering method
always retains the original value of signals.
In particular, the signals of plant secondary
metabolites can be underestimated compared to
high levels of primary metabolites. As an alterna-
tive technique, the so-called Pareto scaling has
become more common. Pareto scaling gives each
variable a variance numerically equal to its initial
standard deviation instead of unit variance.
Hence, Pareto scaling is intermediate between
the extremes of mean-centered and unit-variance
scaling, which gives some weight to minor sig-
nals, and at the same time it provides interpreta-
ble loadings. In recent years variable stability
(VAST) scaling which weights each variable
according to a metric of its stability improved the
class distinction and predictive power of PLS-DA
models for 1H-NMR spectra of urines [5].
2D-NMR Spectroscopy to Compare
Metabolites
In NMR spectroscopy signals are used to dis-
criminate sample groups by multivariate data
analysis. Some metabolites like carbohydrates,
amino acids, or organic acids related to the TCA
cycle can be elucidated merely based on their
chemical shifts in 1H-NMR spectra. However, for
most cases, the spectral complexity and signal
overlap in the 1H-NMR spectra are too high for
identification. Chemical structures of metabo-
lites, especially species-specific plant secondary
metabolites, are confirmed by additional meth-
ods. Diverse two-dimensional (2D) NMR spec-
troscopy gives information on chemical structures.
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
Indeed, one of the advantages of NMR spectroscopy
in plant metabolomics is the power of structure
elucidation of molecules in a complex mixture.
The 2D-NMR spectroscopy available for metab-
olomics can be divided into two types, resolved
and correlated spectroscopies according to the
type of dispersion in the second frequency
dimension. A typical resolved technique is the
2D-1H-1H-J-resolved spectrum. It provides addi-
tional information on spin–spin coupling con-
stants, together with chemical shifts. One of the
difficulties for interpreting a 1H-NMR spectrum
is the interpretation of the splitting pattern of
each signal. If a target signal is detected in a
crowded region overlapping with other signals,
the first step is to identify its splitting pattern.
In addition to this use of J-resolved spectra in
structure elucidation, projected spectra on to the
axis of chemical shift have been applied in sev-
eral studies. The projected spectrum produces a
1
H-NMR spectrum consisting of singlets for each
1
H chemical shift, that is, a fully proton-decoupled
proton NMR spectrum. In the projected spectrum
all signals from macromolecules which have
short T2 relaxation times are readily suppressed
because the pulse sequence of the J-resolved
technique is based on a spin echo sequence. Thus,
baseline shifting caused by proteins, oligo- or
polysaccharides, which might overlap minor sig-
nals or result in overestimation of target signals,
is clearly reduced in the projected spectra and
lead to better separation in multivariate data anal-
ysis. Another promising resolved technique is
diffusion-ordered spectroscopy (DOSY) where
each NMR signal is resolved based on the molec-
ular diffusion coefficient. DOSY can be used for
molecular weight evaluation of unknown, purified
constituents by preparing calibration curves
with known compounds. Although DOSY has a
potential to differentiate metabolites having the
same chemical moieties and different molecular
weights in plant extract, for example, glycosides
from their aglycones, the application in metabo-
lomics has been limited so far to the analysis of
polymers like polysaccharides from insects or
mushrooms, interaction between small molecules
and ligands, and hydrocarbon mixtures [5].
Metabolic Fingerprints
Following the resolved spectra, information
on correlation between protons or carbon–proton
can be obtained by homonuclear and heteronu-
clear experiments. A typical proton homonuclear
technique is COSY that shows which signals in a
proton spectrum have mutual spin–spin cou-
plings. The resulting spectrum has the conven-
tional 1D-NMR spectrum along the diagonal
cross peaks at chemical shifts corresponding to
pairs of coupled nuclei. In general protons within
three bonds are well correlated in the COSY
spectrum but in sp2 spin systems such as olefinic
and phenolic compounds, even the correlation
beyond three bonds is readily detected. A
modified pulse sequence of COSY, long-range
COSY, can selectively detect correlation between
protons far from each other (more than three
bonds). It can show structure confirmation of
connectivity between two molecules which is not
possible to detect in a normal COSY spectrum. It
is one of the most time-consuming tasks to define
NMR signals belonging to the same molecule in
the complex 1H-NMR spectra of a mixture. In par-
ticular, most 1H signals of carbohydrates are dete-
cted in an extremely crowded region (d = 3.0–4.5)
overlapping with each other or with the signals of
amino acid protons close to the amino group. A
powerful 2D-NMR technique, total correlation
spectroscopy (TOCSY), or the Hartmann–Hahn
experiment (HAHAHA) provides information
on unbroken chains of coupled protons in the
same molecule. For example, if anomeric protons
of carbohydrates are defined, the rest of the sig-
nals in the crowded region can be easily assigned
using the correlation in the TOCSY spectrum.
All organic molecules have carbons as their
chemical backbone. The information on the car-
bons is thus crucial for structure elucidation. In
fact measurement of a 13C-NMR spectrum is an
important, if not indispensable, step to elucidate
the chemical structure of a pure compound.
However, the magnetically active 13C isotope has
too low sensitivity for the application to the anal-
ysis of the metabolome since the natural abun-
dance of 13C is only 1.1% of 12C and its
magnetogyric ratio is one fourth of 1H. It results
in 1/57,000 of the 1H sensitivity. Also, the long
175
relaxation time of 13C requires a long measuring
time in order to obtain quantitative results. To
overcome the low sensitivity of 13C, heteronu-
clear correlation techniques are used from which
the information on 13C chemical shifts is indi-
rectly obtained. Direct correlation between car-
bons and protons can be detected by heteronuclear
multiple quantum coherence (HMQC) or hetero-
nuclear single quantum coherence (HSQC) which
gives better sensitivity than HMQC but needs
phase correction. In both HMQC and HSQC
spectra, any carbon attached directly to a proton
is detected; consequently the chemical shift of a
13
C connected to a specific proton can be obtained.
The assignment of C-6 and C-8 of
5,7-dihydroxyflavonoids can easily be learned
from the HMQC spectra of the extract of aerial
parts of Genista tenera. In the HMQC spectra the
unusual upfield shift of those aromatic carbons
compared to other aromatic carbons, caused by
the adjacent oxygen, confirmed the presence of
5,7-dihydroxyflavonoids [5, 13]. Long-range cor-
relations (two and three bonds) between carbons
and protons can be observed by heteronuclear
multiple-bond correlation (HMBC) spectra. It is
an extremely powerful tool for structure eluci-
dation since carbon–carbon correlations can be
indirectly obtained with the aid of HMQC or
HSQC. In addition, correlations between quater-
nary carbons like carboxylic acid and nearby pro-
tons can be seen in the spectra. The long-range
couplings in HMBC spectra can give information
on the connectivity between two moieties which
are connected only by quaternary carbons or
heteroatoms (e.g., O-glycosides). One of the
advantages of NMR-based metabolomics is that
there are numerous combinations of 2D-NMR
experiments that provide information on struc-
tures by diverse correlations. Aiming at specific
correlations, enhancing resolution, or simplifying
spectra HMBC-J-resolved, DOSY-selective
TOCSY, 2D-J-DOSY, and DOSY–HMQC has
been applied to the analysis of complex mixtures.
The combined HMQC (or HSQC)–TOCSY
sequence experiment leads to a 13C-edited TOCSY
spectrum and information on all connectivity of
carbons in each molecule in a mixture [5].
176
Evaluation of Quality
of High Therapeutic-Value Herbals
Case Study 2
Saffron (Crocus sativus) is the most expensive
spice, and its production is extremely laborious,
so low-quality saffron is frequently encountered,
containing styles and petals instead of or in addi-
tion to the stigmata, or even falsified saffron con-
taining dyes and artificial flavors, or other plant
products. The chemical composition of saffron is
the most important indicator of its quality and
value. Several analytical techniques are available
for assessment of various constituents to deter-
mine the quality of this spice. These include
UV–Vis. spectroscopy, near-IR spectroscopy, as
well as various chromatographic techniques. GC
and HPLC techniques are appropriate for quanti-
tative determination of specific metabolites in
saffron. In recent years, 1H-NMR spectroscopy
emerged as a useful method for plant metabolite
fingerprinting with the purpose of sample
classification and determination of discrimina-
tory features. The advantages of 1H-NMR spec-
troscopy combined with chemometric methods
are that (there is no need of separation of sample
constituents, and also there is no need of prior
knowledge about expected sample composition
or external calibration of the response) the infor-
mation content of individual spectra is high and it
is possible to identify discriminating constituents
from spectral features revealed by loading plots
or covariance matrices. 1H-NMR spectroscopy of
plant material extracts also offers good reproduc-
ibility and robustness [14].
The chemical composition of saffron has been
thoroughly studied, and numerous volatile and
Fig. 9.5 Therapeutic ingredients of C. sativus [14]
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
nonvolatile constituents have been identified.
Characteristic constituents of saffron are crocin
(1) and congeners, which are a family of water
soluble glycosides of crocetin (2) contributing to
the red color of saffron, numerous monoterpe-
noids such as safranal (3), responsible for the
aroma of saffron, and picrocrocin (4), which is a
precursor of safranal (Fig. 9.5).
In addition, saffron contains numerous
other glycosylated monoterpenes, carotenoids,
flavonoids, and simple aromatic compounds. A
study, for determination of sample quality and
variability as well as means of unsupervised
detection of adulteration in C. sativus, 1H-NMR
metabolic fingerprints of 63 saffron samples,
consisting of authentic reference samples from
Iran (31 samples) as well as samples purchased
from retail stores in several countries (32 sam-
ples), provides immediate means of unsupervised
classification of saffron samples. The detail of
the experiment is as follows.
Materials and Chemicals
Saffron samples from Iran (n = 31, samples 1–31)
were produced in Khorasan, Iran, near Torbat-e
Heydariyeh, in the area spanning from 34º 20¢ N
to 35º 47¢ N and from 58º 15¢ E to 60º 37¢ E, at
altitudes 1,000–1,750 m. The plantation ages
ranged from 2 years to 9 years with 24 (75%) of
the samples coming from plantations in the range
of 3–5 years. The drying conditions were stated as
“near the heater” (n = 8), “shadow” (n = 19), and
“sun light” (n = 4). The moisture and volatile con-
tent of the samples were below 12%. Commercial
samples (n = 32, samples 32–63) were purchased
in shops and bazaars in Denmark (n = 17, samples
41–45, 47, 49–59), Iran (n = 2, samples 60–61),
Evaluation of Quality of High Therapeutic-Value Herbals
Sweden (n = 4, samples 46, 48, 62–63), and Turkey
(n = 9, samples 32–40). Deuterated methanol (99.8
atom % of deuterium) was obtained from Euriso-
Top (Saclay, France) [14].
Sample Preparation
Grinded saffron samples (10 mg) were extracted
with CD3OD (1,000 ml) by maceration for 1 h at
room temperature with two 10-min periods of
sonication at the beginning and at the end of the
extraction period. The mixtures were centri-
fuged for 5 min.; 700 ml aliquots of the superna-
tant were transferred to 5-mm NMR tubes for
analysis. Two replicates of each extract were
prepared [14].
1
H-NMR Spectroscopy
1
H-NMR data were recorded at 298 K on a
Bruker Avance 600-MHz spectrometer equipped
with a 5-mm inverse probe head and a 25-posi-
tion sample changer. For each analysis, 256 tran-
sients were collected as 64 k data points with a
spectral width of 12 kHz (20 ppm), using 308
pulses and inter-pulse delay of 3.65 s. Each sam-
ple was analyzed twice by a second revolution of
the sample changer. The acquisition was con-
trolled by in-house written scripts running under
XWIN-NMR and ICON-NMR (Bruker Biospin,
Karlsruhe, Germany). The protocol included a
180-s temperature equilibration period, gradient
shimming using the z-axis profile of the 2H-NMR
signal of the solvent, receiver gain adjustment,
and acquisition. The total time for each NMR
analysis was 25–32 min. The free induction
decay signal was zero filled to 128 k and expo-
nentially multiplied with a 0.3-Hz line broaden-
ing factor. The zeroth order phase constants were
manually optimized after Fourier transform.
Gradient-enhanced heteronuclear multiple-bond
correlation (HMBC) spectra were acquired with
2 k × 256 k data points for 7 kHz × 36 kHz sweep
width, with 64 spectra per increment and 65-ms
mixing time. Longitudinal relaxation time con-
stants (T1 values) were determined by an inver-
sion recovery experiment (180º pulse-variable
delay-90º pulse-acquisition) with 16 variable
delays ranging from 15 s to 5 ms and a relaxation
delay of 20 s. The number of acquired data points
177
was 8 k for each spectrum (12-kHz spectral
width, 32 transients each), Fourier transformed
to 16 k with an exponential multiplication line
broadening factor of 1.0 Hz, and a polynomial
baseline correction of the FID (degree 5) to
reduce the water signal. One tenth of all data
points, corresponding to the highest intensities,
were integrated over 30-Hz ranges and the inte-
grals fitted to the function I(t) = b × (1-a × e –t/T1)
in Matlab using function in Curve Fitting
Toolbox (version 2.1, The MathWorks Inc., MA,
USA) [14].
Spectral Analysis
The spectra were imported into Matlab (ver. 7.7,
The MathWorks Inc., MA, USA) using in-house
written routines and calibrated to residual solvent
signals at d 3.31, normalized with respect to the
receiver gain setting, and baseline corrected by
fitting cubic polynomial functions through sig-
nal-free ranges of the spectra. The spectral matri-
ces were prepared by integration of 0.04-ppm
wide buckets in the range 0.5–11 ppm. Regions
corresponding to residual NMR solvent (d 3.33–
3.29 and d 5.05–4.50) were excluded. Vicinal
buckets with a correlation coefficient greater than
0.95 were merged. The data were normalized to
constant sum, Pareto scaled and analyzed using
PLS Toolbox 5.2 (Eigenvector Research, Inc.,
WA, USA) running within Matlab. For statistical
correlation spectroscopy, the spectra were inte-
grated in 0.01-ppm wide buckets resulting in
1,105 variables and analyzed without further nor-
malization or scaling with functions in Statistics
Toolbox (ver. 7.1, The MathWorks Inc., MA,
USA) [14].
Methanol was considered appropriate solvent
for polar (crocins and other glycosides) as well as
nonpolar (terpenoids) constituents, and in order
to minimize sample preparation, saffron was
extracted directly with the deuterated solvent.
Two replicates of each extract were prepared for
1
H-NMR analysis. Longitudinal (T1) relaxation
times were determined for representative samples
in order to choose acquisition parameters that
result in quantitative data, that is, true molar
ratios between the constituents present (e.g.,
Fig. 9.6).
178
Fig. 9.6 Example of 1H-NMR spectrum (600 MHz) of a
saffron extract in methanol-d4 showing the range of T1 val-
ues (red lines, with 95% confidence intervals in blue)
Two spectra (repeats) were recorded for each
sample, resulting in four spectra for each of the
63 saffron samples. One spectrum was excluded
due to low quality, and the final data set consisted
of 251 spectra. In a preparatory phase of this
project, saffron samples were also extracted with
excess of chloroform or methanol–water followed
by evaporation and reconstitution of the residue
with a small amount of the corresponding deuter-
ated solvent, but these sample preparation
methods were found to give poor reproducibility
as judged from large inter-sample variation in
score plots after principal component analysis
(PCA); possibly, this could be attributed to partial
loss of volatile constituents.
The main differences among authentic saffron
samples are thus the relative amounts of picro-
crocin and the sum of different crocetin glyco-
sides. The most abundant crocetin glycoside in
C. sativus stigmata is reported to be the di-(b-d-
gentibiosyl) ester of crocetin (1). Variants include
symmetrical and unsymmetrical crocetin esters
of b-d-glucose, b-d-gentibiose, and b-d-neapoli-
tanose, as well as isomers with a cis-double bond
next to the inner methyl group of crocetin (2). The
presence of the mixture of different crocetin
esters is reflected in the 10 principal components
needed to adequately describe the data set of 1H-
NMR spectra of authentic Iranian saffron extracts.
However, the spectral overlap makes it impossible
to interpret the influence of individual components
separately. This study showed that it is highly
feasible to use unsupervised PCA models of
1
H-NMR metabolic fingerprints to assess the
authenticity of saffron [14].
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
across sample components. The displayed spectrum is the
most relaxed spectrum of an inversion recovery experi-
ment [14]
Molecular Modeling
Molecular modeling may be defined as a tech-
nique for deriving, representing, and manipulat-
ing the structures and reactions of molecules,
with computer and software, and those properties
that are dependent on these three-dimensional
structures.
Case Study 3
In a study the mixture of turmeric, ginger, and
seeds of fenugreek (generally used in joint pain
relief and heart diseases in rural area of Indian
subcontinent) was used for molecular modeling.
The main constituents of turmeric are curcumi-
noids (mixture of curcumin, de-methoxy cur-
cumin, bis-demethoxy curcumin), and those in
ginger are gingrol and hexahydrocurcumin. The
past phytochemical investigations of fenugreek
seeds reveal the presence of diosgenin, trigonel-
line, gitogenin, vicenins 1 and 2, vitexin, querce-
tin, luteolin, kaempferol, sitosterol, etc.; moreover,
the endosperm of the seeds is rich in galacto-
mannan [15].
In an attempt to isolate a mixture of these
spices and spectroscopic characterization of iso-
lated compound, turmeric rhizome, ginger, and
fenugreek seeds were washed to clean, dried in
air oven at 60 °C and grinded to a powder using
an electric grinder to pass a 0.4-mm screen. The
powder was taken in a cleaned round-bottom
flask having methanol and stirred for 12 h. The
mixture was filtered and botanical mass was
To Decipher the Herbal Constituents
Fig. 9.7 1H-NMR spectra of complex compound [15]
washed with hot methanol. The filtrate was dis-
tilled and the residue was redissolved in excess
warm methanol, and clear solution was kept
undisturbed for weeks to give beautiful gray
crystal. The novel compound having chemical
structure (molecular formula C36H57N3O24) was
obtained. Various attempts to obtain the single
crystals have so far been unsuccessful. The 1H-
NMR spectra (Fig. 9.7) of the synthesized com-
pound have well-resolved signals. The compound
shows the resonance with integrated intensities.
The chemical shift of the compound appeared at
3.38–3.64 (8H, m) for OH of alcohol, 2.0(3H, m)
for NH of amine, 3.09–5.03, (8H, m) for CH,
7.27–7.30 (5H, m) for CH of benzene, and 3.39–
1.18 (9H, d) for CH3. The compound was charac-
terized by various spectroscopic techniques and
has triclinic crystal system. The molecular struc-
ture has been optimized by MM2 calculation.
This compound may be used as medicine in the
future (after passing all regulatory norms),
because in Indian subcontinent the mixture of
turmeric, ginger, and fenugreek in powder form
is being used traditionally as pain killer, to treat
heart disease and regulate blood sugar [15].
179
To Decipher the Herbal Constituents
The therapeutic potential of medicinal plants is
also effected by abiotic factors; cultivation prac-
tices and harvest processing pattern affect the
chemical composition and clinical efficacy of
herbals and herbal products. So it is necessary to
establish a method to control the quality of herbs
and herbal products. The most common and suit-
able approach is analyzing chemical markers
which are supposed to be present in the analyte.
Case Study 4
In a study for quantitative analysis of sennosides
A and B in the herbal slimming teas and laxative
formulations in Turkey, the powdered leaves of
Cassia acutifolia (300 g) were extracted with
methanol (3 × 3.5 L, 45 °C), and the combined
extracts were evaporated to yield 44.0-g residue.
The methanolic soft mass was dissolved in water
and partitioned with petroleum ether, ethyl ace-
tate, and n-butanol. n-Butanol extract (9.56 g)
was fractionated on a Sephadex LH-20 column
180
Fig. 9.8 The compounds from the senna leaves [16]
eluting with 70% methanol to give eight main
fractions (named as fraction A–H). The fraction
C was chromatographed over silica gel eluting
with chloroform–methanol–water (100:10:5) to
give fraction “a” (161.3 mg). Further column
chromatography was applied on fraction “a”
using silica gel and eluted with chloroform–
methanol–water (80:20:2–60:40:4). A mixture
of 1 and 5 (101.3 mg) was obtained. Fractions 1
obtained from fraction “a” chromatographed
over Sephadex LH-20 column with methanol
elution to get pure compound 2 (6.3 mg).
Compound 4 (14.6 mg) was obtained from frac-
tion D using Sephadex LH-20 column and elut-
ing with methanol. Fraction D gave compound 3
(8.8 mg) by silica gel column elution with chlo-
roform–methanol–water (70:30:3–60:40:4). The
NMR characteristics of compounds (Fig. 9.8)
were as follows [16]:
Compound (1): Yellow amorphous powder; 1H-
NMR (CD3OD, 400 MHz) and 13C-NMR
(CD3OD, 100 MHz) data were in agreement with
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
data given in the literature for kaempferol 3-O-b-
d-gentiobioside.
Compound (2): Orange amorphous powder; 1H-
NMR (CD3OD, 400 MHz) and 13C-NMR
(CD3OD, 100 MHz) data were in agreement with
data given in the literature for aloe-emodin
8-O-b-d-glucopyranoside.
Compound (3): Yellow amorphous powder; 1H-
NMR (CD3OD, 400 MHz) and 13C-NMR
(CD3OD, 100 MHz) data were in agreement with
data given in the literature for rhein 8-O-b-d-
glucopyranoside.
Compound (4): Yellow orange amorphous pow-
der; 1H-NMR (CD3OD, 400 MHz) and 13C-NMR
(CD3OD, 100 MHz) data were in agreement with
data given in the literature for torachrysone
8-O-b-d-glucopyranoside.
Compound (5): Yellow amorphous powder; 1H-
NMR (CD3OD, 400 MHz) and 13C-NMR
(CD3OD, 100 MHz) data are in agreement with
data given in the literature for isorhamnetine
3-O-b-d-gentiobioside.
To Decipher the Herbal Constituents
Fig. 9.9 UV spectrums and HPLC chromatograms of slimming tea [16]
Senna leaves are one of the herbs, traditionally
used as laxative in case of constipation and herbal
tea to promote weight loss. In aforesaid study for
the quality control of slimming teas claimed for
having senna leaves and including senna extract
enriched by sennoside B was analyzed by HPLC
RP column using gradient elution. The presence
of sennosides A and B in laxative formulation was
proved, but slimming teas were devoid of senno-
sides. Each chromatogram was checked with their
UV spectra for sennosides, and the lack of sen-
nosides was proved completely (Fig. 9.9), but an
additional compound known as rhein 8–O–b-d-
glucopyranoside (the degraded monomer of sen-
noside B, by enzymatic processes) was identified,
which is similar to the standard UV spectra of
rhein 8-O-b-d-glucopyranoside.In another study
it was established that the forced decomposition
of herbal material under high temperature caused
181
oxidative decomposition of the sennosides to
rhein 8-O-b-d-glucopyranoside [16, 17].
The laxative formulations were investigated
with the same method to prove whether they con-
tain sennosides A and B or not. The content of
sennosides was quantitatively analyzed by
HPLC–PDA. The presence of sennosides was
clearly observed on each chromatogram and their
corresponding UV spectra, which proves that all
samples have sennosides A and B (Fig. 9.10).
When the results were summarized, it was
observed that slimming teas did not have sen-
noside B and the percentages of the anthra-
noids were not of a significant value, but
somehow they showed strong laxative effect
like laxative drugs which have quite high sen-
noside content. The team of scientists writes
that these results make us think of the malprac-
tice for herbal teas [16].
182
Fig. 9.10 UV spectrums and HPLC chromatograms of laxative formulation [16]
Metabolic Fingerprints to Distinguish
Plant Species
NMR metabolic fingerprinting has been applied
to diverse fields of plant research, in which the
classification and identification of adulteration of
plant products are of major interests. Using
NMR-based metabolic analysis, it was possible
to discriminate different species of plants, for
example.
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
Case Study 5
Eleven species of Ilex were analyzed and
based on their metabolites; each species could be
discriminated, especially Yerba mate (Ilex para-
guariensis) from its adulterants. The contributing
metabolites were arbutin, phenylpropanoids,
caffeine, and theobromine. Caffeine and theobro-
mine were only found in the I. paraguariensis,
whereas arbutin was found only in other species.
Quality Assurance of Traditional Medicines
The study of flower heads of chamomile
(Matricaria recutita) showed that this technique
can be applied to assess the relative amount of
stalk materials in the different batches of flowers.
Another example of NMR analysis combined
with PCA is the application to characterize 3 dif-
ferent Strychnos species using different parts of
the plants (seeds, roots, leaves, and barks). All
samples were clearly classified based on their
metabolites such as brucine, loganin, Strychnos
icaja alkaloids (icajine, sungucine), as well as
fatty acids. Twelve Cannabis sativa cultivars were
discriminated by analyzing the organic fractions
and aqueous fractions. Tetrahydrocannabinolic
acid (THCA) and cannabidiol acid were the most
important metabolites in organic fractions, while
primary metabolites found in the water fraction
such as glucose, asparagine, and glutamic acid
served as discriminating metabolites [5, 18].
Quality Assurance of Traditional
Medicines
The traditional Chinese medicines (TCM) are
often used as a mixture of several plants, with a
hardly defined composition. Even though there
are regulations for quality control, this is often
restricted only to a certain compound or group of
compounds. NMR-based metabolic fingerprinting
combined with pattern recognition may be a very
good tool for the quality control of TCM.
Case Study 6
Chemotaxonomic analysis including quality
control issues was applied in the study of
Ephedra [5, 19]. Ephedra is one of the oldest
medicinal plants known to mankind. However,
three different species of Ephedra, E. sinica, E.
intermedia, and E. equisetina, are commonly
and widely used without strict distinction as long
as there is a certain amount of ephedrine alka-
loids (0.8% of dry weight). Using NMR-based
metabolic fingerprinting, it was possible to dis-
criminate E. sinica, E. intermedia, and E. dis-
tachya. Not only the ephedrine type of alkaloids
183
but also another secondary metabolite (benzoic
acid analogue) was found to be an important dis-
criminating metabolite. Among nine tested com-
mercial Ephedra materials, one was shown to be
the mixture of two species. There are several
reports on the discrimination of ginseng (Panax
ginseng) preparations, which is one of the most
popular herbal medicines. The first report using
NMR-based metabolomic approach showed the
differences in ginseng products, different ages
(4–6 years old), and differently processed (white
and red) products. PCA of J-resolved 1H-NMR
spectrum of three commercial ginseng prepara-
tions showed striking differences. Loading plots
explained that alanine, arginine, fumaric acid,
inositol, and ginsenosides are important metabo-
lites to differentiate preparations from each
other. Other studies also showed that this
approach can be used for the quality control of
fresh ginseng roots of different ages and different
origins. Recently, another study for the quality
evaluation of TCM was reported by Tarachiwin
and coworkers. A combination of an NMR tech-
nique and multivariate analysis showed the dif-
ferences in two Angelica acutiloba roots, A.
acutiloba Kitagawa (yamato-toki) and A. acuti-
loba Kitagawa var. sukiyamae Hikino (hokkai-
toki), with regard to their geographical and
variety origin. In a study for application of NMR
to commercial preparations of Artemisia, NMR
analysis combined with PCA showed clear dif-
ferences between A. annua and A. afra. A more
targeted analysis combined with LC–MS showed
that the content of artemisinin in the commercial
products is different from the commercial claim.
Other studies of NMR-based methods have been
applied to the characterization of crude extracts
and commercial preparations, including feverfew,
kavakava, Artemisia, and St. John’s wort [5].
The major limitation of NMR is its rather low
sensitivity when compared to other analytical
methods. The probes for high-resolution NMR,
such as cryoprobes and capillary NMR, help to
increase the sensitivity threshold of the technique.
CryoNMR aims to limit the receiver coil noise
and to improve the coil quality factor by cooling
the receiver coil and the preamplifiers to 25 K or
below. With the use of cryogenically cooled
184
probes, the detection sensitivity of NMR is at
least 3–4-fold enhanced in comparison with con-
ventional probes operating at the same magnetic
field. Capillary NMR takes advantage from the
use of small-diameter radiofrequency (RF) coils
for mass-limited samples (usually in the order of
nanomole quantities). By decreasing the diameter
of the receiver coil, the signal to noise (S/N) ratio
improves. Both cryoNMR and capNMR are very
promising analytical approaches for metabolic
fingerprinting studies. The main importance of
chemical characterization of the metabolic com-
pounds is to look for further development of the
metabolomic approaches. As no single analytical
technique is suitable for the detection and
identification of all the metabolites in a biologi-
cal sample, the most common strategy to clean up
or isolate and structurally identify metabolites is
to combine chromatographic techniques to MS/
NMR spectroscopy. The resulting hyphenated
methods, such as various HPLC–NMR tech-
niques, are successfully applied to natural prod-
uct research. An off-line solid phase extraction
may sometimes be required to partially purify or
concentrate the analytes [5].
The next generation of plant metabolome sci-
entists will probably focus on the cellular and
even subcellular compartmentation of metabo-
lism. Researchers have started looking into the
metabolomics of single cells with the use of
laser microdissection (LMD). In a study for
identification of 68 metabolites, more than half of
authors could demonstrate whether they were
enriched or depleted in vascular bundles. LMD
has also been successfully used in combination
with cryoprobe NMR for the analysis of special-
ized cells from the floral and leaf tissues of Dilatris
spp. coming from different herbarium specimens.
The phytochemical pattern of LMD-derived sam-
ples was compared with that of whole leaf extracts,
and some secondary metabolites were identified
and connected with the specific cells. The study
revealed the potential of the method in chemot-
axonomic applications even of plants that are
difficult to raise or to obtain. The same kind of
approach, in combination with mass spectromet-
ric measurements (LMD/NMR/MS), was carried
out on the stone cells of Norway spruce bark [5].
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
Detection of Contaminations
High-resolution nuclear magnetic resonance
(HRNMR) spectroscopy is emerging as an
important tool in the quality control of herbals
and herbal drugs. The use of the 1H-NMR
fingerprint as an example of a rapid and simple
method in cases of the contamination of herbal
products by synthetic substances. In comparison
with HPLC/MS (but similar considerations can
be deduced in part for TLC and HPTLC), in
NMR fingerprinting (apart from the initial invest-
ment) the cost of each analysis can be consider-
ably lowered (less material, minor disposable
solvents) in cases of the examination of several
samples. Furthermore, there is no waste (recov-
ery of the sample is possible) and several sam-
ples can be analyzed in a very short time and in
the same conditions, avoiding the stabilization
and equilibration of the apparatus, as well as its
cleaning. It is not possible to obtain a precise
quantitative analysis, but the amount of a sub-
stance can be considered by adding a known
solution of the standard to the tested sample [20]
or by other proposed methods [21].
Presently large numbers of herbal-based prod-
ucts are on the market; it is difficult to check
every batch of these by regulatory authorities for
their quality. Self-medication of herbal drugs,
especially in cases of analgesic effects, can induce
a consumption of a large quantity of a product, sup-
posed “safe” since it is promoted as “natural and
innovative food supplement”. The presence of an
adulterant can often easily be found by NMR
fingerprint. Therefore, although HPLC remains
the main analytic instrument, NMR fingerprinting
can easily be applied to evidence, a contamina-
tion in complex mixtures such as herbal dietary
supplements or similar products. However, NMR
lacks high sensitivity, since the impurity or adul-
terant must be present in sufficient quantity to be
observed. In this regard simple preliminary extrac-
tion of desired sample by suitable solvent to get
enriched form for NMR studies is very useful to
decipher the degree of contamination. Based on
aforesaid theory and analysis of the P.C. 28 Plus,
a product based on herbal drugs marketed by the
References
Italian company Cosval has been detected as hav-
ing the presence of nimesulide on it [20].
Herbal extracts, presently, are in use as nutra-
ceuticals and cosmaceuticals, and drugs have an
important and growing role in disease prevention
and therapy and are in use extensively, with the
concept of synergistic behavior and polyvalency.
The composition of polyherbal formulation is far
more complex than herbs in itself. Consequently,
their quality control is becoming an increasingly
important issue. The European legislation (direc-
tive 2004/24/EC, concerning traditional herbal
medicinal products) writes for a strict control on
the quality and purity, similar to the case of State
Drug Administration of China. The fingerprint
chromatograms are also accepted by the World
Health Organization (WHO) as an identification
and qualification technique for herbals and herbal
drugs. The power of NMR in elucidating the
structure of isolated natural compounds is well
established. Presently NMR has also become an
important tool for the qualitative and quantitative
analysis of complex mixtures such as herbal for-
mulations. The combination of 1H-NMR and mul-
tivariate analysis, quite often used in metabolomics,
leads to a metabolite fingerprint that can be used
for quality control and identification of the origin
(e.g., geographical or supplier). Compared with
other methods and despite its relatively low intrin-
sic sensitivity, NMR has the advantages of simple
sample preparation, high robustness, and nonse-
lectivity thus providing global information in a
single analysis, along specific structural charac-
terization. Several examples of the application of
1
H-NMR in the quality control of herbals and
herbal products have been reviewed, globally. The
DOSY 1H-NMR method is regarded as a virtual
chromatography for screening adulterations of
herbals and herbal products.
References
1. Khajuria RK, Agarwal SG. Proceedings of approaches
towards evaluation of medicinal plants prior to clini-
cal trials principles of quality control, organized by
the foundation for medical research. Standardization
and chemo profiling of medicinal plants and ISM
preparations. 2006. p. 89–101.
185
2. Nakabayashi R, Kusano M, Kobayashi M, Tohge T,
Keiko YS, Kogure N, Yamazaki M, Kitajima M, Saito
K, Takayama H. Metabolomics oriented isolation and
structure elucidation of 37 compounds including two
anthocyanins from Arabidopsis thaliana. Phytochemistry.
2009;70:1017–29.
3. Chatterjee S, Srivastava S, Khalid A, Singh N,
Sangwan RS, Sidhu OP, Roy R, Khetrapal CL, Tuli R.
Comprehensive metabolic fingerprinting of Withania
somnifera leaf and root extracts. Phytochemistry.
2010;71:1085–94.
4. Knothe G, Kenar JA. Determination of the fatty acid
profile by 1H-NMR spectroscopy. Eur J Lipid Sci
Technol. 2004;106:88–96.
5. Kooy FVD, Maltese F, Choi YH, Kim HK, Verpoorte
R. Quality control of herbal material and phytophar-
maceuticals with MS and NMR based metabolic
fingerprinting. Planta Med. 2009;75:763–75.
6. Pauli GF, Jaki BU, Lankin DC. Quantitative 1H-NMR:
development and potential of a method for natural
products analysis. J Nat Prod. 2005;68:133–49.
7. Verpoorte R, Choi YH, Kim HK. NMR-based metab-
olomics at work in phytochemistry. Phytochem Rev.
2007;6:3–14.
8. Jaki BU, Franzblau SG, Cho SH, Pauli GF.
Development of an extraction method for mycobacte-
rial metabolome analysis. J Pharm Biomed Anal.
2006;41:196–200.
9. Kim HK, Choi YH, Erkelens C, Lefeber AWM,
Verpoorte R. Metabolic fingerprinting of Ephedra spe-
cies using 1H-NMR spectroscopy and principal com-
ponent analysis. Chem Pharm Bull. 2005;53:105–9.
10. Seger C, Sturm S. Analytical aspects of plant meta-
bolic profiling platforms: current standings and future
aims. J Proteome Res. 2007;6:480–97.
11. Rasmussen B, Cloarec O, Tang H, Staerk D,
Jaroszewski J. Multivariate analysis of integrated and
full-resolution 1H-NMR spectral data from complex
pharmaceutical preparations: St. John’s wort. Planta
Med. 2006;72:556–63.
12. Lindon JC, Holmes E, Nicholson JK. Pattern recognition
methods and applications in biomedical magnetic reso-
nance. Prog Nucl Magn Reson Spectrosc. 2001;39:1–40.
13. Rauter AP, Martins A, Borges C, Ferreira J, Justino J,
Bronze M-R, Coelho AV, Choi YH, Verpoorte R. Liquid
chromatography–diode array detection–electrospray
ionisation mass spectrometry/nuclear magnetic resonance
analyses of the anti-hyperglycemic flavonoid extract of
Genista tenera: structure elucidation of a flavonoid-C-
glycoside. J Chromatogr. 2005;A. 1089:59–64.
14. Yilmaz A, Nyberg NT, Mølgaard P, Asili JJW. 1H-
NMR metabolic fingerprinting of saffron extracts.
Metabolomics. 2010;6:511–7.
15. Mishra P. Isolation, spectroscopic characterization
and molecular modeling studies of mixture of
Curcuma longa, ginger and seeds of fenugreek. Int J
Pharm Tech Res. 2009;1:79–95.
16. Demirezer LO, Karahan N, Ucakturk E, Kuruuzum-Uz
A, Guvenalp Z, Kazaz C. HPLC fingerprinting of
sennosides in laxative drugs with isolation of standard
186
substances from some senna leaves. Rec Nat Prod.
2011;5(4):261–70.
17. Goppel M, Franz G. Stability control of senna leaves
and senna extracts. Planta Med. 2004;70:432–6.
18. Choi YH, Sertic S, Kim HK, Wilson EG, Michopoulos
F, Lefeber AWM, Erkelens C, Kricun SDP, Verpoorte
R. Classification of Ilex species based on metabolo-
mic fingerprinting using NMR and multivariate data
analysis. J Agric Food Chem. 2005;53:1237–45.
19. Kim HK, Choi YH, Erkelens C, Lefeber AWM,
Verpoorte R. Metabolic fingerprinting of Ephedra spe-
cies using 1H-NMR spectroscopy and principal com-
ponent analysis. Chem Pharm Bull. 2005;53:105–9.
20. Nicoletti M, Petitto V. Contaminations of herbal prod-
ucts determined by NMR fingerprint. Nat Prod Res.
2010;24(14):1325–9.
21. Li C-Y, Tsai S-I, Damu AG, Wu T-S. A rapid and
simple determination of protoberberine alkaloids in
Rhizoma coptidis by 1H-NMR and its application for
quality control of commercial prescriptions. J Pharm
Biomed Anal. 2009;49:1272–6.
Bibliography
Bradley SA, Krishnamurthy K, Hu H. Simplifying DOSY
spectra with selective TOCSY edited preparation. J
Magn Reson. 2005;172:110–7.
García-Villalba R, León C, Dinelli G, Segura-Carretero A,
Fernández-Gutiérrez A, Garcia-Cañas V, Cifuentes A.
9 NMR Spectroscopy: Herbal Drugs and Fingerprints
Comparative metabolomic study of transgenic versus
conventional soybean using capillary electrophoresis–
time-of-flight mass spectrometry. J Chromatogr.
2008;A.1195:164–73.
Gruenwald J, Brendler T, Jaenicke C. PDR for herbal
medicines. 3rd ed. Montvale: Thomson PDR; 2004.
Jaki BU, Franzblau SG, Cho SH, Pauli GF. Development
of an extraction method for mycobacterial metabolome
analysis. J Pharm Biomed Anal. 2006;41:196–200.
Le Gall G, Colquhoun IJ, Defernez M. Metabolite
profiling using 1H-NMR spectroscopy for quality
assessment of green tea, Camellia sinensis (L.).
J Agric Food Chem. 2004;52:692–700.
Li SH, Schneider B, Gershenzon J. Microchemical analy-
sis of laser-microdissected stone cells of Norway
spruce by cryogenic nuclear magnetic resonance spec-
troscopy. Planta. 2007;225:771–9.
Reily MD, Lindon JC. NMR spectroscopy: principles and
instrumentation. In: Robertson DG, Lindon J,
Nicholson JK, Holmes E, editors. Metabonomics in
toxicity assessment. Boca Raton: CRC Press; 2005.
p. 75–104.
Seger C, Sturm S. Analytical aspects of plant metabolic
profiling platforms: current standings and future aims.
J Proteome Res. 2007;6:480–97.
Sumner LW, Mendes P, Dixon RA. Plant metabolomics:
large-scale phytochemistry in the functional genomics
era. Phytochemistry. 2003;62:817–36.
Tuli R, Sangwan R, editors. Ashwagandha: a model Indian
medicinal plant. New Delhi: Monograph council of
scientific and industrial research; 2009.
 Metals in Herbal Drugs
and Fingerprints
Herbal drugs as polyherbal and nanoherbal are in
use to increase solubility, stability, bioavailabil-
ity, and pharmacological activity. The advantage
of herbal as nanodrug include protection from
toxicity, improving tissue macrophages distribu-
tion, sustained delivery, and protection from
physical and chemical degradation. Silver nano-
particles of Ocimum sanctum extract exhibited
maximum antibacterial activity at a dose of
150 mg in wistar rats. The herbal drug incorpo-
rated antibacterial nanofibrous mat fabricated by
electrospinning provided a potential application
for use of wound dressing. Nanoherbal therapy as
per Chinese herbal practices is widely accepted
in China and recognized globally. Metal ions
such as Cu2+, Zn2+, Mn2+, Fe2+, Ni2+, and Co2+ are
essential micronutrients for plant metabolism,
but the presence of nonessential metals such as
Cd2+, Hg2+, As2+, and Pb2+ can become extremely
toxic. Pollution of environments due to huge
industrial human activities in recent years, espe-
cially marine fauna and flora, have accumulated a
high concentration of metals, which transfer to
different ways to the soil, finally enter into
human body. When their concentrations exceed
the required levels, they become toxic and cause
several health problems. Presence of these ele-
ments is to be measured in parts per million
(ppm), with most sophisticated instruments like
atomic absorption spectrometry (AAS) and
inductively coupled plasma (ICP) devices, and
calculated against the response of an existing
standard for quantification. The present discussion
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_10, © Springer India 2012
10
is mainly focused on the elemental utility, toxicity,
and estimation by latest analytical techniques, to
produce evidence-based herbal drugs.
Atomic Absorption Spectroscopy
AAS determines the presence of minerals in liquid
phase, along their concentration in the range mil-
ligram/liter. In elemental stage, metals absorb
ultraviolet light when they are excited by heat.
Each metal absorbed a characteristic wavelength,
and the AAS looks for a particular metal by
focusing a beam of a specific wavelength through
a flame and into a detector. Thus, atomic absorp-
tion is very much like molecular absorption spec-
trometry where absorption is measured and
calculated to determine the concentration. The
major differences lie in instrument design, espe-
cially with respect to the light source, the sample
container, and the placement of the monochro-
mator. In atomic absorption (AA), the sample of
interest is aspirated into the flame, the metal
(if present) in the sample absorb some of wave-
lengths, thus reducing its intensity. The detector
measures the change in intensity and transfer the
signal to recorder proportional to the absorption.
As concentration of metal goes up, absorbance
also increases. An easy and feasible approach for
routine analysis is to draw a calibration curve by
running standards of various concentrations,
against absorbance, and test samples is evaluated
by comparing absorbance with calibration curve.
189
190
This technique is also known as flame atomic
absorption spectroscopy (FAAS), where a liquid
sample is aspirated and mixed as an aerosol with
combustible gases (acetylene and air or acetylene
and nitrous oxide) The mixture is ignited in a
flame of temperature ranging from 2,100 to
2,800°C (depending on the fuel gas used). During
combustion, atoms of the element of interest in
the sample are reduced to the atomic state. A
light beam from a lamp whose cathode is made of
the element being determined is passed through
the flame into a monochromator and detector.
Free, unexcited ground state atoms of the element
absorb light at characteristic wavelength; this
reduction of the light energy at the analytical
wavelength is a measure of the amount of the
element in the sample.
Instrumentation in AAS
The light source, called a hollow cathode tube, is
a lamp that emits exactly the wavelength required
for the analysis (without the use of a monochro-
mator). The light is directed at the flame contain-
ing the sample, which is aspirated. The flame is
typically wide (4–6 in.), giving a reasonably long
path length for detecting small concentrations of
atoms in the flame. The light beam then enters the
monochromator, which is tuned to a wavelength
that is absorbed by the sample. The detector mea-
sures the light intensity, which after adjusting for
the blank, is output to the readout, much like in a
single beam molecular instrument. Also as with
the molecular case, the absorption behavior fol-
lows Beer’s Law, and concentrations of unknowns
are determined with the same principle. Double-
beam instruments are also in use in AA. In this
case, however, the second beam does not pass
through a second sample container (it is difficult
to obtain two closely matched flames). The sec-
ond beam simply bypasses the flame and is
relayed to the detector directly. This design elim-
inates variations due to fluctuations in source
intensity (the major objective), but does not elim-
inate effects due to the flame (cuvette) or other
components in the sample (blank components),
so adjusted by reading the blank.
10 Metals in Herbal Drugs and Fingerprints
In order to atomize and excite most metal ions
and achieve significant sensitivity for quantita-
tive analysis, flame of temperature 2,300°C is
required, which is obtained using the air–acetylene
mixture. Ideally, pure oxygen with acetylene would
produce the highest temperature (3,100°C), but
such a flame suffers from the disadvantage of
a high burning velocity, which decreases the
completeness of the atomization and therefore
lowers the sensitivity. Nitrous oxide (N2O) used
as the oxidant, however, produces a higher flame
temperature (2,900°C) while burning at a low
rate. Thus, nitrous oxide–acetylene flames are
fairly popular. The choice is made based on which
flame temperature/burning velocity combination
works best with a given element. Since all elements
have been studied extensively, presently the
recommendations for any given element are
easily available in the literature sources and
reference books.
There are two designs of burners for the flame
atomizer that are in common use, known as “total
consumption burner” and a few as “premix
burner.” In total consumption burner, fuel, oxi-
dant, and sample all three meet at the base of the
flame. The fuel and oxidant are forced, under
pressure, into the flame, whereas the sample is
drawn into the flame by aspiration. The rush of
the fuel and oxidant through the burner head cre-
ates a vacuum in the sample line and draws the
sample from the sample container into the flame
with a nebulizing or mixing effect. This type of
burner head is used in flame photometry, but not
useful for AA as resulting flame is turbulent and
nonhomogenous.
The readout pattern is different with AAS,
compared to the molecular spectrometry (as absor-
bance and transmittance, or possibly both). In the
atomic spectrum the wavelength is fixed, and the
absorbance or transmittance is recorded vs. time as
the various solutions are aspirated. As acetylene is
a fuel so well vented, fume hood above the burner
is necessary to remove burned and unburned fuel
from the area, and safety glasses must be worn at
all times operating the instrument. In case of AAS
chemical interferences usually result from incom-
plete atomization caused by an unusually strong
ionic bond, for example, in the analysis of a sample
Atomic Absorption Spectroscopy
for calcium, from CaCl2 and CaSO4, where cal-
cium chloride completely atomizes, but calcium
sulfate does not, to avoid confusion best way is to
match the sample and the standards in terms of
chloride and sulfate.
An element is considered essential when
reduction of its exposure below a certain limit
results consistently in a reduction in a physiologi-
cally important function. More than 40 elements
have been considered essential to life systems for
the survival of both mammals and plants. So an
attempt was made to determine what essential
elements and their levels in medicinal plants by
using AAS. There are three main steps of analy-
sis via AAS that are as follows: sample drying,
sample preparation, and sample digestion. One
additional step is required in case of mercury,
commonly known as mercury reduction. The
drying section may not be applicable for all situ-
ations. During analysis the flame is arranged in
such a way that it is laterally long (usually 10 cm)
and not deep. The height of the flame must also
be controlled by controlling the flow of the fuel
mixture. A beam of light is focused through this
flame at its longest axis (the lateral axis) onto a
detector past the flame. The light that is focused
into the flame is produced by a hollow cathode
lamp. Inside the lamp is a cylindrical metal cath-
ode containing the metal for excitation and an
anode. When a high voltage is applied across the
anode and cathode, the metal atoms in the cath-
ode are excited into producing light with a certain
emission spectra. The type of hollow cathode
tube depends on the metal being analyzed. For
analyzing the concentration of copper in an ore, a
copper cathode tube would be used, and likewise
for any other metal being analyzed. The electrons
of the atoms in the flame can be promoted to
higher orbitals for an instant by absorbing a set
quantity of energy (a quantum). This amount of
energy is specific to a particular electron transi-
tion in a particular element. As the quantity of
energy put into the flame is known and the quan-
tity remaining at the other side (at the detector)
can be measured, it is possible to calculate how
many of these transitions took place, and thus get
a signal that is proportional to the concentration
of the element being measured.
191
Case Study 1
Five medicinal plants Fagonia indica, Argemone
mexicana, Cnicus benedictus, Silybum mari-
anum, and Solanum surattense were analyzed for
elemental determination, using the aerial parts
[1]. Plant material was collected freshly from the
field. All plant material was first cleaned, dried,
and then powdered using an electric blender.
Samples in powder form were used for AAS.
Each plant material (0.25 g) was taken in 50-ml
flask and add 6.5 ml of mixed acid solution, that
is, nitric acid (HNO3), sulfuric acid (H2SO4), and
perchloric acid (HClO4) (5:1:0.5). The sample
boiled in acid solution in fume hood on hot plate
till the digestion has been completed which was
indicated by white fumes coming out from the
flask. Thereafter, few drops of distilled water
were added and allowed to cool. Then these
digested samples were transferred in 50-ml volu-
metric flasks, the volume was made up to 50 ml
by adding distilled water and filtering it using
Whatman filter paper (no. 42), and filtrate was
collected in labeled plastic bottles. The solutions
were analyzed for the elements of interest, utiliz-
ing AAS with suitable hollow cathode lamps.
The percentages of different elements in these
samples were determined by the corresponding
standard calibration curves obtained by using
standard AR grade solutions of the elements.
Mercury (Hg) was analyzed by cold vapor tech-
nique as at room temperature mercury exist at atom
stage, so mercury is measured by atomic absorp-
tion without a heated sample cell. In this process
mercury is chemically reduced to the free atomic
state by reaction of sample with a strong reducing
agent like stannous chloride or sodium borohydride
in a closed reaction system. The volatile free mer-
cury is then driven from the reaction flask by bub-
bling air or argon through the solution. Mercury
atoms are carried in the gas stream through tubing
connected to an absorption cell, which is placed in
the light path of the AAS. Sometimes the cell is
heated slightly to avoid water condensation, but
otherwise the cell is completely unheated.
As the mercury atoms pass into the sampling
cell, measured absorbance rises indicating the
increasing concentration of mercury atoms in the
192
light path. Some systems allow the mercury vapor
to pass from the absorption tube to waste, in
which case the absorbance peaks and then falls as
the mercury is depleted. The highest absorbance
observed during the measurement is taken as the
analytical signal. In other systems, the mercury
vapor is rerouted back through the solution and
the sample cell in a closed loop. The absorbance
rises until an equilibrium concentration of mer-
cury is attained in the system. The absorbance is
then level off, and equilibrium absorbance is used
for quantitation. The sensitivity of the cold vapor
technique is far greater than can be achieved by
conventional flame AA. This improved sensitiv-
ity is achieved, first of all, through a 100% sam-
pling efficiency. All of the mercury in the sample
solution placed in the reaction flask is chemically
atomized and transported to the sample cell for
measurement [1].
Case Study 2
In another study elemental assay was done by
using ASS on some ethnomedicinally important
species of genus Ficus [2]. Flame atomic absorp-
tion methods are referred to as direct aspiration
determinations. They are normally completed as
single element analyses and are relatively free of
interelement spectral interferences. The use of
special light sources produced by the cathode
lamp is emitted from excited atoms of the same
element which is to be determined, and specific
spectrum selection allows the quantitative deter-
mination of individual components of a multiele-
ment mixture.
Material and Methods: The different parts of
plants including leaves, aerial roots, fruits, and
bark were rinsed with tap water and then with
distilled water in order to remove surface con-
tamination and dried at 55–60°C in an electric
oven. The dried plant samples were then homog-
enized in pistil mortar. 0.25 g each of the pow-
dered plant samples digested in 6.5 ml of acid
solution (HNO3–H2SO4–HClO4 in ratio of
5:1:0.5). The corresponding solution was heated
until white fumes had appeared. The clear solu-
tion was diluted up to 50 ml with distilled water
10 Metals in Herbal Drugs and Fingerprints
and filtered with Whatman filter paper no. 1. The
standard working solutions of elements of inter-
est were prepared to make the standard calibra-
tion curve. Absorption for a sample solution uses
the calibration curves to determine the concentra-
tion of particular element in that sample. A Varian
AA240FS AAS was used for the determination
of 13 metals, that is, Na, K, Ca, Mg, Mn, Zn, Ni,
Co, Fe, Cu, Cr, Cd, and Pb. Cathode lamps used
as radiation source. Air–acetylene gas was used
for all the experiments. This method provides
both sensitivity and selectivity since other ele-
ments in the sample generally not absorb the cho-
sen wavelength and do not interfere with the
measurement [2].
In the human body, the elements play vital role
in many physiological reactions and their
deficiency or excess can affect human health [3].
Zn deficiency may contribute to arrested sexual
maturation, growth retardation and hair loss,
delayed wound healing, and emotional distur-
bance. K is helpful in reducing hypertension and
maintaining cardiac rhythm. Magnesium (Mg)
improves insulin sensitivity, protect against diabe-
tes and its complications, and reduce blood pres-
sure. Sodium (Na) involves in the production of
energy and transport of amino acids and glucose
into the body cells. Copper (Cu) plays an impor-
tant role in treatment of chest wounds and prevent
inflammation in arthritis and similar diseases.
Copper (Cu) is an essential redox-active transition
element that play vital role in various metabolic
processes. Being toxic, only trace presence of Cu
is preferred. It is well known that the high content
of transition metal like Cu catalyzes the formation
of hydroxyl (OH−) radicals; hence, excess quan-
tity can cause oxidative stress in plants and conse-
quently increase the antioxidant response. Calcium
overcomes the problems of high blood pressure,
heart attack, premenstrual syndrome, and colon
cancer and keeps the bones strong and reduces the
risks of osteoporosis in old age. Co and Ni are
required in little amount in the human body.
Cobalt (Co) is necessary in very small amounts in
all mammals and is used to treat several different
types of cancer in humans and treat anemia, but
the intake of high amount can cause heart dis-
eases. The health benefits of nickel (Ni) are
Inductively Coupled Plasma
optimal growth, healthy skin and bone structure;
it is involved in iron metabolism, but it is required
in low quantity, otherwise it may cause toxicity.
Chromium (Cr) balances blood sugar levels, regu-
lates hunger, reduces cravings, protects DNA and
RNA, improves heart function, and helps control
fat and cholesterol levels in the blood [2]. Lead
(Pb) is a toxic metal and nonessential element for
human body as it causes a rise in blood pressure,
kidney damage, miscarriages and subtle abortion,
brain damage, declined fertility of men through
sperm damage, diminished learning abilities of
children, and disruption of nervous systems.
Cadmium (Cd) is also toxic. The whole study of
the absence of these heavy metals in samples indi-
cates that most of the plants were collected from
their unpolluted natural habitats and therefore
reflects their natural levels of heavy metals [2].
Further a point of concern that in this study 13
elements were estimated in different parts in dif-
ferent species of Ficus, the present information is
helpful in the synthesis of new modern drugs
with various combinations of plants which can be
used in the cure of many diseases ethnomedici-
nally. The chemical composition of the different
elements in edible fruits of Ficus indicates that
many of these elements are of vital importance in
metabolism and are needed for growth, develop-
ments, and prevention and treatment of many dis-
eases. It is evident that they are important sources
of essential mineral elements in reasonable con-
centrations which are required in treatment of
many diseases [2]. Phosphorus content was ana-
lyzed calorimetrically, using 0.5 ml of digested
sample with 4 ml of distilled water, 3 ml of
0.75-M H2SO4, 0.4 ml of (NH4)6Mo7O24 .4H2O,
and 0.4 ml of 2% ascorbic acid were added and
mixed [4]. The solution was allowed to stand for
20 min. and absorbance was recorded 660 nm.
The Graphite Furnace Atomizer
A non-flame type of atomizer, a high-temperature
graphite furnace, offers some advantages over the
AAS. There are several different designs, but
basically this furnace is a small cylindrically
shaped with a sample injection port at the top.
193
The light to be absorbed enters from one end of
the cylinder and emerges through the other end.
The sample solution (1–100 ml) is injected into
the furnace through the injection port. The high
temperature of the furnace (about 2,500°C) is
reached in stages, ultimately resulting in atomi-
zation as in the flame. The atomized metal
species then absorbs the light, and the absorption
is measured. One obvious difference between
the furnace and the flame is that, contrary to the
flame, the sample is not continuously fed into
the furnace and the sample distribution is neither
homogeneous nor reproducible. Thus, a furnace
offers greater sensitivity (because more atoms
can be placed in the path of the light) and requires
less volume of sample, but sometimes suffers
from lack of accuracy and precision. Thus, the
graphite furnace is the choice of preference only
when the sample size is small and/or there is a
need of greater sensitivity.
Inductively Coupled Plasma
In inductively coupled plasma (ICP), there is an
induction coil. Induction coil is a coil of wire that
has an alternating (oscillating) current flowing
through it. In the ICP, the coil is wrapped around a
quartz tube through which flows a plasma (a col-
lection of charged particles) and induces the mag-
netic field. When we use a stream of argon gas that
has been partially ionized prior to enter into induc-
tion coil, become more ionized due to induced
magnetic field and an extremely hot as flame-like
emission device, is called the ICP. With respect to
the measurement of sample solutions, the proce-
dure is an aspiration, similar to AAS, in which the
solution is aspirated into the flowing argon prior
to entering the quartz tube. The net result is an
extremely high temperature, capable of producing
very intense emissions from atomized and excited
atoms from the sample solution. Being an emission
technique, it is very useful for qualitative analysis,
especially having the intense signals compared to
AAS, with high accuracy. The simultaneous multi-
element analysis of sample is also possible, using
ICP, but the possible disadvantage, perhaps, is the
high cost of the equipment compared to AAS.
194
Fig. 10.1 WD-XRF spectra 300
in overlay mode [5]
250
200
150
100
50
0
WDXRF Spectroscopy
Wavelength dispersive X-ray fluorescence
(WDXRF) spectroscopy has been successfully
validated to analyze silicon (Si), the second most
abundant element on earth; most of its com-
pounds cannot be consumed by plants and ani-
mals. The presence of Si in plants is a result of
an uptake of its soluble forms as orthosilicic acid
[Si(OH)4] and/or its ionized form Si(OH)3O− is
an essential microelement for human health. The
highest concentration of Si in the human body is
in hair and nails. The symptoms of a possible Si
deficiency are cardiovascular and arterial prob-
lems, fragile bones, weakened gums and teeth,
joint deterioration, and digestive disorders.
Silicon does not have a specified recommended
daily allowance (RDA). According to the FDA
suggestions, the total daily intake for men and
women is 40 and 19 mg, respectively. A safe
upper limit is thought to be 50 mg/day. It is
essential to keep proper Si levels during growth
periods, while the daily uptake should be
increased for the elderly, as silicon deficiency is
considered associated with aging. To determine
concentration of silicon and its structural forms
present in herbal drugs, dietary supplements
(tablets and capsules) were studied using WDXRF
and high-resolution solid-state 29Si-NMR. Three
different silicon forms were detected in the
herbal samples, Si(OSi)4, Si(OH)(OSi)3, and
Si(OH)2(OSi)2 [5].
10 Metals in Herbal Drugs and Fingerprints
40 mg/g reference mixture
herb of E. arvense
100° 105° 110° 115°
The analytical method for determination of
silicone was validated before it has been used to
test the actual sample. XRF spectra of 40 mg/g
reference mixture were compared with spectra
recorded for natural samples. Comparison shows
that regardless of sample there is no interference
of signal Ka for Si (Fig. 10.1). A determination of
standardized samples containing known amount
of Si was performed using a calibration curve
established in linearity study. Precision was
assessed by making five determinations of 2 mg/g
reference mixture (mixture of talcum with cellu-
lose), the relative standard deviation was 3.44%,
and the relative standard deviation (RSD) (for
repeatability) was 1.53%. Calibration curve was
plotted which has intercept value negligible,
along with limit of detection ( LOD, in this case
0.24 mg/g) as well as limit of quantification
[LOQ, by multiplying LOD three times (i.e.,
3 × 0.24 mg/g = 0.72 mg/g)]. The analytical
method was verified using three certified stan-
dards of powdered herbs, also.
High-resolution solid-state NMR spectra were
acquired at 298 K on an Avance 400WB spec-
trometer (Bruker, Rheinstetten, Germany) with a
Bruker 4 mm CP/MAS probe. The acronym CP
stands for cross-polarization. The acronym MAS
stands for magic angle spinning (a method for
narrowing signals in the solid-state NMR, for
achieving high-resolution spectra). NMR mea-
surements were carried out on powdered herbs at
the 29Si and 1H resonance frequencies of 79 and
Removal of Heavy Metals from Herbal Drugs
29
Fig. 10.2 Si CP/MAS–NMR spectrum of E. arvense from a herbal teapot bag [5]
400 MHz, respectively. The 29Si chemical shifts
were externally referenced against TMS, using
kaolinite as a secondary standard with a signal at
91.5 ppm. Peak fittings were done using a NutsPro
NMR computer program (Acorn 2007). Mixed
Lorentzian/Gaussian line shapes were applied to
fit experimental spectra and obtain chemical
shifts together with approximate peak areas.
Silicon forms were identified using high-resolu-
tion solid-state 29Si NMR. Because of the low Si
contents, the spectra were very noisy despite long
accumulation times (Fig. 10.2). In the whole
study only Q2, Q3, and Q4 forms were detected
(Table 10.1) [5].
Heavy Metals
There are eight common heavy metals, namely,
arsenic, barium, cadmium, chromium, lead, mer-
cury, selenium, and silver. These are all naturally
occurring substances which are often present in
the environment at low levels. In larger amounts,
they can be dangerous. Generally, humans are
exposed to these metals by ingestion (drinking or
eating). These metals are at least five times denser
than water. These (metallic form vs. ionic form)
are stable (i.e., cannot be metabolized), accumulate
195
in the body, and have highly toxic effects after a
certain limit.
Removal of Heavy Metals
from Herbal Drugs
Heavy metal content is one of the important
factors imparting toxicity in the herbal formu-
lations and needs to be checked and confirmed
that they are well below the WHO prescribed
limits. Some of the hazardous heavy metals as
lead (causing abdominal pain, convulsion,
hypertension and renal dysfunction, loss of
appetite, fatigue, sleeplessness, headache, and
vertigo), arsenic (causing abdominal pain, vom-
iting, sensory changes, numbness and tingling,
muscle tenderness, burning sensation in hand and
feet, excessive darkening of skin, liver injury),
mercury (causing shortness of breath, metallic
taste in mouth, abdominal pain, nausea, vomiting,
headache, weakness, visual disturbance, and
hypertension), and cadmium (causing nausea,
vomiting, abdominal pain, breathing difficulties,
growth impairment, osteoporosis, loss of taste
and smell, and cardiovascular disease). Raw
material used in the production of food, drug,
and Ayurvedic formulation are always tested,
196 10 Metals in Herbal Drugs and Fingerprints

Table 10.1 Silicon forms found in plants and range of their chemical shift [5]
Chemical shift
Symbol (ppm) Silicon site Structural formula
Q1 78–86 Si(OH)3(OSi≡)

Q2 86–94 Si(OH)2(OSi≡)2

Q3 95–107 Si(OH)(OSi≡)3

Q4 107–120 Si(OSi≡)4

and the consignments are rejected having the Case Study 3

higher limit of heavy metals specified by WHO.
Different analytical techniques are in practice
to decipher the quantity of heavy metals. In
Ayurveda these metals have been transformed
to nontoxic forms that are safe for internal use.
The heavy metal-based innovations are in prog-
ress to remove these metals and increase the
acceptability of the raw material grown from
the polluted sites also.
With the aim of designing and developing a
method for the bioremediation of the neem
(Azadirachta indica) extract various methods
described in official books [6, 7] but chelation
with dithizone was selected as the probable
option, due to a number of advantages as the
compound does not chemically react with the
main marker compound (unlike most of the other
Removal of Heavy Metals from Herbal Drugs 197

Table 10.2 Role of pH for removal of heavy metals from neem extract [8]
Volume of Volume of
extract used dithizone solution Heavy metal pH Inference
10 ml 17.5 ml Lead 6–7 Complexation reaction occurs in neutral medium
Cadmium 10–12 Complexation reaction occurs in alkaline medium
Mercury 3.5–4 Complexation reaction occurs in acidic medium

Table 10.3 Heavy metal concentration in neem leaf
extract [8]
Conc. in leaf WHO limit
S. no. Element extract (ppm) (ppm)
1 Lead 16.53 10
2 Cadmium 2.43 0.3
3 Mercury <0.10 1 hazardous elements in improving the quality of the
4 Arsenic <0.50 10
chelating agents), and it has a wide spectrum
of activity and even can chelate out the trace
elements [8].
Neem leaf was purchased from the local mar-
kets of Kolkata. All the chemicals and reagents
were of certified and of AR/LR grade. The pow-
dered dry neem leaf was mixed with distilled
water in the proportion of powder–water (1:8)
and filtered in order to remove the unwanted
materials, fibers, etc. The solution was then sub-
jected to analytical tests to detect the undesirable
elements or impurities. TLC was performed to
confirm the presence of the heavy metals.
Comparison of the Rf value of the herbal extract
and heavy metals proved the presence of lead,
cadmium, and mercury. Stationary phase silica
gel 60 F254, mobile phase: benzene, duration of
TLC development: 40 min for 10 cm, Rf value of
Pb = 0.316, Rf value of Hg = 0.769, Rf value of
Cd = 0.143, at 25°C [8].
UV–Vis. spectrometric analysis (standard curve
method) was used for quantification of the herbal
extract. Standard curves were plotted for lead, cad-
mium, mercury, and arsenic. Specific wavelengths
were used for each heavy metal (like 490 and
510 nm were the official wavelengths for the deter-
mination of cadmium and lead, respectively). The
absorbance hence obtained was compared with the
standard curve by using UV–Vis. spectrometer [8].
Nimbidin (an active constituent of neem extract)
was chosen as the marker compound. From the
analytical studies, it transpires that the heavy metal
impurities, namely, cadmium and lead, need prior-
ity action for their removal. So, the work plan was
designed to facilitate the elimination of these two
neem-based products. The reagents used for this
purpose was dithizone (0.005%) in chloroform.
Trial and error method was resorted for the pur-
pose of volumetric quantification of the reactants
as also the respective pH values (Table 10.2).
Dithizone was added quantitatively to avoid the
occurrence of any untreated reagent. The TLC
method followed the following specifications:
extract loaded, 2 ml; mobile phase, chloroform–
menthol (97:3); stationary phase, silica gel 60F254;
extraction media, methanol; Rf value of sample
(before dithizone) = 0.527; Rf value of sample
(after dithizone treatment) = 0.520; temperature,
25°C [8].
TLC was used for the identification of the
heavy metals, using the principle of comparing
the Rf values of the standard metal solutions
and the herbal extract. Quantity estimation was
done using UV–Vis. spectrophotometric method,
which establishes the presence of lead and cad-
mium, well beyond the WHO limit (Table 10.3).
After the chelation of the heavy metals, studies
were performed to establish that the marker com-
pound remained unaffected with the chelating
agent.
HPLC analysis for rapid data of nimbidin and
related terpenoids from neem before and after
dithizone treatment (mobile phase 20% ace-
tonitrile in isocratic mode, flow rate 1 ml/min,
lmax = 220 nm) HPLC chromatogram (Figs. 10.3
and 10.4) showed there was no significant change
in the mean area (A) and the retention time (RT)
198
Fig. 10.3 HPLC chromatogram of neem extract at initial stage [8]
Fig. 10.4 HPLC chromatogram of neem extract after dithizone treatment [8]
of the chromatogram both before (A = 97528.663
and RT = 4.181) and after (A = 88727.576 and
RT = 3.804) the dithizone treatment. From the
two HPLC chromatograms (Figs. 10.3 and 10.4),
it is reconfirmed that the marker compound, nim-
bidin, remained unaffected by chelation, although
the toxic heavy metals were removed from the
neem extract.
Bhasmas as Nanoherbal Drugs
Bhasmas are the Ayurvedic metallic preparations
in which metal act as a nanocarrier for drug deliv-
ery (Table 10.4), are widely recommended for
treatment of a variety of chronic ailments, and are
taken along with milk, butter, honey, or ghee to
10 Metals in Herbal Drugs and Fingerprints
eliminate the harmful effects of metals and
enhancing their biocompatibility in the body.
Neutron activation analysis of 20 metallic-based
bhasmas such as calcium, iron, zinc, mercury, sil-
ver, potassium, arsenic, copper, tin, and gem-
stones confirmed the purity of these bhasma as
the other elements such as Na, K, Ca, Mg, V, Mn,
Fe, Cu, and Zn were found in mg/g amounts and
Au and Co in ultra-trace (ng/g) amounts. Various
techniques like atomic force microscope (AFM),
transmission electron microscope (TEM), scan-
ning electron microscope (SEM), and energy dis-
persive spectroscopy (EDM) have been employed
for the estimation and characterization of bhasma.
The Swarna bhasma (nanoparticles of gold) anal-
ysis qualitatively through X-ray diffraction and
Fourier transform infrared spectroscopy (FTIR)
References
Table 10.4 Ayurvedic bhasma and basic metal which act as a nanocarrier [9]
S. No. Trade name of bhasma
1 Shanka bhasma
2 Swarna bhasma
3 Mukta
4 Abrak
5 Godanti
6 Loha
7 Trivang
8 Naga
9 Parad
showed that the particle size is about 56 nm con-
taining pure gold which acts as a nanocarrier for
drug delivery. Mineral arsenicals mainly in the
form orpiment (As2S3), realgar (As4S4), and arse-
nolite (As2O3) have been used in traditional drugs
for treatment of various diseases; however, arse-
nic can be highly toxic and carcinogenic [9].
The increased level of environmental pollu-
tions along our highways due to vehicular emis-
sion, extend it for the cultivation, and collection
of medicinal plants, intentional and unintentional,
route to the human body, with the message of
high health risk. The heavy metals in Ayurvedic
products are generally not present as contami-
nants but are added intentionally, with specific
procedure of detoxification (heating; heating and
cooling in buttermilk, cow’s urine, and sesame
oil; and the use of “mineral herbs” or other herbal
products such as tamarind). The equilibrium of
lead, copper, gold, iron, mercury, silver, tin, and
zinc are seen in Ayurveda as essential for normal
functioning of the human body and an important
component of good health; in addition, some
products contain other heavy metals such as
thallium and arsenic. In Ayurvedic practices an
individual herbal extracts or a mixture of herbal
extracts, with vegetable, animal, and mineral
products, can be used as drug and is defined as
the basic principle that anything can be used as a
drug [10]. The use of these and other traditional
formulation is increasing day by day globally,
so there is a need for epidemiological studies
in these areas to assess the potential risks and
for culturally appropriate education to inform
the public of the potential for toxicity associated
199
Basic metal as nanocarrier
Calcium present in sea products
Gold
Pearl
Manganese
Gypsum stones
Iron
Aluminum and zinc
Lead
Mercury
with these products, as the therapeutic efficacy is
associated with lifestyle and geographical condi-
tions also.
References
1. Zafar M, Khan MA, Ahmad M, Jan G, Sultana S, Ullah
K, Marwat SK, Ahmad F, Jabeen A, Nazir A, Abbasi
AM, Rehman Z, Ullah Z. Elemental analysis of some
medicinal plants used in traditional medicine by atomic
absorption spectrophotometer (AAS). J Med Plants
Res. 2010;4(19):1987–90.
2. Khan KY, Khan MA, Niamat R, Munir M, Fazal H,
Mazari P, Seema N, Bashir T, Kanwal A, Ahmed SN.
Element content analysis of plants of genus Ficus using
atomic absorption spectrometer. Afr J Pharm
Pharmacol. 2011;5(3):317–21.
3. Ekinci N, Ekinci R, Polat R, Budak G. Analysis of
trace elements in medicinal plants with energy disper-
sive X-ray fluorescence. J Radioanal Nucl Chem.
2004;260:127.
4. Nahapetain A, Bassiri A. Changes in concentration and
interrelationships of phytate. P, Mg, Cu, Zn, in wheat
during maturation. J Agri Food Chem. 1975;32:1179–84.
5. Pajchel L, Nykiel P, Kolodziejski W. Elemental and
structural analysis of silicon forms in herbal drugs using
silicon-29 MAS NMR and WD-XRF spectroscopic
methods. J Pharm Biomed Anal. 2011;56:846–50.
6. Despande VY, Mendulkar KN, Sadre NL. Male anti-
fertility activity of Azadirachta indica in mice. J
Postgrad Med. 1993;59:215–7.
7. Badani L, Deolankar RP, Kulkarni MM, Nagsampgi
BA, Waugh UV. Biological activities and medicinal
properties of neem. Indian J Malariol. 1987;24:111–7.
8. Ghos A, Chakrabarti P, Roy P, Bhaduri S, Nag T, Sarkar
S. Bioremediation of heavy metals from neem
(Azadiracta indica) leaf extract by chelation with
dithizone. Asian J Pharm Clin Res. 2009;2(1):87–92.
9. Gogtay NJ, Bhatt HA, Dalvi SS, Kshirsagar NA. The
use and safety of nonallopathic Indian medicines. Drug
Saf. 2002;25:1005–19.
200
10. Chaudhary N, Sekhon BS. An overview of advances
in the standardization of herbal drugs. J Pharm Educ
Res. 2011;2(2):53–70.
Bibliography
Al-Qurainy F. Toxicity of heavy metals and their molec-
ular detection on Phaseolus vulgaris (L.). Aust J Basic
Applied Sci. 2009;3(3):3025–35.
Balaji TA, Nair RN, Reddy AGC, Rao AVR, Naidu KS,
Manohar SB. Determination of essential elements in
Ayurvedic medicinal leaves by k0 standardized instrumen-
tal NAA. J Radioanal Nucl Chem. 2000;243(3):783–8.
Bowen HJM. Environmental chemistry of elements.
London: Academic; 1997. p. 6.
Gite VN, Pokharkar RD, Chopade VV, Takate SB.
Evaluation of physicochemical standardization param-
eters of Enicostemma axillare. J Biosci Technol.
2010;1(4):187–90.
10 Metals in Herbal Drugs and Fingerprints
Government of India, Ministry of Health and Family Welfare.
Indian Pharmacopoea. The Controller of Publications,
Civil Lines, Delhi-110054, 1996, A-62–A-64.
Kaladharan P, Kanadan V. Photosynthetic, potential and
accumulation of assimilates in the developing chloro-
embryos of Cyamposis tetragonoloba (L.) Taub. Plant
Physiol. 2001;29(2):408–12.
Kumar A, Nasair AG, Reddy AV, Garg AN. Bhasmas: unique
ayurvedic metallic-herbal preparations, chemical charac-
terization. J Biol Trace Elem Res. 2006;109:231–54.
Liu J, Lu Y, Wu Q, Goyer RA, Waalkes MP. Mineral arseni-
cals in traditional medicines: orpiment, realgar, and arse-
nolite. J Pharmacol Exp Therap. 2008;326(2):363–5.
Ponnusankar S, Santhi KD, Jacob N, Jacob B. Safety mea-
sures with herbals. Indian J Pharm Educ. 2005;7(2):84–7.
Rao AD, Devi KN, Thyagaraju KJ. Ayurtox for
bodydetoxification. Enzyme Inhib. 1998;14:85–6.
Saper RB, Kales SN, Paquin J, Burns MJ, Eisenberg DM,
Davis RB, et al. Heavymetal content of ayurvedic
herbal medicine products. J Adv Med Res.
2004;292:2868–73.
 Pesticide Residues in Herbals
and Detection
Pesticides, applied on crops for protection against
a range of pests, transmit to the different parts of
plants and finally plant products such as infusions,
decoctions, tinctures, essential oils, and extracts,
causing problem to health. Organophosphorus
(OP) pesticides (e.g., malathion, parathion, diazi-
non, fenthion, dichlorvos, chlorpyrifos, ethion)
act as cholinesterase inhibitors (chemicals that
disrupt neuromuscular transmission), resulting in
neurotoxicity. These are usually not persistent in
the environment. Carbamate pesticides affect the
nervous system by disrupting an enzyme that
regulates acetylcholine, a neurotransmitter. In this
case the enzyme effects are usually reversible.
Pyrethroid pesticides, a synthetic version of the
naturally occurring pesticide pyrethrin, which is
found in Chrysanthemums, have been modified
to increase their stability in the environment.
Some synthetic pyrethroids are toxic to the ner-
vous system. Biopesticides are certain types of
pesticides derived from such natural materials as
animals, plants, bacteria, and certain minerals,
for example, canola oil and baking soda have
pesticidal applications and are considered biope-
sticides. These can be categorized as microbial
pesticides, plant incorporated protectants (PIPs),
and biochemical pesticides. Microbial pesticides
consist of a microorganism (e.g., a bacterium,
fungus, virus, or protozoan) as the active ingredi-
ent and used to control many different kinds of
pests, specific for its target pest(s). The most
widely used microbial pesticides are subspecies
and strains of Bacillus thuringiensis, or Bt. Each
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_11, © Springer India 2012
11
strain of this bacterium produces a different mix
of proteins and specifically kills one or a few
related species of insect larvae, while some
Bt’s control moth larvae found on plants, other
Bt’s are specific for larvae of flies and mosqui-
toes. The target insect species are determined by
whether the particular Bt produces a protein that
can bind to a larval gut receptor, thereby causing
the insect larvae to starve. PIPs are pesticidal
substances that plants produce from genetic
material and are added to the plant, for example,
scientists can take the gene for the Bt pesticidal
protein and introduce the gene into the plant’s
own genetic material; then the plant, instead of
the Bt bacterium, manufactures the substance
that destroys the pest. Under these circumstances
pesticides enter inside the animal tissues also and
pesticide residue problem becomes a concern to
the whole biological system, so further discussion
on this chapter spread to the biological samples
from herbals.
Biochemical pesticides are naturally occurring
substances that control pests by nontoxic mecha-
nisms. Conventional pesticides, by contrast, are
generally synthetic materials that directly kill or
inactivate the pest. Biochemical pesticides include
substances, such as insect sex pheromones that
interfere with mating, as well as various scented
plant extracts that attract insect pests to traps.
Because it is sometimes difficult to determine
whether a substance meets the criteria for classifi-
cation as a biochemical pesticide, EPA has estab-
lished a special committee to make such decisions.
201
202 11 Pesticide Residues in Herbals and Detection

Table 11.1 Pesticides and utility [1]

S. No. Pesticide Use
1 Aldrin Against termites and other soil pests and termites attacking building
materials, in grain storage, and for vector control
2 Camphechlor (Toxaphene) Control of insect pests in cotton and other crops
3 Chlordane Against termites and other soil pests and termites attacking building
materials
4 DDT Control of medical and veterinary vectors, such as malaria-transmitting
mosquitoes, plague-transmitting fleas, and trypanosomiasis-transmitting
tsetse flies
5 Dieldrin Control of locusts, termites, human disease vectors
6 Endrin Formerly used against insects and rodents, presently not in use
7 Heptachlor Against termites and other soil pests and termites attacking building
materials
8 HCB Formerly used for seed treatment against fungal diseases, as well as
for industrial purposes
9 Mirex Against leaf-cutting ants and termites in buildings and outdoors, as a fire
retardant

Since the early 1970s, one country after another
restricted or banned the use of persistent organic
pesticides, often with the use of DDT for public
health applications (disease vector control) as the
only exemption. The last known uses for each of
the persistent organic pollutant (POP) pesticides
are summarized in Table 11.1 [1].
Regulatory Norms
In view of the menace, WHO recommends that
every country producing medicinal herbs (natu-
rally grown or cultivated) should have at least
one quality control laboratory to estimate pesti-
cide residues in herbals and herbal products. The
Association of Official Analytical Chemists
(AOAC) official method 985.22 is one of the
methods used in pesticide residue labs world-
wide. The European Pharmacopoeia (EP) and
United States Pharmacopoeia (USP) also define
limits, pesticide residue limit, for herbal drugs.
BP describes for 34 specific pesticides under
Part-1, V.4.6 Pesticide Residue (1995) and offers
methods of analysis under VIII.17. Testing for
pesticide residues in pharmaceutical raw materi-
als is described in the Ayurvedic Pharmacopoeia
of India (API), USP, Pharmacopoeia of the
People’s Republic of China, etc. In a case study
14 organochlorine pesticide residues of DDT group
(p,p¢-DDT, o,p¢-DDT, p,p¢-DDD, and p,p¢-DDE),
HCH isomers (alpha-, beta-, and gamma-HCH),
hexachlorobenzene (HCB), aldrin, dieldrin, endrin,
heptachlor, alpha-endosulfan, and endosulfan
sulfate were evaluated in 127 samples of medicinal
herbs collected in pharmacies (78 samples) and
herb stores (49 samples) in 1996. Samples were
divided between 15 national brands and 7 foreign
brands. Most samples sold in pharmacies con-
tained residues of gamma-HCH (51.3%). All
residues were detected in analyzed samples, with
exception of endrin in herb store samples.
Detection frequency varied between 51.3% for
gamma-HCH and 1.3% for endrin in pharmacy
samples and between 34.7% for HCB and 4.1%
for endosulfan sulfate in herb store samples.
Maximum residue levels were exceeded in 38
(48.7%) pharmacy samples and in 26 (53.1%)
medicinal herb store samples [2].
Analysis of Pesticide Residues
Chromatography (TLC, HPTLC, GLC, HPLC,
etc.) techniques have been recommended as the
principal method for the determination of pes-
ticide residues, but using chromatography, the
separations may not always be complete, pesticides
Analysis of Pesticide Residues 203

Table 11.2 Classification of the biological samples according to moisture and fat contents [3]
Biological sample
Environmental water
Blood (plasma, serum)
Urine
Mammalian milk (animal, human)
Eggs (egg albumin, egg yolk)
Other samples of animal origin (e.g.,
muscle, liver, saliva, hair, fat)
Plant material (e.g., fruit, vegetables,
feeds, medicinal plants, seeds)
Food (e.g., meat and meat products,
fish, milk and milk products, cereals,
maize, rice, sugar, beer, wine, juices,
plant oils)
Character of sample
Aqueous
Aqueous
Aqueous, urea
Aqueous, proteins, lipids
High lipid and albumin content
Various fat, protein, or water contents
Various (high or low) water, plant
pigment, lipid, protein, essential oil,
or wax contents
Various fats, oils, lipids, proteins,
sugars, starches, water, or pigments
Typical analytes
Pesticides industrial contaminants
Medicaments (human, veterinary),
pesticides
Drugs (permitted or prohibited),
industrial contaminants, pesticides
Veterinary drugs, toxic elements,
pesticides, industrial contaminants
Veterinary drugs, industrial
contaminants, pesticides
Drugs (permitted or prohibited),
industrial contaminants, pesticides
Pesticides, toxic elements,
industrial contaminants
Pesticides, industrial contami-
nants, synthetic colorants,
additives, synthetic sweeteners,
antioxidants

may decompose or metabolize, and many of the
metabolic products may be unknown. As a result
of limitations of the analytical techniques and
incomplete knowledge of pesticide interaction
with the environment, it is not possible to apply
an integrated set of methods that will be satis-
factory in all situations. WHO suggests that
plant materials of unknown history should be
tested for groups of compounds rather than indi-
vidual pesticides, and in case where the pesti-
cide to which the plant material is exposed is
known, it can be identified by a validated ana-
lytical method for determination of that particu-
lar pesticide.
Pesticide residues in samples of biological
(plants or animals) origin require isolation in the
form of the matrix; its cleanup to achieve com-
patibility with the mobile phase is necessary.
Stability of an analyte with respect to chemical or
enzymatic degradation during analysis is an
important parameter. The analytical conditions
are adjusted based on water (high, medium, dry)
and fat content. Samples of biological origin (liq-
uid or solid) can be divided generally into eight
groups (Table 11.2), an important division in ana-
lytical work plan for selection of an isolation
method, solvent(s) for extraction, and for the
sample cleaning procedure [3].
Isolation and Purification Methods
Isolation techniques have a significant role in the
pesticide residues’ analysis and quantification.
Commonly liquid–liquid extraction (LLE),
solid phase extraction (SPE) on a mini-column
(cartridge) and/or membrane extraction disk
(MED), solid phase micro extraction (SPME)
as a no-solvent modification of the SPE, extrac-
tion on nonpolar graphitized carbon black
(GCB), by immune-affinity extraction (IAE),
and supercritical fluid extraction (SFE). SFE is
characterized by high isolation and purification
effect. The advantage of SFE is that the super-
critical fluid penetrates excellently into matrices,
resulted into selectivity of analyte extraction,
high extract purity, and the ability to isolate
thermolabile analytes from a matrix. SFE pro-
vides a good possibility for automation and
ensures a minimum impact on the environment.
The technique is suitable for analyte isolation
from dry samples. The water content in the
sample has an influence on recovery of analyte
during extraction, and therefore, samples with
higher water contents are dried by addition of
an inert desiccating material before the extrac-
tion. The disadvantage of SFE is the require-
ment for high-purity carbon dioxide or other
204
extraction medium and high-input investment
for the equipment. SFE has been successfully
used for the isolation of pesticide residues
from cereals, oranges, food products or vegeta-
bles such as pepper, tomato, and cucumber, and
onion [3].
For cleaning gel permeation chromatography
(GPC) and column adsorption chromatography
are the most important ones. The stationary
phase for column chromatography is either by
florisil, Florex, alumina, or silica gel. The sorp-
tion capability of alumina can be modified by
impregnation with water–acetone mixtures
containing silver nitrate, which was used for the
removal of sulfur, a co-extract from a sample
(e.g., onion, garlic). Additional purification on
silver-loaded alumina after GPC is acceptable
for most commodities except for celery, parsnip,
cabbage, and cauli fl ower. Cleaning by GPC
is adequate for GC and/or LC in most cases.
A great advantage of GPC is the long lifetime of
the GPC column. Generally, it can be used for
several months without any adverse effect on
the retention volumes and the cleanup capacity.
GPC is usually used for extract purification
after liquid–liquid extraction and/or matrix
solid-phase dispersion (MSPD). A major advan-
tage of GPC is that the technique carries out
automatically the cleanup step. The sweep
co-distillation technique (SCD) combines a
rapid procedure and low capital cost for the uni-
versal trace residue extractor, with the significant
advantage of a reduced volume of organic sol-
vent. The separation tube of the extractor is
filled with silanized glass wool, and the sample
extract is injected into a heated fused-silica tube
through which an inert gas flows. Compounds
with low and high vapor tensions are forcibly
distilled from the tube together with the vapors
of the organic solvent. Compounds undergoing
easier distillation after condensation in a pattern
tube yield the distillates, which are analyzed by
gas chromatography. The effectiveness of extract
purification by sweep co-distillation is similar
to that of GPC or adsorption chromatography on
florisil. The sweep co-distillation technique is
unsuitable for thermolabile and low-volatile
analytes [3].
11 Pesticide Residues in Herbals and Detection
Hyphenated Techniques for Isolation
and Purification
The isolation (extraction) of an analyte from a
biological sample is connected with a defined
purification effect, and each purification step is
connected with isolation effect. So it is not pos-
sible to define exactly a technique used for the
isolation and cleanup procedure as an exclusive
isolation and/or purification technique. Column
adsorption chromatography is a suitable classical
technique for fast crude extract purification after
LLE and/or MSPD or as additional cleanup step
for extremely co-extractive loaded extracts com-
ing from SPE, GPC, and SCD treatments of bio-
logical samples. As an optimum technique for the
eluate, an additional cleanup step from the GPC
column is provided by adsorption chromatogra-
phy on a florisil column, and the same purification
step is used for the determination of organochlo-
rine pesticides in eggs. The lipids extracted from
whole egg by acetonitrile are separated first by
GPC column and then by a florisil minicolumn.
This cleanup is highly efficient for samples of
plant and animal origin. The combination of the
GPC with an additional cleanup on florisil or on
a silica gel cartridge has been used for GC/ECD
and GC/NPD. The advantages of adsorption
chromatography are robustness, high cleaning
column effectiveness, simplified optimization of
elution conditions, and versatility. Its disadvan-
tage is the necessity to control the adsorbent
properties. Adsorption chromatography on florisil
is a versatile purification technique suitable as
additional cleanup step of crude extracts from the
majority of samples. Some combination possi-
bilities of isolation and cleaning techniques used
in pesticide residues analysis in biological sam-
ples are summarized in Table 11.3 [3].
The simple and fast MSPD technique is used
preferentially for the isolation of pesticide residues
from samples of plant and animal origin with higher
water and relative low fat contents, using polar
solvent (e.g., methanol, acetonitrile, acetone).
The MSPD technique is not used for extraction of
some pesticide groups (e.g., chlorophenoxycar-
boxylic acids, aryloxyphenoxypropanoic acids),
which are better extracted by LLE from the
Analysis of Pesticide Residues 205

Table 11.3 Solvent selection for pesticide residues extraction [3]

Extraction solvent Pesticide group Samples
n-Hexane–acetone (9:1) Pyrethroids Vegetables
Petroleum ether–dichloromethane (3:1) Pyrethroids Vegetables and fruits
Dichloromethane Various pesticides carbamate and urea insecti- Vegetables
cides, sulfonylurea herbicide (chlorsulfuron)
Acetonitrile Organochlorine and organophosphorus Soy oil
pesticides, pyrethroids
Ethyl acetate Organophosphorus and organochlorine Fruits and vegetables
pesticides, pyrethroids

water-organic phase following pH adjustment.
MSPD of pyrethroid pesticide residues in fatty
material (e.g., soy oil) is applied by adsorbing a
fat solution into an Extrelut-3 cartridge and
extracting the pyrethroid residues with acetonitrile.
Pyrethroid pesticides are lipophilic compounds,
and their extraction from fatty matrices (e.g.,
food of animal origin, oils, cereals, oil seeds) is
accompanied by the simultaneous extraction of
considerable amounts of lipid material. MSPD
combined with GPC was used for the effective
separation of lipids from the crude acetonitrile
extract of soy oil. An additional cleanup step on a
florisil column can be used for low fatty samples
such as milk or highly fatty samples after a GPC
cleanup procedure. Classical LLE offers a wide
choice of organic solvents for effective analyte
isolation from the sample. Pure acetone and
methanol, rarely acetonitrile (due to high toxic-
ity), are often used for the extraction of various
pesticide residues from a biological matrix.
Pesticide residues from biological samples of
higher fat content are to be extracted with solvents
like dichloromethane, ethyl acetate, acetonitrile,
etc [3].
The microwave-accelerated solvent extraction
(MASE) is a new isolation technique that utilizes
microwave energy to heat up either the matrix or
the solvent used for extraction. MASE is attrac-
tive because the extraction is fast, typically from
5 to 10 min, and multiple samples can be extracted
in parallel [3].
The bipyridylium herbicides (desiccants)
diquat and paraquat have a cationic nature, and
they were extracted with hot dilute hydrochloric
acid followed by cleanup on an Amberlite GC-50
column and separated by HPLC using NH2 col-
umn. This procedure was described for the deter-
mination of paraquat and diquat in agricultural
products (rice, wheat, maize, potato, peach, cab-
bage). Residues of the polar herbicide bentazone
were selectively extracted by ion pair extraction
(IPE) as an ion pair with tetrabutylammonium
ion into dichloromethane. This technique was
used to clean up potatoes, cucumber, and wheat
grain samples. Extraction of bentazone residues
from plant material (barley, maize, wheat, soy
bean, alfalfa) with acetone or methanol is without
problems, but the effective re-extraction of its
residues into chloroform requires pH adjustment
according to the character of the sample [3].
Contemporary Trends in Pesticide
Residue Analysis
The SPE technique now commonly serves for the
isolation of residues of a wide group of pesticides
and their transformation products originating
from water samples or those from fruits and
vegetables with high water content. It is carried
out not only on nonpolar cartridges (GCB),
polymeric styrene–divinylbenzene (SDVB), and
modified silica gel cartridges (e.g., C8, C18) but
also on membrane extraction disks. These adsor-
bents are currently used as SPE materials for the
enrichment of acidic herbicides, although poly-
meric materials are considered to be more useful
since they permit a broader range of pH conditions.
SPME is a direct, solvent-free extraction technique
for pesticide compounds from aqueous samples
into a fused-silica fiber to which a stationary
206
phase of poly(dimethylsiloxane)/divinylbenzene
or polyacrylate has been bonded. SPME repre-
sents a very simple and rapid technique for the
extraction of various pesticides in environmental
water samples. This technique has a few impor-
tant advantages as solvent purchase and disposal
costs are greatly reduced, fibers are reusable
(fiber lifetime depends on conditions of use), and
versatility, use with any GC or GC/MS. Headspace
SPME has been used for the isolation of dinitroa-
niline herbicides in human blood, urine, or water
samples for GC/ECD without further purification.
Immuno-affinity-based extraction for the isola-
tion of pesticide residues from aqueous samples
replaces the organic solvent for the cleanup of
plant extracts. The results indicate that immune-
affinity chromatography (IAC) with or without
SPE/SAX cleanup offers several advantages over
classical techniques for the cleanup of aqueous
and/or water rich extracts for determination of
some herbicides. Perhaps the most significant
aspect is that no organic solvent other than meth-
anol is required in the procedure. The immune-
affinity cartridges are sometimes robust enough
to be regenerated many times and to be stored for
relatively long times (6–12 months) without
significant loss of activity, also true for cleanup
of plant extracts for the LC or GC determination
of urea and triazine herbicides. The major disad-
vantage of immunoassays is the cross-reactivity
to structurally similar compounds (can be sur-
prisingly used in IAE for the enrichment of a
group of analytes). The production and immobi-
lization of antibodies is still time-consuming and
requires special skills [3].
Modern analytical techniques have replaced
the classical LLE for the analysis, though we
have faced the ecologically harmful waste
of expensive organic solvents. Although LLE
method is still the basis for the isolation of pesti-
cide residues from samples of plant or animal
origin, an increased interest can be seen in the use
of the MSPD technique and the ecologically very
suitable SFE technique. The versatile GPC serves
for the primary purification or final purification
of a crude extract of biological samples with a
higher content of lipids. Extraction procedures
(e.g., LLE) with GPC or high-performance GPC
11 Pesticide Residues in Herbals and Detection
cleanup have achieved good reproducibility of
recovery. GPC is the multi-residual technique for
the cleanup of crude extracts for the determina-
tion of a range of pesticides, including phenoxy
acid herbicides in plants as well as in various
samples of biological origin before application of
GC/NPD, GC/ECD, GC/FPD, and/or GC/MS
(SIM). A comparison between different GC
element selective detectors (ECD, NPD, FPD) in
the sulfur and phosphorus modes, mass spec-
trometry detector in selected ion monitoring
(MSD-SIM), and atomic emission detector (AED)
was performed for the determination of various
pesticide residues from fruit and vegetables
samples. The results demonstrated that the
plant extracts (e.g., oranges, lemons, grapefruit,
pears, plums, lettuces, tomatoes, alfalfa, almonds,
broccoli, cauliflower, green onion, strawberry,
zucchini) can be analyzed by the detectors used
since no interfering co-extracted compounds
were detected [3].
Generally, most of the pesticide compounds
can be identified and/or determined by means
of their retention and spectral characteristics.
However, GC methods, when applicable, still
have the advantages of great separation efficiency,
high speed of analysis, and the availability of a
wide range of high sensitive and selective detec-
tors; LC can often be used as a method of choice
when polar, nonvolatile, or thermolabile com-
pounds are to be analyzed. Optical detectors
(UV, DAD) for HPLC generally need a highly
effective cleanup of the crude extract of the plant
sample. Liquid chromatography with selective
fluorescence detector (LC/FD) often requires
derivatization of the analyte, but it was used
successfully for the determination of glyphosate,
which is a very polar and amphoteric compound,
in blackberry and cereal samples. The LC–MS
technique has been used for the determination of
many pesticides in a variety of high-moisture
crops (e.g., apples, peaches, potatoes, tomatoes,
peppers, spinach, lettuce, snap beans, and sweet
maize). The adsorption chromatography using
florisil column is a rapid and simple technique for
additional cleanup of the extract from a biologi-
cal matrix. Adsorption chromatography has not
lost its position in spite of the significant spread
Analysis of Pesticide Residues
of GPC in pesticide residual analysis, and it
serves as an efficient purification technique for
atypical and complicated samples such as medic-
inal herbs [3].
Currently, pesticide residue analyses are
typically multi-residue procedures with highly
sensitive methods. Consumption of costly and
toxic solvents is being minimized, and fully
automated analytical procedures are expected in
the future. Hyphenated techniques such as
coupled systems (immune-affinity extraction,
immune-affinity chromatography, stationary
phases as restricted access materials, or molecu-
larly imprinted polymers) have become very
popular during the last decade. Most immune-
chromatographic techniques can be classified
into two types: pre-column and post-column
techniques, the pre-column techniques such as
immune-affinity extraction, where antibodies are
used as sample cleanup, and the post-column
technique, where antibodies are used in an immu-
noassay mode. The combination of immunoassays
with chromatographic techniques is very effective.
Frequently used is IAE, which can easily be
combined with many other chromatographic or
electrophoretic techniques; in most cases the
extract is directly suitable for HPLC techniques.
A graphitized carbon black or an octadecyl (C18)
cartridge was used in combination with ion-
exchange SPE cartridges. GCB made it possible
to remove color components but showed little
effect on reducing the matrix enhancement by
itself. Ion-exchange (SCX) cartridges in combi-
nation with or without GCB substantially reduced
the matrix enhancement effect, but did not eliminate
it. Phenylurea and triazine herbicides, including
some metabolites, were isolated from water and
soil extracts using a layered system of two mem-
brane extraction disks, a method called double-
disk solid-phase extraction (DD-SPE). The first
disk consisted of ion exchanger (SAX) of styrene–
divinylbenzene (SDB) particles embedded in
Teflon, and the second disk was a C18 disk. The
combination of SAX and C18 is an effective
method for herbicide isolation and cleanup when
HPLC/DAD is used for detection [3].
Molecular imprinted polymer (MIP) is a stable
synthetic polymer possessing selective recognition
207
sites, which are obtained by using high amounts
of cross-linking monomers in the presence of a
template molecule. Some of these polymers have
selectivity and affinity constants comparable with
those of naturally occurring recognition, such as
monoclonal antibodies or receptors, and for this
reason they have been referred to as plastic anti-
bodies. Two molecularly imprinted polymers
were synthesized with porogen and terbutylazine
as the template and were used as SPE cartridges
for the enrichment of six chlorotriazines in natu-
ral waters and sediment samples. The extracted
samples were analyzed by LC/DAD. Some
advantages of MIP as compared with an immu-
nosorbent are the potentially higher reproduc-
ibility of preparation, sample load capacity, and
stability. On the other hand, some disadvantages
are the site heterogeneity and a slow mass
transfer [3].
Restricted access material (RAM) can be con-
sidered a combination of a reversed-phase mate-
rial and an exclusion chromatographic gel. RAM
can be based on silica, which is modified hydro-
phobically. The outer surface of the particles,
however, is hydrophilic. The pore size is selected
in such a way that the analytes can enter the pores
and therefore are bound to the reversed phase.
RAMs are mostly used for online extraction pro-
cedures with very difficult matrices. Generally,
the analyte separation is performed on a second
column, which is filled with a standard RP mate-
rial. The use of RAM columns in the LC/LC
configuration is a powerful approach for the anal-
ysis of acidic compounds (mecoprop, metsulfu-
ron-methyl, bentazone, bromoxynil, and MCPA)
in water samples containing high levels of organic
matter [3].
Degree of Uncertainty of Analysis
Each analytical result is a combination of more
analytical steps, which comprise (without sam-
pling) several steps of sample adjustment and
finally measurement of analyte in the sample.
The standard way of expressing the variability of
an analytical method is its evaluation as a rela-
tive standard deviation (RSD), which embodies
208
maximum possible influences on dispersion
around the studied value within one laboratory
and also interlaboratory variation. For uncer-
tainty expression in the case when the analytical
method cannot be validated for time or financial
reasons for a wide range of matrices, the analyte
content and employed isolation and purification
techniques, the determined uncertainties can be
compared with variability values according to
the generally valid Horwitz equation [3, 4]
RSD(%) = 2(1 − 0.5 log w)
where RSD = relative standard deviation (%),
w = mass fraction (e.g., kg/kg or g/g).
By comparing individual partial uncertainties,
it is possible to identify significant contribu-
tions to combined (total) uncertainty. Combined
uncertainty is obtained from individual partial
uncertainties (all or significant ones only) accord-
ing to the law of dissemination of uncertainties.
In the case of a complicated analytical procedure,
to which pesticide residue analysis of samples
of biological origin no doubt belongs, uncer-
tainty factors, especially uncertainties that are
connected with sample preparation for chro-
matographic analysis and determination of
repeatability, rank among the significant contri-
butions to the total method. Random errors are
present in each step of an analytical procedure.
The field-to-field variability of residues is the larg-
est source of uncertainty (70–100%), followed
usually by the within-field (30–40%) and ana-
lytical (10–20%) variability. The sample prepa-
ration procedure is generally independent of
the nature of residues, their extraction, and
detection. Therefore, when a particular sample
preparation process is validated for a typical
commodity with its surface, the results may be
considered valid for any analyte and similar
commodity combination, provided that the stability
of the residues during processing is confirmed.
The uncertainty of the analytical steps is also
determined during the method validation. Two
thirds of the calculated Horwitz value can be
used as a guide about the acceptability of the
result [3].
11 Pesticide Residues in Herbals and Detection
Isolation and purification techniques are
mutually complementary and each isolation pro-
cedure is connected with some cleanup effect.
A way of purification of an extract from a bio-
logical sample strictly respects the analyte and
sample properties. A versatile isolation and
purification technique for general use with ana-
lyte and matrix is not known, however it is desir-
able to combine the simple analyte isolation from
a biological matrix with the effective purification
techniques of multi-residual and multi-matrix
character. Most synthetic agrochemicals with a
chiral structure are marketed as racemates
although the desired biological activity may be
derived only from one enantiomere. Some syn-
thetic agrochemicals as pyrethroid insecticides,
aryl-oxy-propanoate herbicides, and triazole fun-
gicides are marketed as the most biologically
active enantiomure isomer. When agrochemicals
have chiral structures, scientists look toward
defining the mode of action, elucidation of meta-
bolic pathway, and definition of the human and
environmental toxicity of each enantiomere.
Separation of chiral compounds is a new sphere
of pesticide residual analysis [3].
Pesticides Residues in Natural
Products
Pesticide residues analysis is intended to ensure
assessment for safety consumption and numer-
ous regulations have set maximum residue lim-
its (MRLs) for pesticides and their relevant
metabolites in a broad range of commodities
(principally for food, feed, and environmental
matrices). Nowadays, chromatography coupled
to MS has emerged as the default tool for
residue analysis because of its improved sensi-
tivity and selectivity provided for quantitative
and qualitative analysis in comparison to
conventional detection systems (i.e., ECD and
FPD). The successful couplings of LC–MS and
MS/MS have become the most used configuration
for large-scale residue analysis according to
the offered capabilities such as simultaneous
identification, confirmation, and quantitative
Current Scenario of Pesticides Residues in Herbals 209

Table 11.4 Selected commodities and their uses in herbal healthcare [6]
S. No. Commodity
1 Beeswax
2 Essential oils
3 Lanolin
4 Propolis
5 Medicinal herbs
Features/properties Pharmaceutical use
Compatibility with skin oils, UV inhibitor Hand and body creams, ointments,
and pill coatings
Aromatic Flavoring
Compatibility with skin oils, water absorbing, Creams and ointments
surfactant, and emulsifying
Antibiotic, anti-inflammatory, healing, Dermal contact, dietary supplement,
antioxidant cough candies
Wide variety of active constituents (i.e., Based on active ingredients, dietary
antibiotic, anti-inflammatory) supplement

determination for residue analysis. However, nat-
ural products were comparatively neglected in
such a trend. Multi-residue methods (MRMs)
are the preferred strategy over single-analyte or
single-class residue methods (SRMs). Analytical
features of the MRMs allow covering a large
range of pesticides in a single analysis. Many
MRMs employing different extraction and
cleanup techniques and a variety of detection
methods have been reported for the determina-
tion of pesticide residues in raw materials. The
selection of analytical method depends mainly
on the matrix composition, the physicochemical
properties of the target analytes, as well as the
chemical nature of interfering moieties present
in the matrices and/or the available instrumenta-
tion. Many herbs and spices that have been
included in the pharmacopeias, like cinnamon
and ginger, are evaluated for pesticide levels
using the newest analytical techniques [5].
Current Scenario of Pesticides
Residues in Herbals
Organochlorines (OCs) such as DDT, aldrin,
dieldrin, endrin, hexachlorocyclohexane (HCH)
isomers, and hexachlorobenzene (HCB) have
been widely used as insecticides since the intro-
duction of pesticides in management strategies
for the protection of crops (Table 11.4) [6]. Due
to its well-known environmental persistence,
most OCs have been currently banned worldwide
and replaced by synthetic pyrethroids and organo-
phosphates (OPs) insecticides.
Most studies still revealed the high inci-
dence of OC pesticides in several herbal drugs
worldwide [5, 7]. However, the main fact can
be divided into (1) findings are still OCs and
OPs insecticides and (2) the occurrence of a
growing amount of pesticides, mainly fungi-
cides, contained in medicinal products which
enlarge the target list of pesticides to search
for. A wide variety of pesticides were found
in a study carried out with Korean herbs [8].
Chlorfenapyr, chlorfluazuron, l-cyhalothrin,
metalaxyl, pyridalyl, fenvalerate, tebuconazole,
and tebufenozide residues were found suggesting
new strategies in pest control for medicinal
plants which include herbicides, fungicides,
and insecticides. However, residues of p,p¢-
DDE were also detected as main contaminants
in several plants [8]. Analytical reports for res-
idues exceeding MRLs are also available [9].
Monitoring campaign in Chinese and Korean
medicinal drugs showed main contamination
with five pesticides: methoxychlor, DDT, g-HCH
(lindane), endosulfan, and procymidone with
residues concentration ranging from 0.044 to
0.501 mg/kg. Intensive monitoring campaigns
have been suggested since the detection rate of
pesticides in 30 different types of drugs was
determined as 3.1% from the 229 samples ana-
lyzed. On critical observation was the detected
amount of procymidone (0.501 mg/kg) and
methoxychlor (0.382 and 0.312 mg/kg) [10].
Other studies showed that, from the eight
detected pesticides (residues between 0.034
and 0.579 mg/kg), four of them were fungi-
cides (captan, chinomethionate, procymidone,
210 11 Pesticide Residues in Herbals and Detection

Table 11.5 Pesticide residues (mg/kg) in dry fruits [12]

Total Total
S. No. Dry fruits HCH DDT Endosulfan
1 Anacardium 0.123 0.140 0.091
occidentale
2 Juglans niger 0.166 0.045 0.021
3 Prunus amygdalus 0.073 0.004 0.032
4 Pistacia vera 0.153 0.022 0.014
5 Cocos nucifera 0.099 0.047 0.017
6 Pinus gerardiana 0.106 0.041 0.034
7 Buchanania lanzan 1.328 0.001 0.079
8 Prunus armeniaca 0.007 0.036 0.037
9 Phoenix dactylifera 0.027 0.007 0.004
10 Vitis vinifera 0.007 ND 0.011
11 Euryale ferox 0.007 0.002 ND

and tolylfluanid) [5]. Moreover, major residues
of chlorothalonil fungicide were also detected
in Brazilian Passiflora leaves [11].
Pesticide Residues in Dry Fruits
The dried seeds of nuts are eaten as raw, roasted,
pureed or as flour, the best source of protein,
fats, and vitamin E. These have cholesterol free
unsaturated fat with magnesium, chromium,
zinc and manganese. A study on pesticide residues
in cashew nut (Anacardium occidentale), walnut
(Juglans niger), coconut (Cocos nucifera), chilgoza
(Pinus gerardiana), chironji (Buchanania lanzan),
makhana (Euryale ferox), resins (Vitis vinifera),
apricot (Prunus armeniaca), almonds (Prunus
amygdalus), date palm (Phoenix dactylifera ),
and pistachio nut ( Pistachio vera ), (Table 11.5)
the dry fruit samples were collected from local
Lucknow market, India, and analyzed as per the
standard procedure of AOAC (2005). 3.0 g of
each dry fruit in triplicate was grinded, taken in
20 ml test tube containing 15 ml petroleum ether.
Test tubes were mixed thoroughly and shaken
on mechanical shaker for 10 min. The extracts
obtained after passing the sample through a filter
paper are transferred to separatory funnel for
liquid–liquid partition. If color present in the
samples, it was removed by cleanup. It was passed
through a glass column (25 cm × 10 mm I.D.)
packed with charcoal and silica gel. Extract after
cleanup was evaporated till dryness and final
makeup done with n-Hexane [12].
Aliquots of above concentrate were injected
to precalibrated GC machine (Nucon 5765)
equipped with 63Ni electron capture detector. A
glass column (1.5 m × 2 mm I.D.) packed with
1.5% OV-17 + 1.95% QF-1 on 100–120 mesh
Chromosorb WHP was used. Operation tempera-
tures were programmed at 195, 200, and 220 °C
for column, injector, and detector, respectively.
Purified nitrogen gas, passing through silica gel
and molecular sieves, was used as carrier gas at
flow rate 60 ml/min. Periodical procedural blanks
were used to check cross contamination. Recovery
studies with purified samples indicated that over-
all recovery value exceeded 85%. Identification
and quantification of DDT and its metabolites
(p,p¢-DDE, o,p¢-DDT, o,p¢-DDD, p,p¢-DDT),
HCH (alpha-, beta-, and gamma-HCH), and endo-
sulfan were accomplished using known amount of
external standards, received from USEPA, pesti-
cides and industrial chemicals repository (MD-8)
Research triangle, NC, USA [12].
In the above study chlordane, endrin, hep-
tachlor, HCB, and hydrogen phosphide were also
focused but not found at or above their reporting
limits (i.e., 0.01 mg/kg for all the pesticides, and
0.001 mg/kg for hydrogen phosphide) [12].
Pesticide Residues in Dry Fruits
TLC Fingerprints
The use of TLC for pesticide residue is still in
practice for qualitative analysis. Pesticide residue
TLC fingerprints are developed, generally using
mobile phase, benzene: methanol and pre-coated
silica gel 60 F254 TLC plate of 0.2 mm thickness,
and detection at UV from 200 to 300 nm [13].
GC Fingerprints
In a case study the presence of organochlorine
pesticides (OCPs) in Lingzhi samples have been
analyzed by a procedure involving sample extrac-
tion, organochlorine pesticide fraction isolation,
and finally GC–ECD analysis on a HP6890 GC
system [14]. About 1 g of selected Lingzhi sam-
ple was extracted by a solvent mixture of 20%
acetone in petroleum ether (v/v) in an Accelerated
Solvent Extractor, ASE200 (Dionex). The extract
was concentrated to 1 ml and applied to florisil
SPE column for cleanup. The OCPs were eluted
with 12 ml 5% ethyl ether in petroleum ether
(v/v), and the eluant was concentrated to 1 ml by
a stream of nitrogen and injected into GC. Two
procedures are compared in recovery experi-
ments, one involved the use of reagent blanks
spiked with known organochlorine pesticide stan-
dards and the other involved the use of standards
spiked in real Lingzhi samples. The second pro-
cedure involved five different runs, each with a
different Lingzhi sample. Overall reasonably sat-
isfactory recovery results (70–130%) were
observed for the pesticides studied [14].
The different classes and wide range of pesti-
cides and agricultural commodities containing
them have fostered the development of new meth-
ods. However, the difficulty to cover all analytes
in one single method and for all the possible
matrices has turned this approach into an almost
impossible task, despite the advances in instru-
mentation. Moreover, new pesticides have been
introduced in the market and are more polar, non-
chlorinated, less persistent, or unstable, leading
to a change in the concept of the analytical and
detection features required [5].
211
The actual trend in pesticide residue analysis
focuses on the use of liquid chromatography cou-
pled to mass spectrometry techniques (LC–MS)
instead of gas chromatography (GC) with selec-
tive detection as electron capture (ECD), flame
photometric (FPD), nitrogen–phosphorus (NPD),
and thermal conductivity (TCD) detectors which
represented the state-of-the-art at the end of the
last century. They are intended for lipophilic (in
many cases obsolete) pesticides like most organo-
chlorine (i.e., p,p¢-DDT, aldrin, dieldrin, lindane).
Moreover, the targeted compounds monitored by
such methods sometimes are neither pesticides of
toxicological relevance or the most commonly
found in a particular sample. Furthermore, MRMs
could not be suitable and additional steps or
SRMs are required.
New trends in pesticide residue analysis have
been focused on the miniaturization of the sample
preparation methodology, moving to the develop-
ment of straightforward, faster, cost-effective,
and environmentally friendly procedures, adapt-
able for routine use in laboratories. Even with the
advent of highly sensitive MS and tandem mass
spectrometry (MS/MS) detection techniques, the
total knowledge of the contribution of pesticides
in pharmaceutical products has not been com-
pletely developed yet, as for food and feed com-
modities or environmental samples.
The widespread presence of the pesticide resi-
dues may be due to one or more of environmental
causes and agricultural practices. Thus, using
cultivated plants under controlled conditions
instead of those collected from the wild can mini-
mize most of these factors and any step toward
pesticide residue prevention and bio-resource
management for better growth and increased
therapeutic potential of herbals and products.
TLC/TPTLC profiles examination together with
hi-tech analysis may be used for quality evalua-
tion and fingerprints for pesticide load. Thus, the
present review with case studies will help in set-
ting up regulatory limit, to ensure the quality as
claimed, for safety of commercial herbals and
their derived products such as herbal dietary sup-
plements and for pesticide residues. This will help
for the comprehensive assessment on pesticide
212
occurrence in matrices particularly not yet well
studied and difficult to handle. An update of
global acceptance on residue analysis methodol-
ogies is a necessary step to be adopted for pesti-
cides which have been introduced recently, as
situation provides opportunities for the develop-
ment of new methods for organic contaminants
residue analysis in pharmaceutical raw materials,
a non closed and challenging issue. TCM and
Ayurvedic products are in use all over the world
with trust in their efficacy and safety. Aromatic
plant drugs including essential oils and natural
aromas have been used extensively in the phar-
maceutical industry as flavoring and to mask the
foul odor or taste of some pharmaceuticals as
excipients.
References
1. Mörner J. Alternatives to POP pesticides for the
protection of plants and building constructions. In:
Alternatives to persistent organic pollutants. The
Swedish input to the IFCS expert meeting on persis-
tent organic pollutants (Manila, 17–19 June 1996).
Rapport från kemikalieinspektionen (Report from the
Swedish National Chemicals Inspectorate). 1996;
4/96.79-16.
2. Lino CM, Guarda LM, Silveira MI. Determination of
organochlorine pesticide residues in medicinal plants sold
in Coimbra, Portugal. J AOAC Int. 1999;82(5):1206–13.
3. Tekel J, Hudecova T, Pecníkova K. Isolation and
purification techniques for pesticide residue analyses
in samples of plant or animal origin. Eur Food Res
Technol. 2001;213:250–8.
4. Horwitz W. Evaluation of analytical methods used for
regulation of foods and drugs. Anal Chem. 1982;
54:67A–7A.
5. Perez-Parada A, Calazzo M, Besil N, Dellacassa E,
Casio V, Heinzen H, Fernandez-Alba AR. Pesticide
residues in natural products with pharmaceutical use:
occurrence, analytical advances and perspectives. p.
357–90. Pesticides in the modern world–trends in
pesticides analysis. Available at http://www.
intechopen.com/source/pdfs/20996/InTech-Pesticide_
residues_in_natural_products_with_pharmaceutical_
use_occurrence_analytical_advances_and_perspec-
tives.pdf. As on 28 Feb 2012.
11 Pesticide Residues in Herbals and Detection
6. Jover E, Bayona JM. Trace level determination of
organochlorine, organophosphorus and pyrethroid
pesticides in lanolin using gel permeation chromatog-
raphy followed by dual gas chromatography and gas
chromatography-negative chemical ionization mass
spectrometric confirmation. J Chromatogr A. 2002;
950(1–2):213–20.
7. Abhilash PC, Singh N. Multiple residue extraction for
organochlorine pesticides in medicinal plants. Bull
Environ Contam Toxicol III. 2008;81(6):604–7.
8. Nguyen TD, Lee KJ, Lee MH, Lee GH. A multi-resi-
due method for the determination 234 pesticides in
Korean herbs using gas chromatography mass spec-
trometry. Microchem J. 2010;95(1):43–9.
9. Abou-Arab AAK, Abou Donia MA. Pesticide residues
in some Egyptian spices and medicinal plants as affected
by processing. Food Chem. 2001;72(4):439–45.
10. Oh CH. Monitoring of residual pesticides in herbal
drug materials of Korea and China. Bull Environ
Contam Toxicol. 2009;82(5):639–43.
11. Zuin VG, Yariwake JH, Lanças FM. Analysis of pes-
ticide residues in Brazilian medicinal plants: matrix
solid phase dispersion versus conventional (European
Pharmacopoeia) methods. J Braz Chem Soc. 2003;
14(2):304–9.
12. Pandey P, Raizada RB, Srivastava LP. Level of organo-
chlorine pesticide residues in dry fruit nuts. J Environ
Biol. 2010;31(5):705–7.
13. Smith AG. Chlorinated hydrocarbon insecticides.
In: Handbook of pesticide toxicology. Wayland J. Hayes
(ed.), San Diego: Academic Press Inc.; 1991. Vol. 2.
14. Tong TC, Lee CS-C, Hu GL, Chang L, Wang X, Fu
PP. A survey of heavy metal and organochlorine
pesticide contaminations in commercial Lingzhi
products. J Food Drug Anal. 2007;15(4):472–9.
Bibliography
Anonymous. The Ayurvedic Pharmacopoeia of India, New
Delhi Department of Ayush, Ministry of Health And
Family Welfare, Government of India; 2009. 70–2.
Anonymous. Pharmacopoeia of India. Edition 4.
Government of India, Ministry of Health, Controller
of Publication, New Delhi; 1996. A-53–55.
The British Pharmacopoeia-1. Author: British Pharma-
copoeia Commission London: Stationery Office;
2002.
Lohar DR, Singh R. Quality control manual for Ayurvedic,
Siddha and Unani Medicine. Ghaziabad. Department
of Ayush, Ministry of Health and Family Welfare.
Pharmacopoeial Laboratory for Indian Medicine.
2008; p. 21–4.
 Radiation Detection in Herbals
Herbs and herbal products are in use as food
supplements because of their unique nutritional
ingredients. Collection of herbs for medicinal
purposes and long-term storage in dried form are
quite common practices. Over the last two decades,
irradiation of herbs and products with g-rays has
emerged as a leading sterilization method against
microbial deterioration or insect infestation during
storage and transport, to preserve hygienic quality,
ensure shelf life, as well as to reduce public health
threat. Unlike other quarantine treatments such as
thermal treatment and fumigation with environ-
mentally disruptive toxic chemicals pose an occu-
pational health hazard, but irradiation technique is
considerably rapid, cost-effective, and offers a
broader spectrum of sanitizing applications for dry
ingredients. In view of the increasing use of irra-
diation technology for preservation of herbs, the
availability of analytical techniques for identifying
irradiated or nonirradiated herbs is of much impor-
tance for regulatory authorities and consumers to
increase confidence in radiation processing tech-
nology. Several detection methods, broadly cate-
gorized as chemical, physical, and biological, have
been developed for the identification of irradiated
herbs and herbal products. Among the physical
techniques, pulsed photo-stimulated luminescence
(PPSL), thermoluminescence (TL), and electron
paramagnetic spin resonance (EPR/ESR), spec-
troscopy are the leading techniques [1].
The thermal pasteurization of liquid (e.g.,
milk, water) is well established and satisfactory
for terminal decontamination/disinfection of such
commodities but inappropriate for solid foods,
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_12, © Springer India 2012
12
dry ingredients, and fresh foods known for raw
characteristics. The radiation pasteurization with
low doses of gamma (g) rays, X-rays, and electron
beam effectively controls food-borne pathogens.
Irradiation leads to the destruction of pathogenic
nonspore-forming food-borne bacteria and para-
sitic organisms, such as trichina, and protects the
consumers from microorganism-related diseases.
The application is based on the fact that ionizing
radiation causes very effective disruption of DNA
molecules in the nuclei of cells, rendering them
inactivate [2]. Irradiation has been examined
thoroughly by joint committees of the World
Health Organization (WHO), the United Nations
Food and Agriculture Organization (FAO), the
European Community Scientific Committee for
Food, Food and Drug Administration (USA), and
by House of Lords Committee. Food absorbs
energy when it is exposed to ionizing radiation;
the amount of energy absorbed is called “absorbed
dose,” which is measured in units called grays
(Gy), kilograys (kGy) (1 kGy = 1,000 Gy). The
energy absorbed by the food causes the formation
of short-lived molecules known as free radicals,
which kill microorganisms and also interact with
other food molecules. Thus, irradiation leads to
check microorganisms, insect gametes, and plant
meristems from reproduction and have preserva-
tive effects as a function of the absorbed radiation
dose (Table 12.1), while chemical- or other radi-
ation-induced changes are minimal [4].
An important reason for the relatively high
sensitivity of DNA to the ionizing radiation is
that DNA molecules are much larger than other
213
214 12 Radiation Detection in Herbals

Table 12.1 Doses for various applications of irradiation [3]

S. No. Preservative effects and types of application Dose (kGy)
1 Killing and sterilizing insects (disinfestations of food) 0.2–0.8
2 Prevention of reproduction of food-borne parasites 0.1–3.0
3 Decrease of after-ripening and delaying senescence of some fruit and vegetables; extension 0.5–5.0
of shelf life of food by reduction of microbial populations
4 Elimination of viable nonspore-forming pathogenic microorganisms (other than viruses) 1.0–7.0
in fresh and frozen food
5 Reduction or elimination of microbial population in dry food ingredients 3.0–10.0

molecular structures inside the cell. The damage
is either direct, caused by reactive oxygen from
hydroxyl radicals (OH−) originating from the
radiolysis of water, or indirect. In the case of
an indirect hit, the damage to the nucleic acids
occurs when radiation ionizes an adjacent mole-
cule, which in turn reacts with the genetic material.
In view of the fact that water is a major compo-
nent of most foods and microbes, it is often the
adjacent molecule that ends up producing a lethal
product. The ionizing radiation causes water mol-
ecule to lose an electron producing H2O+, which
immediately reacts with other water molecules to
produce a number of compounds, including hydro-
gen and hydroxyl radicals, molecular hydrogen,
oxygen, and hydrogen peroxide. Hydroxyl radi-
cals are very reactive and are known to interfere
with the bonds between nucleic acids within a
single strand or between opposite strands.
Although biological systems have a capacity to
repair both single-stranded and double-stranded
breaks of the DNA backbone, the damage occur-
ring from ionizing radiation is random and exten-
sive [5]. Therefore, recovery processes in bacteria
after their radiation damage are unlikely to occur.
The differences in sensitivity to radiation among
microorganisms are related to the differences in
their chemical and physical structure and in their
ability to recover from the radiation injury. The
amount of radiation energy required to control
microorganisms in food and food supplements,
therefore, varies depending on the resistance of
the particular species and the number of organisms
present. Besides inherent abilities of microorgan-
isms, several environmental factors such as the
composition of the medium, moisture content,
temperature during irradiation, and presence or
absence of oxygen significantly influence their
radiation resistance, particularly in vegetative
cells. The actual dose employed is a balance
between that what is needed and that what can
be tolerated by the product without objectionable
changes (e.g., off-flavors, texture changes, and
flavor alterations).
Microorganisms in Herbals
In stored herbals, microorganisms develop favor-
able conditions for themselves; such incidents
are more common with poorly stored products.
From a hygienic viewpoint such contaminated
material should be destroyed irrespective if active
principles have been affected or not. A summarized
discussion on the problem is as below; [6]
Spores of bacteria and molds are always
present in the air, and dried herbs are particularly
liable to be contaminated. Under satisfactory
storage conditions, their presence is not a problem;
however, for some crude drugs, certain pathogenic
bacteria are specifically limited or excluded, for
example, Escherichia coli in digitalis, tragacanth,
and gelatin and Salmonellae in digitalis as well as
cochineal. Certain species of molds, for example,
Aspergillus flavus and Fusarium tricinctum, pro-
duce extremely potent mycotoxins, which in the
past have been the cause of many deaths through
the consumption of overwintered moldy grain in
certain countries. The spores of such species have
been shown not to be common in vegetable drugs
and would be harmless anyway unless allowed to
develop through poor storage. With the exception
of chromogenic species of bacteria, for example,
Bacillus prodigiosus, which produces red patches
Microorganisms in Herbals
in starchy materials, the presence of actively
growing bacteria is not readily visible. However,
bacterial growth is usually accompanied on the
crude drug by growth of molds whose presence is
soon evident by the characteristic smell and by
the mass of clinging particles entrapped in the
mycelial hyphae. In most cases, in order to iden-
tify a particular mold which is proliferating in a
stored product, it is necessary to culture it on a
suitable medium, to obtain fruiting bodies for
examination. However, if the drug to be exam-
ined is infested to an advanced degree, then it
may be possible to make microscopic prepara-
tions directly from the sample. The principal
molds encountered with poorly stored drugs
include the genera Mucor (e.g., the gray mold, M.
mucedo), Rhizopus (e.g., black mold, R. nigri-
cans), Penicillium (e.g., blue mold, P. glaucum),
Aspergillus (e.g., green mold), and Saccharomyces
(yeasts). The spores of Mucor and Rhizopus can
be clearly seen on their stalks with a hand lens,
and the members of this group can usually be
immediately recognized because of the rapidly
growing light-colored, loose-textured colonies
which are usually white or light gray in color.
Aspergillus forms a mop-like head of spores, while
Penicillium produces a flat brush-like head.
The arachnids (mites) are found in stored
drugs usually in countless numbers, on examina-
tion with a hand lens. They can be examined
microscopically by clearing a sample of the powder
containing them with chloral hydrate reagent; the
outline and appendages remain distinct, and the
females can be seen to contain numerous larvae.
A commonly found members of the group are the
flour mite (Tyroglyphus farinae, Acarus siro),
which thrives on starchy and cereal products,
cheese mite (Tyroglyphus dimidiatus), and
smaller mites (Cheyletus eruditus) that are often
found with herbals and/products.
A number of small beetles are a common
source of infestation of stored herbals. Beetles
undergo a complete metamorphosis, from egg to
larva to imago, and are harmful for stored herbals
at both larval and adult stages. The most common
beetle found in many drugs, including gentian,
liquorice, and rhubarb, as well as leafy parts and
seeds, is Stegobium paniceum, the biscuit beetle
215
(name being derived from its frequent occurrence
in the biscuits of the old-time sailors). The Lasio-
derma serricorne is found in stored products
including ginger and liquorice.
A few species of the Lepidoptera attack stored
products, and although damage at the larval stage
only, the infestation can spread rapidly due to the
mobility of the adults. Some species, for example,
Ephestia kuehniella, flour moth, produce larvae
which, when fully grown, remain in the food mate-
rial where they overwinter and pupate. On the other
hand, the grubs of E. elutella, the cocoa moth, leave
the original food containers and during this process
they cover themselves with a web of silky threads.
They enter cracks and crevices where they spin
cocoons and finally emerge as adults.
A general practice to use cool–dry condition
for the storage of herbals and products is not
always safe, for example, the eggs of the flour
mite can survive several months at a temperature
of 0°C and 12 days at −10°C. Irradiation of herb-
als and products is currently in practice since two
decades. These radiations are limited to high-
energy photons (gamma rays of radionuclides
60
Co and, to a much smaller extent, 137Cs or X-rays
from machine sources with energies up to
5 MeV or accelerated electrons with energies up
to 10 MeV produced by electron-accelerating
machines), to produce the desired food preserva-
tive effects, without inducing radioactivity in
foods, food supplements, or packaging materials,
for commercial purpose. The radiation treatment
causes only minimal temperature rise in the
product and can be applied through packaging
materials including those that cannot withstand
heat, meaning that the radiation treatment can be
performed also after packaging; thus, recontami-
nation or reinfestation of the product is avoided.
Long-term animal feeding studies have demon-
strated that radiation-pasteurized/sterilized foods/
food supplements are safe and nutritious also for
humans, irradiated at doses below 10 kGy. The
Directive 1999/3/EC established a community
list of foods and food ingredients that may be
treated with ionizing radiation (EC 1999).
According to this directive, maximum allowed
overall average absorbed dose is 10 kGy for dried
aromatic herbs, spices, and vegetable seasonings.
216
The Food and Drug Administration (FDA), USA,
sets a limit for irradiation treatment of culinary
herbs, seeds, spices, vegetable seasonings, and
blends of these aromatic vegetable substances
that must not exceed 30 kGy. The radiation
pasteurization process for many foods has been
approved or endorsed by many world agencies
and associations such as the FDA, WHO, the
Codex Alimentarius Commission, the American
Medical Association, the Institute of Food
Technologies, and the health authorities in
approximately 40 countries [7].
Safety and Effectiveness of Irradiation
A mild treatment as a radiation dose of 1 kGy
represents the absorption of only enough energy
to increase the temperature of the product by
0.36°C. In fact, heating, drying, and cooking may
cause higher nutritional losses. Moreover, certain
carcinogenic aromatic and heterocyclic ring com-
pounds produced during thermal processing of
food at high temperatures were not identified in
food after irradiation. In general, food macronu-
trients (carbohydrates, proteins, and lipids) and
most micronutrients (mainly water-soluble and
fat-soluble vitamins) are not appreciably affected
by 10-kGy range ionizing dose with regard to
their nutrient contents. However, with higher
radiation doses (above 10 kGy, the exceeding
permitted limit in the EU), the structural proper-
ties of the fibrous carbohydrates can be degraded,
and lipids can become somewhat rancid, leading
to a loss of food quality [7, 8]. Moreover, the irra-
diation of lipids at high doses, and especially in
the presence of oxygen, can lead to the formation
of liquid hydroperoxides. The oxidation products
formed often have undesirable odors and flavors
(rancidity). It is known that the unsaturated fatty
acids are more prone to develop rancidity. Lipid
oxidation can be significantly reduced by freezing
and/by oxygen removal prior to irradiation. Among
micronutrients, thiamine is a subject of concern
as it is relatively sensitive to the effects of radia-
tion. Foods that contain thiamine (e.g., pork) are
suitable as indicators of food safety regarding the
irradiation treatment [7, 8], but minerals remain
12 Radiation Detection in Herbals
stable. Besides the nutritional and sensory values,
the wholesomeness (lack of mutagenicity, terato-
genicity, and toxicity) of irradiated foods has
been studied extensively. Neither short nor multi-
generational feeding studies have produced any
evidence of toxicological effects in mammals due
to ingestion of irradiated foods. In fact, multigen-
erational studies with animals have demonstrated
that the ingestion of irradiated foods is completely
safe and that the nutritive value remains essen-
tially unchanged. The data support the conclusion
that properly processed irradiated foods are whole-
some. Radiolytic changes in foods are minimal
and are predictable from the radiation chemistry
of principal food components. Furthermore,
possible radiolytic products derived, for exam-
ple, from lipids (most of which are saturated
and unsaturated hydrocarbons, aldehydes, and
2-alkylcyclobutanones) are neither unique nor
toxicologically significant in the quantities found
in irradiated foods. Irradiation is necessary for
the safety from microflora, and other benefits are
an increased shelf life (of fruits, vegetables, spices,
and meat), and a suitable alternative to chemical
treatments, especially for the decontamination of
fruits, vegetables, and spices [7].
Irradiation of Spices and Dried
Vegetable Seasonings
The aromatic oil-bearing plant parts, especially
essential oil-rich moieties, are known as spices
often collected from the developing countries
where harvest and storage conditions are poor
with respect to food hygiene. A very small amount
of these spices may be a potential source of
microbial pollution for food stuffs to which they
were added. Most spices are dried in the open
air and can become seriously contaminated by
air- and soil-borne bacteria, fungi, and insects.
Microorganisms of public health significance
such as Salmonella, Escherichia coli, Clostridium
perfringens, Bacillus cereus, and toxigenic molds
can also be present. Bacterial plate counts of
1–100 million per gram of spice are usual. Good
manufacturing practices during harvest and pro-
cessing could improve their hygienic quality, but
Irradiation Dose for Essential Oils and Organoleptic Characters
frequently not to an extent sufficient to obtain
an acceptable microbiological purity level.
Contaminated, dry plant ingredients cause seri-
ous troubles in the food-based industry. Therefore,
many commercial food processors fumigate
spices with methyl bromide to eliminate insects
or with ethylene oxide to eliminate bacteria and
molds. However, it has been found that both
methyl bromide and ethylene oxide are extremely
toxic compounds. Moreover, methyl bromide is
potentially capable of depleting the atmospheric
ozone layer. Ethylene oxide has been banned in
Europe because of safety and environmental
concerns, and its use for the treatment of ground
spice has been banned in the United States. The
US Clean Air Act and the Montreal Protocol of
the Vienna Convention require that any substance
listed as ozone depleting be withdrawn from
production and use by the year 2001. Spices,
herbs, and dried vegetable seasonings are cur-
rently treated with ionizing radiation to eliminate
microbial contamination. It was unambiguously
confirmed that the treatment with ionizing energy
is more effective against bacteria than the thermal
treatment and does not leave chemical residues
in the food product. Thus, ethylene oxide and
methyl bromide treatments can be effectively
replaced by food irradiation, which in fact is less
harmful to the spices than heat sterilization, as it
leads to the loss of thermolabile aromatic vola-
tiles and/causes additional thermally induced
changes (e.g., thermal decomposition or produc-
tion of thermally induced radicals) [7].
Irradiation Dose for Essential Oils
and Organoleptic Characters
Gas chromatography with flame ionization detec-
tion (GC/FID), gas chromatography–mass spec-
trometry (GC/MS), sensory evaluation, and
GC–olfactometry are in use to compare the impact
of irradiation on essential oils. In a case study, pow-
dered black pepper was irradiated with different
recommended doses of gamma rays (5 and 10 kGy,
respectively) and treated with microwaves for dif-
ferent periods (20, 40, and 75 s). The results obtained
indicate that irradiation treatment with controlled
217
doses of gamma irradiation is a safe and suitable
technique for decontamination of black pepper.
In comparison with microwave treatment, irradia-
tion does not result in extensive loss of flavor
compounds. In contrast to the radiation treatment,
the thermal treatment of black pepper (using 130°C
hot dry steam for 4 min, internal temperature of the
treated berries was 95°C) caused a significant
increase in the content of monoterpenes [7, 9].
These changes might have resulted from the forma-
tion of thermal isomerization products of some
terpenes. The qualitative composition of volatile
oils obtained from control, thermally treated sam-
ple, and from irradiated samples of black pepper at
various doses (up to 30 kGy) was identical. Statistical
analysis of the effects of irradiation on the total
content of volatile oils in spices showed no
significant differences between irradiated and non-
irradiated samples. As per Sadecka et al., the most
important changes were observed in the black pep-
per sample irradiated up to the dose of 30 kGy,
which resulted in a threefold increase of caryophyl-
lene oxide concentration and a parallel decrease of
sesquiterpene caryophyllene, in comparison with
an untreated sample [7, 10]. Nevertheless, such a
dose of ionizing energy exceeds three times the
level permitted by the EU legislation (EC 1999).
The observed changes induced in terpenes by irra-
diation could be explained by oxidation or hydroxy-
lation of aromatic rings in terpene molecules. The
products with low moisture content (10%) are prone
to the formation of free radicals by irradiation which
leads to an increased production of oxides and alco-
hols. In addition, the configuration of the side groups
on the terpene skeleton, especially the position of
double bonds and functional groups, can result in a
variety of compounds produced. The research has
demonstrated that gamma irradiation at the dose of
10 kGy (toxicologically and nutritionally confirmed
maximum safe dose) can eliminate microbial load
of spices without causing any significant organolep-
tic or chemical alterations [7].
Studies on dried basil leaf for essential oils by
Antonelli et al. observed that the composition of
essential oils treated at doses of 5 and 10 kGy
was different in comparison to the controlled
sample. They concluded that radiation caused
more evident changes in the composition profiles
218
than the microwave treatment. Subsequently, a
sensory test confirmed significant differences
between the extracts. The panelists preferred the
gamma-treated sample, while the microwaved
sample was least appreciated [7].
Radiation Quantity and Antioxidant
Activity
Spices (anise, cinnamon, ginger, liquorice, mint,
nutmeg, cumin, vanilla, etc.) are well-known
source of antioxidants used in daily life. In a case
study with the nonirradiated samples, the water
extracts of the irradiated spices at 1, 3, 5, and
10 kGy did not show any significant difference in
the antioxidant activity in the radical-scavenging
assay. The antioxidant properties of anise, cara-
way, cumin, and fennel essential oils extracted
from untreated, gamma-irradiated, and micro-
waved seeds was studied. Gamma-irradiation at
10 kGy and microwave treatments did not affect
the antioxidant property of the essential oils
under study. In addition, essential oils extracted
from gamma-irradiated fruits were more effective
antioxidants in sunflower oil than those produced
from microwaved fruits. The capsaicinoid content
in sun-dried and dehydrated paprika samples
that were irradiated at doses from 2.5 to 10 kGy
capsaicin, dihydrocapsaicin, and homodihydro-
capsaicin was increased by about 10% in the
samples irradiated at the dose of 10 kGy [7, 11].
The effects of irradiation by electron beam on
the color and the contents of volatile oils in five-
spice powder (prickly ash, star aniseed, cinna-
mon, clove, and fennel) and chili were assessed
by Lianzhong et al. [12]. Irradiation enhanced
the UV absorption of aqueous extracts of spices,
but the darkening phenomenon of spices due
to irradiation was temporary. In studies for the effect
of gamma irradiation at 10 kGy on the free
radical formation and the antioxidant contents
of aromatic herbs and spices (basil, bird pepper,
black pepper, cinnamon, nutmeg, oregano, pars-
ley, rosemary, and sage), Calucci et al., observed
that in a general increase of quinine radical
content [measured using electron paramagnetic
resonance (EPR) spectroscopy] in all samples
12 Radiation Detection in Herbals
investigated, and in a significant decrease of
the total ascorbate and carotenoids content of
some spices [13]. Results from different studies
have found no significant differences between
EPR spectra of the samples of white pepper,
sweet paprika, and nutmeg irradiated with elec-
tron beams or X-rays at 0–10 kGy irradiation.
Several studies using EPR spectroscopy to inves-
tigate free radicals formed by the gamma-
radiation treatment of ground black pepper,
oregano, all spice, ginger, and clove and to evalu-
ate the influence of the absorbed dose of gamma
radiation on the radical-scavenging potential of
alcoholic extracts, it was observed that radical-
scavenging (antioxidant) activity of spice extracts
was only slightly influenced by the gamma-
radiation treatment [7, 14].
Detection of Irradiated Foods
and Food Supplements
For the international trade of foods and food
supplements, there is a look for simple and reli-
able method to identify irradiated food stuffs.
Numerous studies have dealt with the detection
methods applicable to irradiated herbs and spices
and have also concluded that food irradiation as a
most acceptable radiologically, microbiologically,
and toxicologically safe technology. Nevertheless,
questions focusing on nutrient loss, free radical
and radiolytic by-product formation, and changes
of antioxidant properties during irradiation are
still being discussed in the scientific community.
In 1993, the European Commission gave a man-
date to the European Committee for Standrdization
(CEN) to standardize the methods for the detec-
tion of irradiated foods. These European standards
have been adopted by the Codex Alimentarius
Commission as general methods and are referred
to in the Codex General Standard for Irradiated
Foods in section 6.4 on “Post-irradiation verifi-
cation” (Code of Federal Regulation 2004) [7].
In case of spices, to measure the degree of
irradiation, the most important methods are vis-
cosity measurement, EPR/ESR, and TL. In rela-
tion to the formation of paramagnetic species
due to gamma irradiation, EPR/ESR spectroscopy
Detection of Irradiated Foodsand Food Supplements
represents a unique detection technique for
detection and characterization. In 2000, CEN
issued the EN 1787 EPR method for the detec-
tion of irradiated foods containing cellulose. The
gamma-radiation treatment of plant products
containing cellulose leads to the generation of a
typical three-line EPR signal, attributable to
cellulosic radicals. Later, more standardized
methods were published for the detection of
irradiated foods (i.e., EN 13708, EN 13751, and
EN 13784). EN 1784 and EN 1785 describe
methods for the detection of irradiated food con-
taining fat, and EN 1786 is a method for the
detection of irradiated food containing bone.
Unfortunately, their application is limited by the
lifetime of the radiolytically produced free radi-
cals [14]. Raffi and Stocker observed that, even
though electron spin resonance (RSR) is known
to be a very sensitive method, in the case of
spices, it did not lead to favorable results because
the main radio-induced signal decreased too fast
with the storage time and disappeared before the
maximal usual commercial storage time [15].
Polonia et al. showed a prolonged appearance of
cellulose peaks in dried paprika [16]. This enabled
to include the EPR method in the European
protocol. For the purpose of post-radiation
identification of cellulose-containing foods, the
presence of relatively weak satellite lines of this
“cellulosic” radical species was accepted as an
unambiguous evidence for gamma-radiation treat-
ment. However, it was observed that the EPR
intensity of the cellulosic triplet signal gradually
decreased with the storage time and that the rate
of disappearance was dependent on temperature,
humidity, the presence of oxygen, and other factors.
In general, the cellulosic EPR signal disappeared
within 70–90 days after the irradiation process.
This method was deemed to fail in the verification
of the gamma-radiation treatment, and, conse-
quently, it was recommended that TL techniques
should be used. Yordanov et al. pointed to the
different thermal behavior of EPR signals of
nonirradiated and gamma-radiation-treated foods
containing cellulose, even after a long storage
period after radiation, when the specific cellulosic
EPR signal is extremely low and recommended
219
this technique as a method to identify gamma-
radiation-processed foods [17].
The utilization of EPR spectroscopy and vis-
cometry (with two different sample preparation
methods) to detect irradiated black pepper
samples concluded that the identification of irra-
diation at doses above 8 kGy is possible using
these methods, at least within 1 month of post-
irradiation storage at ambient temperature.
The viscosity measurement was reported to be a
promising method for the identification of irradi-
ated spices [7] and described a method using
heat gelatinization of starch in different spices.
In agreement with their findings, Farkas et al.
reported a dramatic decrease in the dispersion
viscosity of heat-gelatinized suspensions of
several irradiated spices with the starch content
compared to that of unirradiated samples. This
approach may provide a relatively simple diag-
nostic technique for the detection of irradiation
treatment of starch-containing spices. Because
the effect mentioned seems to be related to the
radiodepolymerization of starch in the irradiated
spices, additional analytical techniques have
been tested to investigate the starch damage in
black and white peppers [7]. The colorimetrically
determined reducing sugar content as well as
the alcohol-induced turbidity of hot-water extracts
indicated increased starch damage in the pepper
samples as a function of the irradiation dose.
However, the effect of irradiation had a less
dramatic response in these tests than in the
viscometric test. The moisture content influences
partial radiodepolymerization of starch. According
to the experimental results, the technique of dif-
ferential scanning calorimetry (DSC), measuring
the energy and temperature characteristics of heat
gelatinization of starches, cannot rival the sensi-
tivity of viscometric measurements in the detec-
tion of radiation-induced changes, suggested an
alternative method to that of heat gelatinization
of starch, which is less time-consuming because
it does not require heat gelatinization. In the case
of cinnamon and allspice (15% suspensions,
particle size less than 0.16 mm), the apparent vis-
cosities seemed to be as sensitive to the irradiation
dose as those of heat-gelatinized spices [7].
220
Fig. 12.1 Energy-level presentation of TL process, showing the thermally activated luminescence. N = concentration of
electron traps with energy Ee, M = concentration of hole traps with energy Ep
A comparison of three physical methods
[viscometry, DSC, NIR (near-infrared spectro-
photometry)] used in the identification of irradi-
ated spices (cinnamon and allspice) revealed that
the apparent viscosity test demonstrated the best
response sensitivity when applied to samples
irradiated with different doses. The storage time
did not influence the apparent viscosity values.
The identification limit of the viscometric method
was determined at 2–3 kGy, whereas the limit of
NIR spectrophotometric method was determined
at 4–5 kGy, respectively. These two methods
enabled to distinguish and correctly order the
irradiated samples. Thermal behavior of free
organic radicals induced in irradiated black pepper
was studied and observed that the radical evolution
in the irradiated pepper obeys a single exponential
function and yields a unique time constant. In a
study, the use of TL, EPR spectroscopy, and vis-
cosimetric measurements to determine whether
or not a spice had been irradiated confirmed that
TL, using the EN 1788 (2001) official protocol,
with an alternative method for the extraction of
mineral impurities, led to the proof of irradiation.
EPR can be used as a proof of irradiation just up
to a few weeks after irradiation and useful only
for some spices [7].
In view of the increasing use of irradiation
technology for preservation of herbs and prod-
ucts, the availability of analytical techniques for
identifying irradiated or nonirradiated moiety is
of much importance for both the regulatory
12 Radiation Detection in Herbals
authorities and consumers. Several detection
methods, broadly categorized as chemical (com-
parative antioxidant properties with reference
standard), physical (e.g., viscosity measurement),
and biological (e.g., antimicrobial against the
reference standard), have been developed for
the identification of irradiated foods. Among the
physical techniques PPSL, TL measurements,
and EPR/ESR spectroscopy are in practice.
Thermoluminescence
Thermoluminescence (TL) is the thermally stim-
ulated emission of light from an insulator or a
semiconductor following the previous absorption
of energy from ionizing radiation. The TL process
can be understood in terms of the band structure
model of insulators. In a pure insulator, there are
two relevant energy bands: (1) an almost com-
pletely filled valence band and (2) an almost
empty conduction band. The two energy bands
are separated by a forbidden gap, which means
that between these two bands there are no elec-
tronic energy levels. Transitions of electrons
between the valence band and the conduction
band are allowed, and they produce free electrons
in the conduction band and free holes in the
valence band (Fig. 12.1).
A large number of minerals (e.g., quartz, feld-
spar, calcite, flint) emitting light energy upon warm-
ing is known as thermoluminescence. The TL of
Detection of Irradiated Foodsand Food Supplements
minerals is roughly proportional to the irradiation
dose to which they had been exposed. The TL
method for the detection of irradiated herbs and
herbal products is based on the isolation of min-
erals (mineral debris) from the herb/herbal prod-
ucts and estimate the energy absorbed due to
irradiation, if any, as minerals store energy dur-
ing irradiation by trapping charge carriers in an
excited state. On controlled heating of these
minerals, after isolation, charge carriers are stim-
ulated to return to the ground state, thereby emit-
ting some of the energy as light. This light is
detected by a sensitive photon counter, and light
emission is recorded, known as glow curve. The
first glow curve (TL1) is compared with a second
glow curve (TL2) obtained by a second TL
measurement of the isolated minerals following
their exposure to a defined radiation dose. This
normalization procedure allows for differences in
mineral composition, for example, varying
amounts of feldspar or quartz and/in weight of
the aliquots of isolated mineral grains. A com-
parison of size and shape of the two glow curves
reveals whether the sample from which the min-
eral grains were isolated has been irradiated or
not (Figs. 12.2, 12.3). The TL glow ratio which is
the ratio of integrated TL intensities of glow 1(TL
1) to glow 2 (TL2), evaluated over a defined tem-
perature interval, is typically greater than 0.5 for
irradiated and generally below 0.1 for nonirradi-
ated samples [18]. TL measurement is a time-
consuming and cumbersome method. TL proved
to be the most reliable method as it was success-
fully used to distinguish all kinds of irradiated
samples from the nonirradiated control samples.
Pulse Photo-Stimulated Luminescence
In pulse photo-stimulated luminescence (PPSL),
the release of trapped energy from minerals
exposed to ionizing radiation is optically stimu-
lated by electromagnetic radiation of appropriate
wavelength, and the emission of stored energy is
recorded by a sensitive detector. The theoretical
principle is analogous to the two-stage process of
photon emission from infrared-stimulated feld-
spar where the photo excitation of charge from
221
the ground state of an electron trap to an excited
state is followed by elevation of the electron to
the conduction band by phonon absorption. PPSL
analysis takes just a few minutes and hence allows
for multiple measurements to be performed.
PPSL detection method has been used for rapid
screening of many irradiated foods including
brown shrimps, herbs, spices, seasonings, and
shellfish. In a study, 90% of 45 nonirradiated and
irradiated spices and seasonings were successfully
identified based on simple PPSL measurement.
Irradiated white ginseng powder, pepper powder,
potato, soybean, dried fig, and chestnut were also
successfully distinguished from nonirradiated
samples using PPSL [19].
Electron Paramagnetic Spin Resonance
Spectroscopy
Electron paramagnetic spin resonance (EPR/
ESR) spectroscopy technique is widely used for
identification of foodstuffs, for free radicals, and
molecules with an odd (unpaired) number of elec-
trons. Such moieties can be produced by high-
energy radiation and are detectable by EPR/ESR
method. There is a relation among free radicals
and quality of life and health. But some foodstuffs
have paramagnetic properties naturally, and they
show a single EPR spectral signal before irradia-
tion. Antioxidants are molecules which can safely
interact with free radicals and terminate the chain
reaction before vital molecules are damaged.
Antioxidants may diminish the energy of the free
radical, stop the free radical from reacting at the
potential side, and minimize the damage caused
by free radicals.
In a case study to understand the EPR spectral
properties of nonirradiated and gamma-irradiated
dry plants, cress seeds and mistletoe have been
studied, and the differences between nonirradiated
and irradiated samples were determined [19].
It has been observed that EPR spectra of these
nonirradiated samples have very weak signal, but
these plants have an intense EPR resonance line
after irradiation. Mistletoe and cress seeds were
selected for studies, and each of the samples was
taken in polyethylene bags. These samples were
222
Fig. 12.2 (a, b) Typical TL (glow 1) curves of silicate
minerals isolated from mushrooms: 1 champignon, 2 por-
cini or yellow boletus, 3 chanterelle, 4 black morel, 5
Suillus or slippery Jack, 6 bay boletus, 7 Chinese black
prepared also for irradiation. Irradiation was
performed in air and at room temperature with a
60
Co gamma-ray source. For both of the samples,
irradiation dose rate was from 0.5 to 2.5 kGy.
All EPR measurements were performed at room
temperature on Varian X-Band EPR spectrometer
operating at a microwave frequency of 9.53 GHz,
with 100-kHz field modulation, using a standard
rectangular cavity. Samples were inserted in
quartz tubes (3–4 mm), and reference material
containing Mn2+ was fixed in EPR cavity; this is
due to the fact that position of samples in cavity
12 Radiation Detection in Herbals
mushroom (mun), 8 Chinese morel (cloud ear), 9
Craterellus (black trumpet) [18], where (a) nonirradiated
samples and (b) samples irradiated with 7 kGy of 60Co
gamma rays
affects the intensity of lines. Samples of mistletoe
and cress seeds have a weak EPR signal with
g = 2.0046 ± 0.0005 value before irradiation. Sugar-
like EPR spectrum was not observed after irradi-
ation. Both of the samples have a resonance line
in EPR spectra after irradiation appeared. The
only difference from nonirradiated properties is
intensity (Fig. 12.4).
European Committee has adopted the six pro-
tocols; three of these (CEN, 1997, 2000, 2001a)
use in EPR spectroscopy for irradiation studies,
the first one for bone, the second for cellulose
Detection of Irradiated Foodsand Food Supplements 223

Fig. 12.3 (a) TL glow curves of minerals separated from erals separated from fruit, ramulus, folium, flower, spike,
the irradiated (0–50 kGy) root, rhizome, and bark of ten peel, and whole plant of ten different dry medicinal herbs
different dry medicinal herbs. (b) TL glow curves of min- irradiated at different doses (0–50 kGy) [1]

Fig. 12.4 EPR spectra

of mistletoe (a) nonirradiated
(b) irradiated [19]
224
Scrophularia root
50 kGy
25 kGy
10 kGy
5 kGy
0 kGy
5mT
Artemisiae argyi folium
50 kGy
25 kGy
10 kGy
5 kGy
0 kGy
5mT
Fig. 12.5 ESR spectra of nonirradiated and irradiated
Scrophularia root, Scutellaria root, Artemisiae argyi
folium, and Alisma rhizome. Only one intense singlet
containing foodstuffs, and the third for foodstuffs
containing crystalline sugar. In all protocols only
X-band EPR technique is used. Following these
protocols, the problem that must be solved by
EPR is on a qualitative level, whether an appro-
priate food sample has been irradiated before or
not. For an ideal case, nonirradiated samples con-
taining hard tissues were selected for the herbals
and herbal products, having EPR signal that is
stable. Identification procedure of dry spices and
some plants is based on the fact that, whereas
nonirradiated samples exhibit only one weak
singlet EPR signal, its amplitude significantly
increases after irradiation together with the
appearance of two weak satellite lines. These lines,
originating from a triplet spectrum whose central
line is overlapped on the initial line, are attributed
12 Radiation Detection in Herbals
Scutellaria root
50 kGy
25 kGy
10 kGy
5 kGy
0 kGy
5mT
Alisma rhizome
50 kGy
25 kGy
10 kGy
5 kGy
0 kGy
5mT
line can be seen in ESR spectra of the irradiated
Scrophularia root, Scutellaria root, and Artemisiae argyi
folium [1]
to free radicals of cellulose generated by irradiation.
The obtained triplet EPR spectrum is characterized
with g = 2.006070.0005 and a = 3,070.5 G. Just
the presence of these two satellite lines is consid-
ered in the protocol (CEN, 2000) as unambigu-
ous evidence for previous radiation treatment of
plants [19].
It is imperative to consider the carbohydrate
spectra also for detection of irradiation status of
herbal products using EPR/ESR spectrometry.
ESR spectra of four irradiated samples of
Scrophularia root, Scutellaria root, Artemisiae
argyi folium, and Alisma rhizome did not show
any resemblance with any established EPR/ESR
signals, such as cellulosic and sugar- or carbohy-
drate-like EPR/ESR spectrum, typical for most
of the irradiated plant products [1] (Fig. 12.5).
Detection of Irradiated Foodsand Food Supplements 225

Table 12.2 Suitability of detection methods to identify irradiated dried mushrooms [18]
S. No. Sample TL EPR PPSL
1 Champignon (Agaricus boletus) + + −
2 Porcini (Boletus edulis) + ± +
3 Chanterelle (Cantharellus cibarius) + ± +
4 Black morel (Morchella conica) + − +
5 Suillus (Suillus luteus) + ± −
6 Bay boletus (Xerocomus badius) + ± ±
7 Chinese black mushroom (Lentinus edodes) + − −
8 Chinese morel (Auricularia polytricha) + − +
9 Craterellus (Craterellus cornucopioides) + + +

For irradiated Scrophularia root, Scutellaria
root, and Artemisiae argyi folium, the spectra
were characterized by one intense singlet at
g = 2.0045 ± 0.0007. The nonirradiated line due to
lignin also appeared as a singlet with the same
spectroscopic parameters. Thus, the irradiated
products of these three samples could not be
distinguished from the nonirradiated ones using
X-band ESR spectroscopy. As EPR/ESR spec-
trometry is not calibrated, no definite judgment
about the irradiation status can be made based on
change in ESR spectrum intensity [1].
The life time of the radiation-induced radicals
strongly depends on the storage conditions (as
hvumidity and temperature) before and after irra-
diation, and generally their EPR spectrum disap-
pear within 70–90 days after irradiation. According
to some reports, however, the above satellite EPR
lines are not observed or disappeared very shortly
after irradiation of the food samples. Therefore, the
presence of the triplet EPR spectrum of cellulose
free radicals if recorded is unambiguous evidence
that the sample under study has been irradiated, but
its absence cannot always be considered as the
opposite. In this case, following European protocol
TL should be used (CEN, 2001b) which is time
consuming. In order to extend the period of
identification by using EPR, the central EPR line is
useful as a proof for previous radiation treatment of
herbs and spices, since its intensity remains enor-
mously high even after the disappearance of the
satellite lines. In this approach, the difference in the
intensity of the singlet EPR line recorded before
and after heating of the sample up to 60°C is moni-
tored. In the cases when the results obtained by the
EPR spectrometry are not definitive with respect to
radiation treatment, the method of TL (CEN,
2001b) can be used. It is unambiguous but is very
slow and expensive, and a general recommendation
is to use first EPR and if results are not clear to do
TL (Table 12.2).
Food and food supplement irradiation has
been shown to be a safe and effective process
in controlling microbial contamination without
adverse side effects and chemical residues that
can be used to improve the safety of supply in the
markets. There is a need to standardize analytical
procedures for organoleptic quality of irradiated
foods, and food supplements mainly spices, in
the dependence on the dose used for ionizing
radiation. In this context, it is necessary to stan-
dardize analytical procedures for an unambiguous
identification of gamma-radiation treatment and
its influence on food quality, in order to obtain
results that are intercomparable with the aim of a
better protection and objective consumer informa-
tion, respecting their freedom of choice. Scientists
have to work more to develop a cheap and validated
method to determine the irradiation potential on
food supplements, for example, Cassia angustifolia
extract (BP, USP grade), Coleus forskohlii extract,
Garcinia cambogia extract (as calcium salt), and
Garcinia mangostana extract in market as food
supplement.
226
References
1. Pal S, Kim BK, Kim WY, Kim MJ, Ki HA, Lee KH,
Kang WS, Kang IH, Kang SJ, Song JM. Identification
of g-ray irradiated medicinal herbs using pulsed
photostimulated luminescence, thermoluminescence,
and electron spin resonance spectroscopy. Anal
Bioanal Chem. 2009;394:1931–45.
2. Diehl J. Safety of irradiated foods. 2nd ed. New York:
Marcel Dekker, Inc; 1995, 310, 454.
3. Farkas J. Irradiation for better foods. Trends Food Sci
Technol. 2006;17:148–52.
4. Thayer DW. Food irradiation: benefits and concerns.
J Food Qual. 1990;13:147–69.
5. Razskazovskiy Y, Debije MG, Howerton SB, Williams
LD, Bernhard WA. Strand breaks in X-irradiated crys-
talline DNA: alternating CG oligomers. Radiat Resist
Res. 2003;160:334–9.
6. Mukherjee PK. Quality control herbal drugs-an
approach to evaluation of botanicals. New Delhi:
Business Horizons; 2002, 110048.
7. Sadecka J. Irradiation of spices – a review. Czech J
Food Sci. 2007;25:231–42.
8. Miller RB. Electronic irradiation of foods. New York:
Springer; 2005. p. 8–11.
9. Sadecka J, Kolek E, Peťka J, Suhaj M. Influence of
two sterilization ways on the volatiles of black pepper
(Piper nigrum L.). Chem List. 2005;99:335–8.
10. Sadecka J, Kolek E, Peťka J, Kovač M. Impact of
gamma-irradiation on microbial decontamination and
organoleptic quality of oregano (Origanum vulgare L.).
In: Proceedings of Euro Food Chem XIII, Hamburg;
2005. p. 590–4.
11. Topuz A, Ozdemir F. Influences of gamma irradiation
and storage on the capsaicinoids of sun-dried and
dehydrated paprika. Food Chem. 2004;86:509–15.
12. Lianzhong D, Songmei Z, Qiying G, Yan Z. Study on
irradiation sterilization of spices. Institute of Applied
Technical Physics of Zhejiang Province, China. 1998.
On http://epaper.kek.jp/a98/apac98/6d059.pdf. As on
8 Jan 2012.
13. Calucci L, Pinzono C, Zandomeneghi M, Capocchi A.
Effects of g-irradiation on the free radical and antioxi-
dant contents in nine aromatic herbs and spices. J Agric
Food Chem. 2003;51:927–34.
14. Polovka M, Brezova V, Staško A, Mazur M, Suhaj M,
Šimko P. EPR investigations of gamma-irradiated ground
black pepper. Radiat Phys Chem. 2006;75:309–21.
15. Raffi J, Stocker P. Electron paramagnetic resonance
detection of irradiated food stuffs. Appl Magn Reson.
1996;10:357–73.
16. Polonia I, Esteves MP, Andrade ME, Empis J.
Identification of irradiated peppers by electron spin
resonance, thermoluminescence and viscosity. Radiat
Phys Chem. 1995;46:757–60.
17. Yordanov ND, Gancheva V. A new approach for
extension of the identification period of irradiated
12 Radiation Detection in Herbals
cellulose-containing foodstuffs by EPR spectroscopy.
Appl Radiat Isot. 2000;52:195–8.
18. Malec-Czechowska K, Dancewicz GSAM, Delincee
WSH. Detection of irradiation treatment in dried
mushrooms by photostimulated luminescence EPR
spectroscopy and thermoluminescence measurements.
Eur Food Res Technol. 2003;216:157–65.
19. Yarbasi Z, Karabulut B, Karabulut A. An EPR study
of gamma irradiated medicinal plants cress seeds and
mistletoe. Gazi Univ J Sci. 2011;24(2):203–7.
Bibliography
Bayram G, Delincee H. Identification of irradiated Turkish
foodstuffs combining various physical detection meth-
ods. Food Control. 2004;15:81–91.
CAC (Codex Alimentarius Commission). Codex general
standard for irradiated foods. CODEX STAN 106–1983.
Rev. 1–2003; 2003.
Calucci L, Pinzono C, Zandomeneghi M, Capocchi A.
Effects of g-irradiation on the free radical and antioxi-
dant contents in nine aromatic herbs and spices. J Agric
Food Chem. 2003;51:927–34.
Communication (2001/C 241/03) from the commission
on foods and food ingredients authorised for treatment
with ionizing radiation in the community. Off J Eur
Commun C 241/6–11 (29.08.2001).
EC. Directive 1999/3/EC of the European Parliament and
of the Council of 22 February 1999 on the establish-
ment of a Community list of food and food ingredients
treated with ionising radiation. Off J Eur Commun L
66/24–25; 1999.
EC. Directive 1999/3/EC of the European Parliament and
of the Council of 22 February1999 on the establish-
ment of a Community list of foods and food ingredi-
ents treated with ionising radiation. Off J Eur Commun
L 66/24; 1999.
European Standard EN 1787. Foodstuffs – detection of
irradiated food containing cellulose-method by ESR
spectroscopy. Brussels: European Committee for
Standardization; 2000.
European Standard EN 13708. Foodstuffs – detection of
irradiated food containing crystalline sugar by ESR
spectroscopy. Brussels: European Committee for
Standardization; 2001.
European Standard EN 1788. Foodstuffs-Thermolumin-
escence detection of irradiated food from which silicate
minerals can be isolated. Brussels: European Committee
for Standardization; 2001.
IAEA (International Atomic Energy Agency). Report of
the ICGFI/IOCU seminar on food irradiation and con-
sumers. The Netherlands, Sept. 1993.
Sanderson DCW, Carmichael L, Fisk S. An international
collaborative blind trial of photostimulated lumines-
cence detection of irradiated herbs, spices and season-
ings. SURRC report to MAFF; 1997.
Bibliography
WHO. High-dose irradiation: wholesomeness of food
irradiated with doses above 10 kGy. Report of a joint
FAO/IAEA/WHO study group. WHO technical report
series 890. Geneva: World Health Organization;
1999.
Yordanov ND, Gancheva V. A new approach for extension
of the identification period of irradiated cellulose-
227
containing foodstuffs by EPR spectroscopy. Appl
Radiat Isot. 2000;52:195–8.
Yordanov ND, Aleksieva K. X- and Q-band EPR studies
on fine powders of irradiated plants. New approach for
detection of their radiation history by using Q-band
EPR spectrometry. Radiat Phys Chem. 2004;-
69:59–64.
 Herbal Drugs and DNA Fingerprints
DNA-based molecular technology has a potential
utility in the herbal drug analysis and widely used
for the authentication of plant species of medici-
nal importance, as each plant is like a factory,
capable of synthesizing unlimited number of
complex and unusual chemical substances whose
structures could otherwise escape the imagination
forever. A chemical marker, a group of chemical
compounds, is unique identity for a plant material
to correlate with biological efficacy, as an herbal
drug. In many cases, we do not have sufficient
chemical and pharmacological data of chemical
markers (due to temperature, light, and solvents
as degrade and isomerizes), which creates a con-
fusion for the authenticity of the raw material
itself. The use of genetic technology to study such
turbulences is a right choice to define the raw
material. Deoxyribonucleic acid (DNA) is the
fundamental building components of all living
cells, and the specific arrangement of DNA base
pair sequences (distinct arrangement of adenine,
guanine, thymine and cytosine, known as DNA
nucleotides) guides the production of proteins and
enzymes, the deciding features for shape, color,
and metabolites, via the central dogma theory [1].
The DNA profile for a particular organism is as
unique as a fingerprint. In a living system, DNA
arrangement is uniform, irrespective of the
organ, age, and environment. Similarities of
DNA fingerprints describe the genetic closeness
of tested samples and distinguish from different
families, genera, species, cultivars, and even sib-
lings. DNA forms the basic genotype (genetic
identity) of an organism, which in turn determines
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_13, © Springer India 2012
13
the phenotype (physical features) of an organism;
even clones have the same DNA fingerprint as
their mother plant. Over the years, scientists have
developed many DNA fingerprinting techniques,
for their high level of resolution, throughput, and
reliability. Based on the specificity of the geno-
type of a system, a particular DNA profile ascribes
to a particular organism, as unique as fingerprints,
specific to that individual.
DNA fingerprinting has popularity and legal
acceptability to determine the ancestry of plants,
animals, and other microorganisms. Genotypic
characterization of plant species and strains is use-
ful to distinguish the considerable variation between
strains of the same species. Herbal drugs are con-
sumed in almost every part of the world in the form
of nutraceuticals and ethno-therapeutics. The vary-
ing therapeutic content of different species of plants
has been a problem in the production of standard-
ized herbal drugs, for example, a Centella asiatica
powdered herb is standardized for minimum 2.8%
asiaticoside (of Madagascar origin), but remaining
97.2% of ingredients are unknown and may affect
the solubility, bioavailability, stability, efficacy, and
toxicity, in case of false claim. Efforts are in prog-
ress to link a particular plant from a region for an
assigned therapeutic value, as similar plant from
another region may not share the same potency, so
traceability of raw material to finished product (to
ensure the identity, purity, quality, strength, potency,
and consistency, as well as to establish a worldwide
business) is essential.
Several accidents due to quality of raw materials
used for the manufacturing of herbal drugs have a
231
232 13 Herbal Drugs and DNA Fingerprints

negative media attention, and the major but unin-

tentional cause has been found that some herbs of
different genetic identities are available under the
same generic names and is difficult a reliable
authentication and quality control for herbal raw
materials, collected from different corners of the
world. In such cases, DNA fingerprinting provides
an objective for evaluation of genetic identity of
species, cultivars, or geographic origin, to ensure
for desired genetic uniformity of raw herbal mate-
rials. Chromatographic techniques (TLC, HPTLC,
HPLC, GC, etc.) provide the profiling of various
chemical constituents of the herb, while the use of
DNA fingerprinting and combined use of DNA
fingerprinting and chromatographic fingerprints is
an effective tool in authentication and quality con-
trol of herbal drugs. A few cases of unique impacts
are as follows.
Fig. 13.1 RAPD fingerprints generated by primer OPC-
6. M-100-bp molecular-weight marker [2]. Where
A = Rasayana Churna, B = T. cordifolia, C = E. officinalis,
Authentication of Traditional D = T. terrestris
Formulations
and preservation of voucher specimens. All the
In a study, DNA fingerprint technique was employed chemicals used in the experiments were of ana-
for determination of claims for the presence of herbs lytical grade. The dried plant parts were washed
in an Ayurvedic formulation named “Rasayana with 70% ethanol for 5 min. and then with sterile
Churna” (a polyherbal formulation used as antioxi- deionized water for 1 min. following ethanol,
dant, immunomodulatory, and rejuvenating) [2]. using sonication to avoid surface contamination.
The formulation claims for the presence of three After being air-dried, the sample was cut into
herbs, named dried stem of Tinospora cordifolia, pieces and ground into a powder with liquid
dried fruit of Emblica officinalis, and dried fruit of nitrogen using a mortar and pestle, whereas
Tribulus terrestris, in powder form. To establish the Rasayana Churna was directly ground with liquid
presence of these herbs in the formulation, 120 nitrogen. DNA was extracted from each of the
decamer oligonucleotide primers were screened in samples using a modified CTAB (cetyltrimeth-
the random amplified polymorphic DNA (RAPD) ylammonium bromide) procedure. Primer OPC-6
analysis. Presence of these three herbals was simultaneously generated three distinct amplicons,
established using RAPD reaction with OPC-6, in each specific to one component. The marker with
the formulation, “Rasayana Churna.” Thus, the 600 base pairs (bp) is specific to T. cordifolia, the
present analytical technique is an additional step marker with 500 base pairs is specific to E.
to prove claims for the presence of ingredients, in officinalis, and the remaining marker >1,000 base
addition of chromatographic fingerprints, in the pairs to T. terrestris (Fig. 13.1).
similar type Ayurvedic/traditional formulations.

Experimental Details
Rasayana Churna and the dried herbal materials
were procured from the local market of Pune,
India. Analysis was done after the authenticated
RAPD Reaction
PCR amplifications were tested with RAPD
primers from commercially available kits (OPA,
OPC, and OPG) (Operon Technologies, CA and
USA). One hundred and twenty primers were
Identification of Chemotypes, Ecotypes, and Substitutes
screened for presence of distinct and consistent
bands. On the basis of which, four primers (OPA-
16, OPC-7, OPC-13, and OPG-5) were found
useful. PCR reaction mixture consisted of buffer
Tris–KCl (20 mM Tris–HCl, pH 8.4 and 50 mM
KCl), 2 mM MgCl2, 0.8 mM primer, 1 mM dNTP,
5U/reaction of Taq DNA polymerase (Gibco
BRL), 15 ng template DNA, and sterile deionized
water to a total volume of 25 ml. For DNA
amplification, Eppendorf 2,400 DNA thermocy-
cler was initially programmed for heating at
94°C for 5 min, followed by 45 cycles of 1 min.
at 94°C, 1 min at 36°C, and 2 min. at 72°C.
Immediately after the last amplification cycle,
reaction mixture was kept at 72°C for 5 min. and
then cooled at 4°C for 20 min. PCR-amplified
DNA fragments were separated by electrophoresis
in agarose gel (1.5%) and stained with ethidium
bromide (20 mg 100 ml−1) at 3 V cm−1 for 4–5 h.
A sample without template DNA was included
as a negative control in each experiment to check
contamination. Electrophoretic profile was visu-
alized under UV radiation and photographed with
Bio-Rad. Sizes of DNA fragments were estimated
by comparison with standard ladder 100 bp
(Gibco BRL). Electrophoretic profiles were ana-
lyzed for polymorphism based on the presence/
absence of DNA bands on agarose gel [2].
There are several herbal formulations on the
market which cannot be identified based on their
morphological or histological characteristics but
can easily be analyzed for quality and reliability,
using DNA technology based on similar type
analytical work plan.
Identification of Chemotypes,
Ecotypes, and Substitutes
Therapeutics in plants, commonly known as sec-
ondary metabolites, generate in the mechanism
of defense and protection [3]. The presence and
quantity of these secondary metabolites depends
on the local geo-climate, seasonal changes, and
external conditions (light, temperature, humidity,
and soil fertility) and have impacts on pharmaceu-
tical applications, for example, (1) Panax ginseng,
well known for its characteristic fleshy roots,
233
grows wild in cool and shady forests (extending
from Korea and northeastern China to far eastern
Siberia and is also cultivated on these regions);
the active therapeutic ingredient, saponins (gin-
senosides), is highest in crops of Korean origin
containing 3–4% [4]. (2) T. terrestris grown in
four different localities in south Slovakia was
found having highest levels of saponins (3.7% in
dried whole plants, 1.22% in roots, 0.7% in seeds)
in the temperate climatic region, whereas lower
levels (2.9, 1.04, 0.48% in whole plants, roots,
and seeds, respectively) were found in plants
from warm regions of the country [5]. Due to
variation in saponin contents and ingredients, its
traditional utilization varies in different parts of
world. T. terrestris of Bulgarian origin is used for
treating sexual disorders and for body building,
while of Indian origin, for muscular growth, and
Chinese as diuretic [6]. (3) Cinchona bark collected
from trees grown in the plains contains quinine,
which is therapeutically active, but the same spe-
cies of tree grown on hilltops and slopes looks
morphologically similar but has no active quinine
[7]. (4) Asiaticoside in C. asiatica have assay
0.08–0.6% from India while from Madagascar
have 2.0–3.5% [8]. (5) Aloe vera has variation in
aloin content ranging from 5.53 to 22.76% in dry
extract of exudates [9]. Atractylodes macrocephala
(Baizhu) grown in Jiangning, Jiangshu province,
China, is more effective than those grown in
Zhejiang and Jiangxi provinces. Codonopsis
pilosula (dang shen) grown in Shanxi province is
generally considered to be more potent than those
grown in other provinces [10].
Case Study
Santolina insularis is an essential oil bearing plant
known for antiviral and antibacterial activities and
potent and selective cytotoxic activity against the
human colon carcinoma cell line. The occurrence
of several chemotypes makes the taxonomic
identification of S. insularis difficult. GC–MS essen-
tial oil analyses of four chemotypes (SI1, SI2, SI3,
and SI4) revealed the presence of different per-
centages of Santolina triene, b-pinene, myrcene,
b-phellandrene, artemisia ketone, and cis-chrysan-
234
themol, allowing a chemical discrimination. Single
fragments of the 5S-rRNA-NTS region of approxi-
mately 150, 170, 260, and 280 bp were produced by
SI1, SI2, SI3, and SI4, respectively, and the sequence
alignment of the 5S-rRNA spacer region flanked by
the 3¢-and 5¢-ends of the coding region confirmed a
consistent difference between chemotypes. GC–MS
analyses of S. insularis essential oils and sequencing
of the 5S-rRNA-NTS region allowed the chemical
and genomic characterization of four distinct chemo/
genotypes, indicating the existence of two ecotypes
[11]. The similar analytical design may lead for
proper utilization of desired herbal in a formulation.
Identification of Adulterants
A general experience we all have is that as soon as
we hear of a traditional healing/medicine, we
immediately suspect its efficacy and purity, the
reason is lack of scientific evidences, i.e. the
fingerprints. It is particularly difficult to identify
commercial products in powder and tablet forms,
and they result in intoxications and even deaths.
Adulterants and substitutes may have completely
different or weaker pharmacological actions com-
pared with their authentic counterparts, even dif-
ferent species of the same genus may have totally
different actions, for example, P. ginseng (ren
shen), considered to be “hot,” is used in “yang-
deficient” conditions, while P. quinquefolius (xi
yang shen), considered to be “cool,” is used in
“yin-deficient” conditions in TCM [10]. Using
DNA fingerprint techniques, adulterants can be
distinguished even in processed samples, enabling
the authentication of the drug. Identification at
species, strain, and origin of locality are the main
parameter for quality assurance/control of TCM,
to rely on claims made on these, so DNA finger-
prints are used for authentication.
DNA Fingerprint Techniques
for Identification of Ingredients
One of the most reliable methods for identification
of herbal drugs is analyzing DNA, a genetic
composition, unique for each individual, is sta-
ble, and can be stored at −20°C for a long period
13 Herbal Drugs and DNA Fingerprints
of time (about 3–5 years), hence eliminating
the time constraint in performing the analysis.
A small amount of sample is sufficient for analy-
sis, and this is advantageous for analyzing herbal
raw materials that are expensive or in limited
supply. In terms of the mechanisms involved,
DNA methods can be classified into three types,
namely, polymerase chain reaction (PCR) based,
hybridization based, and sequencing based.
PCR-based methods use amplification of the
region(s) of interest in the genome; subsequent
gel electrophoresis is performed to size and score
of the amplification products. PCR-based meth-
ods have the advantage of requiring tiny amounts
of samples for analysis due to the high sensitiv-
ity of PCR. However, PCR inhibitors (e.g., poly-
phenols, pigments, and acidic polysaccharides)
may be present in DNA samples, thereby ham-
pering amplification. Moreover, PCR is prone to
contamination because of its high sensitivity.
DNA from contaminating bacteria or fungi in
some improperly stored samples may be
co-amplified if the stringency used is not high
enough [12].
All of these methods have expanded the avail-
ability of genetic markers hence facilitating the
creation of genetic maps. Genetic marker, a gene
(DNA sequence) with a known location on a
chromosome and associated with a particular
trait, is described as a variation, which may arise
due to mutation (alteration) in the genomic loci
that can be observed. A genetic marker may be a
short DNA sequence, such as a sequence sur-
rounding a single base pair change (single-nucle-
otide polymorphism, SNP), or a long one, like
minisatellites. Some commonly used types of
genetic markers are RFLP (restriction fragment
length polymorphism), AFLP (amplified frag-
ment length polymorphism), RAPD (random
amplification of polymorphic DNA), VNTR
(variable number tandem repeat), MSP (micro-
satellite polymorphism), SNP (single-nucleotide
polymorphism), STR (short tandem repeat), and
SFP (single-feature polymorphism) [13].
The genetic markers can be further categorized
as dominant and codominant. Dominant markers
allow for analyzing many loci at one time, while
codominant markers are analyzed one locus at a
time [13].
DNA Fingerprint Techniques for Identification of Ingredients 235

Fig. 13.2 Illustration for

PCR to amplify a sample
of DNA [13]

DNA sample is heated

original sample
5' 3'

3' 5'
DNA strands separate

Sample is cooled slowly after one cycle

one
5' cycle
3'
3' 5'
5' 3'
3' 5'
Primers bind after two cycles

Sample is incubated

after three cycles

Primers extended in 3’-5’ direction

Polymerase Chain Reaction can be detected by specific dyes or by radio-labeling

Polymerase chain reaction (PCR), invented by
Kary Mullis, 1983, is a method used to generate
billions of copies of genomic DNA within a very
short time. This amplification is useful in cases
where there are miniscule amounts of DNA avail-
able (Fig. 13.2). Today, PCR finds application in
almost all aspects of biomedicine. PCR has been
used for the detection of many pathogenic organ-
isms, from bacteria to viruses [13].
using gel electrophoresis. The advantage of using
SSRs as molecular markers is the extent of poly-
morphism, which enables the detection of differ-
ences at multiple loci between strains. From a
sample, we can identify the plant species or strain
of interest, by using the coupled chemical and
morphological data. The main advantage of using
SSRs for fingerprinting is that small amounts of
DNA are required compared to the other methods.
A drawback to using SSRs is the need to develop
separate SSR primer sets for each species [13].

Simple Sequence Repeats

Simple sequence repeats (SSRs) also known as
microsatellites, where 1–6 nucleotides in length,
which have a high degree of polymorphism, are
isolated and using hybridized probes followed by
their sequencing. Like any DNA fragment, SSRs
Restriction Fragment Length
Polymorphisms
Restriction fragment length polymorphism (RFLP,
“rif lip”) is used to determine the variation in the
DNA sequence of a genome, that is, detected by
236
cell sample extracted DNA
binding of
radioactive DNA
probe to specific
DNA fragments
membrane
washed free of
excess probe
Fig. 13.3 Diagrammatic illustration of RFLP [13]
breaking the DNA into pieces with restriction
enzymes and analyzing the size of the resulting
fragments by gel electrophoresis. The basic tech-
nique for detecting RFLPs involves fragmenting
a sample of DNA by a restriction enzyme
(Fig. 13.3), which can recognize and cut DNA
wherever a specific short sequence occurs, in a
process known as a restriction digest. The result-
ing DNA fragments are then separated by length
through a process known as agarose gel electro-
phoresis and transferred to a membrane via the
southern blot procedure. Hybridization of the
membrane to a labeled DNA probe determines
the length of the fragments which are comple-
mentary to the probe. An RFLP occurs when the
length of a detected fragment varies between
individuals. Each fragment length is considered
an allele and used for genetic analysis. Analysis
of RFLP variation is an important tool in genome
mapping, localization of genetic disease genes,
determination of risk for a disease, genetic finger-
printing, and paternity testing [13].
13 Herbal Drugs and DNA Fingerprints
cleavage of DNA by
restriction enzyme
separation
transfer to of DNA
a membrane fragments by
(southern blott) electrophoresis
plane A sample plane B
X-ray film
used to detect
radioactive
pattern
DNA comparison
Amplified Fragment Length
Polymorphism
Amplified fragment length polymorphism (AFLP)
is a PCR-based derivative method of RFLP in
which sequences are selectively amplified using
primers. DNA is cut with two restriction enzymes
to generate specific sequences, which are then
amplified suitably. The mere addition or deletion
of bases at the 3¢ end determines the selectivity
and complexity of the amplification. It was devel-
oped in the early 1990s by Keygene, uses restriction
enzymes to cut genomic DNA, and is followed
by legation of adaptors to the sticky ends of the
restriction fragments. A subset of the restriction
fragments are then amplified using primers com-
plementary to the adaptor and part of the restriction
site fragments. The amplified fragments are visu-
alized on denaturing polyacrylamide gels either
through autoradiography or fluorescence method-
ologies. AFLP–PCR is a highly sensitive method
for detecting polymorphisms in DNA. In detail,
DNA Fingerprint Techniques for Identification of Ingredients
the procedure may be divided into three steps:
(a) digestion of total cellular DNA with one or
more restriction enzymes and ligation of restric-
tion half-site-specific adaptors to all restriction
fragments, (b) selective amplification of some of
these fragments with two PCR primers that have
corresponding adaptor and restriction site-specific
sequences, and (c) electrophoretic separation of
amplicons on a gel matrix, followed by visualiza-
tion of band pattern [13].
The AFLP technology has the capability to
detect various polymorphisms in different genomic
regions simultaneously. It is also highly sensitive
and reproducible. As a result, AFLP has become
widely used for the identification of genetic varia-
tion in strains or closely related species of plants,
fungi, animals, and bacteria. The AFLP technol-
ogy has been used in criminal and paternity tests,
in population genetics to determine slight differ-
ences within populations, and in linkage studies to
generate maps for quantitative trait locus (QTL)
analysis. There are many advantages to AFLP
when compared to other marker technologies
including RAPD, RFLP, and microsatellites. AFLP
not only has higher reproducibility, resolution, and
sensitivity at the whole genome level compared to
other techniques but it also has the capability to
amplify between 50 and 100 fragments at one
time. In addition, no prior sequence information is
needed for amplification. As a result, AFLP has
become extremely beneficial in the study of bacte-
ria, fungi, and plants, where much is still unknown
about the genomic makeup of various organisms.
Similar to SSRs, AFLP-based assays are cost-
effective and can be automated [13].
Random Amplified Polymorphism
Random amplified polymorphic DNA (RAPD,
“rapid”) is one of the most commonly used pri-
mary assays for screening the differences in
DNA sequences of two species of plants. RAPD
consists of fishing for the sequence using ran-
dom amplification. Here, plant genomic DNA
is cut and amplified using short single primers
at low annealing temperatures, resulting in
amplification at multiple loci. By running a
237
2-dimensional electrophoresis gel, it is possible
to determine the change in sequence pattern by
superimposing the 2 gels. Once the band of
interest is identified, the gel is cut, and the DNA
is isolated and sequenced. Using this target,
DNA from other cultivars can be assessed using
other techniques such as AFLP or SSRs. It is
also more cost-effective than RFLPs. RAPDs
lack specificity, due to low annealing tempera-
tures and easier reaction conditions [13], for
example:
Authentication of Medicinal Plants
DNA-based techniques have been widely used
for authentication of plant species of medicinal
importance. This is especially useful in cases that
are frequently substituted or adulterated with
other species or varieties that are morphologi-
cally and phytochemically indistinguishable.
Dried fruit samples of Lycium barbarum were
differentiated from its related species using
RAPD markers. The RAPD technique has also
been used for determining the components of a
Chinese herbal prescription, Yu Ping Feng San.
In this study, the presence of three herbs (Astragalus
membranaceus (Fisch.) Bge., Ledebouriella
seseloides Wolff, and Atractylodes macrocephala
(Koidz) in the formulation has been detected
using a single RAPD primer.
Detection of Adulteration/Substitution
RAPD technique was adopted to identify eight
types of dried Coptis rhizomes and one type of
Picrorhiza rhizome, a substitute for the former in
the Chinese herbal market. P. ginseng is often
substituted by P. quinquefolius (American gin-
seng). Sequence characterized amplified region
(SCAR), AP–PCR, RAPD, and RFLP have been
successfully applied for differentiation of these
plants and to detect substitution by other closely
related species. Characterization of Echinacea
species and detection of possible adulterations
have been done using RAPD technique. DNA
fingerprinting and polymorphism in the Chinese
drug “Ku-Di-Dan” (herba elephantopi) and its
substitutes were studied using AP–PCR and
RAPD. The results were used for authentication
of “Ku-Di-Dan” and its substitutes [14].
238
Medicinal Plant Breeding
ISSR–PCR has been found to be an efficient and
reliable technique for the identification of zygotic
plantlets in citrus interploid crosses. Molecular
markers have been used as a tool to verify sexual
and apomictic offspring of intraspecific crosses
in Hypericum perforatum, a well-known anthel-
minthic and diuretic. An attempt has been made
toward marker-assisted selection of fertile clones
of garlic with the help of RAPD markers. RAPD
markers have been successively used for selection
of micropropagated plants of Piper longum for
conservation [15].
DNA-based molecular markers have been used
extensively for a wide range of applications in
food crops and horticultural plants. These appli-
cations include study of genetic variation, culti-
var identification, genotyping, cross-breeding
studies, identification of disease-resistant genes,
identification of quantitative trait loci, diversity
analysis of exotic germplasms, sex identification
of dioecious plants, and phylogenetic analysis.
Recently, the application of DNA-based molecu-
lar markers is being explored in the field of nutra-
ceuticals [16].
Single-Nucleotide Polymorphism
Single-nucleotide polymorphism (SNP, “snip”) is
a DNA sequence variation, occurring when a
single nucleotide – A, T, C, or G – in the genome
(or other shared sequence) differs between
members of a species (or between paired chro-
mosomes in an individual), for example, two
sequenced DNA fragments from different indi-
viduals, AAGCCTA to AAGCTTA, contain a
difference in a single nucleotide. In this case,
we say that there are two “alleles”: C and T.
Almost all common SNPs have only two alleles.
Variations in the DNA sequences of humans can
affect how humans develop diseases and respond
to pathogens, chemicals, drugs, vaccines, and
other agents. SNPs are also thought to be key
enablers in realizing the concept of personalized
medicine. However, their greatest importance in
biomedical research is for comparing regions of
the genome between cohorts (such as with
13 Herbal Drugs and DNA Fingerprints
matched cohorts with and without a disease).
The study of single-nucleotide polymorphisms is
also important in crop and livestock breeding
programs [13].
Short Tandem Repeat
Short tandem repeat (STR) in DNA is a class of
polymorphisms that occurs when a pattern of two
or more nucleotides are repeated, and the repeated
sequences are directly adjacent to each other.
The pattern can range in length from 2 to 10 bp
[e.g., (CATG) in a genomic region] and is typi-
cally in the noncoding intron region, making it
junk DNA. By examining enough STR loci and
counting how many repeats of a specific STR
sequence there are at a given locus, it is possible
to create a unique genetic profile of an individual.
STR analysis has become the prevalent analysis
method for determining genetic profiles in regu-
latory/forensic cases. STR analysis is a relatively
new technology in the field of forensics, having
come into popularity in the mid- to late 1990s.
It is used for the genetic fingerprinting of indi-
viduals. The STRs in use today for forensic
analysis are all tetra- or penta-nucleotide repeats,
as these give a high degree of error-free data
while being robust enough to survive degradation
in nonideal conditions. Shorter repeat sequences
tend to suffer from artifacts such as PCR stutter
and preferential amplification, as well as the fact
that several genetic diseases are associated with
trinucleotide repeats such as Huntington’s disease.
Longer repeat sequences suffer more from envi-
ronmental degradation and do not amplify by
PCR as well as shorter sequences [13].
DNA Barcode
DNA barcoding is a technique to assess inter-/
intraspecific genetic variation among taxa and
is a critical technique employed in biodiversity
genomics. Hebert et al. [17] developed DNA
barcoding as a method for species identification
and recognition in animals using specific regions of
DNA sequence data. The DNA barcoding analytical
Trouble Shooting in DNA Fingerprinting
results are successful in animals but have difficulties
in case of plants. Detailed studies have demon-
strated the utility of barcoding as an effective
tool for plant identification also. Recently, DNA
barcoding has been used as a modern genomics
tool for identifying cryptic plant species as an
important tool for identification. A single gene
region (approx. 600 base pairs) is used as a barcode,
and the same sequence is used for all species to
standardized protocol [18].
In a case study, three DNA regions (rbcL,
matK, and trnL-F) were selected based on the
previous plant barcoding studies and isolated
total genomic DNA from approximately 10 mg
of dried leaf material from each sample using the
kit, NucleoSpin® 96 Plant II (MACHEREY-
NAGEL) [19]. Extracted DNA was stored in ster-
ile microcentifuge tubes at −20°C. The selected
loci were amplified by PCR on a PTC-100 ther-
mocycler (Bio-Rad). DNA was amplified in 20 ml
reaction mixtures containing 1UAmpliTaq Gold
Polymerase with GeneAmp 106PCR Buffer II
(100 mM Tris–HCl pH 8.3, 500 mM KCl) and
2.5 mM MgCl2 (Applied Biosystems, Foster City,
CA), 0.2 mM dNTPs, 0.1 mM of each primer
(0.5 mM for matK), and 20 ng template DNA.
Amplified products were sequenced in both direc-
tions with the primers used for amplification, fol-
lowing the protocols of the University of Guelph
Genomics facility. Products from each specimen
were cleaned using Sephadex columns and run
on an ABI 3730 sequencer (Applied Biosystems,
Foster City, CA). Bidirectional sequence reads
were obtained for all the PCR products. Sequences
were assembled using Sequencher 4.5 (Gene
Codes Corp, Ann Arbor, MI) and aligned manu-
ally using Bioedit version 7.0.9. The sequences
were used in combination with the morphometric
analysis to produce classification trees.
DNA barcoding, a tool for identifying cryptic
species and validating ethnotaxa, has additional
utility in overcoming taxonomic impediments,
identifying cryptic materials such as unknown
leaves and roots. Barcoding was used in the study
of nutmeg to identify species in the Myristicaceae
that are primarily separated by androecium char-
acters in small, short-lived flowers that are only
available for 2 weeks of the year. This study
239
identified several cryptic taxa including population
level differences in Compsoneura associated with
ecotypic differences and vicariance, suggesting
several new cryptic species. DNA barcoding is a
tool that ethnobiologists can employ to (a) validate
ethnotaxa, (b) help overcome hurdles of ambigu-
ity, (c) gain credibility in science, and (d) stimulate
new theory on understanding, preserving biological
and cultural diversity [20].
Genetic Markers
It is established that pieces of DNA that lie near
each other on a chromosome tend to be inherited
together. This property enables the use of a marker,
which can then be used to determine the precise
inheritance pattern of the gene that has not yet
been exactly localized. Genetic markers have to
be easily identifiable, associated with a specific
locus, and highly polymorphic, because homozy-
gotes do not provide any information. Detection
of the marker can be direct by RNA sequencing
or indirect using allozymes. Some of the methods
used to study the genome or phylogenetics are
RFLP, AFLP, RAPD, and SSRs. They can be
used to create genetic maps of whatever organism
is being studied.
Trouble Shooting in DNA
Fingerprinting
DNA fingerprint methods are excellent for
identifications of herbs used, in various forms,
and have logical scientific answers of many que-
ries, but during analysis, DNA degradation is
common for those materials that have been heat-
treated by frying, sun drying, oven drying, or
milling without cooling; treated with various
chemicals; and stored for a long time in various
conditions. Such problem can be overcome by
choosing more degradation-tolerant methods,
one of which is employing multi-copy genes
(e.g., ribosomal and mitochondrial genes) so
that the chances of getting some full-length cop-
ies are higher. The mitochondrial genes are also
suitable because mitochondria are relatively
240
intact during processing. Another approach is to
analyze small regions of DNA by methods such
as SSR analysis which may target sequences as
short as several tens of base pairs of DNA, and
the chances of breakage within the target region
are minimal. Hybridization is also useful in
identification of degraded DNA. The presence of
PCR inhibitors is another problem due to phenolic
compounds, acidic polysaccharides, and pig-
ments, which are usually present in herbs.
Selection of most suitable DNA extraction proce-
dures for the types of samples may help eliminate
the PCR inhibitors. Other possible solutions are
diluting the extracted DNA and adding PCR
enhancers. Contamination by nontarget DNA of
bacteria, fungi, or insects due to improper storage
or by other plant materials in formulations is
another challenge. This creates the biggest prob-
lem for fingerprinting methods because the DNA
of the contaminants will be amplified. This may
be overcome by using methods specific for the
target such as ARMS and SCAR. If the region of
analysis is species-specific and the stringency is
high enough, microarray analysis is also an alter-
native for studying DNA mixtures. However,
DNA degradation by microbes cannot be avoided
in contaminated samples [22].
Commercial Utility of Genetic
Markers in Herbal Technology
Genetic markers have proved their utility in fields
like taxonomy, physiology, embryology, and genet-
ics. As the science of plant genetics progressed,
researchers have tried to explore these molecular
markers techniques for their applications in com-
mercially important plants such as food crops
and horticultural plants and recently in pharma-
cognostic characterization of herbal drugs. These
techniques have become extremely popular for
phylogenetic analysis adding new dimensions for
easy, fast, and automated assistance to scientists
and breeders.
Genome analysis based on molecular markers
has generated a vast amount of information, and
a number of databases are being generated to pre-
serve and popularize it. Molecular markers in
13 Herbal Drugs and DNA Fingerprints
genetics, sometimes called DNA markers, are the
pinpoint of the location of desirable genetic traits
or indicate specific genetic differences, just as
smoke rising into the sky makes it easier to locate
a forest fire. A gene of interest is easier to locate
when a researcher starts with a nearby marker.
Dr. Jim Register said, “To successfully use a
specific marker to follow a specific trait, the
marker must be found close enough to the gene
of interest that variations (alleles) of both the
marker and the gene can be inherited together”
[23]. The experiments carried out by Dr. Jim
Register (Figs. 13.4, 13.5, 13.6 and 13.7) support
the same. Register says, “We are able to use a
particular fragment of DNA as a marker when we
can detect differences in that fragment’s DNA
sequence between multiple plants or plant lines.”
According to Register, these variations in DNA
sequence, called polymorphisms, can be associ-
ated or linked with different forms (alleles) of
nearby genes involved with particular traits.
The polymorphism, or difference, is the clue
researchers need to find the gene of interest, for
example, markers associated with genes involved
in disease resistance have been identified in corn
and soybeans. Differences between the DNA
sequences of these genes can be responsible for
making a plant sensitive or resistant to a particu-
lar disease. And differences in DNA sequences
near the gene can be used as markers to locate the
gene and track the desired results in breeding
programs. Using the latest marker technologies,
scientists are able to determine right in the lab
which plants have economically beneficial traits.
This allows us to select plants based on the traits
they possess even before going to field trials [23].
According to the new European Council legis-
lation, the labeling of food or food ingredients
produced from or containing licensed genetically
modified organisms must indicate the inclusion of
these ingredients where they are present at or
above a level of above 1%. In compliance with the
labeling regulation for GM foods, several coun-
tries in Europe such as Germany and Switzerland
have extensively developed PCR methods for both
identification and quantification purposes. In
response to reports of unlicensed GM ingredients
in food in the international market, the Food
Commercial Utility of Genetic Markers in Herbal Technology 241

Fig. 13.4 Experimental demonstration to identify the genes for evaluation [23]

Fig. 13.5 Experimental demonstration to identify the genes for evaluation [23]
242
Fig. 13.6 SSR markers (microsatellites) to improve the product performance [23]
Safety Authority of Ireland has completed a sur-
vey to determine the levels of GM maize ingredi-
ents in tortilla chips and taco shells on sale in
Ireland, using the PCR technique. Where sufficient
GM DNA was present in the sample, quantitative
analysis was undertaken using real-time PCR.
Primers specific for inserted genes in Roundup
ReadyTM soybean have been found to be suitable
for detection and discrimination of GM soybean
from non-GM products. In another study of
Roundup Ready soybeans, maize, and Cecropin
D capsicum have been successfully discriminated
from non-GM products using primers specific for
inserted genes and crop endogenous genes [23].
Comparative Genetic Analysis
The trend to cultivate medicinal and aromatic
plants is increasing day by day, and there is a
look for better and most convenient crop always,
13 Herbal Drugs and DNA Fingerprints
where DNA fingerprint technique has a potential
role. A detailed study has been conducted for
selection of Mucuna pruriens accessions with
trichome-less pods, as the trichomes present on
the pods cause severe itching, blisters, and der-
matitis, and it discouraged cultivation. Researchers
screened genetic stocks of M. pruriens bean for the
trichome-less trait, along with high seed yield and
l-DoPA content. The highest yielding trichome-
less elite strain was selected and indentified on
the basis of a PCR-based DNA fingerprinting
method (RAPD), using deca-nucleotide primers
[24]. M. pruriens has high industrial demand, as
all parts of it possess the medicinal properties,
but the seeds are more valuable as has l-DoPA
(l-3,4-di-hydroxyphenylalanine) which is used
for the treatment of Parkinson’s disease and
mental disorders. According to Ayurveda, Mucuna
seeds are also used as an astringent, laxative,
anthelminthic, aphrodisiac, alexipharmic, and tonic.
The root is used as a remedy in facial paralysis
Comparative Genetic Analysis
Fig. 13.7 Use of SSR techniques through gel electrophoresis [23]
and nervous disorders. Decoction of the roots
purifies the blood, cures rheumatism, asthma,
cough, stone in the bladder, and improves vitality.
It is also used for fevers, edema, and elephantiasis.
However, the high demand for l-DoPA is
largely met by the pharmaceutical industry
through extraction of the compound from wild
populations, but commercial exploitation of this
compound is hampered because of its limited
availability. After the discovery that Mucuna
seeds contain l-DoPA, its demand in interna-
tional markets has increased many fold and moti-
vated Indian farmers to start its commercial
243
cultivation. Conventional methods of propaga-
tion of this plant are limited to seeds, which cre-
ates problems because the trichomes present on
the pods cause uncontrolled itching. Therefore,
improvement is required in terms of higher yields
of l-DoPA content as well as trichome-less pods.
Researchers were successful to use DNA
fingerprinting technique, random amplified poly-
morphic DNA (RAPD), to differentiate among
the accessions of Mucuna as well as for genetic
characterization of the selected accession, with a
high yield of l-DoPA and specific feature of
absence of hairs on the pods [24].
244
Future Scope of Genetic Markers
in Herbal Drug Research
Chemical profiling establishes a characteristic
chemical pattern for a plant material, its fractions,
or extracts. TLC and HPTLC are routinely used as
valuable tools for qualitative determination of
small amounts of impurities. In order to ensure
efficacy, selection of the correct chemotype of the
plant is necessary even when there are many
known chemotypes of a plant species; selection of
the right chemotype to which clinical effects are
attributed is difficult. Another difficulty encoun-
tered in the selection of the correct plant material
is to establish the identity of certain species that
may be known by different binomial botanical
names in different regions. In view of these limi-
tations, there is need for a new approach that can
complement or in certain situations serve as an
alternative. Molecular markers generally refer to
biochemical constituents including primary and
secondary metabolites and other macromolecules
such as nucleic acid. Secondary metabolites as
markers have been extensively used in quality
control and standardization of botanical drugs.
DNA markers are reliable for informative poly-
morphism as the genetic composition is unique
for each species and is not effected by age, physi-
ological condition, as well as environmental fac-
tors. DNA can be extracted from fresh or dried
organic tissue of the botanical material; hence, the
physical form of the sample for assessment does
not restrict detection. Genetic markers identify
plant at genomic level and establish new standards
in standardization and quality control of botanicals,
have highly polymorphic nature, easily available,
highly reproducible, and allow easy exchange of
data between laboratories.
DNA information is not directly correlated with
the concentration of active ingredients, but genetic
data have its own advantages over other methods
for authentication and identification. DNA meth-
ods are in use to identify components in concen-
trated herbal preparations in which the components
have been grounded, boiled, filtrated, concen-
trated, dried, and blended. Authentication of herbal
drugs is important for ensuring safe, effective,
13 Herbal Drugs and DNA Fingerprints
minimizing unfair trade, and raising consumer’s
confidence. It also plays an important role in the
modernization, industrialization, and internation-
alization of traditional healing practices. DNA
methods are reliable approaches toward authenti-
cation of plant-based drugs, especially for endan-
gered species and those with high market value.
References
1. Breithaupt H. Back to the roots. EMBO Rep.
2003;(1):10–2.
2. Shinde VM, Dhalwal K, Mahadik KR, Joshi KS,
Patwardhan BK. RAPD analysis for determination of
components in herbal medicine. eCAM. 2007;(S1):21–3.
3. Szakiel A, Paczkowski C, Henry M. Influence of envi-
ronmental abiotic factors on the content of saponins in
plants. Phytochem Rev. 2010. doi:10.1007/s11101-
010-9177-x.
4. Park JD, Rhee DK, Lee YH. Biological activities and
chemistry of saponins from Panax ginseng C. A.
Meyer. Phytochem Rev. 2005;4:159–75.
5. S’alamon I, Haba´n M, Baranec T, Haba´nova´ M,
Knoll M. The occurrence of puncture vine (Tribulus
terrestris) and its metabolic characteristics in Slovakia.
Biol Bratislava. 2006;61:25–30.
6. Farley D. Tribulus terrestris: Bulgarian versus the rest
of the world. In: The MuscleTalker Issue 35. 2005.
http://www.muscletalk.co.uk/newsletter-0305.aspx
7. Joshi DD, Uniyal RC. Different chemo types of
Gokhru (Tribulus terrestris): a herb used for improv-
ing physique and physical performance. Int J Green
Pharm. 2008;2:158–61.
8. Jha S. Term Paper BT 012. DNA fingerprinting, p. 16.
At http://www.scribd.com/doc/53022975/1040070108.
On 17 Jan 2012.
9. Joshi DD, Kharkwal H. Commercial manufacturing
of herbal medicines: recent trends to select suitable
chemo-types as raw herb. Indo-Italian workshop on
bacteria & fungi for environmental sustainability;
2010. p. 79–1.
10. Yip YP, Chau CF, Mak CY, Kwan HS. DNA methods
for identification of Chinese medicinal materials.
Chin Med. 2007;2:9. doi:10.1186/1749-8546-2-9.
11. Gnavi G, Bertea CM, Usai M, Maffei ME. Comparative
characterization of Santolina insularis chemotypes by
essential oil composition, 5S-rRNA-NTS sequencing
and EcoRV RFLP-PCR. Phytochemistry. 2010;
71(8–9):930–6.
12. Henry RJ. Plant genotyping: the DNA fingerprinting
of plants. New York: CABI Publishing; 2001.
13. Vasudevan H. DNA fingerprinting in the standardiza-
tion of herbs and nutraceuticals. Sci Creat Quart. 2011.
Available at http://www.scq.ubc.ca/dna-fingerprinting-
in-the-standardization-of-herbs-and-nutraceuticals/.
On 17 Jan 2012.
Bibliography
14. Gardiner JM, Coe EH, Melia-Hancock S, Hoisington
DA, Chao S. Physical and genetic mapping of chro-
mosome 9 s in maize using mutations with terminal
deficiencies. Genetics. 1993;134(3):917–30.
15. Um JY, Chung HS, Kim MS, Na HJ, et al. Molecular
authentication of Panax ginseng species by RAPD anal-
ysis and PCR-RFLP. Biol Pharm Bull. 2001;24:872–5.
16. Ratnaparkhe MB, Gupta VS, Venmurthy MR, Ranjekar
PK. Genetic fingerprinting of pigeonpea [Cajanus
cajan (L.) Millsp.] and its wild relatives using RAPD
markers. Theor Appl Genet. 1995;91:893–8.
17. Bertsch A. Barcoding cryptic bumblebee taxa: B.
lucorum, B. crytarum and B. magnus, a case study.
Beitrage zur Entomologie. 2009;59:287–310.
18. Ming LI, Hui CAO, Paul Pui-Hay BUT, Shaw P-C.
Identification of herbal medicinal materials using
DNA barcodes. J Syst Evol. 2011;49(3):271–83.
19. Newmaster SG, Ragupathy S. Ethnobotany genomics-
discovery and innovation in a new era of exploratory
research. J Ethnobiol Ethnomed. 2010; 6: 2. http://
www.ethnobiomed.com/content/6/1/2
20. Newmaster SG, Velusamy B, Murugesan M,
Ragupathy S. Tripogon cope, a new species of
Tripogon (Poaceae: Chloridoideae) in India with a
morphometric analysis and synopsis of Tripogon in
India. Syst Bot. 2008;33(4):695–701.
21. Newmaster SG, Fazekas AJ, Steeves R, Janovec J.
Testing candidate plant barcode regions in the
Myristicaceae. Mol Ecol Resour. 2008;8(3):480–90.
22. Warude D, Chavan P, Joshi K, Patwardhan B. DNA isola-
tion from fresh and dry plant samples with highly acidic
tissue extracts. Plant Mol Biol Rep. 2003;21:467a–67f.
23. Srivastava S, Mishra N. Genetic markers-a cutting-
edge technology in herbal drug research. J Chem
Pharm Res. 2009;1(1):1–18.
24. Dhawan SS, Rai GK, Darokar MP, Lal RK, Mishra HO,
Khanuja SPS. Comparative genetic analysis of trichome-
less and normal pod genotypes of Mucuna pruriens
(Fabaceae). Genet Mol Res. 2011;10(3):2049–56.
245
Bibliography
Cheng KT, Tsay HS, Chen CF, Chou TW. Determination
of components in Chinese prescription Yu-Ping-Feng
San, by RAPD analysis. Planta Med. 1998;64:563–5.
Ebeler SE, Takeoda GR, Winterhalter P. Authentication
of food and wine. American Chemical Society,
Washington, DC: Oxford University Press; 2007. p. 364.
Ha WY, Shaw PC, Liu J, Yau FC, Wang J. Authentication
of Panax ginseng and Panax quinquefolius using
amplified fragment length polymorphism (AFLP) and
directed amplification of minisatellite region DNA
(DAMD). J Agric Food Chem. 2002;50(7):1871–5.
Heubl G. New aspects of DNA-based authentication of
Chinese medicinal plants by molecular biological
techniques. Planta Med. 2010;76:1963–74.
Hon CC, Chow YC, Zeng FY, Leung FCC. Genetic
authentication of ginseng and other traditional Chinese
medicine. Acta Pharmacol Sin. 2003;24:841–6.
Karehed J, Groeninckx I, Dessein S, Motley TJ, Bremer B.
The phylogenetic utility of chloroplast and nuclear DNA
markers and the phylogeny of the Rubiaceae tribe
Spermacoceae. Mol Phylogenet Evol. 2008;49:843–66.
Lahaye R, Van Der Bank M, Bogarin D, Warner J, Pupulin
F, Gigot G, Maurin O, Duthoit S, Barraclough TG,
Savolainen V. DNA barcoding the floras of biodiver-
sity hotspots. Proc Natl Academy Sci USA. 2008;-
105:2923–8.
Ruzicka J, Lukas B, Merza L, Gohler I, Abel G, Popp M,
Novak J. Identification of Verbena officinalis based on
ITS sequence analysis and RAPD-derived molecular
markers. Planta Med. 2009;75:1271–6.
Singh D, Ahuja PS. 5S rDNA gene diversity in tea
(Camellia sinensis (L.) O.Kuntze) and its use for variety
identification. Genome. 2006;49:91–6.
World Health Organization. Traditional medicine. Fact sheet
N134. Geneva: World Health Organization; 2008.
 About the Author
D.D. Joshi, Ph.D., chemistry (organic), presently
asst. director of Amity Institute of Phytochemistry
and Phytomedicine, Amity University Uttar
Pradesh since March 2005, having 20 years vast
experience in R&D, QC, and QA, scale-up from
R&D to pilot plant to commercial production of
standardized herbal extracts and pure phytochem-
icals, nutraceuticals, cosmaceuticals, and essen-
tial oils, using GLPs, WHO-GMP, and ISO
9001–2000 certified facilities, in different organi-
zations with different job profiles (technical
services to international customers, consumers,
regulatory affairs) on different herbal-based prod-
ucts. Extraction and purification of herbal ingre-
dients with different techniques using hi-tech
D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4, © Springer India 2012
analytical instruments to decipher ingredients,
analytical process development, and validation of
the developed process are the areas of interest of
the author. Before joining Amity group, the author
has served with Sunita Minechem, as R&D and
QC manager; Sanat Products Ltd., as analytical
development scientist; and Alchem International
Ltd as scientist after Ph.D. from ITRC Lucknow,
India. He has first-class academic career through-
out with 20 research publications, 32 patents, two
authored books, besides other industrial activities.
At present, he is actively engaged with academics
as PI with two and co-PI with four government-
financed R&D projects, Ph.D. supervisor, and
patent coordinator to Amity Group.
247
 Index

A Chemotherapy, 7
AAS. See Atomic absorption spectrometry (AAS) Chemotypic, 9
Absorbance, 51, 58, 63, 78, 102, 106, 109, 115, 121, 122, China, 3–5, 15, 19, 20, 40, 93, 116, 128, 138, 144, 145,
126, 130–132, 136, 139, 189–193, 197 185, 189, 202, 233
Alkali flame ionization detector (AFID), 86 Chukh, 94
Alkaloids, 8, 9, 20, 29, 38, 40, 42, 46, 54, 55, 57, 67, 68, Citrus peels, 405
70, 72, 74, 78, 83, 87–89, 165, 172, 183 Citrus pseudolimon, 94
Allergies, 4 Classical Chinese medicine, 4
Allium sativum, 90, 187 Cleavage reaction, 147
Aloe vera, 108, 187, 233 Column
Alzheimer’s disease, 7 selectivity, 62
Ancient practices, 3, 5 specification, 62
Anthraquinones, 29, 38, 40 Commiphora wightii, 113–114
Anti-aphrodisiac, 7 Complex herbal, 3
Anti-diarrhea, 7 Composition and efficacy, 15
Anti-pyretic, 6, 7, 75, 78 Computer aided similarity evaluation (CASE), 28
Antiseptic, 7, 22 Correlation spectroscopy (COSY), 168, 169, 171, 175
Aphrodisiac, 7, 94, 242 Coumarins, 29, 38, 40, 43, 44, 46, 67, 78
A Physical Directory, 1 CP/MAS-NMR spectrum, 195
Aromatherapy, 19, 86, 93–94 Current regulations, 3
Asian continent, 3–8
Aswarista, 20
Atomic absorption spectrometry (AAS), 189–193 D
Atomic mass unit (AMU), 90, 149 Dactylorhiza, 7
Authenticity, 3, 23, 88, 109, 113, 129, 137–138, 178, Datura stramonium, 94
230, 231 De Materia Medica, 6
Ayurvedic pharmacopoeia, 202 Deoxyribonucleic acid (DNA), 15, 20, 107, 108, 113,
Ayurvedic scholars, 5 136, 193, 213, 214, 229–244
Detection, 11, 38, 49, 61, 86, 103, 123, 149, 170, 194,
201–213, 235
B Detector, 11, 17, 61, 63, 64, 66–78, 83–88, 91, 103,
Bathochromic shifts, 103, 105 106, 113, 118, 121, 122, 130, 139, 147–150,
Beer–Lambert law, 102 156, 160, 161, 170, 189–191, 206, 210,
Bhasma, 20, 141, 144, 198–199 211, 221
Bioluminescence, 37–38 Diabetes, 5, 8, 9, 16, 17, 192
Blood circulation, 5 Diffusion-ordered spectroscopy (DOSY), 174, 175, 185
Digitalis purpurea, 114, 126
Dioscorea membranacea, 106
C Direct analysis in real time (DART), 46–47
Capillary columns, 84–86, 90, 91, 161 DNA. See Deoxyribonucleic acid (DNA)
Carbamate, 40, 201, 205 DNA barcodes, 238–239
Carrier gas, 83, 85, 87, 210 Doctrine of signatures, 6
Charak Samhita, 5 DOSY. See Diffusion-ordered spectroscopy (DOSY)
Chemical shift, 74, 164, 171–175, 179, 195, 196 Dragendorff’s reagent, 38, 40, 42
Chemiluminescent (CL), 68 Drug discovery, 12, 13, 29, 37, 40, 74, 99, 133, 163
Chemometric approaches, 27 Dry fruits, 210

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 249
DOI 10.1007/978-81-322-0804-4, © Springer India 2012
250 Index

E Global resurgence, 9
Ecotypic, 9, 239 Glycosides, 20, 29, 40, 42, 43, 54, 66, 67, 72, 74, 77, 78,
Eight principles, 4 83, 88, 92, 104–106, 108, 138, 153, 172,
Electrical signal, 84 174–178
Electrochemical detector (ECD), 66, 69 Graphite furnace atomizer, 193
Electromagnetic radiation, 51, 99, 101, 102, 123, Green synthesis, 110–113
163, 221 Group specific reaction, 55–57
Electromagnetic spectrum, 51, 99, 101, 102 Guggulsterone, 113
Electrophoretic techniques, 3, 207
Electrospray, 18, 61, 71, 73, 77, 147, 150–154, 160
Electrospray ionization (ESI), 18, 61, 71–73, 150, H
153–156, 160 Heart attacks, 8, 192
Embryology, 5, 240 Heart disease, 5, 9, 114, 125, 178, 179, 182
Ephedra, 1, 8, 172, 183 Heart problems, 4
EPR/ESR spectroscopy, 213, 218, 220, 221, 224, 225 Herbals, 3–47, 49–59, 61–78, 83–95, 101–119,
ESI. See Electrospray ionization (ESI) 121–145, 147–161, 163–199, 201–244
Essential micro-elements, 194 drugs, 3–47, 49–59, 61–78, 83–95, 101–119,
Essential oils, 10, 23, 29, 40, 44, 83–85, 89–90, 93, 121–145, 147–161, 163–199, 201–212,
94, 105, 125, 201, 203, 209, 212, 216–218, 231–244
234, 235 and produce, 6, 15, 16, 29, 43, 56, 71, 72, 85, 90,
European healing system, 7 101, 102, 105, 108, 110, 121, 123, 133,
Extraction, 10–11, 16, 17, 40, 43, 45, 61, 72, 77, 78, 86, 147, 154, 164, 189, 190, 201, 214, 215,
87, 89, 105, 109, 126, 137, 156, 158, 166, 220, 230, 239
171–172, 177, 184, 197, 203–208, 211, 220, Hibiscus rosa sinensis, 116
240, 243, 247 High-performance liquid chromatography (HPLC), 9,
11–12, 16–18, 23, 38, 41, 44, 46, 50, 57,
61–78, 86, 100, 106, 107, 113, 116–118,
F 152–156, 158–160, 165, 168, 170, 176, 181,
Family lineages, 4 182, 184, 198, 202, 205–207, 229, 232
Fenugreek, 92, 126, 178, 179 High-performance thin-layer chromatography (HPTLC),
Fingerprint(s) 9, 11, 16, 23, 27, 49–59, 75, 76, 86, 158, 184,
documentation, 35 202, 229, 232, 244
techniques, 27, 229, 232–235, 242 H-NMR, 165–180, 183, 185
Five element, 4, 5 Homeopathy, 20, 21, 114
Five great elements, 5 Homogenizer, 29
Flavonoids, 16, 20, 29, 40, 42, 43, 57, 67–69, 72, 74, 77, HPLC. See High-performance liquid chromatography
87, 88, 103–108, 131, 153–155, 175, 176 (HPLC)
Flavor, 7, 8, 10, 90, 176, 209, 212, 214, 216, 217 HPTLC. See High-performance thin-layer
Food and Drug Administration (FDA), 2, 22, 194, 216 chromatography (HPTLC)
Forskolin, 27, 36, 44 Human anatomy, 5, 6
Forward pharmacology, 13 Hydrophobicity, 65
FTIR spectrum, 129 Hyphenated chromatography, 11, 160
Functional groups, 29, 38, 51, 55, 57, 69, 124–126, 133, Hyphenated techniques, 28, 44, 57, 73, 83, 88, 116,
137, 139, 145, 147, 217 160–161, 204–205, 207
Fused silica open tubular (FSOT), 84

I
G Immune-modulators, 165, 188, 232
Galgal, 94 Immune-suppression, 187
Gas chromatography (GC), 9, 44, 57, 77, 83–95, 127, Immune system, 4, 93, 94, 207
160, 161, 167, 170, 176, 204, 206, 210–212, Indian System of Medicine (ISM), 3, 21, 165
217, 232 Inductively coupled plasma (ICP), 189, 193
Gas chromatography–mass spectroscopy (GC-MS), 9, Ingredients, 1–3, 6, 8–12, 15, 16, 18–23, 27, 29, 34,
11, 73, 85, 86, 88–91, 93, 94, 99, 100, 161, 40, 44, 49, 62, 67, 69, 83, 84, 86, 87, 94–95,
165, 167–170, 233, 234 99, 113–115, 127, 129, 138, 145, 153, 164,
Gastro-intestinal, 4, 7 176, 201, 209, 213–215, 217, 231–237, 240,
Gastrointestinal disorders, 4 242, 244
GC. See Gas chromatography (GC) Interferogram, 121, 122, 133
Genotypic, 9, 231 In vitro, 13, 14, 16, 17, 103, 108
Global regulations, 3 Ion trap detector (ITD), 161
Index 251

g-Irradiation, 217, 218 O

IR radiations, 121, 123, 139 Oestropathy, 3
Orchids, 6, 8
Orchis mascula, 6
J Organochlorines, 209
Japan, 3–5, 21–22, 68, 93, 138, 144, 145 Organophosphorus, 86, 201, 205

K P
Kama Sutra, 94 Pain syndromes, 4
Kampo drugs, 21 Patented medicines, 4
Korea, 5, 138, 233 PCA. See Principal component analysis (PCA)
PCR inhibitors, 234, 240
Pesticide residues, 201–212
L Pharmaceutical market, 4
Lepa, 20 Pharmacology, 2, 5, 12–16, 86, 165, 170
Liquid chromatography-mass spectrometry (LC-MS), Pharmacopoeias, 1, 23
9, 17, 18, 23, 41, 65, 66, 73, 77, 99, 100, 106, Piper nigrum, 94
160, 183, 206, 208, 211 Plant metabolomics, 174
Liver toxicity, 8 Polyherbal, 100, 185, 189, 232
Principal component analysis (PCA), 68, 129–133, 139,
144, 173, 178, 183
M Psychoses, 8
Magnetic resonance imaging (MRI), 163 Pueraria lobata, 68
Magnetogyric ratio, 163, 175 Pulsed photo-stimulated luminescence (PPSL), 213, 220,
MALDI. See Matrix-assisted laser desorption ionization 221, 225
(MALDI) Pyrethroid, 201, 205, 208, 212
Mangifera indica, 114 Pyrolysis (PY), 85, 89
Marker, 9–11, 16, 20, 33, 40–42, 44–47, 76, 117, 118,
165, 196–198, 229, 231, 232, 234, 237–240
Mass spectrometry (MS), 23, 38, 61, 64–66, 70–74, 77, Q
90, 91, 94, 100, 116, 119, 147–161, 170, Quality control, 3, 5, 14–19, 21–24, 27, 35, 57, 59, 66,
208, 211 67, 75, 76, 78, 83, 89, 116, 123, 137, 138,
Matrix-assisted laser desorption ionization (MALDI), 158, 160, 170, 181, 183–185, 202, 229, 230,
150, 156–160 232, 244
McLafferty rearrangement, 147, 167
Micropipette, 30
Mobile phase, 27, 29, 32, 33, 35, 37–45, 53, 58, 61–67, R
69–71, 74–78, 83, 106, 113, 153, 154, 156, Raman scattering, 101, 102
197, 203, 211 Regulatory purposes, 95
Molecular characterization, 15, 89, 147, 155, 240 Relative intensity, 147
Molecular properties, 18, 89, 101, 116, 178, 216, 218, Reproducibility, 15, 29, 34, 39, 49, 50, 64, 65,
220, 221 75, 86, 117, 132, 159, 170, 176, 178,
Moringa oleifera, 128, 188 206, 207, 237
Mover, 29 Respiratory disorders, 4
Multivariate data analysis (MVDA), 67, 171, Retention factor (Rf), 29, 34, 65, 75, 76
173, 174 value, 11, 29, 33–35, 37, 39, 43, 47, 76, 197
Reverse pharmacology, 12–14
Reverse phase, 10, 11, 62–65
N Robustness, 10, 34, 39, 61, 78, 130, 171, 176,
Nanoherbal, 189, 198–199 185, 204
Naturopathy, 3 Rutin, 42, 57, 67, 105
Neurological disorders, 4
New drug discovery, 12, 29, 37, 40, 133, 163
Nicotinamide adenine dinucleotide (NAD), 111 S
Nimbu, 94 Salep, 6–8
Ninhydrin, 38, 56 Sample application, 30, 32, 50
Normal phase, 11, 62–64 Saponins, 20, 29, 36, 40, 42–44, 67, 70, 72, 77, 131, 138,
Nuclear magnetic resonance (NMR), 18, 39, 66, 73, 74, 165, 233
77, 99, 154, 160, 161, 163–185, 195 Seizures, 8
252 Index

Selectivity, 34, 39, 62, 65, 66, 68, 69, 71, 85, 86, 192, Tubers, 6–8
203, 207, 208, 236 Two dimensional TLC, 29, 35–37
Sexual dysfunction, 4
Sharpener, 29
Shikimate pathway, 103 U
Signal/noise ratio, 58, 59, 70, 85, 133 Ultra diluted drugs, 114–116, 126
Silver nanoparticles, 110, 111, 113, 189 Unani, 3, 5, 20, 21, 46, 164
Soft independent modeling of class analogy (SIMCA), UV-radiation, 43, 106, 108, 233
129, 130, 133 UV-Vis. spectrum, 102, 110, 115, 117
Solvent front, 29, 33–35, 39, 43
Stress related syndromes, 4
Stroke, 8 V
Sulfur chemiluminescence detector (SCD), 85 Vanilla planifolia, 7
Support-coated open tubular (SCOT), 84 Vanillin, 7, 42, 47
Synergism, 2, 12, 37, 49, 94, 131, 185 Vedic civilization, 3, 5
Vibrio fischeri, 38
Visualizing reagents, 53
T Vitamin C, 54, 55, 108–110
Tannins, 9, 29, 38, 40, 43, 88–89 Volatile oils, 11, 40, 77, 83, 88, 89, 217, 218
Tanzania, 8
Thermo-ionic detector (TID), 85
Thermoluminescence (TL), W
213, 218–223, 225 Wall-coated open tubular (WCOT), 84
Thin-layer chromatography (TLC), 11, 29–47, 49, 50, Wave number, 121–124, 128, 131, 139
52–57, 59, 77, 78, 86, 113, 114, 116, 158, 184, WD-XRF, 194–195
197, 202, 211, 232, 244 Withanolides, 36, 43, 165, 167, 168
TLC analysis, 37–45, 49, 50, 114, 197
Total correlation spectroscopy
(TOCSY), 175 X
Toxic effects, 89, 108, 195 Xenobiotics, 9
Toxic elements, 203
Traditional Chinese system (TCM), 3
Traditional uses, 4, 6, 7, 50, 153, 156 Z
Transmittance, 108, 121, 122, 139, 190 Zambia, 8
Tuberculosis, 5, 144 Zingiber officinale, 94, 188