Who Guidelines For Herbal Medicines

Standardization, extraction and evaluation of selected plants
4. Standardization and evaluation of

selected plants
4.1. Introduction
A system to ensure that every single medicinal plant, part of a plant or an extract, an isolate
or an enriched portion or a product thereof, being sold has the correct substances in the
correct amount and will induce its therapeutic effect, is known as standardization. Medicinal
plants, being an important aspect of various traditional systems of medicine, have been used
therapeutically around the world. From Ayurveda to Chinese traditional medicine, from
Unani to Tibetan Medicine and from Amazonian to African Medicine, all systems of
traditional medicine, although based on different theoretical and cultural models, integrate
phytotherapy into their doctrine [1]. In high-income countries, however, the widespread use
of phytotherapy declined at the end of the first part of the twentieth century, due to the
development and production of synthetic medicines. During the past few decades, however,
phytotherapy has started to be increasingly used even in industrialized countries. In low and
middle-income countries, phytotherapy never stopped being important, often representing the
only therapeutic system preferred by certain people; in the Indian sub-continent about 70%
of the population (WHO) extensively use traditional and alternative medicines for health care
[2]. The growing use of botanicals by the public has initiated evaluation of the health claims
of these agents and steps are being taken to develop standards for their safety, efficacy and
quality. In addition, the WHO has developed a series of technical guidelines and documents
relating to the safety and quality assurance of medicinal plants and herbal materials as a
minimum requirement.
The present study is hence an effort to standardize the plant materials, prepare a suitable
extract form, and to carry out its evaluation for the development of a herbal dietary
supplement. The entire plant of Cissus quadrangularis Linn., dried exudate of Commiphora
mukul, and ripe or half ripe fresh fruits of Morinda citrifolia were selected for the study. Each
one was standardized as per the WHO guidelines.
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4.2 Experimental
4.2.1 Materials, instruments and chemicals, given separately in list of chemicals,
instruments and equipment Page no. I-III
4.2.2 Collection and authentication
The entire plant of Cissus quadrangularis Linn., was collected from areas in and around
Erode, Tamilnadu, India and from the hilly areas of Pavagada, Karnataka, India, during the
months of April and June, 2008. The fresh ripe or half ripe fruits of Morinda citrifolia Linn.,
and the exudate of the plant Commiphora mukul Engl., (Shuddha Guggul) were obtained
from Alva’s pharmacy, Mizar, near Manipal, Karnataka, during the month of September
2008.
The plant/ plant material was authenticated by Dr. Gopalakrishna Bhat, Department of
Botany, Poorna Prajna College, Udupi, Karnataka. Voucher specimens of the plant/ materials
vide Nos. 514, 563, 571 respectively; have been deposited in the Department of
Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal, India.
4.2.3 Morphology and Microscopy
4.2.3.1 Morphology [ 3]
Macroscopic characters of the medicinal plant material were based on shape, size, colour,
surface characteristics, texture, fracture and appearance of the cut surface.
4.2.3.2 Microscopy
4.2.3.2 .1 Anatomical studies [3,4]

Free hand sections of the specified parts were boiled with chloral hydrate to remove all the
colouring matter and then carefully stained with phluoroglucinol and HCl (1:1). The sections
were mounted as per standard procedures. Photomicrographs were made at the Department
of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal, using different
magnifications.
4.2.3.2.2 Powder analysis [5]

The dried and powered plant materials which passed through sieve no. 60 were used for
powder analysis. The macroscopic characters of the powders were observed first, following
47
which, the powders were examined microscopically by mounting in chloral hydrate solution,
iodine solution and by staining with phluoroglucinol: HCl.
4.2.4 Physical/physicochemical standardisation [3, 4, 5, 6,7]
4.2.4.1 Total ash
About 2 g of the powdered drug was accurately weighed in a tared silica crucible. The
powdered drug was spread as a thin layer at the bottom of the crucible. The crucible was
incinerated at a temperature not exceeding 450°C until free from carbon. The crucible was
cooled and weighed. The procedure was repeated till a constant weight was observed. The
percentage of the total ash was calculated with reference to the air-dried drug.
4.2.4.2 Acid insoluble ash
The ash obtained as described in the determination of total ash was boiled with 25 mL of
hydrochloric acid for 5 min. The insoluble ash was collected on an ash-less filter paper and
washed with hot water. The insoluble ash was transferred into a tared silica crucible, ignited,
cooled and weighed. The procedure was repeated till a constant weight was observed. The
percentage of acid insoluble ash was calculated with reference to the air-dried drug.
4.2.4.3 Water soluble ash
The ash obtained as described in the determination of total ash was boiled for 5 min with 25
mL of hot water. The insoluble matter was collected on an ash-less filter paper and washed
with hot water. The insoluble ash was transferred into a tared silica crucible and ignited at a
temperature not exceeding 450°C. The procedure was repeated until a constant weight was
observed. The weight of the insoluble matter was subtracted from the weight of the total ash.
The difference in weight was considered as water-soluble ash. The percentage of water-
soluble ash was calculated with reference to air-dried drug.
4.2.4.4 Extractive values [8]
4.2.4.4.1 Ethanol soluble extractive
5 g of previously weighed air-dried drug was taken in a stoppered flask to which 100 mL of
95% ethanol was added. It was shaken continuously for 4 h on a magnetic stirrer. Then it
was filtered rapidly taking precautions against loss of the solvent. 25 mL of this filtrate was
evaporated to dryness in a tared flat-bottomed petri dish, dried at 105°C and weighed. The
percentage of ethanol soluble extractive was calculated with reference to the air-dried drug.
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4.2.4.4.2 Water soluble extractive
5 g of previously weighed air-dried drug was taken in a stoppered flask to which 100 mL of
chloroform water was added. It was shaken continuously for 4 h on a magnetic stirrer. Then
it was filtered rapidly taking precautions against loss of the solvent. 25 mL of this filtrate
was evaporated to dryness in a tared flat-bottomed petri dish, dried at 105°C and weighed.
The percentage of water-soluble extractive was calculated with reference to the air-dried
drug.
4.2.4.4.3 Ether soluble extractive
5 g of previously weighed air-dried drug was taken in a stoppered flask and 100 mL of ether
was added to it. It was shaken continuously for 4 h on a magnetic stirrer. Then it was filtered
rapidly taking precautions against loss of the solvent. 25 mL of filtrate was evaporated to
dryness in a tared flat-bottomed petridish, dried at 105 °C and weighed. The percentage of
ether-soluble extractive was calculated with reference to air-dried drug.
4.2.4.5Foreign organic matter [3]
The sample (100-500 g) was spread on a white tile or a glass plate uniformly to form a thin
layer without overlapping. The sample was inspected with the unaided eye or by means of a
lens (5x or above).
The foreign organic matter was separated manually. After complete separation, the matter
was weighed and percentage w/w present in the sample was determined as described in
WHO guidelines.
4.2.4.6 Moisture content by Loss on drying [3]
About 2-5g of accurately weighed drug was dried at 100-105C for 5 h, and then weighed
again. Percentage was calculated with reference to the initial weight.
4.2.4.7 Volatile content [3]
Volatile content of the plant material was determined using Clavenger’s apparatus as
described in WHO guidelines.
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4.2.5 Phytochemical evaluation
4.2.5.1Preparation of extracts
The plant/material was either extracted successively with different organic solvents from
lowest polarity to highest polarity, or exhaustively extracted with ethanol and then
fractionated successively with solvents of lowest polarity to highest polarity.
4.2.5.1.1 Procedure
A. The dried coarsely powdered plant/ material (50 g) was successively in increasing order of
polarity extracted with n-hexane, chloroform, ethyl acetate, n-butanol and methanol by the
hot extraction process using a soxhlet apparatus. After completion of each extraction process
(4 h) the solvent was removed by distillation and the extracts were concentrated in vacuo.
The marc left after each extraction process, was dried so that it was completely free from the
solvent.
B. Plant material (3 kg) was exhaustively extracted with ethanol (95%) in a soxhlet apparatus
for 6 h. After extraction the solvent was distilled from the extract under pressure, till syrupy
consistency after which it was evaporated to dryness under pressure. The solvent free extract
(50 g) was suspended in sufficient water. The suspension was fractionated with various
solvents of ascending polarity (n-hexane, chloroform, ethyl acetate, n-butanol and methanol).
Each fraction was then separated and distilled to remove solvent and concentrated in vacuo
4.2.5.2 Physico-chemical evaluation of the fractions
4.2.5.2.1 Determination of moisture content
Same method as described above in 4.2.4.6 was employed.
4.2.5.2.2 Determination of solubility [9]
100 mg each of various fractions (n-hexane, chloroform, ethyl acetate, n-butanol) obtained
from the ethanolic extract of whole plant of C. quadrangularis were accurately weighed,
dissolved in 10 mL volume of various solvents and solubility was observed.
4.2.5.2.3 Fluorescence analysis [10,11]
The hexane, chloroform, ethyl acetate, butanol fractions and total ethanol extracts were
observed under daylight and under UV[254 nm] and the color was recorded. The test material
was further treated with different reagents namely, 1N Hydrochloric acid, 1N sodium
50
hydroxide (aqueous), ferric chloride, 1N nitric acid, ammonia, iodine, 1N sodium hydroxide
(alcoholic), picric acid and 1N Sulphuric acid, and then observed for any color change in
daylight as well as under UV[254 nm].The change in colour was recorded
4.2.5.3 Preliminary phytochemical screening [7]
Each of the extracts/ fractions was subjected to various phytochemical tests to detect the
presence of various phytoconstituents such as carbohydrates (Molisch’s, & Fehling’s tests),
glycosides (Borntrager’s & Modified Borntrager’s test), saponins (Foam test), flavonoids
(Shinoda’s test), alkaloids (Mayer’s, Dragendroff’s, Wagner’s & Hager’s reagent tests),
sterols (Libermann Burchard test), fixed oils (Spot & Saponification test), tannins and
phenols (Ferric chloride, Lead acetate & Aqueous bromine solution), proteins and amino
acids (Biuret test, Ninhydrin test).
4.2.5.4 Estimation of total phenolic content [12,13]
Total phenolic content was estimated by Folin–Ciocalteu colorimetric method using gallic
acid as a standard phenolic compound.
a. Reagents - 1) Folin–Ciocalteu reagent (0.2 N)

2) Saturated sodium carbonate (75 g/l)
b. Procedure - 100 µl (two replicates) of the samples (1 mg/mL) were mixed with 900 µl of
distilled water and 5mL of 0.2 N Folin–Ciocalteu reagent. After 5 min, 4 mL of saturated
sodium carbonate (75 g/l) were added. The absorbance of the resulting blue-coloured solution
was measured at 765 nm after incubation at 30°C for 1.5 h with intermittent shaking.
Quantitative measurements were performed, based on a standard calibration curve (10, 20,
40, 80, 160 and 320 µg/mL of gallic acid in 95% methanol). The total phenolic content was
calculated as gallic acid equivalents (GAE) in milligrams per gram of dry material by the
following formula.
Where T = total phenolic compounds, mg/g plant extract, in GAE;

C= concentration of gallic acid established from calibration curve, mg/mL;
V = the volume of extract, mL;
M = the weight of ethanolic plant extract, gram.
4.2.5.3 Estimation of total flavonoid content[14]
The total flavonoid content was determined using the method of Meda et al., with minor
modifications.
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a. Reagent – 1) Aluminium trichloride (2%)
b. Procedure - 5 mL of 2% aluminium trichloride was mixed with the same volume of

sample (1 mg/mL). Absorbance at 415 nm was taken after 10 min against a blank sample
consisting of 5 mL of sample solution and 5 mL of methanol without aluminium trichloride.
The total flavonoid content was determined using a standard curve of quercetin at 1–64
µg/mL. The average of three readings was expressed as quercetin equivalents (QE) on a dry
weight (DW) basis.
4.2.5.5 Estimation of tannin content [15].
Tannin content of the samples was determined using the method of Bajaj & Dev Sharma.
a. Principle [16,17]
Phosphomolybdic-phosphotungstic acid (Folin-Denis reagent) is reduced to a blue colour

complex of tungsten and molybdenum oxide in alkaline solution by phenols. The intensity of
the blue colour produced is measured using spectrophotometer at 760 nm. Tannic acid is used
as a standard to calculate the concentration of phenols in the sample and is expressed as
tannic acid equivalent.
b. Reagents – 1) Folin-Denis reagent.

2) Saturated sodium carbonate solution. (35 g in 100 mL water)
c. Extract solution 10 mg of extract was dissolved in 10 mL of methanol.
d. Procedure
1 mL (0 - 1000 µg/mL in distilled water) of the standard tannic acid solution was pipetted
into 10-mL standard volumetric flasks containing 7.5 mL of water. 0.5 mL of Folin-Denis
reagent and 1.0 mL of sodium carbonate solution was added and diluted to the mark with
water. The solution was mixed well and the absorbance was determined at 760 nm after 30
min. Absorbance was plotted against concentration of tannic acid. Determination of samples
was carried in the same manner as standard, where 1 mL of each fraction (1 mg/mL) was
taken instead of standard and total tannin content was expressed in mg tannic acid equivalent
(TAE)/g dry weight (DW) of fraction. The blank consisted of all the reagents without the
sample.
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4.2.6 Marker based standardisation of the selected plants and their extracts
by HPTLC.
4.2.6.1 Cissus quadrangularis
4.2.6.1.1 Estimation of -sitosterol and lupeol in various fractions obtained from

ethanolic extract of the whole plant of C. quadrangularis Linn.
a. Preparation of Standard solution
The stock solutions of standard β –sitosterol and lupeol were prepared by dissolving 1mg
each of accurately weighed standards in 5 mL methanol and transferring into a 10 mL
volumetric flask. The flasks were sonicated for 10 min and the final volume of the solutions
was made up to 10 mL with methanol to get stock solutions containing 100 µg/mL.
b. Sample preparation
10 mg, each of ethanolic extract, n-hexane and chloroform fractions obtained from ethanolic
extract of C. quadrangularis were accurately weighed, dissolved in 5 mL methanol and
transferred to a 10 mL volumetric flask. The flasks were sonicated for 10 min and the
contents were filtered through Whatman No. 1 paper (Merck, Mumbai, India). The final
volume was made up to the mark with methanol. This solution (1 mg/mL) was further used
for HPTLC estimation.
c. Mobile phase - Benzene: Ethyl acetate (9.5: 0.5 v/v).
d. Estimation procedure
HPTLC was performed on 10 x 10 aluminium backed plates coated with silica gel 60 F254.
Standard solutions and sample solutions were applied on the same chromatographic plate as
bands of 2 l volume using Linomat V sample applicator equipped with a 100 μL Hamilton
syringe. Ascending chromatographic development was performed at room temperature (28 ±
2°C), with the mobile phase, in Camag twin-trough glass chamber previously saturated with
mobile phase vapour for 20min.
e. Visualization and Scanning
After development, the plate was dried, sprayed with 10% methanolic sulphuric acid,
followed by heating at 105ºC for 10 min. The plate was then scanned between 254 and 554
53
nm with Camag TLC Scanner- 3.The phytoconstituent(s) content was calculated by the
formula,
AUC of sample x Conc. of std x %purity
AUC of standard x Conc. of sample

4.2.6.2 Commiphora mukul
4.2.6.2.1 Estimation of -sitosterol in hexane and total ethanol extract obtained from
Commiphora mukul Engl.
Preparation of standard/ sample solutions and content estimation were similar to that
followed for the estimation of -sitosterol in C. quadrangularis (4.2.6.1.1).
4.2.6.2.2 Estimation of guggulsterone in hexane and total ethanol extract obtained from
The stock solutions containing 1mg/mL standard guggulsterone E and Z in methanol were
prepared as mentioned above (4.2.6.1.1a).
A 1mg/mL solution of each of n-hexane and ethanolic extract of Commiphora mukul Engl. in
methanol was prepared as discussed before (4.2.6.1.1b).
c. Mobile phase – Toluene:acetone (9: 1 v/v).
HPTLC was performed in accordance with the afore said procedure (4.2.6.1.1d).
e. Visualization and Scanning
After development, the plate was dried, sprayed with 10% methanolic sulphuric acid, and
then heated at 110ºC. The plate was scanned at 366 nm and the content was estimated by
using the same formula given earlier
4.2.6.3 Morinda citrifolia
4.2.6.3.1 Estimation of -sitosterol in hexane, chloroform fractions & ethanolic extract

of fruits of M. citrifoliaLinn.
54
The methodology for preparation of standard/ sample solutions and content estimation was
same as followed in the estimation of -sitosterol in C. quadrangularis (4.2.6.1.1) with same
HPTLC conditions.
4.2.6.3.2 Estimation of gallic acid in various fractions of ethanolic extract of fruits of M.

citrifolia Linn.

A 100 µg/mL stock solution of standard gallic acid was prepared by dissolving 1 mg of
accurately weighed standard in methanol as per the standard procedures.
A 1 mg/mL of sample solution was prepared by dissolving 10 mg each of ethyl acetate and n-
butanol fraction obtained from the ethanol extract of the fruit and ethanol extract in methanol
as per the standard procedures. The Solutions were sonicated (for 10 min), filtered through
Whatman No. 1 paper.
c. Mobile phase – Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2 v/v/v/v)
The estimation was carried out as per the procedure described above. The plates were
scanned as 380nm.
4.2.6.3.3 Estimation of scopoletin in hexane, chloroform fractions & ethanolic extract of

fruits of M. citrifoliaLinn.
A 100 µg/mL stock solution of standard scopoletin was prepared by dissolving 1 mg of

accurately weighed standard in methanol as per the standard procedures.
A 1 mg/mL of sample solution was prepared by dissolving 10 mg each of ethyl acetate and n-
butanol fraction obtained from the ethanol extract of the fruit and ethanol extract in methanol
as per the standard procedures. The Solutions were sonicated (for 10 min), filtered through
Whatman No. 1 paper.
c. Mobile phase – Chloroform:acetone (9.5:0.5 v/v)
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4.2.7 Heavy metal analysis
4.2.7.1 Determination of heavy metal content [3]
Heavy metal content for crude drugs was determined by using the Atomic Absorption
Spectroscopy as per the recommendations of WHO.
4.2.8 Microbiological evaluation
4.2.8.1Determination of micro-organisms [8,9]
Presence of micro-organisms were detected as per WHO guidelines.
4.2.8.1.1Total Aerobic Bacterial Count:
1 g of sample (crude drug powder) was weighed in a sterile test-tube. It was dissolved /
suspended in 9 mL of buffered sodium chloride-peptone solution pH 7.0 (PB). Then the
contents were vortexed to form a uniform suspension. Dilutions were made upto 10-3 in PB. 1
mL of each dilution was taken in a sterile petri plate and to it, 18 mL of liquefied soyabean-
casein digest medium (TSA) was added at a temperature not exceeding 45°C. The contents
were mixed and the plate was allowed to set. These plates were then incubated at 28°C for 5
days. The plates were examined daily for the bacterial count.
The numbers of colonies formed were counted and the results were expressed in terms of
cfu/g or cfu/mL.
4.2.8.1.2 Total Yeast and Mould Count
Sample (crude drug powder, 1 g) was weighed in a sterile test-tube. It was dissolved and
suspended in 9 mL of PB. The content was vortexed to form a uniform suspension. Dilutions
were made upto 10-3 in PB. To 1 mL of each dilution taken in a sterile petri plate, 18 mL of
liquefied Sabouraud’s agar (SAB) was added at a temperature not exceeding 45°C. The
contents were mixed and the plate was allowed to set.This was followed by incubation at
23°C for 5 days and daily examination for the count. Numbers of colonies formed were
counted and the results were expressed as cfu/g or cfu/mL.
Table 4-1: Microbial contamination limits in medicinal plant materials; WHO [9]
Plant material/ Total Aerobic Total Yeast and Mould Enterobacteriaceae
formulation bacterial count count count
Used for internal use NMT 105 /gm NMT 103 /gm NMT 103/gm
NMT: Not More Than
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4.2.8.1.3 Detection of individual pathogens
4.2.8.1.3.1 Preparation of stock
1 g of each of the samples (crude drug powder) was weighed in a sterile test-tube. It was
dissolved / suspended in 9 mL of either Lactose Broth (LB) buffered sodium chloride-
peptone solution pH 7.0 or phosphate buffer (PB). The contents were then vortexed to form a
uniform suspension. The tubes were incubated at 37°C for 2-3 h.
4.2.8.1.3.2 Escherichia coli
i. Primary test
0.1 mL of the stock in LB was transferred to 10 mL of MacConkey’sbroth and incubated at

37°C for 24 h. A subculture was prepared on MacConkey’sagar plate and incubated at 37°C
for 24 h. Growth of red, generally non-mucoid colonies indicated the presence of E.coli.
Gram’s staining was carried out when any colonies were found present. Confirmation was
done by secondary test.
ii. Secondary test
A single colony was picked from the plate and a saline suspension of it was prepared.
IMViC’s test was performed as follows
a. Indole test
2-3 loops full of suspension in peptone water (3 mL) were inoculated and incubated at
37°C for 24 h. 0.5 mL Kovac’s reagent was added. A red colour ring in alcohol phase
indicated positive results.
b. Methyl red test
2-3 loops full of suspension in Glucose phosphate broth (3 mL) were inoculated and
incubated at 37°C for 24 h. Methyl red indicator was added. A red colouration after
addition of methyl red indicated positive results.
c. Voges Proskaur test
2-3 loops full of suspension in Glucose phosphate broth (3 mL) were inoculated and
incubated at 37°C for 24 h. On addition of 1 mL 40% KOH and 3 mL α-naphthol,
presence of pink colour in 2-3 minutes indicated positive results.
d. Citrate test
57
Sub-culture of suspension on Citrate agar slant was prepared and incubated at 37°C
for 24 h. Growth with bluish colouration indicated positive results.
iii. Interpretation:
Positive indole and methyl red test; with negative VP and Citrate test confirmed the
presence of E.coli.
The sample being examined passed the test if colonies of the type described did not
appear in the primary test, or if the secondary tests were negative.
4.2.8.1.3.3 Salmonella spp.
i. Primary test
1 mL of the stock in LB was transferred to 10 mL of tetrathionate bile brilliant green broth

and incubated at 37°C for 24 h. A subculture was prepared on Deoxycolate Citrate Agar and
Xylose Lysine Deoxycolate agar media; and incubated at 37°C for 24 h.
Table 4-2: Salmonella colonies on different media.

Medium Description of colony
Deoxycolate Citrate Agar Well developed, colourless
Xylose Lysine Deoxycolate Well developed, red, with or without black centres
ii. Secondary test
A single colony from the plate was taken and its saline suspension was prepared. Further sub-
culture of suspension was prepared on Triple Sugar Iron agar slant using deep inoculation
technique and incubated at 37°C for 24 h. A change of colour from red to yellow observed in
the deep culture (but not in the surface culture) with the formation of gas with or without
production of hydrogen sulphide, confirmed the presence of salmonella spp.
iii. Interpretation
The material passed the test if no such colonies were detected or if the confirmatory
biochemical tests were negative.
4.2.8.1.3.4 Staphylococcus aureus
i. Primary test
0.1 mL of the stock in PB was transferred to 10 mL of TSA broth and incubated at 37°C for
24 h. A subculture was prepared on Baird Parker’s agar incubated at 37°C for 24 h. Presence
58
of black colonies surrounded by clear zones indicated the presence of S. aureus.

Confirmation was done by secondary test.
ii. Secondary test
Suspension in TSA broth was prepared on single colony. Tubes were then incubated at 37°C
for 24 h. 1: 10 dilution of plasma in saline was prepared and from this 1 mL was taken in a
small tube and 100 μl of above grown culture was added and then incubated at 37°C and
examined for coagulation at 1h., 2 h. and 3 h. The conversion of plasma in a soft or stiff gel
(best seen on tilting the tube to horizontal position) was considered a positive result.
iii. Interpretation:
biochemical test was negative.
4.2.8.1.3.5 Pseudomonas aeruginosa
i. Primary test
0.1 mL of the stock in PB was transferred to 10 mL of TSA and incubated at 37°C for 24 h. A
sub-culture was prepared on Cetrimide agar plate and incubated at 37°C for 24 h. Growth of
colonies which are Gram negative rods on Gram’s staining colonies indicated need for
secondary test.
ii. Secondary test
2-3 drops of freshly prepared oxidase reagent was placed on filter paper strip and smear of
the colony was applied on it. The test was positive if a purple colour was produced within 5-
10 seconds.
iii. Interpretation
biochemical test was negative.
4.2.8.1.3.6 Note
For validation of these tests a control set was always run using the following standard
cultures:
59
 Escherichia coli ATCC 8739
 Salmonella spp.
 Staphylococcus aureus ATCC 6538
 Pseudomonas aeruginosa ATCC 9027
4.3 Results and Discussion
4.3.1 Standardization Fig 4-1: Photo showing stems and leaves

of Cissus quadrangularis
4.3.1.1 C. quadrangularis
4.3.1.1.1 Macroscopy
C. quadrangularis is a rambling shrub, climbing over bushes, with thick fleshy quadrangular
stem, glabrous and constricted at the nodes.
Leaves, alternate coriaceous, broadly ovate to suborbicular, 6.5 x 8 cm with serrate margins.
Flowers are small umbellate cymes, calyx cup shaped obscurely lobed. Petals are greenish
yellow, red tipped and berries are globose.
4.3.1.1.2Microscopy
4.3.1.1.2.1 Transverse section of fresh quadrangular stem
A transverse section of inter- nodes showed an angular outline.

Epidermis shows a single row of cells covered with thick cuticle. Numerous stomata are
found on the epidermis.
Cortex is made of several layers of thin walled parenchyma cells. Some cells contain
chloroplast , starch grains or acicular raphides of calcium oxalate crystals. At the corner
internal to the epidermis three to four layers of compactly arranged sclerenchymatous cells
are seen.
Cork: Next to cortex are 3-4 layers of rectangular cork cells arranged compactly without
intercellular spaces.
Phellogen occurs in the sub-epidermal layer. Collenchyma occurs as discrete strands in the
corners. Individual collenchyma cells are isodiametric with cellulose thickening at their
angles. The endodermis is not distinguishable.
60
f
r a
co n S
c
ck ph
c
cm
e x
p st
Fig 4-2:T.S of stem of C.quadrangularis and its
magnified (10X) parts and powder characteristics.a:
air cavity; c: cuticle; ck: cork; cm: cambium; co:
collenchyma; ep: epidermis; f: fibers; n: calcium oxalate
needles; r: rosettes; ph: phloem; Sc: sclerenchyma; st:
sunken stomata; x: xylem
Vascular bundles are collateral, open and arranged in a ring around the large central pith.
Pith constitutes more than half the thickness of the stem and is composed of large
parenchyma cells, compared to the cortical parenchyma.
The microscopic assessment along with the morphological characters of the plant further
confirm its identity. The characters and their description matched that of the standards given
[18].
Fig 4-3: Fresh ripe or half ripe fruit of
4.3.1.2M.citrifolia
M. citrifolia
4.3.1.2.1Macroscopy
Morinda citrifolia L. (Rubiaceae), known popularly

as Noni, is a small evergreen tree or shrub, native to
South Asia that currently grows throughout the
tropics. Leaves are broadly elliptic, bright green,
glabrous; flowers are white and in dense ovoid heads. The fruits of Morinda citrifolia are
very distinct and easy to recognize. The white tubular flowers form in clusters on the young
fruit. The syncarpous fruit grows to be about 5-10 cm long and turns from a greenish to a
translucent yellowish-white colour when the fruit ripens. The surface of the fruit is covered
with polygonal segments that surround post-floral nectarines, which continue to function
during the development of the fruit.
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4.3.1.2.2 Microscopy
4.3.1.2.2.1 Transverse section of unripe fruit
Morinda citrifolia is a syncarpous many- seeded fruit. A T.S. taken through the fruits showed
the following characters.
1. Pericarp
E
a) Epicarp: A single layer with small
p
cells.
Mc
b) Mesocarp: is the bulk of the fruit and is
made up of parenchymatous cells, in T
which collateral lignified vascular
bundles arranged in a radial manner En
Sc
were found. Some cells of mesocarp
were filled with calcium oxalate crystals
in the form of acicular raphides.
c) Endocarp: not distinct Fig 4.4: Microscopic characters of the fresh

fruit of M citrifolia. Ep: Epicarp; Mc:Mesocarp;
Endocarp; T: Testa; En: Endosperm; Sc:
2. Testa sclerenchyma [Magnification: 10 and 40 X]
Seed of the fruit contains a hard seed coat , the testa. It is thick , lignified and made up of
sclerenchymatous bands represented by tangentially and longitudinally running fibres.
The whole layer is lignified.
3. Endosperm
Made up of thick walled polygonal colorless parenchymatous cells containing fixed oils
and aleurone grains.
4.3.1.2.2.2 Powder characteristics of unripe fruits of M. citrifolia
1. Organoleptic evaluation
Color : Brownish black
Odour: Not characteristic
Taste: Bitter
2. Microscopic characters
62
Endosperm: Polygonal colorless cells filled with aleurone grains & fixed oil.
Calcium oxalate crystals: Scattered

Fig 4-5: Photograph showing
accicularraphides were seen in the powder. shuddhaguggul (Commiphora mukul)
Fibres: Group of lignified sclerenchymatous fibres

of testa layer were seen.
Few layers of parenchymatous cells of the mesocarp

were seen containing calcium-oxalate crystals in the
form of acicular raphides.
The fruits were identified as Morinda citrifolia. As the

fruit of the plant is an aggregate, it was difficult to take a TS, hence the possible sections or
parts of section were taken to see and understand the tissues in the fruit. The findings were
compared with available standards for reconfirmation of the identity.
4.3.1.3C.mukul
4.3.1.3.1 Macroscopy
Commiphora mukul is an oleo-gum-resin obtained as dried exudate by making incisions on

stems of the plantain the winter seasons. It is obtained as yellowish brown to dark brown
tears which are translucent.
Color: Brown to dark brown
Odour: Agreeable, aromatic and balsamic
Taste: Characteristic bitter
Size and shape: 0.5 to 1.00 to 2.5 cm in diameter: Rounded or irregular masses or
agglomerated tears. Tears are somewhat transparent, with waxy surface and are brittle in
nature with fractured surface.. The botanical identity was confirmed by the botanist. The
morphological characters supported its identification.
4.3.2 Physical/physicochemical standardisation [3, 4,5, 6]
In the standardization of herbal material, physical and physico-chemical factors play an

important role in the establishment of purity and quality. Ash values testify for the presence
or absence of foreign matter like silica; extractive values indicate the extractable matters in a
solvent and is an indication of possible exhausted material. Moisture on the other hand
63
indicates percentage content and possible deterioration if present in larger quantity.

4.3.2.1Ash Values
The ash values (total, acid insoluble and water soluble) of all the three selected drugs were
determined in triplicate. A mean of three determinations and its standard error was
calculated. The results were expressed as Mean±SE (table 4-3).
The ash values of all the three crude drugs were within the prescribed limits. C.
quadrangularis, showed higher total ash due to the presence of calcium oxalates. C. mukul
shows higher mineral content which is acid insoluble. The total ash of M. citrifolia must be
due to physiological ash which is why the water soluble ash is greater.
Table 4-3: Ash values of C.quadrangularis, C.mukul and M. citrifolia
Parameters C.quadrangularis C.mukul M.citrifolia

(% w/w) (% w/w) (% w/w)
Total ash 10.48± 1.24 7.5 ± 0.64 5.66 ± 0.22
Acid insoluble ash 0.38± 0.02 1.7 ± 0.13 1.0 ± 0.06
Water soluble ash 2.15± 0.44 5.6 ± 0.42 4.0 ± 0.18
4.3.2.2 Extractive values
The amount of extractable mater in each plant material, extracted by different solvents
namely, water, ethanol and ether was determined by cold maceration, the values are tabulated
below (Table 4-4)
Table 4-4: Extractive values of C.quadrangularis, C.mukul and M. citrifolia

Parameters C. quadrangularis C. mukul M. citrifolia
(%w/w) (%w/w) (%w/w)
Water soluble extractive 10.5 ± 0.82 6.35 ± 0.45 24.9 ± 1.6
Ethanol soluble extractive 4.0 ± 0.22 8.5 ± 0.22 16.9 ± 1.4
Ether soluble extractive 8 ± 1.14 8.10 ± 0.27 8.2 ± 1.8
The results suggest that all the plants /materials have a higher content of water soluble mater
as compared to the alcohol or ether soluble content. It is especially noteworthy that, the fruits
of M. citrifolia showed richer water soluble content than any other plant /material which may
be due to the high carbohydrate, amino acid and glycoside composition. The higher ether
soluble extractive of C.mukul, compared to the other extractives is probably due to the
presence of resins and volatile oils..
64
4.3.2.3 Foreign matter [3]
The foreign matter in the different plant materials was observed by the naked eye and by the
aid of a magnifying lens(Table 4.5). Guggul, being a purified oleo-gum-resin showed little
silicaceous mater. The foreign inorganic matter present in C. quadrangularis can be
attributed to the presence of few roots in addition to the whole plant material. The fruits of
noni were freshly collected hence there was no adherent inorganic mater.
Table 4-5: Weight (%) of foreign organic and inorganic mater in C. quadrangularis, C. mukul & M.
citrifolia
Parameters C. quadrangularis C. mukul (%w/w) M. citrifolia (%w/w)
(%w/w)
Foreign organic Nil 1.062 ± 0.53 (Adherent 1.532 ± 0.42 (fruit stalk and
matter bark and leaves) sepals)
Foreign in-organic 0.55 ± 0.02 (Silicaceous 0.62 ± 0.04 (Silicaceous Nil(Fruits were thoroughly
matter material in roots) material in roots) washed)
4.3.2.4Moisture content [3]
The moisture content of the dried powdered material of all the crude drugs was determined
by loss on drying method and is tabulated below (Table 4-6). Strictly, the test is moisture and
other volatile mater, as it is loss on drying, the selected material showed moisture within
limits. Some of the crude drugs were observed to be highly hygroscopic, such drugs would
have to be stored in air tight containers. M. citrifolia showed slightly higher moisture than
the other two drugs.
Table 4-6: Percentage of moisture in dry powders of C. quadrangularis, C. mukul and M. citrifolia
Parameters C. quadrangularis (%) C. mukul (%) M. citrifolia (%)
Loss on Drying 5.2±24 6.4 ± 0.45 8.80 ± 0.32
4.3.2.5Fluorescence analysis of the powdered crude drugs [10, 11]
The powdered crude drugs were observed under day light and UV[254] light. Change in
colour was recorded. Fluorescence of the powdered crude drugs has been studied as a
pharmacognostic character to distinguish between plants and their species.The powders were
then treated with different reagents as per the descriptions of Chase et al., [10] and Kokoski
et al., [11].
65
4.3.3 Phytochemical evaluation
Herbal drugs are known to contain a wide variety of chemicals which include both primary as
well as secondary plant metabolites. The pharmacological activity of a plant is generally
attributed to a specific chemical entity of the plant or a group of chemicals, for instance,
isoflavonoids in soy are believed to be responsible for the estrogenic activity. It is, therefore,
important to assess the quality of a plant material by evaluating the phytochemicals. While
phenols and polyphenols are responsible for antioxidant potential of plant, certain specific
chemicals like lupeol, guggulsterone E and Z are responsible for the specific activity. The
phytochemicals of a plant tend to vary depending on the geographical source, time, method of
collection etc., hence it is essential to evaluate drugs for their constituents before use.
Table 4-7 Fluorescence analysis of the crud drug powders

Sr. Powder + White light UV light (254 nm)
No. Treatment CQ CM MC CQ CM MC
1 Powder as such Green Brownish Black Dark green Brown Brown
black
2 Powder + 1N HCL Greenish Dark Light Blackish Brown Green
black brown brown brown
3 Powder + 1N Dark Brown Blackish Dark brown Brown Light
H2SO4 brown brown green
4 Powder + 1N Greenish Black Light Brown Black Light
NaOH (Aq.) yellow brown green
6 Powder + Yellowish Yellow Brown Yellowish Parrot Parrot
Ammonia brown brown green green
7 Powder + Iodine Brown Brown Brown brown Chocolate Dark
green brown
8 Powder + 5% Greenish Brown Yellowish Greenish Chocolate Dark
FeCl3 Brown blue brown brown
9 Powder + Acetic Green Light Brown green Black Buff
acid brown
10 Powder + 1N Yellowish Light Light Yellowish Dirty green Parrot
HNO3 brown brown brown brown green
11 Powder + Picric Greenish Brown Yellowish Yellowish Brown Greenish
acid yellow brown brown brown
4.3.3.1 Extraction and fractionation
The yield of each extract/ fraction, its physical state, colour, and consistency was recorded
(Table 4.8). Of all the selected drugs, C. quadrangularis showed the highest lipid soluble
material with a yield of about 24% in n-hexane which accounts for the waxy components of
the plant. The plant was also found to contain substantial quantities of mid-polar to polar
components. Guggul being an oleo-gum-resin showed moderate proportions of non-polar,
mid-polar and polar constituents. The consistency was sticky and semi-solid.
66
Table 4-8: Percentage yield ,colour and consistency of extracts and fractions of C.quadrangularis,
C.mukul and M. citrifolia
Sr. Colour and Consistency Yield (% w/w)
Solvent
No. CQ CM MC CQ CM MC
Dark green Yellow Yellowish green
1. Hexane 23.89 8.98 1.15
( Waxy semisolid) (Sticky Oil) (Sticky semisolid)
Dark green Brown (Sticky Dark green
2. Chloroform 16.37 1.97 0.62
(Sticky semisolid) semisolid) (Sticky semisolid)
Ethyl Dark brown Brown (Sticky Dark brown
3. 18.3 1.55 3.54
acetate (Sticky semisolid) semisolid) (Sticky semisolid)
Dark brown Dark brown Dark brown
4. Butanol 18.7 1.01 3.89
(Sticky semisolid) (Sticky solid) (Sticky semisolid)
Total Dark brown Dark brown Buff 11.8 11.2
5. 7.41
ethanol (Sticky semisolid) (Sticky solid) (Sticky semisolid) 5 5
67
4.3.3.2.3 Fluorescence analysis [11, 12]
Individual fractions of the selected plants, were also observed for fluorescence pre and post treatment with different reagents (Table 4-9,4-10,4-
11)
A. C. quadrangularis:
Table 4.9: Fluorescence analysis of the fractions of C. quadrangularis
Hexane Chloroform Ethyl acetate Butanol
S. Fraction +
UVlight UVlight UVlight UVlight
no treatment White light White light White light White light
(254nm) (254nm) (254nm) (254nm)
Yellowish
1 Dried fraction Dark green Dark green Dark Green Dark brown Dark brown Dark brown Dark brown
green
Yellowish
2 Fraction + 1N HCl Dark green Dark green Dark Green Dark brown Dark brown Dark brown Dark brown
green
Fraction + 1N Yellowish
3 Dark green Dark green Dark Green Dark brown Dark brown Dark brown Dark brown
H2SO4 green
HNO3 green
NaOH green
Ammonia green
Fraction + 1N Greenish Greenish
7 Brown Brown Dark brown Dark brown Dark brown Dark brown
Iodine brown brown
Fraction + 1N 5%
8 Dark green Dark green Dark green Dark green Dark green Dark green Dark green Dark green
FeCl3
Fraction + 1N Yellowish Yellowish Yellowish Yellowish
9 Yellowish green Yellowish green Yellowish green Yellowish green
Picric acid green green green green
Fraction + 1N
Acetic acid
68
B. C. mukul
Table 4.10: Fluorescence analysis of the fractions of C.mukul
S. Fraction +
UVlight UVlight UVlight UVlight
no Treatment White light White light White light White light
(254nm) (254nm) (254nm) (254nm)
Chocolate Chocolate
1 Dried fraction Yellow Green Light brown Dark green Green Buff
brown brown
Yellowish Yellowish
2 Fraction + 1N HCl Greenish Brown Dark Green Dark brown Dark brown Dark brown Dark brown
brown green
Fraction + 1N Yellowish Yellowish
3 Greenish Brown Dark Green Dark brown Dark brown Dark brown Dark brown
H2SO4 brown green
HNO3 brown green
NaOH brown green
Ammonia brown green
Fraction + 1N Greenish Greenish
7 Brown Brown Dark brown Dark brown Dark brown Dark brown
Iodine brown brown
Fraction + 1N 5%
FeCl3
Fraction + 1N Yellowish Yellowish Yellowish Yellowish
9 Yellowish green Yellowish green Yellowish green Yellowish green
Picric acid green green green green
Fraction + 1N
10 Dark yellow Dark green Dark green Dark green Dark green Dark green Dark green Dark green
Acetic acid
69
C. M. citrifolia
Table 4.11: Fluorescence analysis of the fractions of M. citrifolia

S.no Fraction+treatment UV light UV light UV light UV light
White light White light White light White light
(254nm) (254nm) (254nm) (254nm)
1 Dried fraction yellowish green Light green Dark green Light brown Dark brown Dark green Dark brown Dark green
2 Fraction + 1N HCl yellowish green Green Dark green Light buff Dark brown Green Brown Green
3 Fraction + 1N H2SO4 Light green Light brown Dark green Brown Light brown Light green Light brown Light green
4 Fraction + 1N HNO3 Light green Light brown Light green Light brown Brown Dark green Dark brown Green
5 Fraction + 1N NaOH yellowish green Light green Dark green Light brown Light brown Light green Brown Dark green
6 Fraction + 1N Ammonia Light green Light brown Light green Light buff Dark brown Dark green Brown Green
Chocolate
7 Fraction + 1N Iodine Dark green Dark brown Dark green Dark brown Earthy brown Dark brown Dark green
brown
Brownish
8 Fraction + 1N 5% FeCl3 Light orange Dark green Orange Dark green Green Orange Dark green
orange
Yellowish Yellowish
9 Fraction + 1N Picric acid Green Yellowish green Light green Green Parrot green Light green
green green
Fraction + 1N Acetic
10 Light green Buff Green Light brown Light brown Green Light brown Green
acid
70
4.3.3.3 Preliminary phytochemical screening
The results of the study are tabulated below (Table 4.12),
+ sign in the table indicates presence while – sign represents absence of the phytoconstituents
Table 4-12: Preliminary phytochemical screening of various fractions and the extracts of
C. quadrangularis, C. mukul and M. citrifolia
Ethanolic
Hexane Chloroform Ethylacetate Butanol
Extract
Test
C C M C C M C C M C C M C C M
Q M C Q M C Q M C Q M C Q M C
Alkaloids - - - + + + - - - - - - - - +
Carbohydrates - - - - - - - - - - + + + + +
Phytosterols + + + + + + - + - - - - + + +
Fixed oils and

+ + + - - - - - - - - - + - +
fats
Saponins - - - - - - - + - + + + + + +
Phenolic
compounds - - - - - - + - + + + + + + +
and tannins
Proteins - - - - - - - - - - - + + + +
Gums,
- - - - - - - - - - + - - + -
Mucilage
Flavonoids - - - - - - + + + + + + + + +
71
Preliminary phytochemical screening is an important method to determine the basic nature of

chemical constituents present in the crude drugs. It is useful in selection of a method for
extraction and carrying out any specific assay.
Fig 4-6: Standard plot of gallic acid.
4.3.3.4 Estimation of total phenolic content.
[12,13,14]
Phenols and flavonoids have been known for their

antioxidant potential. Total phenolic content is an
estimate of the antioxidant capacity of a plant on
one hand and on the other is a parameter for the
quality of the plant. The total phenolic content was
estimated using gallic acid as a reference standard and the results are given below (Table 4-
13) as gallic acid equivalents.
Table 4-13: Total phenolic content of the extracts and selected fractions of C. quadrangularis, C. mukul
and M. citrifolia
C. quadrangularis C. mukul M. citrifolia

Fraction Abs GAEq Abs GAEq Abs GAEq
Ethyl acetate 0.241 118.78 1.083 53.37

0.76 374.59
n-Butanol 0.24 118.29 0.573 28.24

0.93 458.4
Ethanolic extract 0.217 106.95 0.772 38.05

0.52 256.3
Note: Abs = Absorbance; GAEq = mg Gallic Acid Equivalent/g dry weight of fraction
4.3.3.5 Estimation of total flavonoid content [15].

Fig 4-7: Standard plot of quercetin
Flavonoids have especially become synonymous with
antioxidant constituents and is hence taken as an
indirect measure of the antioxidant potential of the
crude drug. The total flavonoid content was measured
using quercetin as a reference standard and results
have been expressed as quercetin equivalents (Table
4.14)
72
Table 4-14: Total flavonoid content of the extracts and selected fractions of C. quadrangularis, C.
mukul and M. citrifolia

Fraction
Abs QEq Abs QEq Abs QEq
Chloroform 0.039 3.29 0.012 1.02 -- --
Ethyl acetate 0.066 5.57 0.052 4.39 0.032 2.704
n-butanol 0.054 4.56 0.09 7.60 0.037 3.127
Ethanolic extract 0.038 3.21 0.041 3.46 0.041 3.465
Note: Abs = absorbance; QEq = mg quercetin equivalent/g dry weight of fraction
4.3.3.6 Estimation of tannin content[16].

Fig 4-8: Standard plot of Tannic acid
Tannins are one of the most widely distributed 1
TOTAL TANNIN CONTENT
phytoconstituents which are also endowed with 0.8
y = 0.008x
Absorbance
antioxidant activity. Total tannin content is an 0.6 R² = 0.9942

important quality control parameter for the 0.4 Tannic acid
evaluation of botanicals and was estimated using 0.2
tannic acid as a reference standard. Results were 0

0 50 100 150
expressed as tannic acid equivalents (Table 4.15). Conc. (µg/ml)
Table 4-15: Total tannin content of the extracts and selected fractions of C. quadrangularis, C. mukul
and M. citrifolia
Fraction
Abs TAEq Abs TAEq Abs TAEq
Ethyl acetate 0.868 108.14 0.611 60.29 0.609 75.87
n-butanol 0.859 107.01 0.74 76.37 0.826 102.90
Ethanolic extract 0.73 90.94 0.696 70.89 0.699 87.08
Note: Abs = Absorbance; TAEq = mg Tannic Acid Equivalent/g dry weight of fraction
73
4.3.4 Marker based standardisation of the selected plants and their extracts
using HPTLC.
Standardization of any plant remains incomplete until and unless the chemical composition is
assayed. Unfortunately estimation of all the chemical constituents of a plant or an extract is
next to impossible. There is hardly any method to determine the concentration of each
chemical present in an extract. As a possible solution to this problem, marker based
chromatographic assays have been developed. Biologically active phytoconstiuents have been
used as standard markers for the standardization of medicinal plants or their purified or semi
purified extracts by suitable chromatographic techniques. High performance thin layer
chromatography (HPTLC) has been successfully used for this purpose. The simplicity of
operation and reproducibility of results have made HPTLC an important tool in the quality
control of medicinal plants. In the present study all the plant extracts and their fractions were
standardized using bioactive markers.
4.3.4.1 Estimation of -sitosterol, lupeol in various fractions obtained from ethanolic

extract of the whole plant of C. quadrangularis Linn.
The amount of -sitosterol and lupeol was estimated in the hexane, chloroform fraction and
total ethanolic extract as given in Table 4.16
β-sitosterol is one of the most abundant sterols found in plants and has shown profound
biological effects in the reduction of carcinogen induced colon cancer and inflammation [19].
Lupeol, a pentacyclic lupane-type triterpene, abundant in diverse flowering plant families is
shown to have intense biological effects on inflammation, rheumatism and cancer [20].
Inflammation and its causative factors viz., certain kinases have a significant role in the
pathophysiology of osteoporosis. Of the several minor pathophysiological pathways involved
in the pathogenesis of osteoporosis, activation of pro-inflammatory kinases is one. Agents
which suppress such kinases have been shown to have antiosteoporotic activity. C.
Quadrangularis has both of these compounds which were therefore, estimated in the total
extract and in its fractions (n-hexane and chloroform), as bioactive markers.
74
Table 4-16:Estimation of -sitosterol, lupeol in various fractions and the total extract of C. quadrangularis
-sitosterol Lupeol
S.no Fraction
Rf AUC %content Rf AUC %content
1 Standard 0.15 3769.3 98 0.28 8654.3 98
2 Hexane 0.15 2131.2 5.54 0.27 2504.6 2.83
3 Chloroform 0.15 667.7 1.73 0.27 527.1 0.59
4 Ethanolic Extract 0.15 2044.4 5.31 0.27 2030.1 2.29
Fig 4-9: Chromatograms of standard β-sitosterol lupeol, hexane fraction and ethanol extract of C.
quadrangularis
Lupeol
Track 1 ID :Lupeol 3D Chromatographic profile

(Std)
-sitosterol
-sitosterol
Lupeol
-
Lupeol
sitostero
l
Track 2 ID :Betasitosterol Track 3 ID :CQ Hexane fraction

(Std)
-sitosterol
Lupeol
Track 7 ID : CQ Ethanolic Extract
75
4.3.4.4 Estimation of -sitosterol in hexane and total ethanolic extract obtained from
The unsaponified fraction of C. mukul was found to contain -sitosterol along with myricyl
alcohol [21]. The drug has been an Ayurvedic remedy for inflammation [ 22] and the activity
can be partly attributed to -sitosterol among the other responsible constituents. Hence,
guggul was standardized to -sitosterol, a bioactive marker, both in the hexane and total
extract. The content of -sitosterol in the hexane and total ethanolic extracts of Commiphora
mukul was found to be 24.40 and 21.70 % w/w respectively (Table 4-18).
Table 4-18: Estimation of -sitosterol in various fractions and the total extract of C. mukul.
-sitosterol
S.no Fraction
Rf AUC %content
1 Standard 0.18 1345.8 98
2 Hexane 0.18 3354.2 24.49
3 Methanol 0.19 2912 21.70
Fig 4-10: Chromatograms of standard β-sitosterol, hexane fraction and ethanol

extract of C. mukul
3D Chromatographic profile of TLC plate Track 1. ID Standard β-sitosterol
Track 4. ID: Hexane fraction Track 7. ID: Total Ethanol extract
76
4.3.4.5 Estimation of guggulsterone E and Z in hexane and total ethanolic extract of

Guggul has been used since time immemorial in traditional remedies for pain and
inflammation. Extensive research on the dried exudate has revealed its potential as an
antioxidant and antihyperlipedemic agent. Two compounds namely guggulsterone E and
guggulsterone Z have been identified as bioactive constituents. Further Ichikawa and
Agarwal have demonstrated the osteoclastic suppression activity of these compounds [23].
Total extract of shuddhaguggul was therefore standardized to guggulsterone E and Z. The
results are tabulated below (Table 4.18)
Table 4-18: Estimation of guggulsterone E and Z in the total extract of C.mukul.
S.No. Sample Guggulsterone –E Guggulsterone –Z
Rf AUC % Content Rf AUC % Content
1. Standards 0.39 10279.6 95 0.47 12675.7 95
2. Guggul extract 0.39 5284.4 2.52 0.47 1898.6 2.20
Fig 4-11: Chromatograms showing standard peaks of Guggulsterone E & Z and that of total
extract of C. mukul
Track ID 2: Guggulsteron E Track ID 3: Guggulsteron Z Track ID 4:Guggl extract
77
4.3.4.6 HPTLC estimation of -sitosterol in hexane and chloroform fractions & in the
ethanolic extract of the fruits of M. citrifolia Linn.
As discussed earlier, β-sitosterol is one of the abundant sterols which is found in many plants
including M. citrifolia [24,25]. The fruits have been used as a general tonic especially in
arthritic pain. β-sitosterol can be estimated as a marker for the assessment of quality of the
drug. Thus the fruit extract was standardized to β-sitosterol by HPTLC and results are
tabulated below ( Table 4.19)
Table 4-19: Estimation of -sitosterol in various fractions and in the total extract of M. citrifolia.
Track ID Fraction Rf AUC % content
2 -sitosterol 0.23 9229.6 98

3 Hexane 0.23 5366.4 5.69
4 Chloroform 0.23 4370.3 4.64
7 Total ethanolic extract 0.23 2706.3 2.87
Fig 4-12: Chromatograms showing

peaks of -sitosterol in standard,
hexane, chloroform fractions and
ethanolic extract of fruits of M. citrifolia
Linn.
Chromatographic profile of standard and extract
Track 2 ID: -sitostearol

Track 3 ID: MC Hexane
Track 4 ID: MC CHCl3 Track 7 ID: MC MeOH
78
4.3.4.7 Estimation of Scopoletin acid in the ethanol extract of fruits of M. citrifolia Linn.
Scopoletin is one of the abundant coumarin present in the fruits of M. citrifolia and it is
biologically active [26,27]. The constituent is specific to the extract and is known to inhibit
certain cytokines like TNF-α and PGE2. Tumour necrosis factors and prostaglandins are
involved in the pathogenesis of osteoporosis [28] and hence its concentration in the extract
gives vital information. The marker was therefore estimated in the total extract of Morinda
citrifolia fruit.
The total extract of the fruit was found to contain 1.56% of Scopoletin
Fig 4-13: Chromatograms showing standard peaks of Scopoletin and that of total extract of M. citrifolia
Track ID 2: Standard Scopoletin Track ID 4: Scopoletin peak in noni

extract
Table 4-19A: Estimation of Scopoletin in the total extract of M. citrifolia.
2 Scopoletin 0.53 1930.4 98
4 Noni extract 0.53 614.3 1.556
The total extract of the fruit was found to contain 1.56% of Scopoletin
4.3.4.8 Estimation of gallic acid in various fractions obtained from ethanolic extract of
fruits of M. citrifolia Linn.
The ethanolic extract of M. citrifolia and various other fractions were standardized to their
gallic acid content by HPTLC. The ethyl acetate fraction showed the highest percentage of
gallic acid (3.23 %) as compared to the others.
Gallic acid, being an established antioxidant that scavenges free radicals,was used as a
bioactive marker for standardization. The ethyl acetate fraction being partially purified
79
showed a better percentage of gallic acid than that of other fractions including total ethanol
extract.
Table 4-20: Estimation of Gallic acid in various fractions and the total extract of M. citrifolia.
1 Gallic acid 0.56 12576.4 98
7 Ethyl acetate 0.56 4150.3 3.23
8 N-butanol 0.56 734.7 0.57
6 Total ethanolic extract 0.56 1291.5 1.006
Fig 4-14: Chromatograms showing

peaks of Gallic acid in standard,
ethanol extract and its fractions
Chromatographic profile of standard and extract
Track 1 ID: Gallic acid Track 6 ID: MC ethanolic extract
Track 7 ID: Ethyl acetate Track 8 ID: MC N-butanol
4.3.5 Heavy metal analysis
Plants are known to contain many minerals along with some metals. Iron is very commonly
found in thick fleshy green leaves. Most of the traditional Ayurvedic and Siddha formulations
have been found to contain heavy metals in large quantities (much beyond permissible
limits). It is therefore imperative to find and estimate heavy metals in the herbal raw
80
materials. This was carried out by atomic absorption spectroscopy. The results are given
below.
Table 4-21: Estimation heavy metals in individual plant material of C. quadrangularis, C. mukul and
M. citrifolia.
Metals C. quadrangularis C. mukul M. citrifolia
Lead (NMT 10 ppm) Absent Absent Absent
Mercury (NMT 10 ppm) Absent Absent Absent
Arsenic (NMT 5 ppm) Absent Absent Absent
4.3.6 Microbiological evaluation
The total aerobic bacterial yeast and mould count was found to be within the prescribed limits
(WHO guidelines), No pathogenic organisms were observed [3] and the raw material was
found to pass the tests (Table 4.22).
Herbal raw material during its processing (collection, drying, dressing and storage), tend to
get contaminated with bacteria yeast and moulds and sometimes with some potentially
pathogenic organisms. If the material is processed improperly and especially if there is
excess of moisture in the crude drugs they are prone for microbial contamination. Any crude
material, if found contaminated, must not be processed further and must be rejected. The test
is therefore very important in the evaluation and standardization of herbal raw material..
Table 4-22: Microbial load and individual pathogen count in the plant materials of C. quadrangularis,
C. mukul and M. citrifolia.
Total extracts C. quadrangularis C. mukul M. citrifolia
4.3.6.1Total Aerobic Bacterial Count (NMT 1000 cfu/g):
Total bacterial count 600 cfu/g 600 cfu/g 600 cfu/g
4.3.6.2 Total Yeast and Mould Count (NMT 1000 cfu/g):
Yeast and mould 20 cfu/ g 20 cfu/ g 20 cfu/ g
4.3.6.3Detection of individual Pathogens
E.coli Absent Absent Absent
Salomonella spp Absent Absent Absent
S. aureus Absent Absent Absent
P.aeruginosa Absent Absent Absent
81
4.4 Conclusion
Standardization is an important aspect of any herbal formulation development. It is important

to identify and record the physical, physicochemical and chemical properties of each plant
material that is involved in product development. The entire plant of Cissus quadrangularis,
dried, purified, oleo-gum-resin of Commiphora mukul (Shuddhaguggul) and dried half ripe
fruits of Morinda citrifolia, their respective extracts and their fractions were standardized in
accordance with the WHO guidelines. All the evaluated parameters were found to be within
the prescribed limits. The extracts and various fractions of the respective extracts were
standardized using bioactive markers like, β-sitosterol, guggulsterone E, guggulsterone Z and
gallic acid.
4.5 References
1 WHO.guidelines for assessing quality of herbal medicines with reference to contaminants and
residues.WHO Library Cataloguing-in-Publication Data; Spain; 2007.
2 Narayana DBA, Katayar CK, Brindavanam NB. Original system: search, research or re-search. IDMA
Bulletin 1998; 29: 413–416.
3 WHO.Quality control methods for medicinal plant materials. WHO/PHARM/92.559/re-v. 1., Geneva;
1992
4 Khandelwal KR, Pawar AP, Kokate CK, Gokhale SB. Practical Pharmacognosy, NiraliPrakashan; Pune
India; 2001.
5 Brain KR, Turner TD. The practical evaluation of phytopharmaceuticals.WrightScientechnica; Bristol;
1975.
6 Harborne JB. Phytochemical methods- a guide to modern techniques of plant analysis.Springer,
California; 1998.
7 Kokate CK. Practical Pharmacognosy. 3rd Edn., Jain MK. VallabhPrakashan; New Delhi India; 1991
8 Indian Pharmacopoeia, Vol 2 Government of India, Ministry of Health and Welfare, Controller of
Publications; New Delhi India; 1996.
9 Nakamura Y, Yomura K, Kammoto T, Ishimatsu M, Kikuchi Y, Niitsu K, Terabayashi S, Takeda S,
Sasaki H, Arimoto K, Okada M, Sekita S, Satake M, Goda Y. Physicochemical quality evaluation of
natural compounds isolated from crude drugs. J Nat Med 2006; 60: 285-294.
10 Chase CR, Pratt R. Fluorescence of powdered vegetable drugs with particular reference to development
of a system of identification. J Am Pharm Assoc 1949; 38: 324-331.
11 Kokoski CJ, Kokoski R J, Slama FJ. Fluorescence of powdered vegetable drugs under ultraviolet
radiation. J Am Pharm Assoc 1958; 47 (10): 715-717.
12 Singleton VL, Rossi JA.Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid
reagents.Am J Enol Vitic 1965;16: 144-158.
82
13 Tawaha K, Alali F, Gharaibeh M, Mohammad M, Elelimat T. Antioxidant activity and total phenolic
content of selected Jordanian plant species. Food Chem 2007; 104: 1372-1378.
14 Meda A, Lamien C, Romito M, Millogo J, Nacoulma O. Determination of the total phenolic, flavonoid
and proline contents in burkinafasan honey, as well as their radical scavenging activity. Food Chem 2005;
91: 571-577.
15 Bajaj KL, Devsharma, AK. A colorimetric method for the determination of tannins in
tea.MicrochimicaActa1977; 68: 249-253.
16 Salunkhe DK, ChavanJK,KadamSS.Dietary Tannins: Consequences and Remedies. CRC Press; Boca
Raton, FL; 1990
17 Tempel AS. Tannin measuring technique: a review. J Chem Ecology 1982;8:1289-1298.
18 Quality standards of Indian Medicinal Plants. Eds Gupta AK, Tendon N, Sharma M. Vol 2 ICMR; New
Delhi: 2005
19 Maridass M, Ramesh U. Investigation of Phytochemical constituents from Eulophiaepidendreae. Int J
biol tech 2010; 1:1-7.
20 MargarethBC, Mirande J, Sarachine. Biological activities of lupeol, Int j biomed pharm sci2009;3 :46-66.
21 Amjad AM, Mashooda H. Chemical investigation of Commiphora mukul. Pakistan J SciInd Res 1967;
10: 21–23.
22 Kimura I, Yoshikawa M, Kobayashi S, Sugihara Y, Suzuki M, Oominami H, Murakami T, Matsuda H,
Doiphode VV. New triterpenes, myrrhanol A and myrrhanone A, from guggul-gum resins, and their
potent anti-inflammatory effect on adjuvant-induced air-pouch granuloma of mice.Bioorganic and
Medicinal Chemistry Letters 2001; 11: 985-989.
23 Ichikawa H, Aggarwal BB. Guggulsterone inhibits osteoclastogenesis induced by receptor activator of
nuclear factor-kappab ligand and by tumor cells by suppressing nuclear factor-kappab activation. Clinical
cancer research 2006; 12: 662-668.
24 Levand O, Larson HO. Some chemical cons tituents of Morindacitrifolia. PlantaMed 1979; 36: 186-7.
25 Farine JP, Legal L, Moreteau B, Le Quere JL. Volatile components of ripe fruits of Morindacitrifoliaand
their effects on Drosophila. Phytochemistry 1996; 41: 433-8.
26 Kamiya K, Tanaka Y, Endage H, Umar M, Satake T. New anthraquinone and iridoid from the fruits of
Morinda citrifolia L. Chem & Pharm Bull 2005; 53: 1597-1599.
27 Dalsgaard PW, Potterat O, Dieterle F, Paululat T, Kuhn T, Humburger M. Noniosides E-H, new
trisaccharide fatty acid esters from the fruit of Morinda citrifolia L. Planta Medica 2006; 72: 1322-1327.
28 Lean JM, Davies JT, Fuller K, Jagger CJ, Kirstein B, Partington GA, Urry ZL, Chambers TJ. A crucial
role for thiol antioxidants in estrogen-deficiency bone loss. J Clin Invest 2003; 112: 915-923.
83

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Standardization, extraction and evaluation of selected plants

4. Standardization and evaluation of

4.2.2 Collection and authentication

4.2.3 Morphology and Microscopy

4.2.3.2 .1 Anatomical studies [3,4]

4.2.3.2.2 Powder analysis [5]

4.2.4 Physical/physicochemical standardisation [3, 4, 5, 6,7]

4.2.4.1 Total ash

4.2.4.2 Acid insoluble ash

4.2.4.3 Water soluble ash

4.2.4.4 Extractive values [8]

4.2.4.4.1 Ethanol soluble extractive

4.2.4.4.2 Water soluble extractive

4.2.4.4.3 Ether soluble extractive

4.2.4.5Foreign organic matter [3]

4.2.4.6 Moisture content by Loss on drying [3]

4.2.4.7 Volatile content [3]

4.2.5 Phytochemical evaluation

4.2.5.2 Physico-chemical evaluation of the fractions

4.2.5.2.1 Determination of moisture content

Same method as described above in 4.2.4.6 was employed.

4.2.5.2.2 Determination of solubility [9]

4.2.5.2.3 Fluorescence analysis [10,11]

4.2.5.3 Preliminary phytochemical screening [7]

4.2.5.4 Estimation of total phenolic content [12,13]

a. Reagents - 1) Folin–Ciocalteu reagent (0.2 N)

Where T = total phenolic compounds, mg/g plant extract, in GAE;

4.2.5.3 Estimation of total flavonoid content[14]

a. Reagent – 1) Aluminium trichloride (2%)

b. Procedure - 5 mL of 2% aluminium trichloride was mixed with the same volume of

4.2.5.5 Estimation of tannin content [15].

Phosphomolybdic-phosphotungstic acid (Folin-Denis reagent) is reduced to a blue colour

b. Reagents – 1) Folin-Denis reagent.

c. Extract solution 10 mg of extract was dissolved in 10 mL of methanol.

4.2.6.1 Cissus quadrangularis

4.2.6.1.1 Estimation of -sitosterol and lupeol in various fractions obtained from

a. Preparation of Standard solution

c. Mobile phase - Benzene: Ethyl acetate (9.5: 0.5 v/v).

e. Visualization and Scanning

AUC of sample x Conc. of std x %purity

AUC of standard x Conc. of sample

a. Preparation of Standard solution

c. Mobile phase – Toluene:acetone (9: 1 v/v).

e. Visualization and Scanning

4.2.6.3 Morinda citrifolia

4.2.6.3.1 Estimation of -sitosterol in hexane, chloroform fractions & ethanolic extract

4.2.6.3.2 Estimation of gallic acid in various fractions of ethanolic extract of fruits of M.

a. Preparation of Standard solution

4.2.6.3.3 Estimation of scopoletin in hexane, chloroform fractions & ethanolic extract of

A 100 µg/mL stock solution of standard scopoletin was prepared by dissolving 1 mg of

c. Mobile phase – Chloroform:acetone (9.5:0.5 v/v)

4.2.7 Heavy metal analysis

4.2.7.1 Determination of heavy metal content [3]

4.2.8 Microbiological evaluation

4.2.8.1Determination of micro-organisms [8,9]

Presence of micro-organisms were detected as per WHO guidelines.

4.2.8.1.1Total Aerobic Bacterial Count:

4.2.8.1.2 Total Yeast and Mould Count

4.2.8.1.3 Detection of individual pathogens

4.2.8.1.3.1 Preparation of stock

4.2.8.1.3.2 Escherichia coli

0.1 mL of the stock in LB was transferred to 10 mL of MacConkey’sbroth and incubated at

ii. Secondary test