Analytical, Toxicity, Experimental and

Clinical Validation of Ayurvedic

Formulations /Herbal Crude Drugs

K V GOPINATH M Pharm PhD,CPhT

Tirumala Tirupati Devasthanams
TIRUPATI
e-mail:gopinath.karnam@gmail.com
Analytical, Chemical, & Biological
Methodology of Validation/Standardization

 Analytical methods should be scientifically sound (e.g., specific,

sensitive, and accurate) and provide results that are reliable and
reproducible
 Standardization of drug means confirmation of its identity,
quality and purity throughout all phases of its cycle ie shelf-life,
storage, distribution & use by various parameters.
 Different techniques involved in standardization of crude drugs
-Macroscopic methods
-Microscopic methods
-Physical methods
-Chemical methods
-Biological methods
PHYSICAL METHODS
STANDARDIZATION OF HERBAL DRUGS

 Viscosity  Bitterness value

 Melting point  Hemolytic activity
 Solubility  Swelling index
 Moisture content and volatile  Foaming index
matter  Ash value
 Specific gravity  Astringency
 Density  Organoleptic testing
 Optical rotation  Determination of Ash value
 Refractive index  pH estimation
 Particle size determination  Estimation of foreign
 Bulk density materials
CHEMICAL METHODS OF
STANDARDIZATION - HERBAL DRUGS

 Detection of carbohydrates and glycosides

 Detection of fixed oils and fats
 Detection of protein and free amino acids
 Detection of alkaloids
 Detection of phytosterols
 Detection of saponins
 Detection of phenolic compounds and tannins
 Detection of gums and mucilage
 Detection of volatile oils
 Chemical Assays
 Limit test for heavy metals
 Determination of Acid Value, Saponification value, & Iodine value
 BIOLOGIAL/ MICROBIOLOGICAL
/TOXICOLOGIAL STANDARDIZATION

 Bio Assays  PESTICIDES

-Matching bioassay -Fungicides
-Interpolation Method -Herbicides
-Bracketing Method -Insecticides
-Multiple point -Acarcicides
TOXICOLOGICAL STANDARDIZATION -Nematocides
-Determination of pesticides. -Rodenticides
-Determination of arsenic and heavy metals -Bactericides
-Determination radioactive contamination
-Determination of aflatoxins.
BIOLOGIAL/ MICROBIOLOGICAL
/TOXICOLOGIAL STANDARDIZATION

 Toxic residues may arise in crude drugs, foods, spices and cosmetics
as a result of pesticide application and fumigation during cultivation
and storage .
 TLC and GC are available for determination of organochlorine and
urea derivatives, enzymatic methods for organophosporus
compounds, colorimetric methods for urea derivatives and
spectroscopic techniques for paraquat, triazines and heavy metals.
 Toxic residues may be substantially removed or reduced by infusion
process of the dried plant material and storage at 30 ᶿ has been seen
to reduce rapidly the ethylene oxide residues in senna pods.
 In certain instances, necessary test for aflatoxins (poisonous
substance in the spores of the fungus Aspergillus flavus induces
cancer) , and radioactive impurities
BIOLOGIAL/ MICROBIOLOGICAL
/TOXICOLOGIAL STANDARDIZATION

 Acceptable residue level (ARL) :

ARL = ADI*E*60
MUI*100
Where
ADI=maximum acceptable daily intake of pesticides (mg/kg of body
weight),
E= extraction factor, which determines the transition rate of the
pesticides from the plant material into the dosage form,
MDI=Mean Daily Intake of medicinal plant products,
60 in numerator=adult body weight,
100 in denominator=consumption factor.
viscosity

 Viscosity is an expression of the resistance to flow of a system under

an applied stress.
 Eg. Castor Oil 1000 cp; Glycerin, 93% 400 cp ;Water 1.0019 cp
 The unit of viscosity is the poise/centipoise/stokes/centistokes
 Kinematic viscosity = n/p
 Choice of Viscometer: Capillary viscometer, Falling-Sphere
Viscometer, Cup-and- Bob Viscometer, Cone-and plate Viscometer
 Viscosity play a role:
-mixing and flow materials, their packaging container and their
removal prior to use
-Consistency from fluid to semisolid to solid, can affect its
patient acceptability, physical stability, and even bioavailability
- Play a role in selection equipment of processing used in the
manufacture
Melting Point

 The freezing point or melting point of a pure crystalline solid is

strictly defined as the temperature at which pure liquid and solid exit
in equilibrium at 1 atm.
 A crystal that is bound together by weak forces generally has a low
heat of fusion and a low mp, whereas one bound together by strong
forces has a high heat of fusion and a high mp.
 The mp of saturated hydrocarbons increases with increase in
molecular weight ; it is used as a identity, and purity of a compound.
 The mp and solubilities of the xanthenes of pharmaceutical interest.
E.g. The methylation of theophylline to form caffeine and the
lengthening of the side chain from methyl(caffeine) to propyl in the 7
position result in a decrease of the mp and an increase in solubility
Theophylline R=H 270-274 o C 4.5 mole/L X 102
Caffeine (R=CH3) 238 oC 13.3 mole/L X 102
 Solubility

 Definition: quantitatively as the concentration of solute in a saturated

solution at a certain temperature, and in qualitatively as the spontaneous
interaction of two or more substances to form a homogeneous
molecular dispersion
 Concentration Expressions: Molarity, Normality, Molality, Mole
Fraction, Mole percent, %w/w, % w/v,%v/v, milligram percent, ppm
 Solubility depends on chemical, electrical, and structural effects that
lead to mutual interactions between the solute and the solvent.
 Solubility of insoluble substances can improved by Co-solvency,
Solubilisation, pH Modification, Conversion to its salt, Hydro trophy
 Solubility can determined by gravimetric and volumetric method
 The presence of adulterant could be indicated by solubility studies
-E.g. pure Asafoetida is soluble in carbon disulphide
Descriptive terms used to express
solubility in Indian Pharmacopoeia

Descriptive terms Relative quantities of solvent

 Very soluble Less than 1 part

 Freely soluble From 1 to 10 parts
 Soluble From 10 to 30 parts
 Sparingly soluble From 30 to 100 parts
 Slightly soluble From 100 to 1000 parts
 Very slightly soluble From 1000 to 10,000 parts
 Practically insoluble More than 10,000 parts
Bulk Density

 Definition: Mass of powder divided by the bulk volume

 Three-tap method has been found to give most consistent results
 Bulk density of powder depends primarily on particle-size
distribution, particle shape, and the tendency of the particle to adhere
to one another.

Name of the chemical Bulk density True density

(g/cm3) (g/cm3)
Magnesium Carbonate Heavy 0.39 3.0
Magnesium Carbonate Light 0.07 3.0
Talc 0.48 2.7
Density

Density can be defined as mass per unit volume and is expressed

as g/cm3 in cgs system and kg/m3.

Specific Gravity: Also known as relative density defined as the

ratio of the mass of a substance to the mass an equal volume of water
at 40 C or some other specified temperature.

Specific gravity may be determined by the use of various types of

pycnometers, Mohr-Westphal balance, hydrometers etc

Important in interconverting between mass and volume

Determination of Moisture Content &
Volatile Matter

 It is the amount of volatile matter i.e., water drying off from the drug
is known as Moisture content.

 Method: Place about 10 g of drug in a tared evaporating dish. Dry at

105º for 5 hours, and to a constant weight (two successive weighing
should not exceed 0.25%).

 Distillation method

 Importance: Purchase of drugs( aloes, gelatin ,gums, digitalis,

hamamelis leaf, yarrow, hawthorn berries, starch,) which contain
excessive moisture - uneconomical.
-It promotes bacterial growth and product decomposition
- It promotes even enzyme activation along with temperature
 DETERMINATION OF FOREIGN
MATTER

 Foreign matter is a material consisting of moulds, insects, animal

faecal matter and other contaminations such as earth, stones and
extraneous material.
 The amount of foreign matter shall not be more than the percentage
prescribed in the monograph.
Drugs Foreign matter limit

Quillaia Bark 2%
Cascara bark 1%
Valerian root 5% stem bases, 2% other
foreign matter
 DETERMINATION OF FOREIGN MATTER
Weigh 100 –500 g of the drug sample to be examined, or the minimum quantity
prescribed in the monograph, and spread it out in a thin layer. The foreign matter should
be detected by inspection with the unaided eye or by the use of a lens (6x). Separate and
weigh it and calculate the percentage present .
Optical Rotation

 It is the angle through which the plane of polarization of light is

rotated(clockwise or anticlockwise) when the polarized light is
passed through a sample of the liquid.
 It can detected by mean of Polarimeter. The observed rotation is a
function of thickness of the layer(1 cm), its temperature (20 0 C) and
nature of the lamp (sodium vapor lamp) .
 It is quick test to check the identity and purity of the compound,
especially volatile oils.
 The BP distinguishes between nutmeg oils from the East Indies (+10 0
to 250) and west Indies (+250 to 450).
Caraway oil +74 0 to 80 0 Peppermint oil -100 to -300
Lemon Oil +570 to +70 0 Spearmint oil -450 to -600
Terpene less lemon oil -50 to +20 Citronella oil, ceylon -90 to -180
Cinnamon oil 00 to -20 Citronella oil, Java -50 to +20
Refractive index

 It is the ratio between the velocity of light in air and the velocity in
the substance under test.
n = sin i/sin r = velocity of light in air/ velocity of light in the
test substance.
 It can be detected by Abbe refractometer or Leico Auto Abbe
refractometer.
– They measure the n with precision to the fifth decimal place
– The dark and colored samples are easily detected.
– The shadow-line location is determined by the instrument software
– No mechanical components are involved, thus reducing wear with time .
• It is particularly valuable for purity assessment of volatile oils
and fixed oils.
-Oils of Cassia 1.61; Lemon oil 1.474-1.476
-Cinnamon 1.573 – 1.600; Terpeneless lemon oil 1.475-1.485
-Cinnamon leaf 1.53;
Particle size determination

 Micromeritics
 Microscopy, sieving and Sedimentation-Andreasen apparatus
 Applications
- Size and surface area of a particle can be related in a significant way to the
physical, chemical, and pharmacological properties of a drug.
-Clinically, the particle size of a drug can affect its release from dosage forms
that are administered orally, parenterally, rectally and topically.
-The successful formulation of suspensions, emulsions,and tablets, from the
viewpoints of both physical stability and pharmacological response also depend on
particle size.
-In the area of tablet and capsule manufacture, control of the particle size is
essential in achieving the necessary flow properties and proper mixingof granules and
powders.
- Extraction procedures
ASH VALUES AND EXTRACTIVES

 The residue remaining after incineration of a vegetable drug is

the ash (inorganic) content of drug
 Total ash method is used to measure the total amount of
material remaining after incineration.
-Importance and indicates to some extent the amount of extra care taken in
the preparation of the drug
 Acid insoluble ash is the residue obtained after boiling the total
ash with dil. HCl and igniting the remaining insoluble matter.
-This usually consists mainly of silica and a high acid-insoluble ash in drugs
such as Senna, cloves, liquorice, Valerian and tragacanth indicates contamination
with earthy material.
- In case of ginger a minimum % of water-soluble ash is demanded.
 Water soluble ash is the difference in weight between total ash
and residue after treatment of total ash with water.
 Sulphated Ash involves treatment with H2SO4 & ignition at 600oC
 DETERMINATION OF EXTRACTABLE
MATTER-Hot extraction

 place 4 gms powdered material in a conical flask. Add water and

weigh to obtain total weight.
 Shake and allowed to stand for 1hr. attach the reflux condenser
and boil for 1hr.
 Readjust to the original weight with solvent. Shake and filter.
 Transfer the filter to a flat bottomed disk and evaporate to
dryness on a water bath.
 Dry at 105˚ c for 6hrs, cool and weigh immediately.
 Calculate the content of extractable matter in mg per g of air
dried material.
 E.g.
Gentian drug, liquorice Evaluated by % Water soluble
extractives
Valerian drug, asafoetida Evaluated by % of ethanol 70% extractive
ORGANOLEPTIC EVALUATION
-BITTERNESS VALUE

 The bitterness value is determined organoleptically by comparison

with a standard quinineHCl solution .
 This standard is relevant to Centaury, Gentian and Wormwood
 This drugs are used for their bitter effect (appetizer).
 The bitterness value is expressed as units equivalent to the
bitterness of a solution containing 1gm of quinine hydrochloride
in 2000ml.
 0.1gm of quinine hydrochloride is dissolved in 100ml drinking
water and the stock solution is prepared. Then it is diluted and
tested and compared with drug.
 Bitterness value in unit per gm = 2000*c
A*B
Where, A = concentration of stock solution; B = volume of test solution in
tube with threshold bitter concentration; C = quantity of quinine hydrochloride
in the tube with threshold bitter concentration
HAEMOLYTIC ACTIVITY

 Hemolytic activity of plant material is determined by comparison

with that of reference material, Saponin R, having hemolytic
activity of 1000units/g.
Method of preparation of standard: Fill a glass stopper flask to
1/10 of its volume with sodium citrate. Add sufficient volume of
blood freshly collected from healthy ox and shake. This can be
stored for about 8 days at 2-4̊ c. place 1ml of citrated blood in a
volumetric flask with phosphate buffer pH 7.4.
Haemolytic activity = 1000* a/b
Where, 1000 = defined haemolytic activity of Saponin standard
a = quantity of saponin standard that produce total
haemolysis (g)
b = quantity of plant material that produce total haemolysis
(g)
SWELLING INDEX

 Swelling index is defined as the volume in milliliters occupied by 1

g of a drug, including any adhering mucilage, after it has swollen in
an aqueous liquid for 4 h.
 Method: The 1 g of a drug is treated with 1 ml ethanol (96%) and 25
ml water in a graduated cylinder, shaken every 10 min for 1hr and
allowed to stand as specified.
Agar 10
Cetraria 4.5
Fenugreek 6
Fucus 6
Ispaghula husk 40
(0.1gm)
Ispaghula seed 9
FOAMING INDEX

 The foaming ability of an aqueous decoction of plant material

and their extracts is measured in terms of foaming index.

 Cosmetic interest

Astringency/Tannin content
– Tannins(eg. Tannic acid),Krameria, catechu, galls,
Aspidosperma, hamamelis, pomegranate rind, kinos

 The method refers to those polyphenols adsorbed by hide powder

and giving a color reaction with sodium phosphomolybdotungstate
reagent
pH Determination

 pH = -log (H3O+)
 The determination of pH can be made by means of any electrode
whose potential depends on the hydrogen ion activity
 The hydrogen and glass electrodes are typical pH electrodes.
 Two hydrogen electrodes can be combined to measure pH, one
serving as the indicating electrode and the other as the reference
electrode, although this arrangement is not generally used in practice.
 The calomel and the silver chloride electrode are more convenient as
reference electrodes and are typically used with commercial pH
meters.
 Importance:
-Physical, chemical and biological stability of crude drugs are
function of pH
-Qualitative and quantitative analysis
Microscopic Method of Analysis – Crude
Fibres

 Cell wall, cell shape and cell contents- which are of value in
identification and in the detection of adulteration

 Parenchymatous tissue, the epidermis and stomata, epidermal

trichomes, the endodermis, cork tissue, collenchyma, sclereids,
fibres(more resistant cellular material), xylem, phloem, secretory
tissues are important in identity and detection of purity.

 E.g. -wavy walled epidermal cells –stramonium;

-papillose epidermis- coca leaf;
-The lignified, thick walled pericyclic fibres, accompanied by a parenchymatous
sheath of cells containing prisms of calcium oxalate, constitute an important diagnostic
character of senna.
-Ginger rhizome which contain relatively large amounts of oleoresin and starch
Microscopic method of Analysis

 Types of Stomata: There are

several types of stomata,
distinguished by the form and
arrangement of the surrounding
cells.
- Anomocytic (irregular-
celled)
- Anisocytic (unequal-
celled)
- Diacytic (cross-celled)
- Paracytic (parallel-celled)
Microscopic method of Analysis:
Determination of Stomatal Index

 The stomatal index is the percentage of the number of stomata

formed by the total number of epidermal cells, including the stomata,
each stoma being counted as one cell.

S x 100
Stomatal index = ————
E+S

 Where S = the number of stomata in a given area of leaf ; and

 E = the number of epidermal cells (including trichomes) in the same
area of leaf.
- For each sample of leaf make not fewer than ten determinations and
calculate the average index.
Microscopic method of Analysis:
Determination of Palisade Ratio

 Palisade ratio is the average

number of palisade cells under
one epidermal cell.
 For each sample of leaf make
not fewer than ten
determinations and calculate
the average number
 18.4
Palisade ratio —— = 4.5
4
Microscopic method of Analysis:
Determination of Vein-Islet Number

 The mesophyll of a leaf is divided into small portions of

photosynthetic tissue by anastomosis of the veins and veinlets; such
small portions or areas are termed “Vein-Islets”.
 The number of vein-islets per square millimeter is termed the “Vein-
Islet number”.
 This value has been shown to be constant for any given species and,
for full-grown leaves, to be unaffected by the age of the plant or the
size of the leaves.
 The vein-islet number has proved useful for the critical distinction of
certain nearly related species.
Macroscopic, Microscopic and Chemical
test of Analysis With an Example
 Alexandrian senna
-Macroscopic: Seldom exceed 40 mm
in length, grayish – green; more
asymmetric at base;
-Microscopic: Hairs are more
numerous; most of the stomata have 2
subsidiary cells only; Vein-islet
number 25-29.5; stomatal index
usually 12.5
-Chemical Tests: Ether extract of
hydrolysed acid solution of drug gives
with methanolic magnesium acetate
solution:
A pink color in day light, a pale
greenish-orange in filtered UV light
 Tinnevelly senna
-Macroscopic: Seldom exceed 50 mm
in length; yellowish – green, less
asymmetric at base;
Microscopic: Hairs less numerous; the
stomata having 2 or 3 subsidiary cells;
Vein-islet number 19.5-22.5; Stomatal
index usually 17.5
-Chemical Test: Same as in A.senna
An orange color in daylight, a
yellowish-green in filtered UV light
Chemical Method of Analysis

 Water determination by Karl Fischer procedure

 Mechanism:
The reagent consists of a solution of iodine, Sulfur dioxide, and
pyridine in dry methanol. This is titrated against a sample
containing water , which causes a loss of the dark brown color. The
basic reaction is a reduction of iodine by sulfur dioxide in the
presence of water.
 This test is particularly applicable to expensive drugs and
chemicals containing small quantities of moisture.
 E.g. Dry extracts of alkaloid containg drugs, fixed oils ( arachis,
castor oil, olive oil, and sesame oil for parentaral use.
 For crude drugs such as digitalis and ipecacuanha moisture
contentis important.
Assays

Drugs which cannot be assayed by chemical, or physical means

are evaluated by biological methods.
 A crude drug may be assayed for a particular group of
constituents(e.g. The total alkaloids in belladonna or the total
glycosides of digitalis) or specific components ( e.g. reserpine
content of Rauwolfia spp.)
INDICATION FOR BIOLOGICAL EVALUATION
 This is true for the substances having an Interfering obstacles
 When quantity is too small.
 No specific chemical test is available
 When the action of drug is due to a mixture of substance
 Purification of drug is not possible
Assays

 Types of assay employed for crude drugs

Type of Assay Examples
Physical *Cineole in eucalyptus oil (f.p. of o-cresol complex)
*Separation and weighing of active constituents(e.g. Colchicine in
colchicum corm; total balsamic esters of peru balsam
Chemical ‘Total alkaloids’ of many drugs (e.g. acid-base titrations);Non-aqueous
titrations of alkaloid salts(e.g strychnine in nux vomica. Morphine in
opium)
Biological Cardio active drugs, antibiotics, vitamins, anthroquinone derivatives,
mydriatic drugs, saponins, antitumour drugs, antiamoebic drugs,
ginkgolides(anti-PAF activity
Radioimmun Cardinolides (Digitalis lanata); Sennosides(Cassia angustifolia) Morphine
oassay(RIA) (poppy capsules)
Enzyme Quassia, tropane alkaloids (solanaaceae) ;ergot alkaloids
-Immunoassa
y
Bio - Assays

 When the estimation of crude drug or its preparation is done by

means of its effect on living organism like bacteria, fungi, or
animal tissue or entire animal it is known as BIOASSAY.

TYPES OF BIOASSAY

 QUANTAL:-It is all or none phenomenon

 GRADED:-Based on observation that there is a proportionate
increase in the observed response with increase in concentration
or dose.
-Matching bioassay
-Interpolation Method
-Bracketing Method
-Multiple point bioassay
Process Validation and Drug Quality

FDA has the authority and responsibility to inspect and evaluate

process validation

 The cGMP regulations for validating pharmaceutical (drug)

manufacturing require that drug products be produced with a high
degree of assurance of meeting all the attributes they are intended to
possess .

Clinical Validation involve principles of Clinical Trials

Effective process validation contributes significantly to assuring

drug quality

The basic principle of quality assurance is that a drug should be

produced that is fit for its intended use
Process Validation and Drug Quality

Quality, safety, and efficacy are built into the product

Quality cannot be adequately assured merely by in-process and

finished-product inspection or testing

Each step of a manufacturing process is controlled to assure that the

finished product meets all quality
Classification of Pharmaceutical validation

 Class A: Tests designed to establish identity, whether of bulk drug

substance or of a particular ingredient in a finished dosage form.
 Class B: Method designed to detect and quantititative impurities in
bulk drug substances or finished dosage forms.
 Class C: Methods used to determine quantitatively the concentration
of the bulk drug substance or of major ingredient in a finished
dosage form.
 Class D: Method used to assess the characteristic of finished dosage
forms such as dissolution profile and content uniformity
 Analytical Parameters:
-Accuracy; -Precision; -Robustness
-Linearity & Range -Selectivity -Limit of detection
-Limit of quantification -Ruggedness
Approach to Process Validation

Process Validation is defined as a scientific evidence that a process

is capable of consistently delivering quality product

• Stage 1 – Process Design: The commercial manufacturing process

is defined during this stage based on knowledge gained through
development and scale-up activities.

• Stage 2 – Process Qualification: During this stage, the process

design is evaluated to determine if the process is capable of
reproducible commercial manufacturing.

• Stage 3 – Continued Process Verification: Ongoing assurance is

gained during routine production that the process remains in a state
of control.
Approach to Process Validation

 A successful validation program depends upon information and

knowledge from product and process development

 Understand the sources of variation

– Detect the presence and degree of variation

– Understand the impact of variation on the process and ultimately

on product attributes

– Control the variation in a manner commensurate with the risk it

represents to the process and product
 General Considerations for Process
Validation

 An integrated team approach - expertise from a variety of disciplines

 Throughout the product lifecycle, various scientific studies can be
initiated to discover, observe, correlate, or confirm information about the
product and process
 Validation that employs risk based decision making throughout that
lifecycle
 Consistency between batches are goals of process validation activities
 The plan should identify the following items:
-the studies or tests to use,
-the criteria appropriate to assess outcomes,
-the timing of qualification activities,
-the responsibilities of relevant departments and the quality unit, &
- the procedures for documenting and approving the qualification.
 General Considerations for Process
Validation

 A written protocol that specifies the manufacturing conditions,

controls, testing, and expected outcomes is essential for this stage of
process validation
-The manufacturing conditions, including operating parameters,
processing limits, and component (raw material) inputs.
-The data to be collected and when and how it will be evaluated.
-Tests to be performed (in-process, release, characterization) and
acceptance criteria for each significant processing step.
-The sampling plan, including sampling points, number of
samples, and the frequency of sampling for each unit operation and
attribute
-Criteria and process performance indicators that allow for a
science- and risk-based decision about the ability of the process to
consistently produce quality products
 General Considerations for Process
Validation

 Design of facilities and the qualification of utilities and equipment,

personnel training and qualification, and verification of material
sources (components and container/closures), if not previously
accomplished.

 Status of the validation of analytical methods used in measuring the

process, in-process materials, and the product.

 Review and approval of the protocol by appropriate departments and

the quality unit.

 Documentation at each stage of the process validation lifecycle is

essential for effective communication in complex, lengthy, and
multidisciplinary projects