Standardisation

Of
Herbal drugs
By
Dr. M. RAMAIAH,
M. Pharm., Ph.D., IPDRA, DICT(USA)
Professor & HOD
Department of Pharmacognosy&Phytochemistry
Hindu College of Pharmacy,
Guntur, A.P. India
In India, the herbal drug market is about $ one billion and the export of plant
based crude drugs is around $ 80 million
But the most important challenges faced by these formulations arise because
of their lack of complete standardization

The advent of herbal products in the form of standardized extracts was

initiated in 1992 as a result of a European Guaranteed Potency law

Herbal medicines are prepared from materials of plant origin which are prone
to contamination, deterioration and variation in composition. Therefore,
quality control of herbal medicines offers a host of problems. To solve this
problem, first and foremost task is the selection of the right kind of plant
material which is therapeutically efficacious.
Definitions
The process of evaluating the quality and purity of crude drugs by means of
various parameters like morphological, physical, chemical and biological
observations is called Standardisation.

A standardized extract means that the manufacturer has verified that the active
ingredient believed to be present in the herb is present in the preparation and that
the potency and the amount of the active ingredient is assured in the preparation.

Importance of Standardisation
✓ Role in identification of Botanical source
✓ Useful in identification of common adulterants
✓ Significant role in the quality evaluation of crude drugs originating
from different localities
✓ Determination of suitable time of harvest
✓ Role in specification of the conditions for drying, storage, and shelf-
life
 WHO & ICH guidelines for quality standardization of
herbal formulations

Botanical parameters

Organoleptic and Sensory evaluation: includes visual macroscopy/touch/odour/taste

Foreign matter: includes foreign plants, foreign animals, foreign minerals, etc
Microscopy: includes histological observation and measurements

Physico-chemical parameters

TLC/HPTLC finger print

Ash values: Total, acid-insoluble, water-soluble
Extractive values: in hot water, cold water, and ethanol
Moisture content and volatile matter: Loss on drying (LOD), azeotropic
distillation
Volatile oils: By steam distillation, Clavenger apparatus
Pharmacological parameters

Bitterness value
Haemolytic property
Astringent property
Swelling index
Foaming index

Toxicological parameters

Arsenic
Pesticide residues
Heavy metals
Microbial contamination
Aflotoxins
Radioactive contamination
Herbs: These are crude plant material such as leaves,
flowers, from seed, stem, wood, bark, roots, rhizomes or
other plant parts, which may be entire, fragmented or
powered.

Quality: It is status of the drug that is determined by

identity, purity, content & various chemical, physical &
biological properties.

Safety: to assure that drug has no side & hazardous effect.

Purity: Freedom from adulteration or contamination.

Potency: Amount of drug required to produce a given

percentage of its maximal effect, irrespective of the size of
maximal effect

Quality control: the term quality control refers o the sum of

all procedures undertaken to ensure the identity& purity of
a particular pharmaceutical.

Herbal medicine: These include herbs, herbal materials,

herbal preparations & finished herbal products.
Herbal materials: are either whole plant or part of
medicinal plants in the crude state. These include herbs,
fresh juices, gums, fixed oils, essential oil, resins & dry
powders of herbs.

Herbal preparations: These are finished herbal products

produced with extraction, distillation, expression,
fractionation, purification, concentration & fermentation.
 Botanical parameters

Organoleptic and Sensory evaluation:

Sensory evaluation of herbal drug refers to evaluation of drug by colour, odour, taste
including touch, texture etc. Since majority of information on the identity,
purity, & quality of material can be drawn from these observations. They are
primarily important before any other testing is carried out. It is a technique of
qualitative evaluation.
Eg:-
Aromatic odour : Umbelliferous fruits
Sweet taste : Liquorice
Pungent taste : Capsicum, ginger
Fractured surface : Wood & Barks ( quassia & cinchona, cascara,
quillaia)
Ovoid tears shape : Gum accacia
Ribbon shape : Tragacanth
Disc shape : Nuxvomica
Conical shape : Aconite
 Foreign matter
Drugs should be free from moulds, insects, animal
fecal matter, and other contaminants, like dust,
soil, and extraneous matter. Foreign matter
sometimes also consists of parts of the organs of
the plant other then that required for the drug by
by definition or beyond limits set by the WHO
guidelines. The amount of foreign matter should
not more than prescribed limit.

»»» 100-500 g of the crude drug sample should be

weighed, or a quantity prescribed by the WHO
guidelines may be used. Foreign matter may be
detected by inspection with unaided eye or by the
use of a lens of 6X power. Separate the foreign
matter and calculate the percentage present.
Microscopy

Microscopy allows more detailed examination of drug and it can be used to identify
the organised drugs by their known histological characters.
Microscope, by the virtue of its property to magnify, permits the minute structure
under study to be enlarged and can be used to conform the structural details of the
drugs from plant origin. For the effective results, various reagents or stains can be
used to distinguish cellular structure.

Leaf constants
palisade ratio
vein-islet number
vein-termination
stomatal number
stomatal index
 Trichomes

Covering glandular hydathodes or spcl type

Unicellular
unbranched
Multicultural uniseriate
branched biseriate

multiseriate
Stomata 1. Paracytic
Dicotyledonous 2. diacytic
Gramineous 3. anisocytic
Gymnospermous 4. anomocytic

Moss 5. actinocytic
 Physico-chemical parameters

Thin Layer Chromatography ( TLC )

Principle:
The main principle involved in TLC is adsorption. One or more components are
spotted on the thin layer of adsorbent coated on a chromatographic plate. The
mobile solvent flows through because of capillary action i.e. against gravitational
force. The components move according to their affinities towards the adsorbent.
The component with more affinity towards the stationary phase travels slower. The
component with lesser affinity towards the stationary phase travels faster.
Ash values

The ash of any organic material is composed of their non-volatile inorganic

components. Controlled incineration of crude drugs results in an inorganic material
( metallic salts and silica ). This value varies within fairly wide limits an is therefore
an important parameter for the purpose of evaluation of crude drugs.
.
The ash value can be determined by 3 different methods

Ash
values

Acid Water
Total
insoluble soluble
ash content
ash content ash content
Determination of Total ash
Ashing involves an oxidation of the components of the product. A high value is
indicative of contamination, substitution, adulteration, or carelessness in
preparing the crude drug for marketing.

Weigh accurately 2-3 g of the air dried crude drug in the tared platinum or
silica dish and incinerate at a temperature not exceeding 450° C until free from
carbon, cool and weigh. If a carbon free ash content cannot be obtained in this
way, exhaust the charred mass with hot water, collect the residue on an ashless
filter paper, incinerate the residue and filter paper until the ash is white or
nearly so. calculate the % of ash with reference to the air dried drug.
Determination of acid insoluble ash
Ash insoluble in Hcl is the residue obtained after extracting the sulphated or total
ash with Hcl, calculate with reference to 100 g of drug.
Boil the ash with 25 ml of hydrochloric acid for 5 min, collect insoluble
matter in a Gooch crucible or on an ashless filter paper, wash with hot water, ignite,
cool in a desicator and weigh. Calculate the percentage of acid-insoluble ash with
reference to the air dried drug.
This ash value particularly indicates contamination with silicious material e.g., earth
and sand, comparison of this with the total ash value of the same sample will
differentiate between contaminating material and variations of the natural ash of the
drug.
Determination of Water soluble ash

Water soluble ash is the part of the total ash content which is soluble in water. It
indicates incorrect preparation.
Boil the ash for 5 min with 25 ml of water. Collect the insoluble matter in a
Gooch crucible or an ashless filter paper, wash with hot water and ignite for 15 min
at a temp not exceeding 450° C. Substract the weight of the insoluble matter from
the weight of the ash; the difference of weight represents the water soluble ash.
Calculate the % of water soluble ash with reference to their air dried drug.
Eg:-

Drugs Total ash (% w/w) Acid insoluble ash (% w/w)

Agar --- 1.00
Aloe 5.0 ---
Ashoka 11.0 ---
Black catechu 6.0 ---
Cannabis 15.0 15.0
Cardamom 6.0 3.50
Clove 7.0 0.75
Determination of Extractable matter

This method determines the amount of active constituents in a given amount of

medicinal plant material when extracted with solvents. It is employed for that
material for which no chemical or biological assay method exist.
 Determination of water soluble extractive

5 g of the air-dried drug, coarsely powdered have to be macerated with 100 ml of

water in a closed flask for 24 hrs, soaking frequently during the first 6 hrs and
allowing to stand for 18 hrs. Thereafter, filter rapidly taking precautions against
loss of water, evaporate 25 ml of the filtrate to dryness in a tared flat-bottomed
shallow dish, dry at 105° C and weigh. The % of water-soluble extractive with
reference to the air dried drug have to be calculated.

Determination of alcohol soluble extractive

5 g of the air dried and coarsely powdered rug has to be macerated with 100 ml of
ethanol of the specified strength in a closed flask for 24 hrs, shaking frequently
during the first 6 hrs and allowing to stand for 18 hrs. Thereafter, filter rapidly
taking precautions against loss of ethanol. Evaporate 25 ml of the filtrate to
dryness in a tared float-bottomed shallow dish, dry at 105° C and weigh. The %
of ethanol soluble extractive with reference to the air-dried drug has to be
calculated.
Hexane soluble extractive
Weigh the 2 g of the powdered drug and subject to complete extraction in a
continuous extraction apparatus with help of solvent hexane for 20 hrs. Transfer
the hexane solution to a tared porcelain dish, and allow it to evaporate
spontaneously. Then dry it over phosphorus pentoxide for 18 hrs and weigh.
Calculate the % extractive from the weight of drug taken.
Volatile ether soluble extractive
Extract 2 g of the powdered drug, dried over phosphorus pentoxide for not less than
12 hrs and accurately weighed, with anhydrous ethyl ether in a continuous extractive
apparatus for 20 hrs. Transfer the ether solution to a tared porcelain dish, and allow to
evaporate spontaneously. Then dry over phosphorus pentoxide for 18 hrs, and weigh
the total ether extract. Heat the extract gradually, and dry at 105° C to constant
weight; the loss in weight represents the volatile portion of the extract.
Non -Volatile ether soluble extractive
Processed as directed under volatile ether soluble extractive. The weight of the
extract after drying in a dessicator at 105° C to constant weight represents the non-
volatile portion of the extract.
Eg:-

Water-soluble Alcohol-soluble Ether-soluble

Drugs extractive extractive extractive
% w/w % w/w % w/w
Aloe Not less than 25.0 Not less than 10.0 ---
Ginger Not less than 10.0 Not less than 04.5 ---
Linseed Not less than 15.0 --- Not less than 25.0
Senna leaves Not less than 30.0 --- ---

Capsicum --- --- Not less than 12.0

Nutmeg --- --- Not less than 12.0
 Determination of Moisture content

LOD method volumetric titrimetric

azeotropic distillation method Karl Fischer method

Determination of Volatile oil in crude drugs

In order to determine the volume of the oil, the plant material is distilled with water
and distillate is collected in a graduated tube. The aqueous portion separates
automatically and is returned to the distillation flask. If the volatile oil possess a mass
density higher than or near to that of water and/or are difficult to separate from the
aqueous phase due to the formation of emulsion, a solvent with a low mass density
and with a suitable boiling-point may be added to the measuring tube. The dissolved
volatile oils will then float on top of the aqueous phase.
 Pharmacological parameters
Determination of Bitterness value :
As per WHO, the bitter properties of plant material are determined by
comparing the threshold bitter concentration of an extract of the material with that of
a dilute solution of quinine hydrochloride. The bitterness value is expressed in Unit
equivalent to the bitterness of solution containing 1 g of quinine hydrochloride in
2000ml. In this determination safe drinking water is to be used as a vehicle for the
extraction of plant material and for the mouth wash after each testing.

The bitterness value has to be calculate in units per gram by using the following
formula,
2000 C
Bitterness value = A B

A = Conc. of the stock solution (mg/ml)

B = Vol of given plant material solution (in ml) in the test tube
C = Qty of quinine Hcl (in mg) in the test tube with the threshold bitter conc.
Determination of Haemolytic property :

The term saponin is derived from the Latin word sapo meaning soap. Plant
containing saponins has long been used for their detergent property. They are mostly
characterized by their frothing property and also the most characteristic property of
their ability to cause haemolysis, when added to a suspension of blood, saponins
produce changes in erythrocyte membranes, causing haemoglobin to diffuse in to
the surrounding medium. Many medicinal plant materials, especially those derived
from the families Caryophyllaceae, Araliaceae, Sapindaceae, Primulaceae, and
Dioscoreaceae contain saponins.

The Haemolytic activity of plant materials, or a preparation containing saponins, is

determined by comparison with that of a reference material, saponin, which has
Haemolytic activity of 1000 units per gram. A suspension of ox erythrocytes is mixed
with equal volumes of serial dilution of the plant extract. The lowest concentration, to
effect complete haemolysis is determined after allowing the mixtures to stand for a
given period of time. A similar test is carried out simultaneously with reference
saponin.
 1000  A
Haemolytic activity =
B

Where,
1000 = the defined haemolytic activity of saponin in relation to ox blood
A = qty of spn that produce total haemolysis (g)
B = qty of plant material that produce total haemolysis (g)
Determination of swelling index :

The dried ripe seeds of plantago ovata, P. psylium, P. arenaria, P. indica etc contain
mucilage in the epidermis of the testa. The seeds of such types of plant may be
evaluated by measuring the volume of mucilage produced in 24 hrs from 1 g of the
seeds. This evaluation procedure is termed as swelling factor. Many medicinal plant
material are of specific therapeutic utility because of their swelling properties,
especially gums and those containing an appreciable amount of mucilage, pectin or
hemi-cellulose.
The swelling index is the volume in ml taken up by the swelling of 1 g of plant
material under specified conditions. Its determination is based on the addition of water
or swelling agent as specified in the test procedure for each individual plant material.
Using a glass-stoppered measuring cylinder, the material is shaken repeatedly for 1 hr
and then allowed to stand for a required period of time. The volume of the mixture ( in
ml ) is then read. The mixture of whole plant material with the swelling agent is easy
to achieve, but cut or pulverized material requires vigorous shaking at specified
intervals to ensure even distribution of the material in the swelling agent.
Determination of foaming index :

Many medicinal plant material contain saponins that cause a persistent foam
when an aqueous decoction is shaken. The foaming ability of an aqueous decoction of
plant materials and their extract is measured in terms of foaming index.
Reduce about 1 g of the plant material to a coarse powder ( sieve size no.
1250 ), weigh accurately and transfer to a 500 ml conical flask containg 100 ml of
boiling water. Maintain at moderate boiling for 30 min. Cool and filter into a 100 ml
volumetric flask and add sufficient water through the filter to dilute to volume. Pour
the decoction into stoppered test tube and adjust the volume of the liquid with water to
10 ml and shake them for 15 sec and allow to stand for 15 min and measure height of
the foam.
If the height of the foam in test tube is less than 1 cm, the foaming index is less
than 100.
Foaming index = 1000
A

Where, A= the volume in ml of the decoction used for preparing the dilution in the
test tube, where foaming to a height of 1 cm is observed.
 Toxicological parameters
Determination of Arsenic and Heavy metals

The medicinal plant materials can be contaminated with arsenic and heavy metals
which can be attributed to many causes including environmental pollution and traces
of pesticides. As these components even in trace amounts are dangerous, they have
to be removed from the herbal drugs.
For determination, Limit tests have been prescribe in pharmacopoeia.

The current recommendations of the German Minister of Health regarding heavy

metals are as follows:
Lead : maximum 5.0 ppm
Cadmium : maximum 0.2 ppm
Mercury : maximum 0.1 ppm
Determination of Pesticide residues

Pesticide residues produce toxic effects like irritation of the eye, lacrimation,
salivation, sweating, blurring of vision, breathlessness and the systemic effects
includes hypotension, tachycardia, cardiac arrhythmias, vascular collapse, respiratory
paralysis, excitement, ataxia, convulsions. Hence the presence of these contaminants
in medicinal plants must be avoided.
Preparation of test sample
20-50 gm of powder is taken. Acetonitrile-water (650:350) is added and blended for 5
min at high speed and filtered. The filtrate is transferred to a liter separating funnel and 100 ml of
light petroleum ether is added. The contents are shaken for 1 or 2 min and 10 ml of sodium
chloride (400g/4 lit) and 600 ml of distilled water added.

The separating funnel is shaken vigorously for 30-45 sec, and the solvent layer allowed
to separate. The pet.ether layer should be collected, and washed with water thrice. This is treated
with anhydrous sodium sulphate. The extract is then subjected to column chromatography. The
column is packed with activated fluorosil and eluted with pet.ether. Three fractions of 200 ml each
are collected. The first elute contains chlorinated pesticides like alderin, benzene hexachloride,
DDT etc, while the second elute contains dieldrein and the 3 rd elute contains malathion. The
elutes are to be concentrated to 10 ml and then used for Thin Layer Chromatography.

Particulars for TLC

Standard samples : All the reference samples are prepared in pet.ether.
Adsorbent : Pre-coated silica gel G plate (10 20 cm) of uniform thickness (0.2 mm).
Solvent system : N-hexane: acetone (7:3).
Detection : under iodine treatment or UV chamber.
Determination of Microbial contaminants
Test for E. coli
A drug id tested for the presence of E. coli by taking 10 gms of powdered
material and making the volume up to 100 ml with lactose broth. This mixture
should be incubated for 4 hrs at 35-37°C. 1 ml of sample from this is taken and
serial dilutions are made with 9 ml of MacConkey broth of concentrations 100
mg/ml, 10 mg/ml, 0.1 mg/ml and 0.001 mg/ml. These samples are incubated at
37°C for 18 hrs. 1 ml of each is inoculate on MacConkey agar media, and further
incubated for 24 hrs at 35-37° C.
Colony characteristics
Growth of red, generally non-mucoid colonies of gram –ve rods indicting the
presence of E. coli.
Biochemical test
The colonies obtained on the MacConkey agar media are inoculated on peptone
water (5 ml) and the culture incubated for 24 hrs at a temperature of 35-37° C.
After the incubation period, 3 ml of peptone culture is to be pipetted into a test tube
and equal volume of Kovac’s reagent is added. Appearance of a pink ring is the
indication of presence of E. coli.
Test for salmonella
10 gm of powdered material is taken and the volume made upto 100 ml with
lactose broth. The mixture is incubated at 35-37° C for 4 hrs. A further 10 ml of this
sample is taken. 100 ml of tetra methylate brilliant green bile broth is added to it and
incubated at 37-40° C for 24 hrs. 1 ml of sample is taken from it and planted on
xylose lysin deoxycholate agar media. Deoxycholate citrate agar or brilliant green
agar can be used. This is incubated at 35° C for 50 hrs.
Colony characteristics
Small transparent and colourless with an opaque, pink zone indicated the presence
of S. typhi.

Biochemical test
The colonies obtained from the above culture are treated with polyvalent salmonella
antisera. This is known as serotyping or slide agglutination test. Appearance of
clumping of the cells confirms the presence of salmonella species.
Limits specified by WHO for the Microbial contamination in Herbal Drugs

The limits vary according to the use of the material

Plants material harvested under acceptable hygienic conditions:
Per gram maximum 104 cells E. coli
maximum 105 cells Mould propagules
Plant materials which are pre-treated or plant materials to b used for topical dosage forms:
Per gram maximum 107 cells Aerobic bacteria
maximum 103 cells Saccaromeycetes
maximum 102 cells E. coli
maximum 104 cells Other enterobacteria
Other plant material for internal use
Per gram maximum 105 cells Aerobic bacteria
maximum 103 cells Saccaromeycetes
maximum 101 cells E. coli
maximum 103 cells Other enterobacteria
 One example for standard monograph

Adhatoda vasica Nees.

a) Classification
Kingdom : Plantae
Division : Angiospermae
Class : Dicotyledoneae
Order : Tubiflorae
Family : Acanthaceae
Genus : Adhatoda
Species : Vasica Nees

(b) Parts used : Leaves

(c) Phytochemistry : Leaves contain vascine as the major bioactive

pyrralazoquinazoline alkaloid. Also contain other alkaloids viz., vasicol, adhatonine,
vasicinone, vasicinol, vasicinolone; aliphatic hydroketones viz., 37-
hydroxyhexatetracont-1-en-15 one and 37-hydroxy hentetracontan-19-one.
(d) Marker constituent : Vasicine

OH
N Mol. Formula : C11H12N2O
Mol.Wt. : 188.23
N

(e) Therapeutic use : Bronchodilator, expectorant

ANALYTICAL SPECIFICATIONS OF THE CRUDE DRUG

Macroscopic characters
Colour and Appearance : Leaves are simple, petiolate, broad, glabrous, and mostly
elliptic lanceolate to ovate lanceolate. They have slightly crenate margin. Dorsal
surface is green in colour but the ventral one is slightly pale. 8-12 pairs of bilateral
veins which are reticulate.
Taste : Bitter
Odour : Characteristic
 TESTS LIMITS PROTOCOLS
Tests for extraneous material
Foreign matter < 1.0%
Sand & Silica Absent
Insect infestation Nil
Rodent contamination Nil
Physico-chemical analysis WHO
Moisture content < 8.0 % w/w
Ash content < 15.0 %w/w
Acid insoluble ash < 2.0 % w/w
Alcohol soluble extractive value 8-12 % w/w
Successive extractive value
Petroleum ether extractive value 0.7-2.0 % w/w -----
Chloroform extractive value 0.7-2.0 % w/w -----
Methanol extractive value 6-11 % w/w -----
Phytochemical analysis
Vasicine content 0.4% w/w By HPLC
 IDENTIFICATION OF CRUDE DRUG BY TLC / HPTLC

Sample : Adhatoda vasica (Leaves)

Solvent system : 1,4-dioxane : Ammonia solution

Sample preparation : Powdered sample extracted with methanol. The

extract concentrated and the residue dissolved in

methanol.

Detection : Dragendroff reagent (Fig. 1)

Densitometer scan : 254 nm (Fig. 2)

TLC HPTLC

Fig. 1 Fig. 2
S = Vasicine (standard) Rf 0.58
T = Test sample
conclusion
 Authentication

Radio active
contaminants Foreign
organic matter

Microbial Organoleptic
count evaluation

STANDARDIZATION
OF
Pesticide Macroscpoy
residue HERBAL DRUGS and
Microscopy

Marker Volatile matter

component

Chromatograph Ash value

ic profile
Extractive
value
It is estimated that the total value of products from the entire
Ayurvedic production in India is on the order of one billion
dollars (U.S.)

There are today 30 companies doing a million dollars or more

per year in business to meet the growing demand for Herbal
medicine
Some of the standardized extracts manufactured from the Top most
Herbal Companies
 Common Standardized Extract
S.no Name Biological Source Composition Therapeutic Use
1.0 % Vasicine, Respiratory support,
1. Vasaca Adhatoda vasica 2.0% total alkaloids cough-cold aid

2. Amla Emblica officinalis 40% tannins Rejuvenating agent

Andrographis
3. Kalmegh paniculata 10% andrographolides Hepatoprotectant

1.5% withanolides,
4. Ashwagandha Withania somnifera 1.0% alkaloids Adaptogen

5. Shatavari Asparagus racemosus 2.5% shatavarin II & IV Adaptogen

6. Jala Bramhi Bacopa moniera 20% bacosodes A&B Memory support

70% total organic acids, Anti-inflammatory,

7. Boswellia Boswellia serrata 40%β-boswellic acid Arthritis support

8. Senna Cassia angustifolia 20% total seenosides Laxative action

Pain reliever,
9. Capsicum Capsicum annum 75% capsaicinoids Carminative

Skin health, Nervine

10. Bramhi Centella asiatica 8% triterpenes tonic
Thank you