SCREENING OF HERBAL DRUGS

Definition
 Herb is a plant or a part of a plant valued for its
medicinal, aromatic or savoury qualities.
 Herbal drugs - Finished, labelled medicinal products

that contain as active ingredients aerial or

underground parts of plants, or other plant material,
or combinations thereof, whether in the crude state
or as plant preparations.
 Plant material includes juices, gums, fatty oils,

essential oils, and any other substances of this

nature.
 Pharmacognosy is the scientific study of drugs from

natural products.
 Herbal medicine or herbalism is the use of
herbs or herbal products for their therapeutic
or medicinal value.
 Herbal medicines may contain excipients in

addition to the active ingredients.

 Medicines containing plant material

combined with chemically defined active

substances are not considered to be herbal
medicines.
Identified Actives
 Activity fully attributed to compound or class

 Sennosides in senna

Co-actives give some activity, not all

 Salicins in willow do not act alone

Markers strictly speaking are inactive

 No activity of the extract is attributable

 Agnuside in chaste tree

Recent resurgence of interest in plant
remedies
 The effectiveness of plant medicines.
 The preference of consumers for natural therapies,
erroneous belief that herbal products are superior.
 A dissatisfaction with the results from synthetic drugs
 The high cost and side effects of most modern drugs.
 Improvements in the quality, efficacy, and safety of
herbal medicines
 Patients’ belief that their physicians have not properly
identified the problem; hence they feel that herbal
remedies are another option.
 A movement towards self-medication.
Two main approaches

 Random collection and screening of material

eg paclitaxel, vincristine

 Ethnopharmacological knowledge eg
artemisinin
Phytochemical screening involves
 Botanical Identification and selection of medicinal
plants for the proposed Phytochemical Tests

 Extraction of the components (using Soxhlet

extractor and cold percolation techniques)

 Separation of the constituents (using different

chromatographic techniques)

 Identification and quantification of the compounds

with the aid of thin layer chromatography (TLC)

 Subject extract to pharmacologically relevant assays

Botanical Identification
 Botanical identity like phytomorphology,
microscopical and histological analysis
(characteristic of cell walls, cell contents,
starch grains, calcium oxalate crystals,
trichomes, fibers, vessels etc)
 Various histological parameters:
 Leaf constant: - Palisade ratio, Vein islet

number, Vein termination, Stomatal number,

and Stomatal index.
 Name of the plant (English, Regional names, Exact
botanical nomenclature)
 Part of the plant used
 Area of collection
 Distribution details
 Season of Crop
 Time and year of collection
 Pesticide and insecticides
 Condition of the plant (fresh or dry)
 Form of the plant (powdered or intact or cuttings

like etc.)
GENERAL METHODS OF EXTRACTION
Maceration
 Powdered crude material is placed in a

stoppered container with the solvent and

allowed to stand for a period of 24 to 48 hours
with frequent agitation until soluble matter is
dissolved.
 The mixture is then strained, the marc ( the

damp solid material) pressed and may be

macerated again.
 Liquid is clarified by filtration or decantation

after standing
Advantages
 Simple and cheap
 Solvent use is limited
 Can give good and selective extraction

Disadvantages
 Analytes must be sufficiently soluble without

stirring/ slow heating

Percolation
 Performed in a cone shaped vessel, (called
percolator) the narrow side of which is fitted
with a suitable filter and a stopcock,
 Plant material allowed to macerate with the

specified menstrum for a specified period in

the percolator after which the stopcock is
opened so that the percolate drips slowly
from the bottom.
 Plant material is continuously flushed with

fresh solvent
Advantages
 Exhaustive extraction possible
 Mild technique

Disadvantages
 Supervision is necessary
 Slow
 Large amounts of solvent required
Hot continuous extraction (Soxhlet
ext.)
 Method involves continuous extraction by
boiling organic solvents.
 Desired compound has a limited solubility in

a solvent, and the impurity is insoluble in that

solvent
 Advantage - just one batch of solvent is

recycled
Advantages
 Minimum solvent consumption
 Not much supervision is necessary

Disadvantages
 Harsh technique causing thermal

decomposition
 Water and electricity is needed
 Less suitable for large scale operation
 1: Stirrer bar 2: Still pot
(the still pot should not be
overfilled and the volume
of solvent in the still pot
should be 3 to 4 times the
volume of the soxhlet
chamber) 3: Distillation
path 4: Thimble 5: Solid 6:
Siphon top 7: Siphon exit
8: Expansion adapter 9:
Condensor 10: Cooling
water in 11: Cooling water
out
Supercritical Fluid Extraction

 Solvent is replaced with supercritical fluid

Advantages
 Mild technique
 Environment friendly and cheap
 Can give fast extractions
 Extraction can be automated
 With 100 % CO2, there is no need for solvent

removal
 Solvating power variable via pressure
Disadvantages
 Less suitable for very polar products
 Rather complex apparatus is needed
 Involves high pressure
 Less suitable for extraction of leaves
 Fresh plant materials are difficult to extract
Steam distillation
 For temperature sensitive materials like
natural aromatic compounds
 When a mixture of two practically immiscible

liquids is heated, boiling points of the

compounds are depressed, allowing them to
evaporate at lower temperatures
 After distillation the vapors are condensed,

yielding water and the organic compounds.

Advantages
 Selective for volatile oils eg essential oils
 Very simple and cheap technique
 Only water is needed as solvent
 Well suitable for large scale extractions
 Very clean extracts
Disadvantages
 Only suitable for polar volatile oils
 Possible decomposition due to presence of

water and high temperatures

 Quantitative distillation is time-consuming
Constituents of extracts
 Flavonoids
 Terpenes
 Steroids
 Glycosides
 Amines
 Alkaloids
 Antibacterial
 Mycotoxins
 Saponins
 Quinones
 Tannins
 Phenolic compounds
 Fats
 Carbohydrates
 Proteins
 Oleoresins
 Essential oils
Fractionation
 The extracts are then subjected to
fractionation by partition among solvents of
different polarities or can be directly put to
chromatographic separation
CHROMATOGRAPHIC PROCEDURES
 Feature common to all chromatographic
methods is the use of two phases, one
stationary and the other mobile.
 Chromatographic methods may be classified

according to the nature of the stationary phase,

which may be a solid or liquid.
 If the stationary phase is a solid the method is

known as adsorption chromatography;

 if a liquid, as partition chromatography.

 the mobile phase may be either a liquid or a gas

Thin Layer (and Paper) Chromatography
TLC plates are inert supports (glass, plastic,
aluminium) with a thin veneer of
chromatographic media (silica)
• Apply a concentrated drop of sample (•)
with a capillary or dropping tube to bottom
of plate

• Stand plate in a sealed vessel.

• carefully add solvent
(keep solvent level below sample).

• • Allow solvent to adsorb up the plate,

drawing the sample with it.
COMMON ADSORBENTS FOR
CHROMATOGRAPHY
Fluorescence quenching
 When a plant extract is spotted on a fluorescent silica
gel layer and exposed to UV light, it appears as spot
on a fluorescent background, thus causing quenching
and is directly proportional to concentration of the
extract.

 Silica gel GF plate is used as an adsorbent for

fluorescence quenching.

 Solvents taken- hexane toluene, ether, ethyl acetate,

butanol, methanol and water
Structure elucidation
•1930's UV (ultraviolet) light
•1940's IR (infrared) spectroscopy
•1950's NMR (nuclear magnetic resonance)
spectroscopy
•1960's MS (mass spectrometry)
•ESR (electron spin resonance)
•ORD (optical rotatory dispersion)
•CD (circular dichroism)
•Advanced synthetic and biosynthetic technology
•X-ray crystallography
EXAMPLES
Zingiber officinale (ginger)
• Extract effective in the treatment of
inflammations of knee joints, motion sickness

• Extract prepared using acetone as solvent

and by percolation
• Column chromatography on silica gel
• HPLC analysis of the extract and fraction
HPLC fingerprint of Zingiber officinale
extract
LCMS of Column Fraction Of Ginger
Extract
NMR & MS Spectra of Gingerol -6
Screening for activity
 Example of antidiabetic
activity of herbal extract
V. RAJESH, P. PERUMAL & T. SUNDARRAJAN :

ANTIDIABETIC ACTIVITY OF
METHANOLIC EXTRACT OF SMILAX
ZEYLANICA LINN IN STREPTOZOTOCIN
INDUCED DIABETIC RATS.

The Internet Journal of Endocrinology. 2010

Volume 6 Number 1
Materials and Methods
Plant material:
 Fresh leaves were collected from tropical area

in Yercaud and authenticated by horticulturist

Extraction:
 Shade dried, Powdered to obtain coarse

powder and then passed through 60 mesh

sieve.
 Continuous hot extraction.
 The extracts were evaporated to dryness
 Phytochemical screening were performed
Animals
 Healthy adult male Wistar rats between 2- 3m
of age and weighing about 150-200 g
 The animals were housed in polypropylene

cages, maintained under standard conditions

(12 h light: 12 h dark cycle; 30◦C; 35-60%
humidity).
 They were fed with standard rat pellet diet

and water ad libitum.

Acute Toxicity Study
 Normal healthy rats were divided into five
groups of six animals each.
 Different doses (100, 250, 500, 750 and

1000 mg/kg body weight) administered

orally.
 Observed continuously for 2 h for behavioral,

neurological, and autonomic profiles and

after 24 and 72 h for any lethality
INDUCTION OF DIABETES
 Fasted overnight
 Intra peritoneal injection of streptozotocin

dissolved in 0.1M sodium citrate buffer pH4.5

at the dose of 50mg/kg body weight.
 Diabetes was confirmed after 48hrs
 Fasting blood glucose level more than

200mg/dl were considered as diabetic rats

and used for the experimentation.
 Grouped five days after induction of diabetes
Groups
 I: Normal control
 II: Diabetic control
 III: Diabetic rats methanolic extract

400mg/kg
 IV: Diabetic rats Glibenclamide 0.5mg/kg

[orally once a day for 15 days]

Sample collection
 Fasted overnight
 Tail vein sample on the 5th day, 15th day and

20th day post induction to determine blood

glucose by Glucose kit method.
Results
 Preliminary phytochemical studies -
phytosterols, flavonoids and glycosides in
methanolic extract of Smilax zeylanica leaf

 Acute toxicity study – no lethality up to

1000mg/kg.

 Oral leaf extract 400mg/kg body weight-

significant decrease (P<0.05) in blood
glucose level in 15 days of treatment
Several problems not applicable to synthetic drugs
influence the quality of herbal drugs

1. Herbal drugs are usually mixtures of many constituents.

2. The active principle(s) is (are), in most cases unknown.
3. Selective analytical methods or reference compounds
may not be available commercially.
4. Plant materials are chemically and naturally variable.
5. The methods of harvesting, drying, storage,
transportation, and processing (for example, mode of
extraction and polarity of the extracting solvent,
instability of constituents, etc.) have an effect.
STANDARDIZATION
 Standardization of drug means confirmation of its identity
and determination of its quality and purity and detection
of nature of adulterant by various parameters like
morphological, microscopical, physical, chemical and
biological observations.
WHO Guidelines For Herbal Drug Standardization

a. Quality control of crude drugs material,

plant preparations and finished products.
b. Stability assessment and shelf life.
c. Safety assessment; documentation of safety
based on experience or toxicological studies.
d. Assessment of efficacy by ethnomedical
informations and biological activity
evaluations
1.Authentication (Stage of collection, parts of the
plant collected, regional status, botanical
identity like phytomorphology, microscopical
and histological analysis, taxonomical identity,
etc.)
2.Foreign matter (herbs collected should be free
from soil, insect parts or animal excreta, etc.)
3.Organoleptic evaluation (sensory characters –
taste, appearance, odor, feel of the drug, etc.)
4.Tissues of diagnostic importance present in the
drug powder.
5. Ash values.
6.Volatile matter
7.Moisture content determination
8. Chromatographic and spectroscopic
evaluation. TLC,HPTLC, HPLC methods
-qualitative and semi quantitative

9. Determination of heavy metals – e.g.

cadmium, lead, arsenic, etc.

10. Pesticide residue –DDT, BHC, toxaphene,

aldrin

11. Microbial contamination

 Different techniques involved in
standardization of crude drugs
 Macroscopic methods
 Microscopic methods
 Physical methods
 Chemical methods
 Biological methods
Macrosopic examination
Organoleptic evaluation
 Colour, odour, size, shape, taste and special
features including touch, texture etc
 Authentic specimen of the material under

study and samples of pharmacopoeial quality

should be available to serve as a reference
 Simple, quick
 No preliminary treatment is necessary
 May vary from person to person and time to

time
Microscopic evaluation
 To ensure that the plant is of the required
species, and that the right part of the plant is
being used
 Pollen morphology may be used in the case of

flowers to identify the species, and the

presence of certain microscopic structures
such as leaf stomata can be used to identify
the plant part used
 Stinging nettle (Urtica urens) is a classic

example where the aerial parts are used to

treat rheumatism, while the roots are applied
for benign prostate hyperplasia
PHYSICAL STANDARDIZATIONOF HERBAL DRUGS:

 Viscosity
 Melting point
 Solubility
 Moisture content and volatile matter
 Specific gravity
 Density
 Optical rotation
 Refractive index
 Bitterness value
 Hemolytic activity
 Swelling index
 Foaming index
 Ash value
 Astringency
VISCOSITY
 Viscosity of a liquid is constant at a given temperature and
is an index of its composition. Hence, it can be used as a
means of standardizing liquid drugs.

MELTING POINT
 In case of pure photochemical, melting points are very

sharp and constant.

 The crude drugs from plant or animal origin, containing

the mixed chemicals, are described with certain range of

melting point.
 Their purity can be ascertained by determining their

melting points in that range

 for E.g. Cocoa butter- 30-33˚c
SOLUBILTY
 The presence of adulterant could be indicated by solubility
studies
 E.g.pure Asafoetida is soluble in carbon disulphide

MOISTURE CONTENT AND VOLATILE MATTER

 The moisture content of the drug should be minimized in

order to prevent decomposition of crude drug either due to

chemical change or microbial contamination.
 The moisture content is determined by heating a drug at 105˚c

in an oven to a constant weight.

 For the drugs containing volatile constituents, toluene

distillation method is used

 E.g. – Aloe should have moisture content not more than 10%
OPTICAL ROTATION
 Optically active compounds have the property of rotating the
plane of polarized light. This property is known as optical
rotation.
 Normally, the optical rotation is determined at 25˚c using
sodium lamp as the source of light.
 E.g. castor oil has optical rotation from +3.5˚to +6˚

REFRACTIVE INDEX
 When a ray of light passes from one medium to another of
different density, then the ratio of velocity of light in
vacuum to its velocity in substance is termed as refractive
index of second medium.
 It is constant for a pure drug and varied with wavelength of
incident light, temperature and pressure
 E.g. Castor oil has refractive index 1.4758-1.527
ASH VALUES AND EXTRACTIVES
 The residue remaining after incineration is the ash content
of drug
 Total ash method is used to measure the total amount of
material remaining after incineration
 Acid insoluble ash is the residue obtained after boiling the
total ash with dil. HCl and igniting the remaining insoluble
matter.
 Water soluble ash is the difference in weight between total
ash and residue after treatment of total ash with water.
BITTERNESS VALUE
 Medicinal plants having strong bitter taste are therapeutically used as
appetizing agents
 The bitterness is determined by comparing the threshold bitter
concentration of an extract material with that of quinine hydrochloride
 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

 Haemolytic activity of plant material is determined by

comparison with that of reference material, Saponin R,
having haemolytic activity of 1000units/g.
 

Haemolytic activity = 1000* a/b

Where, 1000 = defined haemolytic activity of Saponin standard
a = quantityof saponin standard that produce total
haemolysis (g)
b = quantity of plant material that produce total
haemolysis (g)
SWELLING INDEX
 The swelling index is the volume in ml taken up by the

swelling of 1gm of plant material under specified conditions.

Its determination is based on addition of water or a swelling
agent

FOAMING INDEX:
 The foaming ability of an aqueous decoction of plant material

and their extracts is measured in terms of foaming index.

 
 
CHEMICAL METHODS OF STANDARDIZATION OF HERBAL DRUGS:

 It comprises of different chemical tests and assays. The

isolation, purification and identification of active
constituents are chemical methods of evaluation.
 Quantitative , Qualitative chemical tests are used in
detection of adulteration.
 The chemical evaluation also covers phytochemical
screening carried out for establishing chemical profile of a
drug.
BIOLOGIAL/ TOXICOLOGIAL STANDARDIZATION

 Drugs which cannot be assayed by chemical, or

physical means are evaluated by biological
methods

INDICATION FOR BIOLOGICAL EVALUATION

 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
TOXICOLOGICAL STANDARDIZATION
 Determination of pesticides.
 Determination of heavy metals
 Determination radioactive contamination
 Determination of aflatoxins.
Determination of pesticides

 Pesticide residues - spraying, treatment of

soils during cultivation, and administering of
fumigants during storage

 Test herbal drugs for broad groups

 Samples of herbal material are extracted by a

standard procedure and individual pesticides
are measured by GC, MS, or GC/MS.
Maximum residue limits
MRL = ADI*E*60
MDI*100

 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
Determination of heavy metals
 Mercury, lead, copper, cadmium, and arsenic
- accidental or intentional.
 Determination is based on color reactions

with reagents eg, thioacetamide or

diethyldithiocarbamate and the amount
present is estimated by comparison with a
standard.
 Trace quantities, admixture, or quantitative -

Instrumental analyses (NAA, AAS)

Permissible limit of heavy metals
 Lead 10 ppm
 Cadmium 0.30 ppm
 Arsenic 10 ppm
 Mercury 1 ppm
Radioactive contamination
 The exposure cannot be avoided because of
naturally occurring sources including
radionucleotides occurring in ground and
atmosphere

Health risk depend on

 Specific radionucleotides
 Level of contamination
 Quantity of drug
 No limits are proposed for radioactive

contamination
 Aflatoxins
 Aflatoxins are the poisonous substance in the
spores of the fungus Aspergillus flavus

 The toxin is known to produce cancer in

human beings living in warm and humid region
of the world

 Stored nuts and cereals are contaminated by

the fungus

.
TESTS FOR SPECIFIC MICROORGANISMS
 Bacteria and molds originating in the soil
 Poor methods of harvesting, cleaning, drying,

handling, and storage

 Total aerobic microbial count, the total fungal

count, and the total Enterobacteriaceae count

 Presence of Escherichia coli, Staphylococcus

aureus, Shigella, and Pseudomonas

aeruginosa and Salmonella spp.
Conclusion
Thank you