Quality Control of Herbal Drugs

Introduction:
The term “herbal drugs” denoted by means of plant or part of plants that have been converted
into phytopharmaceuticals by simply means of processes involving collection or harvesting,
drying and storage. Medicinal plants have play an important role in world health. They are
circulated world-wide, but they are most rich in tropical countries. It is noted that about 25%
of all modern medicines are indirectly or directly obtained from higher plants. The use of
herbal drugs as medicine is the ancients form of healthcare known to delicacy and it is used
in all cultures throughout history.
Herbal drugs are the main constituent in usual medicine and a general ingredient in
Homeopathic, Ayurvedic, Naturopathic and in other medicine system. World Health
Organization (WHO) find out that 80% of the world people currently use herbal medicine or
drugs for the most important health cares. WHO has individual herbal drugs as whole,
labelled medicinal products that have robust ingredients, aerial or secret parts of the whole
plant or other plant material or mixture of them.
World Health Organization (WHO) has a set of specific Guidelines for the evaluation of the
safety, efficacy and Quality of herbal drugs or herbal medicines. The identification of purely
active moiety is an important requirement for quality control and dose determination of plant
related dugs. Standardization of herbal drugs means confirmation of its identity, Quality and
purity.

Classification of herbal medicines:

According to WHO herbal medicines can be classified into four categories, based on their
origin, evolution and the forms of current usage. While these are not always mutually
exclusive, these categories have sufficient distinguishing features for a constructive
examination of the ways in which safety, efficacy and quality can be determined and
improved.
1. Indigenous herbal medicines
This category of herbal medicines is historically used in a local community or region and is
very well-known through long usage by the local population in terms of its composition,
treatment and dosage. It can be used freely by the local community or in the local region.
However, if the medicines in this category enter the market or go beyond the local
community or region in the country, they have to meet the requirements of safety and
efficacy laid down in the national regulations for herbal medicines.
2. Herbal medicines in systems
Medicines in this category have been used for a long time and are documented with their
special theories and concepts, and accepted by the countries. For example, Ayurveda, Unani
and Siddha would fall into this category.
3. Modified herbal medicines
These are herbal medicines as described above in categories 1 and 2, except that they have
been modified in some way–either shape, or form including dose, dosage form, mode of
administration, herbal medicinal ingredients, methods of preparation and medical indications.
They have to meet the national regulatory requirements of safety and efficacy of herbal
medicines.
4. Imported products with a herbal medicine base
This category covers all imported herbal medicines including raw materials and products.
Imported herbal medicines must be registered and marketed in the countries of origin. The
safety and efficacy data have to be submitted to the national authority of the importing
country and need to meet the requirements of safety and efficacy of regulation of herbal
medicines in the recipient country.

Advantages and Disadvantage of Herbal Medicines:

Advantages :

1. Herbal medicines can treat minor conditions like scrapes, rashes and burns.
2. Effective with chronic condition.
3. They can also be used to treat migraines, arthritis and depression at a very low cost.
4. The cost of herbal medicines are very low compared to pharmaceutical drugs because
they can be found in local supermarkets or grown at home. According to Christopher
Golden from Harvard University Center for the Environment, if people used herbal
medicines in place of pharmaceuticals, they could save themselves 22-63% of what
they spend on healthcare annually.
5. They may have fewer side effects.
6. Effective with chronic condition.
Disadvantages:

Herbal Medicines may come with many advantages. But it also comes with a set of
disadvantages as well.

1. Herbal medicines take a longer time to work compared to pharmaceutical drugs. If an

individual decides to take the herbal alternative to pharmaceuticals, he or she must be
very patient.
2. Herbal medicines are often self-administered. As a result, there is no dosage or
warnings specified.
3. Inappropriate for many conditions.
4. When Herbal medicines are consumed with pharmaceutical drugs, the two can
interact with each other resulting in injuries to health.
5. Poison risk associated with wild herbs. It may be the case where a certain part of a
plant may be edible and another part may be poisonous. Take rhubarb for example.
The roots of rhubarb are used as a laxative and the stem is edible. However, its leaves
are poisonous. An individual may not be able to identify a poisonous plant. This
would put the individual at the risk of poisoning themselves or others.

Extraction of Herbal drugs:

Extraction, as the term is used pharmaceutically, involves the separation of medicinally active
portions of plant or animal tissues from the inactive or inert components by using selective
solvents in standard extraction procedures.

Extraction techniques of Medicinal plants:

The general techniques of medicinal plant extraction include maceration, infusion,

percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic
extraction by fermentation, counter current extraction, microwave-assisted extraction,
ultrasound extraction (sonication), supercritical fluid extraction, and distillation techniques
(water distillation, steam distillation, phytonic extraction (with hydro fluorocarbon solvents).

For aromatic plants, hydro water and steam distillation), hydrolytic maceration followed by
distillation, expression and effleurage (cold fat extraction) may be employed. Some of the
latest extraction methods for aromatic plants include headspace trapping, solid phase micro
extraction, protoplast extraction, micro distillation.

Parameters for Selecting an Appropriate Extraction Method:

i) Authentication of plant material should be done before performing extraction.

Any foreign matter should be completely eliminated.

ii) Use the right plant part and, for quality control purposes, record the age of plant
and the time, season and place of collection.

iii) Conditions used for drying the plant material largely depend on the nature of its
chemical constituents. Hot or cold blowing air flow for drying is generally
preferred. If a crude drug with high moisture content is to be used for extraction,
suitable weight corrections should be incorporated.

iv) Grinding methods should be specified and techniques that generate heat should be
avoided as much as possible.

v) Powdered plant material should be passed through suitable sieves to get the
required particles of uniform size.

vi) Nature of constituents:

a) If the therapeutic value lies in non-polar constituents, a non-polar solvent may

be used.

b) If the constituents are thermolabile, extraction methods like cold maceration,

percolation and CCE are preferred. For thermostable constituents, Soxhlet
extraction (if nonaqueous solvents are used) and decoction (if water is the
menstruum) are useful.
 c) Suitable precautions should be taken when dealing with constituents that
degrade while being kept in organic solvents, e.g. flavonoids and phenyl
propanoids.

d) In case of hot extraction, higher than required temperature should be avoided.

Some glycosides are likely to break upon continuous exposure to higher
temperature.

e) Standardization of time of extraction is important, as:

• Insufficient time means incomplete extraction.

• If the extraction time is longer, unwanted constituents may also be extracted.

For example, if tea is boiled for too long, tannins are extracted which impart
astringency to the final preparation.

f) The number of extractions required for complete extraction is as important as

the duration of each extraction.

Choice of solvents:
For Successful determination of biologically active compounds from plant material is largely
dependent on the type of solvent used in the extraction procedure.
A property of a good solvent in plant extractions includes:
 Low toxicity
 Ease of evaporation at low heat
 Promotion of rapid physiologic absorption of the extract
 Preservative action
 Inability to cause the extract to complex or dissociate
The factors affecting the choice of solvent are:
 Quantity of phytochemical to be extracted
 Rate of extraction
 Diversity of different compounds extracted
 Diversity of inhibitory compounds extracted
 Ease of subsequent handling of the extracts
 Toxicity of the solvent in the bioassay process
 Potential health hazard of the extractants
Solvents used for active component extraction: Water, Ethanol, Methanol, Chloroform,
Ether, and Acetone.

Water Ethanol Methanol Chlorofo Ether Aceton

rm e
 Anthocya Tannins Anthocyani Terpenoi Alkaloid Phenol
nins Polyphenol ns ds s Flavon
Starches s Terpenoids Flavonoi Terpeno ols
Tannins Polyacetyle Saponins ds ids
Saponins nes Tannins Coumari
Terpenoid Flavonols Xanthoxylli ns
s Terpenoids nes Fatty
Polypeptid Sterols Totarol acids
es Alkaloids Quassinoid
Lectins s
Lactones
Flavones
Phenones
Polyphenol
s
Table: Solvents used for active component extraction

Extraction procedures
a) Plant tissue homogenization: Plant tissue homogenization in solvent has been widely
used by researchers. Dried or wet, fresh plant parts are grinded in a blender to fine
particles, put in a certain quantity of solvent and shaken vigorously for 5 - 10 min or left
for 24 h after which the extract is filtered. The filtrate then may be dried under reduced
pressure and redissolved in the solvent to determine the concentration. Some researchers
however centrifuged the filtrate for clarification of the extract.
b) Serial exhaustive extraction: It is another common method of extraction which involves
successive extraction with solvents of increasing polarity from a non-polar(hexane) to a
more polar solvent (methanol) to ensure that a wide polarity range of compound could be
extracted. Some researchers employ soxhlet extraction of dried plant material using
organic solvent. This method cannot be used for thermolabile compounds as prolonged
heating may lead to degradation of compound.
c) Soxhlet extraction: Soxhlet extraction is only required where the desired compound has
a limited solubility in a solvent, and the impurity is insoluble in that solvent. If the desired
compound has a high solubility in a solvent then a simple filtration can be used to
separate the compound from the insoluble substance. The advantage of this system is that
instead of many portions of warm solvent being passed through the sample, just one batch
of solvent is recycled. This method cannot be used for thermolabile compounds as
prolonged heating may lead to degradation of compounds.
d) Maceration: In maceration (for fluid extract), whole or coarsely powdered plant-drug is
kept in contact with the solvent in a stoppered container for a defined period with
frequent agitation until soluble matter is dissolved. This method is best suitable for use in
case of the thermolabile drugs .
e) Decoction: this method is used for the extraction of the water soluble and heat stable
constituents from crude drug by boiling it in water for 15 minutes, cooling, straining and
passing sufficient cold water through the drug to produce the required volume.
f) Infusion: It is a dilute solution of the readily soluble components of the crude drugs.
Fresh infusions are prepared by macerating the solids for a short period of time with
either cold or boiling water.
g) Digestion: This is a kind of maceration in which gentle heat is applied during the
maceration extraction process. It is used when moderately elevated temperature is not
objectionable and the solvent efficiency of the menstruum is increased thereby.
h) Percolation: This is the procedure used most frequently to extract active ingredients in
the preparation of tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel
open at both ends) is generally used. The solid ingredients are moistened with an
appropriate amount of the specified menstruum and allowed to stand for approximately
4hours in a well closed container, after which the mass is packed and the top of the
percolator is closed. Additional menstruum is added to form a shallow layer above the
mass, and the mixture is allowed to macerate in the closed percolator for 24 h. The outlet
of the percolator then is opened and the liquid contained therein is allowed to drip slowly.
Additional menstruum is added as required, until the percolate measures about three-
quarters of the required volume of the finished product. The marc is then pressed and the
expressed liquid is added to the percolate. Sufficient menstruum is added to produce the
required volume, and the mixed liquid is clarified by filtration or by standing followed by
decanting.
i) Ultrasound Extraction (Sonication): The procedure involves the use of ultrasound with
frequencies ranging from 20 kHz to 2000 kHz; this increases the permeability of cell
walls and produces cavitation. Although the process is useful in some cases, like
extraction of rauwolfia root, its large-scale application is limited due to the higher costs.
One disadvantage of the procedure is the occasional but known deleterious effect of
ultrasound energy (more than 20 kHz) on the active constituents of medicinal plants
through formation of free radicals and consequently undesirable changes in the drug
molecules.

Standardization and quality control of herbal crude drugs:

Standardization of herbal medicines is the process of prescribing a set of standards or
inherent characteristics, constant parameters, definitive qualitative and quantitative values
that carry an assurance of quality, efficacy, safety and reproducibility. It is the process of
developing and agreeing upon technical standards. Specific standards are worked out by
experimentation and observations, which would lead to the process of prescribing a set of
characteristics exhibited by the particular herbal medicine.
According to WHO, standardization and quality control of herbals is the process involved in
the physicochemical evaluation of crude drug covering aspects, such as selection and
handling of crude material, safety, efficacy and stability assessment of finished product,
documentation of safety and risk based on experience, provision of product information to
consumer and product promotion.
Standardization Parameters for Herbal Drugs:
1. Morphological or Organoleptic 7. Total ash value
evaluation 8. Heavy metal
2. Microscopic evaluation 9. Microbial content
3. Macroscopic evaluation 10. Loss on drying
4. Foreign matter 11. Specific gravity
5. Chemical evaluation 12. Density
6. Moisture content 13. Melting point
 14. Pesticide residue 18. Determination of specific optical
15. Purity rotation
16. Extractive value
17. Radioactive contamination

Morphological or Organoleptic evaluation:

It includes the evaluation of herbal drugs by size, shape color, odor, taste and particular
characteristics like touch, texture etc.
This is a technique of qualitative evaluation related to the study of morphological and sensory
report of whole drugs. eg. Fractured surfaces in cascara, cinchona, and quillia bark and
quassia wood are essential characteristics. Umbelliferous fruits have aromatic odour and
liquorice have sweet taste are the example of this type of evaluation. Shape of drug may be
conical (aconite), subcylindrical (podophyllum), cylindrical (sarsapilla), fusiform (jalap).Size
represents thickness, length, breadth and diameter. Color represents external color which
various from white to brownish black are essential diagnostic features. Taste which is a
specific type of sensation feel by epithelial layer of tongue. taste may be sweetish
(saccharic),sour (acidic),salt like (saline),and bitter or tasteless.

Microscopic evaluation :
It involves the detailed assessment of the herbal drugs and it is used to recognize the
organized drugs on the basis of their known histological characters. It is regularly used for
qualitative analysis of organized crude drugs in total and powder form with the help of
microscope. The inner pseudoparenchyma cells are round or oval shape. They contain protein
and fixed oil. Crude drugs are microscopically identified by taking thin TS (Transverse
section), LS (Longitudinal Section) in a bark, wood and leaf. The various parameters
included in microscopy are given bellow.
I. Stomata II. Trichomes III. Leaf Content IV. Quantitative Microscopy

Some Microscopic Identification test are given below:

Sr Name of constituents Procedure for test / Reagents Result
.
no
1 Starch/ Hemicellulose T.S. of Crude drug + 1 Drop of Iodine Solution Blue color
2 Mucilage Ruthenium red Pink color
3 Lignin T.S. of crude drug + 1 drop of phloroglucinol + 1 Pink color
drop of HCL

Chemical Evaluation:
The most of drug contain definite chemical constituents to which their pharmacological and
Biological activity depended. Qualitative chemical test used to identify drug quality and
purity. The identification, isolation and purification of active chemical constituents is
depends chemical methods of evaluation. Preliminary phytochemical investigation is also a
part of chemical evaluation. Some Qualitative chemical test for chemical evaluation crude
drugs are Saponification value and acid value etc.
Some important test used in chemical evaluation:
Sr. no Name of constituents Identification Test
1 Volatile oil 1.Ester value
2. Acetyl value
2 Resin 1.Sulphated Ash
2. Acid value
3 Gums 1.Methoxy determination
2.Volatile acidity

Determination of Foreign Matter:

Herbal drugs should be prepared from the confirmed part of the plant. They should be totally
free from insects or moulds, including visible and excreta contaminant such as stones, sand,
harmful and poisonous foreign matter and chemical residues. Animal objects such as insects
and invisible microbial contaminants, which produces toxins, as well as the potential
contaminants of herbal medicines. Macroscopic evaluation can easily used to determine the
presence of foreign matter, although microscopy is essential in certain special cases (for
example starch intentionally added to “dilute” the plant material).
Determination of Total Ash Content :
The residue after incineration is the total ash content of the crude drug, which simply
represents inorganic salts, naturally found in drug or adhering to it or deliberately added to it,
in the form of adulteration.
Two types of total Ash value:
1. Water soluble Ash value 2. Acid insoluble Ash value.
Some examples of drug with their Total Ash Content :
Sr. No. Drugs Total Ash(%w/w) Acid insoluble Ash(%w/w)

1 Agar - 1.00
2 Bael 3.50 -
3 Cannabis 15.00 5.00
4 Ginger 6.00 1.7(water soluble ash)

Determination of Extractive Values:

The extract obtained by exhausting crude drugs are indicative of approximate measure of
their chemical constituents. The various solvent are used for determination of extractives.
These are classified as fallows.
1. Water Soluble extractives.
2. Alcohol Soluble extractives.
3. Ether Soluble extractives.
Some examples with their Extractive values:
Sr. Water Soluble Alcohol Soluble Ether Soluble
Drugs
no extractives (%w/w) extractives (%w/w) extractives (%w/w)
1 Aloe NLT 25.00 NLT 10.00 -
2 Ginger NLT 10.00 NLT 4.50 -
Capsicu
3 - NLT 12.00
m
4 Nutmeg - - NLT 25.00
NOTE: NLT means Not Less Than.
Determination Of heavy Metals:
Contamination by toxic metals can either be accidental or intentional. Contamination by
heavy metals such as mercury, lead, copper, cadmium, and arsenic in herbal materials of
vegetable origin tend to show much higher levels of microbial contamination than synthetic
products and the requirements for microbial contamination in the European Pharmacopoeia
allow higher levels of microbial contamination in herbal remedies than in synthetic
pharmaceuticals. The allowed contamination level may also depend on the method of
processing of the drug. For example, higher contamination levels are permitted if the final
herbal preparation involves boiling with water.
In general, quantitative and limit tests accurately determine the concentration of heay metals
in the form of impurities and contaminants. The heavy metals like Arsenic, mercury, lead,
thalium, cadmium have been shown to be contaminants of few herbal ingredients. A simple
determination of heavy metals can be found ia many pharmacopieas and it is based on color
reaction with special reagents such as diethyldithiocarbonate or thioacetamide and amount is
determined by coparison with a standards. The methods commonly used for analysis are
inductive coupled plasma (ICP), Netron activation analysis(NAA), Atomic Absorption
Spectrophotometry(AAS).
Radioactive contamination:
The microbial growth in herbal drugs is usually avoided by irradiation. Dangerous
contamination, may be the consequence of a nuclear accident. The WHO, in close co-
operation with several other international organizations, has developed guidelines in the event
of a wide spread contamination by radio nuclides resulting from major nuclear accidents. The
nuclear accident in chernobyl and Fukushima may be serious and depend on the specific
radionuclide, the stage of contamination and the quantity of the contaminant consumed.
Examples of such radionuclides include long lived and short lived fission products, actinides
and activation products. Theirfore, at current no limits are proposed for radioactive
contamination
Pesticides Residue:
Pesticides residue are any particular substance in food, agriculture commodities or animal
feed resulting from the use of a pesticides. Herbal drugs are prone to contain pesticide
residue, which gather from agricultural practices, such as Spraying, behavior of soil during
cultivation and addition of fumigants during storage. The Pesticides contain chlorine in the
molecules, which can be determined by analysis of chlorine, insecticides containing
phosphate can be detected by measuring total organic phosphorus.
The various methods are used to measure pesticides by GC, MS, OR GC-MS. Some simple
methods are also published by the WHO and Europian pharmacopeia has in general limits for
pesticides residue in medicine.
Biological evaluation:
Pharmacological activity of certain drugs has been applied to evaluate and standardize them.
The assays on living animal and on their intact or isolated organs can indicate the strength of
the drug or their preparations. These assays are known as Biological assays or Bioassay.
Moisture content: Checking moisture content helps reduce errors in the estimation of the
actual weight of drug material. Low moisture suggests better stability against degradation of
product.
Determination of volatile oil:
Minimum standards for the percentage of volatile oil present in a number of drugs are
prescribed by many pharmacopoeias. A distillation method is usually employed, and the
apparatus first described by Meek and Salvin in 1937 is still widely used in laboratories; the
receiver for this apparatus is very similar to that for water estimation by heavy entrainment.
The weighed drug is placed in a distillation flask with water or a mixture of water and
glycerin and connected to the receiver (cleaned with chromic acid), which is filled with water
and connected to a condenser. On distillation, the oil and water con-dense and the volatile oil
which collects in the graduated receiver as a layer on top of the water is measured.
Loss on drying:
This is employed in the BP/EP and USP. Although the loss in weight, in the samples so
tested, principally is due to water, small amounts of other volatile materials will also
contribute to the weight loss.
For materials (digitalis, hamamelis leaf, yarrow, haw-thorn berries, starch, aloes, fibres)
which contain little volatile mater-ial, direct drying (100–105°C) to constant weight can be
employed. The moisture balance combines both the drying process and weight recording; it is
suitable where large numbers of samples are handled and where a continuous record of loss
in weight with time is required. For materials such as balsams which contain a considerable
proportion of volatile material, the drying may be accomplished by spreading thin layers of
the weighed drug over glass plates and placing in a desiccator over phosphorus pentoxide.
Vacuum drying over an absorbent may be utilized, possibly at a specified temperature.

Phytochemical screening:
Phytochemical examinations were carried out for all the extracts as per the standard methods.
1. Detection of alkaloids:
Extracts were dissolved individually in dilute Hydrochloric acid and filtered.
Mayer’s Test: Filtrates were treated with Mayer’s reagent (Potassium Mercuric Iodide).
Formation of a yellow coloured precipitate indicates the presence of alkaloids.
Wagner’s Test: Filtrates were treated with Wagner’s reagent (Iodine in Potassium Iodide).
Formation of brown/reddish precipitate indicates the presence of alkaloids.
Dragendroff’s Test: Filtrates were treated with Dragendroff’s reagent (solution of Potassium
Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids.
Hager’s Test: Filtrates were treated with Hager’s reagent
(saturated picric acid solution). Presence of alkaloids
confirmed by the formation of yellow coloured precipitate.
2. Detection of carbohydrates: Extracts were dissolved
individually in 5 ml distilled water and filtered. The filtrates were
used to test for the presence of carbohydrates.
Molisch’s Test: Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test
tube. Formation of the violet ring at the junction indicates the presence of Carbohydrates.
Benedict’s test: Filtrates were treated with Benedict’s reagent and heated gently. Orange red
precipitate indicates the presence of reducing sugars.
Fehling’s Test: Filtrates were hydrolysed with dil. HCl, neutralized with alkali and heated
with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of
reducing sugars.
3. Detection of glycosides: Extracts were hydrolysed with dil. HCl, and then subjected to
test for glycosides.
Modified Borntrager’s Test: Extracts were treated with Ferric Chloride solution and
immersed in boiling water for about 5 minutes. The mixture was cooled and extracted with
equal volumes of benzene. The benzene layer was separated and treated with ammonia
solution. Formation of rose-pink colour in the ammoniacal layer indicates the presence of
anthranol glycosides.
4. Legal’s Test:
Extracts were treated with sodium nitroprusside in pyridine and sodium hydroxide. Formation
of pink to blood red colour indicates the presence of cardiac glycosides.
5. Detection of saponins:
Froth Test: Extracts were diluted with distilled water to 20ml and this was shaken in a
graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of
saponins.
Foam Test: 0.5 gm of extract was shaken with 2 ml of water. If foam produced persists for
ten minutes it indicates the presence of saponins.
6. Detection of phytosterols:
Salkowski’s Test: Extracts were treated with chloroform and filtered. The filtrates were
treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of
golden yellow colour indicates the presence of triterpenes.
Liebermann Burchard test: Extracts were treated with chloroform and filtered. The filtrates
were treated with few drops of acetic anhydride, boiled and cooled. Conc. Sulphuric acid was
added. Formation of brown ring at the junction indicates the presence of phytosterols.
7. Detection of phenols :
Ferric Chloride Test: Extracts were treated with 3-4 drops of ferric chloride solution.
Formation of bluish black colour indicates the presence of phenols.
8. Detection of tannins
Gelatin Test: To the extract, 1% gelatin solution containing sodium chloride was added.
Formation of white precipitate indicates the presence of tannins.
9. Detection of flavonoids
Alkaline Reagent Test: Extracts were treated with few drops of sodium hydroxide solution.
Formation of intense yellow colour, which becomes colourless on addition of dilute acid,
indicates the presence of flavonoids.
Lead acetate Test: Extracts were treated with few drops of lead acetate solution. Formation
of yellow colour precipitate indicates the presence of Flavonoids.
10. Detection of proteins and amino acids
Xanthoproteic Test: The extracts were treated with few drops of conc. Nitric acid.
Formation of yellow colour indicates the presence of proteins.
Ninhydrin Test: To the extract, 0.25% w/v Ninhydrin reagent was added and boiled for few
minutes. Formation of blue colour indicates the presence of amino acid.

Pharmacological Screening:
Pharmacological screening means thorough investigations measure the pharmacological
activity of new or chemically undefined substance, investigate the function of endogenous
mediators, measure drug toxicity and unwanted effects.

Types of Screening:
A. Simple screening
B. Blind screening
C. Programmed screening
A) Simple Screening:
It involves the use of one or two simple tests to find substances having a particular property.
For example, a single test for conc. of glucose in blood can be used to screen compound for
hypoglycemic activity.
B) Blind Screening:
It is used to detect the pharmacological activities of new drugs whose pharmacological
activity is unknown. The chief purposes is to demonstrate whether these new drugs are
worthy of further attention or not.
C) Programmed Screening:
It is used when a new drug of specific type is to be screened for some pharmacological
effects. Examples are screening of certain drugs on the CVS, CNS, kidney, blood etc. It
includes the use of quantitative assay of the compounds and their comparison with standard
drugs that are quite active representative members of their pharmacological class. It also
provides indications of potential side effects.

Methods of screening:
1. Invitro method: Experimental process in a given procedure which is mainly done outside
the body in a controlled condition .

2. Ex-vivo method: Experimental process which is performed outside the living body in an
artificial in vivo environment .This usually lasting up to 24 hours .
3. In-vivo method : Experimental process which is performed in the living body using
laboratory animals .
4. Insilco method : Process which is performed on computer or via stimulator

Bioassay:
Bioassay plays a key role in pharmacological screening and standardization of drugs. It is
defined as the estimation of concentration or potency of a substance (drugs, hormones,
vitamins, toxins, and antitoxin) by measurement of the biological response that it produces.
Pharmacological Applications of bioassay:
 Used in determination of drugs potency
 Screenings of new agents isolated from plants, animals or chemical labs and find their
field of activities
 Establishment of SAR (structure activity relationship)
 Essential in monitoring environmental pollutants
 Determination of the pharmacological activities of a new drug
 Determine the therapeutic advantage of one drug over anther treatments
 Useful in study of new hormonal or other chemically mediated control systems
Principles of Bioassay:
These are the principles of bioassays:
1. The potency of unknown preparations is compared with that of standard product under
identical experimental conditions.
2. Active principle should be identical in both the standard and the preparation to be assayed.
3. Assay experiment should be designed in which the effect observed is not the same as the
desired therapeutic effect.
Pharmacologic profile Test:
A variety of biologic assays at the molecular, cellular, organ, system, and whole animal levels
are used to define the activity and selectivity of the drug. Studies are performed during drug
screening to define the pharmacologic profile of the drug at the molecular, cellular, system,
organ, and whole organism levels.
For example, a broad range of tests would be performed on a drug designed to act as an
antagonist at alpha-adrenoceptors for the treatment of hypertension.Anti-infective drugs may
be tested against a variety of infectious organisms some of which areresistant to standard
agents. Hypoglycemic drugs for their ability to lower blood sugar, etc
Molecular level:
At the molecular level, the compound would be screened for receptor binding affinity to cell
membranes respective receptors, other receptors and binding sites on enzymes. If crystal
structures of the drug and target are available, structural biology analyses or computer
assisted virtual screening might be done to better understand the drug receptor interaction.
For example, studies on liver cytochrome P450 enzymes would be performed to determine
whether the drug of interest is likely to be a substrate or inhibitor of these enzymes or to
interfere with the metabolism of other drugs.
Cellular level:
Effects on cell function would be studied to determine whether the drug is an agonist, partial
agonist, or antagonist at a receptors. Isolated tissues, especially vascular smooth muscle,
would be utilized to characterize the pharmacologic activity and selectivity of the new
compound in comparison with reference compounds. Comparison with other drugs would
also be undertaken in other in vitro preparations such as gastrointestinal and bronchial
smooth muscle.
Systems/ Organism levels: Whole animal studies are generally necessary to determine the
effect of the drug on organ systems and disease models. Cardiovascular and renal function
studies of all new drugs are generally first performed in normal animals.
After Screening via Bioassay:
These studies might suggest the need for further chemical modification to achieve more
desirable pharmacokinetic or pharmacodynamic properties.
For example,
• Oral administration studies might show that the drug was poorly absorbed or rapidly
metabolized in liver so; modification to improve bioavailability might be indicated.
• If the drug is to be administered long-term, an assessment of tolerance development would
be made.
• For drugs related to or having mechanisms of action similar to those known to cause
physical dependence, abuse potential would also be studied.
Lead compound:
The desired result of this screening procedure (which may have to be repeated several times
with analogs or congeners of the original molecules) is called a lead compound.Lead
compound is now a leading candidate for a successful new drug.
Factors affecting Pharmacological response of drugs:
Sex: Difference in drug response among different sex is attributed to difference in the
level of metabolizing enzymes
Age: The level of metabolizing enzymes varies with age and some enzymes may be
lacked in newly born animal.
Diseases: E.g. hypersensitivity to catecholamine action on CVS is increased in
hyperthyroidism.
Environmental: Such as temperature, seasons, nutrition, light and isolation of animal etc.
e.g. the convulsive action of insulin in mice is greatly influenced by temperature

Screening of disease:
The search for unrecognized disease or defect by means of rapidly applied tests, examinations
or other procedure in apparently healthy individuals. Screening is testing for infection or
disease in population or in individuals who are not seeking health care facilities.
Types of Screening:
There are 3 types of screening.
a) Mass screening
b) High risk or selective screening
c) Multiphasic screening
Mass Screening:
It means screening of whole population or a subgroup, as for example all adults. It is offered
to all, irrespective of the particular risk individual. E.g. tuberculosis, AIDS, hypertension,
diabetes, leukemias etc.
High risk or selective Screening:
Screening will be most productive if applied selectively to high-risk groups. The groups
defined on the basis of epidemiological research. e.g. cervix cancer tends to occur relatively
less often in the upper social groups as compare to lower social groups etc
Multiphasic Screening:
It is defined as the application of two or more screening tests in combination to a large
number of people at one time than to carry out separate screening tests for single diseases.
Procedure may include Questionnaire, clinical examination and a wide range of
measurements and investigations include chemical and hematological tests on blood, urine
specimen, lung function assessments, audio and visual tests with appropriate staffing
organization and specialized equipments.

Safety of herbal medicine:

A major benefit of herbal medicine is its long history of safe use. Humans have been taking
herbal medicine for thousands of years. This is like a huge experiment where thousands if not
millions of people are utilizing plants to benefit their health and relieve symptoms, in some
cases herbs like garlic have at least 3,000 years of safe use. Contrast this with many
pharmaceuticals that have only a few years of studies and human use to support their safety.
As it turns out, about 5% of drugs have to be pulled from the market after some years of use.
In many cultures herbal medicines are added to food, and taken daily as tonics and restorative
remedies.
In European studies comparing herb preparations and pharmaceutical drugs, the side effects
of the herbal remedies when compared with pharmaceutical drugs is typically one half, or
equivalent to the rate of side effects for the placebo.
The World Health Organization created one of the largest herb safety databases in the world.
A thorough search of this database shows that very serious side effects or interactions have
been reported internationally over the last 20 years.Very few reports of serious adverse events
associated with herbal medicines can be found after a thorough search of databases and
reports from government agencies such as the centers for disease control (CDC), and the food
and drug administration (FDA), charged with monitoring the safety of the drugs and devices
used in health care.
Safety guidelines:
1. Only herbs recommended in respected herb books
should be used.
2. New or unproven remedies should be avoided.
3. It is better to discontinue the herb consumption if no benefit or result was obtained after a
moderate period of time, or if adverse reactions toke place.
4. Patients or physicians should not engage in drug usage for complex conditions without
knowledge.
5. Drug interactions and contraindications must be considered on an individual basis.
6. It is better to avoid herbal remedies during pregnancy.In overall although medicinal plants
are widely used and assumed to be safe, however, they can potentially be toxic. Where
poisoning from medicinal plants has been reported, it usually has been due to
misidentification of the plants in the form in which they are sold, or incorrectly preparation
and administration by inadequately trained personnel.
Toxicity of harbal Medicine:
Herbal remedies are plant-based, as in fact are many orthodox drugs - some 25% of present
pharmaceutical preparations contain at least one active ingredient extracted from plant
sources. Moreover, thousands of our present drugs were originally derived from plants,
including digitalis (foxglove), aspirin (willow and meadowsweet) and paclitaxel (Taxol).
However, in the case of orthodox drugs the active ingredient is isolated from the plant,
chemically standardized, subjected to critical clinical assessment and then often replaced with
a synthetic analogue.
In contrast, the herbalist uses mixtures of diverse herbal ingredients of varying potency.
Potency depends on the part of the plant used - for example root, stem, leaves or fruits - the
time of the year it is picked, and the actual species of plant used.
1. Gingko biloba: It can cause spontaneous bleeding.There have been several published
incidents of bleeding problems associated with ginkgolide B, a platelet-activating factor
inhibitor that is a component of ginkgo biloba.
2. Garlic: Garlic can lower cholesterol level. It can cause increased INR with warfarin,
increased post-operative bleeding, hypoglycemia and increased bleeding with Aspirin,
subdural haematoma.
3. Ginseng: It increases the body’s resistance to stress and builds up general vitality. It can
cause hypertension and mastalgia. Reports have linked ginseng to potential medication
interactions with warfarin, alprazolam, donezepil and trazodone and digoxin. Ginseng should
be avoided in patients who are on MAO inhibitors, Nifedipine and in cancer therapy.
4. Peppermint oil: Products containing this ingredient have been associated with
bronchospasm, anaphylactoid reaction and duodenal ulcer perforation.
5. Senna extract: It has cathartic properties and is used for constipation. It can cause grand
mal seizures, circulatory failure, hypertension and anaphylactic reaction. Regular use should
be avoided if the patient is taking either thiazide diuretics or corticosteroids to prevent
electrolyte imbalance.
6. Primrose oil: It has been used for various skin ailments as well as for pre-menstrual
syndrome. It can cause undiagnosed temporal lobe epilepsy, especially in schizophrenic
patients and those on drug therapy forepilepsy.
7. Isapghol: It is stated to possess demulcent and laxative properties. It can cause
bronchospasm, asthma and intestinal obstruction. If swallowed dried, ispagula may cause
oesophageal obstruction.
8. Ephedra: Adverse reactions reported range from high blood pressure, irregular heartbeat,
nerve damage, injury, tremor, headache, seizures, heart attack, stroke, and death.
9. Liquorice: Used as an expectorant and carminative infood. Regular high doses should be
avoided in conjunction with steroids including oral corticosteroids.

Stability studies herbal drugs:

The World Health Organization (WHO) recommends that stability data be provided to
support the shelf-life proposed for finished herbal products under the specified conditions of
storage.
The shelf-life of a product is the period during which it remains within acceptable chemical,
physical and microbiological stability if stored correctly. The expiry date is represented on
the product to indicate the end of the shelf life. Finished herbal products are unique compared
to synthetic products due to the varied nature of constituents that may be present even in an
herbal product with only one herbal ingredient. It is often not feasible to determine the
stability of each active ingredient.
The herbal material, in its entirety, is normally regarded as the active ingredient and a mere
determination of the stability of the constituents with known therapeutic activity will not
usually be sufficient. Constituents (markers) for stability determination in herbal products
may be categorized as; chemical, analytical and active markers.
Chemical markers may be defined as chemically defined constituents or groups of
constituents of an herbal medicinal product which are of interest for quality control purposes
regardless of whether they possess any therapeutic activity. The quantity of a chemical
marker can be an indicator of the quality of an herbal medicine. Chemical markers may be
used to evaluate product quality and stability over time and determine the recommended shelf
life. Analytical markers are the constituents or groups of constituents that serve solely for
analytical purposes. Active markers are the constituents or groups of constituents that
contribute to therapeutic activities.
Stability determination of herbal medicinal products:
The principle of a stability study is to provide evidence that an active substance or finished
product varies with time under the influence of a variety of environmental factors such as
temperature, humidity and light. The importance of stability testing is to; evaluate the
efficacy of a drug; provide background information during the development phase of the
product or drug discovery; develop suitable packaging information for quality, strength,
purity and integrity of a product during its shelf-life.
Mechanisms that may indicate a change in stability include; loss of activity; change in
concentration of active component; alteration in bioavailability of product; loss of content
uniformity; loss of elegance; formation of toxic degradation products and loss of packaging
integrity. It is difficult to develop analytical methods for herbal medicines due to the presence
of phytochemical constituents which may be susceptible to enzymatic breakdown of these
plant metabolites. Predictable chemical changes in herbal products includes; hydrolysis;
oxidation (especially in fixed, volatile and essential oils); racemization; geometric
isomerization and polymerization.
General methods for stability testing:
The stability of herbal medicinal products may be determined based
on physical and sensory tests, microbial tests and chromatographic/ spectral tests.
Physical and sensory methods:
Herbal products, like pharmaceutical products, usually undergo physical changes during
storage. These changes though not usually quantitative in nature may be used as a guide to
check if the products are deteriorating. These include evaluation of changes in parameters
such as colour, taste, odour, clarity, specific gravity, total solid residue, viscosity, the
moisture content of powders, dissolution and disintegration tests for capsules and tablets. It
must be noted that some of these methods of assessment such as taste and odour should be
carried out only if they do not affect the safety of the personnel involved.
Microbial tests:
Microbial contamination or load tests and preservative efficacy or challenge tests (where
preservatives are used) of finished herbal products are essential in the determination of
stability and shelf life of the product. Key factors affecting the efficacy of the antimicrobial
preservative added are the active ingredient, excipients, storage conditions, the container and
its closure. The British Pharmacopoeia states that for a product “it shall be demonstrated that
the antimicrobial activity of the preparation as such or if necessary, with the addition of a
suitable preservative or preservatives provides adequate protection from adverse effects that
may arise from microbial contamination or proliferation during storage and use of the
preparation.
Analyses of such parameters with time allows the tracing of stability of the product and
subsequent prediction or estimation of shelf-life. These tests should be done according to
Pharmacopoeia methods (British Pharmacopoeia, United States Pharmacopoeia, European
Pharmacopeia.), WHO methods, or any other internationally recognized methods. The
microbial tests should involve: Total viable aerobic plate count; contaminating fungus (yeast
and mould ); Salmonella spp.; Escherichia coli and Staphylococcus aureus.
Chromatography and spectral methods:
Chromatographic methods used to assess the chemical stability of herbal products include
thin layer chromatography (TLC), high (ultra) performance liquid chromatography
(HPLC,UPLC), high performance-thin layer chromatography (HP-TLC), gas liquid
chromatography (GLC) etc. while spectral methods used include ultraviolet-visible (UV-VIS)
spectroscopy, infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) and mass
spectroscopy (MS). These techniques allow tracing of changes which may occur during
storage of a complex mixture of biologically active substances contained in herbal materials.
Comparisons of appropriate characteristic/fingerprint chromato-grams allow the
determination of the stability of identified active ingredients (if any) and other substances
present in the finished herbal product (which may appear as markers).
References
1. Doris kumadohi, Kwabena ofori-kwakye. Dosage forms of herbal medicinal
products and theirs stabilaty considerations and overview

2. Safety profile of herbal drugs: urgent need for monitoring,

https://www.researchgate.net/publication/267040558

3. Hamid Nasri, Hedayatollah Shirzad. Toxicity and safety of medicinal plants.

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www.jocpr.com
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8. Debjit Bhowmik, Chiranjib, Pawan Dubey, Margret Chandira, K. P. Sampath Kuma.

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