QUALITY CONTROL OF DRUGS OF NATURAL ORIGIN

Quality control of drugs of natural
origin
By – Pooja S Agrawal
M. Pharm.(QA)
For – B. Pharm IV Sem

Objectives
On completion of this lesson, you would be able to know:
➢ Methods of adulteration of Crude Drugs.
➢ Commonly used substitutes in adulteration
➢ Evaluation for determining adulterants

Adulteration
• Adulteration is a practice of substituting original crude drug partially or wholly with
other spurious substances but the later is either free from or inferior in chemical and
therapeutic properties.
• The motives for intentional adulteration are normally commercial one and originate
mainly with the intention of enhancement of profits.
• Some of the reasons that can be cited here are scarcity of drug and its high price
prevailing in market.
• The adulteration is done deliberately, but it may occur accidentally in some cases. It is
also very common with the contraband drugs.
• Adulteration means deterioration, admixture, sophistication, substitution, inferiority
and spoilage

✓ Deterioration is impairment in the quality of drug.
✓ Admixture is addition of one article to another due to ignorance or carelessness or by a
accident.
✓ Sophistication is the intentional or deliberate type of adulteration.
✓ Substitution occurs when some totally different substance is added in place of original
drug
✓ Inferiority refers to any substandard drug, and spoilage is due to the attack of
microorganisms.
❖ During the routine quality control, various tests are conducted for their detection. The
present topic deals with different techniques used in adulterating crude drugs and
laboratory methods for their detection

Adulteration and Evaluation
➢ Adulteration involves incorporation of impurities.
➢ Includes spoilage deterioration admixture.
➢ Genuine drugs are intentionally substituted..
➢ With spurious, inferior, defective or harmful substances.

Types of Adulterants
The different types of adulterants found in market are given below.
1. Substitution with substandard commercial varieties: The adulterants used here may
resemble original crude drug by morphological, chemical or therapeutic characters, but are
substandard in nature and hence cheaper in cost. This is rather the most common practice
of adulteration.
2. Substitution with superficially similar inferior drugs: These inferior drugs used may or
may not have any chemical or therapeutic value as that of original natural drug. Due to
their morphological resemblance to authentic drug, they are marketed as adulterants.
3. Substitution with artificially manufactured substances: It has been also observed that
substances artificially prepared to resemble original drug are used as substitutes.
Generally, this practice is followed for much costlier drugs.

4. Substitution with exhausted drugs: In this type, the same drug is admixed but is devoid of
any medicinally active constituents as they are already extracted out. This practice is more
common in case of volatile oil containing drugs like fennel, clove, coriander, caraway etc.
sometimes, natural characters of exhausted drugs like colour and taste are manipulated by
adding other additives and then it is substituted.
5. Besides these common practices, sometimes other methods are employed like use of
synthetic chemicals to enhance the natural character
6. Presence of vegetative matter from the same plant: Sometimes, the other miniature
plants growing along with medicinal plant are mixed with drug due to their resembling colour,
odour and in some cases constituents.
7. Harmful adulterants: Several times, the wastes from market are collected and admixed
with authentic drugs. This is particularly noticed for liquids or unorganized drugs.
8.Adulteration of powders: Besides the entire drugs, the powdered forms are frequently
found to be adulterated.

Evaluation
• Evaluation of a drug means confirmation of its identity and determination of its quality and
purity and detection of nature of adulteration
• The evaluation of a crude drug is necessary because of three main reasons
(a) biochemical variation in the drug,
(b) deterioration due to treatment and storage, and
(c) substitution and adulteration, as a result of carelessness, ignorance or frand.
• Over the years the nature and degree of evaluation of crude drugs has undergone a systematic
change.
• Initially, the crude drugs were identified by comparison only with the standard description
available.
• Due to advancement in the chemical knowledge of crude drugs, at present, evaluation also
includes method of estimating active constituent present in the crude drug, in addition to its
morphological and microscopic analysis.

• With the advent of separation techniques and instrumentation analysis, it is
possible to perform physical evaluation of a crude drug which could be both
qualitative and quantitative.
• The biological behavior of crude drug extracts constitutes pharmacological
evaluation.
• The crude drugs can be identified on the basis of their morphological, histological,
chemical, physical and biological studies.

1. MORPHOLOGICAL OR ORGANOLEPTIC EVALUATION
• It refers to evaluation of drugs by colour, odour, taste, size, shape and special
features, like touch, texture, etc. It is a technique of qualitative evaluation based on
the study of morphological and sensory profiles of whole drugs
• Organoleptic evaluation means conclusions drawn from impressions on organs of
senses.
• The study of form of a crude drug is Morphology while description of the form is
Morphography.
• Aromatic odour of umbelliferous fruits and sweet taste of liquorice are the examples
of this type of evaluation.
• The ribbon shaped characteristic of tragacanth, disc-shaped structure of nux vomica,
conical shape of aconite, quills of cinnamon etc. are important diagnostic characters.

Continued..
• The general appearance of the lot of a crude drug often indicates whether it is likely to
comply with prescribed standards, such as percentage of seed in colocynth, stalk in
clove, etc.
• Over drying, makes leaf drugs and flowers brittle and cause them to break in transit
making the task of morphological evaluation difficult.
• The wavy shape of Rauwolfia, pungent taste of capsicum and ginger, brown colour of
cinnamon, odour and taste of spice drugs like, asafoetida, black pepper, nutmeg,
cummin, etc. are important diagnostic organoleptic characteristics.

2. MICROSCOPIC EVALUATION
• This method allows more detailed examination of a drug and it can be used to identify the
organized drugs by their known histological characters. It is mostly used for qualitative
evaluation of organized crude drugs in entire and powdered forms.
• Microscope, by virtue of its property to magnify, permits the minute structure understudy to
be magnified to confirm the structural details of the drugs from plant origin.
• For the effective results, various reagents or stains can be used to distinguish cellular
structure.
• Microscopic evaluation also covers study of the constituents by application of chemical
methods to small quantities of drugs in powdered form or to histological sections of the drug
(Microchemistry or chemomicroscopy).

Examples:
• A drop of phoroglucinol and concentrated hydrochloric acid give red stain with lignin.
• Mucilage is stained pink with ruthenium red and also, when treated with corallin soda
and few drops of sodium carbonate solution, cellulose swells and dissolves in cuoxam,
• while N/50 iodine solution stains blue starch and hemicelluloses.
• Histological studies are made from very thin sections of drugs.
• The characteristics of cell walls, cell contents, starch grains, calcium oxalate crystals,
trichomes, fibres, vessels, etc. can be studied in detail
• e.g. lignified trichomes in nux vomica, warty trichomes of senna, wavy medullary rays
of cascara bark, glandular trichomes of mint etc.

• The powdered cloves do not contain sclereid or calcium oxalate crystals, but both of them are
present in powdered clove stalks.
• Powdered clove fruits show presence of starch while it is absent in cloves.
• Presence of non-lignified vessels in powdered ginger indicate adulteration
• Other important histological aspect is the quantitative microscopy and linear measurements.
The various parameters studied here are stomata number and index, palisade ratio, vein-islet
number, size of starch grains, length of fibres, etc
• Senna varieties are distinguished by differing stomata number and palisade ratio. The
diameter of starch grains in Cinnamomum cassia is 10 microns, hence, useful for detecting
adulterants. The number of sclerenchymatous cells per square mm of cardamom is useful for
detecting different varieties of cardamom seed.

(a)Leaf Constants or Diagnostic Characters of Leaves
1.Palisade ratio - is defined as average number of palisade cells beneath each epidermal cell. It
can be determined with powdered drugs.

2.Vein-islet numbers is defined as the number of vein-islets per sq. mm of the leaf surface
midway between the midrib and the margin. Levin in 1929 determined vein-islet numbers
of several dicot leaves
3.Vein-termination number is defined as the number of veinlet terminations per sq. mm of
the leaf surface midway between midrib and margin.
4.Stomata number is the number of stomata per sq. mm of epidermis of the leaf.
5. Stomata index is the percentage which the numbers of stomata form to total number of
epidermal cells: each stoma being counted as one cell
It is calculated by using the following equation
SI = S/E + S * 100
where,
SI = Stomatal Index
S = Number of stomata per unit area
E = Number of epidermal cells in the same unit area

b) Stomata
Epidermis of leaf shows different characteristics, e.g. cuticle Stomata, trichomes, Water pore
cell inclusions, etc.
• A stoma is a minute epidermal opening present on aerial parts of the plants, with
following characteristics. (a) A central pore (b) Two kidney shaped similar cells
containing chloroplasts known as guard cells and varying number of subsidiary
(epidermal) cells covering the guard cells.
• The primary and most important function of stomata is gaseous exchange and the
secondary function is transpiration. It is not essential that each plant must have stomata
• The submerged leaves of aquatic parts do not contain stomata. Generally, stomata present
in green parts of the plant (mostly leaves), but absent in roots. Apart from the leaves, they
are also present in the stems (ephedra), flowers (clove), and fruits (fennel).

However, it is generally observed that stomata are abundantly present in dicot leaves. In
some cases, they are present on the upper surface of leaves, while in others on lower surface
only (coca and cherry).
In some, the stomata are present on both surfaces of the leaves (senna, belladonna, datura,
etc.). The distribution of stomata between upper and lower epidermis in dicot leaves shows
great variation.
Types of stomata Depending upon the type of the guard cells and arrangement of
subsidiary cells, stomata are divided into four types:
1. Moss type
2. Gymnosperms type
3. Gramineous type
4. Dicotyledonous type
Out of these, fourth type of the stomata is of diagnostic significance.

Dicotyledonous stomata are classified into following types depending upon the form and
arrangement of subsidiary cells.
(i)Paracytic or rubiaceous or parallel-celled stomata -These type of stomata comprise two
guard cells covered by two subsidiary cells, the long axes which are parallel to that of stoma,
ex. coca and senna leaves
(ii)Diacytic or caryophyllaceous or cross-called stomata –The guard cells are covered by
two subsidiary cells, as in the case of parasitic stoma, but the arrangement of subsidiary cells
on the guard cell is at right angle to that of stoma, e.g. peppermint, spearmint, and vasaka.

(iii)Anisocytic or cruciferous or unequal-celled stomata The number of guard cells is two,
as in all other cases, but the guard cells are covered by three subsidiary cells, of which one is
markedly smaller than the other two, e.g. belladonna, datura.
(iv) Anomocytic or ranunculaceous or irregular-called stomata In this type, stomais
surrounded by varying number of subsidiary cells resembling other epidermal cells, e.g.
digitalis and lobelia.
Indian Pharmacopoeia recognizes one more type of stomata known as Actinocytic stomata.
(v) Actinocytic or radiate-celled stomata The two guard cells are surrounded by a circle of
radiating subsidiary cells.

(C) Trichomes or Plant hairs
• These are other important diagnostic characters helpful in the identification of drugs and
detection of adulterants. Trichomes are the tubular elongated or glandular outgrowth of the
epidermal cell. Trichomes are also known as plant hairs.
• Trichomes consist of two parts namely root (in the epidermis) and body (outside the
epidermis).
• Trichomes are present in most of the parts of the plant such as leaves (senna and digitalis),
seeds (nux vomica and strophanthus), fruits (Ladies finger), etc. Trichomes are as such
functionless, but sometimes, perform secretory function. The trichomes excrete water and at
times, volatile oil as in case of peppermint
• Trichomes are present in most of the aerial parts of the plant, but are absent on roots.
Depending upon the structure and the number of cells present in trichomes, they are
classified as follows:
1. Covering trichomes or non-globular trichomes or clothing trichomes
2. Glandular trichomes
3. Hydathodes or special type of trichomes.

(d) Calcium Oxalate Crystals
• Several cell contents like aleurone grains, mucilage, tannin, fixed and volatile oil
globules and several other inorganic components like calcium carbonate, calcium
oxalate and silica are frequently found to occur naturally in the vegetable drugs.
• The inorganic crystalline compounds by virtue of their specific shapes can be utilized
for the identification of herbal drugs. Due to this reason they are known as diagnostic
characters of the plant.
• Here a special reference is being given to different forms of calcium oxalate crystals in
plant.
• Calcium oxalate is a dimorphic salt of which both the types occur in the plant body.
The crystals are either monoclinic or tetragonal in shape. Both the types differ in their
characters such as wat content, optical properties etc.
• Monoclinic crystals contain only one molecule of water of crystallization i.e. CaC204,
while tetragonal crystals are CaC204.3H20.

Here are six forms of calcium oxalate crystals in plants as follows:
(1) Cubical (Prisms): As the name indicates, these crystals are cubical in shape i.e. their
height, width and length is equal) and also have three equal axes, all at right angles to one
another.
(2) Rhombic (Diamond Shaped): Here crystals have three axes, each axes are at right to each
other and unequal in length.
(3) Tetragonal: This is characterized by presence of three axes, at right angle to one another,
two axes equal which are lateral, while the third is either longer or shorter and is known as
vertical or principal axis.
(4) Monoclinic: This form has three axes and all the three are unequal. The lateral two at right
angles to each other, while the third. i.e. Principal is at right angles to lateral. The monoclinic
crystals shine more than the tetragonal system.
(5) Acicular: these are excessively long slender forms, with pointed ends and normally
reported in bundles.
(6) Rosettes (Clusters) : These are also aggregate crystals. This type has shape similar to full
expanded rose flowers and hence the name rosettes.

(e) Quantitative Microscopy
Lycopodium spore method for percentage purity
• It is an important analytical technique for powdered drugs, especially when chemical and
other methods of evaluation of crude drugs fail as accurate measures of quality.
• Lycopodium spores are very characteristic in shape and appearance and exceptionally
uniform in size (25 pm). On an average, 94,000 spores per mg of powdered Lycopodium.
• A powdered drug is evaluated by this technique, if it contains (a) well defined particles which
may be counted, e.g. starch grains or pollen grains, (b) single layered cells or tissues, the area
of which may be traced under suitable magnification and actual area calculated or (c) the
objects of uniform thickness, the length of which can be measured under suitable
magnification and actual area calculated.
• The percentage purity of an authentic powdered ginger is calculated using the following
equation
N * W * 94,000 * 100= per cent Purity of drug
S * M * P

N = number of characteristic structures (e.g. starch grains) in 26 fields
W = weight in mg of Lycopodium taken
S = number of Lycopodium spores in the same 25 fields
M = weight in mg of the sample, calculated on basis of sample dried at 105°C
P = 2,86,000 in case of ginger starch grains powder
Lycopodium spore method can be used for evaluation of powdered clove, ginger, cardamom,
nutmeg, umbelliferous fruits, etc.

3. CHEMICAL EVALUATION
• It comprises different chemical tests and assays. The isolation, purification and
identification of active constituents are chemical methods of evaluation. Qualitative
chemical tests such as acid value, saponification value, etc. are also covered under this
technique.
• Preliminary phytochemical screening is a part of chemical evaluation. The qualitative
chemical tests are useful in detection of adulteration.
• Halphen’s test for cotton seed Oil, Van Urk’s reagent for ergot, Vitali’s test for tropane
alkaloids, murex idee test for purine bases, etc. are examples of specific tests.

• The chemical evaluation also covers phytochemicals screening carried out for establishing
chemical profile of a crude drug.
PHYTOCHEMICAL INVESTIGATIONS
The systematic investigations of plant material for its phytochemicals behaviour involve four
different stages.
1. The procurement of raw material and quality control.
2. Extraction, purification and characterization of the constituents of pharmaceutical interest
and in process quality control.
3. Investigations of biosynthetic pathways to particular compounds and
4. Quantitative evaluation.

• The commonly employed technique for separation of active substance from crude
drug is called EXTRACTION Which involves the use of different solvents.
• The plant material used for extraction should be properly authenticated or identified.
The choice of the plant material for extraction depends on its nature and the
components required being isolated.
• The dried powdered plant material is commonly used for extraction.
• The fresh plant parts when used are homogenized or macerated with a solvent such
as alcohol. It is a general solvent for many potential constituents and as such may
give problem in subsequent elimination of pigments, resins, etc.

• Water immiscible solvent, such as light petroleum is used for the extraction of fixed
and essential oils, steroids and aglycones.
• Chloroform and ether are used for the separation of alkaloids and quinines
• The extraction of organic bases like alkaloids usually necessitates basification of
plant material if a water immiscible solvent is to be used while for aromatic acids
and phenols, acidification may be required.
• The glycosides are soluble in water and alcohol, but insoluble in non-polar solvents
• Tannins are phenolic matter soluble in water, alcohol and ethyl acetate. Extraction
itself may be performed by repeated maceration with agitation, percolation or by
continuous extraction using Soxhlet extractor.

Preliminary Phytochemical Screening
• The plant is a biosynthetic laboratory, not only for chemical compounds such as
carbohydrates, proteins and lipids that are utilized as food by man, but also for a multitude
of compounds like glycosides, alkaloids volatile oils, tannins, etc., that exert a
physiological and therapeutic effect.
• The compounds that are responsible for medicinal property of the drug are usually
secondary metabolites. A systematic study of a crude drug embraces, thorough
consideration of primary and secondary metabolites derived as a result of plant metabolism.
The plant material is subjected to preliminary phytochemicals screening for the detection of
various plant constituents on following lines.
• Successive solvent extraction: The air-dried powdered plant material is extracted in Soxhlet
assembly successively with petroleum ether benzene solvent ether, chloroform, acetone,
ethanol and methanol.
• Finally, the drug is macerated with chloroform water. Each time before extracting with the
next solvent, the powdered material is dried in hot-air oven below 500 ºC

• Each extract is concentrated by distilling off the solvent and then evaporating to
dryness on water-bath.
• The extract obtained with each solvent is weighed. Its percentage is calculated in
terms of air-dried weight of plant material. The colour and consistency of the
extract are noted.
• The extracts with different solvents can also be prepared by successively
macerating (co extraction) the powdered drug in order of increasing polarity
• The general approach for extraction of different constituents from fresh plant may
be briefly described in the following chart

Qualitative Chemical Examination
The extracts obtained as above are then subjected to qualitative tests for the
identification of various plant constituents.
1. Detection of alkaloids: The small portions of solvent free chloroform, alcoholic and
water extracts are stirred separately with a few drops of dilute hydrochloric acid and
filtered. The filtrate may be tested carefully with various alkaloidal reagents: Mayer’s
reagent (cream precipitate), Dragendroff’s reagent (orange brown precipitate). Hager’s
reagent (yellow precipitate) and Wagner’s reagent (reddish brown precipitate)
2. Detection of carbohydrates and glycosides
(a) Small quantities (200 mg) of alcoholic and aqueous extracts are dissolved separately
in 5 ml of distilled water and filtered. The filtrate may be subjected to Molisch’s test to
detect the presence of carbohydrates.

(b) Another small portion of extract is hydrolyzed with dilute HCl for few hours in water-bath
and is subjected to Liebermann Burchard’s and Borntrager’s tests to detect presence of
different glycosides.
(c) A small portion extract is dissolved in water and treated with Fehling’s, and Benedict’s
reagents to detect presence of different sugars.
3.Detection of phytosterols The petroleum ether, acetone and alcoholic extracts are refluxed
separately with solution of alcoholic potassium hydroxide till complete saponification takes
place. The saponification mixture is diluted with distilled water and extracted with ether. The
ethereal extract is evaporated and the residue (unsaponifiable matter) is subjected to
Liebermann’s and Burchardts tests.
4. Detection of fixed oils and fats: A small quantity of petroleum ether and benzene extracts is
pressed separately between two filter papers. Oil stains on the paper indicate the presence of
fixed oil

5. Detection of saponins About 1 ml of alcoholic and aqueous extracts is diluted separately
with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes. One cm layer of
foam indicates presence of saponins.
6. Detection of phenolic compounds and tannins Small quantities of alcoholic and aqueous
extracts in water are tested for the presence of phenolic compounds and tannins with dilute
ferric chloride solution (5 per cent), 10% lead acetate and aqueous bromine solutions.
7. Detection of proteins and free amino acids: Small quantities of alcoholic and aqueous
extracts are dissolved in a few ml of water and subjected to Millen’s, Biuret and Ninhydrin tests.
8. Detection of gums and mucilages: About 10 ml of aqueous extract is added to 25 ml of
absolute alcohol with constant stirring. The precipitate is dried in air. Then the precipitate is
examined for its swelling properties and for the presence of carbohydrates

9. Detection of volatile oil: About 50 g of powdered material is taken in a volatile oil
estimation apparatus and subjected to hydro-distillation for the detection of volatile oil. The
distillate is collected in the graduated tube of the assembly in which the aqueous portion is
automatically separated from the volatile oil, if it is present in the drug, and returned back to
the distillation flask.

4. PHYSICAL EVALUATION
• Physical standards are to be determined for drugs, wherever possible. These are rarely
constant for crude drugs, but may help in evaluation, specifically with reference to moisture
content, specific gravity, density optical rotation, refractive index, melting point; viscosity,
and solubility in different solvents. A few of them are described below
(i) Moisture content: The percentage of active chemical constituents in crude drugs is
mentioned on air-dried basis.
• Hence, the moisture content of a drug should be determined and should also be controlled.
The moisture content of a drug should be minimized to prevent decomposition of crude
drugs 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
• Ex, Aloes - not more than 10% w/w
Starch - NMT 15% w/w
Acacia – NMT 15% w/w

(ii) 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.
• Examples. Liquid paraffin - Kinematic viscosity not less than 64 centistokes at 37.8º.
Pyroxylin - Kinematic viscosity, 1100 - 2450 centistokes
(iii) Melting point: It is one of the parameters to judge the purity of crude drugs. In case of
pure chemicals or phytochemicals, melting points are very sharp and constant. Since the crude
drugs from animal or plant origin contain the mixed chemicals, they are described with certain
range of melting point.
• Examples, Bees wax – 62-65 ºC
Cocoa butter – 30-35 ºC
(iv) Solubility: The presence of adulterant in a drug could be indicated by solubility studies.
• Castor oil is soluble only in three volumes of 90 per cent alcohol, while the adulterated
form may show good solubility in alcohol.

• Asafoetida is soluble in carbon disulphide.
• Alkaloidal bases are soluble in chloroform, while alkaloidal salts are soluble in polar solvent.
(vii) Ash Values and Extractives As mentioned earlier, evaluation of drug basically needs its
identification and can be done by morphological or microscopic characters.
Many a time, even if the drug is identified is of substandard quality due to either faulty
collection or incorrect storage.
Thus, to prove its acceptability as a drug, the following tests can be applied to it, wherever
possible.
(a) Ash content The residue remaining after incineration is the ash content of the drug, which
simply represents inorganic salts, naturally occurring in drug or adhering to it or deliberately
added to it, as a form of adulteration

Physiological ash: Total ash of the drug is inclusive of Physiological ash as well as non
physiological ash.
• Physiological ash is derived from the plant tissues while non physiological ash consists of
residue of the extraneous matter, sand, soil, etc. adhering to the herb itself.
• For determining ash, the powdered drug is incinerated to burn out all organic matter.
• Ash value is a criterion to judge the identity or purity of crude drugs.
• Total ash usually consists of carbonates, oxides, phosphates silicates and silica.
• Ex., Aloes – 5.0% w/w
Clove – 7.0% w/w
Ginger – 6.0%w/w

References-
• Pharmacognosy by CK Kokate, AP Purohit, SB Gokhale, Nirali Publication,
Pg. 6.1 – 6.46

QUALITY CONTROL OF DRUGS OF NATURAL ORIGIN

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