SEC_II (Herbal Technology)

Unit_4
(Analytical Pharmacognosy)

Analytical Pharmacognosy: Drug adulteration- types, methods of drug evaluation, biological testing of
herbal drugs, Phytochemical screening tests for secondary metabolites (alkaloids, flavonoids, steroids,
triterpenoids, phenolic compounds).

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Drug adulteration- types, methods of drug evaluation.

The term adulteration is defined as substituting original crude drug partially or wholly
with other similar-looking substances. The substance, which is mixed, is free from or inferior in
chemical and therapeutic property.

Types of Adulterants

Adulteration in simple terms is debasement of an article. The adulteration is done

deliberately, but it may occur accidentally in some cases. Adulteration involves different
conditions such as deterioration, admixture, sophistication, substitution, inferiority and spoilage.
Deterioration is impairment in the quality of drug, whereas admixture is addition of one article
to another due to ignorance or carelessness or by accident. Sophistication is the intentional or
deliberate type of adulteration. Substitution occurs when a 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.

Unintentional Adulteration

Unintentional adulteration may be due to the following reasons:

1. Confusion in vernacular names between indigenous systems of medicine and local

dialects.

2. Lack of knowledge about the authentic plant.

3. Nonavailability of the authentic plant.

4. Similarity in morphology and or aroma.

5. Careless collection.

6. Other unknown reasons.

Name confusion:

In ayurveda, ‘Parpatta’ refers to Fumaria parviflora. In siddha, ‘Parpadagam’ refers

to Mollugo pentaphylla. Owing to the similarity in the names in traditional systems of medicine,
these two herbs are often interchanged or adulterated or substituted. Because of the popularity of
siddha medicine in some parts of south India, traders in these regions supply M. pentaphylla as
Parpatta/Parpadagam and the north Indian suppliers supply F. parviflora. These two can be
easily identified by the presence of pale yellow to mild brown-coloured, thin wiry stems and
small simple leaves of M. pentaphylla and black to dark brown-coloured, digitate leaves with
narrow segments of F. parviflora.

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Lack of knowledge about authentic source:

‘Nagakesar’ is one of the important drugs in ayurveda. The authentic source is Mesua
ferrea. However, market samples are adulterated with flowers of Calophyllum inophyllum.
Though the authentic plant is available in plenty throughout the Western Ghats and parts of the
Himalayas, suppliers are unaware of it. There may also be some restrictions in forest collection.
Due to these reasons, C. inophyllum (which is in the plains) is sold as Nagakesar. Authentic
flowers can be easily identified by the presence of two-celled ovary, whereas in case of spurious
flowers they are single celled.

Similarity in morphology

Mucuna pruriens is the best example for unknown authentic plant and similarity in
morphology. It is adulterated with other similar papilionaceae seeds. M. utilis (sold as white
variety) and M. deeringiana (sold as bigger variety) are popular adulterants. Apart from this, M.
cochinchinensis, Canavalia virosa and C. ensiformis are also sold in Indian markets. Authentic
seeds are up to 1 cm in length with shining mosaic pattern of black and brown colour on their
surface. M. deeringiana and M. utilis are bigger (1.5–2 cm) in size. M. deeringiana is dull
black, whereas M. utilis is white or buff coloured.

Lack of authentic plant

Hypericum perforatum is cultivated and sold in European markets. In India, availability

of this species is very limited. However, the abundant Indo-Nepal species H. patulum is sold in
the name of H. perforatum. Market sample is a whole plant with flowers, and it is easy to
identify them taxonomically. Anatomically, stem transverse section of H. perforatum has
compressed thin phloem, hollow pith and absence of calcium oxalate crystals. On the
otherhand, H. patulum has broader phloem, partially hollow pith and presence of calcium
oxalate crystals.

Similarity in colour

It is well known that in course of time, drug materials get changed to or substituted with
other plant species. ‘Ratanjot’ is a recent-day example. On discussion with suppliers and Non
Timer Forest Product (NTFP) contractors, it came to be known that in the past, roots
of Ventilago madraspatana were collected from Western Ghats, as the only source of ‘Ratanjot’.

Careless collections

Some of the herbal adulterations are due to the carelessness of herbal collectors and
suppliers. Parmelia perlata is used in ayurveda, unani and siddha. It is also used as grocery.
Market samples showed it to be admixed with other species (P. perforata and P. cirrhata).
Sometimes, Usnea sp. is also mixed with them. Authentic plants can be identified by their thallus
nature.

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Unknown reasons

‘Vidari’ is another example of unknown authentic plant. It is an important ayurvedic

plant used extensively. Its authentic source is Pueraria tuberosa, and its substitute is Ipomoea
digitata. However, market samples are not derived from these two. It is interesting to know that
an endangered gymnosperm Cycas circinalis is sold in plenty as Vidari. The
adulterated materials originated from Kerala, India. Although both the authentic plant and its
substitute are available in plenty throughout India.

Intentional Adulteration

Intentional adulteration may be due to the following reasons:

1. Adulteration using manufactured substances

2. Substitution using inferior commercial varieties

3. Substitution using exhausted drugs

4. Substitution of superficially similar inferior natural substances

5. Adulteration using the vegetative part of the same plant

6. Addition of toxic materials

7. Adulteration of powders

8. Addition of synthetic principles

Adulteration using manufactured substances

In this type of adulteration the original substances are adulterated by the materials that are
artificially manufactured. The materials are prepared in a way that their general form and
appearance resemble with various drugs. Few examples are cargo of ergot from Portugal was
adulterated with small masses of flour dough moulded to the correct size and shape and coloured,
first using red ink, and then into writing ink.

Substitution using inferior commercial varieties

In this type, the original drugs are substituted using inferior quality drugs that may be
similar in morphological characters, chemical constituents or therapeutic activity. For example
hog gum or hog tragacanth for tragacanth gum, mangosteen fruits for bael fruits, Arabian senna,
obovate senna and Provence senna are used to adulterate senna, ginger being adulterated with
Cochin, African and Japanese ginger. Capsicum annuum fruits and Japanese chillies are used
for fruits of C. minimum.

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Substitution using exhausted drugs

In this type of substitution the active medicaments of the main drugs are extracted out
and are used again. This could be done for the commodities that would retain its shape and
appearance even after extraction, or the appearance and taste could be made to the required state
by adding colouring or flavouring agents. This technique is frequently adopted for the drugs
containing volatile oils, such as: clove, fennel etc. After extraction, saffron and red rose petals
are recoloured by artificial dyes.

Substitution of superficially similar inferior natural substances

The substituents used may be morphologically similar but will not be having any relation
to the genuine article in their constituents or therapeutic activity. Ailanthus leaves are substituted
for belladona, senna, etc. saffron admixed with saff flower; peach kernels and apricot kernels for
almonds; clove stalks and mother cloves with cloves; peach kernel oil used for olive oil; chestnut
leaves for hamamelis leaves and Japan wax for beeswax are few examples for this type of
adulteration.

Adulteration using the vegetative part of the same plant

The presence of vegetative parts of the same plant with the drug in excessive amount is
also an adulteration. For example, epiphytes, such as mosses, liverworts and lichens that grow
over the barks also may occur in unusual amounts with the drugs, e.g. cascara or cinchona.
Excessive amount of stems in drugs like lobelia, stramonium, hamamelis leaves, etc. are few
example for this type of adulteration.

Addition of toxic materials

In this type of adulteration the materials used for adulteration would be toxic in nature. A
big mass of stone was found in the centre of a bale of liquorice root. Limestone pieces with
asafetida, lead shot in opium, amber-coloured glass pieces in colophony, barium sulphate to
silvergrain cochineal and manganese dioxide to blackgrain cochineal, are few examples in this
adulteration.

Adulteration of powders

Powdered drugs are found to be adulterated very frequently. Adulterants used are
generally powdered waste products of a suitable colour and density. Powdered olive stones for
powdered gentian, liquorice or pepper; brick powder for barks; red sanders wood to chillies;
dextrin for powdered ipecacuanha, are few adulterants.

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Addition of synthetic principles

Synthetic pharmaceutical principles are used for market and therapeutic value. Citral is
added to lemon oil, whereas benzyl benzoate is added to balsam of Peru. Apart from these, the
herbal products labelled to improve sexual performance in men, when analysed, contained
sildenafil. Brand names included Actra-Rx and Vasx,

Biological Methods of Drug Evaluation

When physical or chemical methods of evaluation or quantity of sample are very less are
not able to produce satisfactory result in drugs then the drugs are evaluated by biological
methods of evaluation. These methods are performed on living animals, isolating living organ
and tissue, animal preparation and micro-organism.

Following method is used as:

1. Anti-inflammatory activity of drugs.

2. Antipyretic activity of drugs.
3. Anti-diabetic activity of drugs.
4. Analgesic activity of drugs.
5. Antiulcer activity of drugs.
6. Anthelmintic activity of drug: they are performed on earth-worms.
7. Cardiac activity of drug: the cardiac glycosides are evaluated on frog and pigeon.
8. Microbiological methods: the living bacteria, yeast and molds are used for assaying vitamins
and to determine the activity of antibiotic drugs.

Anti-inflammatory activity of drug:

A. Paw edema method:

Male or female Sprague-Dawley rats with a body weight between 100 and 150 g are
used. The animals are starved overnight. To insure uniform hydration, the rats receive 5 ml of
water by stomach tube (controls) or the test drug dissolved or suspended in the same volume.
Thirty minutes later, the rats are challenged by a subcutaneous injection of 0.05 ml of 1%
solution of carrageen an into the plantar side of the left hind paw. The paw is marked with ink at
the level of the lateral malleolus and immersed in mercury up to this mark.

The paw volume is measured plethysmographically immediately after injection, again 3

and 6 h, and eventually 24 h after challenge. The increase of paw volume after 3 or 6 h is
calculated as percentage compared with the volume measured immediately after injection of the
irritant for each animal.

Effectively treated animals show much less edema. The difference of average values
between treated animals and control groups is calculated for each time interval and statistically
evaluated. The differences at the various time intervals give some hints for the duration of the

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anti-inflammatory effect. A dose-response curve is run for active drugs and ED50 values can be
determined.

B. Cotton wool granuloma method:

Male Wister rats with an average weight of 200 g are anaesthetized with ether. The back
skin is shaved and disinfected with 70% ethanol. An incision is made in the lumbar region. By a
blunted forceps subcutaneous tunnels are formed and a sterilized cotton pellet is placed on both
sides in the scapular region.

The pellets are either standardized for use in dentistry weighing 20 mg or pellets formed
from raw cotton, which produce a more pronounced inflammation than bleached cotton. The
animals are treated for 7 days subcutaneously or orally.

Then, the animals are sacrificed, the pellets prepared and dried until the weight remains
constant. The net dry weight, i.e. after subtracting the weight of the cotton pellet is determined.
The average weight of the pellets of the control group as well as of the test group is calculated.
The percent change of granuloma weight relative to vehicle control group is determined.

Antipyretic activity of drug:

Rabbits of both sexes and of various strains with a body weight between 3 and 5 kg can
be used. The animals are placed into suitable cages and thermocouples connected with an
automatic recorder are introduced into the rectum. The animals are allowed to adapt to the cages
for 60 min.

Then 0.2 ml/kg containing 0.2 µg lipopolysaccharide are injected intravenously into the
rabbit ear. Sixty min later the test compound is administered either subcutaneously or orally.
Body temperature is monitored for at least 3 hrs.

A decrease of body temperature for at least 0.5°C for more than 30 min as compared with
the temperature value before administration of the test compound is regarded as positive effect.
This result has been found after 45 mg/kg phenylbutazone subcutaneous or 2.5 mg/kg
Indomethacin subcutaneous.

Antidiabetic activity of drugs:

A. Streptozotocin induced diabetes:

Male Wister rats weighing 150-220 g fed with a standard diet are injected with 60 mg/kg
streptozotocin (Calbiochem) intravenously. As with alloxan, three phases of blood glucose
changes are observed. Initially, blood glucose is increased, reaching values of 150-200 mg%
after 3 h. Six-eight h after streptozotocin, the serum insulin values are increased up to 4 times,
resulting in a hypoglycemic phase which is followed by persistent hyperglycemia. Severity and
onset of diabetic symptoms depend on the dose of streptozotocin.

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 After the dose of 60 mg/kg intravenous. Symptoms occur already after 24-48 h with
hyperglycemia up to 800 mg%, glucosuria and ketonemia. Histologically, the beta-cells are
degranulated or even necrotic. A steady state is reached after 10-14 days allowing using the
animals for pharmacological tests.

Alloxan induced diabetes: Rabbits weighing 2.0 to 3.5 kg are infused via the ear vein
with 150 mg/kg alloxan monohydrate (5.0 g/100 ml, pH 4.5) for 10 min resulting in 70% of the
animals to become hyperglycemic and uricosuric. The rest of the animals either die or are only
temporarily hyperglycemic.

Analgesic activity of drug:

A. Hot Plate Method:

Groups of 10 mice of either sex with an initial weight of 18 to 22 g are used for each
dose. The hot plate, which is commercially available, consists of a electrically heated surface.
The temperature is controlled for 55° to 56°C. This can be a copper plate or a heated glass
surface. The animals are placed on the hot plate and the time until either licking or jumping
occurs is recorded by a stop-watch. The latency is recorded before and after 20, 60 and 90 min
following oral or subcutaneous administration of the standard or the test compound.

B. Tail immersion test:

Young female Wister rats (170-210 g body weight) are used. They are placed into
individual restraining cages leaving the tail hanging out freely. The animals are allowed to adapt
to the cages for 30 min before testing. The lower 5 cm portion of the tail is marked. This part of
the tail is immersed in a cup of freshly filled water of exactly 55°C. Within a few seconds the rat
reacts by withdrawing the tail. The reaction time is recorded in 0.5 s units by a stopwatch. After
each determination the tail is carefully dried.

The reaction time is determined before and periodically after either oral or subcutaneous
administration of the test substance, e.g., after 0.5, 1, 2, 3, 4 and 6 h. The cut off time of the
immersion is 15 s. The withdrawal time of untreated animals is between 1 and 5.5 s. A
withdrawal time of more than 6 s therefore is regarded as a positive response.

C. Heffner’s tail clip method:

An artery clip is applied to the root of the tail of mice and the reaction time is noted. Male
mice (Charles River strain or other strains) with a weight between 18 and 25 g are used. The
control group consists of 10 mice. The test compounds are administered subcutaneously to fed
mice or orally to fasted animals. The test groups and the control group consist of 7-10 mice.
The drug is administered 15, 30 or 60 min prior testing. An artery clip is applied to the root of
the tail (approximately 1 cm from the body) to induce pain. The animal quickly responds to this
noxious stimulus by biting the clip or the tail near the location of the clip. The time between
stimulation onset and response is measured by a stopwatch in 1/10 seconds increments.

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Antiulcer activit:

A. Pylorus ligation in rats (Shay Rat):

Female Wister rats weighing 150-170 g are starved for 48 h having access to drinking
water ad libitum. During this time they are housed single in cages with raised bottoms of wide
wire mesh in order to avoid cannibalism and coprophagy. Ten animals are used per dose and as
controls. Under ether anesthesia a midline abdominal incision is made. The pylorus is legated,
care being exercised that neither damage to the blood.

No supply or traction on the pylorus occurs. Grasping the stomach with instruments is to
be meticulously avoided; else ulceration will invariably develop at such points. The abdominal
wall is closed by sutures. The test compounds are given either orally by gavages or injected
subcutaneously.

The animals are placed for 19 h in plastic cylinders with an inner diameter of 45 mm
being closed on both ends by wire mesh. Afterwards, the animals are sacrificed in
CO2 anesthesia. The abdomen is opened and a ligature is placed around the esophagus close to
the diaphragm.

The stomach is removed, and the contents are drained in a centrifuge tube. Along the
greater curvature the stomach is opened and pinned on a cork plate. The mucosa is examined
with a stereo micro-scope. In the rat, the upper two fifths of the stomach form the rumen with
squamous epithelium and possess little protective mechanisms against the corrosive action of
gastric juice.

Below a limiting ridge, in the glandular portion of the stomach, the protective
mechanisms are better in the mucosa of the medium two fifths of the stomach than in the lowest
part, forming the antrum.

Phytochemical screening tests for secondary metabolites (alkaloids, flavonoids, steroids,

triterpenoids, phenolic compounds)/ Qualitative analysis of phytochemicals of both fungus
infected and non-infected plant extracts.

Preliminary phytochemical analysis of ethanolic extracts of both fungus infected and

non-infected plants were carried out for the evaluation of presence or absence of the
phytochemicals such as phenolic compounds, flavonoids, steroids, triterpenoids and alkaloids.
Both extracts showed the presence of phenolic compounds, flavonoids, steroids, triterpenoids
and alkaloids.

These are the following Phytochemical screening tests for secondary metabolites:

1. Test for Alkaloids:

Extracts were dissolved individually in dilute HCl and filtered

(a) Wagner’s test:

To about 1-2 mL of the filtrate, 2 mL of Wagner’s reagent was added. Reddish brown
coloured precipitate indicates the presence of alkaloids.
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 (b) Mayer’s test:

To 1.0 mL of the filtrate, 2 mL of the reagent was added. Formation of white or pale
precipitate shows the presence of alkaloids.

(c) Dragendroff’s test:

The filtrate was treated with Dragendorff’s reagent and the formation of orange
precipitate indicates the presence of alkaloids.

2. Test for Flavonoids

(a) Ammonia test:

5 mL of dilute ammonia solution were added to a portion of the crude extract followed by
addition of concentrated H2SO4. Formation of a yellow colouration in the extract indicates
the presence of flavonoids. The yellow colouration disappears after some time.

(b) Shinoda’s test:

The extracts were dissolved in 5 mL of (95%) ethanol. To this, a piece of magnesium

followed by concentrated hydrochloric acid was added drop wise and heated. Appearance of
magenta colour shows the presence of flavonoids.

(c) Zinc–hydrochloride test:

To the extract, a pinch of zinc dust was added followed by addition of concentrated
hydrochloric acid along the sides of the test tubes. Appearance of magenta color indicates the
presence of flavonoids.

(d) Lead acetate test:

The extract was treated with a few drops of lead acetate solution. Formation of yellow
precipitate indicates the presence of flavonoids. Orange to crimson colour shows the
presence of flavonones.

(e) Alkaline reagent test:

The extract was treated with a few drops of sodium hydroxide. Formation of intense
yellow colour, which becomes colour less on addition of few drops of dilute acid, indicates
the presence of flavonoids.

(f) Ferric chloride test:

To the extract, a few drops of neutral ferric chloride solution was added, a blackish
red colour forms, indicating the presence of flavonoids.

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 3. Test for Steroids:

(a) Acetic anhydride test:

2 ml of acetic anhydride was added to 0.5 mL crude extract of plant sample with 2
mL H2S04. The change in colouration from violet to blue or green in samples indicates the
presence of steroids.

(b) Liebermann-Burchard Test:

The extracts were dissolved in 2 mL of chloroform to which 10 drops of acetic acid and
five drops of concentrated sulphuric acid were added and mixed. The change of red colour
from blue to green indicates the presence of steroids.

4. Test for triterpenoids:

(a) Salkowski test:

5 ml of the extract was added to chloroform along with a few drops of conc. sulphuric
acid. The mixture was shaken well and kept aside for some time. Appearance of red colour
in the lower layer indicates the presence of steroids and the formation of yellow colour in the
lower layer indicates the presence of triterpenoids.

5. Test for phenolic compounds:

(a) Ferric chloride test:

A little extract was dissolved in distilled water. To this, 2 mL of 5% ferric chloride

solution was added. Formation of blue, green or violet colour indicates the presence of
phenolic compounds.

(b) Lead acetate test:

A little extract was dissolved in distilled water. To this, a few drops of lead acetate
solution was added. Formation of white precipitate indicates presence of phenolic
compounds.

(c) Dilute iodine solution test:

To 2-3 mL of extract, a few drops of dilute iodine solution was added. Formation of
transient red colour indicates the presence of phenolic compounds.

Prepared by
Dr. S. A. Kowser Alam Laskar
Department of Botany
M.H.C.M.Sc. College, Algapur
08/08/21 [04:55AM]
Haflong

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