Unit 1

BASIC TEST FOR DRUGS

PHARMACEUTICAL SUBSTANCES

1. AMIKACIN SULPHATE

Description

• A white to yellowish white, crystalline powder, almost odorless.

Procedure

• To the solution of 10mg of amikacin sulfate add Sodium hydroxide and mix, then add cobalt
(II) nitrate a violet colour is produced.

• To a solution of 0.05 gm of amikacin sulfate add Anthrone slowly a Bluish violet colour is
produced.

• To the solution of 20 mg of amikacin sulfate add barium chloride a white precipitate is

produced which is practically insoluble in HCL.

2 .CAPTOPRIL

Description

• A white or almost white, crystalline powder; odour, characteristic but faint.

Procedures

• Dissolve 10 mg in hydrochloric acid and add about 1ml of iodine colour of the iodine
disappears immediately and a white turbid solution is produced.
• Dissolve 10 mg in water and add 0.5 ml of lead acetate a white precipitate is produced.
• Dissolve 10 mg in ethanol, add 0.5 ml of tetramethyl ammonium hydroxide/ethanol and
shake. Then add 0.5 ml of triphenyltetrazolium chloride/ethanol and shake again
red color is produced.
3. MAGNESIUM SULFATE

Description

• Brilliant, colourless crystals or a white, crystalline powder; odorless; effloresces in warm and
dry air.

Procedure

• Dissolve 10 mg in 2 ml of water and add 1 ml of ammonia;a white precipitate is produced. Add

1 ml of ammonium chloride; the precipitate dissolves. Add 1 ml of disodium hydrogen
phosphate; a white precipitate is produced.

• Dissolve 10 mg in water and add 3 drops of titan yellow and 2 ml of sodium hydroxide
a distinct pink colour is produced.

• Dissolve 0.05 g in water. Add 1 ml of hydrochloric acid and 1 ml of barium chloride

a white precipitate is produced.

4.VINBLASTINE SULFATE

Description

• A white to slightly yellow, amorphous or crystalline powder; hygroscopic.

• This substance is very toxic and should be handled with care. Melting point: About 200°C.

Procedure

• To about 0.5 mg, add 2 drops of a solution of ceric ammonium sulphate in phosphoric acid
a purplish red colour is produced which darkens with time.

• To about 1 mg, add a freshly prepared 10 mg/ml solution of vanilin hydrochloric acid;
after about 1 minute a pink colour is produced .
• Mix about 0.5 mg with 4-dimethylaminobenzaldehyde, about of glacial acetic acid and about
sulfuric acid a reddish-brown colour is produced which changes to pink after the addition of
about 1 ml of glacial acetic acid.

• Dissolve 10 mg in water. Add 1 ml of hydrochloric acid and 1 ml of barium chloride a white

precipitate is produced which is practically insoluble in hydrochloric acid.

5.LEVAMISOLE

Description

• white crystalline powder, characteristic odour.

• Melting point: about 59degree Celsius

Procedure

• dissolve 0.05g in 20 ml water

• add 1 ml NaOH , boil for 10 mins and cool
• add few drops sodium nitroprusside a red color is formed which fades with time.

MEDICINAL PLANT MATERIALS

1.IPECACUANHA ROOT
• It is the dried root and rhizome of the shrub Cephaelis Ipecuacuanha. The principal alkaloids are
emetine and cephaelin.
Description
• slightly bitter taste

Procedure

• Coarsely powder the root, mix 0.05 g with about 2 ml of hydrochloric acid and 1 drop of
hydrogen peroxide and warm the mixture. An orange colour is produced (rubremetine).
• Coarsely powder the root, mix about 0.2 g with 2 drops of ammonia and 2 ml of
dichloromethane, shake and filter.
• Evaporate to dryness about 1 ml of the filtrate, dissolve the residue in about 0.2 ml of water and
add 3 drops of potassium iodobismuthate/acetic acid an orange precipitate is produced.
• To the remaining filtrate from test 2, add 0.5 ml of ethanol and transfer to a small test-tube. Dip
a strip of filter paper into the test tube vertically and allow the solution to ascend.
• Dry the paper strip in air and expose it to iodine vapors for 30 seconds. Observe under
ultraviolet light at 365 nm; a blue fluorescence appears.

2. SENNA LEAF

• Senna leaf consists of the dried leaflets of Cassia senna L, known Alexandrian or Khartoum
Senna (C. acutifolia Delile) and Tinnevelly Sem (C. angustifolia Vahl), or a mixture of both
species.

Description

• Odour is slight; taste is first mucilaginous and sweet and then slightly bitter.

Procedure

• Before carrying out any tests. powder the leaves to a particle size that allows them to pass
through a sieve no. 45.
• Sprinkle 10 mg of the powdered leaves on the surface of about 1 ml of sulfuric acid without
stirring; within 5 minutes a greenish to brownish colour appears (other colours such as; red
indicate the presence of other species, for e.g. C. auriculata L., C. goratensis Fres.).

3. SENNA FRUIT

• Alexandrian or Khartoum Senna fruit is the dried ripe fruit of Cassia senna L (C. acutifoliaDelile)
and Tinnevelly Senna fruit is the dried ripe fruit of C. angustifolia Vahl. ripe fruit.

Description

Odour slight; taste, first mucilaginous and sweet, then slightly bitter.
Procedure

• Before carrying out any tests, crush the fruit to a fine powder.
• Sprinkle 10 mg of the powdered fruit o n the surface of about 1 ml of sulfuricacid without
stirring: within 5 minutes a greenish to brownish colour appears (other colours such as; red
indicate the presence of other species, Eg; C. auriculata L, C. goratensis Fres.).
•

4.PODOPHYLLUM RESIN

• Podophyllum resin is a mixture of resins obtained from the rhizomes and roots of the
herbaceous plant Podophyllum hexandrum Royle (P. emodi Wall) or P. peltatum L. after
percolation with ethanol and precipitation from water or very dilute acids.

Description

• Light brown to greenish-yellow or brownish-grey masses or an amorphous powder. Darkens

when exposed to light or stored at temperatures above 25°C.
• This material is very toxic and should be handled with care.
• Melting point: About 184°C.

Procedure

• Finely powder the resin, dissolve about 0.2 gm in 10 ml of potassium hydroxide; a clear,
yellow solution is formed which darkens on standing.
• Acidify with hydrochloric acid; the resin precipitates.
• Finely powder the resin, add 04 gm to 2 ml of ethanol , then add 0.5 ml of potassium
hydroxide, shake gently, and allow to stand: the resin of P. hexandrum produces a stiff
jelly whereas that of P. peltatum does not gelatinize.
• Dissolve 10 mg in 2 ml of ethanol and add 1 drop of ferric chloride; a deep, dark green
colour is produced and the solution appears black in reflected light.
• Dissolve 10 mg in 1 ml of ethanol add 4 ml of water and about 1 ml of sulfuric acid and
cool. The resin of P. hexandrum forms an orange to brownish red solution whereas,
that of P. peltatum forms a yellowish-green solution.
 DOSAGE FORMS

1.AMIKACIN SULFATE INJECTION

Description

• The injection is a sterile solution usually containing 250 mg of amikacin sulfate

in 1.0 ml of a suitable vehicle.

Preparation of the sample

• Pool the contents of the ampoules equivalent to 1.0 gm of amikacin sulfate and use directly as
test solution 1, dividing it into 2 equal volumes
• Dilute 1 volume of test solution 1 to 25 ml with water and use it as test solution 2.

Procedures

• To 3 ml of test solution 2, add 1 ml of sodium hydroxide (-80 gm/L) TS, mix and add 2 ml of
cobalt (II) nitrate ; a violet colour is produced.
• To 1 volume of test solution 1, add slowly 2 ml of anthrone ; a bluish violet colour is produced.
• T o 2 ml of test solution 2, add a few drops of barium chloride; a whit precipitate is produced
which is practically insoluble in hydrochloric acid .

2.BECLOMETASONE DIPROPIONATE INHALATION (AEROSOL)

Description

• The inhalation, supplied in a pressurized canister, contains a fine suspension of beclometasone

dipropionate in a suitable propellant, usually equivalent to 50 ug per spraydose.
Preparation of the sample

• Place 25 ml of ethanol in a small beaker and expel under the surface of the solvent 60 spray
doses equivalent to about 3 mg of beclometasone dipropionate. Use this solution as the test
solution.
• Evaporate 10 ml of the test solution and use the residue as test substance 1.
• Evaporate 15 ml of the test solution and use the residue as test substance 2.

Procedure

• Dissolve test substance 1 in about 2 ml of sulfuric acid and allow to stand for 5 minutes; a dark
reddish-brown solution is produced.
• Very cautiously pour the solution into 10 ml of water; a very light bluish grey precipitate is
produced.
• Dissolve test substance 2 in 2.0 ml of ethanol and add 1.0 ml of tetra methyl ammonium
hydroxide/ethanol and 1.0 ml of triphenyl tetrazolu chloride/ethanol , shaking thoroughly after
each addition.
• Allow to stand in the dark for 20 minutes; red color is produced.

.3. CALCIUM GLUCONATE INJECTION.

Description

• The injection is a sterile solution usually containing 100 mg of Calcium Gluconate in 1.0ml of a
suitable vehicle.

Preparation of the sample

• Dilute the contents of 1 ampoule to obtain a concentration of 10mg of calcium gluconate in 1

ml of water and use as the test solution.

Procedure

• Evaporate 1 ml of the test solution to dryness over a water bath , add 5mg of 2-naphthol
dissolved in about 1 ml of sulfuric acid and heat in a water -bath for 1 minute ;a dark blue-green
colour is produced.
• To 2 ml silver nitrate ,add ammonia, or drop by drop , until the initially formed brown
precipitate just dissolves. Add 1 ml of the test solution and heat to boiling for 1-2 minutes; a
silver mirror is produced.
• To 2 ml of the test solution , add 5 drops of ammonium oxalate ; a white precipitate is
produced . To a portion of the precipitate add a few drops of HCl ; the precipitate dissolves.
• To the remaining precipitate add a few drops of acetic acid ; the precipitate is partially
insoluble.

4. MAGNESIUM HYDROXIDE ORAL SUSPENSION.

Description

• The suspension usually contains the equivalent of 55mg of magnesium oxide in 1.0 ml of a
suitable vehicle.

Preparation of the sample

• Take a portion of the suspension equivalent to 0.20 gm of magnesium hydroxide and use
directly as test solution , dividing it into 2 equal volumes.

Procedure

• To 1 volume of the test solution, add 5 ml of HCl and filter.To the filtrate add 5 ml of
ammonium chloride , 3ml of disodium hydrogen phosphate , and 5 ml ammonia ; a white
precipitate is produced which is soluble in acetic acid.
• To 1 volume of the test solution add 5 ml of HCl and 5 ml of NaOH ; a white , gelatinous
precipitate is produced which is insoluble in an excess of NaOH .Add a few drops of iodine the
colour of the precipitate changes to dark brown.

5.PHENYTOIN TABLETS

Description

• Each tablet usually contains 50mg of Phenytion.

Preparation of the sample

• Weigh 1 tablet and calculate the amount equivalent to 0.08gm of phenytion.

• Grind the tablets , weigh out the above calculated equivalent amount as a powdered material
and use directly as the test substance, dividing it into 2 equal parts.
• Melting point: About 298 degree Celsius.

Procedure

• To 1 part of the test substance ,add 4 ml of ethanol ,shake and filter . To the filtrate add 4 drops
of cobalt (II)chloride and 1 drop of ammonia ; a blue – violet colour is produced.
• To half of 1 part of the test substance placed on a white test plate add 2 drops of ammonia and 1
small drop of copper (II) sulphate and mix thoroughly with a glass rod ; a pink precipitate is
formed.
WHO GUIDELINES FOR QUALITY CONTROL OF HERBAL DRUGS

• For the purpose of quality control of herbal drugs, the World Health Organization (WHO)has
prepared the guidelines.
• The objective of the guidelines involves general test methods and the general limits for
contaminants in herbal drugs.

A. BOTANICAL PARAMATERS

1. Sensory evaluation:

Visual microscopy, Colour,Odour, Taste, Fracture are the common tests conducted for
identification of the crude drug.

2.Foreign matter:

• It must be determined if the foreign matter is organic (Moulds, Insects, Animal excreta
etc.) or Inorganic (Stone, soil etc).
• Foreign matter is considered as material not collected from the original plant source
(insects, moulds, or other animal contamination).
• Parts of the organ, or organs from which the drug is derived other than the parts named
in the definition and description.
• Methods to determine the foreign organic matter:
1. Manual method
2. Lycopodium spore method
3. Microscopy
B.PHYSICOCHEMICAL PARAMATERS

1.Chromatographic fingerprint:

• Separation, identification, impurity detection and assay of herbal drug in the formulation or in
the extract are carried out by following methods:
➢ HPTLC
➢ HPLC/Densitometric chromatography
➢ GLC
➢ TLC
• Importance - The herbal drug shows variability in its chemical constituents according to various
locations/weather. To avoid any erroneous identification chromatographic fingerprint remains
the assessment of choice.

2.Ash values:

• The types of ash determined are Total ash, Acid insoluble and water soluble.
• Ash value is used to determine the quality and purity of the drug and to establish its identity.
• Ash contains inorganic radicals like phosphates, carbonates, and silicates of sodium, potassium,
magnesium, calcium, etc.
• These are present in definite amount in a particular crude drug, hence quantitative determination
in terms of various ash values helps in their standardization.
• Ash value is used to determine foreign inorganic matter present as impurity.

3.Total Ash Value:

• The method of total ash is designed to determinethe amount of material that remains after
ignition.
• Ash is classified as:a) physiological ash which is derived from the plant tissue itself.
b) non-physiological ash which is the residue after ignition of extraneous matter (e.g. sand and
soil).
• It is carried out at low temperatures possibly because alkali chlorides, which are volatile at low
temperatures, may be lost. The total ash consists of carbonates, phosphates, silicates and silica.
4.Acid insoluble ash:

• Sometimes, inorganic variables like calcium oxalate, silica, and carbonate content of the crude
drug affects 'Total ash value'. Such variables are removed by treating with acid (as they are
soluble in hydrochloric acid) and acid insoluble ash value is determined.
• Acid insoluble Ash, Water soluble ash and sulphated ash are also evaluated.

5. Extractive values:

• It is useful for evaluation of a crude drug.

• It gives an idea about the nature of the chemical constituents present in a crude drug.
• Useful for estimation of constituents extracted with the solvent used for extraction.
• Employed for material for which as yet no suitable chemical or biological assay exists.
• It can be done by following methods:
• Cold maceration
• hot extraction
• Ethanol

6. Moisture content and volatile matter:

• The moisture content of the drug should be minimized in order to prevent decomposition of
crude drug either due to chemical change or microbial contamination.
• The moisture content is determined by heating a drug at 105°c in an oven to a constant weight.
• E.g. Aloe should have moisture content not more than 10%w/w .
• Moisture content can be determined by following methods:
1. Gravimetric
2. Volumetric
3. Instrumental

7.Volatile oil content:

• Volatile oils are the liquid components of the plant cells, immiscible with water, volatile at
ordinary temperature and can be steam distilled at ordinary pressure.
• Many herbal drugs contain volatile oil which is used as flavoring agent.
• For the drugs containing volatile constituents, toluene distillation method/steam distillation
method is used to determine the volatile oil contents.

C.PHARMACOLOGICAL PARAMATERS

1.Bitterness value:

• Medicinal plants having strong bitter taste are therapeutically used asappetizing agents.
• The bitterness is determined by comparing the threshold bitter concentration of an extract
material with that of quinine hydrochloride .
• The bitterness valueis expressed as unit's equivalent to the bitterness of a solution containing
1gm of quininehydrochloride in 2000ml.

Method for determination: 0.1gm of quinine hydrochloride is dissolved in 100ml drinking water
and the stock solution is prepared. Then it is diluted and tested and compared withdrug.

Bitterness value in unit per gm = 2000xC+ AxB

Where,

A=concentration of stock solution

B = volume of test solution in tube with threshold bitterconcentration

C = quantity of quinine hydrochloride in the tube with threshold bitter concentration

2. Haemolytic property:

• Many medicinal plant materials, of the families Caryophyllaceae, Araliaceae, Sapindaceae,

Primulaceae, and Dioscoreaceae contain saponins
• The most characteristic property of saponins is their ability to cause haemolysis; when added to
a suspension of blood, saponins produce changes in erythrocyte membranes, causing
haemoglobin to diffuse into the surrounding medium.
• The haemolytic activity of plant materials, or a preparation containing saponins, isdetermined
by comparison with that of a reference material, saponin R, which has ahaemolytic activity of
1000 units per gram.

Method of Determination:Calculate the haemolytic activity of the medicinal plant material using
thefollowing formula:

1000 x a/b

Where,

1000= the defined haemolytic activity of saponin R in relation to ox blood

a= quantity of saponin R that produces total haemolysis (g)

b = quantity of plant material that produces total haemolysis (g)

3.Astringent property:

• It is determined by amount of tanninspresent in the drug.

• Tannins (or tanning substances) are substances capable of turning animal hides into leather by
binding proteins to form water insoluble substances that are resistant to proteolytic enzymes.
• This process, when applied to living tissue, is known as an "astringent" action and is the
reason for the therapeutic application of tannins.
• Chemically, tannins are complex substances; usually occur as mixtures of polyphenols that are
difficult to separate and crystallize.

Determination of Tannins: Calculate the quantity of tannins as a percentage using the following
formula:

[T1-(T2-T0)] 500/w

Where,

w= the weight of the plant material in grams

T1=Weight of material extracted in water

T2=Weight of material not bound to hide powder

TO=Weight of hide powder material soluble in water that bind tostandard frieberg Hide powder.

4. Swelling Index:

• The swelling index is the volume in ml taken up by the swelling of 1 g of plant material under
specified conditions.
• Its determination is based on the addition of water or a swelling agent as specified in the test
procedure for each individual plant material (either whole, cut or pulverized).
• It gives an idea about the mucilage content of the drug; hence it is useful in the evaluation of
crude drugs containing mucilage.

5. Foaming index:

• Many medicinal plant materials contain saponins that can cause persistent foam when an
aqueous decoction is shaken.
• The foaming ability of an aqueous decoction of plant materials and their extracts is measured in
terms of a foaming index.
• Calculate the foaming index using the following formula –
1000/a
Where,

a = the volume in ml of the decoction used for preparing the dilution in the tubewhere
foaming to a height of 1 cm is observed.
• Saponins give persistent foam when shaken with water. Hence, plant material/extract containing
saponins is evaluated by measuring the foaming ability in terms of foamingindex.

D.TOXICOLOGICAL PARAMETERS

1.Arsenic and heavy metals:

• Contamination of medicinal plant materials with arsenic and heavy metals can be attributed to
many causes including environmental pollution and traces of pesticides.
• There are different methods to identify the amount and concentration of heavy metals in herbal
drugs. Limit test for arsenic and Limit test for cadmium and lead are few of them.
• The contents of lead and cadmium may be determined by inverse voltametry or by atomic
emission spectrophotometry.

Determination: The following maximum amounts in dried plant materials, which are based on the
ADI values, are proposed: lead, 10 mg/kg; cadmium, 0.3 mg/kg. Stain produced onHgBr2 paper in
comparison to standard stain.

2.Pesticide residues:

Examples of pesticide residues:

1. Chlorinated hydrocarbons and related pesticides: BHC, DDT

2. Chlorinated phenoxyalkanoic acid herbicides: 2,4-D; 2,4,5-T
3. Organophosphorus pesticides: malathion, methyl parathion, parathion
4. Carbamate insecticides: carbaryl (carbaril)
5. Dithiocarbamate fungicides: ferbam, maneb, nabam, thiram, zineb

Determination of pesticides: Pesticides should not be more than 1%, an ARL (in mg of pesticide
per kg of plant material) can be calculated on the basis of the maximum acceptable daily intake of
the pesticide for humans (ADI), as, recommended by WHO, and the mean daily intake (MDI) of the
medicinal plant material.

ARL=ADIxEx60/MDIx100

Where,

ADI = maximum acceptable daily intake of pesticide (mg/kg of body weight);

E = extraction factor, which determines the transition rate of the pesticide from the plantmaterial
into the dosage form;

MDI = mean daily intake of medicinal plant product.

3.Microbial contamination:

• Contamination either at source or during processing is possible.

• Maximum possible limits of each organism are given in various texts.
 WHO limit for number of micro-organisms per gram of material

TYPE OF FINISHED PRODUCT RAW MATERIAL

MICROORGANISM
E.coli 10^1 10^4
Salmonella - -
Total aerobic bacteria 10^3 -
Enterobacteria 10^3 -

4. Aflatoxins:

• Aflatoxins are naturally occurring mycotoxins produced mainly byAspergillusflavus and

Aspergillusparasiticus.
• The presence of aflatoxins can be determined by chromatographic methods using standard
aflatoxins B1, B2, G1, G2 mixtures.
• Determination:
IP method: NMT 2 µg/kg of aflatoxins B1& Total aflatoxins 4 µg/kg
USP method: NMT 5ppb of aflatoxins B1 & Total aflatoxins 20ppb
• Radioactive contamination: The range of radionuclide's that may be released into the
environment as the result of a nuclear accident might include long-lived and short lived fission
products, actinides, and activation products. Microbial growth in herbals is usually avoided by
irradiation. This process may sterilize the plant material but the radioactivity hazard should be
taken into account.

.
 EVALUATION OF COMMERCIAL CRUDE DRUGS INTENDED FOR USE

• Evaluation of a drug means confirmation of its identity and determination of its quality,

purity and detection of the nature of adulteration.

• Evaluation of drug is necessary because -

1. To determine the biochemical variation in the drugs
2. To identify drug deterioration due to treatment and storage.
3. Substitution and adulteration, as a result of carelessness, ignorance and fraud.

Methods of drug evaluation:

1. Organoleptic or Morphological evaluation

2 Physical evaluation

3. Microscopical evaluation

4. Chemical evaluation

5 Biological evaluation

6. Analytical evaluation

1.ORGANOLEPTIC OR MORPHOLOGICAL EVALUATION

Drugs are evaluated by studying the shape, size, fracture,odour, etc. It is a preliminary examination.
Example:

• Colour: Yellowish -green colour of Indian Senna.

• Odour: Aromatic odour of Umbelliferous fruits (Fennel and Coriander).
• Shape: Conical shape of Aconite root.
• Fracture: Short fracture of Cinnamon bark, fibrous fracture of Cascara bark.
• Single and double quills: Cinnamon bark.
• Taste: Sweet taste of Liquorice root, pungent taste of Ginger
2.PHYSICAL EVALUATION
Drugs can be evaluated by various physical methods:

• Moisture content
✓ The presence of moisture in a crude drug can lead to microbial contamination and
decomposition due tothe activation of certain enzymes or the growth of microbes.
✓ Moisture content can be determined by heating the drug at 150°C in an oven to a constant
weight and calculating the weight.
✓ Aloes Not more than 10
✓ Digitalis Not more than 5.

• Viscosity
✓ It is defined as the resistance to the flow of liquid . Unit is poise and centipoise. The viscosity of
a liquid drug isconstant at a given temperature. Hence, any deviation from the viscosity value
will indicate that the drug is adulterated.
✓ Example: Glycerin viscosity is 14.9 poise at 20°C.
• Melting point
✓ It is one of the parameters to judge the purity of crude drugs containing lipids as constituents.
They may be of animal or plant origin and contain fixed oils, fats and waxes Chemicals that are
absolutely pure will have a sharp melting point.
✓ Example: Colophony shows a melting point of 75-85°C.
✓ Bees Wax shows melting point of 62-65°C.
• Solubility:
✓ Solubility of solid or liquid crude drug is fixed at a particular solvent. Any variation indicates
that the drug is adulterated.
✓ Example: Colophony soluble in light petroleum.
✓ Asafoetida is soluble in carbon disulphide.
• Optical Rotation
✓ It is the property by which the substance rotates the plane of polarized light. It is a characteristic
property of volatile oil and fixed oil. Based on the optical rotation, we can find out the purity of
drug.
✓ Example Honey +3 to -15, Caraway oil + 75° to +80°.
• Refractive Index

When a ray of light passes from one medium to another medium of different density, it bents
towards the normal. The ration of the velocity of light in a vacuum to its velocity in the substance is
the Refractive index of the second medium. Example: the Refractive index of Arachis oil is 146 to
147, Clove oil is 1.52 to 1.53.

• Ash values

Ash value is the residue that is left after the crude drug is incinerated or burnt. It indicates all the
inorganic salts of carbonates, phosphates, silicates of sodium, potassium, calcium and magnesium
present in the drug. Ash value is the criterion to judge the identity OR purity of the crude drug.

Example: Clove 7% (Total ash value), Senna 2.5% (Acid insoluble ash value).

Types of Ash values:

➢ Total ash value: Useful for detecting low-grade products exhausted products, excess of
sandy and earthy matter with drug.
➢ Acid insoluble ash value: Used for the determination of earthy matter present in roots,
rhizomes and the leaves.
➢ Sulphated ash value: It indicates the presence of sulphur in the crude drug.
➢ Water-soluble ash value: It indicates the presence ofinorganic salts in the crude drug.
• Extractive values

It indicates the active constituents present in the crude drug. Extracts are prepared by treating the
crude drug with the solvent so that, most of the active constituents get solubilize.Thepercentage of
active constituents present in the drug indicates the extractive value.
Types of extractive values

a) Water-soluble extractive values: Water-soluble extractive value is applied for the drugs which
contain water soluble constituents such as; tannins, sugars, plant acids andmucilage.

Example: Senna leaves, not less than (NLT), Aloe vera NLT 10%

(b) Alcohol soluble extractive value: Alcohol soluble extractive value is applied for the drugs which
contain alcohol soluble constituents such as; tannins, resins and alkaloids .

Example: Ginger NLT 4.5%, Asafoetida NLT 50%.

(c) Ether soluble extractive values: Ether soluble extractive value is applied for the extraction of
volatile oils, fixed oils coloring matter and resins.

Example: Clove NLT 15%, Capsicum NLT 12%.

• Volatile oil content

The effectiveness of several drugs is drug is due to their odorous principle (volatile oils). Such
crude drugs arestandardized on the basis of their volatile oil contents. The weighed quantity of the
drug is boiled with water in a round bottomed flask fitted with Clevenger apparatus. The distillate
collected is volatile oil. The amount thus obtained is recorded from the tube.

Example: Caraway NLT 2.5%, Clove NLT 15%.

• Foreign matter

In a crude drug anything else present other than the part of the drug described in the Biological
source called the foreign matter.

Example: In Senna, the Biological source is the dried leatles of Cassia angustifolia, so in the Senna
the foreign matter is the stalk.
• Swelling Index

It is defined as the volume occupied in ml by 1 gm of drug after ithas swollen in water for 4 hours.
it used only for the drugs containing gums and mucilage.

1 gram of drug together with 1 ml of alcohol (it helps inpenetration of water into the drug) and 25
ml of water transferredin a measuring cylinder. It is shaken after every 10 minute for 1hour and
then the crude drug is made to stand undisturbed for 3 hours. The volume occupied by the drug
after 4 hours is the swelling index.

Example: Swelling index of Ispaghula husk is 40 to 90, Ispaghula seed is 20 to 40.

3.MICROSCOPICAL EVALUATION

• Stomatal Number:
The average number of stomata present per square millimeter of tpidermis of the leaf is known as
stomatal number.
Example: Atropa belladonna upper epidermis is 07 to 10 and lower epidermis is 77 to 115

• Stomatal Index (SI):

It is defined as the percentage proportion of the number of stomata to the total number of
epidermal cells.
SI is calculated by:
Stomatal Index (S) = X 100
Where,
SI = Stomatal Index,
S Number of stomata per unit area,
E Number of epidermal cells.

• Vein-islet Number:
Vein-islet number is defined as number of vein-islets per square millimeter of the leaf between
margin and midrib. E.g.: Andrographis paniculata islet number is 9 to12.
• Vein-termination Number:
Vein termination the number of veinlet terminations per squa millimeter of leaf surface between
margin and midrib. Memecy umbellatum vein termination number is 7 to 9.

• Palisade ratio:
It is defined as the average number of palisade cells beneath each epidermal cells. E.g.: Atropa
belladonna 05 to 70, Digitalis lanata -2.5 to 6.5.

• Lycopodium spore method:

It is a method used to detect the percentage of foreign organic matter in case of the powdered drugs.
This method is used to evaluate if the powdered drug contains pollen grains, starch grains or spores.
Lycopodium spore method can be calculated by the following formula.

% Purity of Drug = N×W×94000×100

______________
S×M×P

Where,
N =Number of the characteristic structure,
W = Weight of Lycopodium taken (mg),
S= Number of Lycopodium spores,
M =Weight of Drug.
P =2,86,000 in case of Ginger.
4.CHEMICAL EVALUATION

Crude drugs are extracted with solvents in their increasing order of polarity (from non-polar to
polar). In this procedure, the active constituents, which are soluble with specific range of polarity,
get extracted in the solvent and the remaining marc is further extracted with the following solvent

TEST INFERENCE

Mayer's Test: Little quantity of filtrate is Yellow to off white

dissolved with few drops of Mayer's precipitate
reagent (Hgl4K2
ALKALOIDS Wagner's Test: Little quantity of filtrate is Reddish brown precipitate
dissolved with few drops of Wagner's
reagent (lodo-potassium iodide)

Dragendroff's Test: Little quantity of Red colour precipitate

filtrate is dissolved with Dragendroff's
reagent (K[BiL,] solution)

Hager's Test: Little quantity of filtrate is Yellow precipitate

dissolved with Hager's reagent (saturated
picric acid solution).

Anthraquinone Glycoside The ammoniacal layer

(Modified Borntrager's Test): Plant show cherry red or pink
extract is prepared with FeCl3 solution. color
Then it is warmed on water bath, cooled
and shaken with benzene. The layer of
benzene was discarded and treated with
NH3 solution.

Cardiac Glycoside: Pink to red colour

GLYCOSIDES Legal's Test: Treat the test extract with
pyridine, methanolic alkali and sodium
nitroprusside.

Baljet Test: Treat the test extract with Orange yellow colour
sodium picrate.
 Saponin Glycoside: 1cm foam develops
Froth Test: Add distilled water to the test
extracts (a
lcoholic or aqueous) and shake.

Steroidal and triterpenoid glycosides: Brown ring develop at the

Liebermann-Burchard's Test: Add junction of two layers
chloroform to the test extract, filter and
Add drops of acetic anhydride and
concentrated HSO4 through the walls
Of test tube.
Ferric chloride Test: Add ferric chloride Blue black colour
solution (5%) to the test extracts.
Shinoda Test: Add a few fragments of Magenta colour
PHENOLIC
Mg metal to the test extracts.Then add
COMPOUNDS
concentrated HCI
Flavanoid Test: Yellow precipitate
Lead acetate Test: Add 10% lead acetate
solution to the test extracts.
Millon's Test: Add Millon's reagent to the White precipitate turns red
PROTIEMS SND test extract on heating
AMINO ACID
Ninhydrin Test: Add ninhydrin reagent to Blue colour
the test extracts and boil.

Sain Test: Sandwich a small quantity of Oily stain on filter papper

FIXED OILS the test extract between two filter filter
AND FATS
paper.

Soap Test: Heat a small quantity of Soapy formation

the test extract with 0.5 N alcoholic KOH
solution.

Molisch's Test: Add alcoholic naphthol Violet ring at the junction

solution and concentrated H2SO4 to the
CARBOHYDRAT
ES test extracts.

Benedict's test: Add benedict's reagent to Orange red precipitate

the test extracts. Heat the mixture on a
water bath

Fehling's Test: Add dilute HCI to the test Deep red colour
extracts and heat. Add equal volumes of
Fehling's A and B solutions
5.BIOLOGICAL EVALUATION

It is employed when theDrugcannot be evaluated satisfactorily by chemical and physical method.

In this method the response produced by the test drug on a living system is compared with that of
standard preparation.

➢ Evaluation of Antidiabetic activity:

• Traditional antidiabetic drugs - MomordicacharentFenugreek and Gudmar.
• Diabetes is induced in animals by Alloxan and Streptozotocin.
• Alloxan causes necrosis of pancreatic islet-B cells which shows180-250 mg/ml fasting blood
glucose levels.
• Diabetes is induced by Streptozotocin (STZ), glucosaminenitrosoureacompound. STZ damages
pancreatic B- resulting in hypoinsulinemia andhyperglyceamia.
• Animals used: Rabbits, Rats, Mice (4 to 7 days).
• Dose: Rats-60 mg/kg mice-50 mg/kg of STZ single oral Injection, 140-180 mg/kg of alloxan for
rabbits at marginal ear vein for 7 days for rats and mice by intraperitoneal route for 2 days.
• Insulin levels are noted by tests like: RIA and ELISA
➢ Evaluation of anti inflammatory activity:
➢ Principle: Anti-inflammatory activity is the reduction of local oedema induced in rat paw by
injecting irritant inflammatory substance. Inflammation is induced bycarrageenan (1% w/v in
saline) and croton oil (1 mg/ear)
➢ Method
• Albino rats or mice are used, oedema is produced in pinna of ear with croton oil (1 mg/ear).
• After inducing herbal extract is applied to the same area.
• Oedema ismeasured by using Verniercalliper and record as 0-no effect+ve-slight, and++ve-
pronounced.
6.ANALYTICAL EVALUATION

• Chromatography is defined as a technique of isolation and identification of components or

compounds or mixture in individual components by using the stationary phase and mobile
phase.
• Plant materials are separated and purified by using various chromatographic techniques.
• Some of them are:

1.TLC-Thin layer chromatography

2.HPTLC-High performance thin layer chromatography

3.HPLC-High performance/pressure liquid chromatography

4.GLC-Gas chromatography

5.CC-column chromatography

6.Gel permeation chromatography

7.affinity chromatography

TLC:

Principle: adsorption

Application:

• It is used to separate non-volatile mixtures.

• It is used in rapid analysis of herbal extracts with minimum sample clean up requirements.

HPTLC:

Principle: adsorption

Application:

• It enables rapid analysis of herbal extracts with minimum mobile phase.

• Qualitative and quantitative analysis of herbal extracts and formulation.
GC

2 types:

• Gas liquid chromatography-

• Gas solid chromatography-

Application:

• Used in both qualitative and quantitative process

• It is most suitable for the separationand analysis of volatile compoundsuch as: essential oil and
fatty oils
• Used in analysis of dairy products for rancidity.

HPLC:

Principle: it is based on adsorption as well as partition chromatography is depending on the nature

of stationary phase(sp).

If sp is solid principle is based on adsorption

If sp is liquid principle is based on partition.

Application:

• Detection of impurities in pharmaceutical industries.

• As it is carried out under high pressure, it provided huge degree of separation and resolution.

CC

Principle: adsorption

Application:

• It is very helpful in separating compoundmixtures.

• It is used to isolate active Ingredients.