III- Physical Examination:

1- Determination of some physical parameters, such as:

a- 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 for standardizing liquid drugs.

b- Melting point:

- In case of pure phytochemicals, melting points are very sharp and constant.

- Crude drugs from plant or animal origin, containing mixed chemicals, are
described with certain range of melting points.
e.g. Colophony (m.p. 75-80 ˚C) and Cocoa butter (m.p. 30-33 ˚C).

c- Solubility:
The presence of an adulterant could be indicated by solubility studies.

e.g. pure Asafoetida is soluble in carbon disulphide.

d- Optical rotation:

- Optically active compounds have the property of rotating the plane of

polarized light. This property is known as optical rotation.

- Normally, the optical rotation is determined at 25 ˚C using sodium lamp as

the source of light, e.g. castor oil has an optical rotation from +3.5˚ to +6˚.

e- Refractive index:
- When a ray of light passes from one medium to another of different density it
undergoes refraction.

- The ratio of velocity of light in vacuum to its velocity in a substance is then

termed as refractive index of the second medium.

- It is constant for a pure drug, but varies with the wavelength of the incident
light, temperature, and pressure, e.g. Castor oil has refractive index of 1.475–
1.527.
2- Determination of foreign matter:

➢ Herbal materials should be free from contamination by moulds, insects, and

other animal contamination, including animal excreta.

➢ No abnormal odour, colour, or signs of deterioration should be detected.

➢ No poisonous or harmful foreign matter or residue should be present.

➢ Any soil, stones, sand, dust and other foreign inorganic matter must be
removed before herbal materials are cut or ground for testing.

➢ Examples of foreign matter:

a) Other parts of the herbal material or materials other than those specified.

b) Any organism, part or product of an organism.

c) Mineral admixtures that are not related to the herbal material, such as
soil, stones, sand, and dust.
3- Determination of ash content:

❑ The ash content is a measure of the total amount of minerals present in a

sample, whereas the mineral content is a measure of the amount of

specific inorganic components in a sample or food, such as Ca, Na, K.

❑ The ash remaining after ignition of herbal materials is determined by 3

different methods: total ash, acid-insoluble ash, water-soluble ash.

❑ The total ash method measures the total amount of material remaining
after ignition.

❑ Total ash includes both “physiological ash”, which is derived from the

plant tissue itself, and “non-physiological ash”, which is the residue of

the extraneous matter (e.g. sand and soil) adhering to the plant surface.
❑ Acid insoluble ash:

is the residue obtained after boiling the total ash with dilute HCl, and
igniting the remaining insoluble matter. This measures the amount of silica
present, especially as sand.

❑ Acid soluble ash = Total ash – Acid insoluble ash

❑ Water-soluble ash:

is the difference in weight between the total ash and the residue after
treatment of the total ash with water.

❑ Besides total ash, it is sometimes useful to determine the ratio of water-

soluble to water-insoluble ash as this gives an indication of the quality
of certain foods, e.g., the content of preservatives in fruits.
❑ The total ash is diluted with distilled water (or demineralized water) then
heated to nearly boiling and the resulting solution is filtered.

❑ The amount of water-soluble ash is determined by drying the filtrate and

the water-insoluble ash is determined by rinsing, drying and ashing the
filter paper.

❑ % Water Soluble Ash =

[Total Ash – Ash after washing with dist. water (= water-insoluble ash)]× 100

Weight of drug
4- Determination of extractable matter:

• It determines the amount of active constituents extracted with different

solvents from a given amount of plant material and herbal formulations.

• Extractive values of crude drugs are useful for their evaluation when their
constituents can not be readily estimated by any other means.
• Different solvents are used, e.g. water, ethanol, methanol, ethyl acetate,
hexane, etc. (like dissolves like)

• The recommended procedure:

- Hot extraction method

- Cold Maceration method

▪ Calculate the content of extractable matter in mg per g of air-dried

material.
5- Determination of moisture and volatile contents:

➢ Drying of Fresh plant material is important to:

i- Prevent growth of microorganisms.

ii- Facilitate grinding into a powder.

iii- Reduce their weight and bulk (to help transportation).

iv- Deactivate hydrolytic enzymes, thus keeping the activity (e.g. glycosides).

➢ Methods for moisture determination:

A- Direct Method:

-This method is used to determine the percentage of water in a sample by

drying a known weight of the sample to a constant weight.

- Herbal drugs should do not be decomposed by heating.

- The sample should not contain volatile material.

(W1–W2 = Moisture + Volatile content)

B- Drying over a hygroscopic material:
- Used for heat sensitive drugs or when they contain volatile constituents e.g.
drying over H2SO4 and anhydrous CaCl2.

- A hygroscopic material such as H2SO4, anhydrous CaCl2 or anhydrous Na2SO4

is used to remove the water content.
Water (moisture) content = W1‒‒W2
C- Azeotropic method (toluene distillation):

- Azeotrope: is a mixture of two or more liquids in such a way that its

components cannot be altered by simple distillation.

- Azeotropes are also called "constant boiling mixtures".

- The word azeotrope is derived from the Greek words zeo (boil) and trope
(turning) combined with the prefix a- (no) to give the overall meaning "no
change on boiling".

- Example: a mixture of benzene, cyclohexane or petroleum ether with water.

- Apparatus :

It consists of a glass flask (A) connected by a tube (D) to a cylindrical tube (B)
fitted with a graduated receiving tube (E) and a reflux condenser (C). The
receiving tube (E) is graduated in 0.1-ml divisions, so that the error of readings
does not exceed 0.05 ml.
- Procedures :

1. Introduce 200 ml of toluene and about 2 ml of water into a dry flask.

2. Distil the mixture for 2 hours, cool, and read off the volume of water (first

distillation).

3. Transfer a quantity of the material expected to give about 2–3 ml of water

(moisture) to the flask with pieces of porcelain and heat gently for 15 minutes.

4. Distil at a rate of 2 drops per second until most of water is distilled, then

increase the rate to 4 drops per second.

5. When water is completely distilled, rinse the inside of the condenser tube

with toluene.
6. Continue distillation for further 5 minutes, then stop heating, allow the

receiving tube to cool to room temperature and dislodge any droplets of water

adhering to the walls of the receiving tube by tapping it.

7. Allow the water and toluene layers to separate, then read off the volume of

water (second distillation).

8. Calculate the content of water :

Percentage of water content (moisture) = (V2 ‒ V1) x 100 / W

Where, W = weight of the material being examined (in grams)

V1= volume of water obtained in the first distillation

V2= total volume of water obtained in both distillations.

D- Loss on drying (gravimetric determination):

1- Place 2–5 g of the plant material in a dried, weighed flat bottle.

2- Dry the sample by one of the following techniques:

- In an oven at 100‒105 °C.

- In a desiccator over phosphorus pentoxide under atmospheric pressure

or reduced pressure and at room temperature.

3- Dry until two consecutive weightings do not differ by more than 5 mg.

4- Calculate the loss of weight in mg per g of the plant material.

E- Determination of moisture by chemical methods (Karl-Fisher
Method):

➢ It is a classic method that uses volumetric titration to determine trace

amounts of water in a sample.
➢ It was developed in 1935 by the German chemist; Karl Fischer.
➢ Principle:

- This reaction involves converting solid iodine into hydrogen iodide in

the presence of sulphur dioxide and water. In this reaction, a single
molecule of water reacts with one molecule of iodine.

- Methanol is most often used as the solvent. Pyridine is often used as a base to
prevent the build-up of sulphuric acid.
SO2 + I2 + H2O + 3 2
N N
HI

N Dry
H SO4-CH3 N
O 2S O
Pyridine salt of methyl CH3OH
sulphate

SO2 + I2 + 3
N
At the end point, when no water is available, the
brown colour of the reagent persists.
6- Determination of volatile oils (essential oils):

➢ Volatile oils have the ability to volatilize at room temperature.

➢ Chemically, they are mixtures of monoterpenes, sesquiterpenes, and their

oxygenated derivatives. Aromatic compounds may also occur.
➢ Volatile oil content (% v/w)= Vol. of oil (ml) × 100 /weight of plant (g.)

Clevenger apparatus
IV- Chemical Examination:

Qualitative, quantitative, and chromatography (TLC, HPLC, GC/MS).

V- Biological Examination:
A) Pharmacological Examination:

1- Determination of bitterness value:

✓ Plants with a strong bitter taste "bitters" are employed as appetizing agents.
Their bitterness stimulates GIT secretions, especially the gastric juice.
✓ The bitter properties are determined by comparing the threshold bitter
concentration of an extract with that of a dilute solution of quinine HCl.

✓ The bitterness value is expressed in unit's equivalent to the bitterness of a

solution containing one gram of quinine HCl in two liters.
2- Determination of hemolytic activity:

➢ Saponins are toxic to cold-blooded animals such as frogs and fish.

➢ In man and other warm-blooded animals, saponins are absorbed by the

intestines to only a small extent. Thus, they are not very toxic on oral

administration.

➢ Saponins are able to cause hemolysis:

when added to a suspension of blood, they produce changes in erythrocyte

membranes, causing hemoglobin to diffuse into the surrounding medium.

➢ The hemolytic activity of herbal materials is determined by comparison

with that of a reference material (a saponin with a hemolytic activity of

1000 units per gram).

➢ Procedures:

✓ A suspension of erythrocytes is mixed with equal volumes of a serial

dilution of the herbal material extract.

✓ The lowest concentration causing complete hemolysis is determined after

allowing the mixtures to stand for a period of time.

✓ A similar test is carried out simultaneously with the reference saponin.

3- Determination of foaming index:

✓ Saponins can cause persistent foam when an aqueous decoction is shaken.

✓ The foaming ability of an aqueous decoction of plant materials and their

extracts is expressed as the foaming index.

✓ Foaming index = 1000/a

a = the volume in ml of the decoction used for preparing the dilution in the
tube where foaming to a height of 1 cm is observed.
4- Determination of tannins:

➢ Tannins are complex phenolic substances, occurs usually as mixtures of

polyphenols that are difficult to separate and crystallize.

➢ Tannins (or tanning substances) are substances capable of turning animal

skins into leather by binding proteins to form water-insoluble substances

that are resistant to proteolytic enzymes.

➢ This process, when applied to living tissues, is known as an “astringent”

action that is the reason for the therapeutic applications of tannins.

➢ Determination of tannins depends on the formation of complexes with skin

powder, which can then be estimated gravimetrically.

5- Determination of swelling index:

➢ Plants containing swelling materials such as mucilage, pectin, hemicellulose

and gums show specific therapeutic values.

➢ Swelling index is the volume in ml taken up by the swelling of 1 g of plant

material.

➢ It is based on the addition of water in a glass-stoppered measuring cylinder

to the plant material.
B) Toxicological Examination:
1- Determination of pesticide residues:

➢ Herbs and herbal products should be free from pesticides or at least present within
safe limits. Such residues are determined by chromatography.

➢ Pesticide residues can accumulate in herbal drugs due to

a- spraying herbs with pesticides.

b- treatment of soils during cultivation.

c- use of fumigants during storage.

➢ Examples:

1. Pesticides containing chlorine, arsenic or lead are detected by measurement of total

organic chlorine, total arsenic and lead, respectively.

2. Insecticides containing phosphate are measured by total organic phosphorus.

3. Dithiocarbamate fungicides are detected by measurement of total bound carbon

disulfide.
2- Determination of toxic heavy metals:

➢ Contamination by heavy metals such as mercury, lead, copper, cadmium,

and arsenic in herbal drugs can be attributed to many causes, including

environmental pollution; causing serious health impacts.

➢ Determination of heavy metals is based on color reactions with special

reagents, such as thioacetamide or diethyl dithiocarbamate, in comparison

with a standard.

➢ The commonly used method is atomic absorption spectrophotometry (AAS).

C) Microbial Examination:
- Pollutants or contaminants are substances which are not allowed to be
present, or if present must be within the permissible limits. They may
produce side effects or poisoning.

- Herbal materials, particularly those with high starch content, are prone to
microbial growth, such as pathogenic bacteria and fungi.

Permissible limits of different microorganisms

Microorganism Limit

Aerobic bacteria ≤103-104 per g/ml

Yeast and moulds ≤ 102 per g/ml

E. coli n.d. per g/ml

Salmonella n.d. per g/ml

Other enterobacteria ≤ 102per g/ml

Pseudomonus aeruginos n.d.per g/ml

Staphylococcus aureus n.d. per g/ml

 Mycotoxins
❑ Mycotoxins are highly toxic secondary metabolites produced under certain
environmental conditions by some fungi that grow on many foodstuffs.
❑ Examples:

Aflatoxins:

➢ They are natural fungal metabolites produced by Aspergillus flavus and

Aspergillus parasiticus, which grow on foodstuffs, such as cereals, beans,

peas, coconuts, and dairy products.

➢ These fungi and their toxins can contaminate different types of crops during

production, harvest, storage or processing.

➢ Aflatoxins are stable to heat and different processing procedures.

➢ Examples of aflatoxins: aflatoxin B1, B2, G1, G2, M1, M2, GM1, and GM2.
 O O
O O

O O
H H

O O

O O H
H3CO H H3CO

Aflatoxin B1 Aflatoxin B2

Maize contaminated by Aspergillus flavus

➢ Exposure to aflatoxins is known to cause both acute and chronic
hepatocellular injury. They are also carcinogenic, especially aflatoxin B1.

➢ Fluoresence can be used to detect the presence of aflatoxins on crops: B1

and B2 give blue fluoresence, while G1 and G2 give green fluoresence.

➢ Carcinogenic effects of aflatoxins:

a) Aflatoxin B1 (AFB1) is oxidized in the liver into AFB1-8,9-epoxide, which is

the major hepatotoxic metabolite.

b) AFB1-8,9-epoxide is neutralized in the

liver by conjugation with glutathione by

glutathione-S-transferase (This enzyme is

abundant in some animals, e.g. mice, but

rats and humans are relatively deficient in it).

c) AFB1-8,9-epoxide reacts with DNA forming an adduct, which is resistant to
DNA repairing processes, causing gene mutations and liver carcinoma.

d) However, the liver can detoxify AFB1 by oxidizing it to other metabolites,

such as AFQ1, which has very little carcinogenic potential and is excreted in
urine with little effect on the body.
➢ Other adverse effects of AFB1-8,9-epoxide:

a) Lipid accumulation in the liver due to decreased lipid transport and reduced
oxidation.

b) Liver failure due to acute aflatoxicosis that is manifested in the form of

jaundice, ascites, portal hypertension, and liver necrosis.

➢ Other chronic effects of aflatoxins:

Immunological Suppression:

In animal models, AFB1 has been shown to impair normal immune functions,
either by reducing phagocytic activity or the number and function of T-cells.

Nutritional Interference:

A dose-dependent relationship was observed between the exposure to

aflatoxins and the rate of growth in children. These mycotoxins were also
shown to interfere with vitamins A and D in animal models.
➢ Inactivation of aflatoxins:

Inactivation of these toxins before ingestion can be achieved by:

a) A synergistic combination of gamma radiation and H2O2 to degrade them.

b) Treatment of corn with ammonia.

c) NaHSO3 soaking for corn.

d) Dichlorvos (2,2-dichlorovinyl dimethyl phosphate (DDVP)) inhibits

aflatoxin B1 biosynthesis.
❑ Treatment

After ingestion, the following should be followed:

▪ Treatment with antibiotics or other drugs has little effect.

▪ UV doses increase the rate and extent of removing AFB1 and AFB2 adducts
from DNA.

▪ Chlorophyll intervention: foods rich in chlorophyll block the bioavailability

of these carcinogens.
▪ Certain types of flavonoids found in grapefruit stimulate the microsomal
activation of aflatoxin B1 to the exo-8,9-epoxide, which is not harmful.
▪ Administration of drugs that induce hepatic detoxification enzymes (e.g.
ethoxyquin and phenobarbital).

• Induction of antibodies formation that allow the body to identify and

remove the toxin upon exposure (e.g. aflatoxin B1-lysine adduct monoclonal
antibody).