EVALUATION OF CRUDE DRUGS, MONO OR POLYHERBAL FORMULATION

 

Example: non volatile ether soluble extractive value of crude drugs

Table 7: Determination of Ether Soluble Extractive (Fixed Oil Content

Drugs

non volatile ether soluble extractive

male fern

Not less than 1.5

Linseed

Not less than 25.0

Microscopical method :
This method allows more detailed examination of drug , and it can be used to identify the organized drug by their known histological character, it is mostly used for qualitative evaluation of organized crude drug in entire and powder form.

Leaf constants:

a.  Palisade ratio:
Determination of Palisade Ratio

Palisade ratio is the average number of palisade cells under one epidermal cell. Place leaf fragments of about 5 × 5 mm in size in a test-tube containing about 5 ml of chloral hydrate solution and heat in a boiling water-bath for about 15 minutes or until the fragments become transparent. Transfer a fragment to a microscopical slide and prepare the mount of the upper epidermis in chloral hydrate solution and put a small drop of glycerol solution on one side of the cover-glass to prevent the preparation from drying. Examine with a 40x objective and a 6x eye piece, to which a microscopical drawing apparatus is attached. Trace four adjacent epidermal cells on paper; focus gently downward to bring the palisade into view and trace sufficient palisade cells to cover the area of the outlines of the four epidermal cells. Count the palisade cells under the four epidermal cells. Where a cell is intersected, include it in the count only when more than half of it is within the area of the epidermal cells. Calculate the average number of palisade cells beneath one epidermal cell, dividing the count by 4; this is the “Palisade ratio” (See Fig 1).

Figure 1: Palisade ratio

For each sample of leaf make not fewer than ten determinations and calculate the average number.

Palisade ratio =18.4/4=4.5

examples

Species

Palisade ratio

Atropa belladonna

6 to 10

Cassia angustifolia

5.1 to 7.5

Datura stramonium

4 to 7

Datura tatula

4 to 7

b. Stomatal no.:
It is average no. of stomata per sq. mm of the epidermis of the leaf ,it is effected by various factor like age of plant, size of leaf, enviourmental condition etc

Determination of Stomatal Number
Place leaf fragments of about 5x5 mm in size in a test tube containing about 5 ml of chloral hydrate solution and heat in a boiling water-bath for about 15 minutes or until the fragments become transparent. Transfer a fragments to a microscopic slide andprepare the mount the lower epidermis uppermost, in chloral hydrate solution and put a small drop of glycerol-ethanol solution on one side of the cover glass to prevent the preparation from drying. Examine with a 40 x objective and a 6x eye piece, to which a microscopical drawing apparatus is attached. Mark on the drawing paper a cross (x) for each stomata and calculate the average number of stomata per square millimeter for each surface of the leaf.

Table 8: Determination of Stomatal Number

Species

number of stomata per sq.mm


upper surface

lower surface

atropa belldona

7.5 to 10 to 17.5

77.5 to 113 to176

cassia angustifolia

180 to 200 to 223

195 to 220 to 257

c. Stomatal index:
Determination of Stomatal Index

The stomatal index is the percentage of the number of stomata formed by the total number of epidermal cells, including the stomata, each stoma being counted as one cell. Place leaf fragments of about 5 × 5 mm in size in a test tube containing about 5 ml of chloral hydrate solution and heat in a boiling water-bath for about 15 minutes or until the fragments become transparent. Transfer a fragment to a microscopic slide and prepare the mount, the lower epidermis uppermost, in chloral hydrate solution and put a small drop of glycerol-ethanol solution on one side of the cover-glass to prevents the preparation from drying. Examine with a 40x objective and a 6x eye piece, to which a microscopical drawing apparatus is attached. Mark on the drawing paper a cross (x)for each epidermal cell and a circle (o) for each stoma. Calculate the result as follows:
Stomatal index = s*100/E+S

Where S = the number of stomata in a given area of leaf ; and E = the number of epidermal cells (including trichomes) in the same area of leaf.

For each sample of leaf make not fewer than ten determinations and calculate the average index.

Examples:

Table 9: Stomatal Index

Species

Stomatal index


upper surface

lower surface

atropa belldona

2.3 to 3.9 to 10.5

20.2 to 21.7 to 23.0

cassia angustifolia

17.1 to 19.0 to 20.7

17.0 to 18.3 to 19.3

d. Vein islet number:
Determination of Vein-Islet Number
The mesophyll of a leaf is divided into small portions of photosynthetic tissue by anastomosis of the veins and veinlets; such small portions or areas are termed “Vein- Islets”. The number of vein-islets per square millimeter is termed the “Vein-Islet number”. This value has been shown to be constant for any given species and, for full-grown leaves, to be unaffected by the age of the plant or the size of the leaves. The vein-islet number has proved useful for the critical distinction of certain nearly related species.

The determination is carried out as follows :
For Whole or Cut leaves
–- Take pieces of leaf lamina with an area of not less than 4 square millimeters from the central portion of the lamina and excluding the midrib and the margin of the leaf. Clear the pieces of lamina by heating in a test tube containing chloral hydrate solution on a boiling water-bath for 30 to 60 minutes or until clear and prepare a mount in glycerol-solution or, if desired, stain with safranin solution and prepare the mount in Canada Balsam. Place the stage micrometer on the microscope stage and examine with 4x objective and a 6x eye piece. Draw a line representing 2 mm on a sheet of paper by means of a microscopical drawing apparatus and construct a square on the line representing an area of 4 square millimeters. Move the paper so that the square is seen in the centre of the field of the eyepiece. Place the slide with the cleared leaf piece on the microscope stage and draw in the veins and veinlets included within the square, completing the outlines of those vein-islets which overlap two adjacent sides of the square. Count the number of vein-islets within the square including those overlapping on two adjacent sides and excluding those intersected by the other two sides. The result obtained is the number of vein-islets in 4 square millimeters. For each sample of leaf make not fewer than three determinations and calculate the average number of vein-islets per square millimeter.

For Leaf Fragments having an area less than 4 square millimeters– Take fragments of leaf lamina each with an area of not less than 1 square millimeter, excluding the midrib and the margin of the leaf. Clear and prepare a mount as stated above. Use a 10x objective and a 6x eyepiece and draw a line representing 1 mm on a sheet of paper by means of a microscopial drawing apparatus and construct a square on this line representing an area of 1 square millimetre. Carry out the rest of the procedure as stated above. The result obtained is the number of vein-islets in 1 square millimetre. For each sample of leaf make no less than 12 determinations and calculate the average number.

Quantitative microscope:

Lycopodium spore method:
It is an important technique for powdered drug ,especially when chemical &other method fail as accurate measure of quality.lycopodium is composed of spores of lycopodium elavatum.I.each spore is tetrahedral in shape,the base is rounded and the three side wall makes the three well marked covering ridge, which join one other at filled with fixed oil. The spore are excepetional uniform in size(25µm) and the shape tetrahedral so that one can always know that a definite no. of spore present in particular weight of lycopodium.on an average 94000 spores per mg of powdered lycopodium are present.using this figure one can calculate theweight of any number of spores under any condition under the microscope.

A powdered drug is evaluated by this technique ,if its contains:
· Well defined particles may be count ed e.g. starch grain or pollen grain.
· Single layered cells or tissue,the area of which may be tracedunder suitable magnification.
· The object of uniform thickness ,the length of which can be measured under suitable magnification and actual area can be calculated

Procedure:
Determine the loss on drying of powdered sample material of both sample &lycopodium powder at 105ºc.

Mix about 100 mg powdered sample drug and 50 mg of lycopodium powder using a small flexible spatula on a glass plate ,with a little suspending fluid.

In this mixture incoroporate a sufficient quantity of suspending fluid (glycerin:mucilage of tragacanth:water::2:1:2 or an oil) until a smooth line paste results .transfer it to stoppered tube by washing with excess of suspending fluid .adjust the final volume so that about 15 to 20 spore are observed in a field using a 4 mm of objective in microscope.

Oscillate the stopered container gently in order to obtain uniformity of the suspension.place one drop of suspension on each of two slide ,spread with a thin glass rod or needle ,apply the cover slip and leave aside for few minutes on the table in order to allow the fluid mixture to settle eventely.

Count the starch chacterstics structureof sample and lycopodium spores under microscope in each of 25 different field selected for observation.

Calculate the %purity  of powdered drug by using the following equation:
% purity of crude drug=n*w*94000*100/s*m*p
N=number of charactersic structure of sample in 25 field
W=weight in mg of lycopodium
94000=number of lycopodium spore mer mg
S= number of lycopodium spore in same 25 field
M=weight in mg of sample ,calculated on the basis of sample dried at 105ºc
P=number of charcterstic sturucture of sample in 1 mg
(p is 2,86000 in case of ginger starch grain powder)

8. Qualitative tests:
a.
detection of alkaloids :
The small portion of solvent free chloroform ,alcoholic and water extract are stirred seperately eith few drop of dilute hcl and filtered .the filterate may be tested carefully with various alkaloidal reagents such as:

Table 10: Detection ofAlkoids

Reagents

Observation

Mayer reagents (pottasium mercuric iodide solution )

Cream precipitate

Dragondroff’ reagent(pottasiun bismuth iodide solution)

Redissh brown ppt.

Wagner’s reagent (iodine pottasium iodide solution)

Redissh brown ppt.

Hager’s reagents

Yellow ppt.

b. Detection of carbohydrate and glycosides:
· Small quantity of alcoholic and aq. Extracts are dissolved seperately in 5 ml of distilled water and filtered. The filterate may be subjected to molish test to detect presense of carbohydrate.
· Another small portion of extract is hydrolysed with dilute sulphuric acid for few hours in water bath and subjected to liebermann- burchard,legals,and brontrager test to detect presense of different glycosides.

c. Detection of saponins :
About 1 ml of alcoholic and aq extract is diluted seperately with disstilled water to 20 ml and shaken in graduated cylinder for 15 minutes.one cm layer of foam indicates presence of saponins.the test solution may be subjected to test for haemolysis.

d. Detection of protein and free mino acids:
Small quantity of alcoholic and aqueous extract are dissolved in few ml of water and subjected to million’s,biuret’s, and ninhydrin test.

e. Detection of volatile oil:
About 50 mg of powdered material is taken in a volatile oil estimation appartus and subjected to hydrodistilation.the disttilte is collected in graduated tube of assembely in which aq. Portion is automatically seperated from the volatile oil, if it present in the drug and returned back to disstilation flask

NOW YOU CAN ALSO PUBLISH YOUR ARTICLE ONLINE.

SUBMIT YOUR ARTICLE/PROJECT AT articles@pharmatutor.org

Subscribe to Pharmatutor Alerts by Email

FIND OUT MORE ARTICLES AT OUR DATABASE


Pages

FIND MORE ARTICLES