c. Histological measurements- This is a quantitative method of evaluation.
It involves-
- Fibre length determination.
- Fibre width determination.
- Epidermal cell area determination.
- Leaf content.

Before these determinations the tissue is disintegrated by the given method. All crude drugs are cut into small pieces before disintegration. Tissues containing a large no. of lignified characters are disintegrated by either of the following methods-

(1) Cut drug material + 5ml HNO3 + Î" to boil + KClO3 + warmâ†' when tissue appears to be almost completely bleached & shows the tendency to disintegrate, apply pressure with a glass rod to the material.
(2) Cut drug material on a small dish + Nitro chromic Acid + Î" until material breaks easily when pressed with a glass rod + wash with water + transfer to the slide + 1 drop of Glycerin & cover with cover slip.

Tissues containing very few lignified characteristic are disintegrated as follows-
Cut drug material + KOH/ NaOH + Î" on a water bath for 15-30 min. until the drug material breaks easily + liq. Is decanted & the drug material is washed with water + mount on the slide.
Then Fibre length, Fibre width etc. are measured by using- Eye piece micrometer, Stage piece micro meter, Camera Lucida.

LEAF CONTENTS- are determined as follows-
Fragments of leaves in a test tube + 5 ml Chloral hydrate + Î" on a water bath for 15min. + transfer the leaf fragments to the slide & observed under microscope using 40X & Eye piece micrometer.
STOMATAL INDEX- is found by using following equation-
S X 100
S.I. = E + S Where as -
S= no. of stomata in given area of leaf.
E= no. of epidermal cells in given area of leaf.
TYPES OF STOMATAS-
i) Anomocytic/ Rannanculous-Stomata are surrounded by a varying no. of cells, generally not different from those of epidermis.
ii) Anocytic/ Cruciferous- Stomata are usually surrounded by Âľ subsidiary cells, one of which is markedly smaller then the others.
iii) Diacytic/ Caryophyllaceous- Stomata are accompanied by two subsidiary cells, the common wall of which is at right angle to stomata.
iv) Paracytic/ Rubiaceous- Stomata has two subsidiary cells of which long axis are parallel to axis of stomata.

(2) PHYSIOCHEMICAL EVALUATION

(1) TLC- Qualitative analysis of any compound/ impurities present in very small amt.
Parameter to notice-
i) Type of Adsorbent- used is coated as thin layer on plate. E.g. Silica gel.
Stationary Phase-
ii) Mobile Phase- passes through stationary phase (sp).
iii) Stationary Phase Activation- 1100 C - 30 min.
iv) Method used may be ascending/ descending.
v) Method & Conc. Used- to preparation standard & sample.
vi) Volume of sample used on plate (sp).
vii) Detecting reagent.
viii) Rf value.

(2) Ash Value-Drug is ignited up to 5000C- 6000C & converted to ash.

Total Ash- PO43- , CO32- , Silicates (SiO2), Silica.
Wt. of crude drug before ignition.
Wt. of ash after ignition (remaining).
Acid insoluble Ash-Dirt & Sand.
Ash + 25 ml HCl, Î" to boil for 5 min.â†' filter through ash less filter paperâ†' ignite the filter paper at â†' temp.â†' coolâ†' weightâ†' Acid insoluble ash value.
Water Soluble Ash-Ash remaining after ignition of crude drug + water + Î" to boil + filter through ash less filter paper + ignite the filter paper at â†' temp.â†' coolâ†' weight.
Water soluble ash = Total ash- W% of water insoluble sub.
Remaining on the ash less filter paper.

(3) Extractive Value-Determination of amt. of active constituents extracted with solvent from a given amt. of medicinal material. Done for those where no. other method of assay is available.
a. Hot Water Extraction- 4gm drug + 100 ml H2Oâ†' reflux for 1 hrâ†' coolâ†' wt. & replace to the original total wt. with solventâ†' shake well, filter through dry filter paper; 25 ml of the filtrate into the dishâ†' evaporate to dryness on water bathâ†' dry at 1050C for 6 hrsâ†' cool in a designator for 30 min.â†' wt.â†' calculate the extractable matter in manufacturing of air dried material.
b. Cold Extraction- drug + maceration + 100 ml solvent for 6 hrsâ†' allow to stand for 18 hrs + solventâ†' filter & evaporate to dryness at 1050C for 6 hrsâ†' cool in a designator for 30 min.â†' wt.â†' calculate the extractable matter in manufacturing of air dried material.

(4) Moisture Content- excess of water in medicinal plants will encourage the microbial growth therefore it has to be control & therefore moisture content determination is an imp. parameter, done by two methods-
a. Loss on drying- 2-5 Gms of drug dried in oven at 1050C for 1 hrsâ†' cool in a designatorâ†' wt.â†' again dry at same condition for ½ hrâ†' wt.â†' continue the process till uniform wt. is achieved.
LOD = wt. before drying- wt. after drying.
b. Azotropic methods- Also called Toluene distillation method. Drug is mixed with Xylene or Toluene & distilled. Water present in the drug get distilled out with the solvent. The total moisture content is calculated as-
% Water Content = 100 (n1-n)/ W

Where as-
W= wt. of drug material in gms
n =no. of ml of H2O obtained after 1st distillation.
n1 = no. of ml of H2O obtained after both distillation.

(5) Vol. Oil Content- Done by using Clevenger's apparatus. Drug + immiscible solvent (Xylene + Toluene) â†' Azotropic mixtureâ†' distillationâ†' water content present in the drug + insolubleâ†' distilled off in receiving beakerâ†' determine total oil content.

(3) PHARMACOLOGICAL EVALUATION

i) Bitterness Value- Done only for those drugs which have a strong bitter taste & used for their bitterness property. The bitter property of the drug is determined by comparing the threshold bitter conc. Of an extract of the material with that of dil. Solution of quinine HCl.
Bitter value is expressed in units' equivalent to bitterness of solution containing 1gm of quinine HCl in 2000ml. Conc. of quinine HCl mentioned is 0.04-0.058 mg/ ml in 9 different test tubes.
Calculate of bitterness value in units/ gm using- 2000 x C
Where, a = conc. of stock solutn. a x b
b = vol. of sample in t.t. with threshold bitter conc.
c = conc. of quinine HCl in t.t. with the threshold bitter conc.

ii) Haemolytic Activity- Done for drugs belonging to the family Caryophyllaceae, Araliaceae, Dioscoreaceae. The drugs' belonging to these families contains saponins. Saponins have the ability to cause haemolysis.
Preliminary Test- plant extract is taken in 4 diff. t.t. in 0.1, 0.2, 0.5 & 1 ml volume + PO4-buffer (at ph 7-9) + 2% Citrated bloodâ†' check the t.t. in which haemolysis occurs.
Haemolysis occurred in t.t. with 0.1 ml extractâ†' main test done for the original plant extract.
Haemolysis occurs in the t.t. with 0.5 ml extractâ†' 2 fold dil. Is done & extract with PO4-buffer.
Total haemolysis occurs in all t.t.â†' 5 fold dilutions. If after 6hrs all 4 t. tubes contain clear red solutionâ†' prepare 10 fold dilutions.
Main Test- 13 diff. t.t. are taken where the ext. of reqd. dilution is taken in diff. amt. + PO4- buffer + 2 % citrated blood + stand for 24 hrs & then check for haemolysis.
Haemolytic Index = 1000 x a/b where a= qty. of saponins that produces total haemolysis, b= qty. of plant material that produces total haemolysis, and 1000= Haemolytic activity of saponins in relation to OX blood.

iii) Astringency-Done for substance containing tannins. It is determined by finding the tanning property. Tannins are one the substance capable of turning animal hide into leather by binding proteins to form water insoluble substance. i.e. that are resistant to proteolytic enzyme.
Plant material taken in a t.t. + heat for 30 min. + transfer to 250 ml volumetric flask + make up the volume with water + keep standing till solid substance settles down + filter + 50 ml filtrate + evaporate to dryness + wt. (T1); 80 ml plant extract + 6 gms hide powder + shake for 60 min. + filter + 50 ml filtrate + evaporate to dryness + wt. (T2); 80 ml water + 6 gms hide powder + shake for 60 min. + filter + 50 ml filtrate + evaporate to dryness + wt. (T0 ).
% Tannic content = [T1-(T2-T0)] x 500 / W where W = wt. of plant material in gms.

iv) Swelling Index-Done for Hemi cellulose, Pectin, Mucilage. The swelling index is the volume in ml taken up by swelling of 1gm of plant material under specific condition.
Swelling agent water as specified for individual plant material. 50ml volumetric flask + 25ml water in a upward direction + shake after 10 min. after 1 hr. + stand for 3 hr. + measured the volume in ml occupied by the extract.

v) Foaming Index- Foaming ability of an aqueous decoction of plant material & their extract is measured in terms of foaming index. 1gm drug + 100 ml boiled water + heat 30 min. + cool & filter into a 100 ml vol. flask + water to make up volume.
Now pore this solutn into 10 diff. stoppered t.t. Diff. vol. i.e. 1ml to 10 ml + adjust the vol. to 10 ml with water + shake well for 15 sec. + allow to stand for 15 min. + measure the height of foam.
- If ht. of the foam in every t.t. < 1 cm â†'then foaming index will be < 100
- If ht. of the foam is 1 cm in any t.t. â†' then foaming index will be & If this is the
1st & 2nd t.t. of the series then intermediate dilution is prepared in similar manner.
- If ht. of the foam in every t.t. > 1 cm â†'then foaming index will be over 1000.
1000/a where a = vol. of decoction in that tube.

About Author / Additional Info:
M.pharma in cognosy and M.Sc in Chemistry