A PRACTICAL HANDBOOK

Of

PHARMACOGNOSY

FOR BACHELOR OF PHARMACY STUDENTS

(As Per PCI Syllabus)

FIRST EDITION
(2023)

Authors

SURENDAR KUMAR
M Pharmacy (Pharmacognosy)

SHIWANK RANA
M Pharmacy (Pharmacology)

PANSHUL SHARMA
M Pharmacy (Pharmacognosy)
NOTION PRESS
India. Singapore. Malaysia.

Published by Notion Press 2023

Copyright © <Shiwank Rana; Surendar Kumar; Panshul Sharma> 2023
All Rights Reserved.

This book has been published with all reasonable efforts taken to make the material error-
free after the consent of the author. No part of this book shall be used, reproduced in any
manner whatsoever without written permission from the author, except in the case of
brief quotations embodied in critical articles and reviews.

The Author of this book is solely responsible and liable for its content including but not
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Preface
For Bachelor of Pharmacy students, "A Practical Handbook of
Pharmacognosy" is a useful handbook. This book was created
in accordance with syllabus framed by the Pharmacy Council
of India, New Delhi.
The topics covered in the syllabus include determining
secondary plant metabolites such as alkaloids, flavonoids,
tannin, glycosides, resins, etc.; identification tests for crude
drugs; microscopy of different crude drugs; determining
stomatal number and index; morphology and histology of
crude drugs originating from natural sources; monograph
analysis; determining phenol content; and determining
aldehyde content. This book also covers a number of
determination techniques, such as ash value determination
and foaming and swelling index determination.
The authors put a lot of work into creating the manuscript
and compiling this book. The 8th edition of the Indian
Pharmacopoeia is used as the standard for chemical test
methodology. This book's procedures and content are
written in such a way that students will find them to be both
simple to comprehend and comfortable to use.
We appreciate Notion Press, India for giving us a platform to
publish the book at the ideal time so that teachers and
students can access it. For the support and inspiration they
provided, "Almighty God" and our parents deserve our
gratitude.
The authors are open to any constructive criticism or ideas
that will help this book become even better.
(Author)
Surendar kumar
(Author)
Shiwank Rana
(Author)
Panshul Sharma
Contents
Unit 1 Page no 1-12
Analysis/evaluation of crude drugs and excipients of
natural origin by chemical tests
Unit 2 Page no 13-20
Determination of stomatal number and index
Unit 3 Page no 21-27
Determination of size of starch grains, calcium oxalate
crystals by eyepiece micrometer
Unit 4 Page no 28-30
Determination of vein islet number, vein islet termination
and palisade ratio
Unit 5 Page no 31-33
Determination of fiber length and width
Unit 6 Page no 34-36
Determination of number of starch grains by lycopodium
spore method
Unit 7 Page no 37-41
Determination of ash value
Unit 8 Page no 42-44
Determination of extractive values of crude drug
Unit 9 Page no 45-48
Determination of swelling and foaming index
Unit 10 Page no 49-50
Determination of moisture content of crude drugs
Unit 11 Page no 51-63
Morphology, histology and powder characteristics
Unit 12 Page no 64-70
Exercise involving isolation & detection of active principles
Unit 13 Page no 71-75
Chromatographic separation of sugars
Contents
Unit 14 Page no 76-77
Thin layer chromatography of herbal extract
Unit 15 Page no 78-85
Analysis of crude drugs by chemical tests
Unit 16 Page no 86-95
Preliminary phytochemical screening of crude drugs
Unit 17 Page no 96-98
Determination of the alcohol content of asava and arista
Unit 18 Page no 99-109
Incorporation of prepared & standardized extract in
various formulations and their evaluation as per
Pharmacopoeial requirements: Herbal cream Lotions
Shampoo
Unit 19 Page no 110-115
Incorporation of prepared & standardized extract in
various formulations and their evaluation as per
Pharmacopoeial requirements: Herbal syrup Mixtures
Tablets
Unit 20 Page no 116-120
Monograph analysis of herbal drugs from recent
Pharmacopoeias
Unit 21 Page no 121-122
Determination of aldehyde content
Unit 22 Page no 123-124
Determination of phenol content
Unit 23 Page no 125-126
Determination of total alkaloids content
References Page no 127-128
Notepad
A Practical Handbook of “Pharmacognosy”

1. Analysis/Evaluation of Crude Drugs and

Excipients of Natural Origin by Chemical
Tests
Background and objective: Unorganized drugs, as the name
suggests, are drugs that show no definite cellular structure.
These are derived from plant, animal or mineral sources by
some process of extraction and followed by purification if
necessary. Unorganized drugs are fairly homogenous and
may be solids, semisolids or liquids. These may be
differentiated by observing the solubility in alcohol and then
applying other physical and chemical standards.

Experiment 1.1
Aim: To identify the chemical characters of given crude drug
“Tragacanth”.
Requirements
Beaker, Measuring cylinder, Test tubes,
Equipments Heating mantle, Weighing balance,
Microscope, Pipette
Hydrochloric acid, Sodium hydroxide
Solution, Fehling’s solution, Barium chloride
Chemicals solution, Lead acetate, Ruthenium red,
Iodine, Potassium hydroxide, Tragacanth
powder
Theory: Tragacanth is an unorganized drug, commonly
known as Gum Tragacanth, Tragcanth. It is a dried gummy
exudation obtained from stem and branches of “Astragalus
gummifer” belong to family Leguminosae. Commercially;
acacia is produced in Turkey, Iraq, Iran & India. In India it is
produced in Punjab, Garhwal. Color of powder is white or

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A Practical Handbook of “Pharmacognosy”

pale yellow and odour is odourless. Taste of the gum

tragacanth is bland and mucilaginous and shape is thin,
flattened ribbon like flakes, more or less curved found in
various sizes. It is soluble in water and insoluble in alcohol.
Collection and preparation of tragacanth: Tragacanth trees
can be cultivated by sowing the seeds in the poor, exhausted
soil containing no minerals and gum is collected by native
from 6-8 years old trees, twice a year in dry weather in
November -April by Incision method.
Chemical constituents: Tragacanthin (8-10%), water Soluble,
bassorin (60-70%), insoluble in water; hydrolysis of
tragacanth are galactouronic acid, d-galactopyranose, and d-
xylopyranose.
Uses: Tragacanth is used as demulcent, suspending agent,
binding agent, Jellies, lotion, astringent & emulsifying agent.
Procedure
1. To 4ml of 0.5% w/v solution, add 0.5ml of hydrochloric
acid and heat for 30 minutes on a water bath. Divide the
liquid into two parts.
(A) To one part, add 1.5ml of sodium hydroxide solution
and Fehling’s solution, warm on water bath: red
precipitate is produced.
(B) To the second part, add barium chloride solution
(10%): No precipitate is obtained (distinction from agar).
2. To a 0.5% w/v solution of the gum, add 20% w/v
solution of lead acetate: A voluminous flocculent
precipitate is obtained (distinction from acacia).
3. Mount a small quantity of powder in ruthenium red and
examine microscopically: Particles do not acquire pink
color (distinction from Indian agar).
4. To 0.1gm of powder, add N/50 Iodine: The mixture
acquires an olive green color (distinction from acacia and
agar).

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A Practical Handbook of “Pharmacognosy”

5. Powder is warmed with 5% aqueous Potassium

hydroxide: Canary yellow-brown colour will obtain;
confirms Indian tragacanth.
Conclusion: From the above morphological characters and
chemical tests the given crude drug is identified as
Tragacanth.

Experiment 1.2
Aim: To identify the chemical characters of given crude drug
“Acacia”.
Requirements
Beaker, Measuring cylinder, Test tubes,
Equipments Heating mantle, Weighing balance,
Microscope, Pipette
Iodine solution, HCl, Lead acetate, Water
(Q.S.), Fehling’s solution A and B, FeCl3,
Chemicals
Hydrogen peroxide, Benzidine solution,
Acacia powder
Theory: Acacia is an unorganized drug, commonly known as
Gum acacia, Gum Arabic, Indian gum, Babul. It is a dried
gummy exudation obtained from stem and branches of
“Acacia arabica” belongs to family Leguminosae.
Commercially; Acacia is produced in Sri-lanka, Sudan, Africa
& India. In India it is produced in Punjab, Rajasthan, and
Maharashtra. Color of the tear is brown to red or Powder is
light brown and odour is odourless. Taste of the acacia is
bland & mucilaginous and shape is irregular brown tears
found in various sizes. It is soluble in water and insoluble in
alcohol.
Collection and preparation of acacia: Acacia trees can be
cultivated by sowing the seeds in the poor, exhausted soil
containing no minerals and gum is collected by native from

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A Practical Handbook of “Pharmacognosy”

6-8 years old trees, twice a year in dry weather in November

or in February-March by Incision method.
Chemical constituents: It consists of arabin, arabic acid, l-
arabinose, d-galatose, d-glucoronic acid, enzyme peroxidase,
gallic acid and chlorogenic acid.
Uses: Acacia is used as demulcent, suspending agent,
binding agent, micro encapsulation, astringent & emulsifying
agent.
Procedure
Test 1. A 10% aqueous solution of acacia fails to produce
any precipitate with dilute solution of lead acetate.
Observation: A clear distinction from agar and tragacanth.
Inference: Acacia present.
Test 2. It does not give any color change with Iodine
solution.
Observation: A marked distinction from starch and
dextrin.
Inference: Distinction from gelatin.
Test 3. It never produces a bluish-black color with FeCl3.
Observation: Solution an apparent distinction from
tannins.
Inference: Acacia is present.
Test 4. Hydrolysis of an aqueous solution of acacia with
dilute HCl yields reducing sugars whose presence are
ascertained by boiling with Fehling’s solution.
Observation: brick-red precipitate of cuprous oxide.
Inference: Reducing sugars is present in acacia.
Test 5. Dissolve about 0.25gm of the coarsely
powdered drug in 5ml of distilled water by shaking in the
cold. Add 0.5ml of hydrogen peroxide and 0.5ml of
benzidine solution, shake and allow standing for few
minutes.
Observation: A deep blue color or greenish blue color is

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A Practical Handbook of “Pharmacognosy”

formed due to the presence of oxidase enzyme.

Inference: Acacia is present.
Conclusion: From the above morphological characters and
chemical tests the given crude drug is identified as Acacia.

Experiment 1.3
Aim: To identify the chemical characters of given crude drug
“Honey”.
Requirements
Beaker, Measuring cylinder, Test tubes,
Equipments Heating mantle, Weighing balance,
Microscope, Pipette
Honey, Ether, 1% Resorcinol , HCl, Molisch’s
Chemicals
reagent, H2SO4 , Fehling’s solution A & B
Theory: Honey is an unorganized drug, commonly known as
Madhu, purified honey, Mel. It is a sugar secretion deposited
in honey comb by the honey bees of “Apis mellifera”, “Apis
dorsata” and other species of Apis; belongs to family
Apidae. Commercially; Honey is produced in New- Zealand,
Australia, and Africa and India. In India it is produced in
Punjab, Rajasthan, and Maharashtra. Color of the honey is
pale yellow to yellowish- brown, odour is pleasant, taste is
sweet and it is syrupy thick liquid, translucent when fresh
and on keeping it becomes granular due to the
crystallization of glucose.
Preparation of honey: Honey starts as flower nectar
collected by bees, which get broken down into simple sugars
stored inside the honeycomb. The design of the honeycomb
and constant fanning of the bees wings causes evaporation,
creating sweet liquid honey. Honey’s color and flavor vary
based on the nectar collected by the bees.

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A Practical Handbook of “Pharmacognosy”

Chemical constituents: Glucose-35%, fructose-45%, sucrose-

2% maltose-1%, gum, traces of succinic acid, acetic acid,
dextrin, formic acid, enzyme (invertase, diastase, inulase).
Uses: Honey is used as demulcent, sweetening agent,
antiseptic, expectorant, good nutrients to infant and
patients, preparation of creams, lotions and soft drinks.
Chemical tests
Fiehe’s test: Take about 3ml of honey + 2ml of ether and
shake thoroughly and allow the 2 layers to separate and
evaporate to dryness. The upper ethereal layer is
separated and put in a china dish and evaporate, to the
residue add 1% resorcinol and HCl.
Observation: (A) Transient pink color (B) Permanent red
color.
Inference: (A) Pure honey (B) Adulterated honey (Invert
sugar).
Molisch’s Test: Take 2ml of sample in dry test tube; add 2-
3 drops of Molisch’s reagent to the solution. Gently
pipette 1ml concentrated H2SO4 along the side of the tube
so that two distinct layers are formed. Observe color
change at the junction of two layers.
Observation: Purple color.
Inference: Presence of carbohydrate.
Reducing Sugar Test: Heat honey; adds a drop of
mixture of Fehling’s solution A & B.
Observation: Brick red color of cuprous oxide.
Inference: Presence of monosaccharide.
Conclusion: From the above morphological characters and
chemical tests the given crude drug is identified as Honey.

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A Practical Handbook of “Pharmacognosy”

Experiment 1.4
Aim: To identify the chemical characters of given crude drug
“Agar”.
Requirements
Beaker, Measuring cylinder, Test tubes,
Equipments Heating mantle, Weighing balance,
Microscope, Pipette
KOH, Ruthenium red, Iodine, HCl, NaOH,
Chemicals Fehling’s solution A and B, Barium chloride,
Agar
Theory: Agar is an unorganized drug, commonly known as
Agar-agar, Vegetable gelatin. It is dried gelatinous substance
obtained from “Gelidium amansi” belongs to family
Galidaceae. Commercially; agar is produced in Japan,
Australia, and New-zealand & India. In India it is produced in
coastal regions of Bengal. Color of the agar is –yellowish-
gray or white or nearly colorless and odour is odourless.
Taste of the agar is mucilaginous and shape is found in
various forms like sheets, strips, flakes or coarse powder.
Sheets are 45-60cm long, 10-15cm wide and strips are 4mm
in width. It is insoluble in cold water or organic solvents and
soluble in boiling water.
Collection and preparation of agar: Red algae grown on
bamboos, is collected in May and October. It is dried,
shaken, bleached washed and remove foreign materials. Boil
with dilute acidified water. On cooling jelly is produced &
dried it to remove excess water for several days.
Chemical constituents: Agar consist of two polysaccharides
namely agarose and agaropectin.
Uses: Agar is used as emulsifying agent, bulk laxative,
Preparation of jellies, Preparation of bacteriological culture,
treatment of chronic constipation.

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A Practical Handbook of “Pharmacognosy”

Chemical tests
Test 1: Boil 1gm of agar with 10ml of water until solution
is affected, cool to room temperature.
Observation: Jelly like mass is formed.
Inference: Agar is present.
Test 2: 0.2% solution of agar; add aqueous solution of
tannic acid.
Observation: No Precipitate is formed.
Inference: Agar is present.
Test 3: Warm little sample in alcoholic solution of
potassium hydroxide.
Observation: Yellow color is produced.
Inference: Agar is present.
Test 4: Mount a small quantity of powder in the solution
of ruthenium red and examine microscopically.
Observation: Particles acquire red or pink color.
Inference: Presence of mucilage in agar.
Test 5: Add 1 drop of N/50 solution of iodine to 10ml of
decoction of agar. Rapidly cool under tap water to room
temperature.
Observation: Crimson or Pale yellow color is produced.
Inference: Agar is present.
Test 6: Add 0.5ml of concentrated HCl to 4ml of 0.5%
solution of agar. Heat it on water bath for 30 minutes,
cool at room temperature and divide into two portions.
(A) Add 3ml of 10% NaOH solution and Fehling’s solution
A and B in equal quantities and warm over water bath.
(B) Add 10% of barium chloride solution.
Observation: (A) Red ppt of cuprous oxide is obtained;
(B) Slight white precipitates of barium sulphate are
obtained.
Inference: Reducing sugars are present.

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Test 7: Incinerate agar to ash, add a drop of HCl observe

under microscope.
Observation: Fragments of diatoms.
Inference: Agar is present.
Conclusion: From the above morphological characters and
chemical tests the given crude drug is identified as Agar.

Experiment 1.5
Aim: To identify the chemical characters of given crude drug
“Gelatin”.
Requirements
Beaker, Measuring cylinder, Test
Equipments tubes, Heating mantle, Weighing
balance, Microscope, Pipette
Soda lime, Million’s reagent, Tannic
Chemicals acid solution, NaOH (1ml of 5%),
Picric acid, Trinitrophenol, Gelatin
Theory: Gelatin occurs in thin sheets, strips or as granular
powder. High grade gelatin is light yellow, semi crystalline
substance. It is odorless and tasteless. In cold water it swells
up and slowly dissolves on warming to form viscous solution.
Uses: Gelatin is used as gelling agent in preparation of
jellies, cosmetics and capsules shells.
Chemical tests
Test 1: When gelatin is heated with soda lime in dry test
tube, ammonia is evolved due to the presence of
nitrogenous compound in gelatin.
Test 2: Gelatin solution is added Million’s reagent to give a
white precipitates, which turns red on heating.
Test 3: Gelatin gives buff white precipitates with tannic
acid.

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A Practical Handbook of “Pharmacognosy”

Test 4: Biuret test to 3ml of test solution of gelatin. NaOH

(1ml of 5%) is added whereby white to whitish buff
colored precipitates is formed which does not dissolve on
heating.
Test 5: Yellow precipitates are formed on adding picric
acid to solution of gelatin.
Test 6: It gives yellow precipitates with tri-nitrophenol in
aqueous solution.
Conclusion: From the above morphological characters and
chemical tests the given crude drug is identified as Gelatin.

Experiment 1.6
Aim: To identify the chemical characters of given crude drug
“Castor oil”.
Requirements
Beaker, Measuring cylinder, Test tubes,
Equipments Heating mantle, Weighing balance,
microscope, Pipette
Chemicals Petroleum Ether, Alcohol, Castor oil
Theory: Castor oil is a vegetable oil obtained from castor
beans. It is a colourless or pale yellow liquid with a distinct
taste and odor. It includes a mixture of triglycerides in which
about 90% of fatty acids are ricinoleates. Oleic acid and
linoleic acid are the other significant components.
Uses: Castor oil is used orally as laxative.
Chemical tests
Test 1: Add 5ml of light petroleum ether (40C-60C) to
10ml of castor oil.
Observation: A clear solution turns turbid on increasing
the petroleum ether about 15ml.
Inference: Castor oil is present.

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A Practical Handbook of “Pharmacognosy”

Test 1: Oil + equal volume of alcohol and cool to 0C for 3

hours.
Observation: A clear solution is obtained.
Inference: Castor oil is present.
Conclusion: From the above morphological characters and
chemical tests the given crude drug is identified as Castor
oil.

Experiment 1.7
Aim: To identify the chemical characters of given crude drug
“Starch”.
Requirements
Beaker, Measuring cylinder, Test tubes,
Equipments Heating mantle, Weighing balance,
Microscope, Pipette
Chemicals Iodine - KI Reagent, Starch
Theory: Amylose forms a colloidal dispersion in hot water
whereas amylopectin is completely insoluble. The structure
of amylose consists of long polymer chains of glucose units
connected by an alpha acetal linkage. Starch-Amylose shows
a very small portion of an amylose chain. All of the
monomer units are α-D-glucose, and all the α-acetal links
connect C1 of first glucose and to C4 of the next glucose. As
a result of the bond angles in the α-acetal linkage, amylose
actually forms a spiral much like a coiled spring.
Chemical tests
Test 1: Amylose in starch is responsible for the formation
of a deep blue color in the presence of iodine. The iodine
molecule slips inside of the amylose coil. Iodine-KI
reagent: Iodine is not very soluble in water; therefore the
iodine reagent is made by dissolving iodine in water in the

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presence of potassium iodide. This makes a linear tri-

iodide ion complex with is soluble that slips into the coil of
the starch causing an intense blue-black color.
Conclusion: From the above morphological characters and
chemical tests the given crude drug is identified as Starch.

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2. Determination of Stomatal Number and

Index
Background and objective: The stomatal number and
stomatal index are important diagnostic characteristics of
dicot leaves. Stomata number is defined as the average
number of stomata per square mm of epidermis of the leaf.
The actual number of stomata per square mm may vary for
the leaves of the same plant grown in different environment
or under different climatic conditions. Meanwhile, stomatal
index is the percentage in which the number of stomata
forms to the total number of epidermal cells.

Experiment 2.1
Aim: Determination of average stomatal number of given
sample of crude drug leaf.
Requirements
Compound microscope, Camera lucida,
Drawing board, Micro slides, Cover glasses,
Equipments Forceps, Water bath, Small watch glass,
Blade, Cello tape, drawing sheet, Dark
colored pencil with sharp lead
Chemicals Chloral hydrate solution
Theory: Stoma (plural-stomata) is a minute epidermal
opening covered by two kidneys shaped guard cells in dicot
leaves. These guard cells, in turn, are surrounded by
epidermal (subsidiary) cells. Stomata perform the functions
of gaseous exchange and transpiration in plants. The nature
of the stomata, as well as, the stomatal number is important
diagnostic characteristics of dicot leaves. Stomata number is
defined as the average number of stomata per sq mm of

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epidermis of the leaf. The actual number of stomata per

square mm may vary for the leaves of the same plant grown
in different environment or under different climatic
conditions.
Procedure
(A) Preparation of lamina: Take a mature leaf. If the leaf is
small, the whole leaf may be taken and if the leaf is large cut
5mm square pieces from the middle portion between the
lamina and midrib.
Fresh leaf
1. Sometimes the epidermis can be easily peeled off in
thick leaves by breaking it into pieces by sheering action.
Separate the epidermis and treat with chloral hydrate.
2. Boil with chloral hydrate in a water bath, the epidermis
separates out.
3. Fold the leaf to gently pull the peel apart to separate a
peeled section from the lower surface of the leaf.
4. Use the forceps to perform this step, allow the peel to
remain in a watch glass holding water for some time.
5. In the watch glass, stain the sample by adding some
drops of safranin through a dropper.
6. Take the peel out after 2-3 minutes. Set it on a clear
glass slide.
7. Add a drop of glycerin on the peel. Put a clear cover slip
over it gently.
8. Excess glycerin and stain can be removed using blotting
paper.
9. Examine the slide first under a low- power and then
under a high power magnification of compound
microscope.
10. Trace the epidermal stomata with the help of camera
lucida.

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Dry leaf
1. Heat the leaf with chloral hydrate in a beaker on a water
bath for 30 minutes.
2. Cut the leaf into two pieces; observe under the
microscope to see whether the stomata are present on
both surfaces and in one.
3. Place the cleared leaf with the veins facing down. Then
the upper epidermis will be visible.
4. Place the other half with veins facing upwards. Then the
lower epidermis will be visible.
5. Add two drops of glycerin and place a cover glass.
6. Label the slides as “upper” and “lower” and trace the
epidermal cells and stomata.
*Note: If the leaf is too thick and dark, procedure to
separate the epidermis is given below.
1. Clear the leaf with chloral hydrate as given in step
number-1. Cut the leaf into two halves.
2. Place one half with the upper surface facing
downwards.
3. Carefully scrape off the upper tissue, with the edge of a
razor blade, without disturbing upper epidermis. Clean it
with a Brush dipped in chloral hydrate solution.
4. The layer of cells remaining is upper epidermis. Turn the
layer upside to trace the cells.
5. Repeat the procedure with the second half, this time
placing the lower surface facing downwards, proceed as
given above.
6. Usually herbs and small shrubs have stomata on both
surfaces. In tree species, stomata are present on the lower
surface. More stomata are present on the lower surface in
dorsiventral leaf, almost the same number in isobilateral
leaf.

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Figure 2.1: Microscopy of leaf stomata

(B) Tracing of cells: In this experiment the number has to be
determined per square millimeter.
1. Adjust the drawing board, if swift camera lucida is used.
It is not necessary to adjust angle with Abbe’s camera
lucida.
2. With the help of stage micrometer, draw a line of
o 1mm
using 10X magnification on a drawing sheet and draw a
10 cm.
3. Replace the stage micrometer with a prepared slide of o
the leaf as given for stomatal number.
4. With the help of camera lucida mark the number of
stomata in the square.
5. Count the stomata. That gives es the number of stomata
per square mm.
6. Take 25 reading and calculate the average.
7. Note the side, for which the stomata number is
determined.
8. Direct counting of stomata can also be done by a
squared eyepiece micrometer, if the drawings
awings are not
required.
Conclusion: Average stomatal
tomatal number of supplied leaf
is_____.

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Experiment 2.2
Aim: Determination of average stomatal index of given
sample of crude drug leaf.
Requirements
Compound microscope, Camera lucida,
Drawing board, Micro slides, Cover glasses,
Equipments Forceps, Water bath, Small watch glass,
Blade, Cello tape, Drawing sheet, Dark
colored pencil with sharp lead
Chemicals Chloral hydrate solution
Theory: Stoma (plural-stomata) is a minute epidermal
opening covered by two kidneys shaped guard cells in dicot
leaves. These guard cells, in turn, are surrounded by
epidermal (subsidiary) cells. Stomata perform the functions
of gaseous exchange and transpiration in plants. The nature
of the stomata, as well as, the stomatal index and stomatal
number are important diagnostic characteristics of dicot
leaves. Stomatal number is defined as the average number
of stomata per sq mm of epidermis of the leaf. The actual
number of stomata per sq mm may vary for the leaves of the
same plant grown in different environment or under
different climatic conditions. It is, however shown that the
ratio of the number of stomata to the total number of
epidermal cells in a given area of epidermis is fairly constant
for any age of the plant and under different climatic
conditions. Stomatal index is the percentage which the
number of stomata forms to the total number of epidermal
cells, each stoma being counted as one cell. Stomatal index
can be calculated by using the following equation:
Stomatal Index (I) = × 100

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Where, S is number of stomata per unit area, E is number of

epidermal cells in the same unit area.
Whilst stomatal number varies considerably with the age of
the leaf and due to changes in environmental conditions,
stomatal index is relatively constant and therefore, of
diagnostic significance for a given species. It is employed for
the differentiation of allied or closely related to species of
same genus in air dried, as well as fresh conditions.
Procedure
(A) Preparation of lamina: Take a mature leaf. If the leaf is
small, the whole leaf may be taken and if the leaf is large cut
5 mm square pieces from the middle portion between the
lamina and midrib.
Fresh leaf
1. Sometimes the epidermis can be easily peeled off in
thick leaves by breaking it into pieces by sheering action.
Separate the epidermis and treat with chloral hydrate.
2. Boil with chloral hydrate in a water bath, the epidermis
separates out.
3. Fold the leaf to gently pull the peel apart to separate a
peeled section from the lower surface of the leaf.
4. Use the forceps to perform this step, allow the peel to
remain in a watch glass holding water for some time.
5. In the watch glass, stain the sample by adding some
drops of safranin through a dropper.
6. Take the peel out after 2-3 minutes. Set it on a clear
glass slide.
7. Add a drop of glycerin on the peel. Put a clear cover slip
over it gently.
8. Excess glycerin and stain can be removed using blotting
paper.
9. Examine the slide first under a low- power and then

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under a high power magnification of compound

microscope.
10. Trace the epidermal stomata with the help of camera
lucida.
Dry leaf
1. Heat the leaf with chloral hydrate in a beaker on a
water bath for 30 minutes.
2. Cut the leaf into two pieces; observe under the
microscope to see whether the stomata are present on
both surfaces and in one.
3. Place the cleared leaf with the veins facing down. Then
the upper epidermis will be visible.
4. Place the other half with veins facing upwards. Then the
lower epidermis will be visible.
5. Add two drops of glycerin and place a cover glass.
6. Label the slides as “upper” and “lower” and trace the
epidermal cells and stomata.
*Note: If the leaf is too thick and dark, procedure to
separate the epidermis is given below.
1. Clear the leaf with chloral hydrate as given in step
number-1. Cut the leaf into two halves.
2. Place one half with the upper surface facing
downwards.
3. Carefully scrape off the upper tissue, with the edge of a
razor blade, without disturbing upper epidermis. Clean it
with a Brush dipped in chloral hydrate solution.
4. The layer of cells remaining is upper epidermis. Turn the
layer upside to trace the cells.
5. Repeat the procedure with the second half, this time
placing the lower surface facing downwards, proceed as
given above.
6. Usually herbs and small shrubs have stomata on both
surfaces. In tree species, stomata are present on the lower
surface. More stomata are present on the lower surface in

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dorsiventral leaf, almost the same number in isobilateral

leaf.
(B) Tracing of cells: In this experiment the number has to be
determined per square millimeter.
1. Adjust the drawing board, if swift camera-lucida is used.
It is not necessary to adjust angle with Abbe’s camera
lucida.
2. With the help of stage micrometer, draw a line of 1mm
using 10X magnification on a drawing sheet and draw a
10 cm.
3. Replace the stage micrometer with a prepared slide of
the leaf as given for stomatal index.
4. Trace epidermal cells and the stomata outside the
square completion on two adjacent sides of the square for
counting purpose.
5. Number the complete epidermal cells and the stomata
within the square.
6. Calculate the stomatal index by using given formula:
Stomatal Index (I) = × 100

Where, I is stomatal index, S is total number of stomata

and E is total number of epidermal cells.
Conclusion: Stomatal number and stomatal index of
supplied leaf is_____.

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3. Determination ofSize of Starch Grains

& Calcium Oxalate Crystals by Eyepiece
micrometer
Background and objective: Measurement of starch grain by
using stage and eyepiece micrometers.

Experiment 3.1
Aim: To measure the diameter of the starch grain of given
crude drug “Ginger”.
Requirements
Equipments Eyepiece, Stage micrometer, Microscope
Chemicals Glycerin, Ginger, Water
Theory: Starch analysis or starch grain analysis is a technique
that is useful in archaeological research in determining
plant taxa on a microscopic level. It can also be used in day-
to-day life by specialists within the pharmaceutical and food
industries in order to determine taxa origins and food
quality. Specifically in regards to archaeology though, the
identification of starch grains, through this context is done
by comparison identification, in which several attributes of
the grains are compared to other known samples in order to
determine the type. This comparison technique, when done
microscopically allows for the specific taxa identification of
starch grains found on specific artifacts, such as ground
stone tools, within soils, through dental calculus, or found in
reference to ceramic vessels. Starch grain analysis can be
helpful as a supplement to other forms of study to
understanding tool use, agricultural activities, as well as
other plant based subsistence strategies, and to reconstruct
plant based diets throughout time.

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A Practical Handbook of “Pharmacognosy”

Figure 3.1: Microscopic image of starch grains

Procedure
Stage micrometer
1. Take the micrometer slide in hand and feel the sides of the
slide.
2. The correct sides show a slight elevation on one side.
3. Place it on the stage of the microscope. View the scale
through the eyepiece at 10 x 10 magnifications to find the
position of the scale on the micrometer.
4. To locate the scale, start viewing from the edge of the
cover glass containing the engraved micrometer scale and
then move to the centre and locate the scale on the
equatorial plane.
Eyepiece micrometer
1. Remove the eyepiece and unscrew the part A.
2. Place the eyepiece micrometer at level B of the eye
piece where there is a hinge or diaphragm (it is the
support on which the eyepiece micrometer rests).
3. Screw part A back and see whether the numbering on
the eyepiece micrometer is in correct position, i.e. zero to
hundred. If numbers are reverse, remove the scale and
place it in a correct manner.
4. The eye-piece
piece can be rotated to make adjustments.

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A Practical Handbook of “Pharmacognosy”

5. Do not introduce the eyepiece micrometer into the

draw tube.
6. Calibration of eyepiece micrometer View through the
eyepiece with the required optical combinations.
7. Adjust the scales of the two micrometers such that both
the scales are superimposed. Rotate the eyepiece to place
the scales in a parallel position or remove the stage
micrometer till the lines coincide with eyepiece scale.
8. Move the stage micrometer such that the “0” readings
of both the micrometer scales coincide or one of the
larger divisions of the stage micrometer coincides with
one of the lines of the eyepiece micrometer scale. Note
the initial readings.
9. Carefully scan the scales to see, which of the two scale
readings exactly coincide on the right side. Note the final
readings and calculate the factor for 1 division of the
eyepiece micrometer.
10. Now the microscope is ready for taking
measurements.
Calibrate eyepiece micrometer by using stage micrometer
and calculate the factor. Mount a little quantity of powdered
sample in glycerin water and measure the diameter of 25
starch grain. Multiply the values by the factor for the next
dimensions of the crystals.

Figure 3.2: Stage and eyepiece micrometer

Calculation for 1mm stage/eyepiece micrometer
1mm =1000µm

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A Practical Handbook of “Pharmacognosy”

1mm = 100 divisions

100 divisions = 1000µm
10 divisions = 100µm
1 division = 100µm
Calculate the average value and give the range for the
dimensions.
Calibration factor calculation
E.g. 2 divisions of stage micrometer = 3 divisions of eyepiece
micrometer
We Know that,
2 divisions of stage micrometer = 0.02mm
Therefore; 0.02mm= 3 divisions of eyepiece micrometer
1 division of eyepiece micrometer = 0.02/3 = 0.0067mm
Conclusion: Average diameter of the starch grain of the
given sample is_____.

Experiment 3.2
Aim: To measure the diameter of the calcium oxalate
crystals of given crude drug “Senna powder”.
Requirements
Equipments Eyepiece, Stage micrometer, Microscope
Chemicals Glycerin, Senna powder, Water
Theory: Calcium (Ca2+) oxalate crystals occur in many plant
species and in most organs and tissues. They generally form
within cells although extracellular crystals have been
reported. The crystal cells or idioblasts display ultra
structural modifications which are related to crystal
precipitation. Crystal formation is usually associated with
membranes, chambers, or inclusions found within the cell
vacuole(s). Tubules, modified plastids and enlarged nuclei
also have been reported in crystal idioblasts. The calcium
oxalate crystals consist of either the monohydrate

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A Practical Handbook of “Pharmacognosy”

whewellite form, or the dihydrate weddellite form. A

number of techniques exist for the identification of calcium
oxalate. X-ray
ray diffraction, Raman microprobe analysis
Microscopy and infrared spectroscopy are the most
accurate. Many plant crystals assumed to be Ca oxalate
ox have
never been positively identified as such. In some instances,
crystals have been classified as whewellite or weddellite
solely on the basis of their shape. Certain evidence indicates
that crystal shape may be independent
dependent of hydration form of
calcium oxalate and that the vacuole crystal chamber
membranes may act to mold crystal shape.

Figure 3.3: Microscopic image of calcium oxalate crystals

Procedure
Stage micrometer
1. Take the micrometer slide in hand and feel the sides of the
slide.
2. The correct sides show a slight elevation on one side.
3. Place it on the stage of the microscope. View the scale
through the eyepiece at 10 x 10 magnifications to find the
position of the scale on the micrometer.
4. To locate the scale, start viewing from the edge of the
cover glass containing the engraved micrometer scale and
then move to the centre and locate the scale on the

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A Practical Handbook of “Pharmacognosy”

equatorial plane.
Eyepiece micrometer
1. Remove the eyepiece and unscrew the part A.
2. Place the eyepiece micrometer at level B of the eye
piece where there is a hinge or diaphragm (it is the
support on which the eyepiece micrometer rests).
3. Screw part A back and see whether the numbering on
the eyepiece micrometer is in correct position, i.e. zero to
hundred. If numbers are reverse, remove the scale and
place it in a correct manner.
4. The eye-piece can be rotated to make adjustments.
5. Do not introduce the eyepiece micrometer into the
draw tube.
6. Calibration of eyepiece micrometer View through the
eyepiece with the required optical combinations.
7. Adjust the scales of the two micrometers such that both
the scales are superimposed. Rotate the eyepiece to place
the scales in a parallel position or remove the stage
micrometer till the lines coincide with eyepiece scale.
8. Move the stage micrometer such that the “0” readings
of both the micrometer scales coincide or one of the
larger divisions of the stage micrometer coincides with
one of the lines of the eyepiece micrometer scale. Note
the initial readings.
9. Carefully scan the scales to see, which of the two scale
readings exactly coincide on the right side. Note the final
readings and calculate the factor for 1 division of the
eyepiece micrometer.
10. Now the microscope is ready for taking
measurements.
Calibrate eyepiece micrometer by using stage micrometer
and calculate the factor. Mount a little quantity of powdered
sample in glycerin water and measure the diameter of 25

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A Practical Handbook of “Pharmacognosy”

calcium oxalate crystals. Multiply the values by the factor for

the next dimensions of the crystals.
Calculation for 1mm stage/eyepiece micrometer
1mm = 1000µm
1mm = 100 divisions
100 divisions = 1000µm
10 divisions = 100µm
1 divisions = 100µm
Calculate the average value and give the range for the
dimensions.
Calibration factor calculation
Example: 2 divisions of stage micrometer = 3 divisions of
eyepiece micrometer
We Know that,
2 divisions of stage micrometer = 0.02mm
0.02mm = 3 divisions of eyepiece micrometer
1 division of eyepiece micrometer =0.02/3= 0.0067mm
Conclusion: Average diameter of the calcium oxalate crystals
of the given sample is_____.

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A Practical Handbook of “Pharmacognosy”

4. Determination of Vein Islet Number,

Vein Islet Termination and Palisade Ratio

Background and objective: Measurement of vein islet

number, vein islet termination and palisade ratio.

Experiment 4.1
Aim: Determination of vein-islet and vein termination
number.
Requirements
Compound microscope, Camera lucida,
Drawing board, Micro slides, Cover glasses,
Equipments Forceps, Water bath, Small watch glass,
Blade, Cello tape, Drawing sheet, Dark
colored pencil with sharp lead
Chemicals Chloral hydrate solution
Theory: Vein islet is the minute area of photosynthetic
tissue encircled by the ultimate division of the conducting
strands. Vein termination number is the number of vein let
terminations per millimeter of leaf surface.
Procedure
1. A piece of leaf was cleared by boiling in chloral hydrate
solution and was placed under microscope camera lucida
and drawings board was arranged. 1mm line was drawn
with help of stage mm.
2. A square was constructed on this line in the centre of
the field.
3. The slide was placed on the stage.
4. The veins included within the square were traced off,
completing the outline of those islets which overlap two

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adjustment side of the square.

5. The average number of vein islet from the four
adjoining square, to get the value for one square mm was
calculated.
6. The number of vein let termination present within the
square was counted.
7. The average number of vein let termination number
from the four adjoining square to get the value for 1
square mm was found known as vein termination number.
Conclusion: Average vein-Islet and vein termination number
of supplied leaf is_____.

Experiment 4.2
Aim: Determination of palisade ratio.
Requirements
Compound microscope, Camera lucida,
Drawing board, Micro slides, Cover glasses,
Equipments Forceps, Water bath, Small watch glass,
Blade, Cello tape, Drawing sheet, Dark
colored pencil with sharp lead
Chemicals Chloral hydrate solution
Theory: Average number of cells beneath epidermal cells
was calculated, known as palisade ratio. It represents
number of palisade cells beneath one epidermal cell, using
four epidermal cells for count. It determine from powdered
drugs with the help of camera-lucida.
Procedure
1. A piece of the leaf was cleared by boiling in chloral
hydrate solution and was placed under microscope
camera lucida and drawings board was arranged.
2. The outline of four cells of the epidermis was traced

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A Practical Handbook of “Pharmacognosy”

using 4mm objective and slide was placed on the stage.

3. Then, palisade layer was focused down and sufficient
cells for covering the tracing of the epidermal cells were
traced off.
4. The outline of those palisade cells which were
intersected by the epidermal walls was completed.
5. The palisade cells under the four epidermal cells
(including cells which are more than half and excluding
cells which are less than half within the area of epidermal
cells) were counted.
6. The determination for five groups of four epidermal
cells from different part of the leaf was repeated.
7. The average number of cells beneath epidermal cells
was calculated known as palisade ratio.
Conclusion: Average palisade ratio of supplied leaf is_____.

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5. Determination of Fiber Length and

Width
Background and objective: Measurement of fiber length
and width.

Experiment 5.1
Aim: Determination of length and width of fibers of
“Cinchona bark” powder.
Requirements
Microscope with mechanical stage,
Equipments
Eyepiece, Stage micrometer
Glycerin water, Phloroglucinol,
Chemicals Concentrated hydrochloric acid, Chloral
hydrate solution, Cinchona bark
Theory: Fiber is composed of plants which resist human’s
digestive enzymes, a definition that includes lignin and
polysaccharides, is of two type’s water soluble and insoluble.
Fibers are of two types; natural and synthetic.
Procedure
Stage micrometer
1. Take the micrometer slide in hand and feel the sides of
the slide.
2. The correct sides show a slight elevation on one side.
3. Place it on the stage of the microscope. View the scale
through the eyepiece at 10 x 10 magnifications to find the
position of the scale on the micrometer.
4. To locate the scale, start viewing from the edge of the
cover glass containing the engraved micrometer scale and
then move to the centre and locate the scale on the

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equatorial plane.
Eyepiece micrometer
1. Place the eyepiece micrometer at level B of the eye
piece where there is a hinge or diaphragm (it is the
support on which the eyepiece micrometer rests).
2. Screw part A back and see whether the numbering on
the eyepiece micrometer is in correct position, i.e. zero to
hundred. If numbers are reverse, remove the scale and
place it in a correct manner.
3. The eye-piece can be rotated to make adjustments.
4. Do not introduce the eyepiece micrometer into the
draw tube.
Calibration of eyepiece micrometer
1. View through the eyepiece with the required optical
combinations.
2. Adjust the scales of the two micrometers such that both
the scales are superimposed. Rotate the eyepiece to place
the scales in a parallel position or remove the stage
micrometer till the lines coincide with eyepiece scale.
3. Move the stage micrometer such that the “0” readings
of both the micrometer scales coincide or one of the
larger divisions of the stage micrometer coincides with
one of the lines of the eyepiece micrometer scale. Note
the initial readings.
4. Carefully scan the scales to see, which of the two scale
readings exactly coincide on the right side. Note the final
readings and calculate the factor for 1 division of the
eyepiece micrometer.
5. Now the microscope is ready for taking measurements.
6. Using stage micrometer calibrates the eyepiece
micrometer. Calculate the factor (average distance
between two lines in microns).
7. Take a little quantity of powder drug (ceylon cinnamon,
cassia bark or cinchona bark) in a test tube and boil with

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clearing agent, chloral hydrate solution.

8. Transfer the cleared powder in a watch glass. Stain the
lignified fibers with the staining reagent (phloroglucinol
and concentrated hydrochloric acid).
9. Mount this treated powder in glycerine water and
observe the slide under low power (Power should be
thinly, uniformly scattered, without overlapping of
particles).
10. Focus a stained fiber (intact fiber).
11. By rotating the scale of eyepiece micrometer, note the
numbers of divisions of the eye piece micrometer covered
by the length of the fiber.
12. Multiply each value by the factor calculated in the first
step to get the value in microns. Then calculate the
average value and write the range for the length and the
width fiber.
Conclusion: Length and width of the fiber present in the
given Cinchona bark powder_____.

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6. Determination of Number of Starch

Grains by Lycopodium Spore Method
Background and objective: Lycopodium spores have uniform
moisture content; hence the weight remains the same. This
is the reason, why these spores are used to evaluate
powdered drugs by comparison. The spores are also
resistant to pressure.

Experiment 6.1
Aim: To determine number of starch grains by “Lycopodium
spore” method.
Requirements
Balance, Watch glass, Small flexible spatula,
Equipments Microscope with mechanical stage or a
counting square
Suspending agent (fixed oil/glycerine :
Chemicals
tragacanth mucilage : water [2:1:2])
Theory: Lycopodium spores are obtained from club moss,
“Lycopodium clavatum Linn”, belongs to family
Lycopodiaceae. The spores are yellow in color, spheroidal,
tetrahedral in shape with reticulate surface. They have
uniform average diameter of 25. One milligram contains
average 94000 spores. They have uniform moisture content;
hence the weight remains the same. This is the reason, why
these spores are used to evaluate powdered drugs by
comparison. The spores are also resistant to pressure.
Procedure
1. Determine the loss on drying for the powder at 105C.
2. Mix a weighed amount of air-dry powder of the drug

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and a weighed amount of lycopodium spores in a small

watch glass (100mg drug and 50mg lycopodium spores).
Mix with a small flexible spatula.
3. Add oil or suspending agent. Mix for 10 minutes till a
smooth paste is obtained.
4. Transfer the suspension to a small glass tube by
draining with the help of a glass rod.
5. Add more suspending agent, washing down the mixture
into the tube. (About 4ml of the suspending agent is
required for 50mg of lycopodium spores). Dilution of the
suspension should give about 10 to 20 spores in a field.
6. This should give about 10 to 20 spores when viewed
less than 4mm objective, when a drop of the mixture is
mounted under a cover glass.
7. Slowly oscillate the glass tube between the two palms
without any air bubbles, until the suspension is uniform.
Take a glass tube with internal diameter of about 2-3mm
and place one drop each on two sides, spread the
suspension on the slide less than the area of the cover slip.
8. Apply a cover slip and leave the slide on an even surface
to settle.
9. Select 25 fields and count the spores and particles in
these fields using 10×40 magnification.
10. Make a similar suspension as above and count
particles in 25 fields on two sides.
11. Take average of four readings.
12. Calculate the percentage of foreign organic matter
from the formula given below:
Percentage of foreign organic matter
𝟗𝟒𝟎𝟎𝟎 × 𝐧 × 𝐰
= × 𝟏𝟎𝟎
𝐬×𝐦×𝐩

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A Practical Handbook of “Pharmacognosy”

Where, m is weight in milligrams (mg) of the sample,

calculated on sample dried at 105C, w is weight in mg of
the lycopodium spores, n is number of particles in 25 fields,
p is number of particles in per mg of the pure foreign matter
dried at 105C, 94,000 is a number of spores in one mg of
lycopodium and S is number of starch grain.
Calculations
 1 mg of lycopodium powder contains spores = 94000
 (w)mg of lycopodium powder contains 94000 x w number
of spores.
 Number of spores in ten fields mix with n number of starch
grains in ten fields. 94000 x w number of spores mix with
𝟗𝟒𝟎𝟎𝟎 × 𝐧 × 𝐰
=
𝐬(𝐧𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐬𝐭𝐚𝐫𝐜𝐡 𝐠𝐫𝐚𝐢𝐧)

 1mg of pure sample of foreign organic matter (FOM)

contains p number of starch grains weight of ginger in the
mixture
𝟗𝟒𝟎𝟎𝟎 × 𝐧 × 𝐰
=
𝐬 × 𝐩(𝐦𝐠 𝐨𝐟 𝐠𝐢𝐧𝐠𝐞𝐫)
 (m)mg of mixture contain
𝟗𝟒𝟎𝟎𝟎 × 𝐧 × 𝐰
=
𝐬×𝐩

 1mg of mixture contains

𝟗𝟒𝟎𝟎𝟎 × 𝐧 × 𝐰
=
𝐬×𝐩×𝐦

 Percentage of foreign organic matter

𝟗𝟒𝟎𝟎𝟎 × 𝐧 × 𝐰
= × 𝟏𝟎𝟎
𝐬×𝐩×𝐦
Conclusion: Number of starch grains in the given sample
is_____.

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7. Determination of Ash Value

Background and objective: The present study is designed to
determine the ash value of the supplied sample. Total ash is
useful in detecting the crude drugs that are mixed with
various mineral substances like sand, soil, calcium oxalate,
chalk powder, or other drug with different inorganic
contents to improve their appearance.

Experiment 7.1
Aim: Determination of total ash value and acid insoluble ash
value of given crude drug powdered “Liquorice”.
Requirements
Silica crucible, Desiccator, Ash less filter
Equipments
paper, Muffle furnace, Weighing balance
Dilute hydrochloric acid , Liquorice
Chemicals
powder
Theory: Ash values are helpful to determine the quality as
well as purity of a crude drug, especially when the drug is
present in powdered form. The object of ashing crude drugs
is to remove the traces of organic matter which may be
interferes in an analytical determination. On incineration,
the crude drugs normally produce ash which is usually
consisting of carbonates, phosphates and silicates of sodium,
potassium, calcium and magnesium. The total ash of a crude
drug reveals the care taken during its preparation. A higher
limit of acid- insoluble ash is incorporated especially in cases
where silica may be present or when the calcium oxalate
content of the drug is very high. Some researchers suggested
mixing of acids like sulphuric acid with the powdered crude
drug before ashing and making the ash sulphated which is
normally less fusible than ordinary ash. The present study is

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designed to determine the ash value of the supplied sample.

Total ash is useful in detecting the crude drugs that are
mixed with various mineral substances like sand, soil,
calcium oxalate, Chalk powder, or other drug with different
inorganic contents to improve their appearance. Acid
insoluble ash is the residue obtained after boiling the total
ash with dilute hydrochloric acid and Igniting the remaining
insoluble matter. This measures the amount of silica
present, especially as sand and siliceous earth.
Procedure
Total ash determination
1. Weigh accurately about 3gm of the powdered drug in
silica crucible.
2. Incinerate the powdered drug.
3. Temperature not exceeding 450C until the sample
was free from carbon.
4. Cool it and keep it in desiccators.
5. Weigh the ash and calculate the percentage of total
ash in contrast to the air dried sample.
6. The unpeeled variety of liquorice root must contain
no more than 10% of total ash.
(W2)
𝐏𝐞𝐫𝐜𝐞𝐧𝐭 𝐭𝐨𝐭𝐚𝐥 𝐀𝐬𝐡 = × 100
(W1)

Where, W1 is weight in grams of silica dish and sample, W2

is weight in grams of silica dish and ash.
Procedure
Acid-insoluble ash determination
1. Boil the total ash obtained as the above procedure for
5 minutes with 25ml of dilute hydrochloric acid.
2. Filter and collect the insoluble matter on ash less filter
paper.

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3. After that wash the filter paper with hot water.

4. Ignite in tared crucible, cool and keep in desiccator.
5. Weigh the obtained residue and calculate acid-
insoluble ash of the crude drug (liquorice).
6. The unpeeled variety of liquorice root must contain no
more than 10% of total ash and 2.5% of acid insoluble
ash.
(W2)
𝐏𝐞𝐫𝐜𝐞𝐧𝐭 𝐚𝐜𝐢𝐝 𝐢𝐧𝐬𝐨𝐥𝐮𝐛𝐥𝐞 𝐚𝐬𝐡 = × 100
(W1)

Where, W1 is weight in grams of silica dish and sample, W2

is weight in grams of silica dish and acid-insoluble ash.
Conclusion: Ash value of supplied sample is_____.

Experiment 7.2
Aim: Determination of sulphated ash value and water
soluble ash value of given crude drug powdered “Clove”.
Requirements
Silica crucible Desiccator, Ash less filter
Equipments
paper, Muffle Furnace, Weighing balance
Sulphuric acid, Distilled water, Clove
Chemicals
powder
Theory: Ash values are helpful to determine the quality as
well as purity of a crude drug, especially when the drug is
present in powdered form. The object of ashing crude drugs
is to remove the traces of organic matter which may be
interferes in an analytical determination. On incineration,
the crude drugs normally produce ash which is usually
consisting of carbonates, phosphates and silicates of sodium,
potassium, calcium and magnesium. The total ash of a crude
drug reveals the care taken during its preparation. A higher

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limit of acid- insoluble ash is incorporated especially in cases

where silica may be present or when the calcium oxalate
content of the drug is very high. Some researchers suggested
mixing of acids like sulphuric acid with the powdered crude
drug before ashing and making the ash sulphated which is
normally less fusible than ordinary ash. The present study is
designed to determine the ash value of the supplied sample.
Water soluble ash is useful in detect the presence of
material exhausted by water. If carbon is still present after
heating at a moderate temperature, the water- soluble ash
may be separated and the residue again ignited. In
sulphated ash the treatment of the drug with sulphuric acid
before ignition, where by all oxides and carbonates are
converted to sulphates.
Procedure
1. Weigh accurately about 3gm of the powdered drug in
silica crucible.
2. Incinerate the powdered drug.
3. Temperature not exceeding 450C until the sample
was free from carbon.
4. Cool it and keep it in desiccators.
5. Weigh the ash and calculate the percentage of total
ash in contrast to the air dried sample.
6. The unpeeled variety of clove buds must contain no
more than 7% of total ash.
7. Boil the total ash obtained as the above procedure
for 5 minutes with 25ml of distilled water.
8. Filter and collect the insoluble matter on ash less
filter paper.
9. After that wash the filter paper with hot water.
10. Ignite in tarred crucible for 15 minutes temperature
not exceeding 450C.
11. Cool and keep in desiccators.

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12. Weigh the obtained residue and calculate water

soluble ash of the crude drug.
13. The difference in weight representing the
water soluble ash.
14. The unpeeled variety of Clove buds must contain no
more than 7% of total ash, 0.75% of acid insoluble ash,
Water soluble ash value not less than 17% and suphated
ash value not more than 11%
(W − W2)
𝐏𝐞𝐫𝐜𝐞𝐧𝐭 𝐰𝐚𝐭𝐞𝐫 𝐬𝐨𝐥𝐮𝐛𝐥𝐞 𝐚𝐬𝐡 = × 100
(W1)

Where, W1 is weight in grams of silica dish and sample (ash),

W2 is weight in grams of silica dish and water soluble ash
and W is weight of total ash.
Procedure
Sulphated ash value determination
1. A silica crucible dish was heated to redness for 10
minutes.
2. Allow to cool in a desiccators and weighed
3. 1gm of the substance being examined was placed in the
dish.
4. Moistened with sulphuric acid.
5. Ignited gently and moistened again with sulphuric acid
and ignited at about 800C.
6. It was then cooled and weight.
7. The percentage of sulphated ash was calculated with
reference to the air dried drug.
M1
𝐏𝐞𝐫𝐜𝐞𝐧𝐭 𝐬𝐮𝐥𝐩𝐡𝐚𝐭𝐞𝐝 𝐚𝐬𝐡 = × 100
M2
Where, M1 is mass in grams of the sulphated ash and M2 is
mass in grams of the prepared sample taken for the test.
Conclusion: Ash value of supplied sample is_____.

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8. Determination of Extractive Values of

Crude Drug
Background and objective: Extractive values are used for
evaluation of crude drugs when they cannot be estimated by
any other method. Extractive values by different solvents
are used to assess quality, purity and to detect adulteration
due to exhausted and incorrectly processed drugs.

Experiment 8.1
Aim: Determination of water-soluble extractive value of
crude drug “Ginger”.
Requirements
Water soluble extractive not less than 10%,
Conical flask (250ml) with stopper,
Equipments
Chloroform water, Shallow flat-bottomed
dish
Chemicals Crude drug ginger, Chloroform water
Theory: Crude drugs contain a number of constituents and
these have a selective solubility in different solvents. Water,
alcohol, alcohol/water mixtures, generally 45%, 60%, 90%
ethanol, ether are used as solvents to prepare ethanol
soluble extractive, water soluble extractive (chloroform
water), ether soluble extractive, etc. Extractive values
indicate the presence of different constituents and TLC
fingerprints can be developed for identification and semi-
quantitative analysis from these extracts. Ginger contains
some water soluble constituents. If the ginger is exhausted
with water or soaked in water for a long time while cleaning,
the water soluble extractive will have a lower value than the

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official limit. However, Ginger exhausted with chloroform

water passes this test.
Procedure
1. Weigh accurately about 5gm of coarsely powdered drug
into a 250 ml conical flask with stopper.
2. Add 100ml of chloroform water.
3. Shake the flask frequently during first 6 hours.
4. Keep it aside without disturbing for 18 hours and then
filter.
5. Pipette out 25ml of the filtrate and evaporate to
dryness in a weighed shallow flat-bottomed dish on a
water bath.
6. Then dry the residue at 105oC to a constant weight.
7. Calculate the percentage of water-soluble extractive.
weight of residue
%𝐚𝐠𝐞 𝐨𝐟 𝐰𝐚𝐭𝐞𝐫 𝐬𝐨𝐥𝐮𝐛𝐥𝐞 𝐞𝐱𝐭𝐫𝐚𝐜𝐭𝐢𝐯𝐞 = X 100
weight of drug

It is expressed as percent w/w of the air-dried drug.

Conclusion: Water-soluble extractive value of Ginger
is_____.

Experiment 8.2
Aim: Determination of alcohol-soluble extractive value of
crude drug “Ginger”.
Requirements
250ml conical flask with stopper, Shallow
Equipments
flat-bottomed dish, Water bath
Chemicals Alcohol (90%), Crude drug ginger
Theory: Crude drugs contain a number of constituents and
these have a selective solubility in different solvents. Water,
alcohol, alcohol/water mixtures, generally 45%, 60%, 90%

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ethanol, ether are used as solvents to prepare ethanol

soluble extractive, water soluble extractive (chloroform
water), ether soluble extractive, etc. Extractive values
indicate the presence of different constituents and TLC
fingerprints can be developed for identification and semi-
quantitative analysis from these extracts. Ginger contains
some water soluble constituents. If the ginger is exhausted
with water or soaked in water for a long time while cleaning,
the water soluble extractive will have a lower value than the
official limit. However, ginger exhausted with chloroform
water passes this test. Alcohol soluble extractive of ginger
(should not be less than 4.5%). Lower values indicate ginger
exhausted with alcohol. Ginger exhausted with alcohol pass
this test. It contains about 1-3% volatile oil and 5-8% resins,
which are soluble in alcohol.
Procedure
1. Weigh accurately about 5gm of coarsely powdered drug
into a 250 ml conical flask with stopper.
2. Add 100ml of chloroform water.
3. Shake the flask frequently during first 6 hours.
4. Keep it aside without disturbing for 18 hours and then
filter.
5. Pipette out 25ml of the filtrate and evaporate to
dryness in a weighed shallow flat-bottomed dish on a
water bath.
6. Then dry the residue at 105oC to a constant weight.
7. Calculate the percentage of alcohol-soluble extractive.
weight of residue
%𝐚𝐠𝐞 𝐨𝐟 𝐚𝐥𝐜𝐨𝐡𝐨𝐥 𝐬𝐨𝐥𝐮𝐛𝐥𝐞 𝐞𝐱𝐭𝐫𝐚𝐜𝐭𝐢𝐯𝐞 = X 100
weight of drug

It is expressed as percent w/w of the air-dried drug.

Conclusion: Alcohol soluble extractive value of Ginger
is_____.

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9. Determination of Swelling and

Foaming Index
Background and objective: The main objective of present
experiment is to measure the swelling and foaming factor of
the given sample. The foaming ability of an aqueous
decoction of plant materials and their extracts are
measured in terms of a foaming index.

Experiment 9.1
Aim: To determine the swelling index of given known drug
“Isapgol seeds”.
Requirements
Equipments Stoppered measuring cylinder (25ml)
Chemicals Isapgol (Plantago ovata) seed, Water (QS)
Theory: Many herbal drugs are of specific for the
therapeutic or pharmaceutical utility because of their
swelling properties especially gums and drug those are
containing an appreciable amount of constituents like
mucilage, pectin or hemicelluloses. The swelling index is
defined as the volume in ml taken up by the swelling of 1gm
of herbal 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
herbal material (either whole, cut or pulverized). Using a
measuring cylinder with glass-stopper, the material must be
shaken repeatedly and then allowed the measuring cylinder
to stand for a required period of time. The volume of the
mixture (in ml) is then read. The mixing of whole herbal
material with the swelling agent is easy to achieve, but cut
or pulverized materials requires vigorous shaking at

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specified interval of time to ensure even distribution of the

material in the swelling agent
Procedure
1. Transfer 1gm of isapgol seed to a 25ml stoppered
measuring cylinder.
2. Fill the cylinder up to 25ml mark with water.
3. Agitate gently occasionally during 24 hours and allowed
to stand.
4. Measure the volume occupied by the swollen.
5. The genuine seed of isapgol occupies a volume of not
less than 10ml and not more than14ml.
Calculation
Swelling index = Final volume of sample - Initial volume of sample
Conclusion: Swelling factor of supplied Isapgol seeds
is_____.

Experiment 9.2
Aim: To determine the foaming index of given crude drug
“Liquorice”.
Requirements
Beaker, Measuring cylinder, Test tubes,
Equipments Heating mantle, Whatman filter paper,
Weighing balance.
Chemicals Liquorice powder, Water (QS)
Theory: Foaming index is a value which is used to express
the quality of the crude drug containing saponin. The
method is based upon the property of saponin to form foam
when shaken with water and this ability to foam is caused by
the combination or non-polar sapogenin and water soluble
side chain. The foam index signifies the dilution of the

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substance or drug to be tested which gives a layer of foam

1cm high if the aqueous solution is shaken for 2-3 minutes,
and then allowed to stand for 15 minutes before reading is
made.
Procedure
1. Weigh accurately about 1gm of coarsely powdered drug
and transferred to 500ml beaker containing 100 ml of
boiling water.
2. Maintain at moderate boiling at 80C-90C for about 30
minutes. Then make it cold and filter it.
3. Add sufficient water through the filter to make the
volume up to 100ml (V1).
4. Cleaned stopper test tubes 10 numbers are taken and
marked with 1 to 10.
5. Take the successive portions of 1m, 2ml up to 1ml drug
in separate tubes and adjust remaining volume with the
liquid up to 10ml in each test tube.
6. After closing the tubes with stoppers, Shake them for 2-
3 minutes and allowed to stand for 15 minutes.
7. Then measure the height of foam.
8. If the height of the foam in each tube is less than 1cm,
the foaming index is less than 100 (significant).
9. If the height of the foam in every tube is more than
1cm, the foaming index is more than 100 (non-
significant).
10. If the height of the foam in each tube is 1cm, the
foaming index is less than 100 then foaming index is
calculated by using formula:

Foaming index = in case of V1

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Where, (a) is volume (in ml) of decoction used for preparing

the dilution in the tube where exactly 1cm or more foam is
observed.
Conclusion: Foaming index of given sample is_____.

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10. Determination of Moisture Content

of Crude Drugs
Background and objective: Moisture content determination
is important, not only to know excess water, but also in
conjunction with suitable temperature moisture will lead to
the activation of enzymes and gives suitable conditions to
the proliferation of living organism.

Experiment 10.1
Aim: Determination of moisture content of given crude drug
“Ginger” powder.
Requirements
Moisture content apparatus, Hot air oven,
Equipments
Crucible, Desiccators, Weighing Machine
Chemicals Ginger powder
Theory: Moisture content is, simply, how much water is in a
product. It influences the physical properties of a substance,
including weight, density, viscosity, conductivity, and others.
It is generally determined by weight loss upon drying.
Moisture content determination is important, not only to
know excess water, but also in conjunction with suitable
temperature moisture will lead to the activation of enzymes
and gives suitable conditions to the proliferation of living
organism. As most vegetable drugs contain all the essential
food requirements for mould, insects and mites,
deterioration can be very rapid, once infestation has taken
place. Various methods for moisture determination are loss
on drying, separation and measurement of moisture,
chemical methods, electrometric methods, and
spectroscopic methods as per Indian Pharmacopoeia (IP).

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Procedure
1. 0.5gm of powder was weighed and placed in an empty
crucible.
2. Kept it in a hot air oven for 15 minutes.
3. Temperature was adjusted to 100C-110C till weight
gets constant.
4. Collected in desiccators and weighed.
5. The loss of weight was regarded as a measure of
moisture content as per IP.
Observation
 Weight of the empty crucible =_____gm
 Weight of the drug taken =_____gm
 Weight of the empty crucible + weight of the drug taken
before drying =_____gm
 Weight of the empty crucible + weight of the drug taken
after drying = _____gm
Calculation
Moisture content = Initial volume - final volume

Final volume
𝐏𝐞𝐫𝐜𝐞𝐧𝐭𝐚𝐠𝐞 𝐦𝐨𝐢𝐬𝐭𝐮𝐫𝐞 𝐜𝐨𝐧𝐭𝐞𝐧𝐭 = × 100
Initial volume
 Percentage moisture content =_____%
Conclusion: Moisture content of the given sample
is_____and percentage moisture content is found to
be_____%.

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11. Morphology, Histology and Powder

Characteristics
Background and objective: Students will able to learn the
protocol of powder microscopy for the identification,
determination of cellular structure of organized drugs and
detection of adulterants.
Intellectual skills: Ability to interpret the tissue components
Motor skills: Ability to prepare thin transverse section of
cinnamon bark and to handle, observe instruments and crude
drug correctly. Labeling various components of cell.

Experiment 11.1
Aim: To study the morphology & powder microscopy of
given crude drugs; “Senna”, “Coriander” and “Clove”.
Requirements
Equipments Watch Glass, Glass Slides, Brush (0-number),
Dropper, Pipette, Spatula, Microscope
Crude Drugs (Senna, Coriander & Clove),
Chemicals Glycerine, Dilute HCl, Phloroglucinol, Sudan
III, Iodine solution
Theory
(A) Senna: Biological source of senna leaf consists of the dried
leaflets of “Cassia acutifolia” also known as Alexandrian
senna and of “Cassia angustifolia” which is commercially
known as Tin-velly senna, belongs to family Leguminosae.
Chemical constituents: Senna contains Sennosides A and B
(2.5%) based on the aglycones sennidin A and B, Sennosides C
and D which are glycosides of heterodianthrones of aloe-
emodin and rhein are present. Senna also contains free
chrysophanol, emodin and their glycosides and free aloe-

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emodin, rhein, their monoanthrones, dianthrones and their

glycosides.
Uses: Senna leaves are used as laxative. It is stimulant
cathartic and exerts its action by increasing the tone of the
smooth muscles in large intestine.
(B) Coriander: Biological source of coriander consists of dried
ripe fruits of “Coriandrum sativum Linn”, belongs to family
Umbelliferae.
Chemical constituents: Coriander consist of about 1% of
volatile oil the chief volatile components are D-(+)-linalool
(coriandrol), along with other constituents like, borneol, p-
cymene, camphor, geraniol, limonene, and alpha-pinenes. The
fruits also contain fatty oil and hydroxycoumarins; includes
acids of petroselic acid, oleic acid, linolenic acid.
Uses: Aromatic, carminative, stimulant, alterative,
antispasmodic, diaphoretic and flavoring agent. It is also used
as refrigerant, tonic, and appetizer, diuretic, aphrodisiac; and
for indigestion, vomiting, flatulence, and other intestinal
disorders.
(C) Clove: Biological source of clove consists of the dried
flower buds of “Eugenia caryophyllus”, belongs to family
Myrtaceae.
Chemical constituents: Clove contains 14-21% of volatile oil.
The other constituents present are the eugenol, acetyl
eugenol, gallotannic acid, and two crystalline principles; α-
and β- caryophyllenes, methyl furfural, gum, resin, and fiber.
Caryophyllin is odourless component and appears to be a
phytosterol, whereas Eugenol is a colorless liquid. Clove oil
has 60–90% Eugenol, which is the cause of its anesthetic and
antiseptic properties.
Uses: Clove is used as an antiseptic, stimulant, carminative,
aromatic, and as a flavoring agent, antiemetic. Dentists use

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clove oil as an oral anesthetic and to disinfect the root canals.

Clove oil can stop toothache. Eugenol is also used as local
anesthetic in small doses.
Morphology: The morphology or organoleptic evaluation
means the study of crude drugs with sense organs which
includes its external morphology, color, taste, odour, etc. The
organoleptic method depend on morphological characters but
for further assurance we use this method by examination of
histological feature because crude drug has his element which
vary from one crude drug to another.
Microscopical evaluation: The crude drugs are evaluated
microscopically by their known histological characters. The
shape, size, relative position of cells and tissues, chemical
nature of cell wall and cell content are determined.
Powder microscopy: Powdered Crude Drug Microscopy of
different plant parts investigates various microscopic
techniques used in the examination of structural and cellular
features in order to determine their botanical origin. These
methods are useful in identifying species with similar
morphological characters. The microscopical examinations of
powdered drugs depend on presence or absence of certain
cells or tissue elements such as: stone cells, fibers or depends
on cells elements such as starch grains, oil droplets, calcium
oxalate, crystals, and aleurones grains.
Procedure
Morphology
Place all three crude drug samples separately in three
different watch glasses and determine their organoleptic
characteristics i.e. color, odor, shape, texture, fracture, size.
Powder microscopy
1. Powder all three crude drugs individually using mortar
pestle and sieve the powdered crude using sieve number

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#80.
2. Now place each powdered drug in different watch
glasses.
3. Small quantity of different plant parts powder is placed
separately on slides, spread powder uniformly over slides
with the help of brush and each slide is mounted 2-3 drops
of: Firstly glycerin with water, then with pholoroglucinol and
lastly with dilute HCl.
4. Then with Iodine solution and each slide was covered
with cover slip then examined under microscope.
5. Repeat this process for each crude drug powder.
6. Different cell components i.e. cork cells, sieve tubes
fibers, lignified fibers, cortex cells, calcium oxalate crystals,
mesocarp, endocarp and stomatal cells are noted and
photography is done by using camera.
Observation
(A) Morphology

Figure 11.1: Morphology of senna and coriander

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(a) Clove
Color: dark brown; odour: aromatic and strong spicy; taste:
aromatic, pungent, bitter and spicy; size: length is 12-17mm
and diameter is 3-4mm; shape: globular, depressed at the
base, reticular wrinkled, bears tri-radiate stigma, with
slender stalk about 4mm long.
(b) Coriander
Color: yellowish- green; odour: fruit becomes fragrant by
drying aromatic odor, especially when bruised; shape:
globular, and composed of two concavo-convex mericarps.
(c) Senna leaf
Color: grayish- green or yellowish- green powder; odour:
faint, characteristic; shape: lanceolateto, ovate lanceolate;
taste: mucilaginous, slightly bitter.
(B) Microscopy
(a) Powder microscopy of senna leaf

Figure 11.2: Microscopy of senna leaf

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(c) Powder microscopy of clove

Figure 11.4: Microscopy of clove

(b) Powder microscopy of coriander

Figure 11.3: Microscopy of coriander

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Conclusion: We have determined morphology and

microscopy of various drugs.

Experiment 11.2
Aim: To study the histology (transverse section) of given
crude drug “Cinnamon”.
Requirements
Compound microscope, Watch glass, Brush,
Equipments Needle, Blade, Glass slides, Cover Slip,
Dropper
Concentrated HCl, Phloroglucinol, Ether:
Chemicals Ethanol (96%) (1:1) Glycerine, Distilled
water, Cinnamon
Theory: Biological source of cinnamon is the dried inner bark
of the coppiced shoots of “Cinnamomum zeylanicum”,
belonging to family Lauraceae.
Chemical constituents: Cinnamon contains about 10% of
volatile oil, tannin, mucilage, calcium oxalate and sugar.
Volatile oil contains 50 to 65% cinnamic aldehyde, along with
5 to 10% eugenol, terpene hydrocarbons and small
quantities of ketones and alcohols.
Uses: It is used as an aromatic, carminative, flavouring
agent, analgesic, antiseptic, anti rheumatic, antispasmodic,
demulcent, digestive, expectorant, stomachic, diaphoretic,
antibacterial, antifungal, etc. It stops vomiting, relieves
flatulence and is given with chalk and as astringents for
diarrhoea and haemorrhage of the womb. It is also used in
the treatment of bronchitis, colds, palpitations, nausea,
congestion, and liver problems.
Microscopical evaluation: The crude drugs are evaluated
microscopically by their known histological characters. The

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shape, size, relative position of cells and tissues, chemical

nature of cell wall and cell content are determined.
Procedure
Transverse section of cinnamon bark
1. Drug is selected for the microscopy evaluation
(transverse section).
2. Before section cutting, soak the drug in hot water
for 3-4 hours, so the hardness of drug changes to soft.
(softening of drug make easy to cut thin sections).
3. A thin section of cinnamon bark was transferred to the
microscopic glass with a brush.
Staining process
1. Clean the platform and issue the apparatus.
2. Take a clean watch glass and add the staining solution
to it. (i.e. one drop of phloroglucinol, one drop of
concentrated HCl).
3. With the help of brush, transfer the section taken from
watch glass containing water to stain solution and keep it
for 2 - 3 minutes.
4. Transfer it to watch glass containing plane water, so
that excess stain is washed away. This section is ready for
mounting.
Mounting process
1. Transfer the section to be mounted on the glass slide
with the help of brush.
2. A drop of glycerine was added to the thin section.
3. Place the clean cover slip over the section with the help
of a forceps and needle.
4. With the help of blotting paper, wipe out excess of
water present outside the cover slip.
5. The slide is ready for observation.
Observation

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Figure 11.5: Transverse section of Cinnamon

Conclusion: On transverse section of Cinnamon we have
observed various types of tissue and cellular structures
under microscope i.e. cortex, pericyclic sclerenchyma,
medullary rays, phloem fibres and oil glands.

Experiment 11.3
Aim: To study the histology (transverse section) of given
crude drug “Fennel”.
Requirements
Compound microscope, Watch glass, Brush,
Equipments Needle, Blade, Glass slides, Cover slip,
Dropper
Conc HCl, Iodine solution, Ether: Ethanol
Chemicals
(96%) (1:1) Glycerine, Dist. water, Fennel
Theory: Biological source of fennel consists of dried ripe
fruits of “Foeniculum vulgar”, belongs to family
Umbelliferae.
Chemical constituents: The best varieties of fennel contain 4
to 5% of volatile oil. The primary constituents of volatile oil
are 50 to 60% of anethole, a phenolic ester; and 18 to 22%

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of fenchone, a ketone. Fenchone is chemically a bicyclic

monoterpene which is a colorless liquid and the odour and
taste is pungent and camphoraceous. The oil of fennel has β-
pinene, anisic acid, phellandrine, and anisic aldehyde. Fennel
also contains about 20% fixed oil and 20% proteins.
Uses: Fennel is used as stomachic, aromatic, diuretic,
carminative, diaphoretic, as a digestive, pectoral, and
flavouring agent. Anethole may have estrogen-like activity
and inhibit spasms in smooth muscles. Fennel can increase
production of bile, used in the treatment of infant colic, to
promote menstruation in women, can increase lactation,
and act as antipyretic, antimicrobial and anti-inflammatory.
Microscopical evaluation: The crude drugs are evaluated
microscopically by their known histological characters. The
shape, size, relative position of cells and tissues, chemical
nature of cell wall and cell content are determined.
Procedure
Transverse section of fennel
1. Drug is selected for the microscopy evaluation
(transverse section).
2. Before section cutting, soak the drug in hot water
for 3-4 hours, so the hardness of drug changes to soft.
(softening of drug make easy to cut thin sections).
3. A thin section of fennel was transferred to the
microscopic glass with a brush.
Staining process
1. Clean the platform and issue the apparatus.
2. Take a clean watch glass and add the staining solution
to it. (i.e. one drop of phloroglucinol, one drop of
concentrated HCl).
3. Using brush, transfer section taken from watch glass,
and keep it for 2-3 minutes in stain solution.

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4. Transfer it to watch glass containing plane water, so

that excess stain is washed away. This section is ready for
mounting.
Mounting process
1. Transfer the section to be mounted on the glass slide
with the help of brush.
2. A drop of glycerine was added to the thin section.
3. Place the clean cover slip over the section with the help
of a forceps and needle.
4. With the help of blotting paper, wipe out excess of
water present outside the cover slip.
5. The slide is ready for observation.
Observation

Figure 11.6: Transverse section of fennel

Conclusion: On transverse section of Fennel, we have
observed various characteristics, under microscope i.e.
carpophores, Raphe, vita (containing oil), reticulate
lignified parenchyma, epidermis, vascular bundles.

Experiment 11.4
Aim: To study the histology (transverse section) of given
crude drug “Clove”.

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Requirements
Compound microscope, Watch glass, Brush,
Equipments Needle, Blade, Glass slides, Cover slip,
Dropper
Concentrated HCl, Phloroglucinol,
Chemicals Ether:Ethanol (96%) (1:1) Glycerin, Distilled
water, Clove
Theory: Biological source of clove consists of the dried
flower buds of “Eugenia caryophyllus”, belonging to family
Myrtaceae.
Chemical constituents: Clove contains 14-21% of volatile oil.
The other constituents present are the eugenol, acetyl
eugenol, gallotannic acid, and two crystalline principles; α-
and β- caryophyllenes, methyl furfural, gum, resin, and fibre.
Caryophyllin is odourless component and appears to be a
phytosterol, whereas eugenol is a colorless liquid. Clove oil
has 60-90% eugenol, which is the cause of its anesthetic and
antiseptic properties.
Microscopical evaluation: The crude drugs are evaluated
microscopically by their known histological characters. The
shape, size, relative position of cells and tissues, chemical
nature of cell wall and cell content are determined.
Uses: Clove is used as antioxidants, helps protect against
cancer, kill bacteria, improve liver health, regulate blood
sugar, promote bone health, reduce stomach ulcer and
alleviate tooth pain.
Microscopical evaluation: The crude drugs are evaluated
microscopically by their known histological characters. The
shape, size, relative position of cells and tissues, chemical
nature of cell wall and cell content are determined.

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Procedure
Transverse section of clove
1. Drug is selected for the microscopy evaluation
(transverse section).
2. Before section cutting, soak the drug in hot water
for 20-30 minutes, so the hardness of drug changes to
soft. (softening of drug make easy to cut thin sections).
3. A thin section of clove was transferred to the
microscopic glass with a brush.
Staining process
1. Clean the platform and issue the apparatus.
2. Take a clean watch glass and add the staining solution
to it. (i.e. one drop of phloroglucinol, one drop of
concentrated HCl).
3. Using brush, transfer section taken from watch glass,
and keep it for 2-3 minutes in stain solution.
4. Transfer it to watch glass containing plane water, to
wash away the stain. This section is ready for mounting.
Mounting process
1. Transfer the section to be mounted on the glass slide
with the help of brush.
2. A drop of glycerine was added to the thin section.
3. Place the clean cover slip over the section with the help
of a forceps and needle.
4. With the help of blotting paper, wipe out excess of
water present outside the cover slip.
5. The slide is ready for observation.
Observation: Follow figure 11.4
Conclusion: On transverse section of clove we have
observed various types of tissue & cellular structures under
microscope i.e. cuticle, epidermis, oil glands, columella,
vascular bundles, and parenchyma.

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12. Exercise Involving Isolation &

Detection of Active Principles
Background and objective: Students will able to learn the
protocol to determine the maximum content of caffeine
among black tea; and process of extraction of volatile oil and
sennosides.
Intellectual skill: Ability to understand the basic principle of
volatile oil separation from crude drugs.
Motor skill: Ability to setup/handle the complete clevenger
assembly.

Experiment 12.1
Aim: To extract caffeine and calculate percentage yield of
caffeine from prepared “Tea” leaves.
Requirements
Beaker (500ml), Glass rod, Funnel, China
dish, Separating funnel, Measuring cylinder,
Equipments
Heating mantle, Weighing balance, Water
bath
Chemicals Lead acetate, 10% H2SO4, Chloroform, Tea
Theory: Biological source of tea consists of prepared leaves
and leaf buds of “Camellia sinensis” and “Thea sinensis”
belongs to the Theaceae family. Extraction, as the term is
used pharmaceutically; involves the separation of
medicinally active portions of plant or animal tissues from
the inactive or inert components by using selective solvents
in standard extraction procedures. The basic principle of
extraction is to grind the plant material (dry or wet) finer,
which increases the surface area for extraction thereby
increasing the rate of extraction. Caffeine which is

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chemically known as 3, 7-dihydro-1, 3, 7-trimethyl-1H-

purine-2, 6-dione or 1, 3, 7-trimethylxanthine having
chemical formula C8H10N4O2 was first discovered in 1827
belonging to the alkaloid family containing nitrogen in their
ring structure. Caffeine; which is the purine based pseudo
alkaloid (not directly derived from amino acid).
Pharmacologically caffeine acts as CNS stimulant, mild
diuretic, a natural pesticide, increase blood pressure,
increase heart rate, stimulate gastric motility, algicidal,
bactericide.
Procedure
1. Accurately weighed tea sample 25gm each with the
help of electronic balance.
2. Transfer tea leaves into marked separate beakers for
each sample add 200ml of distilling water and start boiling
for about 10 minutes with constant stirring.
3. Filter the mixture and collect infusion in a separate
beaker.
4. Now repeat the above procedure two more times by
using the same tea leaves.
5. Collect all the filtrates in the same beaker and add lead
acetate in order to precipitate tannins which are present
in the tea infusion.
6. Again heat the mixture of tea infusion and lead acetate
to complete the process of precipitation.
7. Now filter it with the help of wahtman filter paper to
separate the precipitated tannins from the infusion.
8. Do washing of tannin precipitate in order to make sure
that no caffeine residue left on filter paper. Add 2ml of
10% sulphuric acid into the filtrate.
9. Now transfer the filtrate into separating funnel and add
30ml of chloroform do proper mixing by inverting the

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separating funnel, so many times release air and allow to

stand to separate caffeine now collect the organic layer
same procedure is repeated twice collect all the sample in
the same beaker.
10. For the evaporation of the solvent placed it overnight
in the fuming hood next day/evaporate it till dryness on
water bath, observe the caffeine crystal, calculate its
percentage yield.
Observation/Calculation
 Initial weight of the drug (theoretical yield) =_____gm
 Weight of caffeine (obtained after extraction) = b - a
 Practical yield =_____gm

𝐏𝐞𝐫𝐜𝐞𝐧𝐭𝐚𝐠𝐞 𝐲𝐢𝐞𝐥𝐝 = × 100

Percentage yield =_____%

Conclusion: We obtained white crystalline powder after
extraction and percentage yield of caffeine is calculated
as_____.

Experiment 12.2
Aim: To extract volatile oil from “Clove” using Clevenger
apparatus.
Requirements
Round bottom flask (500ml), Funnel,
Clevenger apparatus, Condenser, Water
Equipments
pipes, Measuring cylinder, Heating mantle,
Weighing balance, Porcelain chips
Glycerine or Paraffin wax, Cloves, Solvent
Chemicals
(250ml)

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Theory: Biological source of clove consists of the dried

flower buds of “Eugenia caryophyllus”, belonging to family
Myrtaceae.
Chemical constituents: Clove contains 14-21% of volatile oil.
The other constituents present are the eugenol, acetyl
eugenol, gallo-tannic acid, and two crystalline principles; α-
and β-caryophyllenes, methyl furfural, gum, resin, and fiber.
Caryophyllin is odourless component and appears to be a
phytosterol, whereas eugenol is a colorless liquid. Clove oil
has 60-90% eugenol, which is the cause of its anesthetic and
antiseptic properties.
Uses: Clove is used as an antiseptic, stimulant, carminative,
aromatic, and as a flavouring agent, antiemetic. Dentists use
clove oil as an oral anesthetic and to disinfect the root
canals. Clove oil can stop toothache. Eugenol is also used as
local anaesthetic in small doses. The extraction of essential
oils is generally carried out by two main techniques:
azeotropic distillation (hydro distillation, hydro diffusion,
and steam distillation) and extraction with solvents.
Distillation: Distillation converts the volatile liquid (the
essential oils) into a vapor and then condenses the vapor
back into a liquid. It is the most popular, and cost effective
method in use today in producing essential oils. Examples
are: Water distillation, steam distillation, hydro diffusion.
Procedure
1. Extraction of clove using clevenger apparatus.
2. Weigh 50gm of crude drug and crush it into coarse
powder form.
3. Soak the drug for 1-2 hours in specified solvent i.e.
water in round bottom flask.
4. Make up the volume of solvent up to 250ml.
5. Fix the complete assembly appropriately.

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6. Now subject the extraction for 3-4 hours.

7. Finally measure & collect the separated volatile oil.
Conclusion: We have obtained 2ml oil from the crude
crushed Clove buds by using clevenger apparatus (hydro
distillation method).

Experiment 12.3
Aim: To extract and detect sennosides from “Senna”.
Requirements
Vacuum filter unit, Electric shaker,
Equipments
Desiccators
Benzene, Ethanol, Methanol, Senna leaves
Chemicals
or pods
Theory: Sennosides are obtained from “Cassia angustifolia”
(Tinne velly senna), “Cassia acutifolia” (Alexandrian senna).
Sennosides are the dimeric anthroquinone glycosides.
Sennoside A and B is a pair of stereoisomer containing rhein
dianthrone (sennedine A and B) as the aglycon.Sennoside D
and E are the dianthrone of aloe-emodin and rhein.
Purgative activity of senna is mainly due to sennoside A and
sennoside B while sennoside C and D exert a powerful
synergistic effect upon the purgative activity.
Procedure
Method (A)
1. Extract coarsely powdered senna leaves or pods with
benzene and 1-5% ethanol to remove pigments and resins.
2. The dried marc extracts with ethanol and concentrates
under at 40C.
3. Alcoholic extract mixed with the solution of calcium
chloride in methanol and the solution is filtered.

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4. Add methanolic ammonia in the filtrate until red brown

color disappears.
5. The precipitated material is filtered and washed with
methanol and dried.
6. The precipitate containing calcium sennoside is
suspended in methanol and acidified with gluconic acid at
40C.
7. Filter the acidified extract which gives yellow mass of
sennoside A.
8. The filtrate when treated with methanolic hydrotropic
acid and subsequently evaporated top reduce sennoside
Method (B)
1. Extract 100gm powdered leaves with 300ml benzene
for 2 hours on electric shaker, filter in vacuum and distill
off the solvent.
2. The dried marc extract with 300ml 70% methanol on
shaker for 4-6 hours, filter under vacuum and the marc re-
extract with 200ml of 70% methanol for 2 hours; filter and
combine methanolic extract.
3. Concentrate methanolic extract and acidify to pH 3.2 by
addition of HCl with constantstirring.
4. Set aside the mixture for 2 hours at 5C. Filter under
vacuum and add 1gm anhydrous calcium chloride in 13ml
denatured spirit with vigorous shaking.
5. Adjust pH of the solution to 8 by addition of ammonia
solution and set aside for 2 hours.
6. Filter the solution by vacuum and dry precipitate over
phosphorous pentoxide in a desiccator.
Chemical tests
Borntrager test: Boil drug with dilute sulphuric acid
(hydrolysis). Filter and cool. Add benzene or CCl4
(immiscible organic solvents). Shake and separate organic

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solvent layer in another test tube. Add strong ammonia

solution, shake slightly and keep the test tubes aside,
lower ammonical layer shows pink or red color.
Thin layer chromatography: Dissolve 1mg sennoside in
1ml solvent containing equal volumes of ethyl acetate, n-
propanol and water (upper layer). The silica gel-G plates
spotted with the sample and eluted in solvent system
ethyl acetate:n-propanol:water (4:4:3).The dried plate is
exposed to vapours of ammonia for 5min till the color
develops. Cover the plate with glass and heat at 110C for
5-10 minutes. sennosides A and B develop two prominent
spots.
Calculation
Weight of the isolated sennoside
%𝐚𝐠𝐞 𝐲𝐢𝐞𝐥𝐝 = × 100
weight of the dried powder taken

Distance travelled by the solute

𝐑𝐟 𝐯𝐚𝐥𝐮𝐞 = × 100
Distance travellrd by the sovent
Conclusion: The percentage yield of isolated sennoside was
found to be_____ and the Rf value of the isolated sennoside
was found to_____.

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13. Chromatographic Separation of

Sugars
Background and objective: Students will come to learn and
understand the extraction technique; and separation of
sugars by paper chromatography.

Experiment 13.1
Aim: To separation of sugars by paper chromatography.
Requirements
Whatman filter paper (number-1), Solvent tank
Equipments
with lid, Pencil, Hot air dryer
Solvents, Spray reagent, Unknown sugar
Chemicals
sample
Theory: The term chromatography comes from the earlier
times when the technique was used for the separation of
colored plants pigments. Chromatography is a technique for
separation of closely related groups of compounds. The
separation is brought about by differential migration along a
porous medium and the migration is caused due to flow of
solvent. Within limits chromatography can be divided into
two types: partition and adsorption chromatography.
Paper chromatography is an example of liquid-liquid
chromatography in this type of chromatography separation
is due to differential partition of solutes between two liquid
phases .One liquid phase is bound to the porous medium for
example, the water bound in the cellulose paper; this phase
is referred to as, the stationary phase. The other liquid
phase, the mobile phase flows along the porous medium. As
the mobile phase flows over the solute mixture, the
individual solutes partition themselves between the

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aqueous stationary phase and the organic mobile phase

relative to their solubility in the two phases. The more
soluble a solute in the mobile phase, the faster it will travel
along the paper, and conversely, the mobile phase must be a
mixture in which the compounds to be separated are soluble
or partially soluble. In paper chromatography solute or
solute mixture is spotted in solution along a base line on a
sheet of filter paper (whatman number-1).
The mobile phase (solvent) is allowed to flow over the spots
either ascending the paper by capillary action or descending
the paper by gravity. The separation is measured in terms of
a unit called Rf (relative rates of flow) with respect to the
solvent front. The Rf value of a compound in a particular
solvent system is constant under identical conditions of the
experiment, e.g.; temperature, pH, etc. Because most
compounds are colorless the spots are visualized after
separation by specific reagent. The location reagent is
applied by spraying the paper or rapidly dipping it in a
solution of the reagent in a volatile solvent. Viewing under
ultraviolet light is also useful since some compound which
absorbs it strongly show up as dark spots against the
florescent background of the paper.
Materials
Paper: Usually whatman number-1 filter paper is used
because of its known
Solvents: Water-saturated phenol + 1% ammonia; n-
butanol-acetic acid-water (4:1:5 v/v) and Isopropanol-
pyridine-water-acetic acid (8:8:4:1 v/v).
Spray reagent
 Ammonical silver nitrate: Add equal volumes of NH4OH to
a saturated solution of AgNO3 and dilute the methanol to
give a final concentration of 0.3M. After spraying the

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developed chromatograms, place them in an oven for 5-10

minutes, when the reducing sugars appear as brown spots.
 Alkaline permanganate: Prepare aqueous solution of
KMNO4 (1%) containing 2% Na2CO3. After spraying with this
mixture, the chromatograms are kept at 100C for a few
minutes, when the sugar spots appear as yellow spots in
purple background.
 Aniline diphenylamine reagent: Mix 5 volumes of 1%
aniline and 5 volumes of 1% diphenylamine in acetone with
1 volume of 85% phosphoric acid. After spraying the dried
chromatograms with this solution the spots are visualized by
heating the paper at 100C for a few minutes.
 Resorcinol reagent: Mix 1% ethanolic solution of resorcinol
and 0.2N HCl (1:1 v/v). Spray the dried chromatograms and
visualize spots by heating at 90C.
Table 13.1: The table below Rf values of some sugars in the
solvents previously mentioned. They are only for
comparative purposes, since Rf Varies with physical
parameters.
Sugar Solvent (a) Solvent (b) Solvent (c)
Glucose 0.39 0.18 0.64
Galactose 0.44 0.16 0.62
Fructose 0.51 0.25 0.68
Ribose 0.59 0.31 0.76
Deoxy ribose 0.73 - -
Lactose 0.38 0.09 0.46
Maltose 0.36 0.11 0.50
Sucrose 0.39 0.14 0.62
Procedure
1. Place sufficient solvent into the bottom of the tank.
Cover the led and allow the tank to be saturated with the

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solvent.
2. Take a sheet of whatman 1 chromatography paper
(about 9 x 10cm) and place it on a piece of clean paper on
a bench.
3. Draw a fine line with a pencil along the width of the
paper and about 1.5cm from the lower edge.
4. Along this line place four equally spaced (about 2cm
apart) small circles with a pencil.
5. Label the paper at the top with the name of each of the
sugars and label the last unknown.
6. Use a fine capillary or tooth pick to place the drops of
the solutions of the sugars, glucose, fructose, maltose,
lactose and the mixture.
7. After spotting, dry the paper with hot air dryer for one
minute, repeat this step again.
8. Place the spotted paper in the chromatographic tank
and make the development by using the ascending
technique.
9. Close the tank with lid, allow the solvent to flow for
about 30-45 minutes.
10. Remove the paper and immediately mark the position
of the solvent front with a pencil.
11. After the chromatogram has dried, spray the paper
with the locating reagent.
12. You need to put the paper on the hot plate at low
temperature or expose it to the hot air dryer, until the
colored spots appear. The colors are stable for some
weeks if kept in the dark and away from acid vapors.
13. Circle the position of each spot with pencil calculates
the Rf value for each spot and also for the spots the
mixture contained.

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Table 13.2: General summary of the behavior of the various

sugars to these reagents are given below.
Sugars (a) (b) (c) (d)
Aldohexoses + + + Pink
Ketohexoses + + + Red
Aldopentoses + + + Blue, green
Ketopentoses + + + -
Deoxy sugars - + + -
Glycosides + - - -
Amino sugars + + + -
Conclusion: By using paper chromatography; sugars was
separated from unknown sugar sample.

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14. Thin Layer Chromatography of

Herbal Extract
Background and objective: Students will come to learn and
understand the principle and method of separation and
identification technique for curcuminoid.

Experiment 14.1
Aim: To separate and identify the curcuminoid present in
“Turmeric” by thin layer chromatography.
Requirements
Glass slides, Capillary tube, Solvent
Equipments
chamber, Hot air oven, UV chamber
Chloroform, Ethanol, Glacial acetic acid,
Chemicals
Silica gel-G, Turmeric (coarsely powdered)
Theory: The term chromatography comes from the earlier
times when the technique was used for the separation of
colored plants pigments. Chromatography is a technique for
separation of closely related groups of compounds. The
separation is brought about by differential migration along a
porous medium and the migration is caused by the flow of
solvent. Within limits chromatography can be divided into
two types: partition and adsorption chromatography.
Procedure
1. The sample is extracted with methanol on a water bath.
Cool and filter.
2. The filtrate is used for thin layer chromatography (TLC)
studies.
3. Adsorbent used for TLC studies is silica gel-G coated on
glass plate.

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4. Mobile phase: chloroform: ethanol: glacial acetic acid

(95:5:1).
5. The solvent system is prepared in the given ratio and
then taken in a chamber and keptfor saturation.
6. The extract is placed as a spot on TLC plate and kept in
the chamber at an angle of 45C.
7. 3/4th movements of solvent the plate is taken out and
mark the solvent front.
8. Then the plate is dried in air and detected in ultra violet
light.
Calculation
Distance travelled by the solute
𝐑𝐟 𝐯𝐚𝐥𝐮𝐞 = × 100
Distance travellrd by the sovent
Conclusion: The Rf value of the given sample of Turmeric
extract was found to be_____.

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15. Analysis of Crude Drugs by Chemical

Tests
Background: Students will come to learn and understand
the meaning and significance of pharmacognostic
parameters and pharmacognostic study of crude drugs
including their analysis by performing various tests.

Experiment 15.1
Aim: Analysis of crude drug “Asafoetida” by chemical
tests.
Requirements
Beaker, Test tubes, Measuring cylinder,
Equipments Dropper, Test tube holder, filter paper,
Heating mantle, Water bath
Asafoetida, Sulphuric acid, Nitric acid (50%),
Chemicals
HCl, Ammonia solution, Water
Theory: Asafoetida also known as Devil’s dung, Heeng and
Gum asafetida is an oleo gum resin of living roots and
rhizomes of “Ferula foetida”, “Ferula rubicals” and other
spices of Ferula belongs to family Umbelliferae.
Description
Color: yellowish brown to reddish brown tears; odour:
intense, penetrating, and persistent alliaceous; taste: bitter,
acrid, and alliaceous; shape: it occurs in two forms tear sand
masses; size: 0.5×4.0cm in diameter; solubility: partly
soluble in alcohol; extra feature: Fresh tears are tough,
dried are hard and brittle. Tears are internally milky white
yellowish, translucent, or opaque mass consists of
agglutinated tears with foreign materials and impurities.

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Chemical constituents: Resin (40%-60%) mainly

asaresinotannol in free or combined form with ferulic
acid,pinene, vanillin and a resene;gum (20%-25%) and
volatile oil (4%-20%) contains isobutyl propanyl disulphide
which gives alliaceous odour to drug.
Uses: Carminatives, laxative, antispasmodic, nervine tonic,
anthelmintic and digestive. It is used to treat flatulence colic,
constipation, asthma, bronchitis, whooping cough and
epilepsy. It is also used as flavoring agent in sauces, pickles
and curries.
Chemical tests/identification tests
Test 1. Fractured surface is treated with sulphuric acid;
then washed with water.
Observation: Reddish brown color, violet color.
Test 2. Asafoetida treated with sulphuric acid.
Observation: Reddish brown color.
Test 3. Fractured surface treated with 50% nitric acid.
Observation: Green color.
Test 4. Asafoetida triturated with water.
Observation: Yellowish orange emulsion.
Test 5. 10ml alcoholic extract of drug + concentrated HCI +
phloro glucinol few drops.
Observation: Pink colour.
Test 6. Asafoetida on burning.
Observation: Yellow flames.
Test 7. (Combined umbelliferone test): 0.5gm drug+ sand
+3ml HCl+3ml water and triturated for several minutes
and boil Add strong ammonia solution to the filtrate.
Observation: Blue fluorescence.
Report: From the above morphological characters and
chemical test the given crude drug was identified as_____.

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Experiment 15.2
Aim: Analysis of crude drug “Benzoin” by chemical tests.
Requirements
Beaker, Test tubes, Measuring cylinder,
Equipments Dropper, Test tube holder, Filter paper,
Heating mantle, Water bath
Chemicals Benzoin, KMnO4, Water
Theory: Benzoin also known as Sumatra Benzoin, Loban,
Siam Benzoin; it is balsamic resin obtained from the incised
stem of syntax benzoin or “Styrax paralleloneurus”, “Styrax
tonkinensis”; belongs to family Styraceae.
Description
Color: grayish brown or grey masses; odour: agreeable and
balsamic; taste: sweetish and slightly acrid; size: varying in
size; shape: tears, masses, lumps.
Uses: Used as an expectorant.
Chemical tests/identification tests
Test 1. 0.5gm benzoin powder heated with 10ml of
KMnO4.
Observation: Odour of benzaldehyde.
Test 2. Alcoholic solution of benzoin + water.
Observation: Milky solution (acidic to litmus).
Test 3. Heat 0.5gm benzoin powder slowly in dry test
tube.
Observation: Evolves irritating whitish fumes which
condense to form whitish crystalline sublimates at upper
part of test tube.
Test 4. Sublimates after cooling.
Observation: Crystals of cinnamic acids.
Report: From the above morphological characters and
chemical test the given crude drug was identified as_____.

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Experiment 15.3
Aim: Analysis of crude drug “Colophony” by chemical tests.
Requirements
Beaker, Test tubes, Measuring cylinder,
Equipments Dropper, Test tube holder, filter paper,
Heating mantle, Water bath
Colophony, KMnO4, Water, Alcohol, Acetic
Chemicals anhydride, Sulphuric acid, Light petroleum,
dil. Copper acetate solution
Theory: Benzoin also known as Rosin, Rosina, Colophonium,
Amber resin; is solid residue obtained after distilling the
oleo-resin from various species of pinus (“Pinus palustris”,
“Pinus longifolia”, and “Pinus radiate” etc) belongs to family
Pinaceae.
Description
Color: amber or pale yellow; odour: turpentine like; taste:
slightly bitter; solubility: alcohol, ether, chloroform, and
light petroleum; insolubility: water; melting point: 75C -
85C; acid value: not less than 150; saponification value:
188-192; ash value: not more than 0.125%; extra features:
brittle and readily fusible glassy masses.
Chemical constituents: Colophony contains 90% of abietic
acid (resin acid), 5%-6% of resene, and 0.5% of volatile oil.
Other acid present are sapinic acid, pimaric acid.
Chemical tests/identification tests
Test 1. Colophony mixed with alcohol.
Observation: Forms milky white solution.
Test 2. Dissolve 0.1gm in 10ml of acetic anhydride by
means of gentle heat cool and add a drop of concentrated
sulphuric acid.
Observation: Bright red changes to violet (for abietic acid).

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Test 3. Dissolve 0.1gm in light petroleum and filter. To this

add 2-3 times dilute copper acetate solution (for
identification of adulteration of colophony).
Observation: Emerald green color of petroleum layer (for
abietic acid.
Report: From the above morphological characters and
chemical test the given crude drug was identified as_____.

Experiment 15.4
Aim: Analysis of crude drug “Aloes” by chemical tests.
Requirements
Beaker, Test tubes, Measuring cylinder,
Equipments Dropper, Test tube holder, Filter paper,
Heating mantle, Water bath
Aloes, Kieselguhr, Borax, Bromine solution,
FeCl3, Dilute HCl, Nitrous acid, Ammonia
Chemicals
solution, NaNO2, Acetic acid, Copper
acetate, Sodium chloride, Alcohol, Water
Theory: Aloes also known as Musabbar, Ghritkumari, Aloe is
the dried juice of the leaves of “Aloe barbadensis” miller
(curaccaoaloes), “Aloe perrbaker”, (socotrine aloes) aloe
ferox miller and its hybrid with “Aloe Africana” miller and
“Aloe spiculata” baker (cape aloes); belongs to family
Lilliaceae. It is obtained by incision of leaves at the base.
Description
Texture: solid waxy; color: dark brown; odour: characteristic
unpleasant odour; taste: bitter; solubility: alkali and glacial
acetic acid; partly soluble in water, chloroform and ether;
extra features: hard and uneven porous fracture.
Chemical constituents: Aloes are the major sources of
anthraquinone glycosides. Aloin is the mixture of three
isomers namely barbaloin, β-Barbaloin and isobarbaloin.

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Uses: Purgative and improve digestion, cosmetics. Ointment

of aloe is used in sunburns, thermal burns, radiation burns,
abrasions and skin irritation. At higher doses of aloes are
abortifacient. Used to prepare compound benzoin tincture in
which it is pharmaceutical adjunct. Stimulates immune
system specially T4 cells.
Chemical tests/identification tests
Test 1. Boil 0.5gm with 50ml of water until nearly
dissolved, cool and add 0.5gm kieselguhr and filter. Follow
below test to the filtrate.
(a) Borax test: Heat 2.5ml solution with 0.1gm borax; add
few drops of this in a test tube having water.
Observation: Green fluorescence.
(b) Bromine test: To 1ml of solution add 1ml of fresh
bromine solution.
Observation: Pale yellow precipitates.
(c) Modified borntrager test: To 5ml of filtrate add 10ml
FeCl3 and 5ml dil. HCl; heat it for 10 minutes and then
filter it. Add benzene to filtrate; separate benzene layer
and strong ammonia solution.
Observation: Pink to red colour to the ammonia layer.
(d) Nitrous acid test: To solution of NaNO2 add acetic acid
and then heat.
Observation: Reddish brown colour.
Test 2. (Cupraloin test for isobarbaloin): 10ml of 0.1%
solution of aloes in distilled water, add 1 drop of 5%
solution of copper acetate, 0.5ml of saturated solution of
sodium chloride, 1ml of alcohol and warm.
Observation: Pale wine red colour.
Test 3. (Bromine test): Add equal quantity of aloe to
bromine solution.
Observation: Bulky yellow precipitate of tetra bromaloin.

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Report: From the above morphological characters and

chemical test the given crude drug was identified as_____.

Experiment 15.5
Aim: Analysis of crude drug “Myrhh” by chemical tests.
Requirements
Beaker, Test tubes, Measuring cylinder,
Equipments Dropper, Test tube holder, Filter paper,
Heating mantle, Water bath
Myrrh, Nitric acid solution, Bromine
Chemicals
solution, Water
Theory: Myrhh is also known as Myrrha, Gum-myrrh and
Bol. it is the oleo-gum-resin obtained from incision from the
stem of “Commiphora molmol”, belonging to family
Burseraceae.
Description
Texture: externally reddish internally brown color: dark
brown; odour: agreeably aromatic; size: 1.5-3.0cm in
diameter; shape: irregular tear or lumps; taste: aromatic,
bitter, acrid; solubility: partly soluble in alcohol and ether
but insoluble in water; extra features: fractured surface in
granular, brittle, and translucent.
Chemical constituents: volatile oil (10%) which are terpenes,
cuminic aldehyde, eugenol, gum (60%), resin (25%-40%)
which contains ether soluble resin acids, α, β, and γ
commiphoric acid. Arabian myrrh is adulterant of myrhh.
Uses: carminatives, antiseptic, uterine stimulant, protective,
used in gargles and mouth washes.
Chemical tests/identification tests
Test 1. Triturate with water.
Observation: Forms yellowish brown emulsion.

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Test 2. (a) Ether extracts of drug with bromine vapours;

(b) moistened with nitric acid solution.
Observation: (a) Produce red colour; (b) red turns to
purple.
Report: From the above morphological characters and
chemical test the given crude drug was identified as_____.

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16. Preliminary Phytochemical Screening

of Crude Drugs
Background: Students will come to learn & understand the
meaning & significance of pharmacognostic parameters &
pharmacognostic study of crude drugs including their
analysis by performing various tests.

Experiment 16.1
Aim: Preliminary phytochemical screening of aqueous
extract of “Neem”.
Requirements
Beaker, Test tubes, Measuring Cylinder,
Dropper, Test tube Holder, Filter paper, Flat
Equipments
bottom shallow dish, Heating mantle,
Water bath
Powdered crude drug (50gm), Calcium
hydroxide, Chloroform, Dilute hydrochloric
acid, Mayer’s reagent, Wagner’s reagent,
Hager’s reagent, Acetic acid, FeCl3,
Potassium chlorate ammonia solution,
H2SO4, Dilute NH3, Fehling’s solution I & II,
Bromine water , Glacial acetic acid,
Chemicals Alcoholic α-naphthol water (1%), Benedict’s
reagent, Benzene, Alcohol, Lead sub-
acetate, Dinitrobenzene, Pyridine, Nitro
prusside, Sodium picrate paper strip,
Hydroxylamine hydrochloride, Alcoholic
KOH, Petroleum ether acetic anhydride,
Ferrous sulphate solution, Magnesium
turnings, Sodium hydroxide, Lead Acetate,

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Millon’s reagent, Dilute copper sulphate,

Ninhydrine solution, Ruthentium red,
NH4OH, Acetone
Theory: Aqueous extract of “Azadirachta indica” (Neem
leaf) was subjected to qualitative chemical analysis. The
various chemical tests were performed on this extract and
aqueous extract for the identification of flavonoids, phenolic
compounds, alkaloids, glycosides, carbohydrates,
carotenoids, proteins, tannin, aminoacids, and sterols as per
Harborne 1998.
Extraction of crude drug
1. Take 50gm of powdered crude drug and macerate it
with 500ml of water for 24 hours.
2. Then occasionally shake with 6 hours time period and
allow it to stand for 18 hours.
3. After filtration evaporate the filtrate to dryness in a tare
flat bottom shallow dish.
Preparation of test solution
1. Take 500mg of extract and dissolve it in 100ml of water.
Stir the solution till the extract is completely soluble in
water.
2. The sample solution is then subjected to various
qualitative tests to reveal the presence or absence of
common phyto-pharmaceuticals.
Evaluation tests
Test for alkaloids
About 2gm of the powdered material was mixed with 1gm
of calcium hydroxide and 5ml of water into a smooth
paste and set aside for 5 minutes. It was then evaporated
to dryness in a porcelain dish on a water bath. To this
200ml of chloroform was added, mixed well and refluxed

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for half an hour on a water bath. Then it was filtered and

the chloroform was evaporated. To this 5ml of dilute
hydrochloric acid was added followed by 2ml of each of
the following reagents.
(A) Mayer’s test: A small quantity of the extract was
treated with Mayer’s reagent. Cream color precipitate
indicates the presence of alkaloids.
(B) Dragendorff’s test: A small quantity of the extract was
treated with Dragendorff’s reagent. Orange brown
precipitate indicates the presence of alkaloids.
(C) Wagner’s test: A small quantity of extract was treated
with Wagner’s reagent. Reddish brown precipitate
indicates the presence of alkaloids.
(D) Hager’s test: A small quantity of extract was treated
with Hager’s reagent. Yellow precipitate indicates the
presence of alkaloids.
Test for purine group (Murexide test)
The residue obtained after the evaporation of chloroform
was treated with 1mL of hydrochloric acid in a porcelain
dish and 0.1gm of Potassium chlorate was added and
evaporated to dryness on water bath. Then the residue
was exposed to the vapour of dilute ammonia solution. No
purple Color was obtained indicating the absence of
purine group of alkaloids.
Test for indole
To the test solution, add acetic acid and trace amount of
anhydrous FeCl3, Under-layer/H2SO4 intense blue at
interface.
Test for quinoline (Thalleioquin test)
To the extract, add 1 drop of dilute sulphuric acid and 1ml
of water. Add bromine water drop wise till the solution
acquires permanent yellow color and add 1mL of dilute
ammonia solution, emerald green color is produced. The

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powdered drug when heated with glacial acetic acid in dry

test tube evolves red fumes, which condense in the top
portion of the tube. The bark, when moistened with
sulphuric acid and observed under ultraviolet light shows
a blue fluorescence due to the methoxy group of quinine
and quinidine.
Test for carbohydrates
(A) Molisch’s test: The extract of the powdered drug was
treated with 2-3 drops of 1% alcoholic α-naphthol and
2mL of concentrated sulphuric acid was added along the
sides of the test tube. A purple color indicating the
presence of carbohydrates.
(B) Fehling’s test: The extract of the powdered leaf was
treated with Fehling’s solution I and II and heated on a
boiling water bath for half an hour. Red precipitate was
obtained indicating the presence of free reducing sugars.
(C) Benedict’s test: The extract of the powdered leaf was
treated with equal volume of Benedict’s reagent. A red
precipitate was formed indicating the presence of
reducing sugar.
Test for anthraquinone glycosides
(A) Borntrager’s test: The powdered drug was boiled with
dilute sulphuric acid, filtered and to the filtrate benzene
was added and shaken well. The organic layer was
separated to which ammonia solution was added slowly.
No pink color was observed in ammoniacal layer showing
the presence of anthraquinone glycosides.
(B) Modified Borntrager’s test: About 0.1gm of the
powdered drug was boiled for 2 minutes with dil. HCl and
few drops of FeCl3 solution, filtered while hot and cooled.
The filtrate was then extracted with benzene and the
benzene layer was separated. Equal volume of dil. NH3
solution was added to the benzene extract. No pink color
was observed in ammonical layer showing the presence of

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glycosides.
Test for cardiac glycosides (for deoxysugar)
(A) Keller-Kiliani test: About 1gm of the powdered leaf
was boiled with 10ml of 70 % alcohol for 2 minutes,
cooled and filtered. To the filtrate 10ml of water and 5
drops of solution of lead sub acetate were added and
filtered, evaporated to dryness. The residue was dissolved
in 3ml of glacial acetic acid. To these 2 drops of ferric
chloride solution was added. Then 3ml of concentrated
H2SO4 was added to the sides of the test tube carefully
and observed. No reddish brown layer was observed
indicating the absence of deoxysugars.
(B) Raymond test: Test solution treated with
dinitrobenzene in hot methanolic alkali gives violet color.
(C) Legal’s test: Test solution when treated with pyridine
made alkaline by sodium nitro prusside solution gives pink
to red color.
Test for cyanogenetic glycosides
Small quantity of the powder was placed in a stoppered
conical flask with just sufficient water, to cover it. A
sodium picrate paper strip was inserted through the
stopper so that it was suspended in the flask and it was set
aside for 2 hours in a warm place. Brick red color was
produced on the paper indicating the presence of
cyanogenetic glycosides.
Test for coumarin glycosides
(A) With ammonia: Take a drop of ammonia on a filter
paper; to this add a drop of aqueous extract of leaves.
Development of fluorescence shows positive test for
coumarins.
(B) With hydroxylamine hydrochloride: To ethereal
extract, added one drop of alcoholic KOH. It was then
heated, cooled and acidified with 0.5N hydrochloric acid.

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Violet color developed upon addition of a drop of 1% w/v

FeCl3 indicated presence of coumarins.
Test for sterol
The powdered drug was first extracted with petroleum
ether and evaporated to a residue. Then the residue was
dissolved in chloroform and tested for sterols.
(A) Salkowski’s test: A few drops of concentrated
sulphuric acid were added to the above solution, shaken
well and set aside. The lower chloroform layer of the
solution turned red in color indicating the presence of
sterols.
(B) Test for Libbermann – Burchard’s: To the chloroform
solution a few drops of acetic anhydride and 1ml of
concentrated sulphuric acid were added through the sides
of the test tube and set aside for a while. At the junction
of two layers a brown ring was formed. The upper layer
turned green indicating the presence of sterols.
Test for saponins
(A) Froth test: 0.1gm of powder was vigorously shaken
with 5ml of distilled water in a test tube for 30 seconds
and was left undisturbed for 20 minutes, persistent froth
indicated presence of saponins.
Test for Tannins
(A) Ferric chloride: Small quantity of the powdered drug
was extracted with water. To the aqueous extract few
drops of ferric chloride solution was added. Bluish black
color was produced indicating the presence of tannins.
(B) Gold beater’s skin test: Add 2% hydrochloric acid to all
small piece of g old beater’s skin, rinses it with distilled
water and place in the solution to be tested for five
minutes. Then give wash of distilled water and transfer to
a 1% ferrous sulphate solution. A brown or black color on
the skin indicates presence of tannin.

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Test for phenol compounds

(A) Ferric chloride test: A small quantity of the powdered
drug was extracted with water. To the alcoholic extract
few drops of ferric chloride solution was added. Bluish
black color was produced indicating the presence of
tannins.
Test for flavonoids
(A) Shinoda’s test: Little of the powdered drug was heated
with alcohol and filtered. To the test solution magnesium
turnings and few drops of concentrated hydrochloric acid
were added. Boiled for five minutes. Red color was
obtained indicating the presence of flavonoids.
(B) Alkali test: To the small quantity of test solution 10%
aqueous sodium hydroxide solution was added. Yellow
orange color was produced indicating the presence of
flavonoids.
(C) Lead acetate: To the test solution add a mixture of
10% lead acetate in few drops added. It gives white
precipitate.
Test for acid
To the small quantity of test solution, few drops of
concentrated sulphuric acid were added. Yellow orange
color was obtained indicates the presence of flavonoids.
Test for protein and amino acids
(A) Millon’s test: Small quantity of acidulous- alcoholic
extract of the powdered drug was heated with Millon’s
reagent. White precipitate turned red on heating;
indicates the presence of proteins.
(B) Biuret test: To one portion of acidulous – alcoholic
extract of the powdered drug one ml of 10% sodium
hydroxide solution and one drop of dilute copper sulphate
solution were added. Violet color was obtained indicating
the presence of proteins.

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(C) Ninhydrine test: To the test solution add Ninhydrine

solution, boil, violet color indicates presence of amino
acid.
Test for sulphur containing amino acid
5ml test solution is mixed with 2ml 40 % sodium hydroxide
and 2 drops of 10% lead acetate solution. Then boil the
solution turned black or brownish due to PLS formation.
Test for terpenoids
Little of the powdered drug was extracted with chloroform
and filtered. The filtrate was warmed gently with tin and
thionyl chloride. Pink color solution appeared which
indicated the presence of terpenoids.
Test for carotenoids ( Carr-Price reaction)
Extract treated with concentrated sulphuric acid and with
a chloroform solution of antimony trichloride. Deep blue
color appeared which indicated the presence of
carotenoids.
Test for volatile oil
Weighted quantity (250gm) of fresh leaves were extracted
and subjected to hydro distillation using volatile oil
estimation apparatus.
Test for fixed oil
A small amount of the powder was pressed in between in
the filter paper and the paper was heated in an oven at
105C for 10 minutes. A translucent greasy spot appeared
indicating the papers
Test for gum
The small quantity of extract was added with few drops of
alcohol to form white precipitate which indicates the
presence of gum.
Test for mucilage
Few ml of aqueous extract was prepared from the
powdered crude drug was treated with ruthentium red.

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Red color was produced indicating the presence of

mucilage.
Test for betacyanins
To 1ml of plant extract, 1ml of 2N NaOH was added and
heated for 5 minutes at 100C. Formation of yellow color
indicated the presence of betacyanins.
Test for anthocyanin
About 0.2gm of plant extract was weighed in separate test
tube; 1ml of 2N sodium hydroxide was added, and heated
for 5 minutes. Observed for the formation of bluish green
color which indicates the presence of anthocyanin.
Test for leucoanthocyanins
To 1ml of plant extract, 1ml of isoamyl alcohol was added.
Formation of red color indicated the presence of
leucoanthocyanins.
Test for quinones
To 1ml of plant extract, 1ml of conc. H2SO4 was added.
Formation of red color indicated the presence of
quinones.
Test for emodins
The dry extract was added to 25% ammonia solution. The
formation of a cherry red color solution indicated the
presence of emodins.
Test for coumarins
To 1ml of plant extract, 3ml of NH4OH and 2mL of benzene
was added. Formation of red color indicated the presence
of coumarin.
Test for resins
The extracts were treated with acetone. A small amount
of water was then added and shaken. Appearance of
turbidity indicates the presence of resins.
Test for phlobatannins
About 2ml of aqueous extract was added to 2ml of 1% HCl

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and the mixture was boiled. Deposition of a red

precipitate was an evidence for the presence of
phlobatannins.
Report: (A) Preliminary phytochemical screening of the
aqueous extract of Neem shows the presence of_____.
(B) Preliminary phytochemical screening of the aqueous
extract of Neem shows the absence of_____.

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17. Determination of the Alcohol

Content of Asava and Arista
Background: Students will come to learn and understand the
meaning and significance of pharmacognostic parameters
and pharmacognostic study of crude drugs including their
analysis by performing various tests.

Experiment 17.1
Aim: To determine the alcohol content of “Asava” and
“Arista”.
Requirements
Digital weighing balance, Beaker, Measuring cylinder,
Distillation flask, Separating funnel, Heating mantle,
Water bath, Gravity bottle, Water
Theory: Ayurveda is a traditional Indian medicinal system
being practiced for thousands of years. More than 1,200
species of plants, nearly 100 minerals and over 100 animal
products comprise the ayurvedic pharmacopoeia asava and
arishta are unique dosage form discovered by ayurveda
having indefinite shelf life and it was said that the “older the
better it is”. Because this dosage form has an inherent
attribute of continuous hydro-alcoholic extraction and
probably formation of natural analogues of the chemical
compounds present in the medicinal plants. The main
objective of this paper is to document this knowledge
available in the traditional literature as well as from the
traditional practices, bring out the technological details,
analyze and list out their medical applications.

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Table 17.1: Relative density with percent ethanol content

Relative density Per cent ethanol Per cent ethanol
at content w/w at content v/v at
25℃ 15.56℃ 15.56℃
0.8158 90 93.3
0.8146 90.5 93.6
0.8131 91 94
0.8118 91.5 94.3
0.8104 92 94.7
0.8090 92.5 95
0.8076 93 95.4
0.8062 93.5 95.8
0.8048 94 96.1
0.8034 94.5 96.5
0.8020 95 96.8
0.8006 95.5 97.1
0.7992 96 97.5
0.7977 96.5 97.8
0.7962 97 98.1
0.7957 97.5 98.4
0.7932 98 98.8
0.7917 98.5 99.1
0.7902 99 99.4
0.7886 99.5 99.7
0.7871 100 100
Procedure
1. Measure 25ml of formulation by measuring cylinder and
transfer to distillation flak having capacity of 500ml.
2. Wash the measuring cylinder with 150ml of water and
add it to the flask.
3. Add some porcelain pieces to the flask and distilled it.
4. Collect about 90ml of distillate from this take 25ml of

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distillate and dilute it to 100ml with water and with the

help of specific gravity bottle determine the specific
gravity of liquid at 25C.
5. Follow the alcohol content table with specific gravity
and determine the v/v alcohol content in sample.
Calculate the percent alcohol content by using multiplication
factor
Procedure
Determine The specific gravity
A tube of known weight (w) was filled first with essential
oil and then with water and the respective weight w1 and
w2 was determined. Then, the specific gravity was
calculated using the following formula:

d=

Report: Alcohol content of Asava and Arista was found to

be_____.

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18. Incorporation of Prepared &

Standardized Extract in Various
Formulations and Their Evaluation as per
Pharmacopoeial Requirements (Herbal Cream,
Lotions & shampoo)

Background: Students will come to learn and understand the

meaning and significance of pharmacognostic parameter
and how to prepare and evaluate various formulations such
as herbal cream, lotions and shampoos.

Experiment 18.1
Aim: To prepare and evaluate 10gm of “Turmeric” and “Aloe
vera” gel herbal cream.
Requirements
Beaker, Measuring cylinder, Test tubes, Heating mantle,
Weighing balance, Pipette
Theory: Creams are viscous semi solid emulsions which are
meant for external application. They usually contain water
soluble base so that it can easily be removed from the skin.
When applied to the skin, creams leave no visible evidence
of their presence on skin. Creams consist of medicaments
dissolved or suspended in water removable or emollient
bases. Creams are of two types: aqueous creams (o/w) and
oily creams (w/o). Therefore, combining immiscible
compounds is possible by mechanical agitation or heat.
Uses: Anti wrinkle properties of herbal ingredients, anti
ageing properties and skin protective properties.

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Table 18.1: Formulation table for herbal cream (10gm).

Ingredients Quantity
White beeswax 5gm
Liquid paraffin 7ml
Borax 2gm
Water 5ml
Perfume 2 drops
Curcumin (turmeric) 200mg
Aloe vera gel 1gm
Methyl paraben 0.02
Ethyl alcohol 2ml (QS)
Note: Curcumin content is 3.14% by dry weight basis
Procedure
1. Melted the bees wax with mineral oil by heating on a
water bath at the temperature of 70C. Here, curcumin is
not soluble in water. So it was mixed with minimum
quantity of ethyl alcohol.
2. This was added to the borax water mixture and heated
to the same temperature.
3. Both the temperature were attained at a temperature
of 70C added the aqueous phase with rapid constant
stirring until cool.
4. Filtered it into a container and labeled.
Evaluation tests
Determination of pH
1gm of prepared tooth paste and add 9ml of freshly boiled
and cooled water. Stir well and make a suspension, pH
was determined by using pH meter.
Spread ability test
About 1gm of sample was weighed and placed at the
center of the glass plate and another glass plate was

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placed over it carefully above the glass plate 100gm

weight was placed up on upper slide so that the
formulation between two slides was pressed uniformly to
form a thin layer, the weight was removed and the excess
of formulation adhering to the slides was scrapped off.
One of the slides was fixed on which the formulation was
placed, the time in which upper slide moves over the
lower plate was taken as measure of spread ability. Spread
ability is calculated by using the formula:
l
𝐒𝐩𝐫𝐞𝐚𝐝 𝐚𝐛𝐢𝐥𝐢𝐭𝐲 = m ×
t
Where, m is weight on the slide, l is length moved on the
slide and t is time taken
Skin irritation test
The ointment was placed on patches and covered with
gauze for 4 hrs, skin was observed for any signs of
redness, inflammation and weeping of scabs.
Skin absorbance test
0.5gm of ointment was rubbed over definite area of skin
for a given time then unabsorbed was collected from skin
and weighed.
Report: Curcumin & aloe vera gel skin herbal cream
prepared and evaluated.

Experiment 18.2
Aim: Preparation and standardization of herbal lotion.
Requirements
Beaker, Measuring cylinder, Test tubes, Heating mantle,
Weighing balance, Pipette
Method of extraction: All the drugs were weighed
accurately & aqueous extraction had been done 10 times of

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the weight of the drug i.e. 5gm in 50ml of water on water

bath at 80C-100C. As the solution concentrated up to
20ml, filtration was done. Residue had been taken & volume
was making up to 40ml, again was boiled. After remaining
20ml was filtered and the same procedure was followed
again.
Table 18.2: Formulation table for herbal lotion (% w/w).
Ingredients Formulation 1 Formulation 2
Extract 1gm 2gm
Glycerin 2gm 2gm
Water QS QS
Sunflower oil 4gm 4gm
Mineral oil 2gm 2gm
Petroleum jelly 1gm 1gm
Cetyl alcohol 1.5gm 1.5gm
Glyceryl monostearate 2mg 2mg
Methyl paraben 0.2gm 0.2gm
Propyl paraben 0.2gm 0.2gm
Fragrance 0.1-1gm 0.1-1gm
Procedure
1. Oil in water emulsion of 1% and 2% of drugs were
formulated.
2. The emulsifier (glyceryl monostearate) and other oil
soluble components (sunflower oil, mineral oil, petroleum
jelly, cetyl alcohol) were dissolved in oil phase (part A) and
heated up to 80C.
3. Extract and water soluble components (glycerin, methyl
paraben, propyl paraben) were dissolved in (part B) and
heated up to 80C.
4. After heating, the aqueous phase was added in portions
to the oil phase with constant stirring until cooling of
emulsifier took place.

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5. Perfume was added when the temperature dropped to

45C ± 5C.
Evaluation tests
Test for thermal stability
Thermal stability of the formulation was determined by
the humidity chamber controlled at 60-70% RH and 37 ±
1C.
Determination of pH
5 ± 0.01gm of the lotion was weighed accurately in a
100ml beaker; 45ml of water was added & dispersed the
lotion in it. The pH of the suspension was determined at
27C using the pH meter.
Determination of total fatty matter
2gm of the sample was weighed in a conical flask, added
25ml of dil. HCl (1% v/v) & refluxed. Poured this into the
separating funnel and 50ml of ethyl ether were added into
it. The separating funnel was shaken well until two layers
were separated. The aqueous layer was separated out and
added 50ml portion of ether twice. All the ether extracts
were combined and filter through the filter paper
containing dried sodium sulphate on it. Distilled off the
ether (filtrate) & dried the material remaining in the flask
at temperature 60 ± 2C to constant mass.
Calculation:
𝐓𝐨𝐭𝐚𝐥 𝐟𝐚𝐭𝐭𝐲 𝐦𝐚𝐭𝐭𝐞𝐫 = 100 ×

Microbial examination of lotion

1gm of material was weighed and aseptically transferred
into the conical flask containing 50ml of dil. Phosphate
buffer at pH 7.2 and pipette out 1ml portion into 3 sterile
plates. Melted soya bean casein digest agar (SCDA)
medium was poured over it (at 45C) and cooled. After

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that plates were rotated to mix properly. Then the plates

were incubated at 30 ± 40C for 74 hrs in an inverted
portion. Average number of colonies was determined by
multiplying the dilution factor.
Determination of water content
10gm of the material was weighed and transferred it into
the flask. 200ml of toluene and few pieces of pumice
stone was added and connected the apparatus with
condenser. The flask was heated until toluene was begin
to boil and refluxed. When the H2O was distilled over
source of heat was removed.
Calculation:
V × D
𝐖𝐚𝐭𝐞𝐫 𝐩𝐞𝐫𝐜𝐞𝐧𝐭 𝐛𝐲 𝐦𝐚𝐬𝐬 = × 100
M
Where, V is volume of water in ml at room temperature
collecting in receiving tube, D is density of water at room
temperature and M is mass in gm of the material taken for
the test.
Patch test
About 1-3gm of material to be tested was placed on a
piece of fabric or funnel and applied to the sensitive part
of the skin e.g. skin behind ears. The cosmetic to be tested
was applied to an area of 1 square meter of the skin.
Control patches (of similar cosmetic of known brand) were
also applied. The site of patch is inspected after 24 hrs. As
there was no reaction the test was repeated three times.
As no reaction was observed on third application, the
person may be taken as not hypersensitive.
Accelerated stability testing
Accelerated stability testing of prepared formulations
conducted at 40 ± 2C temperatures and 75 ± 5% relative
humidity and studied for 90 days.
Report: Herbal lotion was prepared and evaluated.

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Experiment 18.3
Aim: Preparation and standardization of “Methi” and
“Shikakai” shampoo.
Requirements
Methi, Shikakai, Orange peel, Distilled water, Beaker,
Glass rod, Measuring cylinder, Weighing balance, pH
meter, Evaporating dish, Canvas paper, Stop watch
Theory: Herbal shampoos are the cosmetic preparations
that with the use of traditional ayurvedic herbs are meant
for cleansings the hair and scalp just like the regular
shampoo. They are used for removal of oils, dandruff, dirt
etc. Methi’s protein, nicotinic acids and large amounts of
lecithin are highly effective against hair fall and provide
strength from the roots. The seed contains a special
hormone that enhances hair growth and helps repair the
hair structure. Shikakai is excellent for hair as it does not
have side-effects unlike shampoo which are loaded with
chemicals to add more lather. It does not strip your hair’s
natural oils, which means that they are stronger from within
and do not look rough and dry. It helps in controlling hair fall
and also in reducing dandruff naturally due to its
antibacterial action.
Table 18.3: Formulation table for methi & shikakai shampoo.
Ingredients Quantity
Methi 250gm
Shikakai 1gm
Orange peel Handful
Water QS to make 2 liters
Procedure
Crush all ingredients into powder form. Add sufficient

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quantity water to produce 2 liters.

Standardization
Physical appearance/Visual inspection
The formulation prepared was evaluated for the clarity,
color, odor and foam producing ability.
Determination of pH
The pH of 10% v/v shampoo solution in distilled water was
measured by using pH meter at room temperature.
Determination of physico-chemical parameters
Moisture content at 105C: Weigh about 1gm of material
into large weighing bottle and heat on a steam bath under
a jet of air for 30 minutes. Continuous heating at 105C in
oven for 2 hours, cool in desiccators, weight and report
non volatile matter.
Ash content at 600C: Weigh 5ml of material place in a
flat bottom platinum dish and heat on a steam bath under
a jet of air for 1 hour. Remove and add 1gm of ash less
cellulose powder, keep the material in dish and heat in a
1K heating lamp till 600C in muffle furnace. Note the
difference in weight.
Determination of percent of solid content: 4gm of
shampoo was placed in a previously clean dry and
weighted evaporating dish. The dish and shampoo was
weighed again to confirm the exact weight of shampoo.
The liquid portion of the shampoo was evaporated by
placing the evaporating dish on the hot plate. The weight
and thus % of the solid contents of shampoo left after
complete drying was calculated.
Quantitative estimation of selected phyto-constituents
Foam test: Shake the drug/ sample extract vigorously with
water. Persistent foam observed, confirms the presence of
saponins.

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Hemolytic test: Add drug/ sample extract or dry powder

to one drop of blood placed on glass slide. Hemolytic zone
appears.
Saponification test: Add few drop of 0.5N alcoholic KOH
to a small quantity of various extract along with a drop of
phenolphthalein separately and heat on a water bath for 1
hour the formation of alkali indicate the presence of fixed
oil and fats. 5 drop of sample, add pinch of sodium
hydrogen sulphate, pungent odour indicate presence of
glycerine.
Evaluation tests
Net content
At the beginning of experiment mark the outside of bottle
at the surface level of liquid, at the end of experiment
empty the bottle and note the volume of water required
to fill it to the mark.
Dirt dispersion
Two drops of shampoo were added in a large test tube
contain 10ml of distilled water. One drop of India ink was
added; the test tube was Stoppard and shakes it ten times.
The amount of ink in the foam was estimated as none,
light, moderate or heavy.
Wetting time
The canvas was cut into 1 inch diameter discs having an
average weight of 0.44gm. The disc was floated on the
surface of shampoo solution of 1% w/v and the stop watch
started. The time required for the disc to begin to sink was
measured acutely and noted as the wetting time.
Surface tension measurement
Measurements were carried out with a 10% shampoo
dilution in distilled water at room temperature.
Thoroughly clean the stalagnometer using chronic acid
and purified water, because surface tension is highly

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affected with grease or other lubricants. The data

calculated by following equation given below:
R2 = (W3-W1) n1  R1 (W2-W1) n2
Where, W1 is weight of empty beaker; W2 is weight of
beaker with distilled water; W3 is weight of beaker with
shampoo solution; n1 is number of drops of distilled
water; n2 is number of drops of shampoo solution; R1 is
surface tension of distilled water at room temperature
and R2 is surface tension of shampoo solution.
Specific gravity
The two methods are commonly used for determination
the specific gravity of liquid one method use the
hydrometer and instrument that gives a specific gravity
reading directly. A second method called a bottle method
uses a specific gravity bottle that is a flask makes to hold a
known volume of liquid at a specified temperature usually
20C.The bottle is weighed filled with the liquid. Whose
specific gravity is to be found and weight again. The
different weight is divided by the weight of equal volume
of water to give the specific gravity of the liquid.
Test to evaluate foaming ability and foam stability
Foaming ability was determined by using cylinder shake
method. Briefly 50ml of the 1% commercial or formulated
shampoo solution was placed into a 250ml graduated
cylinder; it was covered with one hand and shaken 10
times. The total volume of the foam content after 1
minute of shaking was recorded.
Wetting time test
A canvas paper was cut into one inch diameter discs
having an average weight of 0.44gm. The smooth surface
of disc was placed on the surface of 1% v/v shampoo
solution and the stop watch started. The time required for
the disc to begin to sink was noted down as the wetting

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time.
Determination of water by toluene distillation
Transfer 10-20gm sample to 250ml round bottom flask,
add 50ml of toluene and 2gm of lamprosin and few glass
heads, connect to distillation unit. Distil until no more
water is collected in the receiver. Cool, read the volume of
water under the toluene at room temperature and
calculate % water content.
Volume of water (ml)
𝐖𝐚𝐭𝐞𝐫 𝐩𝐞𝐫𝐜𝐞𝐧𝐭 𝐛𝐲 𝐦𝐚𝐬𝐬 = 100 ×
weight of sample

Report: Methi-shikakai shampoo was prepared and

evaluated. Standardization of the shampoo was performed
by determining the physical parameters such as pH, visual
appearance, % of solid contents, foaming ability, wetting
time test.

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19. Incorporation of Prepared &

Standardized Extract in Various
Formulations and Their Evaluation as per
Pharmacopoeial Requirements (Herbal Syrup,
Mixture & Tablets)

Background: Students will come to learn and understand the

meaning and significance of pharmacognostic parameter
and how to prepare and evaluate various formulations such
as herbal syrup, mixtures and tablets.

Experiment 19.1
Aim: To prepare and evaluate “Orange syrup” as per IP.
Requirements
Beaker, Orange peel (Fresh 250gm), Alcohol (90%)
Theory: Tincture of orange is an alcoholic extract of fresh
bitter orange peel and is prepared by maceration process.
Fresh peel is used for this preparation because it is stable
and contains a higher proportion of volatile oil than the
dried peels. Drying dissipates part of the oil and fresh peels
are more aromatic. Alcohol (90%) is used as the menstrum
for the maceration process. This preparation contains
flavoring oils which are volatile in nature and should be
stored in tightly closed containers in a cool place.
Procedure
(A) Preparation of orange tincture
Take fresh orange peel and cut into thin slices. Weigh the
required quantity of thin slices and macerate it with whole
quantity of alcohol in a covered vessel. Allow to macerate

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for seven days with occasional stirring. Strain the liquid

press the marc and mix the expressed liquid to the
strained liquid. Clarify the combined liquids by filtration.
IP formula: Fresh orange peel in thin slices-250gm,
Alcohol- 1000ml.
(B) Preparation of simple syrup
A 100ml empty beaker was weighed and the weight was
noted. Half the quantity of purified water was placed into
the beaker. Calculated quantity of sucrose was weighed
and added to the water. Sucrose was dissolved by heating
with occasional stirring. After cooling, purified water was
added to make up the required volume.
IP formula: Sucrose-667gm, Purified Water (QS)-1000ml.
(C) Preparation of orange syrup
The measured quantity of orange tincture was mixed with
3/4th quantity of simple syrup. The volume was then made
up with remaining syrup.
IP formula: Tincture of orange-60ml, Simple syrup-
1000ml.
Standardization
Physical appearance/visual inspection: The formulation
prepared was evaluated for the clarity, color, and odour.
Determination of pH: The pH of 10% v/v of syrup was
measured by using PH meter at room temperature.
Report: Orange syrup as per IP was prepared and
standardized.

Experiment 19.2
Aim: To prepare and evaluate 50gm of churna mixture.
Requirements
Ginger powder (16.7gm), Fennel powder (16.7gm),

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Cinnamon powder (8.7gm), Ajwain powder (8.7gm)

Theory: Churna is the best known example of mixture; is a
powdered form of medications used in the treatment of
disorders in the ayurvedic system of medicine. It is most
basic and simplest form of ayurvedic medicine containing a
fine powder of single herb or several herbs.
General method of preparation of churna: All the
ingredients required for the churna should be cleaned
thoroughly and dried well in the shade or in the sun
separately to ensure the complete absence of moisture in
the same. Each ingredient is powdered finely and then,
sieved to remove any coarse particles. The ingredients are
weighed separately and then, mixed together. It should be
noted that some herbs contain a fibrous matter. Hence, the
weight of such herbs may vary before and after drying.
Hence, it is important to powder and weigh them separately
so that the correct quantity of each herb can be present in
the final product.
Procedure
1. Take all the ingredients in given proportion.
2. Mix the powders thoroughly.
3. Pass the mixture through sieve number #60.
4. Pack it in well close container and store it in dry place.
Evaluation tests
Physical parameters
Color examination, odor examination, taste examination,
pH determination, determination of moisture content,
determination of total ash, acid-insoluble ash, water-
soluble ash, sulphated ash, water soluble extractive value
and alcohol soluble extractive value.
Report: 50gm churna was prepared and evaluated.

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Experiment 19.3
Aim: To prepare and evaluate 400mg poly-herbal tablet.
Requirements
Clove, Cinnamon, Lactose, Mannitol, Sodium saccharine,
Talc, Magnesium stearate
Theory: A tablet is a pharmaceutical dosage form. Tablet
may be defined as the solid unit dosage form of medicament
or medicaments with or without suitable excipients and
prepared by either by molding or by compression. It
comprises a mixture of active substance and excipients
usually in powder form, pressed or compacted from a
powder into a solid dose. The compressed tablet is the most
popular dosage form use in today. About two-thirds of all
prescription is dispensed as solid dosage forms, and half of
these are compressed tablets. A tablet can be formulated to
deliver an accurate dosage to a specific site; it is usually
taken orally, but can be administered sublingually, buccally,
rectally, or intra vaginally.
Table 19.1: Formulation table for poly-herbal tablets.
Ingredients F1 F2 F3 F4
Clove 10mg - 100mg -
Cinnamon - 100mg - 100mg
Lactose 290mg 290mg - -
Mannitol - - 290mg 290mg
Sodium saccharine 2mg 2mg 2mg 2mg
Talc 4mg 4mg 4mg 4mg
Magnesium stearate 4mg 4mg 4mg 4mg
Procedure
1. Weigh all the excipients along with API as shown in
table.

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2. Passed through sieve number #20.

3. Mix all ingredients following geometric mixing excluding
glidant and lubricant thoroughly for 15 minutes.
4. Mix the powder blend was thoroughly with talc and
magnesium stearate.
5. Compress 400mg tablet using single rotary punching
machine.
Evaluation tests
General appearance
The general appearance of a tablet, its identity and
general elegance is essential for consumer acceptance, for
control of lot-lot uniformity and tablet-to- tablet
uniformity. The control of general appearance involves the
measurement of size, shape, color, presence or absence
odour, taste etc.
Content uniformity test
Randomly select 30 tablets. 10 of these assayed
individually. The tablet pass the test if 9 of the 10 tablets
must contain less than 85% and not more than 115% of
the labeled drug content and the 10th tablet may not
contain less than 75% and more than 125% of the labeled
content. If these conditions are not met, remaining 20
tablets assayed individually and none may fall outside of
the 85 to 115% range.
Thickness
Dimension of the tablets are measured by using a
calibrated dial caliper. Five tablets sample formulation are
picked out randomly and its thickness is measured
individually. Mean value of thickness is observed.
Weight variation
Twenty tablets were selected at random and weighed
individually. The individual weights were compared with
the average weight for determination of weight variation.

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The percentage deviation was calculated and then

compared with IP limit.
Hardness
Five tablets are randomly selected from each batch and
hardness of tablet is determined by using Monsanto
hardness tester. The mean values for each batch are
calculated and compared to IP standard.
Friability
Friability indicates the ability of a tablet to withstand
mechanical shocks while handling. Friability of the tablets
was determined using Roche friabilator and is expressed in
(%). Twenty tablets were initially weighed and placed into
the friabilator. The friabilator was operated at 25rpm for 4
minutes or run upto 100 revolutions and then the tablets
are weight again. The loss in tablet weight due to abrasion
or fracture is measured astablet friability.
Disintegration time
The disintegration time for all formulations is carried out
using tablet disintegration test apparatus. Six tablets are
placed individually in each tube of disintegration test
apparatus and discs are placed. The water is maintained at
temperature of 37+ 2C and the time taken for the entire
tablet to disintegrate completely is noted.
Dissolution study
Dissolution study of tablet is carried out using phosphate
buffer 6.8 as a dissolution media. The samples are
withdrawn for 8 hours at the interval of 45 minutes. The
absorbance of sample measured on UV
spectrophotometer and percentage release are calculated.
Report: 400mg of poly-herbal tablets were prepared and
evaluated.

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20. Monograph Analysis of Herbal Drugs

from Recent Pharmacopoeias
Background and objective: The monograph is a scientific
work that examines various facets of herbal drug research. It
contains details about the specific plant, such as its scientific
name, alternate common names, morphological and
anatomical traits, chemical components,
adulterants/substitutes, pharmacology, key therapeutic
claims, safety considerations, and dose. It is an exhaustive
record of the specific plant that can be used to monitor its
quality.

Experiment 20.1
Aim: Monograph analysis of “Castor oil”.
Source: Castor oil is the fixed oil obtained from the seeds of
“Ricinus communis linn”, belongs to family Euphorbiaceae.
Description
Color: pale yellowish in color or almost colorless; odour:
slight; taste: at first bland but afterwards slightly acrid and
usually nauseating; solubility: soluble in alcohol, miscible
with ethyl alcohol and with chloroform and with solvent
ether.
Identification: Mixes completely with half its volume of light
petroleum and it is only partially soluble in two volumes
(boiling point 40-60C). Add to an equal volume of alcohol “a
clear liquid is obtained”, cool to 0C for 3 hours the liquid
remains clear. Distinctions from other fixed oil are: wt/ml: at
25C – 0.945-0.965 per gram; acetyl value: not less than
143; acid value: not more than 2; iodine value: 82-90;

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refractive index: 25C – 1.4758-1.4798; optical rotation: not

less than +3.5; saponification value: 177-185.
Storage: Preserve castor oil in a well closed container in a
cool place.
Category: Laxative, pharmaceutical aid.
Dose: 4-16ml.
Procedure
Determination of wt/ml of castor oil
(The wt/ml of a liquid is the weight in grams of 1ml of a
liquid when weighed in air at 25C unless otherwise
specified)
1. Select a clean and dry pycnometer. Calibrate the
pycnometer by filling it with recently boiled and cooled
water at 25C and weighing the contents.
2. Assuming that the weight of 1 ml of water at 25C when
weighed in air of density 0.0012gm per ml is 0.99602gm.
3. Calculate the capacity of the pycnometer (ordinary
deviations in the density of air from the value given do not
affect the result of a determination significantly).
4. Adjust the temperature of the substance to be
examined to about 20C and fill the pycnometer with it.
5. Adjust the temperature of the filled pycnometer to
25C, remove any excess of substance and weigh.
6. Subtract the tare weight of the pycnometer from the
filled weight of the pycnometer.
7. Determine the weight per ml by dividing the weight in
air expressed in grams of the quantity of liquid which fills
the pycnometer at the specified temperature by the
capacity expressed in ml of the pycnometer at the same
temperature.

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Experiment 20.1.1
Aim: To determine the acid value of the given sample of
“Castor oil” and report the purity with the standard level
given by Indian pharmacopoeia.
Principle: Acid value is the number which express in
milligram (mg) of amount of KOH necessary to neutralize the
free acid present in (oil) given substance. Acid value can be
determined by titrating with an ethereal alcoholic solution
of fixed oil with 0.1M KOH using phenolphthalein as an
indicator. End point is the appearance of pink color.
R – COOH + KOH → R – COOK +H2O
Increased acid value can be determined by using the
following formula:
× .
𝐀𝐜𝐢𝐝 𝐯𝐚𝐥𝐮𝐞 =
η = Strength of KOH × titre value
Procedure
1. Dissolve about 10gm of castor oil in 50ml mixture of
equal volume of ether and ethanol previously neutralized
with 0.1M KOH to phenolphthalein. If the sample does not
dissolve and boil solvent.
2. Connect a flask to reflex condenser and warm slowly
with frequent shaking until the sample dissolved.
3. Add phenolphthalein solution and titrate with 0.1M
KOH until the solution remains faintly pink after shaking
for 30 seconds.
4. Calculate the acid value from the following formula:
( )× × .
𝐀𝐜𝐢𝐝 𝐯𝐚𝐥𝐮𝐞 =

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Experiment 20.1.2
Aim: To determine the saponification value of the given
sample of “Castor oil” and report the purity with the
standard level given by Indian pharmacopoeia.
Principle: Saponification value is the number which express
in mg of amount of potassium hydroxide to neutralize free
acids and present in 1gm of the substance.
Procedure
1. Introduce about 2gm of substance accurately weigh to a
200ml conical flask of borosilicate glass fitted with a reflex
condenser.
2. Add 25ml of 0.5N alcoholic potassium hydroxide and
boil in a reflex water bath for 30 minutes. Frequently
rotating the content.
3. Cool and add for 2 drops of phenolphthalein and titrate
immediately against 0.5N HCl.
N
(b − a) × Strength of HCl × 28.05
𝐒𝐚𝐩𝐨𝐧𝐢𝐟𝐢𝐜𝐚𝐭𝐢𝐨𝐧 𝐯𝐚𝐥𝐮𝐞 = 2
Weight of sample taken
Where, (b) is blank value and (a) is titre value.

Experiment 20.1.3
Aim: To determine the refractive index of the given sample
of “Castor oil” and report the purity with the standard level
given by Indian pharmacopoeia.
Principle: The refractive index (n) of a substance with
reference to air is the ratio of the sin of the angle of
incidence to the sin of the angle of refraction of a beam of
light passing from air into the substance. It varies with the
wavelength of light used in its measurement. The refractive
index is usually measured at 25C (±0.5) with reference to

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the wavelength of the sin of sodium (λ = 589.3nm). The

temperature should be carefully adjusted as refractive index
varies significantly with temperature. The Abbe’s
refractometer is convenient for most refractive index
measurements. To achieve accurately the apparatus should
be calibrated against distilled water which has a refractive
index of 1.3325 at 25C.
Uses: Refractive index can be used to identity a substance,
to measure its purity and to determine concentration of one
substance dissolved in another. Refractometer is used to
determine refractive index.
Procedure
1. Clean the nickel prism by means of cotton wool, dipped
in ether, acetone or chloroform. Clamp the lower prism
with the upper prism and view through the eye piece to
get a clean bright field.
2. Without disturbing the light arrangements remove the
lower prism and place 2-3 drops of the given sample of
castor oil, between the two prisms and clamp it again.
Remove excess of liquid if any, using cotton wool. View for
a clear light field arranges.
3. Cross wire such that to the bright field. Look for exact
reading on a graduated scale and not down the refractive
index. Repeat the same for concordant value.
Report: wt/ml of water is_____, wt/ml of castor oil is____,
acid value of castor oil is_____, saponification value of
castor oil is_____, refractive index of water is_____,
refractive index of castor oil (I.P Limit = 1.4758-1.4798)
is_____.

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21. Determination of Aldehyde Content

Background and objective: Students will come to learn and
understand the meaning and significance of
pharmacognostic parameter and how to determine
aldehyde content.

Experiment 21.1
Aim: Determination of aldehyde content in “Lemon oil”.
Requirements
Equipments Stoppered tube
Hydroxylamine hydrochloride reagent in
Chemicals alcohol (60%), 0.5N Potassium hydroxide in
alcohol (60%), Methyl orange, Lemon oil
Procedure
1. Weigh accurately about 10gm of lemon oil in a
stoppered tube. Add to it, 7ml of hydroxylamine
hydrochloride reagent in alcohol (60%) and a drop of
solution of methyl orange.
2. Titrate the liberated acid with 0.5N potassium
hydroxide in alcohol (60%) until the red color changes to
permanent yellow in the lower layer.
3. Calculate the aldehyde content as follow:
4. 1ml of 0.5N potassium hydroxide in alcohol (60%) is
equivalent to 0.07672gm of citrus.
Preparation of standard reagent (Hydroxylamine
hydrochloride)
Dissolve 3.475gm of hydroxylamine hydrochloride in 95ml
of 60% alcohol; add 0.5ml of 0.2% w/v solution of methyl
orange in 60% alcohol and 0.5N potassium hydroxide in
alcohol 60% until yellow color is produced. Make volume

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with sufficient alcohol to 100ml.

Calculation
Weight of citral
𝐏𝐞𝐫𝐜𝐞𝐧𝐭 𝐨𝐟 𝐜𝐢𝐭𝐫𝐚𝐥 = × 100
Weight of lemon oil
Example: 1ml of 0.5N KOH in (60%) alcohol = 0.07672gm of
citrus
5.5ml of 0.5N KOH= 0.42gm of citrus
.
 𝐏𝐞𝐫𝐜𝐞𝐧𝐭 𝐨𝐟 𝐜𝐢𝐭𝐫𝐚𝐥 = × 100 = 4.2%

Report: The aldehyde content of given sample of Lemon oil

is found to be_____.

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22. Determination of Phenol Content

Background and objective: Students will come to learn and
understand the meaning and significance of
pharmacognostic parameter and how to determine phenol
content.

Experiment 22.1
Aim: Determination of “Gallic acid” equivalent in (HAECB).
Requirements
UV Visible spectrophotometer, Shimadzu
Equipments
(model 1800)
Folin-Ciocalteu reagent (1N), Sodium
Chemicals carbonate solution (10%), Gallic acid
solution
Theory: Total phenolic content of the various concentrations
of HAECB was determined by folin-ciocalteu reagent
method. The hydroxyl group (OH) of phenolic compounds
reduces the phosphomolybdic acid to molybdenum blue in
the presence of alkaline medium (present in Folin reagent).
The blue colored complex was then spectrophotometrically
measured at 760nm.
Procedure
1. About 1ml (1mg/ml and 0.5mg/ml) of hydroalcoholic
extract of “Commelina benghalensis Linn” (leaf) (HAECB),
0.5mL of Folin-ciocalteu reagent (1N) were added and
allowed to stand for 15 minutes.
2. Then 1ml of 10% sodium carbonate solution was added
to the above solution.
3. Finally the mixtures were made up to 10ml with distilled

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water and allowed to stand for 30 minutes at room

temperature and total phenolic content was determined
spectrophotometrically at 760nm wavelength.
4. The calibration curve was generated by preparing gallic
acid at different concentration ([5, 10, 15, 20 and
25]µg/ml).
5. The reaction mixture without sample was used as blank.
6. Total phenolic content of HAECB extract is expressed in
terms of mg of Gallic acid equivalent per gm of extract (mg
GAE/gm).
Report: Hydro alcoholic extract of Commelina benghalensis
L. (HAECB) was found to contain of GAE (Gallic acid
equivalent)_____.

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23. Determination of Total Alkaloids

Content
Background and objective: Students will come to learn and
understand the meaning and significance of
pharmacognostic parameter and how to determine alkaloid
content.

Experiment 23.1
Aim: To determine the total alkaloid content of “Cinchona”
extract.
Requirements
Separating funnel, Litmus paper, Distillation
Equipments
flask, Water bath, Dropper
Cinchona extract, 1N Sulphuric acid (10ml),
Water 10ml, Chloroform, Mayer’s reagent,
Chemicals
Ammonia, alcohol, HCl (N/10), Sodium
hydroxide (N/10), Methyl red indicator
Theory: Alkaloids are basic nitrogen containing compounds
obtained from plants, animals and microorganism having a
marked physiological action. The term Alkaloids is derived
from the word alkali-like and they have some of the
characters of natural amines. The definition of alkaloid is the
organic compounds from natural or synthetic origin which
are basic in nature and contain one or more nitrogen atoms
normally in heterocyclic ring and possess specific
physiological action on human animal body when used
therapeutically. Alkaloids found in cinchona bark still play an
important role in medicine for example as anti-malarial and
anti arrhythmic drugs. Six respective derivatives (dihydro
quinidine, dihydro quinine, quinidine, quinine, cinchonine

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and cinchonidine) have been quantified in crude plant

extract. Total alkaloids are determined volumetrically by
acid-base titration and calculated as quinine.
Procedure
Determination of total alkaloids of cinchona extract
1. Introduce 10ml of the cinchona extract into a separating
funnel, add 1N sulphuric acid (10ml) and water (10ml).
2. Shake with 10ml of chloroform and allow separating the
mixture. Shake and allow separating and discarded the
chloroform layer.
3. Transfer the acid wash to the mother liquor and basify
with about 5ml of strong ammonia (test with litmus paper)
4. Shake with successive portions of chloroform (30ml,
20ml, 20ml and 10ml). Test for completion of the
extraction with mayer’s reagent.
5. Wash the combined chloroform extract with 10ml of
water. Transfer the extract to a distillation flask to remove
the solvent on a boiling water bath.
6. Add 5ml of alcohol to the residue and evaporate the
alcohol on a water bath.
7. Dissolve the residue in 2ml of chloroform and add 10ml
standard N/10 hydrochloric acid. Heat on a water bath to
remove the chloroform and back titrate the excess acid
against N/10 sodium hydroxide using 3-5 drops of methyl
red as indicator.
Calculation
Each 1ml of N/10 hydrochloric acid is equivalent to
0.03091gm of quinine
[ × . × ]
%𝐚𝐠𝐞 𝐭𝐨𝐭𝐚𝐥 𝐚𝐥𝐤𝐚𝐥𝐨𝐢𝐝𝐬 =
(% w/v calculated as quinine_____)
Report: The total alkaloid content was determined_____.

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