Sem VI - HDT Journal Final New

Subject code : BP609P HERBAL DRUG TECHNOLOGY(Sem VI)
B-Pharm Sem- VI Manual
Manual
B- Pharmacy Semester VI
Subject – Herbal Drug Technology
Subject Code- BP609P
1
ACADEMIC YEAR 2021-22
Subject code : BP609P
Class: T.Y B.Pharm
Sem : VI th Semester
Sub : HERBAL DRUG TECHNOLOGY
Topic : Practical Manual
Subject teacher : Dr.Manisha Vite
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3
INDEX
SR TOPIC PAGE DATE DATE OF SIGNATURE

NO NO CONDUCTED SUBMISSION
To perform preliminary
1 phytochemical screening of 28/12/21
crude drugs.
Determination of the alcohol
2 content of Asava and Arista 04/01/2022
Evaluation of excipients of
3 natural origin 14/01/2022
Incorporation of prepared and
4 standardized extract in cosmetic 18/01/2022
formulations like creams and
their evaluation.
formulations like lotions and
their evaluation.
formulations like shampoos and
their evaluation.
4

5 standardized extract in
formulations like syrups, 08/02/2022
mixtures and their evaluation as
per Pharmacopoeial
requirements.
5 standardized extract in
formulations like tablets and
their evaluation as per 15/02/2022
Pharmacopoeial requirements.
Monograph analysis of herbal

6 drugs from recent
Pharmacopoeias 22/02/2022
Determination of Aldehyde
7 content 01/03/2022
8 Determination of Phenol content 08/03/2022
9 Determination of total alkaloids 15/03/2022
5
Experiment No.1
Aim - To perform preliminary phytochemical screening of crude drugs.
Reference -
•Trease & Evans. “The Textbook of Pharmacognosy”,16 th Edition, pg-20
•Dwivedi S. “Pharmacopoeal standards for Ayurvedic Formulation” (Council of Research in
Indian Medicine & Homeopathy) pg no -156
•Dr.Vyas N. “A Practical Book of Herbal Drug Technology”, Edition 18, Page No:56
Requirements-
Chemicals- Sulphuric Acid, Conc.HCL, 10% NaoH solution, Nacl, Millon's reagent,
etc
Introduction -
Preliminary phytochemical evaluation is the step after extraction in order to
identify different classes of constituents that can be present in extracts, i.e.
carbohydrates, proteins, lipids, flavonoids, tannins, glycosides, alkaloids and essential
oils. Always choose a solvent of extraction whose solubility and/or polarity is same as
that of the constituents, i.e. use polar solvents only to obtain polar components. After
detecting the particular class, one can perform specific chemical tests for whole crude
drug or individual constituents to confirm any known drug or component. The present
experiment elaborates maximum available tests that can be performed for preliminary
phytochemical screening.
● General Test for Preliminary Phytochemical Screening

•Preliminary Test
Test Observation Inference

Colour Green Senna, Digitalis,Vasaka, Datura,Vinca, Fennel
Brown-red Clove, Cinnamon, Rhubarb, Cinchona
Cream or Rauwolfia, Coriander, Liquorice, Ginger
yellow
Pinkish cream Ergot
Light brown Ipecac
Odour Aromatic Clove, Cinnamon, Fennel, Coriander, Ginger
6
Characteristic Senna, Digitalis, Nutmeg

Taste Sweet Liquorice, Cinnamon
Bitter Senna, Rhubarb, Cascara, Cinchona, Nux vomica,
Ipecac, Datura
Astringent Isapgol, Catechu, Myrobalan
Pungent Ginger, Cinchona
Characteristic Digitalis
Mucilaginous Squill
General Test for Phytoconstituents
Carbohydrates
Test Procedure Observation
Molisch’s test Mix 1 ml reagent in 2 Red to violet ring depending

ml of test solution. on the amount of sugar
Add 1 ml of appears at the
concentrated sulfuric junction of the two liquids
acid.
Iodine test for starch Mix 0.5 ml of iodine Starch gives deep blue
solution with 1 ml of colour
the test solution.
Fehling's test Mix 1 ml of Fehling's Yellow to red precipitate

solution A with 1 ml of
Fehling's solution B
and 1 ml of test
solution. Then, boil it.
Benedict’s test Mix 2 ml of Benedict's Formation of red, yellow

reagent with 2 ml test green coloured precipitate
solution. Boil it in a depending on the sugar
water bath. concentration
7
Barfoed's test Mix 2 ml of Barfoed's Brick-red precipitate of

reagent with 1 ml of monosaccharides
the test solution. Boil it
and wait.
Seliwanoff's test for Mix 2 ml of Deep red colour due to keto

ketohexoses Seliwanoff’s reagent hexoses.
with 1 ml of test
solution. Boil.
Bial's test for pentoses Mix 5 ml of Bial's Green colour precipitate due
reagent with 1 ml of test to pentoses.
solution.
Warm slowly.
Non-reducing sugars Perform Benedict's No characteristic colour

test. formation.
Non-reducing sugars Mix 5 ml of test Red or yellow colour
solution with 5 drops
of concentrated HCl.
Boil it and add 10%
sodium hydroxide
solution. Add 10 ml of
Benedict’s reagent
boil again and wait.
Osazone Test Add 0.5 g of Needle-shaped yellow

phenylhydrazine osazone crystals for
hydrochloride and 01 glucose fructose and
o of sodium acetate in mannose.
5 ml of test solution.
Add 5 ml of glacial
acetic acid. Boil in • Mushroom shaped

water bath. Cool the crystals
solution slowly. for lactose
• Flower-shaped
crystals for maltose
Proteins
8
Biuret test Mix 2 ml test solution Violet to pink colour

with 2 ml Biuret
reagent.
Millon’s test Mix 2 ml test solution Red colour
with 2 ml Millon's
reagent. Boil it.
Xanthoprotein test Mix 2 ml test solution Yellowish white precipitate
with 2 ml concentrated
H₂SO4.
Precipitation test Mix 2 ml test solution Precipitate
with 2 ml of 5%
HgCl2 or 5%
ammonium sulphate.
Lead acetate test Mix 2 ml test solution Black to brown colour
with 2 ml of 40%
NaOH and 0.5 ml lead
acetate solution.
Boil it.
Amino Acids
Ninhydrin test Mix 2 ml test solution Blue or purple colour
with 1 ml of 5%
ninhydrin solution. .
Boil for 5 minutes in
water bath.
Tyrosine test Mix 2 ml test solution Dark red colour
with 1 ml Millon's
reagent and boil the
solution.
Tryptophan test Mix 2 ml test solution Red violet ring will appear at
with 1 ml glyoxylic the junction of two layers
acid and concentrated
H2SO4.
Cysteine test Mix 3 ml test solution Black precipitate
with 0.5 ml of 40%
NaOH and add 10%
lead acetate. Boil it.
Alkaloids
9
Dragendorff’s reagent Mix 2 ml of reagent Reddish brown precipitate

with 2 ml filtrate of
plant drug extract.
Modified Dragendorff’s Mix 2 ml of reagent with Precipitate

reagent 2 ml filtrate of plant
drug extract.
Hager’s reagent Mix 2 ml of reagent with Yellow colour precipitate

2 ml filtrate of plant
drug extract.
Mayer’s reagent Mix 2 ml of reagent with Cream coloured precipitate

drug extract.
Wagner’s reagent Mix 2 ml of reagent with Reddish brown precipitate

drug extract.
Marme’s reagent Mix 2 ml of reagent with Precipitate

2 ml filtrate of plant drug
extract
Sonnenschein reagent Mix 2 ml of reagent with Pale yellow to pink to

2 ml filtrate of plant drug colourless
extract
Bertrand’s reagent Mix 2 ml of reagent with Blue to purple colour

2 ml filtrate of plant drug
extract
Scheibler’s reagent Mix 2 ml of reagent with Yellow to orange

(phosphotungstic acid) 2 ml filtrate of acidified precipitate
plant drug extract
10
Kraut’s reagent Mix 2 ml of reagent with Initially, the red-brown

2 ml filtrate of plant drug layer changes to blue green
extract
Glycosides
General test Test 1: If Test 2 has dark colour
than Test 1 (if sugar content
Extract sample powder is high in Test 2 than Test
with alcohol or water 1). it indicates the presence
and then add Fehling of glycosides
Solution A and B.
Test 2: Note:
Extract sample powder Acid hydrolyzes glycone

with alcohol or water and aglycone moiety and
and then add sulfuric thus, sugar content is
acid and then Fehling increased in Test 2.
Solution A and B.
Cardiac Glycosides
Kedde’s test Mix 1 ml of test Blue to purple colour
solution with 2 ml
reagent.
Baljet reagent Mix 10 ml of test Yellow and orange to deep
solution in red colour
2 ml sodium picrate
solution
Keller- kiliani test for To the alcoholic The initial red-brown layer
digitoxose sugar extract of sample, add changes to blue green
5 ml of water and 0.5
ml of strong solution
of lead acetate. Filter
and treat the clear
filtrate with equal
volume of chloroform
and evaporate to yield
11
dry residue. Add

glacial acetic acid, 0.5
ml of ferric chloride
solution and 2 ml of
concentrated sulphuric
acid.
Legal’s test Mix 1 ml of test Pink or red colour

(Cardenolides) solution with 2 ml
pyridine and sodium
nitroprusside.
Raymond test Mix alcoholic extract of Violet colour changes to
sample in 0.1 ml of blue
Raymond's reagent and
add
2-3 drops of 20%
NaOH solution.
Flavonoids
Shinoda test Add magnesium • Flavones, favonols
powder and a few and xanthones:
drops of concentrated Orange, pink, red
HCI or H2SO4,to 2 of and purple.
sample solution.
• Flavanones and
also flavononols:
Weak pink to magenta
colours, or no colour at
all.
Modified Shinoda test The procedure is same Flavanonols:
as above except the use
of zinc powder Deep-red magenta colour
instead of
magnesium.
Sulphuric acid test Add 3 ml of H2SO4, in • Flavones and
sample flavonols: Deep yellow
colour • Chalcones and
aurone: Red or red-bluish
• Flavanones:
12
Orange to red colours

Lead acetate test Mix test solution with Yellow precipitate
lead acetate
Alkali test Treat test solution with Yellow colouration which
increasing amount of decolourizes after addition of
NaOH. acid
Tannins
Ferric chloride test Mix 2 ml of test Blue, blue-black or blue
solution with 5% of green colour reaction
ferric chloride
solution.
Gelatin-salt test Prepare three test tubes Formation of a precipitate in
of extract solution. the second treatment
To the first. add 1% suggests the presence of
solution of NaCl; tannins and a positive
To the second, add 1% response after addition of
NaCl and 5% gelatin FeCl3 to the third portion
solution; and To the supports this inference
third, add FeCl3
solution.
Lead acetate test Mix test solution with White precipitate
lead acetate solution.
Bromine water test Mix test solution with Discolouration of original
bromine water. solution.
Dilute lodine test Mix test solution with Red colour to solution
dilute lodine solution.
Potassium dichromate test Mix test solution with Rad precipitate
potassium dichromate
solution.
Dilute HNO, test Mix test solution with Red to yellow colour
diluted HNO3 solution.
Triterpenoids
Liebermann-Burchard Mix 2 ml test extract Blue-green to red orange
test with 1 ml chloroform, colour.
1 ml acetic anhydride • A bluish green or blue
and add one drop colour indicates
concentrated H₂SO4. presence of steroids
13
• A pink-violet colour
indicates terpenoids
Noller's test Mix 2 ml test extract Pink colouration indicates the

with small quantity of presence of triterpenoids
tin and thionyl
chloride.
Sannie test Mix 2 ml extract with Brown colour

stannous chloride,
acetic acid and carbon
tetrachloride (6: 50:
50). Heat at 100°C.
Steroids
Liebermann test Mix 2 ml test extract Blue colour
with 2 ml acetic
anhydride. Boil and
add 0.5 ml of H₂SO4.
Zimmermann test Mix 2 ml test extract Violet colour
with 1 ml of 2N KOH
in alcohol and 1 ml 1%
dinitrobenzene in
alcohol. After 10 min
add this mixture to 8
ml alcohol.
Tschugaeff test Mix 1 ml sterol Red colour
solution with 2 ml
glacial acetic acid, 0.5
mg zinc chloride and 1
ml acetyl chloride.
Pinus test Mix 2 ml of test Blue colour
solution with 2 ml of
antimony trichloride in
acetic acid.
Salkowski reaction Mix 10 ml of test Chloroform layer show red
solution in 1 ml of colour and acid layer shows
green
14
CHCl3 and add 1ml fluorescence

concentrated H2SO4.
Saponins
Foam test Shake aqueous Foam lasts for more the 15
solution of a saponin seconds.
containing sample
producing foam which
is stable for 15 minutes
or more
Anthraquinone Glycosides
Borntrager's test Take little quantity of Rose pink colour to
aqueous solution of ammonia layer
sample: add H2SO4
then add CCl4, or ether
in it. Separate the
organic layer and
shake with dilute
ammonia
Schonteten's Test for Take a little quantity of Green fluorescence
anthranols aqueous solution of
sample and add sodium
borate.
P-nitroso dimethyl aniline Take little quantity of • Anthrones change

test for anthrones aqueous solution of colour of solution.
sample and add P-nitroso • Anthraquinones do
dimethyl aniline in it. not change colour.
.
15
Modified anthraquinone Take little quantity of Rose pink colour of

test for C- glycosides aqueous solution of ammonia layer
sample; add ferric
(Modified Borntrager’s chloride solution, HCI
and then add CCI4 or
Test)
ether. Separate the
organic layer and shake
with dilute ammonia.
Cyanogenetic Glycosides
Sodium picrate test Take the aqueous test Hydrogen Cyanide (HCN)
(Grignard test) solution of sample in test turns the paper to brick red
tube and add dilute colour due to formation of
H₂SO4 suspend sodium sodium iso-perpurate
picrate treated filter
paper.
Mercurous nitrate test Mix 2 ml test extract Formation of metallic
solution with 3% mercury
aqueous mercurous
nitrate solution.
Coumarin Glycosides
Odour test Take the odour of Aromatic smell
powder or extract.
Alkali test Mix the test solution Blue green fluorescence
with alkali.
Fluorescence test Take the moist powder Yellow green fluorescence
of drug in test tube,
cover test tube with
alkali moist filter paper.
Heat the test tube and
observe paper under UV
light.
Gum
Test for carbohydrate Take the extract and add Positive test
HCl
to hydrolyze the
polysaccharides.
16
Now, perform Fehling's

or Benedict's test.
Mucilage
Ruthenium red test Treat the powder with Red colour

ruthenium red.
Swelling test Dissolve the Powder swells
powder in
water.
Fatty Oil
Filter paper test Press the powder Permanent oily spot
between filter paper.
Solubility test Mix oil in alcohol. Insoluble
Essential Oil
Sudan red III test Treat test solution with Red colour
Sudan red III.
Tincture alkana test Treat test solution with Red colour
tincture alkana.
Solubility test Dissolve the oil in Completely soluble
alcohol.
Confirmatory Chemical Tests for Organized Crude Drugs
Plant Drug Test Observation
Berberis Bromine test Bright red colour.
Treat berberine
solution with a drop of
bromine water and a
few drops of HCI.
Froehde's reagent Brown or green colour.
Treat berberine
solution with
Froehde's reagent.
17
Potassium Brown-violet colour.

dichromate
(K₂Cr₂O7) test
Treat berberine
solution with sulphuric
acid and crystals
K₂Cr₂O7
Mandelin's reagent Violet colour
Treat berberine
solution with
Mandelin's reagent.
Cinchona Erythroquinine test Red colour which is taken
Take dilute acidic up by chloroform layer after
solution of quinine; addition of 1 ml of
add few drops of chloroform.
bromine water and a

drop of 10% solution
of potassium
ferrocyanide. Then,
add a drop of strong
ammonia solution.
Thalloquin test Emerald green colour.
Take powder and add
one drop of dilute
sulphuric acid and 1 ml
of water. Then, add
bromine water to get
yellow colour and
finally add dilute NH3
solution.
Vapor test Purple vapors are produced.
Take powder and add
glacial acetic acid to it.
Now, heat it.
Cinnamon Ferric chloride test Green colour due to presence
Take 1 ml of oil and of cinnamaldehyde and
add 4 ml of alcohol in eugenol.
it; and then add 1 ml
18
of ferric chloride
solution.
Crystal test Rod shaped crystals

Take 1 ml of cinnamic aldehyde.
chloroform
extract. and add drop
of
10% aqueous phenyl
hydrazine
hydrochloride solution
Tannin test Blackish red colour due
Take 1 ml of aqueous tannins.
solution and add 1 ml
of 5% ferric chloride.
FeCl, test: Blue colour.
Take 2 mi of alcoholic
extract of drug and add
solution of FeCl3.
Eugenol test Crystals of sodium eugenate
Take Chloroform
extract of drug and add
3% NaOH.
Coca Treat the powder with Characteristic smell due
H2SO4 add water, mix methyl benzoate.
and heat.
Colchicum Acid test Yellow colour.
Treat alcoholic
solution of colchicines
with 70 %
H₂SO4.
Iron (III) chloride Red colour.
(FeCl3)
Treat alcoholic
solution of
colchicines with FeCl3
19
Nitric acid (HNO3) Violet colour changes to

test Treat alcoholic Yellow colour.
solution of colchicines
with concentrated
HNO3.
Curare FeCl3 test Green colour.
Treat aqueous solution
of
(+) tubocurarine with
FeCl3.
HgNO3 test: Red colour.
Treat aqueous solution
of (+) tubocurarine
with HgNO₂ solution.
Datura Vitali-Morin reaction Violet colour.
Treat the dilute

solution of
atropine with fuming
HNO3 evaporate to
dryness, dissolve
residue in acetone and
add methanolic KOH
solution.
Duboisia To the aqueous Lemon yellow colour.
solution of atropine,
add AuCl2 and HCL.
Ephedra Dissolve ephedrine in Violet colour solution which
water and dil. HCl. after shaking with solvent
Then, add ether produces purple colour
CuSO4 and in organic layer and blue
NaOH. colour in aqueous layer.
Ergot Sodium bicarbonate Red-violet colour.
test
Take the powder, add

solvent ether and
H₂SO4; shake well and
filter. Treat filtrate
20
with saturated solution

of sodium bicarbonate.
Van Urk's reagent Blue colour
Treat the solution with

Van
Urk's reagent (p-
dimethylamino
benzaldehyde).
Gentian Observe the powder Light blue fluorescence.

under UV light
Ginger Oleoresin Take a small quantity Blue colour.
of ginger oleoresin;
and add a few ml of
70% sulphuric acid and
vanillin. Allow to stand
for 15 min and then,
add water.
Gymnema Prepare the aqueous Foam and precipitate will be
solution of powder, observed.
shake and add dilute
HCI.
Indian squill Stain the transverse Red colour mesophylls
section of squill with observed.
colarin solution or
iodine solution.
Ipecac KCI test Yellow colour changes to
Take the aqueous red due to emetine.
solution of drug
powder, filter and add
0.5 g potassium
chloride.
21
Froehde's reagent test Dark green colour.

Take the powder,
add H₂SO4 and
sodium
molybdate.
Liquorice Add 80% H₂SO4 to Deep yellow colour.
powder.
Lobelia Acetophenone test Characteristic smell of
Boll the solution of acetophenone.
lobeline.
Acid test Red colour
Treat lobelia solution
with H₂SO4 and then
with formaldehyde
Mustard Treat the powder with Yellow colour.
alkali.
Nux vomica Mandelin's reagent Endosperm shows purple
(sulphovanadic acid) colour due to strychnine.
test
Treat strychnine
powder or transverse
Section of Nux-
vomica, with
ammonium vanadate
and H₂SO4.
Nitric acid (HNO3, Endosperm shows yellow
test) colour due to brucine.
Take the TS of Nux

Vomica and add
concentrated HNO3.
Opium General test Dark reddish purple colour

Dissolve the opium due to meconic acid which
powder in water and persists even after the
filter it. Add ferric addition of hydrochloric
chloride solution to acid.
filtrate.
22
Marquis reagent Red-bluish colour.

Add 100 ml of
concentrated (95%-98
%) sulphuric acid to 5
ml of 40%
formaldehyde in 5-10
mg of substance.
Pilocarpus Take a few ml of Blue violet colour in organic
pilocarpine solution; layer and yellow colour in
add few ml of each of aqueous layer.
dilute
H₂SO4, H₂O2,
potassium chromate
solution and
benzene. Mix and
allow to make
separate layers.
Psoralea Alkali fluorescence Yellow fluorescence.
test
Take alcoholic solution
of powder and add
NaOH solution to it.
Fluorescence test Blue fluorescence.
Take alcoholic solution
of powder, add mixture
of propylene glycol,
acetic acid and water
(3:5: 40). Observe
under UV light.
Turmeric Acid test Crimson colour.
Take 1 mg powder and
add sulphuric acid.
Boric acid test Take 1 Reddish brown colour.
ml of aqueous
solution of turmeric
and add boric acid.
Strophanthus Add sulphuric acid to Green colour.
the aqueous solution of
powder.
23
Wild cherry bark Sodium picrate test After few minutes, yellow
Take powder or pieces colour of paper is turned into
of bark in a test tube brick red.
and add water. Put
filter paper previously
dipped in sodium
picrate at the neck of

the test tube.
Guaiacum resin Cut surface of crude drug
test Cut the piece of turns to blue color.
bark and expose the
filter paper
moistened with
copper sulphate to
cut surface.
CONFIRMATORY CHEMICAL TESTS FOR UNORGANIZED CRUDE

DRUGS OR EXCIPIENTS OF NATURAL ORIGIN
Drug Test Observation
Aloe Cupraloin • Curacao aloes: Wine

(Klunge's red colour lasts for
isobarbaloin) test many hours.
Take little quantity of • Cape aloes: Faint
aqueous solution of colour immediately
aloes, add a few changes to yellow.
drops. of saturated • Socotrine aloes: No
CuSO4, a few mg of colour.
sodium chloride and • Zanziber aloes: No
5 ml of 90% alcohol. colour.
Nitric acid test • Curacao aloes: Dark
Add nitric acid to red colour.
aqueous solution of • Cape aloes: Brown
aloes colour changes to
green.
• Socotrine aloes: Light
brown colour changes
to yellow.
24
• Zanzibar aloes:
Yellowish brown
colour.
Nitrous acid test Curacao aloes: Dark pink

Add sodium colour.
nitrite and acetic Cape aloes: Faint pink
acid to aqueous colour. Socotrine and
solution of aloes zanziber aloe Less change in
colour.
Borax Bead Test Green fluorescence is
Heat 5 ml solution with produced.
02 gm borax add few
drops of this solution
in a test tube filled
with water.
Modified Lower ammoniacal layer
Borntrager's test shows pink to red colour.
Take the extract
solution, add FeCl3
heat and filter it.
To filtrate, add
benzene.
Separate benzene layer

and strong ammonia
solution.
Kino Mix in cold water. Partially (80%-90%) soluble.
Take aqueous solution Green.

and add dilute ferric
chloride solution.
Take aqueous solution Violet.
and add dilute alkali
solution.
25
Take aqueous solution Precipitate obtained.

and add mineral acid.
Agar Take aqueous solution Red colour.
and add ruthenium red.
Take aqueous solution Deep crimson to brown
and add N/50 iodine colour.
Take 5% aqueous Reduction takes place due to
solution; and add 5 ml galactose.
of dilute hydrochloric
acid and heat on water
bath for 30 min. Add to
the first part, 3 ml of
10% caustic soda
solution and 2 ml of
Fehling solution. Heat
on water bath.
Take 5% aqueous White precipitate of barium
solution and add 5 ml sulphate.
of dilute hydrochloric
acid and heat on water
bath for 30 min. Add
barium chloride
solution (10%) to
second part.
Prepare ash of Agar, Skeleton and sponge
add dilute hydrochloric spicules of diatoms will be
acid and observe under observed.
microscope.
Take 5% aqueous No precipitate.
solution and add tannic
acid to it.
Gelatin Heat a small quantity Ammonia is evolved.
of gelatin with soda
lime.
Take 0.5% aqueous White precipitate.
solution and add a few
drops of 10% tannic
acid.
26
Take 0.5% aqueous White precipitate which

solution and add becomes red on heating.
Millon's Reagent.
Take 0.5% aqueous Yellow precipitate.
solution and add 10%
picric acid solution to
it.
Acacia Take 5 ml of 2% w/v Flocculent white precipitate.
solution and add 1 ml
of strong solution. lead
subacetate
Take 5% aqueous Deep blue colour.
solution; add 0.5 ml
hydrogen peroxide
solution and 0.5 ml of
1% alcoholic
benzidine. Shake well
and allow to stand for 5
minutes.
Treat powder with No red colour indicates that
ruthenium red solution it is different from agar.
and microscopically.
Take 10 ml of 2% w/v No precipitation indicates it
solution and add 0.2 ml is different from agar and
of 20% w/v lead tragacanth.
acetate solution.
Take 01 g of powder No crimson colour indicates
and add 1 ml of N/50 that is different from agar
iodine to it. and tragacanth.
Take 1 ml of solution, Red precipitate of cuprous
add 4 ml of water and oxide.
dilute hydrochloric
acid, boil for few min
Add Fehling's solution
and heat.
27
Tragacanth Take 4 ml of 0.5% w/v Red precipitate.

solution add 0.5 ml of
hydrochloric acid and
heat it for 30 min on a
water bath:
add 1.5 ml of sodium
hydroxide solution and
Fehling's solution,
Heat solution using
water bath.
Take 4 ml of 0.5% w/v No precipitate indicates
solution: add 0.5 ml of different it is from agar.
heat
for 30 min on a water

bath; add 10% barium
chloride solution.
Take 0.5% w/v Flocculent precipitate is
solution of the gum obtain which indicates it is
and add 20% w/v different from acacia.
solution of lead
acetate.
Treat powder with No pink colour (distinction
ruthenium red solution from Indian tragacanth).
and examine
microscopically.
Treat powder with N/50 Olive green colour
iodine solution. (distinction from acacia and
agar).
Warm powder with Canary yellow colour.
5% aqueous caustic
potash (KOH).
Indian Tragacanth Boil powder with Brown colour.
aqueous KOH.
Treat powder with Pink colour.

solution of ruthenium
red.
28
Mix with any alkali Insoluble.

solution.
Colophony Dissolve 0.1 g powder Purple-red colour changes to
in 10 ml of acetic violet due to reaction of
anhydride with mild abietic acid.
heat, cool and add a
drop of concentrated
sulphuric acid.
Dissolve 0.1 g powder Ether portion shows emerald
in petroleum ether and green colour due to reaction
filter To filtrate, add 2- of abietic acid.
3 ml dilute copper
acetate solution.
Expose alcoholic Colour changes due to acidic
solution of colophony reaction.
to litmus paper.
Guaiacum Treat 10% alcoholic Deep blue colour.
solution with ferric
chloride solution.
Benzoin Heat 0.5 g slowly in a It melts and evolves irritating
dry test tube. whitish fumes which
condense to form a whitish
crystalline
sublimate in the upper part

of the tube.
Heat gently 1 g of Distinct odour of
powder with 5 ml of benzaldehyde.
potassium
permanganate solution
in a test tube.
Titrate 0.1 g of powder No bright green colour
with 5 ml of alcohol (distinction from siam
(95%), filte and to the benzoin).
filtrate, add 0.5 ml of
5% w/v alcoholic
ferric chloride
solution.
29
Dissolve 0.2 g of Reddish-brown colour.

benzoin in ether. Add 1
ml of the ethereal
solution in a porcelain
dish containing1 ml of
concentrated sulphuric
acid.
Shellac Dissolve powder in a Swells to a gelatinous mass.
solution of ammonia in
a closed vessel.
Myrrh Titrate powder with Yellowish emulsion.
water.
Treat dry ethereal Red colouration.
extract with bromine
vapors.
Treat dry ethereal Purple colouration.
extract with nitric acid
solution.
Asafoetida Titrate asafoetida with Yellowish-orange emulsion
water with addition of turning to greenish-yellow.
alkali.
Heating with dilute Reddish-brown colouration.
sulphuric acid.
Take 10 ml of Pink colour.
alcoholic extract and
add few drops of
phloroglucinol and
concentrated
hydrochloric acid .
Boil with dilute Blue fluorescence due
hydrochloric acid and umbelliferone.
ammonia filter into
ammonia.
Gambier (Pale Catechu) Take 10% alcoholic The upper layer exhibits a
extract: add 2 ml of brillian green fluorescence
sodium hydroxide and 2 due to reaction of gambier
ml of light petroleum, fluorescein.
shake and allow to
separate.
30
Mount powdered drug Acicular crystals of catechin.

in lactophenol and
examine under
microscope.
Dip a match stick into The match stick develops a re
an aqueous extract of stain due to formation
catechu. Dry the stick phloroglucinol from catechin.
and dip it into
concentrated
hydrochloric acid,
remove it quickly and
then, warm near a
flame.
Prepared Chalk Treat with dilute Brick red colour to the flame.
introduce on a platinum
wire into the flame.
Treat with concentrated Gas evolved turns to milky
hydrochloric acid. lime water.
Bentonite Treat 1 g powder with 2 A residue of silica is obtained
g of anhydrous sodium and the acid solution after
carbonate; warm the neutralization of the free
residue with water and yields reaction characteristic
filter, acidify the filtrate aluminium.
with concentrated
hydrochloric acid;
evaporate to dryness
and warm the residue
with dilute hydrochloric
acid.
31
Experiment No.2
Aim - To determine Alcohol content of Asava and Aristha

Reference -
•Trease & Evans. “The Textbook of Pharmacognosy”, 16th Edition, pg-25,26.
Requirements-
Apparatus/ Glasswares:- Digital weighing balance, Beaker ,Measuring Cylinder,
Distillation flask, Separating funnel, Heating metal, Water bath, Specific gravity bottle
Chemicals- Distillation assembly, Distilled water, Pumice powder.
Introduction- There are two types of method for determination of Alcohol content in
Asava and Aristha
Method 1: Distillation and Specific Gravity

Method 2: Gas Chromatography
A)Distillation and Specific Gravity Method

Procedure:
• Take accurately measured 25 ml of the preparation being examined at 24.9 0C to 25.10C and
transfer to the distillation flask.
•Add 150 ml of water and a little pumice powder.
•Now start distillation until not less than 90 ml of the distillate is collected into a 100 ml
volumetric flask.
• Dilute this distillate to 100 ml volume with distilled water at 24.9 0C to 25.10C.
•Measure the relative density (Specific Gravity) at 24.9 to 25.1°and calculate the alcohol
content from the density table given in Indian Pharmacopoeia.
Weight per milliliter Specific Gravity
Weight per milliliter:
The weight per milliliter of a liquid is the weight in g of 1 ml of a
liquid when weighed in air at 250, unless otherwise specified.
32
Specific gravity:
The specific gravity of a liquid is the weight of a given volume of
the liquid at 250 (unless otherwise specified) compared with the weight of
an equal volume of water at the same temperature, all weighing being taken
in air.
Method -
Proceed as described underweight per ml. Obtain the specific gravity of the liquid by dividing
weight of the liquid contained in the pycnometer by the weight of water contained, both
determined at 250 unless otherwise directed in the individual monograph.
Alcohol table
Relative Density Percent Ethanol w/w Percent Ethanol v/v at

At 250 at 15.560
15.560
0.8158 90.0 93.3
0.8146 90.5 93.6
0.8131 91.0 94.0
0.8118 91.5 94.3
0.8104 92.0 94.7
0.8090 92.5 95.0
0.8076 93.0 95.4
0.8062 93.5 95.8
0.8048 94.0 96.1
0.8034 94.5 96.5
0.8020 95.0 96.8
0.8006 95.5 97.1
0.7992 96.0 97.5
0.7977 96.5 97.8
0.7962 97.0 98.1
0.7977 97.5 98.4
0.7932 98.0 98.8
0.7917 98.5 99.1
0.7902 99.0 99.4
0.7886 99.5 99.7
0.7871 100.0 100.0
Result:-
Alcohol content of Asava and Arista was found to be ______________
33
Experiment No: 3
Aim- Evaluation of excepients of natural origin.
Reference -
•Wade A. W,“PJ Handbook of pharmaceutical excipients",11 th edition,pg no. 426.
••Trease & Evans. “The Textbook of Pharmacognosy”, 16th Edition, pg-80.
Requirements:
Apparatus/Glasswares- Digital weighing balance, Measuring cylinder, Distillation flask,
Separating funnel
Chemicals - Hydrochloride acid , sodium hydroxide solution, Fehling’s solution, barium
chloride solution, lead acetate
Excipients
•Pharmaceutical excipients can be defined as non active ingredients that are mixed with
therapeutically active compounds to from medicines.
•The ingredients which is not an active compound is regarded as excipients.
•Excipients affect the behaviour and effectiveness of the drug product more and more
functionally and significantly.
•The variability of active compounds, excipients and process are obvious components
for the product variability.
Significance of Natural Excipients

•These polymers such as natural gums and mucilage are biocompatible, cheap and easily
available and are preferred to semi synthetic and synthetic excipients because of their lack of
toxicity, low cost, availability, soothing action and non- irritant nature.
•The traditional view that excipients are inert and do not exert any therapeutic or
biological action or modify the biological action of the drug substance has changed. and
it is now recognized that excipients can potentially influence the rate and/or extent of
absorption of a drug. As herbal excipients are non- toxic and compatible, they have
major role to play in pharmaceutical formulation. Hence, this chapter is an attempt to
34
review herbal excipients, in reference to their sources, preparation, composition and

applications, used in Novel Drug Delivery System.
•Pharmaceutical excipients can be defined as non-active ingredients that are mixed with
therapeutically active compound(s) to form medicines. The ingredient which is not an
active compound is regarded as an excipient. The excipients affect the behaviour and
effectiveness of the drug product more and more functionality and significantly - The
variability of active compounds, excipients and process are obvious components for the
product variability.
•Examples of polymers or derivatives that have been used or investigated as vaccine
adjuvants are Individual saponins derived from South American tree Quillaja
saponaria. Key hole limpet hemocyanin (KLH), a nonheme copper containing protein
found in anthropods. MPL, a monophosphoryl derivative of the lipid a molecule found
in gram-negative bacteria. Leishmania elongation initiation factor (LelF), a protein
produced by the parasite leishmania Ricin, a potent immunotoxin obtained from the
seeds of castor bean plants.
Classification of Excipients
Excipients are commonly classified according to their application and function in drug
products as Binders, Diluents. Lubricants, Glidants, Disintegrants, Polishing Film formers and
coatings agents, Plasticizers. Taste improving agents. Supending agents Preservatives,
antioxidants Printing inks. Dispersing agent. The most widely used natural excipients are from
class as Colorants, Sweeteners Flavorants, Binders, Diluents, Disintegrants and Thickeners
A) PERFUME
Example: Peppermint Oil (Pudina ka tail)
Peppermint Oil is the essential oil obtained by steam distillation of the flowering herb
Mentha piperita L.var. Family Lamiaceae. Peppermint Oil contains not less than 13.0
per cent w/w and not more than 28.0 per cent w/w of menthone, and not less than 32.0
per cent w/w and not more than 45.0 per cent w/w of menthol.
Category: Pharmaceutical aid.
Description: It is a clear, almost colourless to amber yellow liquid, free from sediment,
suspended matter and characteristic of mint and menthol-like odour.
Identification
A. Determine by gas chromatography (2.4.13).
Test solution. A 2.0 per cent w/v solution of oil under examination in ethanol (95 per
cent). Reference solution. A 2.0 per cent w/v solution of Peppermint oil RS in ethanol
(95 per cent). Use chromatographic system described in the Assay. The peaks in the
chromatogram obtained with the test solution corresponds to the peaks in the
chromatogram obtained with the reference solution.
B. Flash Point: 67.0° to 71.0°.
35
Tests
Relative density: 0.890 to 0.910.
Refractive index: 1.4500 to 1.4595.
Optical rotation: 14.0° to -38.5°C
Assay: Determine by gas chromatography
Storage: Store protected from light and moisture.
B) BASE/ VEHICLE
Example: Castor Oil (Arandi ka tail)
Castor Oil is the fixed oil obtained by cold expression from the seeds of Ricinus communis
Linn. Family Euphorbiaceae. It may contain suitable antioxidants.
Category: Irritant purgative, antirheumatic, amavata.
Description: A pale yellowish or almost colourless, transparent, viscid liquid; odour, slight
and characteristic.
Tests
Light absorption:
Absorbance of 1.0 per cent w/v solution in ethanol (95 per cent) at the maximum at about 270
nm, not more than 0.7 and 1.5.
Weight per ml: 0.945 g to 0.965 g.
Refractive index: 1.4758 to 1.4798.
Optical rotation: +3.5° to +6.0⁰.
Acid value: Not more than 2.0.
Iodine value: 82 to 90.
Saponification value: 176 to 187.

Foreign fatty substances.
A. Mixture of 2 ml of the substance under examination and 8 ml of ethanol (95 per
cent) is clear.
B. Shake 10.0 ml with 20.0 ml of light petroleum (60° to 80°) and allow to separate;
the volume of the lower layer is not less than 16.0 ml.
Storage-Store protected from moisture.
Labelling-The label states (1) the name and quantity of any added antioxidant; (2) whether the
contents are suitable for use in the manufacture of parenteral preparations.
36
C) BINDER
Example: Acacia
Acacia is the air-hardened, gummy exudation from the stem and branches of
Acacia nilotica and A. arabica Family Leguminosae, or other species of Acacia.
It is available as pieces (tears) or in the form of a powder.
Category. Pharmaceutical aid, antidiabetic.
Description
Tears-Irregular and broken pieces of varying size, yellowish white, yellow or amber in
colour, with numerous minute fissures; brittle fractured surface, glassy and occasionally
iridescent; odourless.
Powder - A white or yellowish-white powder; odourless; on treatment with water it

dissolves to give a mucilaginous liquid which is colourless or yellowish, dense, viscous,
adhesive and translucent.
Identification
•An aqueous solution is gelatinised by the addition of lead sub acetate solution.
•To 5 ml of a 10 per cent w/v solution add gradually, while shaking, 10 ml of ethanol
(95 per cent). The cloudy liquid, on addition of 0.5 ml of acetic acid, gives a white
precipitate. Filter and add to the clear filtrate 50 ml of ammonium oxalate solution; the
filtrate becomes cloudy. .
•A 10 per cent w/v solution is either dextro-rotatory or slightly levo-rotatory.
Tests
Sterculia gum and agar. To 50 mg of the powdered substance under examination add 0.2 ml
of freshly prepared ruthenium red solution and examine microscopically; the particles do not
acquire a red colour after irrigation with water,
Agar and tragacanth. To 10 ml of a 10 per cent w/v solution add 0.2 ml of lead acetate
solution; no precipitate is produced.
Water-insoluble matter. Dissolve 5 g of fine powder in 100 ml of water in a 250-ml flask,

add 10 ml of dilute hydrochloric acid and boil gently for 15 minutes. Filter by suction while
hot through a sintered-glass crucible, previously tared, wash thoroughly with hot water, dry at
105° and weigh: the residue does not exceed 50 mg.
Microbial contamination 1.0 g is free from Escherichia coli.
37
Sulphated ash Not more than 5.0 per cent.

Acid-insoluble ash Not more than 1.0 per cent, determined on 1.0 g by Method C.
Loss on drying Not more than 15.0 per cent, determined on 1.0 g by drying in an oven
at 105°.
Storage. Store protected from heat, moisture and against attack by insects and rodents.
Analytical Parameters for Perfume, Vehicle and Binder

A. Weight per millilitre Specific Gravity
A. Weight per millilitre: The weight per millilitre of a liquid is the weight in g of 1
ml of a liquid when weighed in air at 250, unless otherwise specified.
Method -
Select a thoroughly clean and dry pycnometer. Calibrate the pycnometer by filling it with
recently boiled and cooled water at 250 and weighing the contents. Assuming that the weight of
1 ml of water at 25 when weighed in air of density 0.0012 g per ml, is 0.99602 g. 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).
Adjust the temperature of the substance to be examined, to about 20° and fill the pycnometer
with it. Adjust the temperature of the filled pycnometer to 25 0,remove any excess of the
substance and weigh. Substract the tare weight of the pycnometer from the filled weight of the
pycnometer. Determine the weight per milliliter dividing the weight in air, expressed in g, 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.
B. Specific gravity: The specific gravity of a liquid is the weight of a given volume of the
liquid at 250 (unless otherwise specified) compared with the weight of an equal volume of
water at the same temperature, all weighing being taken in air.
Method -
Proceed as described under weight per ml. Obtain the specific gravity of the liquid by dividing
weight of the liquid contained in the pycnometer by the weight of water contained, both
determined at 250 unless otherwise directed in the individual monograph.
Refractive Index -
The refractive index (ɳ) of a substance with reference to air is the ratio of the sine of the angle
of incidence to the sine of the angle of refraction of a beam of light passing from air into the
substance. It varies with the wavelength of the light used in its measurement.
38
Unless otherwise prescribed, the refractive index is measured at 250 (±0.5) with reference to
the wavelength of the D line of sodium (λ 589.3 nm). The temperature should be carefully
adjusted and maintained since the refractive index varies significantly with temperature.
The Abbe's refractometer is convenient for most measurements of refractive index but other
refractometer of equal or greater accuracy may be used, Commercial refractometers are
normally constructed for use with white light but are calibrated to give the refractive index in
terms of the D line of sodium light.
To achieve accuracy, the apparatus should be calibrated against distilled water which has a
refractive index of 1.3325 at 25° or against the reference liquids given in the Table
Reference Liquid (ɳ)D20 Temperature C0-

efficientΔn/Δt
Carbon tetrachloride 1.4603 -0.00057
Toulene 1.4969 -0.00056
α-Methylnaphthalene 1.6176 -0.00048
Reference index value for the D line of sodium, measured at 20 0 .
The cleanliness of the instrument should be checked frequently by determining the refractive
index of distilled water, which at 250 is 1.3325.
C Optical Rotation
Certain substances, in a pure state, in solution and in tinctures possess the property of rotating
the plane of polarized light, i.e., the incident light emerges in a plane forming an angle with the
plane of the incident light. These substances are said to be optically active and the property of
rotating the plane of polarized light is known as optical rotation.
The optical rotation is defined as the angle through which the plane of polarized light is
rotated when polarized light obtained from sodium or mercury vapour lamp passes
through one decimeter thick layer of a liquid or a solution of a substance at a temperature
of 25° unless as otherwise stated in the monograph, Substances are described as
dextrorotatory or levoretatory according to the clockwise or anticlockwise rotation
respectively of the plane of polarized light. Dextrorotation is designated by a plus (+)
sign and levorotation by a minus (-) sign before the number indicating the degrees of
rotation.
Apparatus: A polarimeter on which angular rotation accurate 0.05° can be read may be used.
Calibration: The apparatus may be checked by using a solution of previously dried sucrose
and measuring the optical rotation in a 2-din tube at 25° and using the concentrations indicated
in Table read may be used.
Concentration Angle of Rotation

(g/100 ml) (+) at 25°
10.0 13.33
39
20.0 26.61
30.0 39.86
40.0 53.06
50.0 66.23
Procedure:
For liquid substances, take a minimum of five readings of the rotation of the liquid and also for
an empty tube at the specified temperature. For a solid dissolve in a suitable solvent and take
five readings of the rotation of the solution and the solvent used. Calculate the average of each
set of five readings and find out the corrected optical rotation from the observed rotation and
the reading with the blank (average).
Acid Value
The acid value is the number of mg of potassium hydroxide required to neutralize the free
acids in I g of the substance, when determined by the following method:
Weigh accurately about 10 g of the substance (1 to 5) in the case of a resin into a 250
ml flask and add 50 ml of a mixture of equal volumes of alcohol and solvent ether,
which has been neutralized after the addition of 1 ml of solution of phenolphthalein.
Heat gently on a water-bath, if necessary until the substance has completely melted,
titrate with 0.1 N potassium hydroxide, shaking constantly until a pink colour which
persists for fifteen seconds is obtained. Note the number of ml required. Calculate the
acid value from the following formula:
a × 0.00561 × 1000
Acid value = W
Where 'a' is the number of ml of 0.1 N potassium hydroxide required and 'W' is the weight in g
of the substance taken.
Iodine Value
The lodine value of a substance is the weight of iodine absorbed by 100 part by weight of the
substance, when determined by one of the following methods:
lodine Flasks- The lodine flasks have a nominal capacity of 250 ml.
lodine Monochloride Method –
Place the substance accurately weighed, in dry iodine flask, add 10 ml of carbon
tetrachloride, and dissolve. Add 20 ml of iodine monochloride solution, insert the
stopper, previously moistened with solution of potassium iodine and allow to stand in a
dark place at a temperature of about 170 or thirty minutes. Add 15 ml of solution of
potassium iodine and j00 ml water; shake, and titrate with 0.1 N sodium thiosulphate,
using solution of starch as Note the number of ml required (a). At the same time carry
out the operation in exactly the same manner, but without the substance being tested,
and note the number of ml of 0.1 N indicator. sodium thiosulphate required (b).
40
Calculate the iodine value from the formula:-
(b-a) × 0.01269 × 100

Iodine value =
W
Where 'W' is the weight in g of the substance taken.

The approximate weight, in g. of the substance to be taken may be calculated by dividing 20
by the highest expected iodine value. If more than half the available halogen is absorbed, the
test must be repeated, a smaller quantity of the substance being used.
Saponification Value
The saponification value is the number of mg of potassium hydroxide required to
neutralize the fatty acids, resulting from the complete hydrolysis of 1 g of the oil or fat,
when determined by the following method:
Dissolve 35 to 40 g of potassium hydroxide in 20 ml water, and add sufficient alcohol make
1,000 ml. Allow it to stand overnight, and pour off the clear liquor. Weigh accurately about 2 g
of the substance in a tared 250 ml flask, add 25 ml of the alcoholic solution of potassium
hydroxide, attach a reflux condenser and boil on a water-bath for one hour, frequently rotating
the contents of the flask cool and add 1 ml of solution of phenolphthalein and titrate the excess
of alkali with 0.5 hydrochloric acid. Note the number of al required (a). Repeat the experiment
with the same quantities of the same reagents in the mate omitting the substance. Note the
number of ml required (b) Calculate the saponification value from the following formula:
(b-a) × 0.02805 × 1.000

Saponification value =
W
Where 'W' is the weight in g of the substance taken.
pH Value
The pH value of an aqueous liquid may be defined as the common logarithm of the reciprocal
of the hydrogen ion concentration expressed in g per litre. Although this definition des a useful
practical means for the quantitative indication of the acidity or alkalinity of a solution, it is less
satisfactory from a strictly theoretical point of view. No definition of pH as a measurable
quantity can have a simple meaning, which is also fundamental and exact.
41
The pH value of a liquid can be determined potentiometrically by means of the glass electrode,
a reference and a pH meter either of the digital or analogue type.
Report- From the above characters are given excipients is identified
42
Experiment No: 4
Aim- To prepare and evaluate skin lightening cream containing standardized

licorice extract
Reference-
•Trease & Evans. “The Textbook of Pharmacognosy”,16 th Edition, pg-55,
Requirements-
Apparatus/Glasswares- Beaker, funnel, measuring cylinder, glass slides.
Chemicals - acetic anhydride, Conc. Sulphuric acid, Water, glacial acetic acid. Licoric
extract, Stearic acid, Cetyl alcohol, Almond oil, Glycerin, Methyl paraben, Propyl
paraben, Triethanolamine .
Method of Extraction:
Glycyrrhiza glabra extract is obtained from the roots of Glycyrrhiza glabra by solvent
extraction method and then concentrating the extract by rotary evaporator. Glycyrrhiza glabra
extract is preserved by refrigeration and/or freezing.
For this, take accurately weighed 100 gm powder of Licorice root. Extract with 500 ml
water for three hours. Filter and evaporate filtrate to dry mass.
Physico-chemical Evaluation of Extract:

1.Physical characteristics
Colour
Odour
Taste
2.Chemical parameters
Perform chemical tests for Liquorice
Foam test Shake aqueous solution of a Foam lasts for

saponin containing sample more the 15
producing foam which is stable for seconds.
15 minutes or more
43
Liebermann Mix 2 ml test extract with 2 ml Blue colour

test acetic anhydride. Boil and add 0.5
ml of H2SO4
Colour test Add 80% H2SO4 to powder. Deep yellow
colour.
3.TLC
Stationary phase Silica Gel GF 254

Mobile phase Butanol: Glacial Acetic Acid: Water
(7:1:2)
Detection Spray with Anisaldehyde-Sulphuric Acid
Reagent
A) Cream
Method of Preparation of Cream:
Oil in water (O/W) emulsion-based cream.
Dissolve Stearic acid, Cetyl alcohol in Almond oil (Oil Phase-Part A) and heat to 75°C.
Dissolve the preservatives and other water soluble components (Methyl paraben, Propyl
paraben, Triethanolamine, Glycerin and Extract of Liquorice in water (Aqueous Phase-Part B)
and heat to 75°C. Now, add the aqueous phase in small portions to the oil phase with
continuous trituration in porcelain mortar until a smooth cream is formed.
Ingredient Quantity
Licorice Extract 1 gm
Stearic acid 5 gm
Cetyl alcohol 3 gm
Almond oil 2 ml
Glycerin 2 ml
Methyl paraben 0.02gm
Propyl paraben 0.02gm
Triethanolamine q.s.
Pharmaceutical Evaluation of Cream:

Evaluate the cream for
• pH,
• Viscosity,
• Appearance,
44
• Spreadability,
• Homogeneity,
• Extrudability,
• Type of Emulsion and
• Spot pigmentation score
Procedure
• pH: Prepare 10% solution of cream. Measure pH with a digital pH meter.
• Viscosity: Evaluate viscosity using Brookfield viscometer using LV-64

spindle at rote rate to 25 rpm. Immerse the spindle into formulated lotion
and measure the viscosity.
• Appearance: Determine the colour, odour and homogeneity of the cream

visually.
• Spreadability: Determine the spreadability of cream by the parallel plate

method Select two glass slides of 20/20 cm. Place about 1 g of the lotion
formulation over one of the slides. Place the other slide upon the top of the
lotion such that the lotion is sandwiched between the slides and 125 g
weight is placed upon the upper slide so that lotion between the two slides
is pressed uniformly to form a thin layer. Remove the weight and measure
the spread diameter.
• Type of Emulsion Test: Conduct dye solubility and dilution test to

determine the type of emulsion formed.
B) Lotion
To prepare and evaluate skin lightening lotion containing standardized
licorice extract
An extract of Glycyrrhiza glabra is rich of natural antioxidants. The best

natural antioxidants extract of Glycyrrhiza glabra are glycyrrhizin (glycyrrhizic
acid) and flavonoids. The role of Glycyrrhiza glabra extract on skin is mainly
attributed to its antioxidant activity particularly to its potent antioxidants
triterpene saponins and flavonoids. Skin whitening, skin depigmenting, skin
lightening, antiaging anti-erythemic, emollient, anti-acne and photoprotection
effects are mainly attributed to Glycyrrhiza glabra extract.
45
Glycyrrhiza glabra extract is obtained from the roots of Glycyrrhiza glabra by solvent
extraction method and then concentrating the extract by rotary evaporator. Glycyrrhiza glabra
extract is preserved by refrigeration and/or freezing.
For this, take accurately weighed 100 gm powder of Licorice root. Extract with 500 ml water
for three hours. Filter and evaporate filtrate to dry mass.
1. Physical characteristics
Colour
Odour
Taste
2. Chemical parameters
Perform chemical tests for Liquorice

15 minutes or more
Liebermann Mix 2 ml test extract with 2 ml Blue color
test acetic anhydride. Boil and add 0.5
ml of H₂SO4.
Colour test Add 80% H₂SO4 to powder. Deep yellow
colour.
3. TLC

Mobile phase Butanol: Glacial Acetic Acid: Water
(7:1:2)
Detection Spray with Anisaldehyde-Sulphuric Acid
Reagent
Method of Preparation of Lotion:

Dissolve stearic acid and Ceto steryl alcohol in mineral oil (Oil phase-Part A) and heat
to 75°C. Dissolve the preservatives and other water soluble components (Methyl
46
paraben sodium, Propyl paraben sodium, Triethanolamine, glycerin and extract of

liquorice in water (Aqueous phase-Part B) and heat to 75°C.
Prepare the lotion by adding non-polar phase to the polar phase with rapid stirring to avoid
separation of water and oil phase.
Ingredient Quantity
Licorice Extract 100 mg
Mineral oil 12 ml
Stearic acid 10 mg
Cetosteryl alcohol 2 ml
Propylene glycol 2 ml
Triethanolamin 3 ml
Methylparaben sodium 2 mg
Propyl paraben sodium 0.2 mg
Purified water vehicle Qs to 100 ml
Pharmaceutical Evaluation of Lotion:
• pH:
• Viscosity:
• Appearance:
• Spreadability: • Stability Test:
• Sensitivity Test:
• Washability Test:
• Type of Emulsion Test:
Procedure
• pH: Prepare 10% solution of lotion. Measure pH with a digital pH meter.
• Viscosity: Evaluate viscosity using Brookfield viscometer using LV-64

spindle at rote rate to 25 rpm. Immerse the spindle into formulated lotion
and measure the viscosity
• Spreadability: Determine the spreadability of lotion by the parallel plate

method Select two glass slides of 20/20 cm. Place about 1 g of the lotion
formulation over one of the slides. Place the other slide upon the top of the
lotion such that the lotion is sandwiched between the slides and 125 g
weight is placed upon the upper slide so that lotion between the two slides
47
is pressed uniformly to form a thin layer. Remove the weight and measure
the spread diameter.
• Stability Test: Store the formulated lotion at different temperatures and

humidity conditions of 25±2°C / 60±5% RH (at room temperature), 40 ±
2°C/75 ± 5% RH (accelerated temperature) for a period of three months
and studied for pH, viscosity and spreadability
• Sensitivity Test: Apply a portion of lotion on the forearms of 6 volunteers

and keep for 20 minutes. After 20 minutes, note if any kind of irritation or
redness occurs.
• Washability Test: Apply a portion of lotion over the skin of hand and allow
to flow under the force of flowing tap water for 10 minutes. Note the time
when the lotion is completely removed.
• Appearance: Determine the colour, odour and homogeneity of the lotion

visually.
• Type of Emulsion Test: Conduct dye solubility and dilution test to

determine the type of emulsion formed.
C. Shampoo
To prepare and evaluate Shampoo containing standardized extract of Reetha fruit
Sapindus mukorossi, commonly called as soapnut, is an extremely valuable

medicinal plant which possesses several biological and pharmacological
properties.
Sapindus mukorosii (Soapnut) extract is obtained from the fruits of Sapindus mukorosii by
solvent extraction method and then concentrating the extract by rotary evaporator. For this,
take accurately weighed 100 gm powder of Soapnut fruits. Extract with 500 ml water for three
hours. Filter and evaporate filtrate to dry mass.
1. Physical characteristics
Colour
48
Odour
Taste
2. Chemical parameters
Perform chemical test for Reetha

15 minutes or more
3. TLC

Mobile phase Butanol: Water: Acetic Acid (8.4:
1.4: 0.7)
Detection Detection: 630 nm
Method of Preparation of Shampoo:

Add Soapnut extract to 10% gelatin solution and mix by shaking for 20 min. Add lemon juice
(1 ml) and methyl paraben with stirring. Adjust the pH of the solution by adding sufficient
quantity of 1% citric acid solution. Add few drops of rose essential oil to impart aroma to the
prepared shampoo and make the final volume to 100 ml with gelatin solution.
Ingredient Quantity
Reetha extract (10-12 % total 4.5 gm
saponins)
Amla extract 2.5 gm
Lemon juice 1 ml
Methyl paraben 1ml of 0.05%
solution
Gelatin solution q.s.
Citric acid q.s.
Essential oil 0.1 ml
Pharmaceutical Evaluation of Shampoo
• Physical Appearance/Visual Inspection:
• Determination of pH:
49
• Determination of % of Solid Contents:
• Surface Tension Measurement:
• Foaming Ability and Foam Stability:
• Wetting weight Time Test:
• Evaluation of Conditioning Performance:
• Total Saponins Determination:
Procedure
Appearance: Evaluate the shampoo for the clarity, colour, odour and foam producing
ability.
Determination of pH: Measure the pH of 10% v/v shampoo solution in distilled water, by
using pH meter at room temperature.
Determination of % of Solid Contents: Place 4 grams of shampoo in a previously clean, dry
and weighed evaporating dish. Weigh the dish and shampoo again to confirm the exact weight
of the shampoo. Evaporate the liquid portion of the shampoo by placing the evaporating dish
on the hot plate. Calculate the weight and thus % of the solid contents of shampoo left after
complete drying.
Surface Tension Measurement: Measure the surface tension of 10% w/v shampoo distilled
water using Stalagmometer at room temperature.
Foaming Ability and Foam Stability: Determine foaming ability by using cylinder shake
method. Place 50 ml of the 1% commercial or formulated shampoo solution into a 250
graduated cylinder, cover with one hand and shake 10 times. Record the total volume of the
foam content after 1 minute of shaking. Evaluate foam stability by recording the foam volume
after 1 min and 4 minute of shake test.
Wetting weight Time Test: Cut a canvas paper into 1-inch diameter discs having an average
weight of 0.44 g. Place the smooth surface of disc on the surface of 1% v/v shampoo solution
and start the stopwatch. The time required for the disc to begin to sink is noted down as the
wetting time.
Evaluation of Conditioning Performance: Take the hair tress of an Asian woman from a
local salon. Cut it into four swatches of the tresses with approximately the length of 10 cm and
the weight of 5 g. A swatch without washing served as the control. Wash other three tresses
with the formulated shampoos in an identical manner. For each cycle, shake each tress with the
mixture of 10 g of a sample and 15 g of water in a conical flask for 2 minutes and then rinse
with 50 ml water. Afterward, dry each tress at room temperature. Wash tresses for maximum
ten cycles. Evaluate the conditioning performance of the shampoos i.e. smoothness and
50
softness, by a blind touch test, by twenty randomly selected student volunteers. All the students
should blind folded and asked to touch and rate the four tresses for conditioning performance
from score 1 to 4 (1 = poor; 2= satisfactory; 3= good 4p; 4=excellent).
Total Saponins Determination: Determine total saponins by Gravimetry. It should contain
10-12 % total saponins.
51
Experiment No: 5
Aim: To prepared and standardized extract in formulations like syrups,

mixtures and tablets and their evaluation as per Pharmacopoeial
requirements.
Reference:
•Trease & Evans. “The Textbook of Pharmacognosy”,16th Edition, pg-77

Requirements :
Apparatus/Glasswares: Pycnometer, digital pH meter.
Chemicals- Sucrose, licorice dry extract, methyl paraben
A. Syrup
Method of Preparation:
Mix 66.7% w/w of sucrose in required quantity of distilled water to prepare a concentrated
solution of simple syrup. Mix 50 mg of Licorice dry extract ( for standardized Licorice extract
preparation) in 100 ml of simple syrup. Add 0.1% of methyl paraben as preservative, to the
above mixture, Mix well to get visibly clear solution.
Evaluation:
Organoleptic Properties: Evaluate syrup for various organoleptic parameters such as colour,
odour and taste.
Determination of pH: Measure pH by digital pH meter.
52
Specific Gravity: Determine the specific gravity using pycnometer by dividing the weight of
the syrup contained in the pycnometer by the weight of water contained, at 25°C.
Stability Testing: (Optional) Carry out stability testing of the prepared syrup by keeping the
samples at room and accelerated temperature conditions. Take final syrup in six different
amber coloured glass bottles and keep three bottles at room temperatures (37°C) and three
bottles at accelerated temperatures (47°C). Examine the samples for all the physicochemical
parameters, turbidity and homogeneity at the interval of 24 hr, 48 hr and 72 hr for any change.
B. Mixture
Ingredient Quantity
Fluid extract of Senna 2 ml
Fluid extract of Licorice 1 ml
Sulphate of Magnesia 15 ml
Alcohol ( 50%) 4 ml
Water 30 ml
Method of Preparation:
Mix well all ingredients and evaluate for various pharmaceutical parameters.
Evaluation of Mixture:
Organoleptic Properties: Evaluate mixture for various organoleptic parameters such as

colour, odour and taste.
Determination of pH: Measure pH by digital pH meter.
Specific Gravity: Determine the specific gravity using pycnometer by dividing the weight of
the syrup contained in the pycnometer by the weight of water contained, at 25°C.
Stability Testing: (Optional)
Carry out stability testing of the prepared mixture by keeping the samples at room and
accelerated temperature conditions. Take final syrup in six different amber coloured glass
bottles and keep three bottles at room temperatures (37°C) and three bottles at accelerated
temperatures (47°C). Examine the samples for all the physicochemical parameters, turbidity
and homogeneity at the interval after 24 hours. 48 hours and 72 hours for any change.
C. Tablet
Wet Granulation
Ingredients Mg/tablet
53
Senna dry extract 100 mg

Maize starch (Binder) 10 mg
Lactose- MCC (Diluents) 276 mg
Magnesium stearate( 1.5 mg
Lubricant)
Procedure:
Prepare binder solution (10 percent) with water. Mix binder with extract and diluents mixture.
Pass the wet mass through sieve (6-8 mesh). Dry the granules and now add lubricant, Mix well.
Compress Tablets using tablet compression machine.
Evaluation of Tablets:
Thickness: Measure the thickness of tablet using micro meter screw gauge. Measure thickness
of six tablets from each batch and calculate mean thickness value.
Hardness: Measure the hardness of six tablets using the Monsanto hardness tester and
calculate mean value.
Weight Variation: Weigh 20 tablets of each batch using an electronic digital balance.
Calculate the average weight and then percentage.
Friability: Select twenty tablets randomly and weigh. Place tablets in friability test apparatus
and rotate at a speed of 25 rpm for 4 minutes. Weigh the tablets again and calculate difference
in weight and express friability percent.
Disintegration Time: Perform the disintegration test according to IP specifications. Use water
as the disintegrating medium. Keep the temperature of the medium at 37°C. Fill beakers at
volume of 800 ml and take care that the six tablets will always below the level of water at the
highest and lowest position of basket-rack assembly. Introduce the discs over ach tablet to
avoid floating of tablets in the medium. The apparatus is operated until all the tablets will
disintegrate.
Preparation of Stock Extracts
Senna Fluid Extract -
Prepare Senna Fluid Extract as follows -

Mix 1000 g of Senna, in coarse powder, with a sufficient quantity (600 mL to 800 mL) of
menstrqum consisting of a mixture of 1 volume of alcohol and 2 volumes of water to make it
evenly and distinctly damp. After 15 minutes, pack the mixture firmly into a suitable
percolator, and cover the drug with additional menstruum. Macerate for 24 hours, then
percolate at a moderate rate, adding fresh menstruum, until the drug is practically exhausted of
its active principles. Reserve the first 800 mL of percolate, and use it to dissolve the residue
from the additional percolate that has been concentrated to a soft extract at a temperature not to
exceed 60°. Add water and alcohol to make the product measure 1000 mL, and mix.
54
Packaging and storage:

Preserve in tight, light-resistant containers, and avoid exposure to direct sunlight and to
excessive heat. Alcohol content, Method I (611): between 23.0% and 27.0% of C 2H5OH.
Licorice Fluid Extract -

Prepare Licorice Fluid Extract as follows
To 1000 g of coarsely ground Licorice add about 3000 mL of boiling Purified Water, mix, and
al low to macerate in a suitable, covered percolator for 2 hours. Allow the percolation to
proceed at a rate of 1 to 3 mL per minute, gradually adding boiling Purified Water until the
glycyrrhiza is exhausted. Add enough diluted ammonia solution to the percolate to impart a
distinctly ammonical odour, and boil the liquid actively under normal atmospheric pressure
until it is reduced in volume to about 1500 ml. Filter the liquid, evaporate the filtrate on a
steam bath until the residue measures 750 mL, cool, gradually add 250 mL of Alcohol and
enough Purified Water to make the product measure 1000 ml. and mix.
Packaging and storage:

Preserve in tight, light-resistant containers, and avoid exposure to direct sunlight and to
excessive heat. Labeling-The label states the Latin binomial name and, following the official
name, the part of the plant source from which the article was derived.
55
Experiment No: 6
Aim: Monograph analysis of Glycerrhiza as per different Pharmacopoeia
Reference-
•Trease & Evans. “The Textbook of Pharmacognosy”,16 th Edition, pg-78
Requirements-
Apparatus/Glasswares - conical flask , volumetric flask ,beaker, water bath , measuring
cyclinder, Petri plate , pipette,
Chemicals- butyl alcohol, sulphuric acid ,Methanol, Ammonia, Chloroform, Glycerrhizic

acid, Glacial acetic acid, Liquorice, Dragendroff reagent ,Ethyl acetate, Chloroform,
Hydrochloric acid ,Ethanol
Yasti (Indian Pharmacopoeia 2018)

Liquorice root: Mulethi, Glycyrrhiza glabra,
Yasti consists of the dried, unpeeled roots and stolons of Glycyrrhiza glabra Linn. Family
Leguminosae. Yasti contains not less than 30 per cent w/w of glycyrrhizinic acid.
Category: Expectorant, antiulcer, stomachic, anti-inflammatory, kasa.
Description: Odour, characteristics and slightly aromatic, taste, very sweet and faintly
astringent the bark is not bitter
Identification
a) Macroscopic: Root with few branches, up to 1 m long and 0.5 to 3 cm in diameter.

Bark, brownish-grey to brown with longitudinal striations, bearing traces of lateral
roots. Stolons, cylindrical, 1 to 2 cm in diameter and up to several meters long, but may
be cut into length of 10 to 15 cm; similar in external appearance to the root but with
occasional small buds. Fracture of the root and stolon, granular and fibrous. Cork layer,
thin; secondary phloem region, wide, light yellow with radial striations, xylem,
56
compact, yellow, with radiate structure. The stolon has a central pith, which is absent
from the root.
(b)Microscopic: Cork and phelloderm are narrow phloem consisting of bundles of
thick- walled, yellow fibers with narrow lumina surrounded by cells each containing a
calcium oxalate prism, alternating in the external layers with areas of strongly hyaline
keratenchyma; functional sieve tissue near the cambium. Medullary rays
parenchymatous, widening towards the exterior, 3 to 12 cells wide. Xylem composed
of radial rows of tracheids and vessels alternating with bundles of lignified fibres with
crystals sheaths similar to those of the secondary phloem; vessels 30 um to 150 um in
diameter with thick walls (5 um to 10 μm) having reticulate thickenings or numerous
bordered pits with slit-shaped openings associated with lignified xylem parenchyma;
medullary rays, 2 to 5 cells wide. Parenchymatous cells throughtout containing simple, round,
oval or fusiform starch granules 2 um to 20 μm, mostly 5 µm to 12 μm, in diameter; parenchymatous
pits present solely in the stolon.
(c)Thin Layer Chromatography:
Stationary Phase: Silica Gel GF 254.
Mobile Phase: A mixture of 70 volumes of butyl alcohol, 20 volumes of water and 10

volumes of acetic acid.
Test Solution: Add 10 ml of 70 per cent v/v methanol to 1 g of dried yasti powder, heat
by shaking on a water bath for 5 minutes, cool and filter.
Reference Solution: Add 10 ml of 70 per cent v/v methanol to 1 g of dried yasti powder, heat
by shaking on a water bath for 5 minutes, cool and filter. Apply to the plate plate 10 ul of each
solution as bands, 10 mm by 2mm .Allow the mobile phase to rise 8cm. Dry the plate in air and
examine under ultraviolet light at 254 nm and 360 nm, spray with anisaldehyde sulphuric acid
reagent. Heat the plate at 1050 for 10 minutes and examine the plate in day light. The
chromatographic profile of the test solution is similar to that of the reference solution. Mix a
small quantity, in powder, with 0.05 ml of sulphuric acid; the powder particles become orange-
yellow and some fragments change, more slowly, to pinkish red.
(d) Water-soluble extractive not less than 20 per cent, determined by the following method.
Mix 2.5 g of the finely powdered drug with 50 ml of water and allow to stand for 2 hours,
shaking frequently, filter, evaporate 10.0 g of the filtrate to dryness on a water-bath, dry the
residue at 105 0C and weigh.
(e)Acid-insoluble Ash: Not more than 2.0 per cent.
(f)Sulphated Ash: Not more than 10.0 per cent.

57
Assay: Liquid chromatography,
Test Solution: Weigh accurately 1.0 g of the coarsely powdered substance under examination
in a 250 ml conical flask, add 100 ml of 0.1 M ammonia and mix with the aid of ultrasound for
30 minutes. Centrifuge a part of the supernatant liquid and dilute 1.0 ml to 5.0 ml with 0.1 M
ammonia. Filter the solution through a membrane filter with an average pore diameter not
greater than 1.0 um and use the filtrate.
Reference Solution: A 0.005 per cent w/v solution of glycyrrhizinic acid RS in 0.1 M
ammonia.
Chromatographic System:
• A stainless steel column 25 cm x 4.6 mm, packed with octadecyl silane

bonded to porous silica (5 um).
• Mobile phase: A mixture of 6 volumes of glacial acetic acid, 30 volumes
of acetonitrile and 64 volumes of water.
• Flow rate: 1.5 ml per minute, • Spectrophotometer set at 254 nm,
• Injection volume: 20 µl.
Inject the reference solution. The test solution is not valid unless the relative standard
deviation for replicate injections is not more than 2.0 per cent. Inject the reference solution and
test solution. Calculate the content of glycerrhizinic acid. Storage: Store protected from light
and moisture.
Yasti (The Ayurvedic Pharmacopoeia of India )

Yasti consists of dried, unpeeled, stolon and root of Glycyrrhiza glabra
Linn, Family: Leguminosae; a tall perennial herb, upto 2 m high found cultivated
in Europe, Persia, Afghanistan and to little extent in some parts of India.
Synonyms:
Sanskrit: Yastimadhuka, Yastika, Madhuka, Madhuyasti, Yastyahva. Hindi: Mulethi, Mulathi,
Muleti, Jethimdhu, Jethimadh
Marathi: Jesthamadh.
Urdu: Mulethi, Asl-us-sus.
Description:
a) Macroscopic: Stolon consist of yellowish brown or dark brown outer layer, externally
longitudinally wrinkled, with occasional small buds and encircling scale leaves, smoothed
transversely, cut surface shows a cambium ring about one-third of radius from outer surface
and a small central pith; root similar without a pith; fracture, coarsely fibrous in bark and
splintery in wood; odour, faint and characteristics; taste, sweetish.
58
(b) Microscopic:
Stolon: Transverse section of stolon shows cork of 10-20 or more layers of tub cells,
outer layers with reddish-brown amorphous content, inner 3 or 4 rows having thicker.
colourless walls; secondary cortex usually of 1-3 layers of radially arranged
parenchymatous cells containing isolated prisms of calcium oxalate; secondary phloem
a broad band, cells of inner part cellulosic and outer lignified, radially arranged groups
of about 10-50 fibres, surrounded by a sheath of parenchyma cells, each usually
containing a prism of calcium oxalate about 10- 35 u long: cambium form tissue of 3 or
more layers of cells; secondary sylem distinctly radiate with medullary rays, 3-5 cells
wide, vessels about 80-200 u diameter with thick, yellow, pitted, reticulately thickened
walls, groups of lignified fibres with crystals sheath similar to those of xylem
parenchyma of two kinds, those between the vessels having thick pitted walls without
inter-cellular spaces, the remaining with thin walls: pith of parenchymatous cells in
longitudinal rows, with inter-cellular spaces.
Root: Transverse section of root shows structure closely resembling that of stolon
except that no medulla is present; xylem tetrarch; usually four principal medullary rays
at right angles to each other; in peeled drug cork shows phelloderm and sometimes
without secondary phloem; all parenchymatous tissue containing abundant, simple, oval
or rounded starch grains, 2-20 u in length.
Identity, Purity and Strength:

Total ash –Not more than 10 per cent.
Acid-insoluble ash - Not more than 2.5 per cent.
Alcohol-soluble extractive - Not less than 10 per cent.
Water-soluble extractive - Not less than 20 per cent.
Constituents: Glycyrrhizin, glycyrrhizic acid, glycyrrhetinic acid,
asparagine, sugar
Properties and Action:

Rasa: Madhura
Guna: Guru, snigdha
Virya: Sita
Vipika: Madhura
Karma: Vatapittajit, Raktaprasadana, Balya, Varnya, Vrsya, Carksusya
Important Formulations: Eladigutika; Yastimadhuka tails, Madhuyastyaditaila.

Therapeutic Uses: Kasa: Svarabheda, Ksaya: Vrana: Vatarakta
Dose: 2-4 g of the drug in powder form.
59
Liquorice (British Pharmacopoeia 1993)

Definition:
Liquorice consists of the dried, unpeeled roots and stolons of Glycyrrhiza glabra. It contains
not less than 4.0 % of glycyeehizinic acid.
Characteristics:
Odour, characteristics and slightly aromatic. Taste, very sweet, faintly astringent; the bark is
not bitter.
Microscopical:
Root with few branches, up to 1 m long and 0.5 to 3 cm in diameter. Bark, brownish
grey to brown with longitudinal striations, bearing traces of lateral roots. Stolon,
cylindrical, 1 to 1 cm in diameter and up to several meters long, but may be cut into
length of 10 to 15 cm, similar in external appearance to the root but with occasional
small buds, Fracture of the root and stolon, granular and fibrous: Cork layer, thin;
secondary phloem region, wide, light, with radial striations: xylem, compact yellow,
with radiate structure. The stolon has a central pith which is absent from the root.
Microscopical:
Stolon with cork and phelloderm narrow. Phloem consisting mainly of radially arranged
bundles of thick-walled, yellow fibres 700 to 1200 um long and 10 to 20 µm wide with a
narrow lumen surrounded by cells each containing a prism of calcium oxalate crystals, 10 to 35
um long and 2 to 5 um wide, alternating in the external layers with areas of strongly hyaline
keratenchyma; normal sieve tissue near the cambium. Xylem of radial rows of tracheids and
vessels alternating with bundles of partially lignified fibers with crystals sheaths similar to
those of the secondary phloem; vessels 30 to 150 um in diameter with walls 5 to 10 um thick
having numerous bordered pits with slit-shaped openings, associated with lignified xylem
parenchyma. Medullary rays, two to five cells wide. Parenchymatous cells throughout
containing simple, round or oval starch granules 2 to 20 um, mostly 5 to 12 um, in diameter;
Parenchymatous pith present only in the stolon.
Identification:
Thin-Layer Chromatography:
(a) Carry out the method using silica GF254 as the coating substance and as the mobile
phase the upper layer, even if turbid, of a mixture of 60 volumes of ethyl acetate,
27 volumes of 1 M ammonia and 13 volumes of absolute ethanol, shake together
and allow to stand for 5 minutes. Prepare the following. For solution (1): Shake
1.0 g. in No. 180 powder, with 20 ml of chloroform for 15 minutes, filter and
60
reserve the extracted powder for the preparations of solution (2). Evaporate the
filtrate dryness and dissolve the residue in 2 ml of mixture of equal volumes of
chloroform and methanol. For solution (2): Add the extracted powder 30 ml of
0.5 M sulphuric acid and heat under a reflux condenser for 1 hour, allow to cool
and extract with two 20 ml quantities of chloroform; dry the combined chloroform
extracts with anhydrous sodium sulphate, filter, evaporate to dryness and dissolve
the residue in 2 ml of a mixture of equal volumes of glycyrrhizinic acid in 2 ml
of a mixture of equal volumes of chloroform and methanol. For solution (3):
Dissolve 10 mg of glycyrrhetinic acid in 2 ml of a mixture of equal volumes
chloroform and methanol. Apply separately to the plate in three bands, each 20
mm long and not more than 3 mm wide, 10 µl of solutions (1) and (2) and 20 ul
of solution (3).
After the removal of the plate, allow it to dry in air for 5 minutes and
examine under ultraviolet light (254 nm). The chromatogram obtained with
solution (3) exhibits a band corresponding to β -glycyrrhetinic acid with an R,
value of about 01. The chromatogram obtained with solution (2) exhibits a
corresponding band but this is not seen in the chromatogram obtained with
solution (1). Spray the plate with anisaldehyde solution, using about 10 ml for a
plate 200 mm x 200 mm in size, heat at 1000 to 1050 for 10 minutes and examine
in daylight. The β - glycyrrhetinic acid bands become violet blue. One or two
bands with an Rf value of about 0.6, visible in daylight before spraying, become
orange-yellow and several other violet-blue bands appear in the chromatogram
obtained with solutions (1) and (2). The band corresponding to β- glycyrrhetinic
acid in the chromatogram is obtained with solution (3).
(b) Mix a small quantity, in powder, with 0.05 ml of sulphuric acid. The powder
particles become orange-yellow and some fragments change, more slowly, to
pinkish red.
Water-Soluble Extractive: Mix 2.5 g, in No. 180 powder, with 50 ml of water and
allow to stand for 2 hours, shaking frequently, filter, evaporate 10.0 g of the filtrate to
dryness on a water bath and dry the residue at 100 to 105. The residue weighs not less
than 0.1 g (20%).
Acid Insoluble Ash: Not more than 2.0%
Sulphated Ash: Not more than 10.0 %. Use 1 g, in powder.
Assay: Carry out the method for thin-layer chromatography, using silica gel GF254 as
the coating substance and as the mobile phase the upper layer, even if turbid, of a
mixture of 60 volumes of ethyl acetate, 27 volumes of 1M ammonia and 13 volumes of
61
absolute ethanol, shake together and allow to stand for 5 minutes. Apply separately and
quantitatively to the plate two 60 ul quantities of each of the following solutions as
bands 20 mm long and not more than 3 mm wide, but ensuring that part of the plate
remains free from the solutions being examined. For solution (1) mix 1 g, in No. 180
powder, with 25 ml of 1 M hydrochloric acid and 2.5 ml of 1,4-dioxane. Heat under
reflux condenser in a water bath for 2 hours, allow to cool, filter through a hardened
filter paper 9 cm in diameter and discard the filtrate. Rinse the flask and filter with five
20 ml quantities of water and discard the rinsing. Dry the flask and filter at 105 0 for 20
minutes, transfer the filter paper to the flask and add 50 ml of chloroform. Boil under a
reflux condenser in a water bath for 5 minutes and filter the warm chloroform solution
through a filter paper 9 cm in diameter. Repeat the extraction with two 25 ml quantities
of chloroform using the same filter each time. Transfer the filter paper to the flask,
extract with 25 ml of chloroform and filter through another filter paper 9 cm in diameter
Evaporate the combined filtrates to dryness, dissolve the residue in a mixture of equal
volumes of chloroform and methanol and transfer to 10 ml graduated flask. Rinse with
two 10 ml quantities of chloroform and evaporate the rinsing until 2 ml remains.
Transfer this solution to the graduated flask and dilute to 10 ml with a mixture of equal
volumes of chloroform and methanol. For solution (2) mix 50 mg of glycyrrhizinic acid
EPCRS with 25 ml of 1 M hydrochloric acid and 2.5 ml of 1,4-dioxan and proceed as
described for solution (1) beginning at the words 'Heat under a reflux condenser.
Develop the chromatogram twice, allowing the plate to dry in air after each
development. Examine the plate under ultraviolet light (254 nm) and mark the areas
corresponding to B-glycyrrhizinic acid in all four chromatograms. Carefully scrape off
the coating substance from the marked areas and treat each separately in the following
manner. Shake with 5 ml of absolute ethanol for 15 minutes and filter through a small
sintered-glass filter (BS porosity No. 4). Rinse the filter with absolute ethanol and dilute
the filtrate to 10 ml with the same solvent. Measure the absorbance of each of the four
solutions at 250 nm, using in the reference cell a solution prepared by treating in the
same manner an area of the corresponding in position and size to the marked areas of β
-glycyrrhizinic acid but taken from the part of the plate that has remained free from the
solutions being examined. Calculate the content of β -glycyrrhizinic acid from the
absorbance of solution (1) and (2) and the declared content of β-glycyrrhizinic acid in
β -glycyrrhizinic acid EPCRS. Storage Liquorice should be kept in airtight container
and protected from light.
Use: Flavour.
Licorice (USP 26, NF 21)

Licorice consists of the roots, rhizomes, and stolons of Glycyrrhiza glabra Linn or
Glycyrrhiza uralensis Fisher Family Leguminosae. It contains not less than 2.5 per cent
of glycyrrhizic acid (C42H62O16) calculated on the dried basis.
62
Packaging and Storage: Preserve in well-closed containers. Store in a cool, dry place.
Labelling: The label states the Latin binomial name and, following the official name,
the part of the plant contained in the article.
USP Reference Standards: USP Glycyrrhizic Acid RS.
Botanical Characteristics:
Macroscopic: The terrestrial stem is nearly cylindrical, 0.5 to 3.0 cm in diameter, and
over 1 m in length; it is extremely dark brown to red-brown and longitudinally wrinkled.
It often has lenticels, small buds and scaly leaves. The transverse section has a rather
clear border between the phloem and the xylem, and a radial structure that often has
radiating splits.
Microscopic: The transverse section reveals several yellow-brown cork layers, and a
layer of phelloderm that is 1 to 3 cells thick. The cortex exhibits medullary rays, and
obliterated sieve portions radiate alternatively. The phloem exhibits groups of phloem
fibres, which are surrounded by crystal cells, with thick but incompletely lignified walls.
The vessels are accompanied by xylem fibers, which are surrounded by crystals cells,
and by xylem parenchyma cells. The parenchyma cells contain starch grains and often
contains single crystals of calcium oxalate.
Thin-layer Chromatographic Identification Test:
Test Solution: Add 10 mL of a mixture of alcohol and water (7:3) to 2.0 g of pulverized
licorice, heat by shaking on a water bath for 5 minutes, cool, and filter.
Standard Solution: Dissolve 5 mg of USP Glycyrrhizic Acid RS in 1 mL of a mixture

of alcohol and water (7:3).
Application volume: 2 µL
Developing solvent system: A mixture of butyl alcohol, water, and glacial acetic acid
(7:2:1).
Procedure: Proceed as directed in the chapter, except to develop the chromatogram in

an unsaturated chamber to a length of about 10 cm. Examine the plate under UV light
at a wavelength of 254 nm. The chromatograms show a dark purple zone, among other
spot, due to glycyrrhizic acid at an R, value of about 0.4.
63
Loss on Drying: Dry it at 1050 for 6 hours: it loses not more than 12.0% of its weight.
Foreign Organic Matter: Not more than 2.0%.
Total Ash: Not more than 7.0%.
Acid-insoluble Ash: Not more than 2.0%.
Alcohol-soluble Extractives: Not less than 25.0%.
Pesticides Residues: Meets the requirements.
Heavy Metals: 0.003%.
Content of Glycyrrhizin Acid:

Solvent: A mixture of alcohol and water (1:1).
Mobile Phase: A filtered and degassed mixture of diluted acetic acid (1 in 15) and (3:2).
Standard Solution: Dissolve an accurately weighed quantity of USP Glycerrhizic Acid RS in
solvent to obtain a solution having a known concentration of about 0.25 mg per mL.
Test Solution: Transfer about 500 mg of licorice, reduced to a powder and accurately
weighed to a suitable flask, add 70 mL of solvent, shake for 14 minutes, centrifuge, and decant
the supernatant into a 100 mL volumetric flask. Mix the residue with 25 mL of solvent, shake
for 15 minutes, centrifuge, and add the supernatant to the volumetric flask Dilute with solvent
to volume, mix and pass through a membrane filter having a 0.45 um porosity.
Chromatographic System: The liquid chromatography is equipped with a 254 nm detector

and a 4.6 mm x 15 cm column that contains packing L1. The flow rate is about 0.6 mL per
minute. Chromatograph the standard solution, and record the peak areas as directed for
procedure: the column efficiency determined from glycyrrhizic acid is not less than 5000
theoretical plates; the tailing factor for the glycyrrhizic acid peak is not more than 2.0; and the
relative standard deviation for replicate injections is not more than 2.0%
Procedure: Separately inject equal volumes (about 20 µL) of the standard solution and the test
solution into a chromatograph, record the chromatograms, and measure the peak areas.
Calculate the percentage of glycyrrhizic acid (C42H62O16) in the portion of Licorice taken by the
formula: 10,000 (C/W) (ru/rs, where C is the concentration in mg per mL of USP Glycyrrhizic
acid RS in the standard solution; W is the weight, in mg, of licorice taken to prepare the test
solution; and r, and r, are the peak areas for glycyrrhizic acid obtained from the test solution
and the standard solution respectively
Aim: Monograph analysis of Black Pepper as per IP
Synonyms: Kali Mirchi
64
Biological source: Pepper consist of the unripe fruits of Piper nigrum Family –
Piperaceae It contains not less than 2-5% w/w of piperine calculated on dried basis.
Category: Anticonvulsant, bioavailability enhancer, appetizer, anthelmintic.
Description: Globular fruits (black-brown work), aromatic odour, pungent taste.
Identification
Morphology: The fruits are globule or a long 4-6 mm in diameter, one seeded.
Microscopy: pepper powder shows presence of tangential elongated oil glands endocarp has
beaker shaped stone cells abundant polyhedral masses of starch grains.
By TLC
Stationary phase: - Silica gel GF254
Mobile phase: - Benzene: Ethyl acetate: Di-ethyl ether (6:3:1)
Detection: - UV ,Dragendroff’s reagent
Test Solution: Methanolic extract
Standard: Methanol solution of pure piperine
Rf : The Rf value of standard (0.5) matches with Rf value of test sample.

Therefore, the given sample is of piperine.
TEST
Foreign organic matter- not more than 20%
1.Ethanol soluble extractive value
1 gm drug +25ml of water and keep for 24hrs. Filter and Evaporate the filtrate then
weigh the residue.
2.Water soluble extractive value
1 gm drug +25ml of water and keep for 24hrs. Filter and Evaporate the filtrate then
weigh the residue.
3.Total Ash
Incinerator 1g of powder till it is carbon free weigh and calculate the percent The total
ash value found to be
6.Acid Insoluble Ash
Dissolve total ash in 25ml of dil. HCI. filter through ashless filter paper ignite the paper
in crucible and calculate acid insoluble ash.
7.Loss on drying
Heat 5 g powder in oven till constant weight is achieved Initial weight =5gm,But after
drug drying=4.5gm, in oven at 1050C for 3 hours.
65
Subject code : BP609P HERBAL DRUG TECHNOLOGY(SemVI)
Experiment No: 7
Aim- To Determine Total Citral Content in Lemon Oil
Reference-
•Trease & Evans. “The Textbook of Pharmacognosy”,16 th Edition, pg-74.
•Dwivedi S. “Pharmacopoeal standards for Ayurvedic Formulation” (Council of
Research in Indian Medicine & Homeopathy) pg no -220
Requirements-
Apparatus/Glasswares- Measuring Cylinder, beaker, conical flask,
pipette, stirrer
Chemicals - potassium hydroxide, cinnamaldehyde, Potassium hydroxide,

ethanol
Introduction-
Citral is the best example of aldehyde monoterpenoids present in oil of
lemon, lemongrass, orange, lime and verbena of family Rutaceae.
Procedure:
•Take accurately weighed quantity of about 10 g of lemon oil in a stoppered tube.
Add 7 ml of hydroxylamine hydrochloride reagent and 0.1 ml of methyl orange
solution.
•Titrate the liberated acid with 0.5 N KOH till the red colour changes to permanent
yellow in the lower layer.
•Calculate the aldehyde content as follows: 1 ml of 0.5 N potassium hydroxide in
alcohol (60%) is equivalent to 0.07672 gm of citral.
To determine Total Cinnamadehyde Content in Cinnamon Oil

Cinnamaldehyde is the best example of aldehyde monoterpenoids present in oil of
camphora, cassia, cinnamon, tamala of family Lauraceae.
67
Procedure:
•Take 1g of cinnamon oil in conical flask. Add 5 ml hydroxylamine hydrochloride
reagent in 15 ml ethanol (60%) and 0.1 ml of methyl orange solution.
•Shake vigorously and titrate with 0.5 M KOH in ethanol (60%) until red colour
changes to full yellow.
•Continue the titration with vigorous shaking until lower layer retain full yellow colour
of indicator.
•Calculate the aldehyde content as follows: 1 ml of 0.5 N potassium hydroxide in
alcohol (60%) is equivalent to 0.0661gm of cinnamaldehyde.
Prerequisites
Standardization of potassium hydroxide solution:
Prepare 0.1 M HCl and take in burette. Take 20 ml prepared potassium hydroxide
solution in a conical flask. Add few drops of Phenolphthalein indicator and titrate it
hydrochloric acid solution.
Reagents:
•Hydroxylamine Hydrochloride Reagent in 60% Alcohol: Dissolve 3.475 g of
hydroxylamine hydrochloride in 95 ml 60% alcohol.
•Methyl orange indicator: 0.2% w/v solution of methyl orange in 60% alcohol •
•Potassium hydroxide solution: 0.5 N Potassium hydroxide solutions in 60% alcohol.
•Hydrochloric acid solution: 0.1 M Hydrochloric acid solution in water.
Result: The aldehyde content in essential oil was found to be ----- % in

lemon/cinnamon oil.
68
Experiment No: 8
Aim: To estimate total phenolic contents by folin-ciocalteu method
Reference-
•Trease & Evans. “The Textbook of Pharmacognosy”,16 th Edition, pg-66.
•Dr.Vyas N. “A Practical Book of Herbal Drug Technology”, Edition 18, Page No:
112
Requirements-
Apparatus/Glasswares- Beaker, Stirrer, measuring cylinder.
Chemicals- Folin-Ciocalteu reagent, Na2CO3
Principle:
The Folin-Ciocalteu reagent (FCR) or Folin's phenol reagent or Folin-Denis
reagent or Gallic Acid Equivalence method (GAE) uses a mixture of
phosphomolybdate and phosphotungstate for the colourimetric assay of phenolic and
polyphenolic antioxidants. It works by measuring the amount of the substance needed
to inhibit the oxidation of the reagent. However, this reagent does not only measure
total phenols but will react with any reducing substance. The reagent, therefore,
measures the total reducing capacity of a sample, not just the level of phenolic
compounds. This reagent forms part of the Lowry protein assay and will also react
with some nitrogen-containing compounds such as hydroxylamine and guanidine.
Reagents:
•Dilute Folin-Ciocalteu reagent with equal volume of distilled water,
•20% sodium carbonate in water and
•Gallic acid.
69
Procedure:
•Prepare calibration curve of standard Gallic acid (10-100 µg/ml in water).

•Prepare 1 milligram/ml of extract solutions.
•Mix 1 ml of each sample with 0.25 ml of Folin-Ciocalteu reagent and 1.25
sodium carbonate solution.
•Allow the mixture to react for 40 min. at room temperature.
•After the reaction period, mix the contents and measure the blue colour at 725
nm in comparison with standards. Calculate the amount of total phenols from
calibration curve as a Gallic acid equivalent by the following formula ;
T=CXV/M
Where,
T = Total content of phenolic compounds, (milligram per gram of plant extract),
C = The concentration of gallic acid established from the calibration curve

,(milligram per millilitre)
V = The volume of extract (millilitre),
M = The gram weight of plant extra
70
Experiment No: 9
Aim: To estimate total alkaloids in plant extracts
Reference-
•Trease & Evans. “The Textbook of Pharmacognosy”,16 th Edition, pg-25,26.
Requirement-
Apparatus/Glasswares- Beaker, Measuring Cylinder, test tube, pippette,
stirrer.
Chemicals- Atropine solution, chloroform, NaOH

Principle: Bromocresol green (BCG) reacts with alkaloids having nitrogen atom
within ring and forms yellow coloured complex which can be easily measured
colourimetrically. BCG doesn't reacts with alkaloids having nitrogen inside chain and
thus, this method is not useful to determine amine or amide alkaloids.
Reagents:
•BCG Solution (Dissolve 69. 8 mg BCG in 3 ml 2N NaOH and 5 ml distilled water.
Make up the volume up to 100 ml),
•Phosphate Buffer Solution (pH 4.7) and
•Standard Atropine Solution (Dissolve 1 mg atropine in 10 ml distilled water).
Procedure:
• Take0.4. 0.6, 0.8, 1.0 and 1.2 ml atropine solution in a separate test tube.
•Add 5 ml of Phosphate Buffer Solution (pH 4.7) and 5 ml of BCG solution.
•Shake well and extract the yellow coloured complex with chloroform.
•Separate chloroform and make up the volume to 10 ml.
•Measure the absorbance at 470 nm against blank.
71
•Now, prepare the methanolic extract of plant material. Dry and dissolve in 2N
HCI. Filter and wash with chloroform. Adjust the pH neutral with 0.1 N
NaOH.
•Now, add 5 ml of Phosphate Buffer Solution (pH 4.7) and 5 ml of BCG
solution.
•Shake well and extract the yellow coloured complex with chloroform.
•Separate chloroform and make up the volume to 10 ml and measure the
absorbance at 470 nm.
•Calculate concentration of total alkaloids from calibration curve of atropine
standard.
72

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Subject code : BP609P HERBAL DRUG TECHNOLOGY(Sem VI)

B-Pharm Sem- VI Manual

ACADEMIC YEAR 2021-22

Subject code : BP609P

Class: T.Y B.Pharm

Sub : HERBAL DRUG TECHNOLOGY

Topic : Practical Manual

Subject teacher : Dr.Manisha Vite

SR TOPIC PAGE DATE DATE OF SIGNATURE

Incorporation of prepared and

Monograph analysis of herbal

9 Determination of total alkaloids 15/03/2022

Aim - To perform preliminary phytochemical screening of crude drugs.

● General Test for Preliminary Phytochemical Screening

Test Observation Inference

Characteristic Senna, Digitalis, Nutmeg

General Test for Phytoconstituents

Test Procedure Observation

Molisch’s test Mix 1 ml reagent in 2 Red to violet ring depending

Fehling's test Mix 1 ml of Fehling's Yellow to red precipitate

Benedict’s test Mix 2 ml of Benedict's Formation of red, yellow

Barfoed's test Mix 2 ml of Barfoed's Brick-red precipitate of

Seliwanoff's test for Mix 2 ml of Deep red colour due to keto

Non-reducing sugars Perform Benedict's No characteristic colour

Osazone Test Add 0.5 g of Needle-shaped yellow

acetic acid. Boil in • Mushroom shaped

Biuret test Mix 2 ml test solution Violet to pink colour

Dragendorff’s reagent Mix 2 ml of reagent Reddish brown precipitate

Modified Dragendorff’s Mix 2 ml of reagent with Precipitate

Hager’s reagent Mix 2 ml of reagent with Yellow colour precipitate

Mayer’s reagent Mix 2 ml of reagent with Cream coloured precipitate

Wagner’s reagent Mix 2 ml of reagent with Reddish brown precipitate

Marme’s reagent Mix 2 ml of reagent with Precipitate

Sonnenschein reagent Mix 2 ml of reagent with Pale yellow to pink to

Bertrand’s reagent Mix 2 ml of reagent with Blue to purple colour

Scheibler’s reagent Mix 2 ml of reagent with Yellow to orange

Kraut’s reagent Mix 2 ml of reagent with Initially, the red-brown

Extract sample powder Acid hydrolyzes glycone

dry residue. Add

Legal’s test Mix 1 ml of test Pink or red colour

Orange to red colours

Noller's test Mix 2 ml test extract Pink colouration indicates the

Sannie test Mix 2 ml extract with Brown colour

CHCl3 and add 1ml fluorescence

P-nitroso dimethyl aniline Take little quantity of • Anthrones change

Modified anthraquinone Take little quantity of Rose pink colour of

Now, perform Fehling's

Ruthenium red test Treat the powder with Red colour

Potassium Brown-violet colour.

bromine water and a

Crystal test Rod shaped crystals

Nitric acid (HNO3) Violet colour changes to

Treat the dilute

Take the powder, add

with saturated solution

Van Urk's reagent Blue colour

Treat the solution with

Gentian Observe the powder Light blue fluorescence.

Froehde's reagent test Dark green colour.

Take the TS of Nux

Opium General test Dark reddish purple colour

Marquis reagent Red-bluish colour.

picrate at the neck of

CONFIRMATORY CHEMICAL TESTS FOR UNORGANIZED CRUDE