Lecture 4

• Chemical standardization methods
• General Methods of extractions
• General reactions for identification of
different chemical classes.
• Detection of different groups by TLC

chemical methods of standardization of
herbal drugs:
• It comprises of different chemical tests and assays. The
isolation, purification and identification of active
constituents are chemical methods of evaluation.
• Quantitative chemical tests such as acid value,
saponification value etc., are also covered under this
technique. Qualitative chemical tests are used in detection
of adulteration.
• The chemical evaluation also covers phytochemical
screening carried out for establishing chemical profile of a
drug.
• Toxic botanicals.

CHEMICAL EXAMINATION
• Detection of alkaloids
• Detection of carbohydrates and glycosides
• Detection of phytosterols
• Detection of fixed oils and fats
• Detection of saponins
• Detection of phenolic compounds and tannins
• Detection of protein and free amino acids
• Detection of gums and mucilage
• Detection of volatile oils

Extraction / Aim of Extraction:
• Is to separation medicinally active portions of plant
from the inactive or inert components by using
selective solvents in standard extraction procedures.
• The products so obtained from plants are relatively
impure liquids, semisolid. These include classes of
preparations known as decoctions, infusions, fluid
extracts, tinctures extracts and powdered extracts.

General Methods of Extraction of Medicinal
Plants
1.Maceration:
In this process, the whole or powdered crude drug is
placed in a container with the solvent and allowed to stand
at room temperature for a period of at least 3 days with
frequent shaking until the soluble matter has dissolved.
The mixture then is filtered, the marc (solid material) is
pressed.

Methods of Extraction of Medicinal Plants
 maceration
 infusion
 percolation
 digestion
 decoction
 hot continuous extraction (Soxhlet)
 aqueous-alcoholic extraction by fermentation
 counter-current extraction,
 microwave-assisted extraction,
 ultrasound extraction (sonication),
 supercritical fluid extraction,
 phytonic extraction (with hydrofluorocarbon solvents).

2.Infusion:
Fresh infusions are prepared by macerating the crude
drug for a short period of time with cold or boiling water.
These are dilute solutions of the readily soluble
constituents of crude drugs.
3.Decoction:
In this process, the crude drug is boiled in a specified
volume of water for a defined time; it is then cooled and
filtered. This procedure is suitable for extracting water-
soluble, heat-stable constituents.

Infusion & Decoction:
Sr.
No.
Infusion Decoction
1. Cold or boiling water is
used as menstruum.
Drug is boiled in water.
2. Drug having soft tissue is
used.
Drug having hard tissue is
used.
3. Drug constituents may be
volatile.
Drug constituents should be non
volatile.
4. Final volume is adjusted. Final volume is not adjusted.
5. When boiling water is used
as menstruum, precaution
are taken to prevent the
escape of heat by covering
the vessel with a cloth .
No such precaution is required.
11MANJUL P. SINGH

4.Digestion:
This is a form of maceration in which gentle heat is
used during the process of extraction.
It is used when moderately elevated temperature is
not objectionable. The solvent efficiency increased.

5.Percolation:
This is the procedure used most
frequently to extract active ingredients in
the preparation of tinctures and fluid
extracts.
A percolator is generally used. The solid
ingredients are moistened with an
appropriate amount of the specified
menstruum and allowed to stand for
approximately 4 hours in a well closed
container, Additional menstruum is added
and stand for 24 in the closed percolator.

6 . Hot Continuous Extraction (Soxhlet):
In this method, the finely ground crude drug is placed
in a porous bag made of strong filter paper, which is
placed in chamber E of the Soxhlet apparatus. The
extracting solvent in flask A is heated and its vapors
condense in condenser D.
The condensed extractant drips into the thimble
containing the crude drug, and extracts it by contact.
When the level of liquid in chamber E rises to the top
of siphon tube C, the liquid contents of chamber E
siphon into fl ask A. This process is continuous and is
carried out until a drop of solvent from the siphon
tube does not leave residue when evaporated.
The advantage of this method, compared to previously
described methods, is that large amounts of drug can
be extracted with a much smaller quantity of solvent.

General Reactions
for
Identification of Different chemical
classes

(1) Tests for Alkaloids:
1. Dragendroff’s test:
1 ml of extract, add 1 ml of Dragendroff’s reagent
(potassium bismuth iodide solution). An orange-red
precipitate indicates the presence of alkaloids.
2.Mayer’s test:
1 ml of extract, add 1 ml of Mayer’s reagent (potassium
mercuric iodide solution). Whitish or cream colored

3.Hager’s test:
1 ml of extract, add 3 ml of Hager’s reagent
(saturated aqueous solution of picric acid
4. Wagner’s test:
1 ml of extract, add 2 ml of Wagner’s reagent
(iodine in potassium iodide). Reddish brown colored

Tests for Glycosides:
Tests for free sugars:
The extract is hydrolyzed with mineral acid and then tested
for the glycone and aglycone moieties.
• Raymond’s test:
Test solution when treated with dinitrobenzene in hot
methanolic alkali, gives violet color.
• Legal’s test:
Treat the extract with pyridine and add alkaline sodium
nitroprusside solution, blood red color appears.
• Bromine water test
Test solution when treated with bromine water gives

Test for Saponin Glycosides:
• Froth Test:
Place 1ml solution of drug in water in a semi-micro tube
and shaken well and noted for a stable froth.
• Hemolysis test:
Add 0.2 ml solution of saponin (prepared in 1% normal
saline) to 0.2 ml of v/v blood in normal saline and mix
well, centrifuge and note the red supernatant compare
with control tube containing 0.2ml of 10% blood in
normal saline diluted with 0.2ml of normal saline.

Test for Anthraquinone Glycosides:
Borntrager's test:
• Boil the test material with 1ml of dilute sulphuric
acid in a test tube for 5min (anthracene
glycosides are hydrolyzed to aglycone and sugars by
boiling with acids)
• centrifuge or filter while hot, filtrate, cool and shake
with an equal volume of dichloromethane
the aglycones will dissolve preferably in
dichloromethane
• separate the lower dichloromethane layer and
shake with half its volume with dilute ammonia.

200mg of powdered herb with 4ml of
alcohol KOH for 2-3' in t.t. dilute with 4ml
of water and filter,
acidify with HCl. Cool, add few drops of
H2O2 and shake well with 5ml of ether,
and allow to separate into t.t.
and shake with 2ml of dilute solution of
ammonium hydroxide cherry-red
color in the aqueous.

Kedde’s test:
Extract the drug with chloroform, evaporate to dryness,
add one drop of 90% alcohol and 2 drops of 2% 3,5-dinitro
benzoic acid(3,5-dinitro benzene carboxylic acid -Kedde's
reagent) in 90% alcohol. Make alkaline with 20% sodium
hydroxide solution. A purple color is produced.
The color reaction with 3, 5-dinitrobenzoic acids depends
upon the presence of an β- unsaturated-o lactones in the
aglycone.

Keller killiani test [test for Deoxy sugars]:
Extract the drug with chloroform and evaporate it to
dryness. Add 0.4ml of glacial acetic acid containing a
trace amount of ferric chloride. Transfer to a small test
tube; add carefully 0.5ml of concentrated sulphuric acid
by the side of the test tube,

Tests for Flavanoids:
Shinoda test:
Dry powder/extract + 5ml 95% ethanol + few drops conc.
HCl + 0.5 g magnesium .
Lead acetate test:
Small quantity of extract + lead acetate solution

Sodium hydroxide test:
Plant extract + NaOH
Ferric chloride test:
2-3 ml of alcoholic extract + 5% Fecl3 Deep blue – black
colour Geletin test : 2-3 ml of alcoholic extract + Geletin
10% + NaOH (10%)

Test of Triterpenoids:
Liebermann -Burchard’s test:
2 mg of dry extract was dissolved in acetic anhydride,
heated to boiling, cooled and then 1 ml of concentrated
sulphuric acid was added along the sides of the test
tube.

Tests of Steroids
(a) Liebermann-Burchard’s test:
2 mg of dry extract was dissolved in acetic anhydride, heated to
boiling, cooled and then 1 ml of concentrated sulphuric acid was
added along the sides of the test tube.
Formation of green colour indicates the presence of steroids.
(b) Salkowski reaction:
2 mg of dry extract was shaken with chloroform, to the chloroform
layer sulphuric acid was added slowly by the sides of test tube.
Formation of red colour indicated the presence of steroids.

Test of Tannins
To 1-2 ml of the ethanolic extract, few drops of 5% w/v
FeCl3 solution was added.

Detection
of Different groups
by
Thin Layer Chromatography

(1) TLC of Alkaloid:
Solvent system:
Toluene-ethyl acetate-diethylnitrile (70:2O: 10), is suitable
for the major alkaloids of most drugs.
Stationary phase:
The principal alkaloids OF the most common alkaloid drugs
can be identified by Silica gel 60 F254 precoated TLC plates
Adsorbent.

Detection of Alkaloid:
UV-254nm some alkaloid types such as indoles,
quinolines, isoquinolines, purines.
UV-365 nm Blue, blue-green or violet
fluorescence of alkaloids, e.g: Boldo folium.
Yellow fluorescence, e.g. colchicine.

(2) TLC of Flavanoids:
Solvent System:
• Different solvent system can be used,
ethyl acetate-formic acid-glacial acetic acid-water(100-11-
11-26 v/v)
or formic acid - water – ethyl acetate mixed in different
proportion with or without ethyl methyl ketone are
suitable for the TLC screening of polar flavonoids
glycosides.
• For less polar flavonoids aglycones we would use a mobile
phase composed of:
Toluene-ethyl formiate -formic acid (50-40-10 v/v) or
Toluene- dioxane - glacial acetic acid(90-25-4 v/v).
Stationary phase: Silica gel, polyamide

Detection of Flavanoids:
• The solvent must be thoroughly removed from silica gel layer
before detection UV-254nm
• All flavonoids cause fluorescence. UV-365nm,Depending on
the structure type, flavonoids shows dark yellow, green or
blue fluorescence, which is intensified and changed by the
use of various spray reagent.
• Spray Reagents Fast blue salt reagent (FBS)-Detection of
phenolic compounds.
• Natural products reagents (NP/PEG) - The plate is sprayed
with 1% methanolic diphenylboric acid, β - ethylamino ester
(= diphenylboryloxyethylamine , NP), followed by 5%
ethanolic polyethylene glycone-4000spray.

(3) TLC of Anthracene Derivatives:
Solvent System:
Aloin, frangulin A/B, glucofrangulin A/B, rhein, aloe-
emodin and rliaponticoside are applied as 0.1% methanolic
solutions. Solutions Sennasides A and B are prepared as a
0.1% solution in methanol-water (1: 1). A total of 10 111 of
each reference solution is used for TLC.
Stationary Phase:
Chromatography is performed on silica gel 60 F254
precoated

Detection Anthracene Derivatives:
• UV 254 nm All anthracene derivatives quench
fluorescence.
• UV 365 nm All anthracene derivatives give yellow or red-
brown fluorescence.
Spray reagents:
Potassium hydroxide After spraying with 5% or 10%
ethanolic KOH, anthraquinones appear red in the visible
and show red fluorescence in UV-365 nm.

(4) TLC of Cardiac Glycoside:
Solvent System:
Ethyl acetate-methanol-water (100:13.5:10) solvents. A
generally applicable solvent system for cardiac glycosides
Ethyl acetate-methanol-ethanol-water (81 : 11 :4: 8).
The addition of ethanol increases the Rf values of strongly
polar compounds.
Stationary System:
Adsorbent Silica gel 60 F254 precoated.

Detection of Cardiac Glycoside :
Without chemical treatment UV-254 nm very weak
fluorescence quenching of all cardiac glycosides UV-365 nm no
fluorescence at all.
Spray reagents:
Specific detection of the y-lactone ring of cardenolides:
• Kedde reagent Immediately on spraying, cardenolides
generate a pink or blue-violet (vis) colour. The colour fades
after a few minutes, but can be regained by repeated spraying.
• Raymond reagent also give red, red-orange or violet (vis)
cardenolide-specifics colors.

(5) TLC of Coumarin:
Solvent System:
• For coumarin Aglycones solvent Toluene-ether (l:l,
saturated with 10% acetic acid)
• For glycosides Ethyl acetate-fortnic acid-glacial acetic acid-
water (100:11:11:26).
Stationary Phase:
Adsorbent Silica gel 60 F254 precoated TLC plates.

Detection of Coumarin :
• UV-254 nm distinct fluorescence quenching of all coumarins.
• UV-365 nm run intense fluorescence (simple
coumarins) yellow, brown, blue or blue-green fluorescence
(furano- and pyranocoumarins).
• The non-substituted coumarin fluoresces in UV-
365 nm only after treatment with KOH- reagent or ammonia
vapour.
Spray reagents:
The fluorescence of the coumarins are intensified by spraying
with Concentrated ammonia vapour has
the same effect.

(6) TLC of Saponin:
Solvent System:
The solvent which is suitable for separation of the saponin
mixtures:
• Chloroform - glacial acetic acid - methanol - water
(64:32:12:8) solvents.
• Chloroform-methanol-water (70:30:4) ginsenosides
(Ginseng radix).
Stationary Phase:
Adsorbent Silica gel 60 F254 precoated TLC plates.

Detection of Saponin:
• Without chemical treatment With the exception of
glycyrrhizin and glycyrrhetic acid (Liquiritiae radix), no
saponins are detectable by exposure to UV-254 or UV-365
nm.
Spray reagents:
Hemolytically active saponins are detected as
. Hemolysis may occur
immediately, after allowing the TLC plate to stand or after
drying the plate in a warm airstream.

(7) TLC of Triterpenes:
Solvent System:
Ethyl formate-toluene-formic acid (50:50: 15)
Toluene-chloroform-ethanol (40:40:10)
Stationary Phase:
Silica gel 60 F254 precoated plates

Detection of Triterpenes:
• UV-254 nm calfeic acid, its derivatives and isoflavones
show quenching.
• UV-365 nm caffeic acid, its derivatives and isoflavones
fluoresce blue.
Spray Reagents:
Anisaldehyde-sulphuric acid reagent Tile sprayed TLC is
heated for 6 min at 100°C; evaluation in vis.: triterpenes
blue-violet (Cimicifugae rhizoma) and red to red-violet
(Ononidis radix).

(8) TLC of Lignans:
Solvent System:
Chloroform-methanol-water (70:30:4)
Cloroform-metllanol( 90:lO)
Stationary Phase:
Silica gel 60 F254 -precoated plates

(9) TLC of essential oil:
Solvent System:
Toluene-ethyl acetate (93:7). This system is suitable for the
analysis and comparison of all important essential oils.
Stationary Phase:
Silica gel 60 F254 precoated TLC plates.

Detection of essential oil:
• Without chemical treatment UV-254nm Compounds
containing at least two conjugated doulble bonds quench
fluorescence and appear as dark zones against the light-
green fluorescent background of the TLC plate.
• UV-365 nm No characteristic Fluorescence of terpenoids
and propylphenols is noticed.
Spray reagents:
Anisaldehyde-sulphuric acid 10 min/110°C; evaluation in
vis.: essential oil compounds show strong blue, green, red
and brown colouration. Most of the compounds develop
fluorescence under UV-365 nm.

Lecture 4

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Lecture 4