Phytochemical Investigations
CHAPTER-IV- QUALITATIVE PHYTOCHEMICAL INVESTIGATIONS
4.1. INTRODUCTION
Phytochemistry is the branch of chemistry deals with the chemical nature of the
plant or plant products (chemistry of natural products). Plants contain many chemical
constituents which are therapeutically active or inactive. A spectrum of natural
compounds like triterpenoids, alkaloids, glycosides, tannins, flavonoids, essential oils and
other similar secondary metabolites which exert physiological activities are synthesized
in the plant, in addition to the carbohydrates, proteins and lipids utilized by man as food
material1.
A systematic and complete study of crude drugs should include a thorough
investigation of both primary and secondary metabolites derived as a result of plant
metabolism2. The qualitative chemical tests are to be performed for establishing profile
of a given extract for its nature of chemical composition.
4.2 EXPERIMENTAL METHODS
The selected plant extracts were subjected to quantitative chemical investigation
to test for the presence of various phytochemicals in extracts3.
4.2.1 Test for carbohydrates
• Molisch’s Test: To 2ml of extract 2-3 drops of alpha naphthalene solution in
alcohol was added, shaken for 2 min and 1 ml of concentrated sulphuric acid was
added slowly from the sides of the test tube. A deep violet colour at the junction
of two layers indicates the presence of carbohydrates.
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Test for reducing sugars
• Fehling’s Test: Fehling’s A and Fehling’s B solutions, each 1ml were mixed and
boiled, for 1ml and 2 ml of extracts were added heated in boiling water bath for
10 min, appearance of yellow and then brick red precipitate indicates the presence
of reducing sugars.
• Benedict’s Test: Equal volume (2ml each) of Benedict’s solution and extracts
were mixed in a test tube and heated in boiling water bath for 10min the changes
in colour to yellow, green and red indicates the presence of reducing sugars.
Test For monosaccharide
• Barfoed’s Test: Equal volume (2ml each) of Barfoed’s reagent and extract were
mixed in a test tube heated in boiling water bath for 2 min and cooled. The
appearance of red precipitate show the presence of monosaccharides.
Test for pentose sugar
• Bial’s Test: To 1 ml of boiling bial’s reagent, 1ml solution of extract were added.
The appearance of green or purple colour shows the presence of pentose sugar.
Test for Hexose Sugars
• Seliwanoff’s Test: To 3 ml of seliwanoff reagent 1ml of plant extract was added
and heated on water bath for 2 min. The change in colour to red indicates the
presence of hexose sugar.
• Cobalt chloride Test: To 3 ml of extract 2 ml of cobalt chloride solution was
added, boil or 2 min and cooled. To this 2-3 drops of sodium hydroxide solution
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was added and observed for the change in colour. The appearance of greenish
blue and purplish colour are upper layer greenish blue and lower layer purplish
indicates the presence of glucose, fructose or mixture glucose and fructose
respectively.
4.2.2 Test for proteins
• Biuret’s Test: To 3 ml of extract 1 ml of 4% w/v sodium hydroxide and 1ml of
1% w/v copper sulphate were added . The change in colour of the solution to
violet or pink indicates the presence of proteins.
• Millon’s Test: To 3 ml extract 5 ml of millon’s reagent was added and heated the
appearance of white precipitate which changed to brick red on heating indicates
the presence of proteins.
• Xanthoprotein Test: To 3 ml of extract 1ml of concentrated sulphuric acid was
added. The appearance of white precipitate which turn to yellow on boiling and
orange on addition of ammonium hydroxide (1ml) indicates the presence of
proteins containing tyrosine tryptophan.
• Ninhydrin Test: To 3 ml extract 3 drops of 5% v/w lead acetate solution was
added and boiled on water bath for 10 min. The change in colour of solution to
purple or blue indicates the presence of amino acids.
4.2.3 Test For Tannins and Phenols
• Ferric chloride Test: To 3 ml of extract, 3 ml of 5% w/v ferric chloride solution
was added. The blue – black colour indicates the presence of tannins and phenols.
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Phytochemical Investigations
• Lead acetate Test: To 3ml of extract, 3 ml of lead acetate solution was added.
The occurrence of white precipitates indicates the presence of tannins and
phenols.
4.2.4 Test For Glycosides
• Legal test: To 2ml of the extract, 1ml of pyridine and 1 ml of sodium nitro
prusside were added. The change in colour pink or red indicates the presence of
cardiac glycosides
• Keller-killiani test: To the test tubes containing 2 ml of extract 1 ml of glacial
acetic acid, 3 drops 5% W/V ferric chloride and concentrated sulphuric acid were
added and observed, disappearance of reddish brown colour at the junction of
two layers and bluish green in upper layer indicates the presence of cardiac
glycosides.
• Borntrager’s Test: To the test tubes containing 2 ml of extract 2 ml of dilute
sulphuric acid was added, boil for 5 min and filtered. To the filtrates, equal
volumes of chloroform was added and mixed well. organic layers were separated
and ammonia was added to this. Pinkish red colour of the ammonia layer
indicated the presence of anthraquinone glycosides.
4.2.5 Test For Saponins
• Foam Test: The extract (2g) was shaken vigorously with 20 ml of water and
observed for persistent foam, which indicates the presence of saponins.
• Haemolysis Test: Sodium chloride( 18% v/w, 2 ml) was shaken in six test tubes.
The three test tubes 2 ml of chloroform, methanol and ethanol- water (8:2) were
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added, to the other three test tubes 2 ml of extract were added, few drops of blood
was added to all the test tubes, mixed and observed for the haemolysis under
microscope.
4.2.6 Test for flavonoids
• Shinoda Test: To the dry extract (2g), 5 ml of ethanol (95% v/v), 5 drops of
hydrochloric acid and 0.5g of magnesium turnings were added. Appearance of
pinkcolour indicates the presence of flavonoids.
4.2.7 Test for Triterpenoids
• Salkowaski Test : To 2 ml of extract 5 drops of concentrated sulphuric acid was
added, shaken and allowed to stand. Appearance of greenish blue colour indicates
the presence of triterpenoids.
• Libermann burchard Test: To 2 ml of the test solution, 10 drops of acetic
anhydride was added and mixed well. To this 5 ml of concentrated sulphuric acid
was added from the sides of the test tube, appearance of greenish blue colour
indicates the presence of triterpenoids.
4.2.8 Test for Alkaloids
To the 10 g of dry extracts, 20 ml of dilute hydrochloric acid was added, shaken well
and filtered. The following tests were performed using the filtrate.
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• Mayer’s Test: To 3 ml of the filtrates, 1ml of Mayer’s reagent (potassium
mercuric iodide) was added. The appearance of white precipitate indicates the
presence of alkaloids.
• Wagner’s Test: To 3 ml of filtrate, 1ml of Wagner’s reagent (iodine in potassium
iodide) was added. The appearance of reddish brown precipitate indicates the
presence of alkaloids.
• Hager’s Test: To 3 ml of filtrate, 1ml of Hager’s reagent (saturated picric acid
solution) was added. The appearance of yellow precipitate indicates the presence
of alkaloids.
• Dragendroff’s Test: To 3ml of the filtrate, 1ml of Dragendroff’s reagent
(potassium bismuth iodide) was added. The appearance of brick red precipitate
indicates the presence of alkaloids.
4.2.9 Test for lipids:
To 10 ml of extract 0.5N alcoholic potassium hydroxide was added along with a
drop of phenolphthalein. This mixture was heated on water bath for 1 h and observed
for the formation of soap or partial neutralisation of alkali.
4.3 RESULTS AND DISCUSSION:
Qualitative phytochemical screening of three selected plant extracts are shown in
Table :4.01
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Phytochemical Investigations
From the results of qualitative phytochemical screening of hexane extract of
[Link] Linn leaf extract it was found to contain phytosterol, glycosides,
phenolic compounds.
Ethyl acetate extract of [Link] Linn leaf extracts revealed the presence
of phytosterols, triterpenoids, glycosides, flavonoids, alkaloids, phenolic compounds.
Ethanolic extract of [Link] Linn leaf extract revealed the presence of
glycosides, saponins, flavonoids, tannins, carbohydrates and phenolics.
Hexane extract of [Link] Linn leaf extract revealed the presence of
phytosterols, flavonids.
Ethyl acetate extract of [Link] Linn leaf extract revealed the presence of
glycosides, flavonoids, alkaloids, phenolic compounds.
Ethanolic extract of [Link] Linn leaf extract revealed the presence of
glycosides,flavonoids,saponins, tannins, carbohydrates, phenolic compounds.
Hexane extract of [Link] Linn leaf extract revealed the presence of
triterpenoids, phenolic compounds.
Ethyl acetate extract of [Link] Linn leaf extract revealed the presence of
glycosides, flavonoids, tannins, carbohydrates, alkaloids, phenolic compounds.
Ethanolic extract of [Link] Linn leaf extract revealed the presence of
glycosides, alkaloids, flavonoids, saponins, tannins, carbohydrates, phenolic
compounds.
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Phytochemical Investigations
Table: 4.01: Nature of phytoconstituents present in Annona squamosa Linn,
Annona reticulata Linn, Annona muricata Linn leaf extracts
Tests [Link] [Link] [Link]
H.E E.A.E E.E H.E E.A.E E.E H.E E.A.E E.E
Phytosterols + + - + - - - - -
Triterpenoids - + - - - - + - -
Glycosides + + + - + + - + -
Saponins - - + - - + - - -
Flavonoids - + + + + + - + +
Tannins - - + - - + - + +
Carbohydrates - - + - - + - + +
Alkaloids - + - - + - - + +
Phenolic compounds + + + - + + + + +
+=presence , - = absence
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Phytochemical Investigations
4.4 REFERENCES:
1. Kokate C K, Purohit A P and Gokhale S B . Pharmacognosy, 20th Edn , Nirali
prakashan, Pune. 2002:108-109.
2. Sofawora E A. Medicinal plants and Traditional medicine in Africa, Wiley
Chichester. 1982: 256.
3. Tona L. Anti amoebic and phytochemical screening of some congolese
medicinal plants. J Ethano pharmacol; 61:57-65.
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