Phytochemical Screening

Phytochemical screening of W. somnifera leaves extract was carried out using standard
procedures provided by (Edeoga et al. 2005, Pradeep et al. 2014 and Rajasudha and Manikandan
2019).

Edeoga, H., Okwu, D. and Mbaebie, B. (2005). Phytochemical constituents of some Nigerian
medicinal plants. African Journal of Biotechnology, 4(7): 685-688.

Pradeep A, Dinesh M, Govindaraj A, Vinothkumar D, Ramesh Babu NG (2014) Phytochemical

Analysis Of Some Important Medicinal Plants. International Journal of Biological &
Pharmaceutical Research, 5(1): 48-50.

Rajasudha V and Manikandan R (2019) Phytochemical screening and High-performance liquid

chromatography (HPLC) profile of different extracts of Euphorbia hirta (Linn). Journal of
Pharmacognosy and Phytochemistry, 8(1): 45-50

2.4.1. Test for Alkaloids (Wagner’s Test): The extract was treated with few drops of Wagner’s
reagent (0.5 g of iodine and 1.5 g of potassium iodide were dissolved in 5 ml of distilled water
and the solution was diluted to 20 ml with water) and formation of reddish brown precipitate
indicates the presence of alkaloids.
2.4.2. Test for Terpenoids (Salkowki's test): Extract was dissolved in 2 ml of chloroform and
treated with 2 ml of concentrated sulphuric acid to form a layer. Reddish brown colouration at
the interface indicates presence of terpenoids.

2.4.3. Test for Flavonoids (Alkaline reagent test): Extract was treated with few drops of 20%
sodium hydroxide solution. Formation of an intense yellow color, which turns colorless on
addition of dilute hydrochloric acid, indicates the presence of flavonoids.

2.4.4. Test for Sterols (Liebermann-Burchard’s test): Extract was diluted with chloroform and
filtered. Few drops of acetic anhydride was added to the filtrate and boiled. The cooled filtrate
was treated with few drops of concentrated sulphuric acid. Formation of a brown ring at the
junction indicates the presence of phytosterols.
2.4.5. Test for Phenols (Ferric chloride test): Extract was treated with 3-4 drops of 5% ferric
chloride solution. Formation of bluish black color indicates the presence of phenols.

2.4.6. Test for Saponins (Foam test): The extract was diluted with distilled water and warmed
on water bath. Development of persevering forth affirms the vicinity of saponins. Further the
frothing was mixed with 3 drops of olive oil and then shaken vigorously. Formation of emulsion
indicates the presence of saponins.

2.4.7. Test for Tannins (Braymer's test): Extract was boiled with 20 ml water and then filtered.
The filtrate was treated with 10% alcoholic ferric chloride solution and formation of brownish
green to blue-black color indicates presence of tannins.

2.4.8. Test for Cardiac glycosides (Keller Kelliani's test): The extract was dissolved with 4 ml
of distilled water and then treated with 2 ml of glacial acetic acid containing few drops of ferric
chloride solution. Further, 2 ml of concentrated sulphuric acid was added carefully without
mixing the solution. Formation of a brown ring at the interface indicates the presence of
deoxysugars (characterisitics of cardenolides).
2.4.9. Test for Carbohydrates (Benedict’s test): Extract was treated with Benedict’s reagent
(cupric citrate complex) and heated gently. Orange-red/brick red/rusty brown precipitate
indicates presence of reducing sugars.
2.4.10. Test for Proteins (Biuret test): Extract was dissolved in 4 ml of distilled water and then
treated with an equal volume of 1% sodium hydroxide solution followed by 3 drops of aqueous
copper II sulphate solution. A color change from blue to purple/violet indicates presence of
proteins.
2.4.11. Test for Quinones: Extract was treated with concentrated HCl and formation of yellow
coloration or precipitate indicates the presence of quinones.

2.4.12. Test for Thiosulphates: Aqueous extract was treated with few drops of 20% barium
chloride solution. Formation of white precipitate (barium sulphate) indicates presence of
thiosulphates.
Table 1. Qualitative analysis of the phytochemicals found in leaf extract of Withania somnifera.

Chemical constituents Results

Alkaloids +++

Terpenoids -

Flavonoids +

Sterols +++

Phenols +++

Saponins ++

Tannins ++

Cardiac glycosides +++

Carbohydrates ++

Proteins -

Quinones +

Key: +++ strong presence, ++ moderate presence, +slight presence, - total absence