This document describes methods for conducting phytochemical screening tests on plant extracts to identify their chemical constituents. It discusses extracting plants using a Soxhlet apparatus with various solvents and then testing the extracts using several chemical tests to detect the presence of phytochemicals like alkaloids, anthraquinones, catechins, coumarins, flavonoids, phenols, quinones, saponins, steroids, tannins, terpenoids, sugars, glycosides, xanthoproteins and fixed oils. The objective is to determine the primary phytochemicals responsible for the biological activities of herbal medicines.

1. Phytochemical Screening Tests
By
Halavath Ramesh
Research Scholar
Department of Chemistry
Loyola College-Chennai
University of Madras
E-mail:halavathramesh39@gmail.com

2. Introduction
• Phytochemicals are chemical compounds produced by plants.
• Challenges in the field phytochemistry including isolating specific compounds and
determining their structure and identifying what specific Phytochemical is primarily
response in biological activity.
• In India Herbal medicines play an important role in the treatment of various diseases by
trational methods practiced such as Ayurveda , Unani and Siddha.
• Over 2,48,000 species of the higher plants have been identified 12,000 plants are known to
have medicinal properties.
• The knowledge of pharmacology in essential for understanding action of drugs on animals
and human system.
• The specific natural products of the primary and secondary metabolites are very important
for chemists.

3. Hot maceration method using Soxhlet apparatus
• Freshly collected plant materials were dried in shade and then
coarsly powdered in a blender.
• 100g of the coarse powder was extracted successively with
250 ml of various solvents in a soxhlet apparatus for 24 h. All
the extracts were filtered through whatman No.41filter paper
separately and all the extracts were subjected to qualitative
tests for the identification of various phytochemical
constituents as per the standard procedures.
• The extract were concentrated in a rotary evaporator. The
concentrated extracts were used for further analysis.

4. Preliminary Phytochemical Screening of different extracts
The chemical tests for various phytoconstituents present in the extracts were
carried out as described below :
Test for Alkaloid:
The test solution was mixed with little amount of dilute hydrochloric acid and
Mayer’s reagent. Formation of a white precipitate indicates the presences of
alkaloids.
Test for Anthraquinone(Borntrager’s Test):
A few drops of magnesium acetate solution were added to the test solution. Pink
colour formation shows the presences of anthroquinone.

5. Test for Catechin:
Take 2ml of test solution, a few drops of Echrlich reagent and
concentrate hydrochloric acid were added. Appearance of pink colour
indicates the presences of catechin.
Test for Coumarin:
Take 2ml of test solution, a few drops of alcoholic sodium hydroxide
were added.Appearance of yellow colour indicates the presence of
coumarin.
Test for Flavonoid(Shindo’s test):
Take 2 ml of the test solution, a few magnesium turnings and a few
drops of concentrate hydrochloric acid were added and boiled for 5
minutes. Appearance of red or orange red colour indicates the
presences of flavonoid.

6. Test for Phenol:
Take 2 ml of the test solution, a few drops of ferric chloride solution were added.Bluish
green or red colour indicate the presences of phenol.
Test for Quinone:
The test solution was treated with a few drops of concentrate sulphuric acid or
aqueous sodium hydroxide solution.Colour formation indicate the presence of
quinoid compound.
Test for Saponin:
The test solution was shaken with water. Copious lather formation indicates the
presences of saponin.

7. Test for Steroid(Libermann-Burchard Test):
Take 2 ml of the test solution , a few drops of chloroform,3-4 drops of acetic anhydride
and one drop of concentrate sulphuric acid were added. Appearance of purple colour,
which changes to blue or green colour , shows the presences of steroid.
Test for Tannin:
The test solution was mixed with basic lead acetate solution.Formation of a white
precipitate indicates the presences of tannin.
Test for Terpenoid(Noller’s Test):
The Test solution was warmed with a piece of tin and a few drops of thionyl
chloride.Violet or purple colouration indicates the presence of terpenoid.

8. Test for Sugar:
The test solution was mixed with equal volumes of Fehling’s solution A and Fehling’s
solution B and heated. Formation of red colour is the indication of the presence of sugars.
Take 2 ml of the test solution, a very small quantity of anthrone and few drops of
concentrate sulphuric acid were added and heated. Green to purple colouration indicates
the presence of sugars.
Test for Glycoside:
The extract was mixed with a little anthrone on a watch glass.One drop of concentrate
sulphuric acid was added and made into a paste and warmed gently over the water
bath.Dark green colouration indicates the presence of glycosides.

9. Test for Xanthoprotein:
To the test solution, a few drops of concentrate nitric acid and few ml of ammonia were
added. Appearance of a red precipitate indicates the presence of xanthoprotein.
Test for Fixed oil(Spot test):
A small quantity of power/extract was pressed between the filter papers. Formation of
grease spot indicate the presence of fixed oils and fats.

11. Bibliography:
1. Anonymous.1990.Phytochemical investigation of certain medicinal plants
used in Ayurveda.Central Council for Research in Ayurveda and siddha, New
Delhi.
2. Lala PK: Lab manuals of Pharmacognosy CSI Publishers and
Distributers,kolkata,1993.
3. Brinda,P., Sasikala,P. And Purushothaman,K.K. 1981.Pharmacognostic
studies on merugan kizhangu.Bull.Med.Ethnobot.Res.3:84-96.