I work on effect of plant seeds extraction on antibiotic resistance bacteria
Respected Sir,
We can follow the following procedure
Preparation of extracts for preliminary phytochemical screening
Hot maceration method using Soxhlet apparatus
Freshly collected plant materials were dried in shade and then coarsely powdered in a blender. 100 g 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.41 filter paper separately and all the extracts were subjected to qualitative tests for the identification of various phytochemical constituents as per the standard procedures (Brinda et al., 1981; Anonymous, 1990 and Lala, 1993). The extracts were concentrated in a rotary evaporator. The concentrated extracts were used for further analysis
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 presence 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 presence of anthroquinone.
Test for Catechin
To 2 ml the test solution, a few drops of Echrlich reagent and concentrate hydrochloric acid were added. Appearance of pink colour indicates the presence of catechin.
Test for Coumarin
To 2 ml of the 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)
To 2 ml 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 presence of flavonoid.
Test for Phenol
To 2 ml the test solution, a few drops of ferric chloride solution were added. Bluish green or red colour indicates the presence 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 indicates the presence of quinoidcompound.
Test for Saponin
The test solution was shaken with water. Copious lather formation indicates the presence of saponin.
Test for Steroid (Libermann- Burchard Test)
To 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 presence of steroid.
Test for Tannin
The test solution was mixed with basic lead acetate solution. Formation of a white precipitate indicates the presence 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.
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.
To 2 ml 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.
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 powder/extract was pressed between the filter papers. Formation of grease spot indicates the presence of fixed oils and fats.
Anonymous. 1990. Phytochemical investigation of certain medicinal plants used in Ayurveda. Central Council for Research in Ayurveda and Siddha, New Delhi.
Lala PK: Lab manuals of Pharmacognosy CSI Publishers and Distributers, Kolkata. 1993.
Brinda, P., Sasikala, P. and Purushothaman, K.K. 1981. Pharmacognostic studies on Merugan kizhangu. Bull. Med. Ethnobot. Res.3: 84-96.
Good Luck
Dear Ali
First you to go for possiblr extraction and next follow the methods of Harnborne or Frnasworth or Kokate for phytochemical screeniang for analysis of phytoconstituents
Respected Sir,
We can follow the following procedure
Preparation of extracts for preliminary phytochemical screening
Hot maceration method using Soxhlet apparatus
Freshly collected plant materials were dried in shade and then coarsely powdered in a blender. 100 g 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.41 filter paper separately and all the extracts were subjected to qualitative tests for the identification of various phytochemical constituents as per the standard procedures (Brinda et al., 1981; Anonymous, 1990 and Lala, 1993). The extracts were concentrated in a rotary evaporator. The concentrated extracts were used for further analysis
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 presence 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 presence of anthroquinone.
Test for Catechin
To 2 ml the test solution, a few drops of Echrlich reagent and concentrate hydrochloric acid were added. Appearance of pink colour indicates the presence of catechin.
Test for Coumarin
To 2 ml of the 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)
To 2 ml 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 presence of flavonoid.
Test for Phenol
To 2 ml the test solution, a few drops of ferric chloride solution were added. Bluish green or red colour indicates the presence 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 indicates the presence of quinoidcompound.
Test for Saponin
The test solution was shaken with water. Copious lather formation indicates the presence of saponin.
Test for Steroid (Libermann- Burchard Test)
To 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 presence of steroid.
Test for Tannin
The test solution was mixed with basic lead acetate solution. Formation of a white precipitate indicates the presence 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.
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.
To 2 ml 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.
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 powder/extract was pressed between the filter papers. Formation of grease spot indicates the presence of fixed oils and fats.
Anonymous. 1990. Phytochemical investigation of certain medicinal plants used in Ayurveda. Central Council for Research in Ayurveda and Siddha, New Delhi.
Lala PK: Lab manuals of Pharmacognosy CSI Publishers and Distributers, Kolkata. 1993.
Brinda, P., Sasikala, P. and Purushothaman, K.K. 1981. Pharmacognostic studies on Merugan kizhangu. Bull. Med. Ethnobot. Res.3: 84-96.
Good Luck
Seeds always rich in fatty acid, terpenoids and steroids. So, please go for specific test using modern intstumetal method.
Dear Ali
You can also do phytochemical test with out soxhlet by - aproximately 2g of seed powder sample was weighed and dissolved in different solvents like Hexane, petroleum ether, methanol, chloroform and water separately and was allowed to stay overnight for 24 hours. After overnight incubation the sample was filtered by Whattman filter paper, the filtrate was used for Phytochemical screening.
Dear Mr Khaled,
Screening is basically an identification method that give you the answer of positive or negative to indicate presence of the compound that you screen for. Simple screening method as mentioned by Dr Tresina is adequate for the purpose.
Best of Luck.
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