Screening of Phytochemicals in a crude plant extract

Detection of Flavonoids

 1. Ferric chloride test : Add few drops of neutral ferric chloride solution to 1 mL of an alcoholic solution of a crude extract (dissolve a little part of crude extract in ethyl alcohol and filter) using a test tube. Formation of blackish red colour indicates the presence of flavonoids.

2. Shinoda’s test : To 1 mL of an alcoholic solution of the crude extract, add a small piece of magnesium ribbon or magnesium foil and few drops of concentrated HCl using a test tube. Change in colour from red to pink shows the presence of flavonoids.

3. Zinc-HCl reduction test : Add a pinch of zinc dust and few drops of concentrated HCl to 1 mL of an alcoholic solution of crude extract using a test tube. The appearance of magenta colour indicates the presence of flavonoids.

 4. Lead-acetate test : Add few drops of aqueous basic lead acetate solution to 1 mL of alcoholic extract using a test tube. The appearance of a reddish brown bulky precipitate indicates the presence of flavonoids.

Detection of Steroids

 1. Salkowski test : Add 1 mL of concentrated sulphuric acid to 5 mL of a chloroform solution of crude extract (dissolve a little part of crude extract in chloroform and filter) using a test tube. After shaking, the test tube allows standing for 5 min. Lower layer turning into red in colour indicates the presence of steroids.

 2. Liebermann-Burchard test : Add few drops of acetic anhydride to 5 mL of a chloroform solution of crude extract followed by adding 1 mL of concentrated sulphuric acid through the wall of the test tube and allow to stand for 5 min. Formation of a reddish brown ring at the junction of the two layers and a green colour in upper layer indicate the presence of steroids.

Detection of Terpenoids

Fresh plant material is treated with 5 mL of 1% aqueous hydrochloric acid for about 4 h. The extract thus obtained is treated with 1 mL of Trim-Hill reagent (10 mL of acetic acid, 1 mL of 0.2% copper sulphate in water and 0.5 mL of concentrated hydrochloric acid) in a test tube followed by heating in a water bath. The appearance of a blue colour indicates the presence of diterpenoids while green colour indicates the presence of monoterpenoids.

Detection of Tannins

 1. Ferric chloride test : Add few drops of ferric chloride solution to 1 mL of aqueous solution of crude extract (dissolve a little part of the crude extract in 1 mL of distilled water and filter) using a test tube. A blackish precipitate indicates the presence of tannins.

 2. Gelatin test : Add gelatin (gelatin dissolves in warm water immediately) solution to 1 mL of aqueous solution of crude extract using a test tube. Formation of white precipitate indicates the presence of tannins.

 3. Lead acetate test : Add few drops of aqueous basic lead acetate solution to 1 mL of aqueous solution of crude extract using a test tube. Reddish-brown bulky precipitate indicates the presence of tannins.

Detection of Glycosides

 1. Kellar Kiliani test : Add 1 mL of aqueous solution of crude extract (dissolve a little part of the crude extract in 1 mL of distilled water and filter) to 1 mL of glacial acetic acid using a test tube. After cooling, add few drops of ferric chloride solution to it. Transfer these contents to a test tube containing 2 mL of concentrated sulphuric acid. Formation of a reddish brown ring at the junction of two layers shows the presence of glycosides.

 2. Sulphuric acid test : Add 1 mL of aqueous solution of crude extract to 1 mL of concentrated sulphuric acid and allow to stand for 2 min. The formation of reddish colour indicates the presence of glycosides.

3. Molisch’s test : Add a mixture of Molisch’s reagent (alpha-naphthol dissolved in ethanol) and concentrated sulphuric acid (1:1) to was added to 1 mL of aqueous solution of crude extract. Formation of the reddish-violet ring at the junction of two liquids indicates the presence of glycosides.

Detection of Saponins

Mix a little part of the crude extract with 20 mL of distilled water and then agitate in a graduated cylinder (test tube also can be used) for 10 min. Foam formation indicates the presence of saponins.

Detection of Alkaloids

1. Mayer’s test : Add 1 mL of Mayer’s reagent (potassium mercuric iodide solution) to 1 mL of an acidic solution of crude extract (dissolved a little part of crude extract in 5 mL chloroform; add diluted hydrochloric acid or diluted sulphuric acid to the chloroform solution and the acid layer is used). Cream colour precipitate indicates the presence of alkaloids.

 2. Wagner’s test : Add 1 mL of Wagner’s reagent (iodine in potassium iodide) to the 1 mL of an acidic solution of crude extract. The formation of reddish-brown precipitate indicates the presence of alkaloids.

 3. Dragendorff’s reagent test : Add 2 mL of Dragendorff’s reagent (a solution of potassium bismuth iodide prepared from basic bismuth nitrate, tartaric acid, and potassium iodide) and 2 mL of diluted hydrochloric acid to the acidic solution of crude extract. An orange-red colour precipitate indicates the presence of alkaloids.

Detection of Phenols

 1. Ferric chloride test : Add 1 mL of ferric chloride solution to 5 mL of an aqueous/ alcoholic solution of crude extract (dissolve little part of crude extract in 5 mL of water or ethyl alcohol and filter) using a test tube. Formation of an intense colour indicates the presence of phenols.

 2. Ellagic acid test : Add few drops of 5% glacial acetic acid and 5% sodium nitrite solution to 2 mL of an aqueous/ alcoholic solution of crude extract using a test tube. Formation of muddy or brown precipitate indicates the presence of phenols.

Detection of Quinones

Add 5 mL of an aqueous/ alcoholic solution of crude extract (dissolve little part of crude extract in 5 mL of water or ethyl alcohol and filter) to 5 mL of alcoholic potassium hydroxide solution. The appearance of a range of red to blue coloration indicates the presence of quinones.

Detection of Lignin

 1. Labat test : Add 5 mL of an aqueous/ alcoholic solution of crude extract (dissolve little part of crude extract in 5 mL of water or ethyl alcohol and filter) to 1 mL of gallic acid. Development of olive green colour indicates the presence of lignins.

 2. Lignin test : Add 5 mL of an aqueous/ alcoholic solution of crude extract to 1 mL of 2% furfuraldehyde using a test tube. Formation of red colour indicates the presence of lignin.

 Detection of Coumarins

One g of dry plant powder is placed in a test tube in the presence of a few drops of distilled water. The test tube is covered with paper soaked in diluted sodium hydroxide solution and boiled. Evolution of yellow fluorescence indicates the presence of coumarins after examination under UV light.

Estimation of total phenol content

Folin-Ciocalteu method is widely used to estimate the total phenol content in a plant extract. In this method, a methanolic solution of the extract (1 mg/ml) is added to 2.5 ml of 10% Folin-Ciocalteu reagent dissolved in water and 2.5 ml 7.5% Na₂CO₃ or NaHCO3. Blank is similarly prepared which contains 0.5 ml methanol, 2.5 ml 10% Folin-Ciocalteu reagent dissolved in water and 2.5 ml of 7.5% of Na₂CO₃ or NaHCO3. Thereafter, the samples are incubated in a thermostat at 45 °C for 45 min. The absorbance is determined using a spectrophotometer at λmax 765 nm. The samples should be prepared in triplicate for each analysis so that a mean value of absorbance can be obtained. The same procedure should be repeated for the standard solution of gallic acid and the calibration line is construed. Based on the measured absorbance, the concentration of phenolics is read (mg/ml) from the calibration line. Thereafter, the total phenol content in a plant extracts is expressed in terms of gallic acid equivalent (mg of GA/g of extract).

Estimation of total flavonoid content

The aluminium chloride colorimetric assay is used to estimate total flavonoid content in an extract. In this method, the sample contained 1 ml of a methanol solution of the extract in the concentration of 1 mg/ml and 1 ml of 2% AlCl3 solution is dissolved in methanol. The samples are incubated for 1 h at 25 °C. The absorbance is determined using spectrophotometer at λmax 415 nm. The samples should be prepared in triplicate for each analysis so that a mean value of absorbance can be obtained. The same procedure is repeated for the standard solution of quercetin and the calibration line is construed. Based on the measured absorbance, the concentration of flavonoids is read (mg/ml) on the calibration line. Thereafter, total flavonoid content in a plant extracts is expressed in terms of quercetin equivalent (mg of QU/g of extract). Rutin can also be used as a standard in place of quercetin and the results can be expressed in terms of rutin equivalent (mg of RU/g of extract).

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