Phytochemical screening

The samples were screened for carbohydrates, alkaloids, flavonoids, phytosterols and steroids, anthocyanin and betacyanin, phenols and tannins, saponin, glycosides, and proteins.

Detection of carbohydrates:
Molisch's test: Treat the test solution with few drops of alcoholic alpha napthol. Add 0.2ml of con. Sulfuric acid slowly through the sides of the test tube, a purple to violet color ring appears at the junction Benedict's test: Treat the test solution with few drops of Benedict's reagent (alkaline solution containing cupric citrate complex) and upon boiling on water bath, reddish brown precipitate forms if reducing sugars are present. Benedicts reagent: Sodium citrate (173 g) and sodium carbonate (100 g) were dissolved in 800 ml distilled water and boiled to make it clear. Capper sulphate (17.3 g) dissolved in 100 ml distilled water was added to it. Fehling's test: Equal volume of Fehling's A (Copper sulfate in distilled water) and Fehling's B (Potassium tartarate and Sodium hydroxide in distilled water) reagents are mixed and few drops of sample is added and Boiled, a brick red precipitate of cuprous oxide forms, if reducing sugars are present. Barfoeds test: To 1 ml of filtrate, 1 ml of Barfoeds reagent was added and heated on a boiling water bath for 2 min. Red precipitate indicated presence of sugar. Barfoeds reagent Copper acetate, 30 5 g was dissolved in 1-8 ml of glacial acetic acid. Camnelisation: Carbohydrates when treated with strong sulfuric acid, they undergo charring with the dehydration along with burning sugar smell. Selwinoffs test: Hydrochloric acid reacts with ketose sugar to form derivatives of furfuraldehyde, which gives red colored compound when linked with resorcinol. Add compound solution to about 5ml of reagent and boil. Fructose gives red color within half minute. The test is sensitive to 5.5mmol / liter if glucose is absent, but if glucose is present it is less sensitive and in addition of large amount of glucose can give similar color. Iodine test: Put one milliliter of the aqueous extract in test tube and add 1 drop mixture of iodine solution. If the color changes, it denotes the presence of polysaccharide

Detection of proteins
The extract (100 mg) was dissolved in 10 ml of distilled water and filtered through whatman No.1 filter paper and the filtrate was subjected to tests for proteins and amino acids. Millons test: To 2 ml of filtrate, few drops of Millons reagent were added. A white precipitate indicated the presence of proteins. Millons Reagent Mercury (1g) was dissolved in 9 ml of fuming nitric acid. When the reaction was completed, equal volume of distilled water was added.

Biuret test: An aliquot of 2 ml of filtrate was treated with one drop of 2 % copper sulphate solution. To this, 1 ml of ethanol (95%) was added, followed by excess of potassium hydroxide pellets, pink colour in the ethanolic layer indicated the presence of proteins. Biuret Test: Small quantity of ethanolic extract was dissolved in a few mL of water. To this test solution 4% NaOH solution and a few drops of 1% CuSO4 solution was added. Appearance of violet colour showed presence of proteins. Ninhydrin test: Two drops of ninhudrin solution (10 mg of ninhydrin in 200 ml of actone) were added to two ml of aqueous filtrate. A characteristic purple colour indicated the presence of amino acid. Hopkins Cole test: 2 Drops of Hopins Cole reagent was added to 2 ml of aqueous filtrate. A characteristic violet colour indicates the presence of protein Xanthoproteic Test: To the small quantity of ethanolic extract 1ml. of conc. H2SO4 was added. This resulted in the formation of white precipitate which on boiling turned yellow. On addition of NH4OH, yellow ppt. turned orange.

Detection of Phenolic compounds and Tannins:

Ferric chloride test: The extract (50 mg) was dissolved in 5 ml of distilled water. To this, few drops of neutral 5% ferric chloride solution were added. A dark green colour or deep blue black colour appeared indicated the presence of phenolic compounds. Ferric chloride reaction: Take 1 ml for the solution in a test tube, add acetic acid and then a few drops of 1% ferric chloride reagent. If it turns brownish green or blue black, then it may contain phenolic composition or tannin. Ferric chloride - potassium ferricyanide reaction: Put the solution on the filter paper, dry and spray ferric chloride - potassium ferricyanide reagent on it. If it turns blue, then it may Gelatin test: The extract (50 mg) was dissolved in 5 ml of distilled water and 2 ml of % solution of gelatin containing 10 % sodium chloride was added to it. White precipitate indicated the presence of phenolic compounds. Lead acetates test: The extract (50 mg) was dissolved in distilled water and 3 ml of 10% lead acetate solution was added. A bulky white precipitate indicated the presence of phenol compounds. Alkaline reagent test: An aqueous solution of the extract was treated with 10% ammonium hydroxide solution. Yellow fluorescence indicated the presence of flavonoids. Alkaline reagent test: Test solution with sodium hydroxide solution gives yellow to red precipitate within short time. Dil. HNO3 Test: On addition of dilute HNO3 solution to the ethanolic extract, reddish colour appeared contain tannin. Vanillin - hydrochloric acid: Put the solution point on the filter paper, get points for the solution on the filter paper after drying, and spray it with vanillin -hydrochloric acid reagent. If it immediately shows varying degrees of red, then it contains phenolic composition. . Vanillin Hydrochloride test: Test solution when treated with few drops of vanillin hydrochloride reagent gives purplish red color.

Mitchells test: With iron and ammonium citrate or iron and sodium tartarate. Tannins give a water-soluble iron-tannin complex, which is insoluble in solution of Ammonium acetate

Detection of Flavonoids
Shinoda Test: To the ethanolic extract, added 5 ml of 95% ethanol and few drops of conc. HCl. To this solution 0.5 g of magnesium turnings were added. Observance of pink coloration indicated the presence of flavonoids. crimson red or occasionally green to blue color appears after few minutes With Lead Acetate: To the small quantity of ethanolic extract lead acetate solution was added. Formation of yellow precipitate showed the presence of flavonoid. With Sodium Hydroxide: On addition of an increasing amount of sodium hydroxide, the ethanolic extract showed yellow coloration, this decolorized after addition of acid Zinc Hydrochloride reduction test: To the test solution add a mixture of Zinc dust and conc. Hydrochloric acid. It gives red color after few minutes. Alkaline reagent test: To the test solution add few drops of sodium hydroxide solution; formation of an intense yellow color, which turns to Colorless on addition of few drops of dil. acid, indicates presence of Flavonoids. Aluminum chloride reaction: Get a certain drop of test solution on the filter paper, dry it, and then spray some aluminum chloride reagent on it. If there appears yellow spots or yellow-green fluorescence under ultraviolet light, it shows it contains flavonoids. Ammonia test: Put some test solution on a filter paper or a silica gel plate, and then fumigate it with ammonia. If there is a yellow or yellow fluorescence under UV light then there is flavonoids. If there appears yellow spots or yellow-green fluorescence under ultraviolet light, it shows there are flavonoids inside. Testing for coumarin and lactones: One milliliter of ethanol extract was stirred and placed separately in a test tube. The result of the solution is seen in the following experiments. Hydroxamic acid iron response: In the test tube, add 70% alcohol solution of hydroxylamine hydrochloride and 10% sodium hydroxide solution in 2 drops of boiling water in home bath. Wait a few minutes to get response. Let it cool, and then add 1% hydrochloric acid with adjusted pH of 3~4, and 2 drops of 1% ferric chloride reagent. If the solution turns red or purple, it contains coumarin or lactones. Opened loop - closed loop response: In the test tube, add 2 drops of 1% sodium hydroxide solution, and heat in boiling water for 3 min to get clear solution. Also add 4 drops of 2% hydrochloric acid to the solution. If the solution becomes cloudy, that means it contains coumarins and lactones. Fluorescence response: Put the test solution on silica gel chromatography plate, dry under UV light observation, and then spray 1% potassium hydroxide reagent. If there is present blue - green fluorescence, and fluorescence enhancement, then it may contain coumarins.

Detection of Sterols, Phytosterols & Triterpenoids

Libermann- Buchard test: Extract is treated with few drops of acetic anhydride, boil and cool, con. Sulfuric acid is added from the sides of the test tube, shows a brown ring at the junction of two layers and the upper layer turns green which shows the presence of Steroids and formation of deep red color indicates the presence of triterpenoids.

Salkowski test: Treat extract in Chloroform with few drops of cone. Sulfuric acid, shake well and allow standing for some time, red color appears at the lower layer indicates the presence of Steroids and formation of yellow colored lower layerindicates the presence of Triterpenoids.

Detection of steroids and phytosterol:

To 1 ml of plant extract, equal volume of chloroform and 3 drops of concentrated sulphuric acid were added. Formation of brown ring indicates the presence of steroids and formation of bluish green colour indicates the presence of phytosterols.

Detection of triterpenes:
To the ethanolic extract chloroform and conc. H2SO4 was added. Appearance of red colour indicated the presence of triterpenes

Detection of Saponins by Foam Test:

The extract (50 mg) was diluted with distilled water and made up to 20 ml. The suspension was shaken in a graduated cylinder for 15 min. A one to two cm layer of foam indicated the presence of saponins.

Detection of Alkaloids:
The ethanolic extract was evaporated in a test tube. To the residue dilute HCl was added, shaken well and filtered. With the filtrate following tests were performed: Mayers test Alkaloids give cream color precipitate with Mayer's reagent [Potassium mercuric iodide solution]. Mayers Reagent: Mercuric chloride (1 35 g) was dissolved in 60 ml of water and potassium iodide (5.0 g) was dissolved in 10 ml of water. The two solutions were mixed and made up to 100 ml with water Dragendorffs test Alkaloids give reddish brown precipitate with Dragendorffs reagent [Potassium bismuth iodide solution]. A prominent yellow precipitate indicated the test as positive Dragendorffs reagent: Stock solution :Bismuth carbonate (5.2 g) and sodium iodide (4 g) were boiled for a few min with 50 ml glacial acetic acid. After 12hrs, the precipitated sodium acetate crystals were filtered off using a sintered glass funnel. Clear, reddish brown filtrate, 40 ml was mixed with 160 ml ethyl acetate and 1 ml water and stored in amber coloured bottle Working Solution: Stock solution 910 ml) was mixed with 20 ml of acetic acid and made up to 100 ml with water. Wagner's test: Alkaloids give a reddish brown precipitate with Wagner's reagent [Solution of iodine in potassium iodide].

Wagners reagent: Iodine (1.27 g) and potassium iodide (2 g) was dissolved in 5 ml of water and made up to 100 mL with distilled water. Hager's test: Alkaloids give yellow color precipitate with Hager's reagent [saturated solution of Picric acid]. Tannic acid test: Alkaloids give buff color precipitate with 10% Tannic acid solution.

Detection of anthocyanin and betacyanin :

To 2 ml of plant extract, 1 ml of 2N sodium hydroxide was added and heated for 5 min at 100C. Formation of bluish green colour indicates the presence of anthocyanin and formation of yellow colour indicates the presence of betacyanin.

Detection of Glycosides
For detection of glycosides, 50 mg of extract was hydrolysed with concentrated hydrochloric acid for 2 hrs on water both, filtered and the hydrolysate was subjected to the following tests The extracts were tested for free sugars The extract is hydrolyzed with mineral acid and then tested for the glycone and aglycone moieties. Raymonds test Test solution when treated with dinitro- benzene in hot methanolic alkali, gives violet color. Legals test Treat the extract with pyridine and add alkaline sodium nitroprusside solution, blood red color appears. Bromine water test Test solution when treated with bromine water gives yellow precipitate. Tests for glycosides: Borntragers Test: To the 3ml of ethanolic extract, dil. H2SO4 was added. The solution was then boiled and filtered. The filtrate was cooled and to it equal volume of benzene was added. The solution was shaken well and the organic layer was separated. Equal volume of dilute ammonia solution was added to the organic layer. The ammonical layer turned pink showing the presence of glycosides. Legals Test: To the concentrated ethanolic extract few drops of 10% NaOH were added, to make it alkaline. Then freshly prepared sodium nitroprusside was added to the solution. Presence of blue or pink coloration indicated the presence of glycosides in the extract. Chemical tests for specific glycosides: Test for Saponin Glycosides: Froth Test: Place 1ml solution of drug in water in a semi-micro tube and shaken well and noted for a stable froth. Hemolysis test: Add 0.2ml solution of saponin (prepared in 1% normal saline) to 0.2ml of v/v blood in normal saline and mix well, centrifuge and note the red supernatant compare with control tube containing 0.2ml of 10% blood in normal saline diluted with 0.2ml of normal saline. Test for Anthrauinone Glycosides: Borntrager's test: Boil the test material with 1ml of dilute sulphuric acid in a test tube for 5min (anthracene glycosides are hydrolyzed to aglycone and sugars by boiling with acids) centrifuge or filter while hot (if centrifuged hot, the plant material can be removed while anthracene aglycones are still sufficiently soluble in hot water, they are however

insoluble in cold water), pipette out the supernatant or filtrate, cool and shake with an equal volume of dichloromethane (the aglycones will dissolve preferably in dichloromethane) separate the lower dichloromethane layer and shake with half its volume with dilute ammonia. A rose pink to red color is produced in the ammonical layer (aglycones based on anthroquinones give red color in the presence of alkali). Modified Borntrager's test: Boil 200mg of the test material with 2ml of dilute sulphuric acid, 2ml of 5% aqueous ferric chloride solution for 5min and continue the test as above. As some plant contains anthracene aglycone in a reduced form, if ferric chloride is used during the extraction, oxidation to anthroquinones takes place, which shows response to the Borntrager's test. Test for Cardiac Glycosides: Testing for cardiac glycosides: One milliliter of ethanol extract was stirred and placed separately in 3, 5 - dinitro-acid, alkaline picric acid and FeCl3-glacial acetic acid.The results are shown separately. The extracts produced purple colour; the solution turned red or salmon pink in 15 min; the solutionis divided into two- the upper turned blue-green, and purple or brown interface for the ring, meaning it contains cardiac glycosides. Keddes test: Extract the drug with chloroform, evaporate to dryness, add one drop of 90% alcohol and 2 drops of 2% 3,5-dinitro benzoic acid(3,5-dinitro benzene carboxylic acid-Kedde's reagent) in 90% alcohol. Make alkaline with 20% sodium hydroxide solution. A purple color is produced. The color reaction with 3, 5-diinitrobenzoic acids depends upon the presence of an - unsaturated-o lactones in the aglycone. Keller killiani test [test for Deoxy sugars] Extract the drug with chloroform and evaporate it to dryness. Add 0.4ml of glacial acetic acid containing a trace amount of 5% ferric chloride. Transfer to a small test tube; add carefully 0.5ml of concentrated sulphuric acid by the side of the test tube, blue color appears in the acetic acid layer. Test for Cyanogenetic Glycosides: Place 200mg of drug in a conical flask and moisten with few drops of water, there should be no free liquid at the bottom of the flask (the test will not work if there is any liquid in the flask as the hydrogen cyanide produced will dissolve in the water rather than, come off as a gas to react with the paper). Moisten a piece of picric acid paper with sodium carbonate solution (5% aqueous) and suspended by means of cork in the neck of the flask, warm gently at about 37C. Observe the change in color. Hydrogen cyanide is liberated from cyanogenetic glycoside by the enzyme activity and reacts with sodium picrate to form the reddish purple sodium isopicrate To 2 ml of plant extract, 1 ml of glacial acetic acid and 5% ferric chloride was added. To these 3 drops of concentrated sulphuric acid was added. Presence of greenish blue colour indicates the presence of glycosides. Testing for anthraquinones Take the ethanol extract and do the following experiment separately. Alkaline reaction One milliliter of the ethanol extract was stirred and placed in aqueous ammonia. If the solution was red, by adding acid, the red subsided; meaning it contains anthraquinones. Magnesium acetate reaction One milliliter of the ethanol extract was stirred and placed in 1% methanol solution of magnesium acetate. The solution turned orange-red, purple and other colors, meaning it contains anthraquinones.

Detection of Fixed Oils and Fats

Stain test: Press the small quantity of extract between two filter papers, the stain on I filter paper indicates the presence of fixed oils.

Saponification test: Add a few drops of 0.5N of alcoholic potassium hydroxide to small quantities of various extracts along with a drop of Phenolphthalein separately and heat on a water bath for 1-2 hrs. The formation of soap or partial neutralization of alkali indicates the presence of Fixed oils and Fats.

Detection of Gum and Mucilage:

The extract (100mg) was dissolved in 10 ml of distilled water and to this, 25 ml of absolute alcohol was added with constant stirring. White or cloudy precipitate indicated the presence of gums and mucilages.

Detection of organic acid:

Take the water extract and do the following experiments separately. pH test strip reaction If the solution is put in a wide range of pH test paper, and found to be acidic, then it may contain organic acids or phenolic. Bromophenol blue reaction Put the solution point on the filter paper, and then spray reagent 0.1% blue bromophenol and 70% ethanol. If it produces a blue background with yellow spots, then it may contain organic acids.