HDT Practical Exp.1

Practical Experiment- Preliminary Phytochemical Screening

1. By Mrs. Poonam N. Chougule Assistant Professor and HOD of Pharmacognosy department AMCP, Peth - Vadgaon . Herbal Drug Technology Practical

2. Experiment No. 1 AIM:- To perform preliminary phytochemical screening of crude drugs. Requirement- Ninhydrine solution, molish’s reagent, Hcl acid, freeze dryer, filter paper, N- haxane , chloroform, Acetic acid, Ferric chloride etc. Theory- Medicinal plant contain bioactive non-nutrient and biologically active compounds known as phytochemicals which contain a broad spectrum of chemical structures and protective disease preventative properties. Thus, conducting preliminary phytochemical screening of plants is an imporatant aspect in determining the chemical constituents in plant materials. Preliminary phytochemical screening of plants is also necessary for the discovery and devlopment of Novel therapeutic agents with improved efficacy.

3. Procedure- 1. Wash the entire plant and then dry it in the shade at room temperature until all the plants parts became well dried. After drying, the plants materials is then powdered well by using grinder. Place it in to a well closed container. 2. The phytochemical extraction is done by using organic solvent extraction as well as aqueous extraction. The organic extraction is performed by soxhlet extraction method. Take 20gm of dried plant powder, place it into a glass thimble and then extract with 250ml of different solvents separately (ethanol, methanol and acetone). The extraction processes is carried out till the solvent in siphon tube of soxhlet apparatus become colorless. After this heat the extract on hot water bath at 35 ᵒ C until the all solvent evaporated. Keep the dried plant crude extract in refrigerator at 2- 8 ᵒ C for their future use.

4. The aqeuous extraction is done by taking 5gms of the plant powder and mixes it with 200ml of distilled water in a beaker. Heat the mixture on a hot plate at 30 ᵒ - 40 ᵒ C and mix it with continuous stirring for 20 minutes. Filter the mixture by using whatman filter paper , filter and filtrate obtained is used for the further phytochemical analysis. Phytochemical qualitative analysis- The aqueous, ethonolic , acetone and methanolic extracts of plants are screened for the presence of the phytochemical classes by using the standard following methods-

5. Tests for Proteins Millon’s test- Mix 2 ml of Millon’s reagent with the entire plant crude extract, if white precipitate appeared, which upon gentle heating turned into red color indicates the presence of protein in the plant. Ninhydrin test- Boil 2 ml of 0.2% Ninhydrin solution with the entire plants crude extract, if violet color appears it indicates the presence of proteins and amino acids.

6. Test for Carbohydrates Fehling’s solutions test- boil a mixture of fehling solutions A and B with equal volumes and add it to crude plant extract. If a red color precipitate appears it indicates the presence of reducing sugars. Benedict’s reagents test- boil 2 ml of benedict’s reagent with a crude extract, a reddish brown color indicates the presence of the carbohydrates. Molisch’s solution test- shake 2 ml of Molisch’s solution with crude plant extract then add 2 ml of concentrated sulfuric acid and pour it carefully along the side of the test tube. A violet ring appeared at the inter phase of the test tube indicates the presence of the carbohydrate. Iodine test- 2ml of iodine solution is mixed with crude plant extract. Purple or dark blue colors prove the presence of the carbohydrate.

7. Tests for Phenols and tannins 2ml of 2% solution of ferric chloride is mixed with crude extract. Black or blue- green color indicates the presence of the tannins and phenols. Gold beater skin test- (Ox intestine) A small piece of goldbeater skin + soaked in 20% Hcl + rinse with water + placed in drug solution for 5mins, skin pieces washed with water + Kept in ferrous sulphate solution. It produces a brown or black colour appear on Skin by tannins.

8. Test’s for Flavonoids Shinoda test- Mix the pieces of magnesium ribbon and concentrated hydrochloric acid with crude plant extract after few minutes pink colored scarlet appears which indicates the presence of flavonoids . Alkaline reagent test- Mix 2ml of 2% NaOH solution with plant crude extract, intensive yellow color is formed, which turns into colorless when added to 2 drops of diluted acid, this result indicate the presence of flavonoids .

9. Test’s for Saponins Add five ml of distilled water to crude plant extract in a test tube and shake it vigorously the foam formation indicates the presence of saponins .

10. Test’s for glycosides Liebermann’s test- mix 2ml of acetic acid and 2 ml of chloroform with entire plant crude extract. Cool the mixture and to it add concentrated sulphuric acid, green color indicates the entity. Salkowaski test- Add concentrated sulphuric acid (about 2ml) to the entire plant crude extract. A reddish brown color produced indicates the entity of steroidal aglycone part of the glycoside. Keller Killani test- add a mixture of Acetic acid glacial (2ml) with 2 drops of 2% Fecl3 solution to the plant extract and concentrated sulfuric acid. A brown ring produces between the layers which indicate the entity of cardiac steroidal glycosides.

11. Test for Steroid Mix two millimeter of chloroform and concentrated sulfuric acid with the entire plant crude extract. The lower chloroform layer produces red color that indicates the presence of steroids.

12. Test for terpenoids Mix two ml of chloroform with the plant extract and evaporate it on the water bath then boil it with 2ml of H2SO4 concentrated. A grey color produced indicates the entity of terpenoids .

13. Thank you…