PHYTOCHEMICAL ANALYSIS OF CENTELLA ASIATICA L . LEAF EXTRACTS

Saranya S, Aswathy V Nair, M. Priyanka Prathapan, Neethu A.S and * Neethu S. Kumar. Post Graduate Department and Research Centre of Botany, Mahatma Gandhi College, Affiliated to University of Kerala, Kesavadasapuram, Thiruvananthapuram695 015, Kerala, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Phytochemicals are plant derived chemicals, which may protect human from a host of numerous diseases. These chemicals are naturally occurring in medicinal plant leaves, vegetables and roots that have defense mechanism and protect from various diseases. Phytochemicals are primary and secondary compounds. Chlorophyll, proteins and common sugars are included in primary constituents and secondary compounds have terpenoids, alkaloids and phenolic compounds 7 . Terpenoids exhibit various important pharmacological activities i.e., anti-inflammatory, anticancer, anti-malarial, inhibition of cholesterol synthesis, anti-viral and anti-bacterial activities 8 . Terpenoids are very important in attracting useful mites which would consume the herbivorous insects 9 .Alkaloids are used as anaesthetic agents and are found in medicinalplants 10 .The present study is aimed to assess the phytochemical constituents in methanol, acetone, chloroform and aqueous extracts of the leaf extracts of C. asiatica L.

Preparation of plant extracts:-
The dried powdered leaves were extracted with four different solvents such as water, acetone, methanol and chloroform. For aqueous extraction, five grams of the powdered leaves were mixed with 50ml of distilled water, boiled for about two hours and filtered. Whereas acetone, methanol and chloroform extracts were prepared by mixing five grams of powdered leaf samples with 50ml of each solvent separately in an orbital shaker incubator for about 48 hours at room temperature. Extracts were filtered, concentrated, dried and stored in the refrigerator at 4°C for further use.

Phytochemical Analysis:-
The major secondary metabolites like carbohydrates, tannins, steroids, terpenoids, alkaloids, flavanoids, glycosides, saponins, quinons and phenolic compounds were assessed 11 .The presence of phytochemicals extracted in various solvents was confirmed by standard protocols.

Test for Alkaloids:-Wagner's reagent test
A fraction of the extract was treated with 3 -5 drops of Wagner's reagent and observed for the formation of reddish brown precipitate (or colouration) which indicates the presence of alkaloids.

Test for Carbohydrate:-Molish's test:-
To 2 ml of Molish reagent , 2 ml of the extract were added and shaken well. To this 2 ml. of conc. H 2 SO 4 was added through the sides of the test tube. Appearance of a reddish violet ring at the junction of the two layers indicate the presence of carbohydrates.

Fehling's Test:-
The filtrate was treated with 1 ml. of fehlings A and B and heated in a boiling water bath for 5 -10 minutes. Appearance of a reddish orange precipitate shows the presence of carbohydrates.

Detection of Flavanoids:-
The extracts were treated with conc. H 2 SO 4 and observed for an yellowish orange colour for the presence.

1830
Test for cardiac Glycosides:-Added 1ml. of extract with 1 ml.of glacial acetic acid and 2-3 drops of 5% ferric chloride solution. To this mixture were added 0.5 ml. dilute HCl. Appearance of a green ring which first turns to violet and then to brown at the interface indicates the presence of cardiac glycosides.
Test for protein:-Biuret Test:-One ml of 40% Nacl. and 2 drops of 1% Cuso 4 were added to the leaf extracts. Appearance of a violet colour confirms the presence of proteins.
Test for phenolic Compounds:-Ferric chloride Test:-Two ml of diluted extracts were treated with dil. Fecl 3 solution. Appearnce of a violet colour indicate the presence of phenol like compounds.

Test for quinones:-
A small amount of the extract was treated with conc. Hcl. and observed for the formation of a yellow precipitate colouration.

Test for Saponines:-
Two ml of the extracts were diluted with 20 ml of distilled Water, shaken vigoursly and was observed for a stable persistent froth.

Test for steroids:-
To1ml. of solvent extract in a test tube acetic anhydride was added and kept in a boiling water bath for 5min, then cooled followed by the addition of 1ml of Con. H 2 SO 4 along the sides of the test tube. Appearance of a green color indicate that the occurrence of steroids Test for tannins:-To the extracts were added a few drops of 10% ferric chloride solution. Appearance of a green/yellow colour indicates the presence of tannins.

Test for terpenoids:-Salkowiski's Test:-
Two ml of the extracts were mixed with 1 ml of chloroform and of conc. H 2 So 4 solution. A reddish brown colour at the inter phase indicates the presence of terpenoids.

Conclusion:-
The present study clearly indicates that the methanol crude extracts of C asiatica (L). exhibited the highest zone of inhibition than compared to the other extracts like water, chloroform and acetone. Many evidences gathered in earlier studies had already confirmed the identified phytochemicals to be bioactive. Several studies in turn confirmed their contribution to the medicinal as well as physiological properties of the plant towards the treatment of different ailments. Medicinal plants thus play a vital role in preventing various diseases. The antidiuretic, antiinflammatory, antianalgestic, anticancer, anti-viral, anti-bacterial and anti-fungal activities of the medicinal plants are due to the presence of the above mentioned secondary metabolites. Phytochemical analysis of the medicinal plants are hence commercially significant in both research institutes as well as pharmaceutical companies for the manufacturing of new drugs for the treatment of various illness. Studies conducted by previous workers and the present study show nearly similar results related to the phytochemical constituents in the leaf extracts of C. asiatica (L). It would not be surprising therefore to use plant samples to cure certain types of illness in humans and animals. This obtained information will therefore serve as a primary platform for further phytochemical and pharmacological studies related to the concerned plant. Hence it can be concluded that the leaves of this herb would direct to the establishment of some compounds that could be used to invent new and more potent anti microbial drugs of natural origin. Therefore future research should be addressed on the application of using leaves of the aforesaid medicinal herb as natural remedied and to protect against infectious diseases.