Collection and Identification of Plant material:
The plants namely Trichosanthes dioica Roxb (Local name: Patol), Benincasa hispida (Local name: Chichinga), Trichosanthes cucumerina linn. (Local name: Chalkumra) were collected from Savar kancha Bazar, Dhaka and Bhoirab, Bramhanbaria, Bangladesh in December 2011 and was identified at the Bangladesh National Herbarium, Mirpur, Dhaka where the voucher specimen no: 33767 have been deposited for future reference. The specimen samples were kept in the Laboratory of Natural Products research in the Department of Pharmacy, Jahangirnagar University, Bangladesh.
Chemicals and drugs:
All chemicals and reagents used were of analytical grade. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical was obtained from Sigma Aldrich Co. (St. Louis, USA). Molisch reagent, sodium hydroxide and Fehling’s solution, Mayer’s reagent, Hagers reagent, Wagners reagent, Dragendroffs reagent, sulphuric acid, ferric chloride, concentrated HCl.
Drying and Pulverization:
The fresh leaves of the plants were first washed with water to remove adhering dirt and then cut into small pieces, sun dried for 4 days. After complete drying, the entire portions were pulverized into a coarse powder with the help of a grinding machine and were stored in an airtight container for further use. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced.
Cold extraction:
From the three plants about 200 g of dried, ground plant material was taken in a clean, flat bottomed 3 glass container and soaked in 500 ml of 95% methanol. The container with its contents was sealed and kept for a period of 7 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then it was filtered through Whatman filter paper (Bibby RE200, Sterilin Ltd., UK).
Extraction with Methanol:
The concentrated methanol extract was made slurry with water. The slurry was taken in a separating funnel and few ml Methanol (50 ml) was added to the aqueous solution and the mass was shaken vigorously in a separating funnel. Then the funnel was allowed to stand for few minutes for the complete separation of the layers. The organic layer was collected. The process was repeated two times.
Extraction with petroleum ether:
Petroleum ether (50 ml) was added to the Methanolic aqueous solution and the mass was shaken vigorously in a separating funnel. Then the funnel was allowed to stand for few minutes for the complete separation of the layers. The organic (upper layer) layer was collected. The process was repeated two times.
The filtrate (methanol and petroleum ether extract) obtained was evaporated with rotary evaporator at 400 C. The extract was transferred to a closed container for further use and protection.
Phytochemical screening:
Testing the presence of different chemical groups in the plant extract is the preliminary phytochemical studies. To identify the chemical constituents of plant extracts, standard procedures were followed. In each test 10% (w/v) solution of extract in methanol and Pet ether extracts were taken unless otherwise mentioned in individual test.
Determination of Carbohydrates:
Two drops of molisch’s reagents were added to about 5 mg of the extract in 5 ml aqueous solution in a test tube. 1 ml of conc. H2SO4 was allowed to flow down the side of the inclined test tube so that the acid formed a layer beneath the aqueous solution without mixing. A red ring was formed at the common surface of the two liquids which indicated the presence of carbohydrate. On standing or shaking a dark-purple solution was formed. Then the mixture was shaken and diluted with 5 ml of water. Dull violet precipitate was formed immediately.
Determination of Glycosides: A small amount of an alcoholic extract of the fresh or dried plant material was taken in 1ml of water. Then, a few drops of aqueous sodium hydroxide were added. A yellow color was considered as an indication for the presence of glycosides.
A small amount of an alcoholic extract of the plant material was taken in water and alcohol and boiled with Fehling’s solution. Brick-red precipitate was considered as an indication for the presence of glycosides.
Determination of Alkaloids:
Mayer’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Mayer’s reagent was added. Yellow color precipitate was formed and that was indicated as the presence of alkaloids.
Hager’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Hager’s reagent was added. Yellow crystalline precipitate was formed and that was indicated as the presence of alkaloids.
Wagner’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Wagner’s reagent was added. Brownish-black precipitate was formed and that indicates the presence of alkaloids.
Dragendroff’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Dragendroff’s reagent was added. Orange brown precipitate was formed and that indicates the presence of alkaloids.