4.1 Maintenance and sub-culturing of endophytic fungi
The cultures were procured from already maintained repository in laboratory. The maintenance of the cultures involved preparation of Potato dextrose agar (PDA) plates, sub culturing of cultures and long term preservation. 4.1.1 Preparation of PDA (Potato dextrose agar) plates 39g of PDA was dissolved in 1000 ml double distilled water, stirred to mix properly and was transferred into 250 ml Erlenmeyer flasks and autoclaved at 121˚C at 15 psi for 15 min. Aseptically, 25ml of sterilized PDA was poured into pre-sterilized 90mm glass petri-plates and allowed to solidify at room temperature. The plates were stored in incubator at the temperature 26 ± 2°C until further use. 4.1.2 Sub culturing …show more content…
4.4.1 Wagner’s Test (Kodangala et al., 2010)
It is a specific test to detect the presence of alkaloids. Wagner’s reagent was prepared by dissolving 1.27g of iodine and 2g of potassium iodide in 100ml distilled water. To the 100μl of ethyl acetate fraction of each endophytic fungi, 4-5 drops of wagner’s reagent was added and observed for the presence of reddish-brown precipitate formation. Vinblastine and methanol was used as positive and negative control respectively
4.4.2 Hager’s Test (Kodangala et al., 2010)
Briefly, to the 100μl of ethyl acetate fraction, 4-5 drops of Hager’s reagent (saturated solution of picric acid) was added and observed for the presence of yellow precipitate formation. Vinblastine and methanol was used as positive and negative control respectively.
4.5 Thin Layer chromatography …show more content…
For the preparation of TLC plates, 20 x 15 x 5 glass plates were properly washed and coated with silica gel (Merck) of 0.5mm thickness. The coated plates were activated by incubating at 100ºC for 3h prior to use. The sample and positive control (Vinblastine, Sigma, 1mg/ml) were spotted on to activated TLC plate just 1cm above the subordinate edge of plate with the help of capillary tube and allowed to air dry. Simultaneously, the TLC chamber was saturated with different solvent systems (Binary and tertiary) consisting of mixture of solvents of different polarities and ratios for 20 min. The TLC plate was kept in saturated TLC chamber in such a way that the applied spot is above from the solvent level and the plate was allowed to develop. When the solvent front reaches up to the desired level, the TLC plate was taken out and allowed to air dry. The Chromatogram was developed by keeping the TLC plate in iodine chamber. Vinblastine was used as standard for the comparison of Rf value and alkaloidal spectrum. Retention factor (Rf) value of each band was obtained as the ratio of distance move by solute to that of solvent
The cultures were procured from already maintained repository in laboratory. The maintenance of the cultures involved preparation of Potato dextrose agar (PDA) plates, sub culturing of cultures and long term preservation. 4.1.1 Preparation of PDA (Potato dextrose agar) plates 39g of PDA was dissolved in 1000 ml double distilled water, stirred to mix properly and was transferred into 250 ml Erlenmeyer flasks and autoclaved at 121˚C at 15 psi for 15 min. Aseptically, 25ml of sterilized PDA was poured into pre-sterilized 90mm glass petri-plates and allowed to solidify at room temperature. The plates were stored in incubator at the temperature 26 ± 2°C until further use. 4.1.2 Sub culturing …show more content…
4.4.1 Wagner’s Test (Kodangala et al., 2010)
It is a specific test to detect the presence of alkaloids. Wagner’s reagent was prepared by dissolving 1.27g of iodine and 2g of potassium iodide in 100ml distilled water. To the 100μl of ethyl acetate fraction of each endophytic fungi, 4-5 drops of wagner’s reagent was added and observed for the presence of reddish-brown precipitate formation. Vinblastine and methanol was used as positive and negative control respectively
4.4.2 Hager’s Test (Kodangala et al., 2010)
Briefly, to the 100μl of ethyl acetate fraction, 4-5 drops of Hager’s reagent (saturated solution of picric acid) was added and observed for the presence of yellow precipitate formation. Vinblastine and methanol was used as positive and negative control respectively.
4.5 Thin Layer chromatography …show more content…
For the preparation of TLC plates, 20 x 15 x 5 glass plates were properly washed and coated with silica gel (Merck) of 0.5mm thickness. The coated plates were activated by incubating at 100ºC for 3h prior to use. The sample and positive control (Vinblastine, Sigma, 1mg/ml) were spotted on to activated TLC plate just 1cm above the subordinate edge of plate with the help of capillary tube and allowed to air dry. Simultaneously, the TLC chamber was saturated with different solvent systems (Binary and tertiary) consisting of mixture of solvents of different polarities and ratios for 20 min. The TLC plate was kept in saturated TLC chamber in such a way that the applied spot is above from the solvent level and the plate was allowed to develop. When the solvent front reaches up to the desired level, the TLC plate was taken out and allowed to air dry. The Chromatogram was developed by keeping the TLC plate in iodine chamber. Vinblastine was used as standard for the comparison of Rf value and alkaloidal spectrum. Retention factor (Rf) value of each band was obtained as the ratio of distance move by solute to that of solvent