A.To about 1 g of the sample,in powder,add 20 ml of ethanol,boil in a water-bath and filter.To the filtrate,add 300 mg of decolorizing charcoal,stir and filter (solution A). To 1 ml of solution A,add 2 drops of a 2 per cent w/v solution of 3,5-dinitrobenzoic acid in methanol and 2 drops of a 5.7 per cent w/v solution of potassium hydroxide in methanol:a purplish red colour develops.
Carry out the test as described in the Thin-layer Chromatography,using silica gel GF254 as the coating substance and a mixture of 85 volumes of chloroform and 15 volumes of absolute ethanol as the mobile phase but allowing the solvent front to ascend 15 cm above the line of application.
Apply separately to the plate ,5 ul of each of the following solution. prepare solution A by boiling 1 g of the sample,in powder,with 20 ml of ethanol on a water-bath for 5 minutes,adding 300 mg of decolorizing charcoal,stirring,and filtering.Evaporate the filtrate under reduced pressure until dryness,and dissolve the residue in 1 ml of warm ethanol(80 per cent).For solution B,dissolve 2 mg of andrographolide in 1 ml of ethanol.After remove of the plate from the chromatographic chamber,allow it to dry in air,and examine under ultraviolet light (254 nm),marking the quenching spots.The chromatogram obtained with solution A shows a quenching spot (hRf values 52 to 56),corresponding to the andrographolide spots from the solution B and other four spots of different hRf value Table1 ; see also Fig 3.Spray the plate with a 2 per cent w/v solution of 3,5-dinitrobenzoic acid in methanol and then with an excess of a 5.7 per cent w/v solution of potassium hydroxide in methanol;the spot due to andrographolide is dark violet.Two dark violet spot due to the spot numbers 4 and 9 in table 1 and other violet and dark violet spots are also observe Table 1;see also Fig3