DETERMINATION OF DIOSGENIN IN PLANTS

Diosgenin, one of the main sapogenins present in the roots of Dioscorea spp., has been studied for its determination. A simple method was proposed as follows: (1) Hydrolysis and extraction: Procedure: Cut the roots of the plant to about 1mm in size. Weigh accurately 0.2-0.4g, add 20 ml 2N sulphuric acid and heat under reflux for 4 hours in a glass. joint apparatus. Filter through paper, wash the residue with 1% sodium carbonate solution and water until the washing is neutral. Dry the paper and residue at 80℃ for about 2 hours. After cutting the sapogenins into small pieces, extract them in a Soxhlet apparatus with benzene for an hour. (2) Chromatographic separation: The benzene extract was first studied by thin layer chromatography on alumina. From this result, a suitable solvent system for column adsorption chromatography was designed to separate diosgenin from the other constituents. Procedure: Make benzene extract to 50 ml in a volumetric flask, pipette out 5 ml, add to the top of an alumina column (1×5 cm). Wash the column with 15 ml fresh benzene in fractions, then elute with ether. Discard the first 4ml and collect the next 14 ml in an Erlenmeyer flask. Evaporate the elute to dryness, collect diosgenin as white needle crystals. (3) Colorimetric determination: Diosgenin was determined by the colorimetric method, based on the Liebermann-Burchard reaction. Huang's modified Liebermann reagent was used. Procedure: Add 5 ml modified Liebermann reagent to the diosgenin residue, stopper the flask loosely, put the flask in a 70±0.5℃ water bath for exactly 4 minutes, then cool in ice water immediately. Measure the colour with a photometer (480 mμ filter), use the reagent as blank. Calculate the results from a calibration curve. Using the above method, five samples have been assayed. The diosgenin contents are: Dioscorea nipponica Makino 1.45% D. deltoidea Wall 2.74% D. panthaica Prain et Burkill 3.45% D. nigrascens R. Kunth 2.39% D. althaeoides R. Kunth 0.90% The average percent deviation of six determinations of D. nipponica is ±1.8%.