Study On Extraction, Separation And Purification Of Glabridin And Glycyrrhizic Acid

Glabridin as the active ingredient has been developed in Glycyrrhiza glabra L in our country or abroad, but the high content of glycyrrhizic acid has been abandoned in the licorice, caused a tremendous waste of licorice resources.To improve the effective utilization of licorice, realize the comprehensive utilization of licorice resources have great significance.A novel HPLC method has been devised for analyzing the contents of glabridin and glycyrrhizic acid.The method of gradient of eluate is rapid and can obtain satisfied separation results.The condition for extraction of glabridin and glycyrrhizic acid has been established. The ultrasound method was be used to extract glabridin and glycyrrhizic acid. The optimum conditions are as follows:solvent is 60% ethanol, extracting time is 25 min, the ratio of solvent to solid is 30:1 (mL/g),extraction 3 times,the maximum yield of glabridin and glycyrrhizic acid are 0.33% and 6.42%.By comparing the separation effect, dichloromethane and 10% ethanol solution was be used to extracted of glabridin and glycyrrhizic acid in licorice root sample. The contents of glabridin and glycyrrhizic acid are 12.90% and 17.85%.The adsorption and desorption properties of glabridin on macroporous resins were optimized. HPD100 resin was selected as the experiment resin; 30% ethanol was used as adsorption solution; feed concentration is 0.5-0.6 mg/mL;the flow rate of adsorption is 3 BV/h and elution rate is 7 BV/h; desorption eluent is 8 BV; the method of gradient of eluate was used 40% ethanol to removed impurities, then collected 70% ethanol as product. After purified, the content of glabridin is 23.90%. Then used AB-8 resin purified glabridin again, the content of glabridin increased from 23.90% to 35.10%.The research established the adsorption and desorption properties of glycyrrhizic acid on macroporous resins.AB-8 resin was selected as the experiment resin; feed concentration is 1.7 mg/mL;the flow rate of adsorption is 2 BV/h and elution rate is 7 BV/h; desorption eluent is 8 BV; the method of gradient of eluate was used 20% ethanol to removed impurities,then collected 50% ethanol as product.After purified, the content of glabridin is 61.94%.High speed counter current chromatography (HSCCC) was used for isolation and purification of glabridin. N-hexane-ethyl acetate-acetonitrile-methanol-water (5:6:1:2:2) has been selected by analytical HSCCC solvent system.After purified, the retention of the solid phase was 43% and the content of glabridin is 95%.Glabridin was isolated and purified by Pre-HPLC.The mobile phase is 70% ethanol;flow rate is 10 mL/min; the temperature is 30℃;the wavelength is 283 nm. After purified, the content of glabridin is 95%,the yield of glabridin is 52%.The inhibition of tyrosinase by glabridin and glycyrrhizic acid were determined. The result showed that glabridin has strong inhibition of tyrosinase, the inhibition of tyrosinase increased with the increasing content of glabridin. But for glycyrrhizic acid, the inhibition of tyrosinase is not very obvious.