Studies On Extraction, Purification And Hydrolysis Of Radix Puerariae Isoflavones

The technology of extracting, purifying and enzymic hydrolysis of Radix Puerariae isoflavones was studied in order to give some scientific message for comprehensive utilization and find out a new method to produce Radix Puerariae isoflavones. Total isoflavones from Radix Puerariae were obtained through subcritical water extraction, ultrasonic extraction. And make a comparison between the two methods. Then the isoflavones extracts were purified by ultrafiltration. Next, make use of hydrolysion acid and "Amano" β-glucosidase to hydrolyse total isoflavones into puerarin and daidzein. Finally, the high purified puerarin and daidzein were obtained from the hydrolyzate by HSCCC.The appropriate conditions of subcritical water extraction were obtained by single factor and orthogonal experiment.The results indicated that the optimum extraction parameters were as follows:extraction time30min, temperature130℃, pressure0.9MPa, liquid-solid ratio25mL/g,. Under these conditions, the yield of Radix Puerariae total isoflavones in the extracts were7.85%.The appropriate conditions of ultrasonic extraction were obtained by single factor and orthogonal experiment.The results indicated that the optimum extraction parameters were as follows:extraction time45min, temperature50℃,70%ethanol solution, microwave400W, liquid-solid ratio30mL/g,. Under these conditions, the yield of Radix Puerariae total isoflavones in the extracts were6.76%.Comparison in subcritical water extraction and ultrasonic extraction, the result indicated subcritical water extraction was more effective and environmentally friendly than ultrasonic extraction.The appropriate ultrafiltration conditions were obtained in the experiments of one-factor design. The ultrafiltration memberane made from polyether sulfone, whose molecular weight cut-off (MWCO) was5000,was used.The ultrafiltration pressure was68kPa,and the ultrafiltration temperature was40℃. After the processing of the ultrafiltration, The isoflavones content of extract was increased from24.9%to36.2%in dry matter and the rejection rate of total solid cotent was80.6%.The appropriate conditions for hydrolysion of the preliminary purification Radix Puerariae total isoflavones by the hydrochloric acid were obtained by single factor and orthogonal experiment.The results indicated that the optimum hydrolyzation parameters for puerarin which was in the form of glycoside were as follows:hydrolysis time7h, hydrochloric acid concentration4mol/L, temperature70℃, under these conditions, the yield of puerarin were92.8%. The optimum hydrolyzation parameters for daidzein which was in the form of glycoside were as follows: hydrolysis time7h, hydrochloric acid concentration4mol/L, temperature50℃, under these conditions, the yield of daidzein were95.1%.The appropriate conditions for hydrolysion of the preliminary purification Radix Puerariae total isoflavones by the "Amano" β-glucosidase were obtained by single factor and orthogonal experiment.The results indicated that the optimum hydrolyzation parameters for puerarin which was in the form of glycoside were as follows:hydrolysis time120min, enzyme concentration40mg/L, the pH4.5, temperature45℃, under these conditions, the yield of puerarin were93.9%. The optimum hydrolyzation parameters for daidzein which was in the form of glycoside were as follows: hydrolysis time120min, enzyme concentration40mg/L, the pH4.5, temperature50℃, under these conditions, the yield of daidzein were96.2%.Make a comparison between this two hydrolysis methods, the result indicated the reaction conditions of "Amano" β-glucosidase hydrolysis was more gentle. And the rection time was less,the yield of puerarin and daidzein were much more than hydrochloric acid hydrolysis.When Radix Puerariae total isoflavones hydrolyzate were purified by HSCCC, two solvent system were used:ethyl acetate-n-butanol-water (4:1:5, v/v/v) and chloroform-methanol-water (4:3:2, v/v). The first solvent system was used for separating puerarin and the second for daidzein. The two isolated component showed high purified as determined by high-performance liquid chromatography analysis, the purify were99.0%and92.5%.