Extraction, Purification, Separation And Determination Of Soybean Isoflavone

As a mixture of various components, soybean isoflavone has been proven to posses various biological and pharmacological activities such as antioxidation, anticancer, estrogenoid and antiestrogen, prevention of cardiovascular diseases and relief of menopausal syndrome. However, each component shows differences in the above activities. Because of these, soybean isoflavone can be used in fields of functional food and medicine.In this thesis, four aspects of research in soybean isoflavone were done. First, different methods to extract soybean isoflavone were compared. The results of orthogonal experiment showed that the yield of soybean isoflavone was 4.07mg/g when the defatted soy flakes were soaked in 70% ethanol 6 times as much as the weight of raw material for 5 hours at 50℃, and that the yields were 4.23mg/g and 4.36mg/g when the defatted soy flakes were soaked in 50% ethanol 8 times as much as the weight of raw material for 1 hour at 70℃ under ultrasonic conditions (25kHz,l60W) without and with stirring at 300r/min, respectively. And three methods have no significant effects on antioxidative activity of soybean isoflavone.Second, three methods to purify soybean isofavone were studied. The results indicated that the contents of soybean isoflavone in purified products increased from 2.74% to 31.6%, from 11.9% to 57.0% and from 40.9% to 71.2% by ethyl acetate extraction at 25℃, adsorption on macroporous resin LSA-8 eluted with 70% ethanol at l.0BV/h and centrifugation at 2×103 r/min for 30 min at 40℃, respectively. The yields of soybean isoflavone were 31.1% and 32.3% by extractionand centrifugation, respectively.Third, three column chromatographies to separate the main monomers of soybean isoflavone were compared. The results showed that genistin, daidzin, genistein and daidzein could be separated by elution of silica gel (300-400 mesh) column with chloroform-methanol (5:1,V/V) at l.0BV/h, and that 90.3% daidzin and 92.0% genistin were obtained by gradient elution of polyamide column with 20% methanol and 40% methanol at l.OBV/h, and 95.7% genistin and 95.1% daidzin by elution of sephadex LH-20 gel column with 95% methanol at 2.0BV/h.Fourth, a systemic study on qualitative and quantitative analysis of soybean isoflavone was carried out. Soybean isoflavone and its monomers can be qualitatively detected by color reactions, fluorescence reactions, two dimensional paper and thin layer chromatographies. Genistin, daidzin and genistein all have maximal absorption peak at about 260nm while daidzein at about 248nm by ultraviolet scan, and these components can be evidently identified by adding moving reagents. Thin layer chromatography, ultraviolet spectrophotometry and high performance liquid chromatography (HPLC) can quantitatively determine soybean isoflavone and its monomers. In the three methods, HPLC has the highest precision, accuracy and sensitivity for quantitative determination of genistin, daidzin, genistein and daidzein.Based on the physical and chemical properties of soybean isoflavone, systemic studies were performed on the extraction, purification, separation and determinations of soybean isoflavone. The extraction and purification conditions for soybean isoflavone were optimized, and the separation and qualitative and quantitative determination methods for soybean isoflavone and its monomers were also established. The results can not only offer references on rational and effective utilization of soybean isoflavone, but also have definite theoretical and practical significances.