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目的通过制备键合β-环糊精琼脂凝胶微球(AG-β-CD)作为分离介质,对大豆异黄酮粗品中大豆苷元进行高效分离纯化。方法利用琼脂为原料,通过乳化、交联并键合功能基团β-环糊精(β-CD)制备AG-β-CD;将其作为一种分离介质,通过考察流动相、洗脱体积流量和微球负载量3个方面,确定AG-β-CD分离纯化大豆苷元的工艺并进行验证,质谱和核磁共振鉴定其结构;通过与其他2种黄酮类物质表没食子儿茶素没食子酸酯(EGCG)和葛根素在C18反相柱色谱保留行为比较,结合auto DOCK4.0进行分子模拟,以及流动相乙腈体积分数变化对3种黄酮类物质在AG-β-CD固定相上的保留时间的变化曲线证明其色谱机制。结果大豆异黄酮粗品中主要成分为大豆苷元(57.14%),AG-β-CD的负载量为1.33 mg/m L(大豆异黄酮粗品总量/柱体积),体积流量为2 BV/h时,通过20%乙醇洗脱2 BV、40%乙醇洗脱1.33 BV、70%乙醇洗脱6~7 BV,得到质量分数≥95%(平均质量分数96.98%)的大豆苷元,收率为97.86%。色谱机制实验表明AG-β-CD具有亲水和反相双重保留与分离作用。结论 AG-β-CD能够高效地分离纯化大豆苷元。
OBJECTIVE To isolate and purify daidzein from soybean isoflavone by high performance liquid chromatography (AG-β-CD). Methods Ag-β-CD was prepared by emulsifying, cross-linking and bonding β-cyclodextrin (β-CD) with agar as a separation medium. The mobile phase was eluted Flow cytometry and microspheres loading, the process of the separation and purification of daidzein AG-β-CD was confirmed and verified by mass spectrometry and nuclear magnetic resonance. By comparing with other two flavonoids epigallocatechin gallate The retention behavior of EGCG and puerarin in C18 reversed-phase column chromatography was compared with that of auto-DOCK4.0, and the retention of three flavonoids on the AG-β-CD stationary phase was determined by the mobile phase acetonitrile volume fraction The time curve shows the chromatographic mechanism. Results The main components of soy isoflavones were daidzein (57.14%), AG-β-CD loading of 1.33 mg / m L (total volume of crude soy isoflavones / column volume), volume flow rate of 2 BV / h , 2 BV was eluted with 20% ethanol, 1.33 BV with 40% ethanol and 6 ~ 7 BV with 70% ethanol to obtain the daidzein with the mass fraction ≥95% (average mass fraction 96.98%), the yield was 97.86%. Chromatographic experiments show that AG-β-CD has both hydrophilic and reversed-phase retention and separation. Conclusion AG-β-CD can efficiently isolate and purify daidzein.