Simultaneously determination of daidzin and its metabolite in rat intestinal bacteria test solution in vitro by HPLC
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摘要: 目的 建立HPLC法测定体外大鼠肠道菌液中的大豆苷及其代谢物，研究大鼠肠道菌群对大豆苷的代谢情况。 方法 采用大鼠离体粪便温孵法，将大豆苷与大鼠肠道菌群培养液进行厌氧温孵。温孵样品采用甲醇沉淀蛋白处理后进行HPLC法分析，色谱条件：色谱柱为Diamonsil C18柱；流动相为0.2%乙酸水溶液与甲醇，梯度洗脱；检测波长为260 nm；柱温为25℃；进样量为10 μl。 结果 大豆苷及其代谢物大豆苷元分别在98.0~490、57.6~288 μg/ml浓度范围内线性良好，高、中、低3个浓度的相对回收率在96.6%~114.0%之间，相对标准偏差小于9.9%，表明本法的准确度和精密度符合要求。大豆苷在肠道菌液中代谢生成大豆苷元，其消除半衰期为37.6 min。 结论 所建立的HPLC法适合同时测定体外大鼠肠道菌液中的大豆苷及其代谢物，大豆苷在体外代谢的主要途径为糖苷键断裂转化成对应苷元。Abstract: Objective To establish an HPLC method for simultaneously determination of daidzin and its metabolite in rat intestinal bacteria test solution in vitro, and study the metabolism of daidzin by rat intestinal bacteria. Methods The incubation of daidzin was carried out at 37℃ with rat intestinal bacteria test solution. The samples were precipitated by methanol followed by a separation with HPLC method. The conditions were as follows:the chromatographic column was Ultimate C18; the mobile phase was 0.2% acetic acid and methanol (gradient elution); the detection wavelength was 260nm, and the column temperature was 25℃. Results Daidzin and daidzein showed good linearity with the range of 98.0~490μg/ml and 57.6~288μg/ml, respectively. The accuracy of daidzin and daidzein was ranged from 96.6%~114.0% and the precision of the two analytes was within 9.9%. Daidzin was transformed to daidzein in the rat intestinal bacteria test solution, and its elimination half-life was 37.6 min. Conclusion The established HPLC method was suitable for the determination of daidzin and its metabolite. Daidzin could be metabolized by rat intestinal bacteria in vitro,and the main metabolic pathway was transforming daidzin into corresponding aglycone through breaking the glucoside bond
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Key words:
- intestinal bacteria /
- metabolism in vitro /
- daidzin /
- HPLC
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