游离金雀异黄素和大豆苷元大鼠尿液排泄动力学研究

Investigation of urinary pharmacokinetics of free genistein and daidzein in rats

目的建立大鼠尿液中游离金雀异黄素（genistein,GT）和大豆苷元（daidzein,DD）同时定量的分析方法,获得其口服后大鼠尿液排泄动力学参数,为大豆异黄酮生物利用度研究和评价提供新的方法。方法大鼠单次灌胃给予GT和DD混悬液（15.0 mg/kg）,在给药后不同时间段收集尿液,尿液经冷冻干燥后,加1.0 m L甲醇溶解提取目标化合物。采用高效液相色谱法（high performance liquid chromatography,HPLC）法测定游离GT和大DD的浓度,采用Krommasil C18色谱柱,以甲醇-水（52∶48）为流动相,流速1.0 m L/min柱温为30℃,检测波长254 nm。结果建立的高效液相色谱法-紫外法（HPLC-UV）方法专属性强,在0.5~10.0μg/m L浓度范围内具有良好的线性关系,良好的精密度及较高的准确度。尿液排泄动力学曲线图上第4.5 h和13.5 h别显示小肠和肝肠循环2个吸收峰,游离GT和DD清除半衰期分别为（7.5±0.59）h和（8.5±1.2）h,72 h尿液累积量分别为（45.2±7.2）μg和（56.1±12.2）μg。结论所建立的方法可靠,简便快速,可用于比较和评价大豆异黄酮类制剂的口服吸收生物利用度和代谢动力学研究。

Objective To offer an alternative approach to evaluate the bioavailability of isoflavone by urinary excretion, establishing an HPLC method for the determination of free genistein and daidzein in rat urine and obtaining the parameters of urinary pharmacokinetics after their oral administration. Method The rats were orally administered with a single dose of genistein and daidzein（15 mg/kg）. Urine samples were collected from different time periods. The samples were lyophilized and then extracted ultrasonically by 1.0 m L methanol. HPLC analysis was performed by a reversed-phase HPLC column（Kromasil 100 A, C18）. The elution conditions for HPLC were methanol and water at the ratio of 52 %（v/v）for isocratic elution, the column was maintained at 40℃, flow rate was 1.0 m L/min, and detection was set to 254.0 nm. Results An excellent liner relationship was obtained by developed HPLC-UV method in the range of 0.5~10.0 μg/m L with high specificity,good precision and high accuracy. A plot of urinary isoflavone concentration versus time suggests that there were two peaks at 4.5 h and 13.5 h related to absorption in intestine and enterohepatic circulation. The half-time in urine was found to be（7.5±0.59） h and（8.5±1.2） h for genistein and daidzein respectively. The cumulative excretions of genistein and daidzein for 72 h were（45.2 ± 7.2） μg and（56.1 ± 12.2） μg, respectively.Conclusion The method is reliable, simple, rapid and suitable for comparison and evaluation of oral bioavailability of isoflavone pharmaceutics as well as pharmacokinetic study.