雷公藤多苷对大鼠与人肝微粒体CYP3A酶活性的体外抑制作用

发布时间：2018-07-09 21:30

  本文选题：雷公藤多苷 + 体外代谢模型　； 参考：《中国药房》2015年04期

【摘要】：目的:建立液相色谱-串联质谱(LC-MS/MS)法测定大鼠和人肝微粒体中1′-羟基咪达唑仑的含量,研究雷公藤多苷对体外大鼠与人肝微粒体细胞色素P45(0CYP)3A酶活性的抑制作用。方法:将不同质量浓度的雷公藤多苷(0.5、1.0、2.0、5.0、10.0、50.0、100.0μg/ml)分别与大鼠和人肝微粒体(0.4 mg/ml)共同孵育20 min后终止反应,采用LC-MS/MS法测定孵育液中咪达唑仑的代谢产物1′-羟基咪达唑仑的浓度以评价CYP3A酶活性并计算酶活性抑制率,采用Graph Pad Prism 5.0软件进行拟合并计算半数抑制浓度(IC50)。色谱柱为Agilent Eclipse XBD-C18,流动相为水-甲醇(梯度洗脱),流速为0.3 ml/min,柱温为30℃,进样量为2μl。采用电喷雾电离(ESI),定量离子为m/z 342.01→324.1(1′-羟基咪达唑仑)、m/z 383.01→337.2(内标氯雷他定)。结果:1′-羟基咪达唑仑检测浓度在0.01~3μmol/L范围内与其和内标物峰面积比值呈良好线性关系;精密度试验RSD均小于10%,提取回收率为97.92%~101.65%,方法回收率为96.70%~104.10%,稳定性试验RSD均小于10%。雷公藤多苷对大鼠和人微粒体中CYP3A的IC50分别为(48.610±1.32)μg/ml和(4.754±1.12)μg/ml;在0.5~100.0μg/ml范围内雷公藤多苷对CYP3A酶活性有抑制作用,且与质量浓度呈正相关。结论:所建立的LC-MS/MS法可用于检测大鼠和人肝微粒体中1′-羟基咪达唑仑的含量。雷公藤多苷对体外大鼠和人肝微粒体药物代谢酶CYP3A均存在抑制作用,且对人肝微粒体CYP3A的抑制作用明显强于大鼠。
[Abstract]:Aim: to establish a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for the determination of 1-hydroxy-midazolam in rat and human liver microsomes and to study the inhibitory effect of tripterygium wilfordii polyglycosides on cytochrome P45 (0CYP) 3A enzyme activity in rat and human liver microsomes in vitro. Methods: different concentrations of tripterygium wilfordii polyglucoside (0.51.02.00.0.0.0.0.100.0 渭 g/ml) were incubated with rat and human liver microsomes (0.4 mg/ml) for 20 min, respectively, and the reaction was terminated after incubating with different concentrations of tripterygium wilfordii for 20 min. In order to evaluate the activity of CYP3A and calculate the inhibition rate of CYP3A, LC-MS / MS method was used to determine the metabolite of midazolam in incubation solution. The half inhibitory concentration (IC50) of midazolam was calculated using Graph Pad Prism 5.0 software. The chromatographic column is Agilent Eclipse XBD-C18, the mobile phase is water-methanol (gradient elution), the flow rate is 0.3 ml / min, the column temperature is 30 鈩，

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