肠道菌群变化对黄芪甲苷的药代动力学影响

Effect of intestinal flora changes on pharmacokinetics of astragalosideⅣ

探究肠道菌群变化对黄芪甲苷在2型糖尿病大鼠体内的药代动力学影响。前期实验采用高糖高脂饲料联合低剂量链脲佐菌素(STZ)腹腔注射建立2型糖尿病大鼠模型,分为模型组、黄芪甲苷组、小檗碱组及黄芪甲苷与小檗碱联合用药组,每组5只。大鼠灌胃2周后,取其粪便进行肠道菌群16S rRNA测序实验。完成前期实验1 d后,在4组大鼠中开展黄芪甲苷的药代动力学实验。以人参皂苷Rb1(ginsenoside Rb1)为内标,血浆样品经甲醇沉淀蛋白,采用UPLC-MS/MS测定不同时间点黄芪甲苷血药浓度。采用Waters Acquity UPLC BEH-C_(18)色谱柱(2.1 mm×100 mm,1.7μm),以乙腈(A)-含0.2%甲酸的5 mmol·L^(-1)甲酸胺水溶液(B)为流动相,梯度洗脱,流速0.4 mL·min^(-1),进样量5μL,柱温40℃;质谱条件为电喷雾离子源(ESI),正离子模式,多反应监测。方法经特异性、线性范围、准确度、精密度、稳定性、稀释效应等确证适合用于血浆中黄芪甲苷的测定。绘制血药浓度-时间曲线,并用DAS 3.2.8软件计算相关药代动力学参数。结果显示,黄芪甲苷入血吸收浓度在03.95 h呈正比趋势增长,3.95 h后黄芪甲苷浓度开始逐渐下降,36 h后代谢完全。小檗碱组、黄芪甲苷组、黄芪甲苷与小檗碱联合用药组分别较模型组黄芪甲苷的血药浓度-时间曲线下面积AUC_(0-t)、药峰浓度C_(max)增加,但无显著性差异。提示药物改变肠道菌群后,但并不会必然导致皂苷类成分的药代动力学特征发生变化。

The effect of intestinal flora changes on the pharmacokinetics of astragalosideⅣin rats with type 2 diabetes mellitus was explored in this study.The rat model in preliminary experiment was established by high-sugar and high-fat diet combined with the intraperitoneal injection of low-dose streptozotocin(STZ).Rats were divided into model group,astragalosideⅣgroup,berberine group and combination group(five rats in each group).After two weeks of gavage,the rats′feces was taken for 16 S rRNA sequencing of intestinal flora.Pharmacokinetic experiments were performed on astragalosideⅣin the four groups one day after the preliminary experiment.Plasma samples were precipitated in methanol with ginsenoside Rb1 as an internal standard,and the plasma concentrations of astragalosideⅣat different time points were determined by UPLC-MS/MS.The chromatographic separation was performed on a Waters Acquity UPLC BEH-C_(18)column(2.1 mm×100 mm,1.7μm)via gradient elution.The mobile phase was acetonitrile(A)and 5 mmol·L^(-1)ammonium formate solution with 0.2%formic acid(B).The flow rate was 0.4 mL·min^(-1),the injection volume 5μL and the column temperature 40℃.The mass spectrometry was carried out with electrospray ionization source(ESI)in multiple reaction monitoring and positive ion modes.The specificity,linearity range,accuracy,precision,stability and dilution effect of the method all met the requirements for the determination of astragalosideⅣin plasma.Plasma concentration-time curves were plotted and relevant pharmacokinetic parameters were calculated by DAS 3.2.8.The results showed that the concentration of absorbed astragalosideⅣincreased within 0-3.95 h and began to decline since 3.95 h.After 36 h,the metabolism was complete.The area under the plasma concentration-time curve(AUC_(0-t))and the peak concentration(C_(max))of astragalosideⅣwere increased in the three administration groups compared with the model group,but without significant difference,which suggested that the pharmacokinetic characteristics of saponin components would not necessarily change after the drug-induced alteration of intestinal flora.