养阴通脑颗粒有效成分在局灶性脑缺血大鼠体内药动学与药效学结合研究

Combined pharmacodynamics and pharmacokinetic in vivo study on active ingredients of Yangyin Tongnao Granules in rats with focal cerebral ischemia

目的:研究养阴通脑颗粒主要有效成分——葛根素、川芎嗪、阿魏酸在局灶性脑缺血再灌注模型大鼠体内的药动学与药效学的相应变化,建立药代动力学-药效动力学模型(PK-PD模型),为养阴通脑颗粒临床应用提供实验依据。方法:将SD大鼠随机分为正常组、模型组、给药组(葛根素50mg/kg、川芎嗪150mg/kg、阿魏酸150mg/kg),采用线栓法制作大鼠脑缺血再灌注模型后尾静脉注射给药,于不同时间点眼眶取血,应用HPLC检测3种有效成分的血药浓度。ELISA法检测转化生长因子-β1(TGF-β1)、缺氧诱导因子-1α(HIF-1α)的含量。建立PK-PD模型,应用DAS 3.2.6进行模型拟合以及数据分析。结果:高效液相色谱-二极管阵列检测器测定葛根素、川芎嗪、阿魏酸方法稳定,3种有效成分线性回归方程分别为川芎嗪Y=0.0139X-0.0032(R^2=0.9999),葛根素Y=0.0156X-0.0006(R^2=0.9999),阿魏酸Y=0.0690X-0.0203(R^2=0.9994),表明具有良好的线性关系。方法学考察稳定,日间、日内精密度RSD均<10%,回收率均在90%~120%之间,稳定性符合药物体内分析要求;与模型组比较,给药组15min后TGF-β1、HIF-1α含量显著降低(P<0.01);PK-PD拟合表明,给药组与两个药效指标拟合度良好,实测值和模型预测值相近。结论:高效液相色谱-二极管阵列分析方法专属性强,各成分分离度好,分析时长适宜,操作简单快速,总量统计矩可将川芎嗪、葛根素、阿魏酸的各项药动学参数整合。经养阴通脑颗粒有效成分治疗后,给药组TGF-β1、HIF-1α含量均有所改善,建立PK-PD模型,能够评价和预测中药多成分多效应之间的相关性。

Objective： To study changes of pharmacokinetics and pharmacodynamics of Yangyin Tongnao Granules＇ main active ingredient included puerarin, ligustrazine and ferulic acid in vivo focal cerebral ischemia rat model, and establish pharmacokinetics-pharmacodynamics（PK-DD） model, which will provide experimental basis for clinical application of Yangyin Tongnao Granules. Methods： Sprague-Dawley（SD） rats were randomly divided into control group, model group and medication group（puerarin 50 mg/kg, ligustrazine 150 mg/kg, ferulic acid 150 mg/kg）. The cerebral ischemia rat model was established by string, then treating with tail vein administration. Taking blood at different time points, blood concentration of three active ingredient were detected by HPLC. Contents of TGF-β1 and HIF-1α were detected by ELSISA. The PK-PD model was set up and the data was analyzed and conducted model fitting by DAS 3.2.6. Results： Puerarin, ligustrazine and ferulic acid were detected by HPLC-DAD method stably, and the linear regression equation of three active ingredients were： ligustrazine Y=0.0139 X-0.0032（R^2=0.9999）, puerarin Y=0.0156 X-0.0006（R^2=0.9999）, and ferulic acid Y=0.0690 X-0.0203（R^2=0.9994）, which indicated a good linear relationship. Methodology examination was stable. Daytime and intraday precision RSD were less than 10%, all recovery rate were between 90% and 120%, stability meets the in vivo drug analysis requirements. Comparing with model group, contents of TGF-β1 and HIF-1α were significantly reduced（P〈0.01）. PK-PD model fitting showed that the drug-administered group had good fit to the two pharmacodynamic indicators, the measured values and predicted were closed. Conclusion： HPLC-DAD method has specific, good separation of components, proper analysis time and simple and fast operation. Total statistical moment can integrate various pharmacokinetic parameters of Puerarin, ligustrazine and ferulic acid. After treating with active ingredients of Yangyin Tongnao Granules, contents of TGF-β1 and HIF-1α of medication group are improved. Setting up the PK-PD model can predict and evaluate the relation between multi-components and multi-effects of Chinese materia medica.