基于大鼠心肌缺血模型的荭草活性成分PK-PD相关性研究

Polygonum orientale is commonly used for treating cardiovascular diseases in the folk and the minority in Guizhou province, which has strong resource advantages with regional feature and potential for development. In our previous study, we have found the anti-myocardial ischemia active ingredients of Polygonum orientale mainly consist of phenolic acids and flavonoids, such as isoorientin, orientin, catechuic acid, vitexin and so on. Moreover, we have investigated the pharmacokinetic study of effective ingredients in Polygonum orientale both normal rats and the rat model of myocardial ischemia. Unexpectedly, the results about metabolism in vivo and relevant transport process of effective ingredients in Polygonum orientale between them were great changed. In the current study, we will further study the difference of the pharmacokinetics of anti-myocardial ischemia active ingredients in Polygonum orientale between normal and model rats. Then, the mechanism of pharmacokinetic difference were investigated from the expression of transporter proteins in small intestine and liver of rats. Moreover, combined with hypoxia-reoxygenation model of myocardial cells, we will build up the PK-PD model of anti-myocardial ischemia active ingredients in Polygonum orientale based on the rat model of myocardial ischemia and cellular level with multi-index. Finally, our study would further clarify the substance of multi-effect ingredients directly responsible for anti-ischemic effect , investigate the mechanism of traditional Chinese medicine pharmacokinetic differences under disease state and its scientific connotation correlation with PK-PD , and provide the scientific basis for the development of innovative drugs and the further development of medicinal resources of Polygonum orientale.

荭草为贵州省少数民族常用药物，用于心血管疾病的治疗，具有较强地域特色资源优势和发展潜力。申请者在前期工作中明确了荭草保护心肌缺血的活性成分主要为异荭草素、荭草素、儿茶酸、牡荆素等酚酸类和黄酮类化合物；并探讨了荭草有效成分在正常大鼠和心肌缺血模型大鼠体内药代动力学特性，结果提示正常大鼠与心肌缺血模型大鼠的体内代谢与相关转运过程发生明显变化。本课题在前期工作基础上，从整体和离体水平比较荭草抗心肌缺血活性成分在正常大鼠和心肌缺血模型大鼠体内的PK差异，进而从小肠和肝脏中外排转运蛋白和内流转运蛋白表达的差异探讨PK差异的机制；结合心肌细胞缺氧复氧模型，建立基于多指标整体动物及细胞水平的荭草抗心肌缺血活性成分的PK-PD结合模型，阐明荭草多效应成分在体内与抗心肌缺血作用直接相关的物质，探讨疾病状态下中药药代动力学差异机制及其PK-PD相关性的科学内涵，为荭草药用资源深度开发提供科学依据。

荭草为贵州省少数民族常用药物，主要用于治疗心血管疾病，具有较强的地域特色资源优势和发展潜力。前期研究已经明确了荭草抗心肌缺血损伤的活性成分主要为黄酮类和酚酸类化合物。但有关其活性成分在不同机体状态下的体内过程及作用机制研究等尚不明确，制约了荭草深层次开发利用。本项目在心肌缺血大鼠模型（MI）建立的基础上，开展了口服荭草提取物后正常和模型大鼠的血浆指纹图谱差异研究，发现荭草提取物在MI大鼠体内的入血成分数量较正常组减少，而原型成分的代谢产物数量比正常组多。基于指纹图谱的差异，进一步开展了不同机体状态下的药动学研究，研究发现单次给药时，与正常组相比，病理状态下原儿茶酸和牡荆素在体内吸收降低，消除增加；而荭草素、异荭草素等4个成分消除减慢，使得体内的蓄积增加，作用时间延长，吸收总量增加；其差异的机制可能与肝脏CYP2C19、CYP1A2酶活性和表达降低，CYP3A2、CYP2D4的酶活性或表达增加有关。多次给药病理状态下各成分吸收均降低，吸收时间延长，清除率有增加趋势，吸收总量降低；其差异的机制可能与CYP3A2、CYP1A2的酶活性或表达量增加、小肠外排转运蛋白P-gp的表达上调有关。在此基础上，结合各药效指标的动态变化规律，量化了荭草活性部位中各入血成分的PK-PD关系，阐明了活性成分与药效的相关性。体内过程研究显示，六个成分可迅速分布至各组织脏器中，模型状态下在各组织中分布的量较低，且排泄量也较低，均不足给药量的5%。肠吸收特性研究显示，在模型状态下，各成分的主要吸收部位下移至结肠段，且6个成分均可能是转运蛋白P-gp的底物。细胞药动学研究显示，在整体细胞中，荭草素、牡荆素、槲皮苷、异荭草素与细胞核和线粒体的亲和力均较高，山奈素-3-O-β-D-葡萄糖苷与线粒体亲和力较高。提示荭草保护心肌细胞中，这些化学成分可能主要是通过保护线粒体或者作用于细胞核而发挥药理效应。上述研究结果为荭草创新药物研制及药材资源深度开发提供了科学依据。