基于代谢酶和转运子的茵陈蒿汤促进胆红素代谢的作用机理研究

As a classic prescription in the treatment of jaundice, Yinchenhao decoction has a satisfactory clinical effect. But the mechanism of removing jaundice and the pharmacodynamic substance foundation of Yinchenhao decoction is not clear, presently. In this project, we speculate the choleretic effect of Yinchenhao decoction would be related to the glucuronidation process of its active ingredients in rats based on the correlation of Yinchenhao decoction and bilirubin metabolic processes. And the mechanism is maybe related to adjustment of key bilirubin metabolic enzymes and transporters. This hypothsesis will be confirmed by the following researches. At first, the in-vivo metabolic spectrum of Yinchenhao decoction in normal rats and jaundice rats will be studied by HPLC-Q/TOF technology. Then taking Yinchenhao decoction as a model drug, using both HPLC-MS/MS and enzymolysis biology technologies to establish a method for pharmacokinetics of chinese medicines which mainly remove jaundice and are metabolized in phase Ⅱ. In addition, it also will be applied to analyze the pharmacokinetics of Yinchenhao decoction in normal rats and jaundice rats. At last, the induction effects of Yinchenhao decoction, its active ingredient and its major metabolites on the key bilirubin metabolic enzymes (UGT1A1 and β-GCD) and transporters (Mrp2 and Bsep) will be studied. And the relationship of major active ingredients and metabolites’ concentration at site of action with its choleretic effect will also be studied to explore the mechanism and pharmacodynamic substance foundation of Yinchenhao decoction in vivo. This study will lay the foundation for further researches of other different Chinese medicines which have the same effects of curing jaundice.

茵陈蒿汤作为治疗黄疸的经典方，临床疗效良好，但其利胆退黄作用机制和药效物质基础目前尚不明确。本项目基于茵陈蒿汤和胆红素体内代谢规律的相关性，推测茵陈蒿汤有效成分体内的葡萄糖醛酸化过程可调节“黄疸”大鼠体内胆红素代谢关键酶和转运子的活性，促进胆红素代谢。研究内容为：采用HPLC-Q/TOF技术建立茵陈蒿汤在正常和黄疸大鼠体内的代谢物谱；采用现代分析与生物酶解技术，以茵陈蒿汤为模型药物，建立以Ⅱ相结合反应为主要代谢途径的利胆退黄中药的药代动力学研究方法，并用于茵陈蒿汤在正常与黄疸大鼠体内药代动力学差异研究，阐明其体内药代动力学规律。最后，通过研究茵陈蒿汤、有效成分及其主要代谢物对胆红素代谢关键酶（UGT1A1和β-GCD）和转运子（Mrp2和Bsep）的调节作用，结合药效作用部位的效应成分含量与利胆作用的相关性，探索其体内药效物质基础及作用机制，为其他利湿退黄中药的体内过程研究提供思路和方法。

背景：茵陈蒿汤作为治疗黄疸的经典方，临床疗效良好，但其利胆退黄作用机制和药效物质基础目前尚不明确。本项目基于茵陈蒿汤和胆红素体内代谢规律的相关性，推测茵陈蒿汤有效成分体内的葡萄糖醛酸化过程可调节“黄疸”大鼠体内胆红素代谢关键酶和转运子的活性，促进胆红素代谢。研究内容：采用多组分含量测定技术首次建立茵陈蒿汤14种成分的含量测定方法，并进行其物质基础研究。采用UPLC-QTOF技术建立茵陈蒿汤体内代谢物库，探索茵陈蒿汤中主要成分的Ⅱ相代谢规律。并采用生物酶解结合HPLC-MS/MS分析技术，建立了茵陈蒿汤中10种原型药物和4种Ⅱ相代谢产物在大鼠血浆中的含量测定方法，并进行茵陈蒿汤在正常大鼠和黄疸大鼠体内药动学差异研究。最后结合茵陈蒿汤及其有效成分（栀子苷和大黄酚）、其代谢物栀子苷元-葡萄糖醛酸结合物对ANIT诱导的黄疸大鼠利胆作用中对大鼠肝脏主要代谢酶UGT1A1和9种转运体的调节作用，初步探索其利胆作用的机制。研究结果：1）茵陈蒿汤提取液中主要成分为栀子苷、京尼平龙胆双糖苷、咖啡酸、大黄酸、绿原酸等，分别占14种成分总含量的71.0%、17.2%、4.8%、2.0%和2.1%。2）体内代谢研究显示大鼠灌胃茵陈蒿汤后，体内共检测到11种原型化合物和19种代谢物，其中8种为II相代谢物。3）采用体内外代谢技术制备了2种主要II相代谢物，经结构确证，分别为栀子苷元-1-O-葡萄糖醛酸苷和大黄素-3-O-葡萄糖醛酸苷。4）药代动力学研究显示8种有效成分及4种II相代谢物在正常和胆汁淤积模型大鼠中药物动力学行为存在较大差异，可能与不同模型大鼠肝脏的代谢酶和转运体的含量水平相关。5）药理实验研究显示茵陈蒿汤对胆汁淤积大鼠有较好的利胆作用，主要表现在增加胆汁流量，促进胆红素代谢。6）其有效成分栀子苷、栀子苷元-葡萄糖醛酸结合物也有较好的利胆作用，可能是茵陈蒿汤利胆作用的物质基础，作用机制与其对关键代谢酶Ugt1a1和转运体的调节作用相关，本研究将为其它利湿退黄中药的体内过程研究提供新的思路与研究方法。