基于药代动力学和毒代动力学的黑豆汁制何首乌减毒增效机制研究

Radix Polygoni Multiflori Preparata is commonly used in chinese medicine clinical as a reinforcing agent.However, there are reports about the patients diagnosed with toxic hepatitis following ingestion of it. Through our previous studies, its efficacy, toxicity have been relatively clear.Moreover , it was showed attenuation and synergia in its medicine compatibility with Black Soybean. On the other hand, the analysis method of its chemical composition in vitro and in vivo has been established. In this study,according to its characteristics that effective dose can cause liver toxicity, pharmacokinetic combined with toxicokinetic will be applied to study attenuation and synergia mechanism in its compatibility with Black Soybean. Rats are administrated drug for 1day, 7 days, 21 days, 90 days, to study its liver toxicity resulting process by tracking the liver toxicity biomarkers in blood.The end of administration, chemical composition metabolism and transport changes in vivo, gene expression changes of transporter proteins and enzymes in intestinal and liver, will be researched and compared in non-toxic effect groups and toxic effect groups. On this basis, Black Soybean's regulatory role to these parameters will be observed. By the way of both combined, it would be to get evidence of "toxic" from the opposite direction by "attenuated" ,and to obtain adjuvant evidence about "efficacy" by "synergia". Through the above research, the correlation among these parameters and efficacy and toxicity are going to be detemined. And attenuation and synergia mechanism for the medicine compatibility of Radix Polygoni Multiflori Preparata and Black Soybean will be explained . And further,the study on the interaction between drug molecules with enzymes or transporter protein will be carried out to found molecular mechanisms of toxicity and efficacy, also attenuated and synergia in drug compatibility .

（制）何首乌作为传统补益中药在临床广泛应用，但近几年关于服用（制）何首乌导致肝毒性的报道屡见不鲜。课题组前期研究对制何首乌药效、毒性及黑豆汁减毒增效作用等已较为明确，并建立了体内外成分分析方法。本课题根据制何首乌有效剂量可致肝毒性的特点，结合药代动力学和毒代动力学的研究方法，以有效剂量给大鼠灌胃1天、7天、21天、90天，跟踪检测血液中肝毒性生物标识物，考察其肝毒性产生的进程，研究和比较在制何首乌有效剂量的无毒效应和有毒效应状态下，大鼠体内化学成分代谢和转运的变化，及肠道和肝脏转运蛋白和酶的基因表达变化，同时观察黑豆汁对各项参数的调节作用，两者有机结合，以"减毒"反证"有毒"，以"增效"佐证"药效"，判断各项参数变化与制何首乌药效和毒性的相关性，阐释黑豆汁制何首乌减毒增效的作用机制。进一步通过研究药物分子与酶及转运蛋白的相互作用，发现药物毒性、药效机制及配伍减毒增效机制。

本课题选用同一批次生何首乌进行实验，清蒸制首乌及黑豆制首乌由实验室自行进行炮制，保证后期实验可平行比较。我们首先对生首乌及其炮制品进行了体外物质基础分析比较，在此基础上，对生、制何首乌灌胃大鼠体内代谢产物进行了分析鉴定，发现体内代谢产物种类基本一致，但含量、蓄积分布有明显差异，进一步对尿液中部分代谢产物进行分离纯化。在以上工作基础上，建立体内原型及代谢产物LC-MS/MS分析方法，结合药代动力学及毒代动力学实验，跟踪跟踪主要肝毒性指标产生的进程，研究转运蛋白及酶的基因表达变化，分析体内药物代谢、吸收、分布及蓄积情况：灌胃给予等量的何首乌提取液时，生首乌于3天开始显示毒性作用，制首乌7天开始显示。肝脏转运蛋白1d 无明显变化，14 d时P-gp、MRP3及UGT1A1有明显的变化，P-gp、MRP3升高，UGT1A1下降。而肠道转运蛋白于14d 的时MRP2降低，MRP3升高。结果显示配伍黑豆汁具有明显的影响作用。体内药代及毒代结果显示，血浆中大部分原型及代谢产物特征基本一致，而New E（二苯乙烯苷代谢产物）在1 d 未检测到，14 d含量较高，有明显的蓄积作用。二苯乙烯苷及大黄素主要分布于肝脏、肺脏，且具有一定的蓄积作用。大黄素-8-O-葡萄糖苷主要在生何首乌组组织中可检测到，且配伍黑豆汁可降低大黄素8-O葡萄糖苷含量，其余大部分代谢物主要分布在肝肾中。Usssing chamber结果提示，大黄素可能是P-gp及MRP2的底物、P-gp的诱导剂、是MRP3的抑制剂，大豆苷元可 可增加大黄素的肠道吸收。体外实验表明，生首乌、蒸制首乌、黑豆制首乌和黑豆提取液对CYP450都有一定程度的抑制作用，而蒸制首乌和黑豆制首乌对CYP450的抑制作用较生首乌减弱，表明炮制后的首乌能够降低对CYP450酶的影响。生首乌提取液与黑豆提取液配伍后，对CYP450酶亚型的影响较生首乌有减弱趋势，提示用黑豆炮制何首乌的合理性。何首乌和黑豆中的单体成分对CYP2E1、CYP2C9、CYP1A2、CYP2C19和CYP2D6有一定抑制作用，对CYP3A4无显著影响；炮制后含量增加的游离蒽醌类成分、没食子酸以及含量降低的二苯乙烯苷对CYP450都有一定的影响，表明何首乌炮制前后的药理功效的差异与其成分变化相关。