基于何首乌固有肝毒性假说的物质基础及肝毒性作用机制研究

According to the fact that a series of cases of Polygonum multiflorum Thunb and its preparations-induced hepatotoxicity were reported, cautions on the intake of them had been paid by CFDA. The hepatotoxic components of P. multiflorum and its mechanism have not been clarified. This brought out a lot of conversation on intrinsic and idiosyncratic drug-induced liver injury of P. multiflorum. Topics take “ establishment of liver injury model---liver toxicity component---potential hepatotoxic compounds---hepatotoxicity screening on in vitro and in vivo models---hepatotoxic compounds---elucidating the mechanism of hepatotoxicity” the research model to validate the found of the hypothesis..By use of the model organism zebrafish, the liver toxicity component (D) was obtained. 35 compounds were isolated using LC-MSn and modern liquid chromatography. 24 compounds were evaluated against models of hepatotoxicity in vitro and in vivo. 14 compounds of them exhibit prominent toxicity. These compounds included 8 anthraquinones and 6 anthrones. The hepatotoxic components of P. multiflorum and its mechanism have not been clarified. Thus, the steps we will be taking are as follows: (1) Isolation and identification of hepatotoxic compounds; (2) Evaluation of hepatotoxicity; (3) To explore the mechanism of hepatotoxicity and relationship between chemical structure and hepatotoxic effects. Finally, intrinsic hepatotoxic compounds of P. multiflorum were obtained and elucidated successfully, which can ensure clinical medication safety and rational.

何首乌及其制剂引起肝损伤的风险已引起国家药监总局的关注，而其肝损伤物质基础及作用机制还不清楚，进而引发其固有毒性和特异质毒性的争议。本研究拟采用“建立肝毒性评价模型—获得肝毒性组分—获得潜在肝毒性化合物—肝毒性模型筛选—获得肝毒性成分—阐明肝毒性作用机制”研究模式来验证假说的成立。.前期研究中，采用模式生物斑马鱼对何首乌进行肝毒性初步筛选，获得了肝毒性组分（D），利用LC-MSn和现代色谱学等手段，分离到35个化合物，对其中24个化合物进行初步的体内外肝毒性评价，获得14个肝毒性成分，分别为8个蒽醌和6个蒽酮类化合物。然而毒性成分是什么？毒性作用机制如何？目前还不清楚。为此，本课题拟开展：（1）肝毒性成分的分离与鉴定；（2）肝毒性成分的评价与筛选；（3）肝毒性成分作用机制及结构和毒性的关系研究。通过本课题研究，明确何首乌固有肝毒性成分，阐明肝毒性作用机制，从而更好地指导临床合理及安全用药。

近年来关于何首乌及其制剂所引起的肝损伤风险，已经引起了国家药品监督管理总局的高度关注，而其肝损伤的物质基础及作用机制依旧不清楚，进而引发其固有肝毒性和特异质肝毒性的争议。本项目基于何首乌及其毒性组分在体外和体内均表现一定的肝毒性作用，采用“建立肝毒性评价模型—获得肝毒性组分—获得潜在肝毒性化合物—肝毒性模型筛选—获得肝毒性成分—阐明肝毒性作用机制”的研究模式，对何首乌中的潜在肝毒性化学成分及作用机制，进行系统研究。.首先，采用大孔吸附树脂、反相硅胶柱色谱、凝胶Sephadex LH-20以及制备型高效液相色谱等技术，对何首乌中具有肝毒性作用特征组分进行系统的化学成分研究。通过测定化合物的红外光谱、紫外光谱、质谱、一维和二维核磁波谱数据以及CD谱等，鉴定其结构和构型。目前，从何首乌中共分离得到97个化合物，主要为蒽醌（单核和双核蒽醌）、二苯乙烯苷以及生物碱类成分，其中29个为新化合物。其次，以三种体外细胞和模式生物斑马鱼为评价模型，对分离得到的单体化合物进行毒性评价。结果表明部分单核和双核蒽醌以及萘类成分在三种体外细胞和模式生物斑马鱼模型中，均表现较强的毒性。再次，对具有肝毒性的双核蒽醌成分进行选择性富集与合成。建立一种快速获得大黄素-大黄素二蒽酮的制备方法；并以大黄素为原料，成功合成大黄素-大黄素二蒽酮。然后，对肝毒性成分进行肝毒性作用机制研究。结果发现大黄素和大黄素-大黄素二蒽酮均能显著促进肝细胞凋亡，且对人肝细胞HepG2细胞线粒体有氧呼吸的影响更为显著，进而可能造成细胞能量代谢紊乱，抑制ATP功能，诱导线粒体途径凋亡发生或增加氧自由基产生造成氧化损伤，进而诱导肝脏毒性。最后，对蒽醌进行体内肝毒性评价。大黄素-大黄素二蒽酮在小鼠急性肝毒性评价模型中，未见明显毒性反应。大黄素在大鼠体内肝毒性评价模式中，表现较强的肝毒性。.以上结果共发表论文12篇，其中SCI论文7篇，总影响因子>20；并申请中国专利2项，培养研究生3名。