基于大鼠心脏毒性指数的生附子毒性预测与皂化煮制减毒机制研究

Aconitum materials are good folk and clinical medicines for treatment of rheumatic illnesses. However, severe aconite poisoning is always caused by the irrational use or processing methods, which results in loss of life and property. Currently, the legal toxicity control system of Aconitum materials is relatively simple just determining total contents of three strong toxic compounds and not emphasizing the differences of their toxic strengths. Moreover, other compounds contributing to the total toxicities of materials are always neglected, which results the early-prediction accuracy of materials are not well. In this project, the fresh Fuzi (lateral root of Aconitum carmichaelii Debx) were selected as model material and we were to use the in vitro and in vivo model of cardiotoxicity and the developed knockout-fishing/spectral efficacy integration and characterization approach for establishment of the integrated identification strategy of the toxic chemical group based on the toxicity contribution index (TCI), which helped to comprehensively pick out the cardiotoxic profiles in fresh Fuzi. In the meantime, the cardiotoxicity prediction approach of Fuzi was also constructed by summing the values of the product for the toxicity strength and the corresponding concentrations of all toxic compounds. This approach could be applied for early-prediction of the total toxicities of the fresh and processed Fuzi from different origins. Moreover, the changes of classical boiling and novel boiling saponification were monitored to elucidate the scientific connotation for decreasing the toxicity and provide a good hint for development of novel processing attenuated approaches.

乌头类药材在民间和临床上是治疗风湿骨病顽疾的良药，但常因不合理的用药及炮制方式而引发乌头中毒事件，造成重大区域性财产和生命损失。目前，乌头类药材毒性控制方法主要关注三个强毒性成分的总量，却忽略各自毒性强度的差异，且未体现其他成分对药材整体毒性的贡献，致使药材毒性无法准确预测。故本课题拟选强毒的生附子作为模式药材，利用体内外心脏毒性模型和成分群缺失-捕获/谱效集成表征技术，建立基于毒性贡献指数的有毒物质（群）的识别体系，明确生附子的心脏毒性物质谱；通过桥接毒性成分的强度和浓度而构建基于多指标体系的附子心脏毒性预测方法，早期评估不同产地生附子及炮制品的毒性强度；通过监测经典水煮和新型油脂共煮方式对生附子毒性变化的规律，系统揭示新型皂化煮制减毒的科学内涵，为新型炮制减毒工艺的开发提供线索。

生附子等乌头类中药是一类常用的毒效双性中药，其具有良好的强心及抗风湿作用，然而使用不当常引发恶性心脏毒性及神经毒性，造成重大财产和生命损失。因此本课题围绕生附子等乌头类中药的毒性评价、减毒机制和限量标准，展开系列研究，对乌头类中药的炮制规范、新药研发等合理应用具有重要意义。1）首先基于电喷雾萃取电离质谱技术建立了乌头类中药煎煮体系实时在线监测方法，全过程表征了生附子煎煮过程中化学成分的具体变化。2）建立了总加权毒性指数概念及计算方法，准确评价并预测了生附子煎煮体系的整体毒性，并获得安全煎煮时间。3）系统比较了生附子分别与猪蹄、鸡肉、瘦猪肉和牛肉共煮时的减毒效果，发现与猪蹄共煮时减毒效果最佳，整体毒性可降低至68%，安全煎煮时间缩短至59.01 min。4）首次揭示了毒性二酯型乌头碱类成分水煮减毒的分子机理为内部亲核取代机理，发现脂肪组织中的脂肪酸类成分可对该反应起到显著催化效果，从而提出了新颖有效的促进减毒存效的乌头类药材炮制方法。5）建立在线萃取电喷雾电离技术，对各种剂型的含乌头类中药的中成药进行直接分析，并基于总加权毒性指数，系统评价了20种市售含乌头类中药的中成药的毒性强弱并建立了毒性乌头碱类成分限量标准。6）建立了基于大鼠原代心肌细胞模型的乌头类中药心脏毒性和药效评价平台，系统评价了附子中9个主成分的心脏毒性和强心作用。7）探究了民间煮附子不能断火和加冷水的科学内涵，发现断火和加冷水可明显延长解毒时间。在本项目资助下，共发表学术论文7篇，其中SCI收录5篇，中科院一区论文3篇，IF>5论文2篇，中文核心期刊2篇，申请国家发明专利2项。本项目负责人顺利从助理研究员晋升为副研究员，获得国家级人才项目中国科协“青年人才托举工程”资助，数次在第二届中国中药资源大会等国内外会议作学术报告。本项目共顺利培养1名博士研究生和2名硕士研究生，参与学生多次获得“博士生优秀奖学金”等各类校级及院级奖学金。