中药材中隐蔽型真菌毒素的产生规律及体内毒性释放机制研究

Mycotoxins have become one of the major exogenous contaminants that affect the quality and safety of Traditional Chinese Medicine (TCM). But the related research is just focused on the detection and prevention control of parent mycotoxins in TCMs at present. However, recent reports have proved modified mycotoxins were found in TCMs. Due to its unclear pharmackinetices, metabolism, and toxicity release in vivo, it poses a serious threat to the clinical safety for TCMs usage. Therefore, based on our previous studies, this project firstly aims to simulate the formation conditions of modified mycotoxins by using a dual-pathways for co-culturing "easily-contaminated TCMs-poisonous fungi" and "plant cell-parent mycotoxins". To clarify the formation rule of modified mycotoxins in the TCMs, the samples collected from co-culture matrix were analyzed by high resolution mass spectrometry and multiple mass defect filter data processing technology. Secondly, protection and de-protection strategies are employed in modified mycotoxins synthesis. Highly reactive sugar units are constructed and used to achieve parent mycotoxins glycosylation at different reaction sites. These synthesized chemicals which were easily found and required special attention in TCMs will solve the problem for reference substance lacking. Thirdly, different cell lines are used to test the toxicity of modified mycotoxins for cells in vitro. Besides, the metabolic profiles and distribution of mycotoxins in different tissues are investigated. The metabolism in vitro incubation models is also performed. The metabolism information both in vivo and vitro is used to clarify the release mechanism of modified mycotoxins through producing prototype or other forms. This project focuses on the urgent quality and safety issues of TCMs to conduct the critical studies. The project implementation will provide a scientific basis for the early screening and warning, prevention and control of the modified mycotoxins from the source for TCMs as well as for the establishment of limit standards.

真菌毒素已成为影响中药质量安全的主要外源性污染物之一，现阶段研究仅局限于中药材中真菌毒素原型的检测和监控。但近年报道证明，中药材中存在隐蔽型真菌毒素污染，由于其在中药材中的形成规律、体内代谢转化和毒性释放机制不明确，严重威胁了中药的临床安全用药。本项目拟在前期研究基础上，采用“易污染药材-产毒真菌”和“植物细胞-真菌毒素”双途径侵染共培养，模拟隐蔽型真菌毒素形成条件，结合高分辨质谱和MMDFs解析技术，阐明其在中药材中产生规律；利用保护与脱保护策略，构建高反应活性糖单元，实现真菌毒素不同反应位点糖苷化，高效合成中药材中易产生的需重点监控隐蔽型毒素，解决对照品匮乏难题；开展其不同细胞系毒性及组织脏器的代谢谱研究，推断其体内毒性和分布，结合体外孵育模型，阐明隐蔽型毒素的体内毒性释放机制。本项目聚焦中药急迫的质量安全问题，为中药中隐蔽型毒素的早期筛查、预警、源头防控及限量标准的制定提供科学依据。

本课题重点针对中药中隐蔽型真菌毒素，通过系统和深入的研究：（1）建立了冰冻-SPE净化结合LC-MS方法对种子类中药材中31种原型及隐蔽型真菌毒素进行了多残留测定；（2）对种子类药食同源中药材中真菌毒素进行了风险评估；（3）构建了基于CdSe@ZnS量子点电致发光传感器快速检测真菌毒素的方法；（4）对麦芽生产工艺中真菌多样性进行了动态分析；（5）对麦芽生产工艺中镰刀菌属及链格孢属真菌毒素进行了动态监测；（6）对麦芽生产工艺过程中隐蔽型毒素D3G及Z14G产生规律进行了研究；（7）建立了生物样本中Z14G及其代谢产物的分析方法；（8）对Z14G及ZEN在SD大鼠中的吸收、分布及转化进行了研究；（9）合成了重点监控隐蔽型真菌毒素Z14G；（10）对Z14G的细胞毒性及毒性释放机制进行了研究。通过该项目的实施，取得了如下成果：（1）共发表文章17篇，其中SCI文章11篇（中科院一区文章7篇），其中，影响因子IF≥10的文章3篇，最高影响因子为14.908，中文文章6篇。（2）申报发明专利 2 项。（3）培养博士2人，1人已毕业，1人在读；培养硕士研究生5名，均已毕业。（4）项目研究成果在相关学术会议以及公众号《药材安全学习与创新》上进行了科普性介绍，且在国内外研讨会进行了研究成果的推广与介绍。上述研究成果为中药中隐蔽型毒素的早期筛查、预警、源头防控及限量标准的制定提供了科学依据。