基于CCS策略的中成药龙胆泻肝丸基原物种鉴定研究

Substandard and counterfeit natural herbal products (NHP), including traditional Chinese patent medicines (TCPM), health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants and/or adulterants. An efficient method for the supervision of herbal species in the multi- ingredient natural herbal product was greatly needed. ..To improve the pharmacopoeia, we have established a method of verifying multiple herbal species composition within a TCPM based on DNA barcoding and use of single-molecule, real-time (SMRT) sequencing technique. The TCPM such as Longdan Xiegan Wan, a classical multi-ingredient herbal prescription widely used in China for several hundred years, was chosen to test the methodology. A SMRT sequencing and species detection pipeline was established using reference TCPM samples. The application of this method was tested on experimental mixtures and commercial TCPM products from the marketplace. Successful SMRT sequencing results should recover circular-consensus sequencing (CCS) reads belonging to the internal transcribed spacer 2 (ITS2) and psbA-trnH regions. Repeatablity and reliablity of this method would be tested for detecting mixtures of multiple herbal species in the natural herbal products. In addition, the method will be confirmed it is sensitive enough to detect all species in the herbal product...This method represents the first trial of SMRT sequencing for the verification of multiple species mixtures in a natural herbal product and has the potential for becoming a valuable quality control tool for monitoring the species composition of multi-ingredient natural herbal product.

中成药基原物种鉴定是中药鉴定学的难点,采用显微和化学方法常常难以实现。CCS（Circular Consensus Sequencing）策略在生命科学研究中发挥重要作用，成为国际前沿，项目组基于CCS策略已获得中药材DNA序列，由此鉴定其基原物种，从而提出科学问题“以龙胆泻肝丸为例，基于CCS策略可否鉴定中成药的基原物种”。龙胆泻肝丸含10味中药材，成分复杂，不易鉴定，本项目通过购买其处方药材，制作对照中成药，经提取DNA、PCR扩增《中国药典》推荐的标准DNA条形码，采用CCS策略鉴定龙胆泻肝丸处方药材的基原物种，建立标准方法。再采购市场上流通的龙胆泻肝丸，按标准方法对其基原物种进行鉴定，并通过实时荧光定量PCR等技术验证市售产品中法定药材或非法混伪品的情况，追溯市售中成药的基原物种。本项目对创建中成药基原物种鉴定新方法具有理论指导作用，为中成药质量监管和用药安全提供科学依据。

中成药基原物种鉴定是中药鉴定学的难点,采用显微和化学方法常常难以实现。高通量测序技术包括CCS（Circular Consensus Sequencing）策略在生命科学研究中发挥重要作用，项目组基于高通量测序技术包括CCS策略已获得中药材DNA序列，由此鉴定其基原物种，从而提出科学问题“以龙胆泻肝丸为例，基于高通量测序技术包括CCS策略可否鉴定中成药的基原物种”。本项目收集龙胆泻肝丸组方涉及的龙胆、柴胡、黄芩、炒栀子、泽泻、木通、盐车前子、酒当归、地黄、炙甘草等10味药材，制作对照中成药，经提取DNA、PCR扩增《中国药典》推荐的标准DNA条形码，采用高通量测序技术鉴定龙胆泻肝丸处方药材的基原物种，建立标准方法。再采购市场上流通的龙胆泻肝丸，按标准方法对其基原物种进行鉴定。高通量测序结果显示，测序的灵敏性足够检出组方中各味药材，且具有较好的稳定性和重复性。市售样本中检出非组方规定物种川木通和泽泻。针对组方药材木通及其混伪品川木通、关木通ITS2序列设计特异性引物和TaqMan探针，构建木通、川木通、关木通荧光检测体系，发现市售样品存在川木通，结果经克隆测序和数字PCR验证无误。上述研究表明高通量测序技术能有效监控中成药物种组成。本项目对创建中成药基原物种鉴定新方法具有理论指导作用，为中成药质量监管和用药安全提供科学依据。.申请人主持撰写的DNA测序技术指导原则已纳入2020年版《中国药典》，并完成英文版的撰写。申请PCT国际专利1项（申请号：PCT/CN2021/138005），发表论文6篇，包括英文论文3篇，其中中科院一区论文2篇，出版专著1部。受邀在香山科学会议、国家药典委员会《中国药典》药品检验技术应用及发展研讨会、山东省中药材产业大会等作特邀报告3次。负责的研究生课程“本草基因组学”入选2020年北京协和医学院精品课程，培养博士后2人、博士生3人。