整合G-qudruplex核酸比色探针和化学表型分析的重楼混伪品遗传特征可视化检测与质量控制研究

"Various related species of the same genus" which used as one kind of medicine has been a traditional problem in the research about species sorting and quality control of traditional Chinese medicine (TCM). In the revision of China Pharmacopoeia (CP), a dispute about the division of Lonicerae Japonicae Flos and Lonicerae Flos has been triggered, and the situation is not optimistic for Paridis Rhizoma. In the study of a previous project of NSFC, we found a phenomenon that in addition to 2 species of genus Paris L. which recorded in CP , 9 species of the same genus also be used as Paridis Rhizoma. The shape and properties of the medicinal herbs herbs is similar and the chemical composition overlaps dramatically, leading to lack of specificity of indicators used in the identification and content determination in CP. " Qualified adulterants " and " Unqualified statutory species " are widespread. It has caused confusion to the authenticity of identification and quality control work for Paridis Rhizoma. . To solve this problem, applicants will introduce G-qudruplex DNA colorimetric probe technique, carried out comparison analysis on the non conservative sequence of ITS area, design specific DNA colorimetric probe, acquire visual identification signal of specific DNA fragments from statutory species, build a reliable、fast、intuitive and economical visual genetic detection system for identification of Paridis Rhizoma. According to the analysis of chemical phenotype based on the interspecific distribution characteristics of steroidal saponin from Paris L., index componet for the quality control will be optimized. This research will greatly enhance the technical level of authenticity of identification and quality control technology, It can also provide reference for the explore of common problems existing in the quality evaluation of TCM characterized by “Various related species of the same genus”.

“同属近缘种”入药是困扰中药材品种整理和质量控制的传统难题，在药典修订中已引发了金银花与山银花的分列之争，但重楼的情况更不容乐观。前一个自然基金研究发现：除2个药典法定种外，9种重楼属植物也大量混用入药，其商品药材的形态高度趋同而化学成分交叉重叠，导致药典鉴别及含量测定指标缺乏专属性，“合格的混用种”与“不合格的法定种”广泛存在，给真伪鉴别和质量控制工作带来了混乱。申请者针对该问题，尝试引入G-qudruplex(G-四聚体)核酸比色探针技术，对非保守的ITS区序列进行比对分析，设计特异性比色探针，获取法定种的特征DNA片段的可视化识别信号，从而构建一个可靠、快捷、直观、成本合理的重楼真伪鉴别可视化基因检测体系；并基于重楼甾体皂苷的种间分布规律开展化学表型分析，优化其质控指标成分。该研究将显著提升重楼真伪鉴定和质量控制的技术水平，并为探索“同属近缘种”入药的中药品质评价共性问题提供借鉴。

本课题以分子生药学和化学分类学为指导，将重楼法定基原的真伪鉴定作为研究重点，以提升重楼的品种整理和质量控制技术水平。通过4年的研究，本项目收集了13种、19变种，共计32个类群的重楼属植物的样本和种质资源，覆盖了商品药材主流基基原；建立了保存活体植株的“四川省重楼种质资源圃”；测定了17个类群的ITS全序列并提交Genebank数据库；发表了2个分类群（宽瓣球药隔重楼、红果五指莲）的地理分布新记录；修正了2个分类群（西畴重楼、亮叶重楼）的生物学特征和分类学地位；建立了一套能准确鉴别《中国药典》法定品种云南重楼和七叶一枝花及其混伪品的荧光可视化真伪鉴定方法；建立了一套对云南重楼两个基因型和七叶一枝花进行快速鉴别的多重PCR 鉴别体系；阐明了重楼药材生长过程中，重楼皂苷类成分的动态分布规律及其对药材质量的影响，为控制和提高药材质量提供了重要参考；开展了球药隔组重楼的生药学研究及质量评价，纯化鉴定出球药隔重楼原变种3种甾体皂苷单体化合物，分析了球药隔组3个类群的化学特征及其药材质量，建立了鉴别球药隔重楼及其混用品（亮叶重楼、卵叶重楼）的方法；阐释了球药隔重楼凝血活性的药效物质基础和作用机理，为其质量控制提供了新的依据，揭示了作为止血药物的科学内涵。基于上述研究结果，已发表论文13篇，其中SCI期刊4篇，中文期刊9篇，申报发明专利2项，获得部省级科技奖1项（中国民族医药学会科学技术三等奖），获得“创青春”四川省大学生创新创业大赛创业计划竞赛铜奖1项，支持企业申报并获批“崇州重楼”国家农产品地理标志，培养硕士研究生3名。这些成果丰富了人们对于重楼属种质资源多样性的认识，完善了重楼属的经典分类学结论，提升了重楼药材真伪鉴定的技术水平，丰富了重楼质量控制的科学指标和技术选择，推动了重楼产业的品牌化和规范化发展，也为“同属近缘种”中药材质量控制共性问题的探索提供了新的研究思路和模式。