中药贝壳杉烷二萜类成分及其同分异构体快速识别和分析体系的构建与应用

Currently, the active ingredients of traditional Chinese medicine (TCM) identified by LC-MS method mainly based on characteristic fragment ions combined with for manual inspection brought many defects such as time-consuming, exertion and omission. Simultaneously, it was difficult to differentiate the isomerides with the same nucleus. In response to the above problems and based on preliminary studies, our task focus on the holistic quality control of TCM with an ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) method for rapid identification and analysis of active components and isomerides in I.rubescens, I.serra, I.amethystoides. Firstly, a few ent-kauraniods and isomerides are selected as carrier to study their Mass spectrometry cracking characteristic, meanwhile, we introduce relative abundance ratios to calculate, set chemomtrics as guidance to improve the definiteness of identification for identification isomerides well and truly. Secondly, we also study a new UPLC-Q-TOF-MS/MS oriented mass defect filtering (MDF) approach combined with MS database searching and diagnostic characteristic ions method for identificate compounds in TCM. Finally, the holistic quality of TCM will be evaluated by means of semi-quantitative analysis and chemometric analysis. Simultaneously, main reasons leading to differences in different samples will be analyzed and this system will be applied for routine analysis of TCM.. This task will not only offer a new view for the apply of LC-MS method to theTCM but also provide valuable bases for identificating isomeride in other species TCM.

本课题针对目前液质联用方法在鉴定中药有效成分时主要依靠特征离子碎片结合人工检识存在费时、费力、容易遗漏的缺点，以及难以鉴定相同母核的同分异构体的问题，在前期研究工作基础上以UPLC-Q-TOF-MS/MS为研究手段，选用富含贝壳杉烷二萜成分的冬凌草、溪黄草、香茶菜为研究载体，以贝壳杉烷类二萜成分及同分异构体为研究对象，建立中药有效成分及其同分异构体快速识别和分析的方法，达到全面质量控制的要求。首先总结质谱裂解规律，并建立以相对丰度比法为主的同分异构体鉴定方法；然后建立以质量亏损过滤技术为基础，结合质谱库检索和特征离子碎片的液质联用方法，并用于中药定性研究；最后通过半定量分析和化学计量统计学实验结果，对中药的质量进行全面评价，并分析导致质量差异的主要化学成分，并将其应用于中药常规分析。这些研究将为液质联用技术在中药分析中的应用提供新的视角，为其他类别中药化学成分异构体的鉴定提供有价值的依据。

中药的质量控制是一项系统工程。本课题针对中药体内外化学成分不明确以及难以鉴定的难题，选择富含贝壳杉烷二萜成分的3种常用香茶菜属中药为研究载体，采用液质联用技术，对香豆素类成分进行了系统分析，建立省时、有效的中药常规分析方法，同时研究了主要贝壳杉烷二萜成分的体内外代谢研究和药代动力学研究，以其对中药的质量进行整体评价，同时上述这些研究方法的建立和应用能为更好地探索中药的物质基础提供信息基础和理论支持。已正式发表论文8篇，SCI收录6篇，CSCD论文2篇，待发表3篇；培养硕士研究生6名，已毕业3名。主要研究内容和成果如下：.1）系统总结了贝壳杉烷二萜对照品的质谱裂解规律，创新性建立相对丰度比法区分贝壳杉烷二萜的同分异构体，并应用于并鉴定出代表性中药中贝壳杉烷二萜成分；2）利用液质联用技术，同时测定代表性中药中主要贝壳杉烷二萜成分的含量，采用统计学软件进行了质量评价，并对正品和伪品进行了有效区分；.3）对冬凌草甲素和冬凌草乙素的体内外代谢轮廓进行了阐述，并且比较了不同种属间的代谢差异；.4）建立了生物样品中主要贝壳杉烷二萜的定量测定方法，分别对3种药材在大鼠体内的吸收和排泄动力学进行了研究；.5）初步建立了贝壳杉烷二萜成分的质谱库，可利用库检索技术对贝壳杉烷二萜成分进行识别和鉴定。 . 本课题对中药及生物体复杂体系中化学成分群表征方法进行了有益探索，其研究结果为普适性的中药质量控制方法学体系的初步建立奠定了坚实的基础，为效应物质基础上中药质量控制模式的建立提供了科学依据。..