2D-LC/MS技术在中药药效成分发现中的应用研究

A number of effective compounds with novel chemical structures have been isolated from Chinese herbal medicines (including those popularly used species) in the recent years. The discovery of these compounds is not only critical for comprehensively interpreting the effective components of traditional Chinese medicines (TCMs), but also provides valuable lead compounds for drug discovery. However, the research process to discover these novel compounds is usually laborious, time-consuming, and inefficient. In order to improve the chance and efficiency of obtaining novel compounds from complicated herbal extracts, the present project aims to establish a new research strategy based on two-dimensional liquid chromatography coupled with mass spectrometry (2D-LC/MS). This strategy will be applied to rapid and efficient discovery of novel compounds from representative Chinese herbal medicines containing different classes of chemical structures, including Curcumae Longae Rhizoma, Puerariae Lobatae Radix, Bufonis Venenum, Drynariae Rhizoma, Coptidis Rhizoma, and Notoginseng Flos. Firstly, a state-of-the-art 2D-LC/MS system for TCM chemical analysis will be established. The extracts of Chinese herbal medicines will be analyzed by high peak capacity two-dimensional liquid chromatography methods in comprehensive or heart-cutting mode. High 2D orthogonality will be achieved to allow good resolution of previously oversighted minor compounds from major ones, and thus allows global chemical profiling of TCMs. Based on mass spectrometry structural information, the TCM chemical constituents will be characterized, and novel compounds containing new skeletons or new substitution patterns will be rapidly and accurately defined. Secondly, these target novel compounds will be efficiently purified by preparative liquid chromatography, and their bioactivities will be evaluated according to clinical therapeutic effects of the source TCMs to find out novel effective components. This project will provide a new reliable research strategy and methodology for comprehensive elucidation of effective components of TCMs.

近年来，中药（包括常用中药）不断发现结构新颖的药效成分，对于全面阐明中药药效物质具有重要意义，也为新药研究提供了先导分子。然而，这些新颖成分的发现过程耗时费力，目标性不强。为了提高从复杂的中药提取物发现新颖药效成分的效率，本项目将探索建立基于二维液相色谱-质谱联用技术的研究方法体系，并应用于姜黄、葛根、蟾酥、骨碎补、黄连、三七花等具有不同结构类型的代表性中药。 首先，构建适合中药特点的2D-LC/MS分析系统，采用全二维或中心切割模式，优化正交条件，建立高峰容量的二维液相色谱分析方法，尤其是凸显常规方法忽略的微量成分，获得中药的整体化学轮廓；再利用各种质谱信息推测结构，快速精准地发现新颖成分（新骨架、新取代方式）。其次，针对性地制备纯化目标新颖成分，开展与中药临床功效相关联的生物活性研究，从而进一步发现中药的新颖药效成分。本项目将为中药药效物质基础研究提供新颖可靠的方法学。

发现新颖活性成分对于全面阐明中药药效物质具有重要意义。为了提高从复杂体系发现新颖成分的效率，本项目将2DLC/MS技术引入中药分析，建立了适合中药复杂体系的快速分离和检测方法，凸显微量成分，发现并定向分离得到新颖活性成分。在此基础上，建立靶向代谢组学方法，提升中药整体质量控制水平。..首先，基于商品化仪器，建立了成熟的2DLC/MS分析策略。通过阀切换、固定相、流动相、洗脱程序的开发，优化了适合黄酮、生物碱、皂苷、酚酸等类型中药化学成分的2DLC分离方法。例如，中心切割-全二维液相色谱（HC-2DLC）解决了大量微量成分共存的问题；基于流动相的全二维液相色谱（MPD-2DLC）解决了多类型成分共存的问题；多中心切割-全二维液相色谱（MHC-2DLC）解决了复方成分过于复杂的问题。对甘草、葛根、猪牙皂、葛根芩连汤等中药和复方开展研究，在40分钟内实现数百个成分的分离，明显提高了中药新颖成分的发现效率。..其次，为了高效发现新结构，建立了中性丢失（NL）、母离子扫描（Prec）、关键离子过滤（KIF）等高选择性质谱检测方法，识别新颖结构，并开展导向制备。例如，建立MNPSS方法，从姜黄发现了846个萜取代姜黄素类新成分，采用统计分析识别新结构碎片，定向分离得到2个新骨架化合物terpecurcumins X/Y。从黄连分离得到14个新颖的生物碱-奎宁酸杂合物；从黄芩分离得到4个新成分；利用生物催化获得源于蟾酥的17个蟾蜍甾烯衍生物。这些成分具有抗肿瘤、抗氧化、抗乙酰胆碱酯酶等活性，为诠释相应中药的药效成分奠定了重要基础。..最后，基于化学成分系统分析，建立了LC/MS/MS靶向代谢组学方法，用于中药材及中成药的质量评价。例如，对95批甘草中151个化学成分进行定量分析，发现27个品种特异性的化学标志物，可用于区分三种药用甘草，解决了药材性状难以区分的问题。这是迄今最大规模的药用植物化学成分定量分析研究，提高了中药的整体质量控制水平。..研究结果在AC、JCA、ASC等知名杂志发表SCI论文13篇，培养研究生10名。

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