天然单胺氧化酶抑制剂的快速识别及跟踪分离研究

During isolation the natural monoamine oxidase inhibitors which are considered to be potential for treating Alzheimer's disease, Parkinson's disease and depression, due to the lack of method for rapidly identifying responsible compounds, researchers need to separate many inactive compounds together with responsible compounds from extracts, resulting for a long study period and the high cost. To solve this problem, a Thin-layer chromatography (TLC) bioautographic method for the detection of monoamine oxidase inhibitors in plants will be developed in this project. The method will have rapid, sensitive, low-cost advantages, and can avoid the time consuming of isolating already known inactive compounds which exist together with responsible compounds in extracts. In this project, we will focus on screening the new substrates and chromogenic agents which are selectively suitable for TLC bioautographic method. The alkaloidal extracts from Corydalis, Aconitum and Senecio genus, as well as the alkaloidal extracts from Traditional Chinese Medicine and Tibetan Medicine will be tested for their inhibitory activities against monoamine oxidase by using this new TLC bioautographic method. The bioactivity-guided chromatographic fractionation will be subsequently executed to obtain the responsible compounds in above active extracts by combining this new TLC bioautographic method and common chromatographic method. This is a new idea for quickly finding lead compounds from natural resources. The succeed of this project will make it possible to discover natural monoamine oxidase inhibitors in a large scale.The experiences obtained from this project will be helpful for constructing new TLC-bioautographic methods for screening other enzyme inhibitors.

在从天然提取物中跟踪分离与阿尔茨海默病、帕金森综合症、抑郁症治疗有关的单胺氧化酶抑制剂时，由于缺乏快速识别责任化合物的方法，研究者常需伴随分离许多与活性无关的非责任化合物，致使研究周期长、费用高。针对这一问题，本项目拟建立一种薄层色谱生物自显影方法，用于单胺氧化酶抑制剂先导物的快速识别及跟踪分离研究。着重是通过研究新型酶反应底物和显色剂，构建实验耗时少、薄层背景明显、重现性好的薄层色谱生物自显影方法。应用此方法，拟对西北分布较广的紫堇属、乌头属和千里光属植物的生物碱提取物以及常用中、藏药的生物碱提取物进行广泛分析，快速识别并跟踪分离其中的单胺氧化酶抑制剂，期望发现有价值的新药先导物。本项目的成功实施将使大规模、高通量地发现天然单胺氧化酶抑制剂成为可能，并将为今后开展其他类酶抑制剂的快速发现提供借鉴。

本项目通过研究新型酶反应底物、显色剂以及改进现有方法的手段，建立了高通量、低费用的薄层色谱生物自显影方法用于单胺氧化酶抑制剂的识别和跟踪分离研究。新方法具有针对性强的、责任化合物直观可见、耗时少、重现性好、薄层背景明显利于观察的优点。利用此方法，对常用中药、藏药和西北分布较广的乌头属、紫堇属植物以及植物内生菌中存在的单胺氧化酶抑制剂进行快速寻找和跟踪分离，发现了十多个有活性的化合物。其中新化合物Desmodeleganine表现出最强的单胺氧化酶抑制活性，其IC50与阳性药磷酸异丙烟肼接近。利用计算机辅助药物设计手段对部分活性好的化合物进行了分子对接研究，阐明了其与酶的结合位点。四年来共发表论文22篇，其中SCI收录论文16篇，影响因子2.0以上5篇， 申请国家发明专利7项，其中3项已授权、培养硕士研究生7名。顺利的完成了计划书中的任务。本项目是快速寻找天然先导物的新思路，也为今后开展其它类酶抑制剂的研究提供了借鉴。建立的薄层色谱生物自显影方法，为研究经费不足的研究者从事单胺氧化酶抑制剂的发现研究提供了捷径。