绞股蓝中具有降脂活性三萜类化合物的快速发现及其构效关系研究

Based on previous work, this proposed research is intended to intensively investigate the hypolipidemic effect of triterpene saponins (Gyp) in the active fraction of Gynostemma pentaphyllum by integrating methods and technologies belonging to different discipline including chemistry of TCM, molecular biology, computer aided drug design (CADD), and pharmacology. The specific research contents of this proposal is made up of four parts: 1) Isolate and elucidate the structures of the Gyp by employing chromatographic and spectroscopic methods; 2) Screen lipid-lowering constituents of Gyp by high throughput screening methods on lipids accumulation model in vitro (HepG2 cell). Choose the Gyp constituents with potent hypolipidemic activities and evaluate the binding degree between active Gyp and AMPK by CADD; 3) Investigate the activation against intracellular AMPK or p-AMPK of the selected constituents based on the above results; 4) Select one to two agonist of AMPK or p-AMPK from the Gyp constituents with obvious lipid lowering activity and confirm their effects in the rat hyperlipidemia model after enough preparation. In this way the fruits of this study will establish solid scientific foundation, thereby, helping not only deeply investigate this abundant and traditional medicinal plant resource in a more reasonable way, but also develop new lead compounds of independent intellectual property and bright developing future. Meanwhile, it is expected that through these collaborative efforts, new general methods will emerge in guiding the studies of antilipemic agents from the Gyp constituents.

利用先进的快速识别技术、提取分离方法和结构鉴定手段以及成熟的体内外降脂药理学筛选模型，对有良好降血脂临床药用基础且资源十分丰富，尚未开展系统研究的绞股蓝的主要降脂活性成分-达玛烷型四环三萜类（Gyp）及其衍生物进行深入的分离制备和体内外药理活性研究。包括以关键调脂酶-腺苷活化蛋白激酶（AMPK）为分子靶标，利用FlexX程序对不同Gyp成分与受体AMPK相互结合程度进行评价，同时，结合体外降脂活性筛选和不同Gyp成分激活HepG2细胞内AMPK的结果，分析结构与活性之间的关系，确定Gyp类化合物降脂作用点和规律性，优选活性最佳且作用靶点明确的三萜类成分（包括微量成分）。足量制备后，通过大鼠高脂血症模型确证其体内降血脂作用，并对其简单的结构修饰。寻找其中降脂活性最优的且结构新颖的化合物，为合理开发利用这一资源丰富的药食同源品奠定物质基础，并为遴选具有新药开发前景的先导化合物提供科学依据。

绞股蓝Gynostemma pentaphyllum (Thunb.) Makion为葫芦科绞股蓝属多年生草质藤本植物，又称“南方人参”，主要产于秦岭和长江以南广大地区。具有滋补保健、抗癌防衰、增强体质和改善脂质代谢等多种功能。课题组研究发现绞股蓝中三萜类化合物是其降脂活性的主要活性物质。本研究采用反复硅胶柱层析法、重结晶法、反相ODS色谱法和半制备液相色谱法等，对绞股蓝总皂苷水解产物和总皂苷进行系统分离，从酸水解产物中分离出17个化合物，1个新化合物；从总皂苷中分离出30个化合物。并且结合理化性质和光谱学数据鉴定了它们的化学结构。实现了绞股蓝三萜类成分的快速识别与快速分离。以降血脂有效成分绞股蓝达玛烷型四环三萜类化合物为研究对象，通过建立体外HepG2肝细胞脂肪堆积模型，确定绞股蓝三萜类为降脂活性成分。进一步利用高脂血症、2型糖尿病之间的联系，开展绞股蓝三萜的降脂、降糖活性筛选，并应用动物实验、分子生物学、计算机辅助模拟药物设计等方法深入探索其作用机制。通过上述研究，分别在国际期刊Journal of Asian Natural Products Research、 Chinese Herbal Medicines（中草药（英文版））上发表2篇学术论文，另外在权威期刊《中草药》、核心期刊《药物评价研究》上发表2篇学术论文，授权国家发明专利1项，培养硕士研究生4名。本项目的研究结果，阐明了绞股蓝降脂、降糖的活性物质，为合理开发利用这一资源丰富的药食同源品奠定物质基础，并为遴选具有新药开发前景的先导化合物提供科学依据。