垂盆草中保护肝损伤降倍半萜苷类成分的追踪分离及作用机制研究

Sedum sarmentosum is a traditional Chinese medicine with the actions of disinhibiting dampness and relieving jaundice, and can be used to treat jaundice, acute and chronic hepatitis. Previous studies indicated that the methanolic extract from S. sarmentosum showed more potent hepatoprotective effect. The molecular weight and characteristic fragment ions of the chemical constituents of the methanolic extract were analyzed by HPLC-MS, and it was concluded preliminarily that megastigmane glycosides (MG) might be the most abundant ingredients in this extract. In this project, firstly, MG of the methanolic extract will be enriched and purificated by macroporous resin to obtain the effective fraction with high content of MG. Secondly, MG will be isolated from the effective fraction under the guidance of HPLC-MS investigation. Thirdly, MG isolated from the effective fraction will be tested for their hepatoprotective activities, which will be helpful to elucidate hepatoprotective substances of the effective fraction. Finally, we will further investigate the acute toxicity, hepatoprotective activity and mechanism of action of the effective fraction using liver injury model in mice, and its mechanism of action will be investigated by assay of oxidative stress, inflammatory response, hepatocyte apoptosis and so on. These studies will contribute to the exploitation of S. sarmentosum medicinal resource and the development of new Chinese medicine for the treatment of liver injury.

鉴于垂盆草的功效（利湿退黄）与主治（湿热黄疸和急慢性肝炎），在前期研究中，我们筛选出垂盆草保肝活性部位为甲醇萃取部位，并通过LC-MS对该部位化合物的分子量和特征碎片峰进行分析，初步预测该部位可能主要含megastigmane型降倍半萜苷（MG）类成分。本项目在前期工作基础上，首先对甲醇活性部位中MG类成分进行富集纯化，获得富含MG类成分有效部位。接下来，在LC-MS导向下针对性地分离该部位中MG类化合物，明确该部位中MG类成分。然后，对分离到的MG类化合物进行保肝活性筛选，初步确认有效部位的保肝药效物质基础。最后，以富含MG有效部位为研究对象，采用动物肝损伤模型，研究该部位的急性毒性，保肝药效及作用机制。运用ELISA、Western blot等方法，从抗氧化抗炎、抗凋亡等途径，揭示其保护肝损伤的作用机理。以上研究为垂盆草药用资源的开发利用和防治肝损伤中药新药的研制提供化学和药理学基础。

垂盆草Sedum sarmentosum为景天属植物垂盆草的干燥全草，具有利湿退黄，清热解毒的功效，用于湿热黄疸，急慢性肝炎湿热瘀结症等。垂盆草虽为药典收载品种，但其指标化学成分并不明确。目前文献报道的有关垂盆草保肝研究主要在粗提物上，其药效成分及作用机制还有待进一步研究。本课题对垂盆草开展了以下三方面的研究：1.垂盆草不同萃取部位的体内保护肝损伤活性研究。2.垂盆草活性萃取部位的化学成分研究。3.化合物对肝细胞损伤的保护作用及其机制研究。结果表明，垂盆草活性萃取部位高剂量组显著改善大鼠血清中ALT、AST、ALP、γ-GTP、TBIL、DBIL、ALB和TP肝功能指标的异常，显著改善大鼠肝组织的病理损伤。同时，可降低肝组织中ROS的含量，提高SOD与GSH-Px活性，下调炎症因子TNF-α和IFN-γ的表达，上调IL-4的表达；对活性部位进行系统化学成分分离和结构鉴定，从中共分离鉴定34个化合物；运用肝细胞损伤模型对分离的部分化合物进行体外保肝活性测定，4个化合物具有体外保肝作用，其中3个是降倍半萜；进一步研究了活性较好的化合物对肝细胞上清液中多种酶含量的影响及其保肝作用机制，结果表明化合物可能通过激活Nrf2抗氧通路和抑制NF-κB炎症通路发挥保肝作用。本项目研究阐明了垂盆草保肝传统功效的物质基础及其作用机制，有助于该药材资源的进一步开发利用，为其质量标准体系的制定提供了科学依据。