龙须藤抗RA活性三萜单体的分离筛选及其作用机理研究

Research on anti-RA activity and its action mechanism of n-butanol extraction part from Bauhinia championii (Benth.) Benth have been finished in National Natural Science Foundation of Youth Science Fund. More than fifty monomeric compounds have been isolated and identified from it. And it was found that some triterpenoids monomeric compounds had outstanding anti-RA in vitro as well. In present, there are seven journal articles, including three SCI articles, and obtain 2 patents. In new project, based on original research, isolation and identification of triterpenoids are continued done to get more and sufficient amount of samples with special characteristic. And investigation of effect of triterpenoids monomeric compounds on LPS-induced synoviocytes by high-throughput screening, expression of relevant target genes on TLR4/NF-κB signaling pathway and β-arrestins will be done based on idear of combination vitro and vivo researches. Than anti-inflammatory and immune regulation effect of active ingredient were evaluated in collegen-induced arthritis rat model as well as its mechanism. Finally study on quantitative structure-activity relationship among the structural characteristics, geometric configuration, activity data and target of drug action, so as to obtain a clear “structure-activity-target” relationship of Bauhinia championii (Benth.) Benth triterpenoids. These findings will deepen understanding of natural drug Bauhinia championii (Benth.) Benth's traditional effect and provide a scientific basis for the prediction of new anti-RA compounds.

已完成龙须藤正丁醇部位抗RA活性及机制研究，从中分离、鉴定50余个单体，并发现部分三萜单体的显著体外抗RA活性。已发表论文7篇，其中SCI收录3篇；申请专利2项。本项目拟在前期基础上，进一步分离鉴定并富集典型特征的痕量/微量三萜单体，并基于体外试验和体内研究相结合的思路，通过RTCA高通量筛选各单体对LPS刺激滑膜细胞的影响，观察其对TLR/NF-κB信号通路及β-arrestins蛋白等的影响，再结合CIA大鼠模型对优选单体进行进一步抗炎、免疫效应评价及机制研究；继之对各三萜的结构特征、空间构型及相应活性数据、作用靶点进行定量构效关系（QSAR）研究，以揭示龙须藤三萜“结构-活性-靶点”间的内在关联。该项目可深化对民族药龙须藤传统功效科学内涵的认识，并为从中发掘新型抗RA先导化合物提供科学依据。

羊蹄甲属植物龙须藤是畲医病证论治中针对痹症治疗的特色药，临床治疗类风湿性关节炎（Rheumatoid Arthritis，RA）疗效确切。本项目在前期基础上，采用经典的化学分离手段结合现代高速逆流色谱、HPLC-DAD-Q/TOF-MS、UPLC /MSn及制备型HPLC等技术进一步分离鉴定并制备出龙须藤单体化合物，并基于体外试验和体内研究相结合的思路，通过RTCA高通量筛选各单体对LPS刺激滑膜细胞的影响，观察其对TLR/NF-κB信号通路及β-arrestins蛋白等的影响，再结合CIA大鼠模型对优选单体进行进一步抗炎、免疫效应评价及机制研究。项目研究结果显示从龙须藤中分离鉴定并制备出33个单体化合物，其中3个化合物为新化合物，24个化合物为首次从龙须藤中分离得到；采用高通量筛选及MTT法对分离得到的单体化合物进行体外抑制滑膜细胞增殖的活性筛选，除化合物18、22、24、25、27和30外，其他化合物均对大鼠原代滑膜细胞具有抑制作用，其中化合物32、33抑制作用最强；同时通过体外和体内实验研究证实了化合物32、33显著抑制炎症细胞因子的抗炎作用，并显著降低大鼠足肿胀、改善大鼠滑膜组织增生的抗类风湿性关节炎作用，其作用机制可能与调控β-arrestins介导的TLR4/NF-κB信号通路相关靶基因和炎症介质表达有关。初步对化合物32、33进行结构及活性分析，探讨“结构-活性-靶点”间的内在关联。研究仍有待进一步深入，但已获得的结果为深化对民族药龙须藤传统功效科学内涵的认识，为其在抗类风湿性关节炎方面的应用提供科学依据。