新型抗RA二萜生物碱的定向发现及其作用机制研究

Rheumatoid arthritis (RA) is a widely prevalent chronic systemic disease, which can cause severe cartilage and bone damage even disability. Its pathogenesis is still fully unknown, and the ideal drugs are unavailable yet. Hence it is significant to search new effective drugs for the treatment of RA from traditional Chinese medicines (TCM). Herbal drugs derived from the species belonging to the genus Aconitum have exhibited remarkable therapeutic effects on RA, but the clinical application is limited due to their toxicity. It was found in our previous studies that vilmorine A-type and vilmoraconitine-type diterpenoid alkaloids isolated from Aconitum vilmorinianum Kom. possess noticeable anti-rheumatic acivities and less toxicity, which prompted us to search similar ingredients from the sibling species of A. vilmorinianum base on the strategy of LC-MS/MS combined with high speed countercurrent chromatography methods. The anti-rheumatic activities of the isolated ingredients will be screened by their inhibition effects on the fibroblast-like synoviocyte (FLS) cells proliferation induced by tumor necrosis factor-alpha (TNF-α) and their therapeutical effects on adjuvant-induced arthritis in rats. And the pharmacological mechanism of two types of ingredients will be explored by their effects on preventing NF-κB signal path activation in TNF-α-activated FLS cells. This study is supposed to provide new lead compounds for the treatment of RA and facilitate the exploitation and utilization of herbal drugs from Aconitum. Furthermore, this study will provide a new idea for the in-depth research of toxic TCMs.

类风湿关节炎（RA）发病率高危害大，是病因不明的难治之症，至今尚无理想治疗药物，从中草药发现新型RA治疗药物意义重大。临床及基础研究显示药用草乌抗RA药效明确，但毒性限制其应用。我们前期发现黄草乌中vilmorine A类和vilmoraconitine类新颖二萜生物碱有显著抗RA作用且毒性低。研究拟在此基础上，根据植物亲缘关系，设计基于LC-MS/MS和HSCCC技术的微量成分高效分离策略，从四种草乌中定向快速分离两类成分。结合成纤维样滑膜细胞（FLS）模型和大鼠关节炎模型筛选目标成分抗RA活性，并通过活性成分对TNF-α诱导FLS细胞NF-κB通道活化的影响研究其抗RA作用机制。项目有望发现抗RA先导化合物，为抗RA创新药物研发奠定基础，促进我国丰富药用草乌资源开发与利用，创新性开拓传统有毒中药研究思路，解决毒性中药应用瓶颈。

源于乌头属和翠雀属植物的药用草乌具有明确的抗类风湿关节炎（RA）作用，对药用草乌开展深入细致的成分研究，极有可能发现新颖的抗RA药物先导化合物。本课题系统研究了八种药用草乌的生物碱类成分，从中分离鉴定了111个化合物，包括11个新的二萜生物碱类化合物。但研究未得到vilmorine和vilmoraconitine类新化合物。体外抗炎活性筛选显示所得新二萜生物碱未表现出具备深入研究抗RA药理作用的活性。本课题还采用大鼠佐剂性关节炎（AA）模型，通过多种药效学指标，对两种药用草乌的抗RA作用和机制进行了研究。动物药理实验验证了两种药用草乌具有显著的抗RA作用，可缓解AA大鼠的相关症状，其药效或与降低关节中相关炎症因子的表达有关。此外，为了探索创新高效的天然产物分离策略，本课题设计了基于2D NMR中HMBC技术的去重软件工具MatchNat，用于从复杂的中药提取混合物中快速识别已知成分。通过对两种药用草乌的二萜生物碱研究中的应用，检验了本课题开发的去重软件MatchNat的实用性，证实该软件作为去重工具可有效提升中药成分研究效率，促进中药新颖活性成分的发现。本课题的研究结果将对国产药用草乌的开发提供重要的参考依据，本课题开发的去重软件也将为中药成分的研究提供实用的创新的研究策略。本项目共发表论文13篇，其中SCI收录论文10篇，中文核心期刊论文3篇。