芫花抗类风湿关节炎显效成分（群）的发现及其协同作用机制研究

Xiaoluotong from proved recipe for anti-rheumatoid arthritis was composed of Caulis Genkwa and Mung Bean. Caulis Genkwa due to its resource limit was replaced by Flos Genkwa in Xiaoluotong. In our preliminary work, replaced Xiaoluotong was almost equal to the old one for anti-rheumatoid arthritis. Previous research demonstrated that effective part of Flos Genkwa had significantly anti-rheumatoid arthristis effect and inhibited the NO release and phagocytosis from LPS-induced-RAW264.7 cells. The effective part was better than the mixture of four ingredients in treating rheumatoid arthristis. It indicated that there was synergy effect between each ingredient. However, synergy mechanisms are unclear which are urgently required to further research. In the project, UPLC-Q-TOF-MS technology will be used to comprehensively analyze the chemical ingredients (group) in Flos Genkwa and their metabolites in vivo. “Capture selected-natural ingredients/ metabolites—efficacy recognition” pattern, cell model and animal model will be introduced to discover the effective ingredient. “Capture selected-natural ingredients/ metabolites—mult-signal pathway” pattern, cell technology and biotechnology will be applied to explore the anti-rheumatoid arthristis mechanisms and synergy mechanisms. The project will provide foundation for TCM’s mult-component and multiple pathways.

源于验方的抗类风湿关节炎（RA）良药-消络痛由芫花条、绿豆组成，鉴于芫花资源可持续利用，以芫花替代芫花条，替代方与原方具等同抗RA作用。前期研究发现，芫花有效部位（4种主成分含量>90%）的抗RA作用显著，其对LPS诱导的RAW264.7的NO释放量及吞噬功能作用强于4种主成分单体的加合，各成分具协同作用，但协同机制尚不清楚，亟待进一步研究。本研究运用UPLC-Q-TOF-MS技术全面表征芫花固有化学成分（群）及体内代谢产物；采用“固有成分/代谢产物选择性捕捉-活性检识”模式，以细胞模型和动物模型检识捕捉的各成分（群）活性，探索固有成分（群）/体内代谢产物与芫花整体药效的关系，发现抗RA的显效成分（群）；采用“固有成分（群）选择性捕捉-多信号通道联用检识”模式，用细胞和分子生物学技术，探索显效成分（群）抗RA的作用途径，阐明协同作用的机理，为揭示中药多成分多途径的作用特点提供依据。

消络痛是由芫花条、绿豆组成的验方，是抗类风湿关节炎（RA）良药，鉴于芫花资源可持续利用，以芫花替代芫花条，替代方与原方具等同抗RA作用。前期研究发现，芫花有效部位（4种主成分含量>90%）抗RA作用显著，其对LPS-RAW264.7的NO释放量及吞噬功能作用强于4种主成分单体的加合，各成分具协同作用，但协同机制尚不清楚。本研究采用UPLC-Q-TOF-MS技术表征芫花固有化学成分（群）及体内代谢产物、固有成分/代谢产物选择性捕捉-活性检识模式、固有成分（群）选择性捕捉-多信号通道联用检识模式，完成了①芫花中化学成分（群）及其体内代谢产物的表征；②芫花中化学成分（群）抗RA 显效成分群的发现；③芫花显效成分（群）抗RA协同作用机制研究。研究获得了重要结果①芫花中化学成分（群）及其体内代谢产物的全面表征：建立芫花体外全成分的指纹图谱，全面定性表征芫花固有化学成分（群），建立芫花后的体内吸收成分（血）指纹图谱、代谢产物（胆汁、尿、粪便）指纹图谱，全面定性表征各吸收成分（群）和代谢产物成分（群）；②发现了芫花中化学成分（群）抗RA显效成分群，以芫花素为最佳显效成分；③对芫花素体内、外抗RA药效及机制进行研究，发现芫花素可调节炎症因子水平，协同抑制滑膜病变组织中NF-κB及JAK2/STAT3通路的激活，从多通路协同治疗佐剂型大鼠关节炎。研究获得了重要数据①芫花中14个黄酮类成分、8个二萜类及30个体内代谢产物成分；②各成分对LPS-RAW264.7细胞分泌NO、IL-1β、IL-6和TNF-α的影响，芹菜素-7-O-β-D葡萄糖苷、木犀草素、芹菜素和芫花素均具有显著抑制作用，以芫花素最佳；③采用WB及免疫组化法，研究了芫花素体内外对炎症通路NF-κB及免疫相关通路JAK2/STAT3协同作用机制，阐明协同作用的机理，为揭示中药多成分多途径的作用特点提供依据。