二至丸中抑制组织蛋白酶K活性的物质基础研究  

Study on material basis of cathepsin K targeted inhibitor in Erzhi Wan

作　　者：蒋益萍 金玉娥[2,3] 张志玮 夏天爽 徐佳乐 薛黎明 JIANG Yiping;JIN Yue;ZHANG Zhiwei;XIA Tianshuang;XU Jiale;XUE Liming(Department of Pharmacognosy,Naval Medical University,Shanghai 200433,China;Institute of Chemical Toxicity,Shanghai Municipal Center for Disease Control and Prevention,Shanghai 200336,China;Shanghai institutes of Preventive Medicine,Shanghai 200336,China)

机构地区：[1]海军军医大学药学院生药学教研室,上海200433 [2]上海市疾病预防控制中心化学品毒性检定所,上海200336 [3]上海市预防医学研究院,上海200336

出　　处：《药学实践与服务》2023年第2期91-96,118,共7页Journal of Pharmaceutical Practice and Service

基　　金：上海市卫生健康委员会中医药重点项目(2020YZ001);上海市‘医苑新星’青年医学人才培养资助计划(沪卫人事〔2021〕99号);国家自然科学基金项目(81503493)。

摘　　要：目的考察传统补肾经典名方二至丸中抑制组织蛋白酶K的活性部位和活性成分。方法采用高通量荧光方法筛选二至丸中正丁醇、二氯甲烷、乙酸乙酯和石油醚提取部位,以及二至丸主要活性成分对组织蛋白酶K(CTSK)与荧光合成底物Z-FR-MCA结合活性的抑制率,和对CTSK与硫酸软骨素A(CSA)复合物形成活性抑制率。进而考察二至丸不同提取部位和活性成分抑制CTSK生理底物I型胶原蛋白降解活性,并采用分子对接和底物结合实验验证潜在CTSK抑制剂。结果二至丸的正丁醇和石油醚提取部位对CTSK与CSA复合物形成抑制率超过90%,石油醚提取部位对CTSK与底物Z-FR-MCA结合抑制率超过90%,正丁醇提取部位对CTSK的胶原降解抑制率超过95%、石油醚提取部位为58.6%。30个有效成分中11个显示对CTSK与CSA复合物形成抑制率超过50%,有5个成分对CTSK与荧光底物Z-FR-MCA结合活性抑制率超过50%。最终筛选确定4个成分墨旱莲皂苷Ⅸ、表儿茶素没食子酸酯、女贞苷和蟛蜞菊内酯,对胶原降解抑制率超过50%,其中墨旱莲皂苷Ⅸ抑制胶原纤维与CTSK的结合率最高达60%,但均与CTSK活性位点分子对接不成功。结论二至丸中存在抑制组织蛋白酶K的活性物质,主要存在于正丁醇部位,但活性成分不是活性位点抑制剂,可能通过与其他位点结合,间接抑制CTSK与寡多糖结合,进而降低了CTSK的胶原降解活性。Objective To investigate the active ingredients and components that inhibiting cathepsin K activity in Erzhi Wan,a classic kidney-tonifying formula.Methods Then-butanol,dichloromethane,ethyl acetate and petroleum ether parts and 30 active components in Erzhi Wan were screened by established high throughput fluorescence methods of inhibit the binding activity of CTSK with Z-FR-MCA substrate,the formation of CTSK and chondroitin sulfate A(CSA)complex activity,and the activity of substrate type I collagen degradation by CTSK.Molecular docking and insoluble collagen substrate binding assays were applied to verify the potential CTSK inhibitors.Results The n-butanol and petroleum ether parts of Erzhi Wan inhibited the formation of CTSK and CSA*complex by more than 90%,the petroleum ether part inhibited the binding of CTSK to substrate Z-FR-MCA by more than 90%,the collagen degradation inhibition rate of CTSK in n-butanol part was more than 95%and that in petroleum ether part was 58.6%.Among the 30 active components,11 showed that the inhibition rate of CTSK and CSA*complex formation was more than 50%,and 5 components with the inhibition rate of Z-FR-MCA binding activity more than 50%.Finally,there were four components including eclalbasaponinⅨ,(-)-epicatechin gallate,nuezhenoside and wedelolactone.The inhibition rate of collagen degradation was more than 50%.Eclipta saponin IX inhibited the binding rate between collagen fibers and CTSK,up to 60%,but all of them failed to dock with CTSK active site.Conclusion There are active components that inhibiting cathepsin K in Erzhi Wan,which mainly exists in the n-butanol ingredients,but the active components is not an active-site inhibitor.It might inhibit the binding of CTSK with oligosaccharides by binding to other sites of CTSK,and then reduce the collagen degradation activity of CTSK.

关 键 词：二至丸 骨质疏松 组织蛋白酶K 活性部位 潜在抑制剂 

分 类 号：R285[医药卫生—中药学]