| 二至丸中抑制组织蛋白酶K活性的物质基础研究 |
| 投稿时间：2022-02-09 修订日期：2022-11-05 点此下载全文 |
| 引用本文：蒋益萍,金玉娥,张志玮,夏天爽,徐佳乐,薛黎明.二至丸中抑制组织蛋白酶K活性的物质基础研究[J].药学实践杂志,2023,41(2):91~96,118 |
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| 基金项目: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) |
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| 中文摘要: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。 |
| 中文关键词:二至丸 骨质疏松 组织蛋白酶K 活性部位 潜在抑制剂 |
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| Study on material basis of cathepsin K targeted inhibitor in Erzhi Wan |
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| Abstract: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 eclalbasaponin Ⅸ, (-)-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. |
| keywords:Erzhi Wan osteoporosis cathepsin K active fraction potential inhibitor |
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