药用红树木榄胚轴中抗HBV复制的腈类化合物定向发现及其对靶点NTCP作用研究

HBV entry inhibitors represent a new generation of antivirals for the treatment of HBV infection, by blocking the HBV entry into the target cells. Sodium taurocholate cotransporting polypeptide (NTCP) was recently indentified and recognized as a fuctional target for HBV. Jing people is the only national marine fisheries of China. Mangrove Bruguiera gymnorrhiza is Jing's treatment of hepatitis B traditional herb medicine. In our previous study, we have discovered alcohol extract of the hypocotyl of B. gymnorrhiza (AEHB) show activities on anti-duck hepatitis B virus and rich of characteristic cyclohexylideneacetonitrile derivatives. High-dosage AEHB has an obvious protective effect on the liver function and liver tissue. Compared with control group, protein expression of NTCP in High-dosage AEHB were down-regulated.AEHB were fractionated to yield three active parts (Y6, H4 and H8) which contain cyclohexylideneacetonitrile derivatives and have protective effects potent on treatment of HBV carriers. Initial searching for bioactive metabolites from Part Y6 has led to the isolation of seven cyclohexylideneacetonitrile derivatives (of which 4 compounds are new) showing protective effects potent as a HBV inhibitor in cell model system..In order to obtain highly efficient and specific anti-HBV drugs with independent intellectual property, this project plans to systematically investigate the active anti-HBV cyclohexylideneacetonitrile derivatives from the hypocotyl of B. gymnorrhiza. The contents include: 1) targeted isolating cyclohexylideneacetonitrile derivatives from the Part H4 and Part H8 by HPLC-DAD(UV)-MS and various modern chromatography techniques; 2) determining the structures of cyclohexylideneacetonitrile derivatives by MS, 1D and 2D NMR, CD, ORD and chemical methods; 3) examining the activity of the isolated compounds based on cell model system and duck hepatitis B model system of cyclohexylideneacetonitrile derivatives; 4) studying the action targets of lead compounds through Sodium taurocholate cotransporting polypeptide (NTCP) as a funtional receptor of HBV on model cell system.The current research would provide fundamentally cyclohexylideneacetonitrile derivatives as lead compound for HBV infection management.

乙型肝炎病毒（HBV）进入抑制剂是颇具前景的新机制治疗乙型肝炎药物。最近发现，钠离子-牛磺胆酸-协同转运蛋白（NTCP）为HBV感染肝细胞所需的特异性靶点。红树木榄是广西京族医生治疗乙型肝炎的传统药物。在前期研究中，申请人发现木榄胚轴提取物具有抑制模型鸭体内HBV复制和保护肝细胞活性，高剂量组会抑制NTCP蛋白生成。申请人发现3个富含环亚己基己腈类化合物的活性部位（Y6、H4和H8），进一步从Y6中分离到7个具有较显著抗HBV复制的环亚己基己腈类活性化合物。本项目拟在前期工作基础上，运用HPLC-DAD(UV)-MS方法从H4和H8部位定向追踪环亚己基己腈类特征活性成分，运用现代波谱技术和化学手段鉴定其结构，借助体内和体外活性模型系统评价环亚己基己腈类化合物抗HBV复制活性，并探讨其对靶点蛋白NTCP作用，获得腈类先导化合物，研究成果为海洋来源的治疗乙型肝炎新型药物提供坚实物质基础。

红树生长在热带和亚热带沿海潮间带区域，是海洋植物的重要组成部分。京族医药曾记录红树木榄具有清热解毒、消肿散结、止咳平喘的功效，主治哮喘、淋巴结肿大、急慢性肝炎等。研究发现红树木榄胚轴正丁醇部位具有极显著的抑制乙肝病毒复制活性，运用现代波谱技术和化学手段鉴定获得了16个环亚己基己腈类化合物的结构，其中5个为新化合物。测试了环亚己基己腈类化合物抗乙型肝炎病毒复制活性，获得2个具有强抗乙型肝炎病毒复制的环亚己基己腈类化合物。获得1个抑制HBV复制的环亚己基己腈类先导化合物Menisdaurin F。通过项目的实施，发表研究论文5篇，其中SCI收录论文3篇，中文核心期刊论文2篇。申请国家发明专利1件。获广西科学技术奖自然科学奖三等奖1项（项目负责人排名第一）。主编出版著作《海洋红树植物药用研究》1部；培养硕士研究生2名。