民族药苦石莲抗流感病毒活性组分的发现与作用机理研究

Caesalpinia minax Hance(called Ku Shi Lian in China), a species belonging to Fabaceae family, has been used to treat the influenza for hundreds years in Zhuang National Medicines, while, its mechanism and bio-active substances for its pharmaceutial effect have not been studied systematically up to now. It is believable that the discovery on its effective components and the special mechanism will provide the new structures and researching data for the current anti-flu medicines researching. In our continous work, C. minax was found with many ways on mechanism for anti-flu activity for the first time, including showing the treatment on the pneumonia caused by virus infection;the significant inhibitory activity against influenza virus neuraminidase (NA) and having protective function for the infected cells. Chemical study exhibited active fractions being rich in cassane diterpenoids. According to these results,we give a hypothesis for this project that Ku Shi Lian might have multi-ways for its anti-influenza mechanism, and the cassane diterpenoids as the main effective constituents could show their anti-flu activity by the target protein NA way. In order to demonstrate the inherent scientificity of the theory, this project will continue to combine the chemical, pharmacological, analytical, and computers technologies, to study and elusidate the reasonable scientific connotation on the effective substances and mechanism of this folk medicine, in addition, new anti-influenza leading compounds could be found. Beside that, this project will achieve integrated research level for this national medicine, and also provide the valuable and important significance for its exploition and the utilization on its resources.

壮药苦石莲民间单方制剂用于治疗流感。对其药效物质基础、作用机理进行深入研究既可发现并找到其独特的活性组分、关键靶点，又可为当前抗流感新药研究提供先导结构，意义深远。前期发现：苦石莲富含卡萨烷活性组分群，对高致病甲型流感病毒神经氨酸酶(NA)有明显的抑制活性，经计算机模拟分子对接，卡萨烷衍生物与NA具有高度的亲和性，提示NA是该类成分发挥药效的关键靶标。此外，苦石莲对病毒感染造成的肺炎有治疗作用，对感染细胞有保护功能。项目认为：苦石莲存在抗病毒、抗炎、免疫调节等多途径抗流感机制，结构特异的卡萨烷是其重要药效组分。为对该民族药的药效物质基础、作用机理做详细阐明，项目将继续结合中药化学、分析、药理、计算机设计等方法对其抗流感物质基础和作用机理做深入研究，总结卡萨烷衍生物作用NA的构效关系规律，为新型NA抑制剂设计提供必要前提。项目还可实现对该民族药研究的一体化，对其创新研发、资源利用意义重大。

本项目以壮药苦石莲为研究对象，结合中药化学、分析、药理、计算机辅助设计等方法对该民族药的活性物质基础做了化学研究，对其抗炎、抗病毒、免疫活性做了活性评价，对相关机制进行了探讨，结合生物信息学，从结构就结合关键靶蛋白间的机制做了探讨。取得了以下重要进展：明确了苦石莲富含的卡萨烷二萜组分群是有效的抗病毒、抗炎活性组分，深入的物质基础研究则从活性部位中分离鉴定了48个单体化合物，其中40个为二萜衍生物。结构涵盖新骨架化合物2个，另有新结构21个。采用LC-TOFMS技术，对活性部位的卡萨烷组分进行了系统的定性研究，鉴定出51个二萜化合物，包括41个呋喃型、10个呋喃内酯型卡萨烷二萜，7个为未见文献报道的新成分。Caesalmin C作为活性部位中含量较高，活性较好的成分，故确认为相关研究指标成分。以卡萨烷型二萜作用H5N1、H7N9的NA活性发现，卡萨烷对H5N1的NA抑制活性最为明显，但对H7N9活性弱或没活性。计算机3D模拟分析显示卡萨烷与靶蛋白NA晶体具有高度的亲和性，构效关系分析可知，成环结构、-OAc官能团、-OH 、双键是活性主体基团，结构引入胍基基团，活性增加明显。苦石莲活卡萨烷组分兼有抗炎活性，可抑制巨噬细胞释放NO，动物实验表明，卡萨烷可抑制完全佐剂致类风湿关节炎，其抗炎药效与阳性对照药雷公藤苷相当；卡萨烷对外源性刺激导致的炎症反应有抑制作用，可抑制LPS诱导的巨噬细胞炎症因子的释放，机制在于抑制IL-1β、IL-6、IL-18、TNF-α的mRNA表达；核内可介导NF-κB/IκB信号通路，明显抑制p65磷酸化。根据相似原理筛选出相关性最高的潜在抗炎靶点-糖皮质激素受体（GR），卡萨烷类二萜成分与GR活性腔有很好的匹配，能与活性口袋中Met601、Met604及Leu608形成疏水作用，同时，还可与Thr739、Gln642、Asn564、Arg611形成氢键作用，从而使复合物的结构更加稳定，有利于激活受体，参与免疫调节和炎症反应。至此项目已发表科研、会议论文7篇，其中SCI统计源论文4篇，中文核心期刊论文1篇，会议论文2篇，另有待发表SCI论文1篇；培养硕士研究生5名，已获硕士学位4人；申请发明专利1项。项目丰富了民族药苦石莲在化学、药效、机制等相关研究上的内容，意义深远。