南药青天葵“清热解毒”药效物质及其免疫调节作用机制研究

Clearing heat and detoxicating herbs play very important roles in clinic for their immunomodulatory activities. Nervilia fordii (Hance) Schltr. is an eatable traditional herb with definitely function of clearing heat and detoxicating in clinic, but its immunomodulatory mechanism and its bioactive components of clearing heat and detoxicating are not explored. From former research, we isolated 25 compounds including 5 new compounds and 6 first isolated compounds, we also discovered that the flavonoids of Nervilia fordii (Hance) Schltr. demonstrated very attractive anti-inflammatory activities through immune pathway. Base on the references, we proposed that the polysaccharides and tannis of Nervilia fordii (Hance) Schltr. should also possessed anti-inflammatory activities through immune pathway. Therefore, we want to do a further phytochemical research of Nervilia fordii (Hance) Schltr. which was bio-guided by two sophisticated immunomodulatory activities screening platforms,LPS-induced RAW264.7 macrophage releasing NO,and in vitro immune hemolysis. Base on this project,the immunomodulatory mechanism of Nervilia fordii (Hance) Schltr. and its bioactive components of clearing heat and detoxicating would be investigated.

对免疫系统的调节作用是清热解毒类中药临床发挥药效的重要机制之一。临床实践表明南药青天葵具有明确的清热解毒作用。但其清热解毒药效物质及其免疫调节作用机理尚未阐明。我们前期从青天葵中分离纯化了25个化合物，其中5个新化合物，6个首次分离的化合物，并发现青天葵黄酮类化合物具有良好的免疫抗炎活性，结合文献报道，我们推测青天葵多糖类物质和鞣质类物质可能具有较好的免疫调节活性。基于前期工作基础及假设，本项目拟通过LPS诱导RAW264.7小鼠巨噬细胞释放NO的细胞炎症模型和体外细胞溶血免疫分子模型为活性分离导向，深入研究青天葵具有免疫调节活性的有效部位及有效单体物质，从免疫细胞、分子水平上阐明青天葵清热解毒药效物质基础及其作用机制。

对免疫系统的调节作用是清热解毒类中药临床发挥药效的重要机制之一。临床实践表明南药青天葵具有明确的清热解毒作用。本项目通过LPS诱导RAW264.7小鼠巨噬细胞释放NO的细胞炎症模型和体外细胞溶血免疫分子模型为活性分离导向，深入研究青天葵具有免疫调节活性的有效部位及有效单体物质，从免疫细胞、分子水平上基本阐明了青天葵清热解毒药效物质基础及其作用机制。     . 通过LPS诱导RAW264.7巨噬细胞炎症模型活性为分离导向，从青天葵地上部分的60%乙醇提取部位分离并鉴定了26个化合物，其中有8个新化合物。采用LPS诱导RAW264.7小鼠巨噬细胞释放NO的炎症细胞模型，测定了18个黄酮类化合物的抗炎活性，结果表明其中5个化合物具有不同程度的抑制NO释放活性。对nervilifordin G和nervilifordin H的抗炎作用机制开展初步研究，结果表明nervilifordin G 和nervilifordin H能够抑制LPS诱导的RAW264.7小鼠巨噬细胞内NO和PGE2的释放，同时抑制iNOS的mRNA和蛋白表达，表明nervilifordin G 和nervilifordin H可通过抑制NO和PGE2的合成而发挥抗炎作用。另外，nervilifordin G和nervilifordin H不仅降低了炎症因子IL-6、TNF-α的释放量，还抑制了mTOR通路中的关键因子ERK1/2、p70S6K mRNA和蛋白表达，表明其可能是通过下调ERK1/2、p70S6K的mRNA和蛋白表达水平来降低炎症因子IL-6、TNF-α的释放量，从而抑制炎症反应。. 通过体外细胞溶血抗补体活性为分离导向，从青天葵粗多糖部位分离得到14个均一多糖，其分子量在980~1300 kD，主要由葡萄糖、鼠李糖、阿拉伯糖、半乳糖等单糖组成。以体外细胞溶血免疫分子模型测定这14个均一多糖的抗补体活性，结果有7个均一多糖在经典途径和旁路途径均有显著地抗溶血活性，1个均一多糖只在旁路途径具有显著抗溶血活性，活性效果甚至强于阳性对照药肝素钠，这表明青天葵多糖可在人体抗体产生之前的感染早期或初次感染即可发挥作用，因此多糖类物质也是青天葵发挥免疫抗炎作用的重要活性物质。