两种思茅藤属植物中新颖甾体结构及其免疫抑制机理研究

An ideal plant system is of great significance to reveal the mystery of the plant kingdom, and drug discovery. Apocynaceae and Asclepiadaceae are two important familis in the flora. Apocynaceae direct and progressive changes in the evolution to Asclepiadaceae. Epigyneae is at the highest position in the evolution system of Apocynacea. Epigynum Wigth belongs to the evolutionary highest family, therefore, it is a genus at a junction status evolution to Asclepiadaceae from Apocynaceae. Epigynum auritum is the only species distributed in China in the genus. As we know, cardiac glycoside is the important bioactive constituent of Apocynaceae, and pregnane is the main constituent of Asclepiadaceae. The chemical perspective of E. auritum remains largely unknown. In our preliminary studies on E.auritum, we discovered three kinds of new steroid natural products with deformed or degraded skeletons. Our studies revealed that these compounds significantly inhibited concanavalin-stimulated spleen lymphocyte proliferation of Balb/c mouse. Remarkably, the activity of androsta-4,6,8(9),14(15)-tetraene-3,11,16-trione was found similar to standard immunosuppresive agents tritolide and dexamethasone. We propose to launch an in-depth study on E. auritum and its kin, E. graciliflorum, mainly distributed in Laos, to find new types of steroid natural products, and to study their immune inhibitory activity. Besides the inhibitory effect of the obtained compounds on the transformation between T cells and B cells, their activity on NK cells and macrophages will also be investigated. Chemical syntheses will be carried out to obtain active compounds in amounts enough for studies on animal models. Our work will reveal the immunosuppresive mechanism and structure-activity relationships of these compounds, and to provide novel leads for drug discovery and development.

作为一个处于从夹竹桃科向萝藦科进化交界地位的种属，思茅藤属植物的化学研究在本课题组之前几乎是空白。本组曾从思茅藤属中唯一分布在中国的植物思茅藤中分离得到三种骨架变形或降解的新类型甾体，其中多个甾体对伴刀豆蛋白刺激的Balb/c小鼠脾淋巴细胞增殖有明显的抑制，特别是雄甾-4,6,8(9),14(15)-四烯-3,11,16-三酮与现有免疫抑制物质雷公藤内酯和地塞米松活性相当。此研究提示：处于植物进化交界位置的植物属种中更易产生非常规骨架的次生代谢产物，其结构及生物活性都值得深入研究。本项目将从思茅藤及分布在老挝的另一同属植物中发现更多类型的新甾体，系统研究其免疫抑制活性，除常规针对T细胞和B细胞转化抑制方面的探讨，还将进行NK细胞、巨噬细胞等活性的研究，并结合化学合成累积活性甾体数量，开展动物实验，探讨作用机理，解析构效关系，为寻找新药提供先导化合物。

项目共完成两种思茅藤属植物（Epigynum auritum 和E. cochinchinensis）植物的化学成分研究，共获得不重复单体化合物96个，其中新颖结构化合物22个，8个新颖多共轭体系雄甾烷类化合物，5个5元D环孕甾糖苷化合物、8个6元吡喃D环孕甾糖苷化合物、1个D环开环孕甾糖苷化合物，和一个三萜类化合物。完成思茅藤提取物不同萃取部位和色谱分离组分分离工作，分析其总皂苷、总黄酮和总多糖含量分析，利用UPLC-MS/MS对获得的甾体成分进行二级质谱的数据采集分析，为后续结构的推测鉴定以及活性物质分离提供依据。完成了思茅藤不同极性部位的体外免疫抑制实验，免疫抑制活性与甾体含量相关，分离得到的甾体化合物具有对淋巴细胞较好的免疫抑制活性，细胞周期显示思茅藤样品显著抑制细胞由 S 期向 G2/M期的转变，显著抑制前炎症因子 IL-2 及 TNF-α的产生。完成巨噬细胞抑制实验，思茅藤样品MCI80%和100%极性段具有较好的抑制活性，抑制IL-6 及 TNF-α产生，而且以剂量依赖的形式减少了细胞内 COX-2 和iNOS 的表达，其中14个化合物（12个甾体和2个酚酸）具有巨噬细胞具有较好的抑制活性，TNF-α、 IL-1β、 PGE2等因子产生。2个单体化合物对急性白血病细胞具有显著的增殖抑制活性，Hoechst33258 染色CCRF-CEM细胞核光亮，皱缩，出现凋亡小体；80μM 下与 DXM 效果相当，细胞周期阻滞发生在G0/G1期；对细胞活性氧的产生呈现剂量反应关系。此外，思茅藤提取物能显著降低卡拉胶诱导的爪肿胀和二甲苯诱导的耳肿胀，急性毒性试验表明思茅藤甲醇提取物LD50大于5000mg/kg体重，为实际无毒，亚急性毒性表明高剂量会引起肝脏、肾脏和脾脏毒性。发表挂基金号相关研究论文11篇，其中SCI研究论文8篇，受理发明专利9项；人才培养方面， 2人晋升副教授，培养毕业硕士研究生6名。