两种青牛胆属植物中抑制小胶质细胞活化成分的发现及其初步作用机制研究

Tinospora sinensis，which stems is always called “kuan jin teng” ，and Tinospora sagittata, which tuberous root is also named “jin guo lan” in Chinese pharmacopoeia, both belong to the genus Tinospora and have played an important role in folk medicine to treat against rheumatic and inflammatory disease. Twenty six compounds have been isolated from the acrial part of T. sinensis including13 diterpenes,7 alkaloids and 6 lignins and the inhibitory activities of most compounds were assessed by LPS-activited murine microglial BV-2 cells by our research group. It’s the first time to find alkaloid constituents with inhitory effects on microglial activation from the genus of Tinospora, with 2 compounds whose activity is better than positive control minocycline. There are still substantial of alkaloids existed in the petroleum extract and butanol part of T. sinensis and the same genus plants T. sagittata, according to UPLC-Q-TOF-HRESIMS analysis. In view of the above findings, further study would be carried out to isolated more alkaloids from the two plants by the using of personal compound data library established by our research group. Then, LPS-induced microglial cells model would be employed to evaluate the inhibitory effects on microglial activation for all the isolated alkaloids. And the preliminary mechanism of the active compounds will be also studied. All the researches will hopefully result in the discovery of good leading compounds for alleviating microglial activation and provide good reference for using Tinospora plants resource to treat neuroinflammation and related ailments, such as Alzheimer’s disease.

中华青牛胆的茎藤又名“宽筋藤”，青牛胆的干燥块根亦为历版《中国药典》收载的“金果榄”，它们均属于青牛胆属植物，在民间广泛用于治疗风湿炎症。我们课题组，前期研究发现从中华青牛胆中分离鉴定的生物碱类化合物能够显著抑制小胶质细胞过度活化，其中2个化合物的活性高于阳性对照米诺环素。并通过UPLC-Q-TOF-HRESIMS分析，发现其剩余部分及同属植物青牛胆提取物各萃取部位均富含生物碱类化合物。本课题拟在前期的工作基础上，完善已建立的生物碱类化合物PCDL数据库，进而对两种植物中生物碱进行精准分离鉴定；并采用LPS诱导活化的BV-2小胶质细胞模型，测试化合物对小胶质细胞NO、TNF-α及IL-6释放的影响；进一步对活性显著的生物碱进行初步的作用机制研究。该研究有望发现具有减轻小胶质细胞活化介导的神经系统疾病（如阿尔茨海默病）的先导化合物，为青牛胆属植物在神经炎症相关疾病方面的应用提供参考。

本项目采用植物化学与药理学相结合的研究手段，基本完成了中华青牛胆、青牛胆及波叶青牛胆植物中包括生物碱在内的植物化学成分的系统分离鉴定及抗神经炎症活性筛选工作。从三种植物中分别分离鉴定了25，56，45个单体化合物，结构类型包括生物碱类，萜类，苯丙素类等，除去各个植物重复分离的5个化合物，总共获得了121个单体化合物，其中新化合物26个，这为开发利用青牛胆属植物化学资源提供了丰富的物质基础。建立完善了LPS激活小鼠BV-2小胶质细胞释放NO的模型，成功筛选到具有潜在抗神经炎症活性化合物17个。运用了ELISA、Western blot等分子生物学和药理学的研究手段，初步探索了3个单体化合物体外发挥抗神经炎症药理活性的作用机制。阐明了2个活性化合物通过抑制炎症相关蛋白iNOS，COX-2的表达，降低炎症因子TNF-ɑ及IL-6的释放达到抗炎活性，为后续抗神经退行性疾病相关药物的研发提供了活性先导化合物。