具有免疫调节活性的山橙属植物新颖吲哚生物碱的发现及其作用机制研究

People of lower immunity are susceptible to be infected with bacteria, fungi, and viruses. Natural sources of immunoregulation is high efficient and low toxic, which has a unique efficiency and mechanism. Therefore, new immune enhancing agent from natural products is an importent pathway to discover the immunoregulation drugs.The indole alkaloids with immuno-enhancing activity have further potential applications value.. We have long been carrying out the plants alkaloids, bioactivity research and new drug research. A series of active alkaloids with novel structures were reported. Previous study found that the total alkaloids from Melodinus plants have significant immunoregulation activity. Chemical investigation on leaves of M. khasianus led to the isolation of 36 indole alkaloids, including 11 new ones, most of which are aspidospermidine-type and vicamine-type alkaloids. This provides the foundation guarantee for further immunoregulation screening and mechanism researchs.. The project will study the chemical constituents of two Melodinus plants ( M. khasianus and M. hemsleyanus). The Melodinus plants have been proven to be good resources of indole alkaloids, and the crude extracts have significant activity of promoting immuno regulation. Firstly, indole alkaloids of the Melodinus plants will be systemic studied by chemical methods, and then the purified alkaloids will be tested for the immuno systems. Based on the bioactivity results, the significant bioactive alkaloids will further study the mechanism of immunoregulation. It is expected to provide lead compounds and the scientific basis for the development of immunoregulation drugs. This project will provide the chemistry and pharmacodymics basis for the development of Melodinus plants.

免疫力低下使机体易患细菌、真菌、病毒等感染性疾病，甚至诱发重大疾病。天然来源的免疫调节药物具有独特的疗效和作用机制，且多具有高效低毒的特性。其中促进免疫调节的吲哚生物碱的发现具有进一步的应用潜力。. 申请人近年积累了生物碱识别、分离、纯化和结构鉴定的经验，并发现一系列结构新颖或具有药理活性的生物碱，前期研究发现山橙属总生物碱具有促进免疫调节的活性，该属植物生物碱含量丰富、结构多样，获得了多个白坚木和长春曼胺骨架的生物碱，包括11个新颖结构生物碱，为进一步的活性筛选和机制研究提供了基础保障。. 本项目选择前期筛选具有免疫调节活性的2种黔产山橙属植物，LC-MS/MS指导下系统的开展化学成分研究，以获得大量结构多样化和新颖性的吲哚生物碱，并对所得化合物进行深入的免疫调节活性筛选、构效关系和作用机制研究。为合理开发该属植物资源、研发免疫调节药物奠定坚实的化学和药效学基础。

本研究共完成了采自贵州的景东山橙、川山橙、思茅山橙和绣花络石的化学成分研究，共分离得到不重复单体化合物132个, 其中有9个新颖单萜吲哚生物碱，6个双吲哚生物碱和1个新颖木脂素类化合物。利用UPLC-MS/MS，建立山橙属生物碱液质指纹图谱研究，并上传至GNPS分析平台。通过脾淋巴细胞和巨噬细胞建立细胞实验模型，完成山橙属植物提取物以及单体化合物的免疫抑制活性研究，结果表明生物碱成分具有良好的免疫抑制活性，可通过减少iNOS和COX-2的表达，抑制MAPK和NF-κB通路相关蛋白的磷酸化水平，可减少巨噬细胞中炎症因子NO、IL-6、TNF-α和IL-1β的分泌，从而免疫疾病的发生。针对人体急性淋巴白血病细胞开展药理学研究，筛选出一个具有应用前景的单体化合物（19-甲氧基水甘草碱），并开展作用机制研究。从民族药应用开发角度出发，开展了景东山橙总生物碱的急性毒性试验以及28天亚急性毒性实验。急毒试验表明其LD50大于2000mg/kg体重，亚急性毒性实验表明高剂量（1000>mg/kg）长期服用会造成器官损伤。此外，在安全剂量下对景东山橙总生物碱进行皮肤超敏反应抑制活性评价。结果表明总生物碱组分以剂量依赖的形式缓减二甲苯诱导的小鼠耳肿胀，以及卡拉胶诱导的爪肿胀。同时，该组分也通过抑制MAPK和NF-κB通路中相关蛋白的磷酸化水平、提升PPAR-γ的表达情况来缓减DSS诱导的结肠炎症。