马钱族中抗胶质瘤干细胞的吲哚生物碱结构与活性研究

Tumor stem cells (TSCs) are widely acknowledged they are target cells for the therapy of cancer, which controlled the tumor occurrence, tumor migration and recurrence. Glioblastoma stem cells (GSCs), isolated from brain tumor, are the only type of tumor stem cell. GSCs can be long-term cultured in vitro and keep stable in biological characteristics and genomic integrity, and can be suitable for drug screening. Glioblastoma multiform is currently incurable and confer a poor prognosis with less than 15 months survival time. Indole alkaloids are important groups of the natural products, and their structures diversity play a very important role in new drugs discovery..Our group has long been carrying out the plants alkaloids, and reported the isolation and structure identification, cytotoxic activities of novel skeletal alkaloids. Besides, the applicants has reported series cytotoxic activities of novel alkaloids, and established tumor stem cell-based screening technology. Moreover, the Strychneae plants are rich in indole alkaloids and their structures are complicated and variable. Previous study found that the chemical investigation on gardneria ovate and strychnos nitida led to the isolation of 35 indole alkaloids, including 13 new ones, most of which are Gardneramine-type and Ajmalicine-type alkaloids. This provides the foundation guarantee for further screening and mechanism research..The project will study four-six Strychneae plants, which have been proven to be good resources of indole alkaloids. Firstly, indole alkaloids of the Strychneae plants are systemic studied by chemical methods and HPLC-UV data, and then the purified alkaloids are evaluated their anti-cancer activity against human glioma stem cell lines. Furthermore, choosing the bioactive alkaloids are studied the mechanism and activity in vivo on glioblastoma of 1-2 promising candidates. Hopefully, it is expected to provide lead compounds for glioblastoma therapy agents in this project.

肿瘤干细胞是肿瘤治疗研究的靶细胞，控制着肿瘤的发生、转移和复发。胶质瘤干细胞是从脑胶质瘤中分离出的肿瘤干细胞，也是目前唯一能在体外长期培养且保持遗传特性不改变的肿瘤干细胞，适用于药物的筛选。脑胶质瘤病人的生存期不到15月，缺乏治疗药物。吲哚生物碱是天然药物中的成药系数较高的类群，是抗癌药物研究的重点和热点。.申请人具有生物碱化学成分研究的丰富经验，并报道系列结构新颖或药理活性的生物碱;合作研究组建立了抗胶质瘤干细胞活性筛选体系。研究发现马钱族植物中富含吲哚生物碱，且结构新颖复杂多样，已获得多个蓬莱葛任碱和阿吗碱型骨架的生物碱，含13个新颖结构，为进一步的活性筛选和体内评价提供基础保障。.本项目拟在前期工作基础上，对富含吲哚生物碱的马钱族植物进行系统的吲哚生物碱化学成分研究；对其开展抗胶质瘤干细胞活性筛选，并对活性生物碱探讨其分子作用机制，以及体内抗肿瘤功能评价，期望发现新型抗肿瘤先导分子。

项目完成了离药蓬莱葛等6种植物中吲哚生物碱系统的化学成分研究，获得了化合物单体132个，其中单萜吲哚生物碱118个，包括25个新颖的吲哚生物碱，结构类型涉及到蓬莱阁碱型、白坚木碱型、长春蔓定胺型、山橙碱型等。对离药蓬莱葛和景东山橙的总碱液质图谱进行了指认，并根据分离得到的吲哚生物结构类型建立了各自的分子网络库。.对分离鉴定的吲哚生物碱单体进行抗肿瘤、抗炎和免疫抑制活性研究。结果显示，获得了20个吲哚生物碱分别对9株不同的肿瘤细胞株具有较好的抑制活性，且具有选择性，其抑制活性优于阳性对照顺铂。其中11-methoxytabersonine (25)对MOLT-4 细胞株的抑制作用最好，IC50 值为0.71 μM，比阳性对照顺铂约小了一个数量级。肝癌细胞和人正常T细胞共培养显示蓬莱葛碱19(E)-18-demethoxygardneramine（16）为选择性细胞毒活性化合物，仅对肝癌细胞MHCC97有较好的杀伤抑制作用，同时对人正常T细胞无细胞毒活性。Angustine（31）为细胞毒活性化合物，对人正常T细胞和肝癌细胞MHCC97均有杀伤抑制作用。细胞划痕实验表明4个蓬莱阁中生物碱19(E)-18-demethoxygardneramine (1), 4,1,11b-Ethanylylidene-2H-pyrido[2',3':5,6] oxepino[2,3-b]indole, gardneramine oxindole deriv (2), gardneramine (3), and 10-deoxysarpagine (7)可以明显抑制CT26细胞向划痕区的迁移。获得了6个单吲哚生物碱和2个双吲哚生物碱可以显著抑制LPS诱导的小鼠RAW 264.7细胞中炎症因子IL-6和TNF-α的表达，浓度依赖性地降低了IL-6和TNF-α的升高，通过抑制细胞中细胞因子的表达而有效地改善炎症。获得了19个吲哚生物碱对 CoA 刺激的 T淋巴细胞和脾细胞增殖均具有较显著的抑制作用，且呈浓度依赖性。.在此项目的资助下，发表相关研究论文15篇，其中SCI研究论文12篇，包括1篇已接收发表的SCI论文，申请专利2项；人才培养方面，项目负责人入选云南省“高层次人才培养支持计划”青年拔尖人才专项，协助培养硕士研究生2名，预计今天夏季毕业。