南海岛屿典型植物内生真菌代谢组差异扫描及新活性物质发现

Fungi derived from special habitats have been shown in recent years to produce a plethora of new bioactive secondary metabolites. Due to geographical isolation, oceanic island ecosystems are vastly different from other ecosystems. However, endophytic fungi living the special niche, especially their secondary metabolites, have rarely been investigated. In a previous study, 127 endophytic strains have been isolated from three representative plants in South China Sea Islands, indicating that islands plants are a rich source of endophytes. Thus, exploring and mining the fungal potential metabolic capabilities, providing useful bioactive compounds, would be highly desirable. The main purpose of this project is to isolate new bioactive compounds and analogues from the endophytic fungi through metabolome differential scanning. Firstly, the endophtic fungal metabolome will be explored using a small-scale fermentation procedure with different kinds of media combined with HPLC-DAD/MS, and 2D NMR. Secondly, based on the results of dereplication, the endophytes sensitive to culture medium with interesting metabolic fingerprinting will be large-scale cultured; the differentially expressed compounds and the analogues will be isolated and identified. Finally, the bioactivities of isolated compounds such as anti-inflammatory, antitumor or antibacterial property, together with structure-activity relationships will be studied. The results of the project will provide scientific information for the discovery of new lead compounds. In addition, it will establish foundation for the further systematic research on oceanic islands fungi.

特殊生境真菌的新活性物质发现几率往往较高，其中，海洋岛屿生态系统因海域隔离，显著有别于其他生态系统。然而，目前海岛植物内生真菌次生代谢物的研究却很少有报道。申请者等的前期工作对南海三种典型海岛植物调查，从中分离得到127株真菌，本项目着力于如何快速从这些真菌中发现新结构活性物质，通过基于代谢组差异扫描设计思路，寻求新天然有机小分子发现方法上的突破。拟对获得菌株不同培养条件少量发酵，运用HPLC-DAD/MS和2D NMR等技术进行代谢组差异分析；结合天然产物数据库排重结果，对培养条件敏感、指纹图谱特殊的菌株放大发酵，跟踪分离鉴定这些差异表达的化合物及其类似物；进行抗炎、抗肿瘤和抑菌等活性测试，获得新活性化合物并探讨构效关系。本项目的完成将为新活性先导化合物的发现提供方法借鉴，同时也为海洋岛屿真菌资源进一步系统性研究奠定基础。

本项目针对我国南海岛屿植物内生真菌次生代谢产物进行研究。处于孤立分布的海洋岛礁，植物生态系统特殊，栖居大量代谢途径特异的植物内生真菌，是有待开发的新生物活性物质的重要生物资源。本研究对分布在我国西沙群岛、南沙岛礁以及近海岸海岛典型植物的内生真菌分布、类群、次生代谢物质进行了调查，重点基于次生代谢组差异扫描，对代谢指纹特殊、活性显著的菌株进行规模放大发酵，分离确定化合物结构。研究的海岛植物包括草海桐、海岸桐、海人树、木麻黄、蔓荆等，从中分离得到植物内生真菌1000余株，种属包括子囊真菌和高等担子真菌，初步构建了种属多样、来源特殊的海岛植物内生真菌菌种资源库。对筛选获得的5株真菌大量发酵获得粗提取，综合运用各种层析技术（正反相硅胶、Sephadex LH-20凝胶、HPLC），从中分离鉴定单体小分子化合物115个，运用1D和2D NMR、质谱（EIMS、ESIMS）、紫外光谱、红外光谱、X-ray晶体衍射、圆二色谱、量子化学计算以及必要的有机化学反应，确定了这些化合物单体的平面和空间结构，结构类型包括聚酮类、糖苷类、生物碱类、萜类及甾体类，其中新结构化合物47个。生物活性测试表明，Aspergillus terreus土曲霉SC1550来源的2个含有丁内酰亚胺的生物碱asperimide C和D具有显著抗炎活性，IC50值分别是0.78 ± 0.06和1.26 ± 0.11 μM，另外，从高等真菌耙齿菌Irpex sp. SC4004中分离得到一组具有显著抗细胞增殖的直链型三萜类糖苷物质。本项目的研究结果，证实了基于代谢组差异扫描策略的真菌次生代谢物研究方法的可行性和有效性，同时也揭示了我国海洋岛屿岛礁生态系统孕育丰富的特殊真菌资源，是新结构类型活性物质的重要潜力资源，值得进一步深入系统研究。