湛江红树林可培养内生真菌多样性及Stemphyliums sp. SCSIO 40436次生代谢产物分离与鉴定

Diversity of culturable endophytic fungi from Zhanjiang mangroves and secondary metabolites of the isolate Stemphyliums sp. SCSIO 40436

摘要:

【背景】红树林生态系统位于陆地和海洋的交汇地带，具有巨大的生态价值和经济效益。红树林内生真菌种类丰富，次生代谢产物结构多样、活性优异，是小分子天然产物和药物研发的重要来源。【目的】从湛江红树林植物样品分离、鉴定和筛选内生真菌，分离筛选菌株Stemphyliums sp. SCSIO 40436次生代谢产物并进行活性筛选。【方法】基于ITS序列鉴定种属研究内生真菌多样性；通过高效液相色谱和滤纸片扩散法筛选大米和燕麦固体培养基培养菌株的化学多样性及抑菌活性。应用硅胶、凝胶和反相色谱等色谱学方法分离纯化；应用高分辨质谱、核磁、X射线单晶衍射、圆二色谱等波谱手段确定化合物的平面结构与立体构型；采用微量肉汤稀释法测定分离化合物的最小抑菌浓度。【结果】从湛江高桥红树林植物样品分离纯化获得52株内生真菌，通过ITS序列比对归属于29个属。筛选到菌株Stemphyliumsp. SCSIO 40436，从其发酵粗提物中分离得到5个聚酮化合物stemphol (1)、macrosporin (2)、altersolanol A (3)、alterporriol E (4)和alterporriol D (5)，获得化合物1和3单晶结构。Stemphol (1)对革兰氏阴性菌鲍曼不动杆菌Acinetobacter baumannii ATCC 19606的最小抑制浓度达到8 μg/mL。【结论】湛江红树林植物分离的多株内生真菌ITS比对相似度低，为潜在新种，丰富了红树林内生真菌的多样性；红树林内生真菌Stemphyliumsp. SCSIO 40436产活性抗菌化合物stemphol (1)，首次发现stemphol对革兰氏阴性菌鲍曼不动杆菌的良好抑制活性。

Abstract:

[Background] Mangroves, trees and shrubs adaptive to salt, grow along the coast at tropical or subtropical latitudes. Mangrove endophytic fungi are the second largest community of marine fungi, which are rich in secondary metabolites with diverse chemical structures and excellent bioactivities. Therefore, mangrove fungi have proven to be an essential source of natural products and novel drug leads. [Objective] To study the diversity of culturable endophytic fungi from Zhanjiang mangroves and to identify bioactive secondary metabolites from the isolate Stemphyliums sp. SCSIO 40436. [Methods] Endophytic fungi isolated from Zhanjiang mangrove forests were identified based on internal transcribed spacer (ITS) sequence. The production of secondary metabolites on rice and oat solid media was evaluated by HPLC-DAD and filter paper diffusion method. The crude extract was subjected to column chromatography with silica gel and Sephadex LH-20, medium-pressure liquid chromatography, or semi-preparative RP-HPLC. The metabolite structures including absolute configuration were determined by spectroscopic methods (HRESIMS, NMR), X-ray crystallographic analysis, and comparison of electronic circular dichroism. Antimicrobial activities were measured against four indicate strains via the broth microdilution method. [Results] A total of 52 strains of culturable endophytic fungi were isolated from the Zhanjiang mangrove forests and categorized into 29 genera. The strain Stemphylium sp. SCSIO 40436 was found to be prolific in metabolites. Extensive chromatographic separation of the ethyl acetate extracts afforded five polyketides: stemphol (1), macrosporin (2), altersolanol A (3), alterporriol E (4), and alterporriol D (5). The structures of 1 and 3 were confirmed by X-ray crystallographic analysis. Stemphol (1) exhibited a good activity against the Gram-negative bacterium Acinetobacter baumannii ATCC 19606 with a minium inhibitory concentration (MIC) value of 8 μg/mL. [Conclusion] A total of 52 endophytic fungal strains are isolated from the Zhanjiang mangrove forests, among which several strains shared <95% similarity of ITS rDNA sequences, which enriches the diversity of mangrove endophytic fungi. Stemphol (1) exhibits a moderate activity against the Gram-negative bacterium A. baumannii.