红树林内生真菌多样性及其次级代谢产物GH-9-1抗肺癌活性的研究

摘 要：目的 分析湛江观海长廊红树林内生真菌多样性，筛选具有抗肺癌活性的内生真菌次级代谢产物，对其中1株内生真菌Penicillium sp. GH-9发酵产物4-甲基-3-甲氧基-2,4-戊二烯酸（GH-9-1）进行抗肺癌活性研究。方法 利用微生物培养和发酵技术对红树林内生真菌进行分离纯化及液体发酵，通过16S rRNA基因序列构建系统发育树分析红树林内生真菌多样性，利用高效液相色谱（HPLC）检测技术对其次级代谢产物进行特征峰追踪分离，根据核磁共振数据鉴定化合物结构，采用CCK-8法、流式细胞术、Western blot技术评价单体化合物的体外抗肺癌活性。结果 从湛江市观海长廊采集6种红树林植物组织标本及红树林根部土壤标本，共分离得到130株内生真菌，从真菌Penicillium sp. GH-9的发酵产物中获得其主要特征性代谢产物4-甲基-3-甲氧基-2,4-戊二烯酸（GH-9-1）。化合物GH-9-1可抑制非小细胞肺癌细胞A549和H460细胞生长增殖，并可诱导A549和H460细胞发生凋亡和细胞周期阻滞。结论 湛江市观海长廊红树林内生真菌具有丰富的多样性，从其中1株内生真菌Penicillium sp. GH-9的次级代谢产物分离得到的GH-9-1具有显著的抗肺癌活性。

Study on biodiversity of mangrove endophytic fungusand anti-lung cancer activity of their secondary metabolites GH-9-1

Abstract: Objective The purpose of this study was to investigate the diversity of mangrove endophytic fungus. These secondary metabolites of endophytic fungus with anti-lung cancer activity were screened and the anti-lung cancer activity of 4-methyl-3-methoxy-2, 4-pentadienic acid (GH-9-1), a fermentation product of Penicillium sp. GH-9, was analyzed. Methods Mangrove endophytic fungus were isolated and purified by microbial culture and fermentation. The 16S rRNA gene sequencing was employed to explore the diversity. High performance liquid chromatography was carried out to isolate the secondary metabolites. Their structures were identified from Nuclear Magnetic Resonance Spectroscopy. CCK-8 assay, flow cytometry and Western blot were performed to evaluate the anti-lung cancer activity. Results Six species of mangrove and mangrove root soil samples were collected from Guanhai Corridor in Zhanjiang, and a total of 130 endophytic fungus were isolated. The main metabolite, 4-methyl-3-methoxy-2,4-pentadienoic acid (GH-9-1), was obtained from the fermentation products of Penicillium sp. GH-9. Conclusion Mangrove endophytic fungus collected in the Guanhai Corridor in Zhanjiang are genetically diverse and the compound GH-9-1 isolated from the secondary metabolites of Penicillium sp. GH-9 showed significant anti-lung cancer activity.