摘要
目前，恶性肿瘤依然是威胁人类生命的主要杀手，而且随着抗生素和免疫抑制剂的大量应用等原因，深部真菌感染也逐渐成为许多疾病死亡的原因，所以，世界各国都投入大量的人力、财力，加强对抗肿瘤、抗真菌药物的研究。海洋由于其生态环境和生物多样性与陆地有很大差别，已成为新药开发的宝贵资源；而红树林作为一种特殊的海洋生态环境，是一类尚未很好地开发和利用的微生物资源库和新化合物库。
本文在国家海洋863课题和国家自然科学基金的资助下，以福建省的红树林植物为研究对象，分离红树植物内生真菌，并在菌株鉴定基础上分离纯化其代谢产物，研究化合物的抗菌、抗肿瘤活性，试图为红树植物内生真菌资源的开发和利用提供依据。
我们从福建九龙江口浮宫镇红树植物桐花树树皮分离获得两株内生真菌。经鉴定，HTF3菌株为小穴壳菌属(Dothiorella sp．)，HTF5菌株为小孢盘多毛孢(Pestalotiopsis microspora)，这是首次从红树植物中分离到这两种内生真菌。我们还首次测定了Dothirella sp．的ITS序列并正准备在GenBank中登记注册。
从两个菌株的发酵液中共分离纯化得到10个单体化合物，应用NMR谱和MS谱对化合物进行结构解析，发现从HTF3菌株中分离的5个单体化合物中有4个是未见报道的新结构化合物：2-[3’，5’-二羟基-2’-(7’-羟基)辛酮基苯基]-乙酸乙酯(dothiorelone A)、2-[3’，5’-二羟基-2’-(6’-羟基)辛酮基苯基]-乙酸乙酯(dothiorelone B)、2-[3’，5’-二羟基-2’-(8’-羟基)辛酮基苯基]-乙酸乙酯(dothiorelone C)和6，8-二羟基-1(R)(5’-羟己基)-异苯并二氢吡咯-3-酮(dothiorelone D)。另一个虽为已知的cytosporone B(dothiorelone E)，但为我们首次从红树林内生真菌中分离获到。活性检测
一摘要一
中发现5个化合物均表现出不同程度的抗肿瘤活性，其中dothioreloneE活
性最强且有较广的肿瘤谱。这5个化合物均没有抗细菌活性，但dothiorelone
E有较强的抗真菌活性，真菌谱较广，且产量达到10mg/L，有作为药物开
发的前景。该成果已申请专利并已公开。本文还分析了类似化合物的结构
与活性的关系，并对可能的作用机理进行了讨论。
从内生真菌HTFS菌株发酵液和菌体中共分离出5个单体化合物，其中
有3个酚类物质，这是首次报道从红树内生真菌代谢物中同时分离到这三
种多酚化合物，推测它们是作为芳香化合物的代谢中间物被分离到的，所
以HTFS菌株可能具有分解芳香化合物的独特能力。从菌体中分离到的2个
脂类物质可能是组成细胞膜的成分。
本文的研究结果表明，红树林内生真菌确是一类值得更好地开发的真
菌资源，是新颖结构和新生理活性的次级代谢产物的一个重要来源。
With the development of molecular biology, the research for new antitumor and anti-fungal compounds has deepened into the mechanism of cancer and fungal disease. On the other side, people begin to search more active leading compounds from new environments beyond terrestrial ones. The marine microorganisms are attractive for being as a potential resource, for a variety of chemically interesting and biologically significantly secondary metabolites have being purified from marine environments and marine microbial secondary metabolites. Mangroves are special woody plant communities in the intertidal zone of tropical and subtropical coasts. They are proved to be a natural microorganisms and new metabolites storage, and are worth of well investigating.
Supported by the National High Technology Research and Development Program of China and the National Natural Science Foundation of China, we studied two endophytic fungi dothiorella sp. (HTF3) and Pestalotiopsis microspora (HTF5) collected from mangrove plant Avicennia marina at the estuary of Jiulong River, Fujian Province. It's the first time to get such two genera of fungi from mangrove trees. We replicated the whole DNA of HTF3 and got its ITS sequence for the frist time. The two endopytic fungi were fermented and their culture broth was extracted with ethyl acetate (EtoAc). The extracts were subjected to column chromatography on silica gel and HPLC. The two endophytic fungi afforded ten compounds which structures were elucidated by ESI 1D- and 2D-NMR spectra.
Five compounds were isolated from the fermentation broth of HTF3, four of them were identified as novel compounds:2-[3',5'-dihydroxy-2'-(7'-hydroxy-
octanoyl)-phenyl]-acetic acid ethyl ester (dothiorelone A), 2-[3',5'-dihydroxy-2'-(6'-hydroxy-octanoyl)-phenyl]-acetic acid ethyl ester (dothiorelone B), 2-[3',5'-dihydroxy-2'-(8-hydroxy-octanoyl)-phenyl]-acetic acid ethyl ester (dothiorelone C) and 6,8-dihydroxy-l-(5-hydroxy-hexyl)- isochroman- 3-one (dothiorelone D), together with a known cytosporone B (dothiorelone E), which was separated from mangrove endophytic fungi metabolites for the first time. Their structure skeletons were very rare in natural products. They all had antitumor activities, while dothiorelone E was more interesting, for it showed the highest activities, its ICso values were 4 u g/mL and 0.0625 u g/mL against human epidermal carcinoma of oral cavity, KB cell line and human Burkitt's lymphoma, Raji cell line, respectively. Furthermore, it showed broad activities against fungi, the MIC against Candida albicans Aspergillus niger Trichoderma sp. and Fusarium sp were 0.156mg/mL 0.125mg/mL 62.5 U g/mL and 62.5 u g/mL, respectively. It was
high-yielded (10mg/L) and was potent for further research. We also analyzed the structure-activity relationships of these analogs.
Three phenolic compounds, 4-hydroxy-benzoic acid 2,5-dihydroxy- benzal dehyde and 2,5-dihydroxy-benzoic acid, accompanied with two lipids were purified from the fermentation broth of stain HTF5. It's the first time to get such phenolic metabolites from a mangrove endophytic fungus.
Our study indicated that mangrove endophytic fungi were great resources for new fungi and new structural metabolites with bio-activities. They are worth further researches.
引文
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