【作者】 张翼；

【导师】 王斌贵；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2007， 博士

【摘要】 海洋微生物拥有丰富多样的次生代谢途径,其中海洋生物内生真菌次生代谢产物研究日益受到天然产物化学界的重视。本论文以菌丝体生物量、发酵产物重量、抗菌与细胞毒活性、薄层色谱分析结果以及高效液相色谱分析结果等为评价依据对采自青岛沿海的13株海藻内生真菌在四种液体培养基上的静置发酵产物进行了综合评价,并从中选择了黑曲霉Aspergillus niger EN-13(分离自褐藻囊藻Colpomenia sinuosa)和杂色曲霉A. versicolor EN-7(分离自褐藻鼠尾藻Sargassum thunbergii)两株真菌进行了30升规模发酵(分别采用GPYM培养基和PDB培养)和化学成分的研究,对分离得到的大部分化合物进行了初步的生物活性筛选。发酵提取物采用常规的硅胶柱层析、反相硅胶柱层析,凝胶Sephadex LH-20柱层析、制备薄层层析、半制备高效液相色谱以及重结晶等分离手段,得到单体化合物。利用各种现代波谱技术(IR、UV、EI-MS、FAB-MS、HR-ESI-MS、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、HMBC等)并结合化学方法从两种菌株发酵提取物中鉴定了55个化合物的结构。其中从菌株A. niger EN-13分离鉴定了31个化合物,发现9个新化合物,包括2个鞘酯类化合物(AN-1~2)、3个萘并-γ-吡喃酮类化合物(AN-3~5)、3个苯乙基取代的α-吡喃酮类化合物(AN-17, AN-19~20)和1个甾体Diels-Alder加成产物(AN-21),另有1个新的天然环二肽(AN-27)被分离鉴定;从菌株A. versicolor EN-7分离鉴定了24个化合物,发现2个新化合物,为蒽醌AV-12与AV-17,另外,从前一菌株(A. niger EN-13)中鉴定的2个新鞘酯类化合物(AN-1~2)在A. versicolor EN-7中也被再次分离到。对大部分单体化合物进行了抗菌活性、DPPH自由基清除活性和细胞毒活性测试。结果显示新化合物AN-1、AN-5和AN-20具有弱或中等强度的抑制白色念珠菌生长的活性,AN-4、AN-5、AN-21显示了弱或中等强度的抑制黑曲霉生长的活性,AV-12、AV-17显示了弱的抑制大肠杆菌生长的活性。在DPPH自由基清除活性筛选中,AN-5显示了中等强度的活性,其EC50为109.3μM,与阳性对照BHT相近(EC50为81.8μM)。其它部分已知化合物在抗菌和DPPH自由基清除活性的筛选中也显示了弱或中等强度的活性。在针对人肝癌细胞株SMMC-7721和人肺腺癌细胞株A549的体外细胞毒活性筛选中,所测样品均未显示显著活性。更多还原

【Abstract】 Marine microorganisms possess highly diverse metabolism pathways. Among them, the research on the secondary metabolites of marine endophytic fungi have drawn the interest of more and more natural product chemists. In this dissertation, on the basis of mycelia’biomass, weight of fermentation extracts, antimicrobial and cytotoxic activities, the results of TLC and HPLC analysis, the static fermentation extracts of 13 endophytic fungal strains (isolated from seaweeds collected along Qingdao coastline) on 4 kinds of liquid cultural mediums, were comprehensively evaluated. And two strains Aspergillus niger EN-1 (isolated from the brown algae Colpomenia sinuosa) and A. versicolor EN-7 (isolated from the brown algae Sargassum thunbergii) were selected as aimed strains to be fermented on 30 liters of GPYM and PDB cultural medium respectively for further investigation of chemical constituents. Most of the isolated compounds were preliminary screened on their bioactivities.The compounds were isolated from the fermentation extracts by repeated column chromatography (CC) on silica gel, RP-18, and Sephadex LH-20, as well as by preparative thin layer chromatography (PTLC), semi-preparative HPLC and recrystallization. The structures of these compounds were elucidated by means of spectroscopic methods including IR, UV, EI-MS, FAB-MS, HR-ESI-MS, 1D-NMR (1H-NMR, 13C-NMR, DEPT) and 2D-NMR (1H-1H COSY, HSQC, HMBC) as well as chemical method. Totally, 55 compounds were structurally elucidated. From the extracts of A. niger EN-13, 31 compounds were isolated and structurally elucidated. Among them, Nine were found to be new compounds, including two new sphingolipids (AN-1~2), three new naphtho-γ-pyrones (AN-3~5), three new phenethyl-α-pyrone derivatives (AN-17, AN-19~20), and a new steroidal Diels-Alder adduct (AN-21). Additionally, a new naturally reported cyclodipeptide (AN-27) were also elucidated. From the extracts of fungus A. versicolor EN-7, 24 compounds were isolated and structurally elucidated. Among them, two were found to be new compounds as new anthraquinones (AV-12, AV-17). In addition, the two new sphingolipids (AN-1~2) from fungus A. niger EN-13 were reisolated from fungus A. versicolor EN-7.Most of the pure compounds were screened for their antimicrobial, DPPH scavenging and cytotoxic activities. It was revealed that the new compounds AN-1, AN-5 and AN-20 exhibited moderate or weak inhibitory activity against Candida albicans, compound AN-4, AN-5 and AN-21 exhibited moderate or weak inhibitory activity against Aspergillus niger, compound AV-12 and AV-17 showed weak inhibitory activity against Escherichia coli. In the DPPH scavenging assay, compound AN-5 showed moderate free radical scavenging activities with an EC50 of 109.3μM, compared with the 81.8μM for positive control BHT. Part of the isolated known compounds also exhibited moderate or weak activity in the antimicrobial and DPPH scavenging assay. None of the tested compounds showed remarkable activity in the in vitro cytotoxic assay against tumor cell lines SMMC-7721 and A-549.更多还原