【作者】 武文斌；

【导师】 瞿伟菁； 朱瑞良； 张雯；

【作者基本信息】 华东师范大学 ， 植物学， 2014， 博士

【摘要】 植物内生真菌(Endophytic fungus)能够合成与药用植物相同或相似的生物活性物质,或者影响药用植物某些活性物质的产量,具有良好的前景和开发利用价值。本论文以从植物内生真菌天然产物中寻找具有抗菌抗肿瘤活性的化合物为主线,选定传统药用植物蒺藜(Tribulus terrestris L.)作为研究对象,从蒺藜内生真菌的分离鉴定、活性及相关化合物的检测和活性菌株代谢产物的分离鉴定等几个方面展开了相关研究,得到以下结果：一、首次从蒺藜植株内分离纯化得到64株内生真菌,经形态学和分子生物学手段鉴定分为2门5纲8目10属。其中,以茎点属(Phoma)和链格孢属(Alternaria)为主,分别占分离到的总菌株数的39.06%和28.12%。选取其中21株代表性菌株作为研究的重点内容。二、从黄酮类皂苷类化合物的检测和生物学活性的检测两方面入手,筛选21株蒺藜内生真菌中的活性菌株。1.选取宿主植物蒺藜的主要药用成分——黄酮类和皂苷类化合物作为检测指标,筛选能够合成类似产物的蒺藜内生真菌。使用SPE-HPLC和GC-MS联用技术检测发酵液中的黄酮类和皂苷类化合物,建立了检测这两类化合物的技术路线。同时,实验结果显示发酵液中无与蒺藜相同的黄酮类化合物；在茎点霉属真菌几-16的发酵液中发现了皂苷的前体物质鲨烯和甾醇类化合物17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradeca hydro-1H-cyclopenta[a]phenanthren-3-ol,但是经反复传代培养后,JL-16不再合成这两种化合物。2.从抗细菌、抗真菌和抗肿瘤三个方面评价蒺藜内生真菌的生物学活性。以大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)作为革兰氏阴性细菌和革兰氏阳性细菌的代表,评价了21株蒺藜内生真菌发酵产物的抗细菌活性。结果显示,PDB培养基发酵液对大肠杆菌和金黄色葡萄球菌都具有抑制作用(抑菌圈直径大于10mm)的菌株占总菌株数的19.05%；察氏培养基发酵液对大肠杆菌和金黄色葡萄球菌均具有抑制作用的菌株占总菌株数的23.81%。采用稻瘟菌(Magnaporthe oryzae)模型评价21株蒺藜内生真菌发酵产物的抗真菌活性。结果显示PDB培养基发酵液中MIC值相对较小(发酵液浓度低于10%)的菌株占总分离菌株的19.05%；察氏培养基发酵液中MIC值相对较小的菌株占总分离菌株的47.61%。以前列腺癌PC-3细胞株和乳腺癌4T1细胞株为模型,评价了21株蒺藜内生真菌发酵产物的细胞毒活性。对于4T1细胞株,经PDB发酵液处理抑制率高于50%的有9株,占总菌株数的42.86%；经察氏发酵液处理抑制率高于50%的有8株,占总菌株数的38.10%。对于PC-3细胞株,经PDB发酵液处理抑制率高于50%的有10株,占总菌株数的47.62%,；经察氏发酵液处理抑制率高于50%的有6株,占总菌株数的28.57%。其中,蒺藜内生真菌JL-3、JL-7w、JL-13、JL-14、JL-16和JL-17的细胞毒性最强,通过对它们的PDB发酵液进行分步萃取,并对所有粗提物进行细胞毒活性评价,结果发现,JL-7w的PDB发酵液的氯仿提取物对于PC-3细胞的抑制作用最强,IC50值为55.15μg/mL。通过以上三个指标发现JL-7w具有良好的生物学活性,需要进一步研究。三、针对具有良好生物学活性的JL-7w进行详细的菌种鉴定和代谢产物分析。1.通过孢子形态鉴定、特异性基因序列分析及紫外指纹图谱分析等手段对链格孢属真菌进行鉴定,确定JL-7w为细极链格孢菌(Altemaria tenuissima)。2.通过反复硅胶柱层析和凝胶柱层析等手段在JL-7w的发酵液中分离得到新化合物Altertoxin Ⅳ和己知化合物Altertoxin Ⅱ,并运用高分辨质谱、核磁共振和单晶衍射等方法确定了Altertoxin Ⅳ的绝对构型。通过Altertoxin Ⅱ和Altertoxin Ⅳ的细胞毒性检验发现,Altertoxin Ⅳ对肿瘤细胞PC-3无明显的抑制作用,而Altertoxin Ⅱ对于肿瘤细胞PC-3有强烈的抑制作用,ICso值为14.28μM。更多还原

【Abstract】 Endophytic fungi can produce the same or similar bioactive agents as medicinal plants, or relay on the phytochemicals produced by the plants. It is urgent to isolate and study associated endophytes from the host plants. The main purpose of this dissertation was to investigate antimicrobial and anticancer compounds from the natural products of endophytes. To continue the previous work in our lab, Tribulus terrestris L., a traditional medicinal plant, was selected for further investigation. In the present study, the research was performed on several aspects including isolation and identification, activity detection, investigation on secondary metabolites of endophytic fungi from T. terrestris L.. The main contents of this dissertation are summarized as follow:1. A total of64endophytic fungi were isolated by culture-dependent method, identified by morphological and molecular methods and categorized into10genera. Among them, Phoma and Alternaria were the dominant genera, accounted for39.06%and28.12%respectively of the total strains. Among them,21strains were selected as representative strains for a further study.2. Screening strains by detection of flavonoids and saponins compounds and biological activities.(1) Flavonoids and saponins were selected as test indicators, as which were the main active ingredients of T. terrestris L. Analysis of the products from21endophytic fungi using SPE-HPLC and GC-MS showed that JL-16could produce squalene and17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-ol, both of which are precursors of saponins. However, on subculturing the fungus JL-16in axenic medium, the endophyte tended to lose their ability to produce the two secondary metabolites.(2) Antibacterial, antimycotic and cytotoxic activities of the endophytes were also evaluated. Antibacterial activity of21endophytic fungi was evaluated by Staphylococcus aureus and Escherichia coli, which are the representative of Gram-negative and Gram-positive bacteria. The strains with their inhibition zone diameter of the PDB and Czapek broth greater than10mm were accounted for19.05% (4/21) and23.81%(5/21) respectively of the total strains. Antimycotic activity of21endophytic fungi was evaluated by Magnaporthe oryzae model. The strains, of which the PDB broth and the Czapek broth with the MIC (minimum inhibitory concentration) of Magnaporthe oryzae lower than10%, were accounted for19.05%(4/21) and47.61%(10/21) respectively of the total strains. Cytotoxic activities of21endophytic fungi were evaluated by PC-3(CRL-1435) and4T1(CRL-2539) cells. The PDB fermentation broth with the inhibitory action on4T1cancer cells over50%was accounted for42.86%of the total PDB broth (9/21). And the Czapek fermentation broth with the inhibitory action on4T1cancer cells over50%was accounted for38.10%of the total Czapek broth (8/21). The PDB fermentation broth with the inhibitory action on PC-3cancer cells over50%was accounted for47.62%of the total PDB broth (10/21). And the Czapek fermentation broth with the inhibitory action on PC-3cancer cells over50%was accounted for28.57%of the total Czapek broth (6/21). JL-3, JL-7w, JL-13, JL-14, JL-16and JL-17exhibited strongly cytotoxic effect. Crude extracts were obtained by fractional extraction and evaluated in PC-3cancer cells. Trichloromethane (TCM) extracts of JL-7w had the most effective activity against PC-3cells with the IC50value of55.15μg/mL. In summary, JL-7w exhibited strongly biological activities.3. Particular identification and chemical analysis of JL-7w.(1) The test of JL-7w resulted to be Alternaria tenuissima, which was confirmed by morphological characterization, endopolygalacturonase gene analysis and UV fingerprint spectra.(2) A new secondary metabolite, named altertoxin IV, together with altertoxin II, were isolated from the fermentation broth of JL-7w using repeated silica gel column and gel column chromatography. The new structure was characterized by HR-ESI-MS, multinuclear NMR spectroscopy, and single crystal X-ray diffraction method. Altertoxin II and Altertoxin IV were tested for their anti-cancer activities in vitro on the PC-3cells. Altertoxin IV had little anti-cancer activity at the tested (highest) concentration of2.67mM; however, Altertoxin Ⅱ showed moderate anti-cancer activity with the IC50value of14.28μM.更多还原