【作者】 郭蕾；

【导师】 秦路平； 吴锦忠； 韩婷；

【作者基本信息】 第二军医大学 ， 生药学， 2009， 硕士

【摘要】 内生真菌是指那些在其生活史中的某一段时期内生活在植物组织内,对植物组织没有引起明显病害的真菌。近年来的研究表明,内生真菌能够产生许多结构新颖、生理活性独特的或与宿主植物相同和相似的天然生理活性物质。基于此,本课题选择生境独特的粗疣合叶苔Scapania verrucosa Heeg.为实验材料,开展内生真菌分离、鉴定及其代谢产物的生物活性研究,并对活性菌株进行化学成分分离和鉴定。主要结果如下:1.通过组织分离法,采用PDA、KN、PY、察氏和萨氏5种分离培养基,从粗疣合叶苔中共分离纯化得到49株内生真菌。2.以经典形态学分类方法,对其中10株产孢的内生真菌进行显微形态特征的观察和鉴定,分别属于青霉属Penicillium和毛壳菌属Chaetomium,其余39株内生真菌在本试验条件下不产生孢子,占总分离菌株数的79.6%,对这些无孢类群进行ITS序列测序,并与序列相似性较高的已知菌株进行比较分析,确定了它们的分类地位。分离得到的粗疣合叶苔49株内生真菌分属于2个纲(Sordariomycetes, Eurotiomycetes)、4个目(Hypocreales, Eurotiales, Sordiriales, Xylariales)、5个科(Hypocreaceae, Trichocomaceae, Clavicipitaceae, Chaetomiaceae, Xylariaceae)、7个属(Hypocrea sp., Penicillium sp., Tolypocladium sp., Chaetomium sp., Xylaria sp., Nemania sp., Creosphaeria sp.),其中16株鉴定到种的水平,25株鉴定到属的水平,8株放在科的水平。毛壳属Chaetomium和Creosphaeria属为优势种群,均占总菌株数的18.36%。3.就分离到的内生真菌进行发酵培养,对其代谢产物进行生物活性筛选。稻瘟霉模型活性初筛的结果显示,粗疣合叶苔分离得到的49株内生真菌中,能抑制稻瘟霉孢子萌发的菌株数为40株,占总分离菌株数的81.6%。进一步的抗真菌及体外抗肿瘤活性测试发现,粗疣合叶苔内生真菌的代谢物具有良好的抗菌及体外抗肿瘤效果,8株内生菌对新生隐球菌和红色毛癣菌均有明显的抑菌活性,菌株T11、T35对白色念珠菌、新型隐球菌、红色毛癣菌和烟曲霉均具有很好的抑制作用。此外,8株内生菌的代谢产物对肿瘤细胞均有不同程度的细胞毒活性,尤其对HL-60都有较好的细胞毒作用。因此,粗疣合叶苔内生真菌能产生抗真菌和细胞毒活性物质,是开发抗菌抗肿瘤等药物的潜在资源。4.运用GC-MS鉴定了内生真菌T37低极性部分的28种化学成分,同时鉴定了粗疣合叶苔挥发油中的49种化学成分。宿主含有大量的倍半萜,而T37主要为烷类,酮类,酚类和酯类化合物,二者的化学成分在数量和种类上存在较大的差异。内生菌T37产生了与寄主植物成分不同的次生代谢产物,并且这些物质也同宿主一样表现出一定的抗真菌谱和细胞毒活性。5.在活性追踪指导下,对菌株T37、T31、T35的乙酸乙酯提取物进行分离和结构解析。采用常压柱层析和薄层层析等技术,共分离得到15个单体化合物,通过对化合物质谱、红外光谱、紫外光谱、核磁共振谱等波谱鉴定,并与标准品、文献值以及图谱库对照,确定了其中10个化合物的结构,分别为麦角甾醇、过氧麦角甾醇、甘露醇、2,3-二甲氧基-5-甲基苯酚、methyl curvulinate、curvulin、O-methylcurvulinic acid,柄曲霉素、Chaetofusins A和Chaetofusins B。其中,4个化合物为首次分到,化合物Chaetofusins A和Chaetofusins为两个结构特殊的新的生物碱类化合物,其余5个化合物结构需经进一步鉴定。粗疣合叶苔内生真菌具有良好的抗真菌和细胞毒作用,同时也蕴藏着较丰富的结构新颖的活性物质,是潜在的天然药物新资源。更多还原

【Abstract】 Endophytes are referred to fungi colonizing in healthy plant tissues without causing overt symptoms in or apparent injury to the host. Research results in recent years show that endophytes can produce some metabolites same or similar to those from the host plant, and metabolites with the novel structure and specific bioactivity. In this paper, we selected Scapania verrucosa Heeg., which grows in special environment as our materials, to isolate and identify its endophytes, and screen the bioactive strains from them. Then, we isolated and elucidated the secondary metabolites of three bioactive strains. The main results are as follows:1. Taking five different separating culture mediums, a total of 49 endophytic fungi were isolated from Scapania verrucosa Heeg. by tissue isolation method.2. After morphology observation, 10 strains of endophytic fungi with coiniophore were identified. They were subjected to Penicillum spp. and Chaetomium spp.. The rest strains (75.6%) without coindiophore were identified by PCR method to recognize the sequence of the rDNA ITS regions. In comparison with similar taxa, the sequence was grouped together.Forty-nine fungal strains in 7 genus(Hypocrea sp., Penicillium sp., Tolypocladium sp., Chaetomium sp., Xylaria sp., Nemania sp., Creosphaeria sp.) were classified, identified, and belonged to 2 classes(Sordariomycetes, Eurotiomycetes), 4 orders(Hypocreales, Eurotiales, Sordiriales, Xylariales) and 5 families (Hypocreaceae, Trichocomaceae, Clavicipitaceae, Chaetomiaceae, Xylariaceae) by morphological and molecular method. Chaetomium sp. and Creosphaeria sp. were dominate genus in endophytes from Scapania verrucosa which occupy 18.36 and 18.36 % of the all strains, respectively.3. The bioassay of the ethyl acetate extract of the fermentation broths of the 49 endophytes from Scapania verrucosa was studied. Biological model Pyricularia oryzae was efficiently applied to the primary screening of the antifungal and antitumor in vitro agents from the secondary metabolites of the microorganisms. Forty (81.6 %) strains of endophytes from S. verrucosa could induce morphological abnormality of P. oryzae. Antimicrobial activity against four fungi and cytotoxic effect against two cancer cell lines of 8 endophpytes extracts with better anti-P. oryzae activity were studied further. The results showed that endophytes from S. verrucosa exhibited antifungal activity and cytotoxic effect in some degree. 8 endophytes all had obvious antifungal activity on Cryptococcus neoformans and Trichophyton rubrum. The strain T11 and T35 could effectively inhibit all the tested fungi. The antitumor study in vitro showed that 8 strains displayed cytotoxic effect against one or more sensitive tumor cell lines and had stronger cytotoxic effect against HL-60 cell. Endophytes from S. verrucosa have antifungal activity and cytotoxic effects, and could be a potential resource for the development of antifungal and antitumor agent.4. A comparison of the constituents of the ether extracts between S. verrucosa and its endophytic fungus Chaetomium fusiforme was investigated by gas chromatography-mass spectrometry (GC/MS). 59 compounds were found in S. verrucosa. and 49 ones were characterized, while the number of identified constituents of the cultured endophyte extract was 28. The components of S. verrucosa were predominantly sesquiterpenes, whilst its endophytic fungus C. fusiforme produced alkanes, ketones, phenols and esters mainly. Although the extracts of S. verrucosa and its endophyte showed little correlation in chemical composition, both of them demonstrated antifungal and antitumor activities. The fungal strain T37 produced prolific secondary metabolites much differently from its host plant.5. The components of the ethyl acetate extract of active strains T37、T31 and T35 were isolated by bioassay-guided methods. Fifty chemical compounds were obtained by normal pressure column chromatography and TLC. Based on the data of spectral (MS, 1H-NMR, 13C-NMR) and comparison with related literatures, the chemical structures of the ten are clearly explained, which may be ergosterol, ergosterol peroxideone, mannitol, 2,3-dimethoxy-5-methylphenol, methyl curvulinate, curvulin, O-methylcurvulinic acid, sterigmatocystin, and two new unusual alkaloid compounds named Chaetofusins A and Chaetofusins B. Identification of other five components needs to be studied further.Endophytes from S. verrucosa have antifungal activity and cytotoxic effects, and produce metabolites with the novel structure and bioactivity. They could be a potential resource for natural medicine.更多还原