四株海洋来源真菌活性次级代谢产物的研究

发布时间：2019-05-19 18:25

【摘要】：海洋真菌作为海洋微生物的重要组成部分,其遗传背景复杂,次生代谢产物具有化学多样性丰富、产量高等特点,在药物先导化合物的发现、石油降解及海洋环境的修复等方面发挥着重要作用。近年来由于对随着对陆生微生物天然产物研究的深入,由于化合物的重复分离,从陆生微生物中寻找寻找新的活性天然产物的难度越来越大。所以,天然产物学家们把他们的目光集中到海洋微生物上,特别是海洋真菌,由于其的生存的特殊环境,被认为是新的活性天然产物的重要源泉。在这个前提下以及作为我们前期海洋真菌活性天然产物发现工作的延续,我们采用化学和生物学相结合的方法,开展对海洋真菌活性次生代谢产物的研究工作。具体内容包括：菌株的分离与纯化；真菌菌株的筛选及“天才”真菌的获得；目标真菌的发酵条件摸索；真菌发酵、提取及化合物分离；单体化合物的结构鉴定；单体化合物活性的初步评价及构效关系的分析。本论文对15个海洋来源样品进行了微生物的分离,最终分离得到90株真菌和46株放线菌。通过TLC. HPLC指纹图谱和HPLC-MS的化学筛选结合抗肿瘤、抗菌、抗病毒及糖苷酶抑制活性等活性筛选,最终分离筛选到4株海洋“天才”真菌,对其发酵条件进行了系统的摸索后进行大规模的发酵提取,然后对其次生代谢产物的结构和活性进行了深入研究。综合采用薄层色谱、硅胶柱色谱、凝胶柱色谱和半制备高效液相等色谱法从菌株茎点属真菌Phoma sp. OUCMDZ-1847中共分离得到18个化合物(1-18),其中包括3个新的硫代二酮哌嗪类化合物(1-3)、10个已知的硫代二酮哌嗪类化合物(4-13)、两个简单的二酮哌嗪类化合物(14-15)、一个苯的衍生物(16)、一个吲哚衍生物(17)和一个嘌呤类化合物(18)。从曲霉属真菌Aspergillus sp.OUCMDZ-1583中分离得到23个化合物(19-41),包括1 8个新化合物(19-36)和5个己知的化合物(37-41)。从菌株OUCMDZ-1635中分离得到3个新的化合物(42-44),包括一个以一对互变异构体存在的生物碱类化合物(42)、一个倍半萜类化合物(43)和一个甾体类化合物(44)。从菌株OUCMDZ-1857中分离得到2个己知的生物碱类化合物(45,46)。通过核磁共振谱(NMR)、质谱(MS)、紫外光谱(UV)、红外光谱(IR)、圆二色谱(CD)等现代波谱和光谱学方法结合X-射线单晶衍射和化学沟通反应等方法确定了化合物的平面结构以及绝对构型。最后,运用体外生物活性筛选模型,采用生物活性评价方法评价了所获得的单体化合物的抗肿瘤、抗流感病毒H1N1、抗菌和α糖苷酶抑制活性。结果表明：结果显示化合物2,4,5,11和12对HL-60, HCT-116, A549, K562和MGC-803五株肿瘤细胞株的显示有较好的肿瘤细胞毒活性。化合物19,20,23,28,29,32-36,和39-41具有一定的糖苷酶抑制活性,IC50分别是2.36,1.65,1.30,2.37,2.70,1.36,1.54,2.21,2.26,0.027,1.65,1.19和1.74 mM,阳性药阿卡波糖的IC50为0.95mM。另外,化合物36和39具有一定的抗H1N1病毒活性,IC50分别为172.4/μM和175.5μM,阳性药利巴韦林的IC50为137.3μM。综上,本文对4株海洋真菌的次生代谢产物进行了系统研究,共分离鉴定了46个化合物,包括24个新的化合物,并对这些化合物的活性进行了系统的评价,其中5个硫代二酮哌嗪类化合物具有较强的细胞毒活性,13个化合物具有一定的糖苷酶活性,2个化合物具有抗病毒活性。
[Abstract]:As an important part of marine microorganisms, marine fungi play an important role in the discovery of the drug-leading compound, the oil degradation and the repair of the marine environment. In recent years, because of the in-depth study of the natural products of the terrestrial micro-organisms, the difficulty of finding new active natural products from the terrestrial microorganisms is becoming more and more difficult due to the repeated separation of the compounds. As a result, natural product scientists focus their attention on marine microorganisms, particularly marine fungi, which are considered to be an important source of new active natural products due to their special environment. In this premise and as a continuation of the discovery of the natural products of marine fungal activity in our early stage, we use a combination of chemical and biological methods to carry out research on the secondary metabolites of marine fungi. The specific content includes the isolation and purification of the strain, the screening of the fungus strain and the obtaining of the "genius" fungus, the fermentation condition of the target fungus, the fungus fermentation, the extraction and the compound separation, and the structural identification of the monomer compound; The preliminary evaluation of the activity of the monomer compound and the analysis of the structure-effect relationship. In this paper,15 marine-derived samples were isolated and 90 fungus and 46 actinomycetes were isolated. By tlc. the HPLC fingerprint and the chemical screening of the HPLC-MS are combined with the activity screening of the anti-tumor, the antibacterial, the anti-virus and the glycosidase inhibitory activity, and finally the 4 strains of marine "genius" fungi are separated and screened, and the fermentation conditions are carried out for large-scale fermentation and extraction, The structure and activity of the secondary metabolites were further studied. The fungi, Phoma sp., were isolated by thin-layer chromatography, silica gel column chromatography, gel column chromatography and semi-preparative high performance liquid chromatography. A total of 18 compounds (1-18) are obtained from the total of OCMDZ-1847, which comprises 3 new thiodione-like compounds (1-3),10 known thiodiketone-like compounds (4-13), two simple diketone-like compounds (14-15), and a benzene derivative (16), And a terin derivative (17) and a methotrexate compound (18). 23 compounds (19-41), including 1 8 new compounds (19-36) and 5 well-known compounds (37-41), were isolated from Aspergillus sp. OCMDZ-1583 of the genus Aspergillus. Three new compounds (42-44) are isolated from the strain OCMDZ-1635, and include an alkaloid compound (42), a polysemide compound (43) and a donor compound (44) present in a pair of tautomers. Two known alkaloid compounds (45,46) were isolated from the strain OCMDZ-1857. The planar structure and the absolute configuration of the compound were determined by means of NMR, MS, UV, IR, CD and other methods such as X-ray single crystal diffraction and chemical communication. Finally, an in vitro biological activity screening model was used to evaluate the anti-tumor of the obtained monomer compound, the anti-influenza virus (H1N1), the anti-bacterial and the anti-glycogenase-inhibiting activity by the method of biological activity evaluation. The results showed that the results showed that the compounds 2,4,5,11 and 12 had good cytotoxic activity on the tumor cell lines of HL-60, HCT-116, A549, K562 and MGC-803. Compounds 19,20,23,28,29,32-36, and 39-41 had a certain glycosidase inhibitory activity with an IC50 of 2.36, 1.65, 1.30, 2.37, 2.70, 1.36, 1.54, 2.21, 2.26, 0.027, 1.65, 1.19, and 1.74 mM, with an IC50 of 0.95 mM for the positive drug acarbose. In addition, compounds 36 and 39 have a certain anti-H1N1 virus activity, IC50 of 172.4/. mu.M and 175.mu. M, respectively, and the IC50 of the positive drug ribavirin is 137.3. m Including 24 new compounds and a systematic evaluation of the activity of these compounds,5 of which have strong cytotoxic activity,13 compounds have a certain glycosidase activity and 2 compounds have anti-viral activity.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R915

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