两株中国南海柳珊瑚来源真菌次级代谢产物及其抗菌活性研究

发布时间：2018-06-17 01:10

本文选题：柳珊瑚来源真菌 + Microsphaeropsis　；参考：《中国海洋大学》2015年硕士论文

【摘要】：海洋中的微生物具有极其丰富的物种多样性,而且生物活性独特,是发现海洋来源药物的前沿研究领域。海洋微生物中蕴含了大量有特殊药理活性的新颖化合物,结构类型多样,显示了广阔的药用前景。本研究以两株中国南海柳珊瑚来源的真菌为研究对象,以抗菌活性筛选为指导,进行次级代谢产物研究,以期获得具有抗菌活性的化合物,从而为海洋活性天然产物以及海洋药物先导化合物的筛选和发现提供基础资料。本研究对花柳珊瑚来源真菌小球壳孢菌Microsphaeropsis sp.R.A10-14发酵液和蕾二岐灯芯柳珊瑚来源真菌曲霉Aspergillus sp.XS-2009-0066化学表观遗传修饰发酵液中抗菌活性化合物进行追踪分离,综合运用正、反相硅胶柱层析,Sephadex LH-20凝胶柱层析及半制备HPLC等分离手段,NMR.MS.IR.UV.CD等波谱学技术,并结合化学方法,分离并鉴定了23个化合物。从真菌Microsphaeropsis sp.RA10-14分离鉴定获得11个化合物。其中包括3个聚酮类化合物(1-3),4个色原酮类化合物(4-7),2个异香豆素类化合物(8,9),1个香豆素类化合物(10)和1个苔黑粉化合物(11),其中化合物1-5是新化合物。化合物1和2是一对C-11位差向异构体,初步的生合成途径推导该类化合物可能是由聚酮类化合物转化而来。化合物1和2的相对构型是运用1D NOE照射实验及耦合常数来确定的,绝对构型是通过Mosher方法确定的。抗菌活性评价结果显示,化合物1和2具有强的抗细菌活性,尤其是化合物1对铜绿假单胞菌(Pseudomonas aeruginosa),巴西诺卡菌(Nocardia brasiliensis),嗜根考克式菌(Kocuria rhizophila), 巨大芽孢杆菌 (Bacillus megaterium)活性显著,MIC值分别为0.19,0.19,0.19,0.19μM,强于化合物2的抗细菌活性,推测该类化合物C-11位的S构型能增强抗菌活性,R构型降低活性。异香豆素类化合物9表现出较好的抗菌活性,而化合物8活性较小,推测异香豆素类化合物中吡喃环比呋哺喃环更能增强化合物的活性。另外,抗真菌评价结果显示,化合物1和2表现出中等的抗真菌活性。除此之外,微藻克生活性评价结果显示,化合物1-3对青岛大扁藻显示了中等强度的抑制活性。对于菌株Aspergillus sp. XS-2009-0066,本课题组此前从中发现了吲哚尘物碱、联苯醚、核苷衍生物等结构类型的化合物。本研究试图运用化学表现遗传修饰方法改变该菌的代谢。在该菌株培养基中添加DNA甲基转移酶抑制剂-5-氮杂胞苷(5-AZA)及组蛋白去乙酰化酶抑制剂-伏立诺他(SAHA),从发酵液中分离得到6个与前期发酵条件下不同的化合物——没药烷倍半菇类化合物(1217)和6个本课题组前期曾从该菌中分离得到的化合物(18-23)。抗菌活性测试结果显示,化合物14对金黄色葡萄球菌(Staphyloccocus aureus)表现出较强的抗菌活性,MIC值为3.13 μM。而化合物17对金黄色葡萄球菌几乎没有抗菌活性,推测,该类化合物C-7位羟甲基是重要的药效团。综上所述,通过抗菌活性筛选和化学筛选,从花柳珊瑚来源小球壳孢菌Microsphaeropsis sp. RA10-14中分离获得了多种结构类型的海洋天然产物,具有抗菌活性,为海洋抗菌药物先导化合物的筛选和发现提供了化合物基础；运用化学表观遗传修饰方法调控海洋真菌的次级代谢,成功激活了海洋真菌沉默基因的表达,获得了新的结构类型次级代谢产物。
[Abstract]:The microorganism in the ocean is extremely rich in species diversity and unique in biological activity. It is a frontier research field for discovery of marine source drugs. The marine microorganism contains a large number of novel compounds with special pharmacological activity, with a wide variety of structure types and a broad prospect of drug use. Two strains of Chinese South China Sea willow corals have been found in this study. The source of fungi, as the research object, guided by the screening of antimicrobial activity, studies secondary metabolites in order to obtain compounds with antibacterial activity, thus providing basic information for the screening and discovery of marine active natural products and marine drug precursor compounds. PSIs sp.R.A10-14 fermentation broth and bud two wick Liu Shanhu source fungi Aspergillus oryzae Aspergillus sp.XS-2009-0066 chemical epigenetic modified fermentation broth were traced and separated, integrated use of positive, reverse phase silica column chromatography, Sephadex LH-20 gel column chromatography and semi preparation of HPLC and other separation methods, NMR.MS.IR.UV.CD and other spectroscopy 23 compounds were isolated and identified by chemical methods. 11 compounds were obtained from the Microsphaeropsis sp.RA10-14 isolation and identification of fungi, including 3 polyketones (1-3), 4 chromoones (4-7), 2 coumarin compounds (8,9), 1 coumarins (10) and 1 moss black powder compounds (11). Compound 1-5 is a new compound. Compounds 1 and 2 are a pair of C-11 dislocation isomers. The initial biosynthesis pathway derives that this kind of compound may be converted from polyketones. The relative configurations of compounds 1 and 2 are determined by 1D NOE irradiation experiments and coupling constants. The absolute configuration is determined by the Mosher method. The results of activity evaluation showed that compounds 1 and 2 had strong anti bacterial activity, especially compound 1 to Pseudomonas aeruginosa (Pseudomonas aeruginosa), Nocardia Brazil (Nocardia brasiliensis), root cork (Kocuria rhizophila), Bacillus giant bacillus (Bacillus megaterium), and MIC value of 0.19,0.19,0.19,0.19. M, which is stronger than the anti bacterial activity of compound 2, speculates that the S configuration of the C-11 bit of this compound can enhance the antibacterial activity and the R configuration reduces the activity. The compound coumarin compound 9 shows better antibacterial activity, and the compound 8 has smaller activity. The results of fungal evaluation showed that compounds 1 and 2 showed moderate antifungal activity. In addition, microalgal life evaluation showed that compound 1-3 showed moderate inhibitory activity to Qingdao algae. For strain Aspergillus sp. XS-2009-0066, indoles, diphenyl ethers and nucleoside derivatives were found in this group. This study tried to change the metabolism of the bacteria by means of chemical expression genetic modification. In the medium, the DNA methyltransferase inhibitor -5- nitrocytidine (5-AZA) and the histone deacetylase inhibitor volino (SAHA) were added to the fermentation broth, and 6 of the fermentation conditions were isolated from the fermentation broth. Different compounds - myrrh half mushroom compounds (1217) and 6 compounds (18-23) which had been isolated from the bacteria in earlier period (18-23). Antibacterial activity test results showed that compound 14 showed strong antibacterial activity to Staphylococcus aureus (Staphyloccocus aureus), MIC value was 3.13 mu M. and compound 17 to Staphylococcus aureus There is almost no antibacterial activity of staphylococci. It is speculated that the C-7 methyl methyl is an important pharmacophore. To sum up, a variety of structural types of marine natural products have been obtained from Microsphaeropsis sp. RA10-14 of corals derived from corals, which have antibacterial activity and are resistant to the sea. The screening and discovery of microbial drug precursor compounds provide the basis for compounds, and the use of chemical epigenetic modification to regulate secondary metabolism of marine fungi has successfully activated the expression of the silencing genes of marine fungi and obtained new secondary metabolites of structural types.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q936

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