白茅内生菌C.globosum的次生代谢产物研究

【作者】 李明；

【导师】 申丽；

【作者基本信息】 扬州大学 ， 中药学（专业学位）， 2015， 硕士

【摘要】 植物内生菌具有可再生性、生长周期短、代谢易于控制和菌种易于选育等优势,且与宿主植物长期协同进化,拥有特殊的代谢途径,能够产生结构新颖和活性突出的次生代谢产物。因此,从植物内生菌次生代谢产物中筛选先导化合物,并进行其结构的修饰与优化,己成为新药研发行之有效的途径。球毛壳菌Chaetomiun globosum IFB-WQ是南京大学功能生物分子研究所从白茅(Imperata cylindrical)中分离获得的一株内生真菌。前期实验发现,该菌株固体发酵产物中含有丰富的细胞松弛素化合物,因此,本学位论文对该菌株固体发酵产物的化学成分进行了深入研究,以期为新药研制提供先导化合物。菌株IFB-WQ固体发酵产物的乙酸乙酯相和正丁醇相经硅胶柱粗分离得到各极性组分：以1H-NMR谱和TLC示踪,组合使用硅胶柱层析、Sephadex LH-20凝胶柱层析、薄层层析、高效液相色谱等方法对组分进行分离纯化,共分离获得7个化合物。采用高分辨质谱、1D和2D核磁共振谱等现代波谱技术及文献比对的方法对化合物进行结构鉴定,上述化合物分别被鉴别为N-benzoylphen-ylalaninyl-N-benzoylphenylalaninate (1)、dihydrocytoglobosin D (2) chaetoglobosin F (3)、cytochalasin A (4)、Chaetoglob-osin E (5)、uracil (6)和5-methyluracil (7),其中化合物2的结构未见文献报道。采用MTT法测定了化合物1、3、4和5的体外细胞毒活性,结果发现：化合物3、4和5对人宫颈癌细胞株HeLa具有较强的抑制活性,其ICso值分别为32.47、51.7和7.20μg/ml,阳性对照顺铂的IC5o值为6.42μg/ml;化合物3、4和5对人肝癌细胞株HepG2也具有较强的抑制活性,其IC50值分别为14.45、7.33和3 .60μg/ml,阳性对照顺铂的IC5o值为4.90μg/ml。此外,还采用改良Ellman法测定了化合物3抑制乙酰胆碱酯酶的活性,结果发现,化合物3对乙酰胆碱酯酶具有中等抑制活性,其EC5o值为7.06mg/ml,阳性对照他可林EC5o值为0.7μg/ml。依据发现先导化合物和发展先导化合物的研究思路,本学位论文对特境微生物--植物内生菌球毛壳菌C. globosum IFB-WQ固体发酵产物的化学成分进行了研究,结果发现多个药理活性成分,为后续研究提供了化合物来源。更多还原

【Abstract】 Endophytes have some advantages, such as renewability, short growth cycle, metabolism and breeding easily to be controlled and so on. During a long term process of co-evolution with the host plant, endophytes formed a special metabolic pathway, which could produce secondary metabolites with a variety of bioactivites and structures. Therefore, screening lead compounds from endophyte secondary metabolites and studying on modification and optimization of the structures became an effective approach in developing new drugs.Chaetomiun globosum IFB-WQ was an endophyte separated from Imperata cylindrical by institute of Functional Biomolecules, Nanjing University. Previous research had showed that there are abundant cytochalasin compounds in the extract of solid culture of C. globosum that aim to provide lead compounds for the development of new drugs. Then chemical constituents of solid culture of this strain had been carried out in the dissertation. EtOAc extract and n-butyl alcohol extract of strain IFB-WQ were chromatographed on silica gel column and several different polar mixtures was obtained. Guided by 1H-NMR spectrum and TLC, further separation and purification was performed using several methods such as silica gel column chromatography, Sephadex LH-20 gel column chromatography,HPLC etc. Finally, seven compounds had been obtained and structures of them were determined by modern spectrum technology such as HR-ESI-MS, 1D and 2D NMR and comparing with references. Compounds 1-7 were identified as N-benzoylphenylalaninyl-N-benzoylphenylalaninate (1), dihydrocytoglobosin D (2), chaetoglobosin F (3), cytochalasin A (4), chaetoglobosin E (5), uracil (6) and 5-methyluracil (7), in which compound 2 had not been reported yet. In vitro cytotoxicity to human cervical cell line HeLa and human gastric cancer cell line HEPG2 of compounds 1,3,4 and 5 were evaluated by MTT. The results showed that compound 3,4 and 5 displayed moderate inhibitory activity against HeLa cells with an IC50 value of 32.47,51.7 and 7.2μg/ml respectively, while the IC50 value of positive control cisplatin was 6.42μg/ml. Compounds 3,4 and 5 obviously could inhibit proliferation of HepG2 cells with an IC50 value of 14.45,7.33 and 3.60μg/ml respectively, while the IC50 value of positive control cisplatin was 4.90μg/ml. In addition, AchE inhibitory activity of compound 3 was tested using modified Ellman method. Compound 3 had moderate AchE inhibitory activity with EC50 value of 7.06mg/ml and the EC50 value of positive control tenakrin was 0.7μg/ml.According to the idea of discovering and developing lead compounds from special environment microorganism, looking for lead compounds from special environment microorganism-endophyte C. globosum was conducted in this dissertation. Several bioactive compounds had been found through our research, which several compounds for further research.更多还原