特殊生境球毛壳菌Chaetomium globosum次生代谢产物研究
发布时间：2018-09-03 13:23
【摘要】：天然产物最初来自于药用动植物,是新药及新药先导化合物的重要来源。自从发现动植物内共生菌在与宿主长期协同进化过程中,形成特殊的代谢途径,能产生结构新颖、活性显著的化学成分,且其具有易于培养以及能够大规模发酵等特点,而成为天然活性成分的新来源。毛壳菌(Chaetomium)是真菌毛壳菌科(Chaetomiaceae)中的一个大属,有100多个种。研究表明,该属真菌能产生萜类、留体类、细胞松弛素类、氧杂蒽酮类和缩酚酸环醚类等多种结构类型的活性化合物,其次生代谢产物值得深入研究。球毛壳菌Chaetomium globosumML-4是青岛农业大学王凤舞博士课题组从青岛海域牡蛎(Oyster)中分离得到的一株共生菌,球毛壳菌Chaetomium globosum IFB-WQ是南京大学功能生物分子研究所从药用植物白茅(Imperatacylindrical)中分离得到的一株内生菌。本学位论文对Cglobosu.ML-4发酵液和C.glbosum IFB-WQ固体发酵产物的化学成分分别进行了研究,以期为新药研发提供先导化合物。Cglobosu.ML-4发酵液经乙酸乙酯萃取得粗浸膏3.2g,C.globosu IFB-WQ固体发酵产物的氯仿甲醇提取物先后经乙酸乙酯和正丁醇萃取得粗浸膏26g和17g。粗浸膏经硅胶柱层析粗分离后,以TLC和1H-NMR谱为指导,综合运用硅胶柱层析、Sephadex L H-20凝胶柱层析、反相柱层析、制备薄层层析以及高效液相色谱等方法对组分进行分离纯化,结果从菌株ML-4中分离得到5个化合物(1～5),从菌株IFB-WQ中分离得到6个化合物(1～2和6～9);采用高分辨质谱、ID和2D核磁共振谱等波谱技术及文献比对的方法对化合物结构进行鉴定,上述化合物分别被鉴定为iso-chaetoglobosinVb(1)、chaetoglobosinV(2)、tyrosol(3)、5-methyluracil(4)、uracil(5)、chaetoglobosinFex(6)、chaetoglobosin W(7)、cytoglobosin I(8)和 cytoglobosinAb(9)。其中化合物iso-chaetoglobosinVb(1)和chaetoglobosin V(2)同时从两株C.globosum中分离得到。本学位论文首次报道了cytoglobosinAb(9)的波谱数据,iso-chaetoglobosinVb(1)的绝对构型有待进一步鉴定。采用MTT法测定了化合物1、2、6和9对人肝癌细胞株SMMC-7721的体外细胞毒活性,结果发现,化合物2对SMMC-7721细胞具有较强的增殖抑制活性,其IC50值为60.5μg/mL,阳性对照顺铂的IC50值为7.92μg/mL;化合物2对SMMC-7721细胞的增殖抑制活性呈现明显的浓度依赖性。化合物1、2、6、7、8和9为细胞松弛素类化合物,文献表明,该类化合物具有抗肿瘤、免疫抑制、抗氧化等多种生物活性。我们前期研究发现,该类化合物对MPP+诱导的PC12细胞损伤具有显著的保护作用,是潜在的抗帕金森病(PD)天然药物,上述细胞松弛素化合物的抗PD活性将在后续实验中做进一步研究。
[Abstract]:Natural products originally come from medicinal plants and animals, and are important sources of new drugs and new drug leading compounds. Since it has been discovered that the symbiotic bacteria in plants and animals form special metabolic pathways during their long-term coevolution with the host, they can produce chemical constituents with novel structure and remarkable activity, and they are easy to culture and can be fermented on a large scale. And become a new source of natural active ingredients. (Chaetomium) is a large genus of (Chaetomiaceae), which has more than 100 species. The results showed that the fungi could produce active compounds of terpenes, residues, cytochalasins, oxyanthracones and phenolic acid cyclic ethers, and the secondary metabolites were worthy of further study. Chaetomium globosumML-4 is a symbiotic bacterium isolated from (Oyster) of oyster from Qingdao Agricultural University. Chaetomium globosum IFB-WQ is an endophytic bacterium isolated from (Imperatacylindrical), a medicinal plant, by the Institute of functional Biomolecular, Nanjing University. The chemical constituents of Cglobosu.ML-4 fermentation broth and C.glbosum IFB-WQ solid fermentation product were studied in this dissertation. In order to provide the lead compound. Gglobosu.ML-4 fermentation broth extracted by ethyl acetate to obtain crude extract 3.2g C. Globosu IFB-WQ solid fermentation product chloroform methanol extract was extracted by ethyl acetate and n-butanol to obtain crude extract 26g and 17g respectively. The crude extract was separated by silica gel column chromatography, followed by TLC and 1H-NMR spectra. The components were separated and purified by silica gel column chromatography with Sephadex L H-20 gel column chromatography, reversed-phase column chromatography, thin layer chromatography and high performance liquid chromatography. Results five compounds (1 / 5) were isolated from strain ML-4 and 6 compounds (1 / 2 and 6 / 9) were isolated from strain IFB-WQ. The structure of the complex was identified, The compounds were identified as iso-chaetoglobosinVb (1) chaetoglobosin V (2) tyrosol (3) 5-methyluracil (4) uracil (5) chaetoglobosinFex (6) chaetoglobosin W (7) cytoglobosin I (8) and cytoglobosinAb (9). The compounds iso-chaetoglobosinVb (1) and chaetoglobosin V (2 were isolated from two C.globosum strains at the same time. The absolute configuration of the spectral data of cytoglobosinAb (9) iso-chaetoglobosin Vb (1) is reported for the first time in this dissertation. The cytotoxic activity of compounds 1, 2 and 9 on human hepatoma cell line SMMC-7721 in vitro was determined by MTT assay. The results showed that compound 2 had strong inhibitory activity on proliferation of SMMC-7721 cells. Its IC50 value was 60.5 渭 g / mL, and the IC50 value of cisplatin positive control was 7.92 渭 g / mL. Compound 2 showed a significant concentration-dependent inhibitory activity on the proliferation of SMMC-7721 cells. Compounds 1, 2, 2, 6, 7, 8 and 9 are cytochalasins, which have been shown to have many biological activities, such as antitumor, immunosuppressive, antioxidant, and so on. Our previous studies have shown that these compounds have a significant protective effect on PC12 cell damage induced by MPP and are potential natural drugs against Parkinson's disease (PD). The anti-PD activity of the above mentioned cytochalasin compounds will be further studied in subsequent experiments.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915
[Abstract]:Natural products originally come from medicinal plants and animals, and are important sources of new drugs and new drug leading compounds. Since it has been discovered that the symbiotic bacteria in plants and animals form special metabolic pathways during their long-term coevolution with the host, they can produce chemical constituents with novel structure and remarkable activity, and they are easy to culture and can be fermented on a large scale. And become a new source of natural active ingredients. (Chaetomium) is a large genus of (Chaetomiaceae), which has more than 100 species. The results showed that the fungi could produce active compounds of terpenes, residues, cytochalasins, oxyanthracones and phenolic acid cyclic ethers, and the secondary metabolites were worthy of further study. Chaetomium globosumML-4 is a symbiotic bacterium isolated from (Oyster) of oyster from Qingdao Agricultural University. Chaetomium globosum IFB-WQ is an endophytic bacterium isolated from (Imperatacylindrical), a medicinal plant, by the Institute of functional Biomolecular, Nanjing University. The chemical constituents of Cglobosu.ML-4 fermentation broth and C.glbosum IFB-WQ solid fermentation product were studied in this dissertation. In order to provide the lead compound. Gglobosu.ML-4 fermentation broth extracted by ethyl acetate to obtain crude extract 3.2g C. Globosu IFB-WQ solid fermentation product chloroform methanol extract was extracted by ethyl acetate and n-butanol to obtain crude extract 26g and 17g respectively. The crude extract was separated by silica gel column chromatography, followed by TLC and 1H-NMR spectra. The components were separated and purified by silica gel column chromatography with Sephadex L H-20 gel column chromatography, reversed-phase column chromatography, thin layer chromatography and high performance liquid chromatography. Results five compounds (1 / 5) were isolated from strain ML-4 and 6 compounds (1 / 2 and 6 / 9) were isolated from strain IFB-WQ. The structure of the complex was identified, The compounds were identified as iso-chaetoglobosinVb (1) chaetoglobosin V (2) tyrosol (3) 5-methyluracil (4) uracil (5) chaetoglobosinFex (6) chaetoglobosin W (7) cytoglobosin I (8) and cytoglobosinAb (9). The compounds iso-chaetoglobosinVb (1) and chaetoglobosin V (2 were isolated from two C.globosum strains at the same time. The absolute configuration of the spectral data of cytoglobosinAb (9) iso-chaetoglobosin Vb (1) is reported for the first time in this dissertation. The cytotoxic activity of compounds 1, 2 and 9 on human hepatoma cell line SMMC-7721 in vitro was determined by MTT assay. The results showed that compound 2 had strong inhibitory activity on proliferation of SMMC-7721 cells. Its IC50 value was 60.5 渭 g / mL, and the IC50 value of cisplatin positive control was 7.92 渭 g / mL. Compound 2 showed a significant concentration-dependent inhibitory activity on the proliferation of SMMC-7721 cells. Compounds 1, 2, 2, 6, 7, 8 and 9 are cytochalasins, which have been shown to have many biological activities, such as antitumor, immunosuppressive, antioxidant, and so on. Our previous studies have shown that these compounds have a significant protective effect on PC12 cell damage induced by MPP and are potential natural drugs against Parkinson's disease (PD). The anti-PD activity of the above mentioned cytochalasin compounds will be further studied in subsequent experiments.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915
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