特殊生境球毛殼菌Chaetomium globosum次生代謝產(chǎn)物研究
發(fā)布時間：2018-09-03 13:23
【摘要】：天然產(chǎn)物最初來自于藥用動植物,是新藥及新藥先導化合物的重要來源。自從發(fā)現(xiàn)動植物內(nèi)共生菌在與宿主長期協(xié)同進化過程中,形成特殊的代謝途徑,能產(chǎn)生結(jié)構(gòu)新穎、活性顯著的化學成分,且其具有易于培養(yǎng)以及能夠大規(guī)模發(fā)酵等特點,而成為天然活性成分的新來源。毛殼菌(Chaetomium)是真菌毛殼菌科(Chaetomiaceae)中的一個大屬,有100多個種。研究表明,該屬真菌能產(chǎn)生萜類、留體類、細胞松弛素類、氧雜蒽酮類和縮酚酸環(huán)醚類等多種結(jié)構(gòu)類型的活性化合物,其次生代謝產(chǎn)物值得深入研究。球毛殼菌Chaetomium globosumML-4是青島農(nóng)業(yè)大學王鳳舞博士課題組從青島海域牡蠣(Oyster)中分離得到的一株共生菌,球毛殼菌Chaetomium globosum IFB-WQ是南京大學功能生物分子研究所從藥用植物白茅(Imperatacylindrical)中分離得到的一株內(nèi)生菌。本學位論文對Cglobosu.ML-4發(fā)酵液和C.glbosum IFB-WQ固體發(fā)酵產(chǎn)物的化學成分分別進行了研究,以期為新藥研發(fā)提供先導化合物。Cglobosu.ML-4發(fā)酵液經(jīng)乙酸乙酯萃取得粗浸膏3.2g,C.globosu IFB-WQ固體發(fā)酵產(chǎn)物的氯仿甲醇提取物先后經(jīng)乙酸乙酯和正丁醇萃取得粗浸膏26g和17g。粗浸膏經(jīng)硅膠柱層析粗分離后,以TLC和1H-NMR譜為指導,綜合運用硅膠柱層析、Sephadex L H-20凝膠柱層析、反相柱層析、制備薄層層析以及高效液相色譜等方法對組分進行分離純化,結(jié)果從菌株ML-4中分離得到5個化合物(1～5),從菌株IFB-WQ中分離得到6個化合物(1～2和6～9);采用高分辨質(zhì)譜、ID和2D核磁共振譜等波譜技術及文獻比對的方法對化合物結(jié)構(gòu)進行鑒定,上述化合物分別被鑒定為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)同時從兩株C.globosum中分離得到。本學位論文首次報道了cytoglobosinAb(9)的波譜數(shù)據(jù),iso-chaetoglobosinVb(1)的絕對構(gòu)型有待進一步鑒定。采用MTT法測定了化合物1、2、6和9對人肝癌細胞株SMMC-7721的體外細胞毒活性,結(jié)果發(fā)現(xiàn),化合物2對SMMC-7721細胞具有較強的增殖抑制活性,其IC50值為60.5μg/mL,陽性對照順鉑的IC50值為7.92μg/mL;化合物2對SMMC-7721細胞的增殖抑制活性呈現(xiàn)明顯的濃度依賴性�；衔�1、2、6、7、8和9為細胞松弛素類化合物,文獻表明,該類化合物具有抗腫瘤、免疫抑制、抗氧化等多種生物活性。我們前期研究發(fā)現(xiàn),該類化合物對MPP+誘導的PC12細胞損傷具有顯著的保護作用,是潛在的抗帕金森病(PD)天然藥物,上述細胞松弛素化合物的抗PD活性將在后續(xù)實驗中做進一步研究。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R915
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