两株内生真菌次级代谢产物的研究

发布时间：2018-06-07 23:14

本文选题：冬凌草 + 内生真菌　；参考：《郑州大学》2017年硕士论文

【摘要】：背景:从天然产物中筛选生物活性成分是开发新药的有效途径之一,但是从药用植物中发现活性化合物的几率一直在降低。而植物内生真菌与宿主植物可能产生类似活性化合物,且种类繁多,具有巨大的开发潜力。因此,从可持续发展的层面上看,从植物内生真菌的代谢产物中寻找活性化合物是开发新药的一条有效的途径。冬凌草系唇形科香茶菜属多年生草本植物碎米桠(Rabdosia rubescens)的干燥地上部分,主要化学成分包括贝壳杉烷二萜类、黄酮类、挥发油及多糖类等,其中以冬凌草甲素为代表的二萜类化合物对食管癌、结肠癌、贲门癌均有一定疗效。我们将河南道地药材冬凌草作为研究对象,对其内生真菌进行分离纯化得到球毛壳菌。球毛壳菌经过发酵的次级代谢产物乙醇提取物有一定的抗癌活性。且文献报道的从球毛壳菌次级代谢产物里分离出的化合物已达200多个,具有不同程度的抗菌抗癌活性。因此我们选取冬凌草内生真菌中的球毛壳菌进行研究以期发现具有抗肿瘤活性的化学成分。红树林(Mangrove),查阅文献报道表明,扩展青霉菌(Penicillium expansum)次级代谢产物中所分离得到的倍半萜类化合物和苯酚三聚体类化合物,具有良好的逆转多药耐药及细胞毒活性。因此我们选取扩展青霉菌进行发酵以期从次级代谢产物中发现具有抗肿瘤活性的化学成分。目的意义:本文分别对球毛壳菌(chaetomium globosum)和扩展青霉菌(Penicillium expansum)的次级代谢产物进行化学成分研究,以期得到具有较好抗癌活性和抗菌活性且结构新颖的单体化合物,这对发现结构新颖生物活性化合物以及对植物内生真菌的研究具有一定意义。方法:本文分别采用固体和液体发酵法对球毛壳菌(chaetomium globosum)和扩展青霉菌(Penicillium expansum)进行发酵,发酵所得次级代谢产物用正相硅胶柱、MCI、大孔吸附树脂、HPLC和Sephadex LH-20等方法进行化学成分的分离和纯化,通过IR,UV,1H-NMR,13C-NMR等现代技术对所分离得到的单体化合物进行鉴定并确认结构。用MTT法对经分离鉴定的化合物进行抗癌活性测定。用纸片法、倍比稀释法和琼脂稀释法进行抗菌活性研究。结论:(1)从冬凌草(Rabdosia rubescens)内分离出16种内生真菌,其中1-8号菌属于暗孢科,9号菌、10号菌属于瘤座孢菌科,11号菌属于炭棒科,12号菌属于黑盘孢菌科,13号菌、14号菌属于球壳孢菌科,15号菌属于鲜壳孢菌科,16号菌属于半壳孢菌科。针对PC-3(前列腺癌细胞)、MGC-803(胃癌细胞)对其发酵粗提物进行抗癌活性测试,5号菌和9号菌的发酵粗体物对PC-3的IC50分别为302μg/ml,136μg/ml,11号菌和15号菌的发酵粗体物对MGC-803的IC50值分别为181μg/ml,123μg/ml。(2)从球毛壳菌(chaetomium globosum)的次级代谢产物中分离鉴定出10个单体化合物,分别鉴定为:stigmasta-4,6,8(14),22-tetraen-3-one(1)、4-methyl-5,6-dihydro-2H-pyran-2-one(2)、(E)-6-hydroaxy-4,6-dimethyl-3-heptene-2-one(3)、glyceroyl monopaimitate(4)、、4-羟基-2′-脱氧嘧啶核苷(5)、胞腺嘧啶脱氧核苷(6)、麦角甾醇过氧化物(7)、bungein A(8)、β-谷甾醇(9)、邻苯二甲酸二丁酯(10)。(3)从扩展青霉菌(Penicillium expansum)的次级代谢产物中分离得到10个化合物,过氧麦角甾醇(11)、环(甘氨酸-亮氨酸)(12)、anthraquinone aversin(13)、5-methoxysterigmatocystin(14)、6,8-di-O-methyl averufin(15)、环(甘-苯丙)二肽(16)、环(L-苯丙-反-4-羟基-L-脯)二肽(17)、Brevianamide K(18)Brevianamides Q(19)、brevianamides W(20)。(4)从扩展青霉菌内分离出来的单体化合物Brevianamide W对MGC-803的IC50值为43.4μg/ml,5-methoxysterigmatocystin对PC细胞的IC50值为56.7μg/ml。
[Abstract]:Background: screening bioactive ingredients from natural products is one of the most effective ways to develop new drugs, but the probability of discovery of active compounds from medicinal plants has been decreasing. Plant endophyte and host plants may produce similar active compounds with great potential for development. Looking for active compounds from the metabolites of endophytic fungi is an effective way to develop new drugs. Rabdosia rabdosia is the dry ground part of the perennial herb (Rabdosia rubescens) of the perennial herb of the genus lipido. The main chemical components include two terpenoids, flavonoids, volatile oils and polysaccharides, and so on. The two terpenoids, represented by oridonin, have a certain effect on esophageal, colon and cardia cancer. We have isolated and purified the endophytic fungi from Henan oridonin, which had a certain anticancer activity through the ethanol extracts of the secondary metabolites of the fermentation. And more than 200 compounds have been separated from the secondary metabolites of S. oridote, which have different degrees of antiseptic and anticancer activity. Therefore, we have selected the fungus in the endophytic fungi of oridonin to find out the chemical constituents of the antitumor activity. Mangrove. The sesquiterpene and phenol triad compounds isolated from the secondary metabolites of Penicillium expansum have a good reversal of multidrug resistance and cytotoxic activity. Therefore, we choose to ferment the Penicillium to discover antitumor chemical constituents from secondary metabolites. In this paper, the chemical composition of secondary metabolites of Chaetomium globosum and Penicillium expansum was studied in order to obtain the monomers with good anti-cancer activity and antibacterial activity with novel structure. The study of bacteria is of certain significance. Methods: in this paper, solid and liquid fermentation methods were used to ferment Chaetomium globosum and Penicillium expansum respectively. The secondary metabolites obtained by fermentation were separated by normal silica gel column, MCI, macroporous adsorption tree fat, HPLC and Sephadex LH-20 to separate chemical components. And purification, through the IR, UV, 1H-NMR, 13C-NMR and other modern technology to identify the isolated monomers and identify the structure. MTT method was used to determine the anticancer activity of the isolated compounds. The antibacterial activity was studied by paper method, double dilution method and agar dilution method. Conclusion: (1) from Rabdosia rubescens (Rabdosia rubescens) 16 endophytic fungi, of which No. 1-8 bacteria belong to the dark spores, 9 bacteria and 10 bacteria belong to the family cyclosporaceae, 11 bacteria belong to the charcoal stick family, the 12 bacteria belong to the spporaceae, 13 and 14 belong to the family sporospora, and the 15 bacteria belong to the fresh shell spores, and 16 bacteria belong to the hemicarporaceae. The antitumor activity of the fermented crude extracts was tested. The IC50 of PC-3 was 302 mu g/ml and 136 mu g/ml respectively. The IC50 value of MGC-803 was 181 mu g/ml, and 123 mu g/ml. (2) was separated from the secondary metabolites of S. ducrum (Chaetomium globosum) for 10 monomers. Compounds were identified as stigmasta-4,6,8 (14), 22-tetraen-3-one (1), 4-methyl-5,6-dihydro-2H-pyran-2-one (2), (E) -6-hydroaxy-4,6-dimethyl-3-heptene-2-one (3), glyceroyl monopaimitate (4), 4- hydroxyl -2 '- deoxy pyrimidine (5), cytosine pyrimidine deoxynucleoside (6), ergosterol peroxide (7), bungein A (8), and beta valerol (9). ), dibutyl phthalate (10). (3) separation of 10 compounds from secondary metabolites of Penicillium extended (Penicillium expansum), peroxy ergosterol (11), ring (Glycine leucine) (12), anthraquinone aversin (13), 5-methoxysterigmatocystin (14), 6,8-di-O-methyl averufin (15), ring (Gan - phenylpropyl) two peptide (16), ring (L- benzene). Two peptide (17), Brevianamide K (18) Brevianamides Q (19), brevianamides W (20), brevianamides W (20). (4) Brevianamide W of the monomer separated from the Penicillium extended to MGC-803 IC50 value is 43.4 micron.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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