两株南极海洋真菌次级代谢产物的研究

发布时间：2018-06-01 21:14

本文选题：南极真菌 + 青霉属　；参考：《第二军医大学》2017年硕士论文

【摘要】：极地自然环境条件苛刻恶劣,具有严寒、强风、强紫外辐射等特点,因而高等动植物罕见,微生物如细菌、真菌、放线菌等资源丰富多样。为了适应极地特殊的自然生态环境,极地微生物在基因组成与表达、酶学及相关代谢调控方面形成独特的分子生物学特性,因而可产生结构新颖、活性独特的次级代谢产物,并具有丰富的生物学活性,如抗肿瘤、抗菌活性等,可成为新型化学药物的重要来源。在本课题组前期研究中,采用PTP1B抑制活性为指征及TLC香兰素显色分析方法筛选得到真菌Penicillium sp.S-1-18。采用含0.3%酵母提取粉,0.5%胰蛋白胨,0.3%麦芽糖,1%葡萄糖及5%蔗糖的培养基,在初始pH 7.0,18°C,180 r/min的条件下培养14 d,发酵结束后滤出菌体,菌液部分浓缩并用乙酸丁酯萃取,最终得到总浸膏约60 g。在此基础上展开对代谢产物的鉴定和生物学活性研究。首先采用石油醚和二氯甲烷对总浸膏进行分步萃取,进而对二氯甲烷相浸膏进行系统分离纯化,分别采用减压柱层析、Sephadex LH 20凝胶柱层析、反相ODS柱层析、正相硅胶柱层析及高效液相等色谱分析方法,最终分离得到9个化合物,分别是butanolide A(1),guignarderemophilane F(2),xylarenone A(3),penicyclone A(4),4(3H)-喹唑啉酮(5),环-(L-脯氨酸-L-苯丙氨酸)(6),环-(L-脯氨酸-4R-羟基-L-脯氨酸)(7),callyspongidipeptide A(8)和N-(2-羟基丙酸)-2-氨基苯甲酸(9)。其中化合物1和2为新化合物,其绝对构型分别通过改良的Mosher法和ECD/CD比对法进行了确定。对该菌株中所得化合物进行生物学活性评价,结果显示化合物1有一定的PTP1B抑制活性,其IC50为27.4μM,此外化合物4、5和8有微弱的抑制鲍曼不动杆菌活性,IC50值均为64μM。与此同时,采用TLC显色指征法筛选出一株南极海洋来源真菌Penicillium sp.S-2-10。以TLC显色及产物浸膏量为参考,对其发酵条件进行初步优化,最终确定选用含1.0%NaCl的GPY培养基,在24°C,180 r·min-1条件下振荡培养12 d进行规模发酵。发酵90 L后用纱布过滤菌体和菌液,用二氯甲烷与甲醇等体积萃取菌体,用乙酸乙酯萃取菌液,合并萃取液浓缩得到约16 g总浸膏。采用Sephadex LH 20凝胶柱层析、正相硅胶柱层析、反相ODS柱层析及高效液相等色谱分析方法对总浸膏进行系统分离纯化,通过核磁(1H-NMR和13C-NMR等)及质谱(MS)等波谱分析方法,并与相关文献比较,确定了7个化合物结构,分别为benzoic acid,2-(2,6-dihydroxybenzoyl)-3-hydroxy-5-(hydroxymethyl)-methyl,methyl ester(10),大黄酚(11),volemolide(12),ergosta-6,8(14),22-trien-3-ol(13),2-aminophenoxazin-3-one(14),2-苯并噻唑酮(15)和苯甲酸(16)。其中,化合物12和13首次从青霉属真菌中获得。对所分得的化合物进行生物学活性评价,发现化合物12具有微弱的肿瘤细胞抑制活性,对SMMC-7721肿瘤细胞的增殖抑制IC50为42.70μM,对SGC-7901肿瘤细胞的增殖抑制IC50为49.98μM。化合物11、12、13和15有微弱的抑制鲍曼不动杆菌活性,IC50值为64μM。
[Abstract]:The polar natural environment is harsh and harsh, with the characteristics of severe cold, strong wind, strong ultraviolet radiation and so on. Therefore, higher plants and animals are rare, microbes such as bacteria, fungi, actinomycetes and other resources are rich and diverse. In order to adapt to the special natural ecological environment in the polar regions, microbes in polar regions form unique molecular biological characteristics in gene composition and expression, enzymology and related metabolic regulation, so that secondary metabolites with novel structure and unique activity can be produced. It has abundant biological activities, such as anti-tumor, anti-bacterial activity and so on, which can become an important source of new chemical drugs. In the previous study of our group, Penicillium sp. S-1-18 was obtained by using PTP1B inhibition activity as the indication and TLC vanillin color analysis method. The culture medium containing 0.5% tryptone, 0.3% maltose, 1% glucose and 5% sucrose was cultured for 14 days at the initial pH of 7.0 ~ 18 掳C ~ (-1) r/min. After fermentation, the bacteria were filtered out, and the bacteria solution was partially concentrated and extracted with butyl acetate. Finally, the total extract was obtained about 60 g. On this basis, the identification and biological activity of metabolites were studied. At first, petroleum ether and dichloromethane were used to extract the total extract step by step, and then the phase extract of dichloromethane was systematically separated and purified. The gel column chromatography was performed by vacuum column chromatography, Sephadex LH 20 column chromatography, and reverse phase ODS column chromatography, respectively. Nine compounds were isolated by normal phase silica gel column chromatography and high performance liquid chromatography. The results are as follows: (1) butanolide A ~ (1) ~ (1) guignar deremophilane (F ~ (2) ~ (3) ~ (3) ~ (-) ~ (3) penicyclone A ~ (4) ~ (4) H _ (3) H _ (1) -quinazolinone (5), ring ~ (-L) -proline -L ~ (phenylalanine) ~ (6), ring ~ (-L) -proline -4R -hydroxy--L ~ (proline) -7callyspongidipeptide A8) and N-( 2-hydroxypropionic acid) -2-aminobenzoic acid. Compounds 1 and 2 are new compounds, and their absolute configurations are determined by modified Mosher method and ECD/CD comparison method, respectively. The biological activity of the compounds obtained from the strain was evaluated. The results showed that compound 1 had a certain PTP1B inhibitory activity, its IC50 was 27.4 渭 m, in addition, the IC50 values of compounds 4, 5 and 8 were both 64 渭 M and 64 渭 M, respectively, against Acinetobacter baumannii (Acinetobacter baumannii). At the same time, a strain of Antarctic marine fungus Penicillium sp. S-2-10 was screened by TLC colorimetric method. With the reference of TLC color development and product extract quantity, the fermentation conditions were preliminarily optimized. Finally, the GPY medium containing 1.0%NaCl was selected, and the culture medium was oscillated under 24 掳C ~ (180) r min-1 for 12 days for large-scale fermentation. After 90 L fermentation, the bacteria were filtered by gauze, the bacteria were extracted by dichloromethane and methanol, the bacteria were extracted by ethyl acetate, and the extract was concentrated to get about 16 g total extract. The total extract was systematically separated and purified by Sephadex LH 20 gel column chromatography, normal phase silica gel column chromatography, reversed phase ODS column chromatography and high performance liquid chromatography. Compared with the related literatures, seven compounds were identified as benzoic acidin2-dihydroxybenzoyl-3-hydroxy-5-hydroxymethyl-methyl-methylmethyl-methyl (10), chrysophanol 11volemolide (12C), ergosta-6 chrysotrium (22-trien-3-ol13trien-2-aminophenoxazin-3-one) and benzothiazolone (15) and benzoic acid (16). Compounds 12 and 13 were isolated from Penicillium for the first time. The biological activity of the obtained compounds was evaluated. It was found that compound 12 had weak tumor cell inhibitory activity. The inhibition of IC50 on SMMC-7721 tumor cells was 42.70 渭 m, and on SGC-7901 tumor cells was 49.98 渭 M. The IC50 values of compounds 11, 12, 13 and 15 against Acinetobacter baumannii were 64 渭 M.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

【参考文献】