本文選題：南極真菌 + 青霉屬��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：極地自然環(huán)境條件苛刻惡劣,具有嚴寒、強風、強紫外輻射等特點,因而高等動植物罕見,微生物如細菌、真菌、放線菌等資源豐富多樣。為了適應(yīng)極地特殊的自然生態(tài)環(huán)境,極地微生物在基因組成與表達、酶學(xué)及相關(guān)代謝調(diào)控方面形成獨特的分子生物學(xué)特性,因而可產(chǎn)生結(jié)構(gòu)新穎、活性獨特的次級代謝產(chǎn)物,并具有豐富的生物學(xué)活性,如抗腫瘤、抗菌活性等,可成為新型化學(xué)藥物的重要來源。在本課題組前期研究中,采用PTP1B抑制活性為指征及TLC香蘭素顯色分析方法篩選得到真菌Penicillium sp.S-1-18。采用含0.3%酵母提取粉,0.5%胰蛋白胨,0.3%麥芽糖,1%葡萄糖及5%蔗糖的培養(yǎng)基,在初始pH 7.0,18°C,180 r/min的條件下培養(yǎng)14 d,發(fā)酵結(jié)束后濾出菌體,菌液部分濃縮并用乙酸丁酯萃取,最終得到總浸膏約60 g。在此基礎(chǔ)上展開對代謝產(chǎn)物的鑒定和生物學(xué)活性研究。首先采用石油醚和二氯甲烷對總浸膏進行分步萃取,進而對二氯甲烷相浸膏進行系統(tǒng)分離純化,分別采用減壓柱層析、Sephadex LH 20凝膠柱層析、反相ODS柱層析、正相硅膠柱層析及高效液相等色譜分析方法,最終分離得到9個化合物,分別是butanolide A(1),guignarderemophilane F(2),xylarenone A(3),penicyclone A(4),4(3H)-喹唑啉酮(5),環(huán)-(L-脯氨酸-L-苯丙氨酸)(6),環(huán)-(L-脯氨酸-4R-羥基-L-脯氨酸)(7),callyspongidipeptide A(8)和N-(2-羥基丙酸)-2-氨基苯甲酸(9)。其中化合物1和2為新化合物,其絕對構(gòu)型分別通過改良的Mosher法和ECD/CD比對法進行了確定。對該菌株中所得化合物進行生物學(xué)活性評價,結(jié)果顯示化合物1有一定的PTP1B抑制活性,其IC50為27.4μM,此外化合物4、5和8有微弱的抑制鮑曼不動桿菌活性,IC50值均為64μM。與此同時,采用TLC顯色指征法篩選出一株南極海洋來源真菌Penicillium sp.S-2-10。以TLC顯色及產(chǎn)物浸膏量為參考,對其發(fā)酵條件進行初步優(yōu)化,最終確定選用含1.0%NaCl的GPY培養(yǎng)基,在24°C,180 r·min-1條件下振蕩培養(yǎng)12 d進行規(guī)模發(fā)酵。發(fā)酵90 L后用紗布過濾菌體和菌液,用二氯甲烷與甲醇等體積萃取菌體,用乙酸乙酯萃取菌液,合并萃取液濃縮得到約16 g總浸膏。采用Sephadex LH 20凝膠柱層析、正相硅膠柱層析、反相ODS柱層析及高效液相等色譜分析方法對總浸膏進行系統(tǒng)分離純化,通過核磁(1H-NMR和13C-NMR等)及質(zhì)譜(MS)等波譜分析方法,并與相關(guān)文獻比較,確定了7個化合物結(jié)構(gòu),分別為benzoic acid,2-(2,6-dihydroxybenzoyl)-3-hydroxy-5-(hydroxymethyl)-methyl,methyl ester(10),大黃酚(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首次從青霉屬真菌中獲得。對所分得的化合物進行生物學(xué)活性評價,發(fā)現(xiàn)化合物12具有微弱的腫瘤細胞抑制活性,對SMMC-7721腫瘤細胞的增殖抑制IC50為42.70μM,對SGC-7901腫瘤細胞的增殖抑制IC50為49.98μM。化合物11、12、13和15有微弱的抑制鮑曼不動桿菌活性,IC50值為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 擄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 擄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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R915

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