【作者】 王慧；

【导师】 朱伟明；

【作者基本信息】 中国海洋大学 ， 药物化学， 2012， 博士

【摘要】 耐酸微生物是可以生存在低pH环境中的极端微生物,由于其生存环境的特殊性,其次生代谢产物中可能存在结构新颖且具有生物活性的化学成分。目前国际上对于耐酸微生物次生代谢产物研究报道较少,该项研究工作前景诱人。“天才菌株”是指那些次生代谢产量较大,并且能够产生系列新结构的生物活性菌株。从酸性环境获得耐酸天才菌株,相信有望寻找到生物活性物质。本文对采自酸性地质环境的云南建水紫陶土壤和锰矿土壤以及河南云台山国家地质公园泥土共27个样品中分离纯化得到121株耐酸真菌。以P388肿瘤细胞增殖抑制、抗甲型流感病毒H1N1活性的测试等作为生物活性筛选模型,以TLC和HPLC指纹图谱作为化学筛选模型,采用生物活性测试和化学多样性评价集成筛选这样的方法,最终从耐酸真菌中筛选得到4株耐酸天才真菌,并对其次生代谢产物开展了研究工作。首先通过目标菌株发酵条件的优化,包括培养基,培养时间等,确定了最佳的发酵条件。经过规模化发酵方法分别对4株耐酸天才真菌进行发酵,以乙酸乙酯对发酵液进行萃取,获得发酵提取物。采用天然产物化学的研究方法,即采用正相和反相硅胶柱色谱、制备薄层色谱、Sephadex LH-20凝胶柱色谱、HPLC(高效液相色谱)等分离纯化手段,共分离得到67个化合物,具体为从产紫青霉菌(Penicillium purpurogenum) JS03-21中分离得到18个化合物(1-17,67)；从菌核青霉菌(Penicillium sclerotiorum) ZZ07-5中分离得到19个化合物(18-36)；从产紫青霉菌(Penicillium purpurogenum) ZZ05-4中分离得到17个化合物(37-53)；从灰黄青霉菌(Penicillium griseofulvum) ZZ10-35中分离得到13个化合物(54-66)。综合运用有机质谱、核磁共振(1H NMR,13C NMR, DEPT, HMQC,1H-1H COSY, HMBC, NOESYY)、CD(圆二色谱)、紫外光谱、红外光谱等现代波谱学技术,以及X-射线单晶衍射、化学法对化合物进行结构鉴定,确定了62个化合物结构,其中28个为新结构。新化合物结构类型有azaphilone类(1-4,21-27,35),苯并呋喃酮类(6-9),萘醌类(10),倍半萜酯类(14-15),内酯类(37-40),脂肪酸(41-42),聚酮类(55-57)。已知化合物的结构类型涉及azaphilone类(18-20,28-29,31-34),倍半萜酯类(16-17),苯的衍生物(11-13,43-46),呋喃衍生物(47-50),色酮(30),萜类(35),内酰胺(51-52),蒽醌(58),四氢萘酮类(59-62)等等。运用体外及体内生物活性筛选模型,采用MTT法、SRB法等方法评价所获得的单体化合物的抗肿瘤活性和抗流感病毒H1N1活性；采用琼脂稀释法评价化合物对致病菌的抑菌活性。抗甲型H1N1流感病毒的活性测试表明,化合物2,3,6,11,27对病毒有强效的抑制活性,IC50分别为61.3、64.0、85.3、58.6、25.5μM,与阳性对照药利巴韦林相当。对于化合物2还进行了动物体内实验,化合物2可以减轻甲型流感病毒感染引起的小鼠肺部炎症,对小鼠起到保护作用,体内评价有效。抗菌实验的结果表明四个倍半萜酯类(14-17)化合物对白色念珠菌均具有中等强度的抑制作用,14、15还对铜绿假单胞菌、产气肠杆菌具有中等强度的抑制活性。为探究氨基酸侧链在抗真菌活性上的作用,化合物17在水解后得到化合物1a-hydroxyconfertifolin,1α-hydroxyconfertifolin经测试无抗白色念珠菌活性,证实氨基酸部分对于化合物17的抗白色念珠菌活性是必不可少的。另外,经测试化合物9具有弱的抗产气肠杆菌活性,MIC76.7μM。抗肿瘤活性测试结果表明：化合物19-21,23-24对神经胶质瘤细胞A172、U87,一对乳腺癌细胞MCF-7、MCF-7/Adr,骨肉瘤细胞MG-63这5种肿瘤细胞均有中等的抑制活性,IC50在2.6-22.3μM；化合物18对乳腺癌细胞MCF-7有弱的选择性抑制活性；化合物27对A1-72、U87、MCF-7、MG-63有中等的抑制活性。化合物57对HL-60细胞增殖有强抑制活性,抑制率达89.1%。最后初步探讨了酸调节作用对耐酸真菌次生代谢产物的影响。在不同pH,不同酸性培养基条件下对耐酸真菌进行小试发酵,通过比较菌株次生代谢产物的产量、TLC色谱、HPLC-UV指纹图谱、化合物以及生物活性等,分析酸调节作用对耐酸真菌的次生代谢产物化学多样性的影响。研究证实了证实了低pH条件能够增加耐酸真菌代谢产物的化学多样性,低pH可以激活某些沉默基因。更多还原

【Abstract】 Aciduric microorganism are extremophiles that can survive in low pH environment. Aciduric microorganism could produce compounds with novel structures and biological activity in the secondary metabolites because of possessing special ecology, specific physiological mechanism. Fungi capable of surviving at low pH conditions have attracted much attention in recent years for their diverse secondary metabolites with a range of biological activities. But up to date, few studies have been done about secondary metabolites of aciduric microorganism, the research work are worthy of further Study."Talented strains" refer to those strains possessing large metabolic production, producing a series of new compounds with biological activity. The special acid environment where aciduric microorganism live could active silent gene and induce other specific metabolic pathway. Therefore, our study was carried out to search novel bioactive compounds from the secondary metabolites of aciduric talented strains under acid environment. Studies include the screening for aciduric talented strains, isolation and structural elucidation of the secondary metabolites, preliminary bioactivity evaluation of compounds, the impact of acid regulation for secondary metabolites of aciduric talented strains.121strains of aciduric fungi have been isolated from acid soil collected in Jianshui, Yunnan province and Yuntaishan National Geological Park from Jiaozuo, Henan, China. Using SRB methods, eighteen active aciduric strains were screened out of two121strains, on the bioassay of P388cell lines. The EtOAc extracts of these fungi were evaluated with the combinatory method of chemical screening (TLC and HPLC) and bioactive evaluation (cytotoxicities and antiviral activity). Four aciduric fungi were determined as aciduric talented strains because of their metabolic production and cytotoxicity or antiviral activities.First of all, fermentation conditions of target strains were optimizated, including medium, fermentation time and so on. After fermentation of four aciduric talented strains, the whole broths were extracted with ethyl acetate to give ferment extracts. Then the EtOAc extracts were subjected to extensive silica gel column chromatography, Sephadex LH-20and HPLC to give compounds. Sixty-seven compounds were isolated from four aciduric talented strains.18compounds were isolated from Penicillium purpurogenum JSO3-21(1-17,67);19compounds were isolated from Penicillium sclerotiorum ZZ07-5(18-36);17compounds were isolated from Penicillium purpurogenum ZZ05-4(37-53);13compounds were isolated from Penicillium griseofulvum ZZ10-35(54-66). By means of modern spectral analysis (NMR, MS, CD, UV, IR), quantum chemical calculations and chemical methods, the structures (see Fig.0-1) of sixty-two compounds were respectively determined, among them twenty-eight were new structures. Types of new compounds include azaphilones (1-4,21-27,35), benzofuranones (6-9), naphthoquinone (10), sesquiterpene derivatives (14-15), lactones (37-40), fatty acids (41-42), polyketides (55-57). In addition, the chemical structure types of compounds reported are involved in azaphilones (18-20,28-29,31-34), sesquiterpene derivatives (16-17), benzene derivatives (11-13,43-46), furan derivatives (47-50), chromone (30), terpene (35), lactams (51-52), anthraquinone (58), tetralones (59-62).The activity of compounds against influenza A virus (H1N1) was evaluated by the CPE inhibition assay. Compounds2,3,6,11,27exhibited stronge anti-H1N1activity with IC50values of61.3、64.0、85.3、58.6、25.5μM, respectively. Compound2was effective in vivo evaluation, too. Compound2could reduce lung inflammation of mice caused by influenza virus infection, play a protective role in mice. Using the agar dilution method the compounds were evaluated on anti-pathogenic microorganisms activity. In antifungal assay, compounds14-17showed moderate antifungal activity against C. albicans with MIC values of3.3,1.3,1.2and1.3μM, respectively. And compounds14and15showed moderate antimicrobial activity against E. aerogenes and P. aeruginosa with MIC values of2.4and2.6μM, and1.2and2.6μM, respectively. In order to evaluate whether the amino acid side chain attributed to the antimicrobial activity, compound17was subjected to basic hydrolysis to produce1α-hydroxyconfertifolin (17a), but no anti-C. albicans activity for17a was observed, indicating that the esterification with amino acid moiety is indispensable for the antifungal activity against C. albicans. Besides, compound9has weak antifungal activity against E. aerogenes with MIC76.7μM. Compounds were evaluated for their cytotoxicities against several cancer cell lines such as HL-60, P388, K562, K562, A549, A172, U87, MCF-7, MCF-7/Adr, MG-63by the MTT or SRB method. Compounds19-21,23-24showed moderate cytotoxicity against A172, U87, MCF-7, MCF-7/Adr, MG-63. Compound57exhibited strong cytotoxicity against HL-60cell line with inhibitory rate of89.1%.To further explore the pH and acid conditions effects on chemodiversity of secondary metabolites of aciduric talented strains, secondary metabolites from four aciduric talented strains under different culture conditions containing different pH and medium were analyzed by comparing weight, TLC, HPLC, bioactivities and compounds. The secondary metabolites contained in the extract of aciduric talented strains fermented at pH2showed a more extensive chemical diversity and better bioactivities than those at pH7. Aciduric talented strains could produce different compounds by fermented in different culture conditions. It has been reported that in general the secondary metabolites can be regulated by external pH. In some cases, the pH effects were larger than carbon and nitrogen effects, and certain silent genes can be activated by low pH.更多还原