【作者】 胡德军；

【导师】 赵凤生；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2008， 博士

【摘要】 植物内生菌是具有高度多样性的微生物资源,代谢产物多种多样,包括杀虫、抗菌、抗肿瘤等活性物质,是目前人们寻找新型抗菌、抗肿瘤天然药物的重要资源。本研究利用上海来益生物药物研究开发中心的植物内生菌资源,通过抗肿瘤和抗菌模型的筛选,在获得的活性菌株中,对其中的3株进行了菌种鉴定,并对它们的次级代谢产物进行分离、纯化及活性研究。主要研究工作包括以下几个方面:对1280株植物内生真菌进行了筛选,经模型初筛、复筛,共获得28份具有高且稳定的抗肿瘤活性的菌株发酵样品,8份具有较强抗菌活力的菌株发酵样品,选取其中3株抗肿瘤活性菌株HCCB1614、HCCB1778及HCCB2310进一步研究。根据菌株的形态、培养特征、18S rDNA以及ITS序列分析结果,对活性菌株HCCB1614、HCCB1778及HCCB2310进行菌种鉴定,结果分别鉴定为拟茎点霉属、链格孢霉属以及扁棒壳菌属。对菌株HCCB1778的代谢产物进行研究,分离纯化得到5个次级代谢产物,分别为HCCB1778-1~5。利用质谱和核磁共振波谱对这些产物进行结构鉴定,确定HCCB1778-1为6,8-二甲基-四氮奈-2,4(3H,10H)二酮,分子量242;HCCB1778-2为4’,7-二羟基异黄酮,分子量254;HCCB1778-3为4’,5,7-三羟基异黄酮,分子量270,对A549、LoVo细胞均具有很强的抑制作用,其中HCCB1778-3对LoVo细胞的抑制率最高,可达78.95%,目前HCCB1778-3已经开发为药物;HCCB1778-4为链格孢毒素(Altertoxin-I),分子量为352,具有较强的诱变作用和一定的抑制血小板聚集的作用;HCCB1778-5(新化合物)化学命名为1,4,9,12-四羟基-1,2,12,12a-四氢苝-3,10(11H,12bH)-二酮,为HCCB01778-4同系物,分子量352,是首次分离得到的新化合物,具有一定的抗肿瘤活性。对菌株HCCB2310次级代谢产物的分离研究得到两个化合物,分别为3,22,24-三羟基-齐果墩烷-12-烯和琥珀酸。为了快速分离得到菌株HCCB1614活性成分,本研究利用传统方法和高速逆流色谱法结合对其活性代谢产物进行了分离。选取两相溶剂系统为正己烷-乙酸乙酯-甲醇-水(1:3:3:3),体系上相为固定相,下相为流动相。成功地从内生真菌HCCB1614的次级代谢物中快速分离得到一细胞毒活性组分。经高效液相色谱分析,其纯度为99%以上。其化学结构由1H-NMR和13C-NMR鉴定确认为Cytochalasin N。从菌株HCCB1778次级代谢产物中分离得到的Altertoxin I (ATX I)是一种链格孢霉菌产生的真菌毒素,一直被认为是一种重要的食品和粮食的污染物。最近研究表明Altertoxin I具有很好的抗凝血作用,能够阻止血小板聚集,IC50为29μM,因此具备作为药物开发的潜力。为了得到足够量的Altertoxin I用于进一步的研究和开发,本研究首次建立了一种利用高速逆流色谱一次性分离制备ATX I的方法。实验利用溶剂体系正己烷-乙酸乙酯-甲醇-水(2:5:5:6)对发酵产物的萃取物进行分离,利用HPLC分析以及质谱和核磁共振波谱对目标产物进行鉴定。结果表明该方法每次可以分离得到毫克级的目标产物。本研究从18L发酵产物中共分离得到45mg AXT I,纯度可以达到95%以上。因此该方法将为以后AXT I的快速分离制备提供有效的帮助。更多还原

【Abstract】 Endophyte is a special and important group of microorganisms with taxonomic diversity, which can produce various bioactive substances, including insecticidal, antimicrobial and antitumor compounds etc. In this research work, a large amount of endophytes had been screened based on several models and dozens of strains showed activities. Three of them were taxonomically identified and their secondary metabolites were studied.The fermentation samples were prepared from 1280 strains of endophytes. After primary and secondary screening on two models, 28 samples showed high and stable antitumor activities while 8 samples showed strong antibiotic activities. Among them, the strains HCCB1614, HCCB1778 and HCCB2310 were selected for further study.According to their morphology, cultural characteristics, 18S rDNA and ITS sequences, the strains HCCB1614, HCCB1778 and HCCB2310 were indentified as Phomopsis sp., Alternaria sp. and Ascospermum sp., respectively.Five compounds from the metabolites, named as HCCB1778-1～5 respectively, were purified by means of solvent extraction, silica gel chromatography, gelatum LH-20, and HPLC preparation. MS and NMR analyses showed that the molecular weights of these compounds are 242, 254, 270, 352, 352, respectively, and their molecular structures were identified as HCCB1778-1: 6, 8, 10- trimethylbenzopteridine- 2, 4 (3H, 10H)- dione; HCCB1778-2: 4’, 7- dihydroxyisoflavone; HCCB1778-3: 4’, 5, 7- trihydroxy- 7 methoyisoflavone; HCCB1778-4: Altertoxin-I; HCCB1778-5: 1, 4, 9, 12- tetrahydroxy- 1, 2, 12, 12a- tetrahydroperylene- 3, 10 (11H, 12bH)- dione; respectively. Compounds HCCB1778-3 and 5 showed strong cytotoxyties against A549 and LoVo cell lines. HCCB1778-4, which is a kind of cytotoxins, hosts strong mutagenicity and inhibition of platelet aggregation. HCBB1778-5 is a new compound. Meanwhile, two compounds isolated from the fermentation broth of strain HCCB2310 were identified as 3, 22, 24-trihydroxy-olean-12-ene and succinic acid.High speed counter-current chromatography (HSCCC) was applied to the separation of anti-tumor constituents from an endophyitic fungi HCCB1614 isolated from Trachelospermum jasminosides. A biphasic solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:3:2.5, v/v) was successfully performed. The bioactive compound purified and collected was analyzed by HPLC. A white powder was obtained in purity above 99% with the method. The chemical structure of the bioactive compound was identified by MS, 1H NMR and 13C NMR.Altertoxin I (ATX I), isolated from strain HCCB1778, is one of the common mycotoxins produced by genus Alternaria which is a common food pathogen of fruits and grains, diminishing the value of human foodstuffs. Recent research found that it can be used for the prophylaxis and therapy of deseases in which high blood platelet aggregations occur. To prepare enough quantity of pure ATX I for further research of mutagenicity, toxicology tests and anti-platelet aggregation, a novel method using preparative high-speed counter-current chromatography (HSCCC) was developed for the first time. The ethyl acetate crude extracts of the acetone washes obtained from fermentation liquor of Alternaria sp. was separated by a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (2:5:5:6, v/v). Collected fractions were analyzed by HPLC and identified by EI–MS and NMR analysis. The technique can isolate target compound at milligram amount in one run, which produced 45mg ATX I with purity better than 95% from 18L fermentation culture. The recovery field of ATX I from the crude extracts was 86.8%. This one-step purification method provided a simple and effective tool for obtaining a relatively large amount of ATX I for the purpose of various studies.更多还原