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海洋红藻来源鲜红青霉和长柄木霉次级代谢产物化学多样性及生物活性研究
刘慧
Subtype硕士
Thesis Advisor王斌贵
2016-05-18
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline生物工程
Keyword海洋真菌 次级代谢产物 鲜红青霉 长柄木霉 生物活性
Other Abstract    天然产物是新药开发的重要来源。海洋具有低温、高盐、低光照以及寡营养等 特殊的环境,是新结构新颖和活性优良的天然产物的重要来源。本文开展了海洋来 源青霉属和木霉属真菌的次级代谢产物多样性及生物活性的研究。从采集到的 6 株 海藻中总共分离得到 17 株内生真菌。对 17 株真菌利用PDB、MH2 和大米培养基进 行静置培养,然后对其粗体物进行 HPLC 和 TLC 分析,最终选择鲜红青霉 EN-480 和长柄木霉 EN-502 进行规模发酵。
    菌株 EN-480 是来自石花菜 Pterocladiella tenuis 的内生真菌,分子生物学鉴定其 为鲜红青霉(Penicillium chermesinum)。利用大米培养基对其静置发酵得到粗提物,再 利用各种分离技术如反相柱层析、凝胶柱层析、pTLC 和 pHPLC 等对其次级代谢产 物进行分离,总共分离并鉴定 11 个单体化合物(PC 1–PC 11),其中有 9 个为新化 合物,包括 5 个新的混源萜(PC 1–PC 5)和 4 个新的倍半萜类(PC 6–PC 9)化合 物。对新化合物进行了抗菌活性和抗卤虫活性测试,发现化合物 PC 1 和 PC 2 对藤 黄微球菌(Micrococcus luteus)表现出明显的抗菌活性,MIC 值均为 8 µg/ml。但是倍 半萜类化合物没有表现出抗菌活性。对该类混源萜进行构效分析,发现 C-5'双键或 者环己烷-2,5-二烯酮对抗菌活性是必须的,而且 C-5'的-COOH 取代会降低抗菌活性。 混源萜类和倍半萜类化合物均没有表现出抗卤虫活性。
    菌株 EN-502 是从冈村凹顶藻(Laurencia okamurai Yamada)中分离得到的一株内 生真菌,通过分子生物学鉴定确定其为长柄木霉(Trichoderma longibrachiatum)。利用 大米培养基进行规模发酵,得到粗提物。对粗提物利用硅胶柱层析、反相柱层析以 及凝胶柱层析进行分离,最终分离并鉴定得到 7 个单体化合物(TL 1–TL 7),其中 化合物 TL 1 是新的倍半萜化合物。同时也对菌株 EN-502 利用 YPM 培养基进行了小 规模的两步法发酵,先摇床培养 7 天,然后再静置培养 28 天,最终从中分离得到 3 个已知的单体化合物(TLY 1–TLY 3)。
    通过本文的研究,进一步丰富了天然产物化学的研究内容,为开发海洋真菌来 源的次级代谢产物提供了理论基础。发现的具有明显抗菌活性的混源萜类化合物为 研究次级代谢产物的生物活性以及药用价值提供了依据。; Natural products are important source of new drug development. There are more and more natural products, including novel structures with potent bioactivities, isolated from marine source, because of special environment of sea, such as low temperature, high salt, and low light, drawing more and more attention of researchers. Metabolites of Penicillium and Trichoderma genus of marine source was carried out on structures and bioactivities diversity research in this dissertation. 17 endophytic fungi stains were isolated from different alga, which was inoculated statically in 1L flasks with PDB, MH2 and rice culture media, respectively. The crude extract was analyzed by HPLC and TLC, EN-480 and EN -502 were choosen for scale fermentation.
    The endophytic fungus Penicillium chermesinum EN-480 was isolated from the inner tissue of marine red alga Pterocladiella tenuis, which was cultured statically by rice solid medium. The EtOAc extract of culture filtrate of P. chermesinum EN-480 was fractioned by RP-18, Sephadex LH-20, preparative TLC, and preparative HPLC to yield 11 compounds (PC 1–PC 11), including 5 new meroterpenoids (PC 1–PC 5) and 4 new esquiterpenoids (PC 6 –PC 9). Compounds PC 1–PC 9 was evaluated for their antimicrobial activities and anti-insect activities, and compounds PC 1 and PC 2 exhibited obvious antibacterial activities against the human opportunistic pathogen Micrococcus luteus, with an MIC value of 8 μg/mL, but none of the compounds exhibited activity at a concentration of 100 μg/mL (lethal rate <60%).
    The endophytic fungus Trichoderma longibrachiatum EN-502 was isolated from the inner tissue of marine red alga Laurencia okamurai Yamada, which was cultured statically by rice solid medium. The crude extract was purified by column chromatography (CC), Lobar LiChroprep RP-18, preparative TLC and Sephadex LH-20 yielding seven isolates (TL 1–TL 7), including one new sesquiterpene (TL 1). Meanwhile, the strain was cultured by YPM medium, shaking cultured at first 7 days and then statically cultured 28 days, whose crude extract was fractioned by column chromatography (CC), Lobar LiChroprep RP-18 and Sephadex LH-20, yielding 3 sterol compounds (TLY 1–TLY 3). Unfortunately, all of them are known compounds.
    The study on second metabolites from marine algal-derived endophytic fungi EN-480 and EN-502, enriching the research of marine natural products and providing theoretical basis for development of marine second metabolites. Two compounds exhibited obvious Abstract antibacterial activities in the dissertation, which are potential source for application of marine natural products.
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/116979
Collection实验海洋生物学重点实验室
Affiliation中国科学院海洋研究所
Recommended Citation
GB/T 7714
刘慧. 海洋红藻来源鲜红青霉和长柄木霉次级代谢产物化学多样性及生物活性研究[D]. 北京. 中国科学院大学,2016.
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