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海鞘内生真菌棒曲霉次级代谢产物及其抗菌活性研究
宋琦
Subtype硕士
Thesis Advisor王斌贵
2019-05-24
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学硕士
Keyword海鞘内生真菌,次级代谢产物,抗菌活性
Abstract

   本文对棒曲霉Aspergillus clavatus AS-107的次级代谢产物与抗菌活性进行了较系统的研究。棒曲霉AS-107分离自印度尼西亚海域海鞘的新鲜组织。对其进行大米固体培养基静置培养发酵并提取得到次级代谢产物粗提物,后借助多种色谱手段分离纯化、运用综合波谱技术鉴定了单体化合物的分子结构,并对这些化合物进行了抗菌活性评价。

  利用反相与正相硅胶柱层析、制备薄层层析以及葡聚糖凝胶柱层析等色谱方法从发酵产物中获得了22个单体化合物(AC1-AC22),通过分析其理化性质(状态、溶解度、熔点和显色特征等)、综合波谱数据(NMR, MS, IR, UV等)以及与相关文献比对确定其结构。获得的化合物结构类型主要包括喹唑啉酮生物碱类、多肽类、香豆素类、混源萜类、甾醇类等。其中AC1AC2AC3为新化合物,通过ECD计算确定了AC1AC3的绝对构型,而AC2则是借助X-射线单晶衍射实验确证了其化学结构。

  对所分离得到的单体化合物进行了抗细菌活性测试,选择了2株人类致病细菌和5株水产致病细菌作为病原指示菌。测试结果表明化合物AC1AC11AC13AC15对大肠杆菌有较为显著的抑制作用,其MIC值分别为8848 μg/mL;化合物AC1AC2对藤黄微球菌有较强的抗菌作用,其MIC值均为8 μg/mL;化合物AC13对鳗弧菌的抑菌活性MIC值为8 μg/mL;化合物AC1对嗜水气单胞菌的抑菌作用比较显著,其MIC值均为4 μg/mLAC11对哈氏弧菌的抑制作用较强,其MIC值为8 μg/mL;化合物AC11AC13AC15对铜绿假单胞菌有明显抑制作用,其MIC值分别为844 μg/mL。其中,化合物AC1AC2AC13对这7株病原细菌均具有一定程度的抑制作用。此外,分析所测化合物的构效关系发现,AC1与同类化合物AC15相比,酯键打开而开环会增强其抗菌活性;AC14AC3相比,C-27位构型不同导致化合物的抗菌活性差异较大。

  本文研究结果进一步丰富了海洋天然产物的化学多样性,所获得的化合物AC1AC2AC11AC13AC15具有较好的抗细菌活性,为海洋生物资源的利用提供了理论和应用基础。

Other Abstract

Abstract

  In this paper, the secondary metabolites of Aspergillus clavatus AS-107, which was isolated from fresh tissues of ascidian in Indonesian seawaters, were systematically studied. The crude extracts of secondary metabolites were obtained from static culture and fermentation on rice solid medium. The compounds in the culture extracts were separated and purified by various chromatographic methods and the chemical structures were identified by comprehensive spectroscopic analysis. In addition, the antimicrobial activities of the isolated compounds were evaluated.

  The metabolites from the culture of the fungal strain A. clavatus AS-107 were purified by reverse phase silica gel column chromatography, normal silica gel column chromatography, preparative thin layer chromatography (TLC), and Sephadex gel column chromatography. Twenty-two compounds (AC1-AC22), including quinazolinone alkaloids, polypeptides, coumarins, terpenoids, and sterols were obtained and identified. The structures were determined by analyzing their physical properties (state, solubility, melting point and color characteristics in the reaction), comprehensive spectral data (NMR, MS, IR, UV, etc.), and by comparing with relevant literatures. Among them, AC1, AC2 and AC3 are new compounds. Moreover, the absolute configurations of AC1 and AC3 were determined by ECD calculation, while AC2 was confirmed by single-crystal X-ray diffraction experiment.

  The antimicrobial activity of the isolated compounds was tested using two human pathogenic bacteria and five aquatic pathogenic bacteria as pathogenic indicators. The results showed that compounds AC1, AC11, AC13 and AC15 had significant inhibitory effect on Escherichia coli with MIC values of 8, 8, 4 and 8 μg/mL, respectively. Compounds AC1 and AC2 had strong antimicrobial activity against Micrococcus luteus with the same MIC value of 8 μg/mL. Compound AC13 had bacteriostatic activity against Vibrio anguillarum with MIC value of 8 μg/mL, while compound AC1 had significant bacteriostatic effect on Aeromonas hydrophila with MIC value of 4 μg/mL. Compound AC11 had certain inhibitory effect on Vibrio harveyi with MIC value of 8 μg/mL. Compounds AC11, AC13 and AC15 have obvious inhibitory effect on Pseudomonas aeruginosa with MIC values of 8, 4 and 4 μg/mL, respectively. The above results indicated that compounds AC1, AC2 and AC13 had inhibitory effects on all of the seven tested pathogenic bacteria, which deserve to be further studied. Based on the analysis of the structure-activity relationship of the tested compounds, it showed that compared with compound AC15, AC1 had stronger antibacterial activity when the ester bond was opened. Compared with compound AC3, the different configuration at C-27 of AC14 led to variation of antibacterial activity.

  The results of this study further enriched the chemical diversity of marine natural products. The obtained compounds AC1, AC2, AC11, AC13 and AC15 had good antimicrobial activity, which provided the theoretical and applied basis for the possible development of marine bioresources.

Subject Area生物学
MOST Discipline Catalogue理学
Language中文
Table of Contents

1 引言 …………………………………………………………………..1

1.1 绪论 …………………………………………………………….…………1

1.2 喹唑啉酮类生物碱 …………………………………………………...…..2

1.2.1 2-取代喹唑啉酮类生物碱 ……….….….….….….….….….….….….2

1.2.2 3-取代喹唑啉酮类生物碱 ……….….….….….….….….…..….….…3

1.2.3 2, 3-取代喹唑啉酮类生物碱 …………………………………………6

1.3 小结 ………………………………………………………………………19

2 海鞘内生真菌Aspergillus clavatus AS-107次级代谢产物及其抗菌活性研究……………………………………………………………….21

2.1 单体化合物结构鉴定……………………………………………………...21

2.1.1 新化合物结构鉴定…………………………………………………....21

2.1.2 已知化合物结构鉴定………………………………………………....27

2.2 实验部分……………………………………………………………….......38

2.2.1 主要实验仪器、材料和试剂…………………………………………38

2.2.2 菌株AS-107基本信息……………………………………………......39

2.2.3 菌株规模发酵培养…………………………………………................41

2.2.4 真菌次级代谢产物的提取与分离…………………………................42

2.2.5 单体化合物的物理性质及波谱数据………………………................42

2.2.6 单体化合物的生物活性测试……………………………………........52

3 结论语与创新点…………………………………………………….55

3.1 结论语……………………………………………………………………...55

3.2 创新点……………………………………………………………………...55

参考文献…………………………………………………………………………..57

附录:部分化合物谱图…………………………………………….………….....65

致谢………………………………………………………………………………..73

作者简历及攻读学位期间发表的学术论文与研究成果………………………..75

 

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156898
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
宋琦. 海鞘内生真菌棒曲霉次级代谢产物及其抗菌活性研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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