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蕲艾内生真菌中抗海洋水产病害菌活性代谢产物研究
石小杉
Subtype博士
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理学博士
Keyword蕲艾 内生真菌 次级代谢产物 水产致病菌 抗水产病害菌活性
Abstract

水产养殖业为粮食安全做出了巨大贡献,是大农业中发展最快的产业之一。但随着养殖规模的扩大和集约化程度的提高,病害的发生频率和影响程度也越来越大,尤其是近年来由水产致病菌导致的病害也在不断大规模爆发,迫切需要研究、发现新的抗海洋水产病害菌活性物质。研究发现许多天然产物对常见的水产动物致病菌具有显著的体外抑制效果,在水产养殖领域的运用中具有一定的研究价值。而真菌来源的天然产物结构新颖多变,具有重要的药理学意义,在医药领域已日益凸显其广阔的应用前景。

微生物次级代谢产物在微生物种内、种间及跨界交流过程中扮演重要角色,从与海洋生物亲缘关系较远的物种中有可能更容易发现具有水产病害菌抑制活性的化合物。内生真菌是具有生物活性的新型天然产物的重要来源,我们前期筛选发现药用植物蕲艾(Artemisia argyi)内生真菌具有显著的抗水产病害菌活性,但目前蕲艾内生真菌代谢产物的化学成分尚未见研究报道。因此,我们以蕲艾为研究对象,研究其内生真菌次级代谢产物,致力于发现具有抗水产病害菌的活性先导化合物。

我们从蕲艾中共分离得到12株内生真菌,对这12株内生真菌使用大米、PDBMH2培养基进行小规模发酵筛选,结合HPLCTLC分析以及抗水产病害菌活性筛选结果,选取拟康宁木霉菌QA-3(Trichoderma koningiopsis QA-3)和绿木霉菌QA-8(T. virens QA-8)作为目标菌株进行规模发酵。拟康宁木霉菌QA-3和绿木霉菌QA-8大米发酵提取物均对水产病害菌溶藻弧菌(Vibrio alginolyticus)、嗜水气单孢菌(Aeromonas hydrophilia)和副溶血弧菌(V. parahaemolyticus)有抑制活性,此外,拟康宁木霉菌QA-3发酵提取物对迟缓爱德华氏菌(Edwardsiella tarda)和哈氏弧菌(V. harveyi)具有一定的抑制活性。

从拟康宁木霉菌QA-3的粗提物中共分离鉴定单体化合物38个,其中新化合物18个,包括15个新的聚酮类化合物(TKO1TKO15)1个新的α-吡喃酮类化合物(TKO16)2个新的萜类化合物(TKO17TKO18)。抗海洋水产病害菌活性测试结果表明,化合物TKO1对创伤弧菌(V. vulnificus)的抑制活性(MIC=4 µg/mL)优于阳性对照氯霉素(MIC=8 µg/mL)化合物TKO6溶藻弧菌(V. alginolyticus)的抑制活性与阳性对照氯霉素相当(MIC=1 μg/mL),化合物TKO8哈氏弧菌(V. harveyi)有抑制活性(MIC=4 μg/mL),化合物TKO11迟缓爱德华氏菌(Ed. tarda)有一定的抑制活性(MIC=2 μg/mL),而化合物TKO29对滕黄微球菌(Micrococcus luteus)表现出很强的抑制活性(MIC=1.0 µg/mL),均与阳性对照氯霉素的活性相当。

从绿木霉菌QA-8的发酵粗提物中共分离鉴定单体化合物43个,其中新化合物20个,包括12个新的杜松烷型倍半萜(TV1TV12,其中三个为成盐化合物(TV8TV10TV12))8个新的胡萝卜烷型倍半萜(TV13TV20)。抗海洋水产病害菌活性测试结果表明,化合物TV6迟缓爱德华氏菌(Ed. tarda)和鳗弧菌(V. anguillarum)具有较强的抑制活性,MIC值分别为1.02.0 µg/mL,阳性对照氯霉素的MIC值分别为0.51.0 µg/mL化合物TV7TV8对被测试菌株具有较广谱的抑制活性,MIC值为0.564 μg/mL,而化合物TV11TV12除了对滕黄微球菌(M. luteus)未表现出活性外,对其余7株受试菌株均有一定的抑制活性,MIC值为0.516 μg/mL化合物TV14–TV18TV23–TV25都对滕黄微球菌(M. luteus)表现出较强的抑制活性,MIC值为0.532 μg/mL,其中化合物TV14TV16的活性等于或优于阳性对照氯霉素。

综上,从上述两株蕲艾来源木霉属真菌的发酵提取物中,共分离鉴定出81个化合物,发现38个新化合物。抗菌生物活性评价结果表明,部分化合物具有进一步研究价值,具有开发成为抗水产病害菌活性先导化合物的潜力。本论文的研究内容不仅丰富了天然产物的化合物类型,也能够为后续天然产物在抗水产病害菌方面的研究提供一定的参考价值。

Other Abstract

Aquaculture industry has made great contribution to food security and is one of the fastest growing industries in large-scale agriculture. However, with the expansion of the scale of aquaculture and the increase of intensification, the frequency and degree of disease occurrence are also increasing. Especially in recent years, the diseases caused by aquatic pathogenic bacteria are also continuously erupting on a large scale. Therefore, it is urgent to study and discover new compounds of antimicrobial activity against aquatic pathogens. The natural products have ideal inhibitory effect on marine-derived pathogens and have research value in the field of aquaculture. The chemical structures of natural product from fungal kingdom are novel and variable, and with significant pharmacological values as well.

Microbial secondary metabolites play an important role in the communication of those within microbial species, microbial interspecies, and cross-border species. It is likely easier to find active substances for anti-marine pathogens from the species that genetically far from marine organisms. Endophytic fungi have been an essential resource of the novel and bioactivity natural products, and the organic extract of the fungal culture exhibited antimicrobial activity against several marine-derived pathogens in our primary screening, but the chemical constituents of endophytic fungi from A. argyi have not yet been reported. Therefore, we studied the secondary metabolites of endophytic fungi isolated from A. argyi, with the aim to discover new leading compounds of antimicrobial activity against aquatic pathogens.

A total of 12 endophytic fungal strains were isolated from the medicinal plant Artemisia argyi. The 12 strains were initially fermented in PDA, MH2 and rice solid medium for screening, and the crude extracts were evaluated for the activity against marine aquatic pathogenic bacteria, and HPLC and TLC analysis were also performed. As a result, two fungal strains, Trichoderma koningiopsis QA-3 and T. virens QA-8 were selected for further chemical investigation. In the antibacterial activity tests, the crude extracts of QA-3 and QA-8 exhibited antimicrobial activities against marine aquatic pathogens Vibrio alginolyticus, Aeromonas hydrophilia and V. parahaemolyticus. In addition, the crude extracts of QA-3 also showed some inhibition against Edwardsiella tarda and V. harveyi.

Chemical study of the fungal strain T. koningiopsis QA-3 resulted in the isolation of a total of thirty-eight secondary metabolites, with eighteen of them are new compounds, including fifteen new polyketides (TKO1–TKO15), one new α-pyrone derivatives (TKO16) and two new terpenoids (TKO17 and TKO18). The activity of TKO1 against V. vulnificus was stronger than that of the positive control chloramphenicol (MIC, 4.0 μg/mL vs 8.0 μg/mL). TKO6 showed strongest activity against V. alginolyticus (MIC=1.0 µg/mL), while TKO8 against V. harveyi (MIC=4.0 µg/mL), TKO11 against Ed. tarda (MIC=2.0 µg/mL), and TKO29 against M. luteus (MIC=1.0 µg/mL), which are comparable to that of the positive control chloramphenicol.

Chemical investigation of the fungal strain T. virens QA-8 resulted in the isolation of a total of forty-three secondary metabolites with different planar structures, with twenty of them are new compounds, including twelve new cadinane sesquiterpenes (TV1–TV12three of which are halogenated compounds) and eight new carotene-type terpenoids (TV13–TV20). Compound TV6 had activity against Ed. tarda and V. anguillarum with MIC values 1.0 and 2.0 µg/mL, respectively, which is comparable to that of the positive control (chloramphenicol, with MIC values 0.5 and 1.0 µg/mL, respectively). Compounds TV7 and TV8 had inhibitory activity against all tested pathogenic strains with MIC values ranging from 0.5–64 µg/mL, while compounds TV11 and TV12 have activity against all other seven tested strains, except for M. luteus, with MIC values ranging from 0.5–16 µg/mL. Compounds TV14–TV18 and TV23–TV25 have inhibitory activity against M. luteus with MIC values ranging from 0.5–32 µg/mL, and the activity of compounds TV14 and TV16 were similar to or stronger than that of the positive control.

In conclusion, a total of 81 metabolites with diverse scaffolds were isolated and identified from the extracts of the above fungal strains, and 38 of them were new compounds. The evaluation of antimicrobial bioactivity showed that several compounds had potential to be developed as anti-aquatic bacterial agents. Our study not only enriched the structure types of natural products, but also provided some reference value for the follow-up research on discovering new types of anti-aquatic bacterial compounds.

Subject Area化学 ; 生物学
MOST Discipline Catalogue理学
Pages191
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156789
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
石小杉. 蕲艾内生真菌中抗海洋水产病害菌活性代谢产物研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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