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海洋来源白黄笋顶孢霉与咖啡青霉 次级代谢产物及其生物活性的研究
曹晋
Subtype博士
Thesis Advisor王斌贵
2019-05
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海洋真菌 内生真菌 白黄笋顶孢霉 咖啡青霉 次级代谢产物 生物活性
Abstract

 

海洋来源内生真菌次级代谢产物的研究如今受到广泛关注。由于与陆地环境的不同,海洋中的真菌面临的生存环境要复杂很多。红树林植物、海藻、海绵以及海底沉积物是海洋真菌的主要来源,处于这种环境下的真菌一方面要适应环境不同而带来的压力,另一方面又要应对同一环境中各种生存者带来的挑战。次级代谢产物作为真菌用于交流的工具,对其进行系统研究,对于发现结构新颖、活性显著的天然产物具有十分重要的意义。

本论文通过对海藻及红树林植物来源内生真菌的分离和筛选,利用TLCHPLC综合分析了菌株代谢产物的多样性,并对部分菌株的粗提物进行了抗菌活性测试,最后选择了白黄笋顶孢霉TK-43Acrostalagmus luteoalbus TK-43)和咖啡青霉MA-314Penicillium coffeae MA-314)两株真菌进行次级代谢产物的研究。通过多种分离方法,从中得到60个单体化合物,其中15个新化合物,结构类型主要包括吲哚二酮哌嗪类与异香豆素类化合物等。

从分离自刺松藻的白黄笋顶孢霉TK-43A. luteoalbus TK-43)中分离得到42个化合物(AL1aAL39),包括一系列新的具有氮甲氧基取代的吲哚二酮哌嗪类化合物(AL1aAL7),1个新的硫代吲哚二酮哌嗪类化合物(AL9)以及1个新的氧杂蒽酮类化合物(AL19),并且通过单晶培养,得到了化合物AL1AL6以及AL19的晶体结构。目前关于氮甲氧基取代的吲哚二酮哌嗪化合物的报道较少,只有两个同类型化合物在之前被发现。此外,化合物AL1AL3为三对吲哚二酮哌嗪对映异构体,对其进行手性拆分得到了三对平面结构相同但绝对构型完全相反的单体化合物(AL1a/AL1bAL2a/AL2bAL3a/AL3b),并通过ECD计算方法完成了对单体化合物的绝对构型的确定。该类吲哚二酮哌嗪的异构现象只在AL1AL3中被发现,且手性拆分得到的单体结构稳定,不存在绝对构型相互转化的现象,因此其异构化的产生应处于生物合成阶段,而与后期的分离纯化过程无关。从红树林植物拉关木来源的咖啡青霉MA-314P. coffeae MA-314)的代谢粗提物中分离鉴定结构18个(PC1PC18),其中包括一个新的六元内酯类化合物(PC1)以及两对异香豆素类对映异构体(PC2PC3)。

对所分离得到的部分化合物根据其结构类型选择了不同的生物活性测试实验,包括抗菌活性实验,抗乙酰胆碱酯酶活性实验以及抗氧化活性实验。在抗菌活性实验中,化合物AL2b对辣椒根腐病菌(Fusarium solani)有微弱的抑制活性,MIC值为32 μg/mL,但其对映异构体AL2a因为构型的差异并未表现出明显活性。同样,相比较于其差向异构体AL3aAL3b未表现出明显活性,化合物AL4对番茄早疫病菌(Alternaria solani)表现出较弱的抑制活性,MIC值为32 μg/mL。化合物PC1对两株植物致病真菌苦瓜枯萎病菌(Fusarium oxysporum f. sp. momordicae nov. f.)与苹果炭疽病菌(Colletotrichum gloeosporioides)表现出良好的抑制活性,MIC值均为1 μg/mL,略微高于阳性对照两性霉素BMIC=0.5 μg/mL)。抗乙酰胆碱酯酶活性实验中,化合物AL1a表现出优于其对映异构体AL1b以及差向异构体AL2aAL2b的活性,IC50值为2.3 μM通过分析构效关系发现,当该类化合物C-3C-6的绝对构型不同时,其表现出的活性要优于相同时,并且在C-3位置为R构型时,活性要优S构型。抗氧化活性实验中,新化合物PC2PC3并未表现出明显的抗氧化活性,但是已知化合物PC4表现出一定的活性,IC50值为656 μM,这一现象说明该类化合物C-4位置的羟基取代会有效减弱其抗氧化活性。

本论文对上述两株海洋来源内生真菌的次级代谢产物进行了系统的研究,从结构鉴定以及活性测试方面对其进行了综合评价,所分离得到的一系列结构新颖且具有一定生物活性的化合物为后续的海洋真菌天然产物研究以及新型海洋药物的开发提供了研究基础。

Other Abstract

Abstract

The study of secondary metabolites from marine endophytic fungi has received extensive attention in recent two decades. Compared to terrestrial fungi, marine fungi live in very different environments. Mangrove plants, algae, sponges and seabed sediments are the main sources of marine fungi. On the one hand, marine fungi have to adapt to the pressure brought by environments. On the other hand, they need to coexist with competitors peacefully. Secondary metabolites are important tools for fungi to communicate with each other, so it`s interesting to research on fungal metabolites to get structure unique and biological active natural products.

In this thesis, through screening of endophytic fungi from alga and mangrove plants by TLC, HPLC and bioactivity test, the chemical diversity of secondary metabolites of two fungi, Acrostalagmus luteoalbus TK-43 and Penicillium coffeae MA-314, was selected for chemical investigation. As a result, a total of 60 metabolites, including 15 new compounds were isolated through a variety of separation methods, and their bioactivity was evaluated.

Briefly, fouty-two compounds (AL1aAL39), including a series of new N-methoxy-containing indolediketopiperazines (AL1aAL7), a new sulfide indolediketopiperazine (AL9) and a new xanthone (AL19), were isolated from the culture extract of Acrostalagmus luteoalbus TK-43, an endophytic fungus obtained from the marine green alga Codium fragile. And the crystal structures of compounds AL1AL6 and AL19 were obtained by single crystal culture. At present, there are few reports on indolediketopiperazines with N-methoxy, and only two compounds of the same type have been found before. Compounds AL1AL3 were three pairs of indolediketopiperazine enantiomers and the optical resolution by chiral HPLC successfully afforded individual enantiomers AL1a/AL1b, AL2a/AL2b and AL3a/AL3b, respectively. The absolute configurations of these compounds were determined by TDDFT-ECD calculations. The structures of these compounds obtained by chiral resolution are stable and the absolute configuration can not spontaneous change. Therefore, the generation of isomerism should be in the biosynthesis stage, and not be affected by the later separation and purification process. Eighteen metabolites (PC1PC18), including a new δ-lactone (PC1) and two pairs of new isocoumarin derivatives (PC2 and PC3) were isolated from Penicillium coffeae MA-314, an endophytic fungus obtained from the fresh inner tissue of the leaf of marine mangrove plant Laguncularia racemosa.

According to the structural features of the isolated compounds, different bioactivity models, such as antimicrobial, anti-acetylcholinesterase (anti-AChE) and antioxidant activities were selected to evaluate the activity of the metabolites. In the antimicrobial activity experiment, compound AL2b showed weak inhibitory activity against Fusarium solani (MIC=32 μg/mL), but its enantiomer AL2a showed no obvious activity. Compound AL4 showed weak inhibitory activity against Alternaria solani (MIC=32 μg/mL), while its epimers AL3a and AL3b showed no activity. Compounds PC1 exhibited potent activity against Fusarium oxysporum f. sp. momordicae nov. f. and Colletotrichum gloeosporioides, with MIC values of 1 μg/mL, which was close to that of the positive control, amphotericin B (MIC=0.5 μg/mL). In the anti-acetylcholinesterase (anti-AChE) activity test, compound AL1a exhibited superior activity with IC50 value of 2.3 μM to its enantiomer AL1b (IC50=13.8 μM) and its epimers AL2a (IC50=78.8 μM) and AL2b (IC50=49.2 μM). It suggests that compounds of those having different absolute configurations at C-3 and C-6 generally showing better anti-AChE activity than those possessing same absolute configuration at C-3 and C-6. In addition, the 3R derivative usually has stronger activity than that of the 3S-isomer. In the antioxidant activity test, the new compounds PC2 and PC3 showed no significant antioxidant activity, but the known compound PC4 showed moderate activity with IC50 value of 656 μM. This phenomenon indicates that the hydroxyl substitution at the C-4 of these compounds can effectively weaken their antioxidant activity.

In summary, the secondary metabolites and their bioactivity of the above two strains of marine endophytic fungi were studied in detail. A series of novel compounds with bioactivities obtained from these fungi provided the research foundation for the future research on natural products of marine fungi and the development of new marine drugs.

MOST Discipline Catalogue理学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156871
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
曹晋. 海洋来源白黄笋顶孢霉与咖啡青霉 次级代谢产物及其生物活性的研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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