实验海洋生物学重点实验室知识库

硫代衍生物广泛存在于海洋微生物中，而海洋真菌是研究最多的海洋微生物群体之一。大多数海洋真菌与宿主共生，并可以从宿主中分离出来。其中，海洋植物来源的真菌在海洋微生物中占据着重要地位，尤其红树林和海藻是占比最大的真菌来源之一。

本论文从海藻及红树林植物中分离出不同种属的内生真菌，并对这些内生真菌进行了发酵筛选，结合化学指标以及生物指标筛选出3株代谢产物丰富的真菌：芽枝状枝孢菌（Cladosporium cladosporioides）MA-299，变幻青霉（Penicillium variabile）EN-394，长梗木霉（Trichoderma longibrachiatum）EN-586进行规模发酵。综合运用多种色谱技术对真菌发酵粗提物进行分离纯化，运用各种波谱手段对分离得到的单体化合物进行结构鉴定，共分离鉴定57个单体化合物，新化合物22个。结构类型包括硫代环内酯类、烯内酯类、甾体类、萜类以及聚酮类等，并对部分单体化合物进行了抑菌、酶抑制、抗肿瘤、抗氧化等生物活性测试。

对芽枝状枝孢菌C. cladosporioides MA-299进行发酵培养时添加硫源，促进了硫代环内酯的合成。最终从菌株MA-299中分离鉴定32个单体化合物，发现新化合物13个，包括7个硫代环内酯类（CC1*–CC4*, CC6*–CC8*）和6个非硫代环内酯类（CC9*–CC11*，CC14*–CC16*）化合物，并首次获得了硫代12元环内酯类化合物的单晶结构（CC1*、CC7*），对硫代环内酯类已知化合物pandangolide 3进行了结构修正，并分析、总结了不同取代类型的12元环内酯类化合物中硫链的取代位置。化合物CC9*是首次发现的具有5/9双环环系的环内酯类化合物，并通过单晶衍射实验进一步确证了其分子结构以及绝对构型；从变幻青霉P. variabile EN-394中共分离鉴定结构17个，发现新化合物5个，包括2个新的烯内酯类化合物（PV1*、PV2*），1个新的甾体类化合物（PV7*）和2个新的聚酮类化合物（PV10*、PV11*）。其中PV7*通过X-ray单晶衍射实验确定了绝对构型；从长梗木霉T. longibrachiatum EN-586中共分离鉴定结构8个，其中新化合物4个，主要类型包括萜类（TL1*）和聚酮类（TL4*–TL6*）。

对分离到的单体化合物进行了抑菌、酶抑制、抗肿瘤、抗氧化等生物活性测试。结果表明，硫代环内酯类化合物抗水产病害和植物病害病原菌的活性都明显优于非硫代环内酯类（CC1*–CC8* vs CC10*–CC17），提示硫元素在化合物抑菌活性中起关键作用。化合物CC9*表现出与硫代环内酯类相当的抑菌活性，可能是由于化合物CC9*中形成的C-C双键及C-2与C-6之间连接形成的5/9双环体系的作用。烯内酯类化合物PV6对鲇鱼爱德华氏菌（Edwardsiella ictarda）有明显的抑制活性，PV7*对多株水产病害菌如鲇鱼爱德华氏菌（E. ictarda）、鳗弧菌（Vibrio anguillarum）、铜绿假单胞菌（Pseudomonas aeruginosa）和植物病原真菌如苹果轮纹病菌（Physalospora piricola Nose）、小麦根腐病菌（Bipolaris sorokiniana）、苦瓜枯萎病菌（Fusarium oxysporum f. sp. momodicae）、玉米小斑病菌（Helminthosporium maydis）都有显著抑制活性，最小抑制浓度（MIC）介于1.0–4.0 μg/mL之间。此外，化合物CC1*、CC5、CC6*、CC12、CC14*对乙酰胆碱酯酶（acetylcholinesterase）有一定的抑制活性，半抑制浓度（IC₅₀）分别为 4.69 μM、7.51 μM、1.06 μM、1.79 μM、40.26 μM。清除自由基DPPH（1,1-diphenyl-2-picrylhydrazyl）活性实验表明，化合物PV2*，PV3有一定的自由基清除活性，其IC₅₀分别为19.35 μg/mL、12.15 μg/mL。

本论文通过对3株海洋植物来源的内生真菌进行次级代谢产物的分离鉴定和生物活性研究，发现了一系列结构新颖、活性良好的硫代环内酯类、烯内酯和其他类型的化合物，丰富了海洋真菌代谢产物的研究内容，为药物先导化合物的发现和某些真菌代谢产物的应用提供了一定的基础和依据。

Sulfide derivatives are widely found in marine microorganisms, while marine fungi are one of most studied communities among the marine microorganisms. Most of the marine-derived fungi are symbiotic with their hosts and can be separated and obtained from the hosts. Among them, marine plants-derived fungi play an important role in marine microorganisms, especially, endophytic fungi from mangroves and alga are the most important ones.

In this thesis, different species of endophytic fungi from algal and mangrove plants were isolated, and, based on the results from chemical and biological screening, three strains Cladosporium cladosporioides MA-299, Penicillium variabile EN-394, and Trichoderma longibrachiatum EN-586 were selected for scale-up fermentation and chemical investigation. After fermentation, a variety of chromatographic techniques were used to isolate and purify the metabolites, and various spectral methods were used to identify the structure of the isolated compounds. As a result, a total of 57 compounds were isolated and identified, and 22 of them are new compounds. The structure types of the isolated compounds covered from sulfur-containing macrolides to tetraene and triene lactones, steroids, terpenes and polyketones, and some of these compounds were tested for antimicrobial activity, enzymatic inhibitory activity, antitumor, antioxidation, and other biological activities.

Sulfur source was added to fermentation medium for C. cladosporioides MA-299 to promote the synthesis of sulfur-containing macrolides. Briefly, 32 compounds were isolated and identified from the metobolites of C. cladosporioides MA-299, among them, 13 compounds were reported for the first time, including seven sulfur-containing macrolides (CC1*–CC4*, CC6*–CC8*) and six non-sulfur lactones (CC9*–CC11*, CC14*–CC16*). The crystal structures of sulfur-containing 12-membered macrolides (CC1* and CC7*) were described for the first time, resulting in the structure revision of pandangolide 3, in which the sulfur side chain was corrected from C-3 to C-2. CC9* is the first reported macrolide with bicyclo 5/9 ring system, and its structure as well as absolute configuration was determined by X-ray crystallographic analysis. For P. variabile EN-394, 17 compounds were isolated and identified from the metabolites of the fungal strain, including two ene lactones (PV1* and PV2*), one steroid (PV7*), and two polyketones (PV10* and PV11*) discovered for the first time. The absolute configuration of PV7* was determined by X-ray crystallographic analysis. Eight metabolites were isolated and identified from the fungal strain T. longibrachiatum EN-586, with four new compounds including a terpenoid (TL1*) and three polyketones (TL4*–TL6*).

The identified compounds were tested for antimicrobial activities, enzymatic inhibitory activities, antitumor activities, and antioxidation activities. The results revealed that the antimicrobial activities of sulfur-containing macrolides, against aquatic bacteria and plant pathogenic fungi, were generally better than those of non-sulfur-containing compounds (CC1*–CC8* vs CC10*–CC17), indicating that sulfur substitution plays a key role in the antimicrobial activity of macrolides. CC9*, a nonsulfur-containing macrolide, also exhibited comparable bioactivities, possibly due to the presence of the double bond and the formation of bicyclic system via connection of C-2 and C-6. The triene lactone PV6 showed obvious inhibitory activity against Edwardsiella ictarda, while PV7* has significant inhibitory activities against a variety of aquatic pathogenic bacteria, such as Edwardsiella ictarda, Vibrio anguillarum, Pseudomonas aeruginosa, and plant pathogenic fungi, such as Physalospora piricola Nose, Bipolaris sorokiniana, Fusarium oxysporum f. sp. momodicae, and Helminthosporium maydis, with MIC of 1.0–4.0 g/mL. In addition, compounds CC1*, CC5, CC6*, CC12, and CC14* showed inhibitory activity against acetylcholinesterase, with IC₅₀ of 4.69, 7.51, 1.06, 1.79, and 40.26 μM, respectively, whereas PV2* and PV3 exhibited obvious scavenging activity of DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals, with IC₅₀ values of 19.35 and 12.15 μg/mL, respectively.

In summary, some sulfur-containing macrolides, ene lactones and other kinds of compounds with novel structures and significant bioactivities were identified from three strains of endophytic fungi that were obtained from marine algae and mangrove plants. The results from this work enriched the chemical diversity of marine fungal metabolites and provided basis for discovery of leading compounds.