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Journal of Tropical Oceanography >

2024 , Vol. 43 >Issue 5: 41 - 48

DOI: https://doi.org/10.11978/2023158

Marine Biology

Studies on secondary metabolites of endophytic fungus Aspergillus terreus GXIMD 03158 isolated from mangroves Acanthus ilicifolius L.

  • AN Fan , 1, 2 ,
  • JIANG Yue 1, 2 ,
  • WANG Yu 1, 2 ,
  • CAO Guangping 1, 2 ,
  • GAO Chenghai 1, 2 ,
  • LIU Yonghong 1, 2 ,
  • YI Xiangxi , 1, 2 ,
  • BAI Meng , 1, 2
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  • 1. Institute of Marine Drugs / Faculty of Pharmacy, Guangxi University of Chinese Medicine, Nanning 530200, China
  • 2. Guangxi Key Laboratory of Marine Drugs, Nanning 530200, China
YI Xiangxi. email: ;
BAI Meng. email:

Received date: 2023-10-23

  Revised date: 2023-12-07

  Online published: 2023-12-14

Supported by

Natural Science Foundation of Guangxi(2023GXNSFAA026313)

Natural Science Foundation of Guangxi(2021GXNSFBA220072)

Natural Science Foundation of Guangxi(2020GXNSFGA297002)

Natural Science Foundation of Guangxi(2021GXNSFDA075010)

Initial Scientific Research Foundation of Introduced Doctors in 2020 of Guangxi University of Chinese Medicine(2020BS024)

Special Fund for Bagui Scholars of Guangxi(05019055)

Guangxi University of Chinese Medicine “Guipai Traditional Chinese Medicine Inheritance and Innovation Team” Project(2022A007)

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Abstract

This work studies the secondary metabolites with antibacterial activity from Aspergillus terreus GXIMD 03158, an endophytic fungus isolated from mangroves Acanthus ilicifolius L. The secondary metabolites of the strains were separated and purified by various chromatographic techniques, and the structures of monomer compounds were identified by comprehensive analyses of 1H, 13C NMR and HREIMS date. Ten compounds were isolated from the metabolites of A. terreus GXIMD 03158, and identified as dankasterones A (1), (14α, 22E)-14-hydroxyergosta-4, 7, 22-triene-3, 6-dione (2), steresterone B (3), ergosta-4, 6, 8(14), 22-tetraen-3-one (4), herbarulide (5), demethylincisterol A3 (6), isochaetochromin B1 (7), isochaetochromin B2 (8), (E)-7, 9-diene-11-carbonyl stearic acid (9) and aspergilfuranone A (10). The minimal inhibitory concentrations (MIC) of all compounds against Staphylococcus albus, Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus and Vibrio parahaemolyticus were determined by broth microdilution method, the results showed that compound 6 exhibited significant antibacterial activity against S. albus, S. aureus and S. epidermidis, and the MIC values were 3.12, 6.25 and 3.12 μg·mL-1, respectively; compound 9 exhibited significant antibacterial activity against S. albus and S. aureus, and the MIC values were 1.56 and 3.12 μg·mL-1, respectively. This study provides a theoretical basis for the research on development and utilization of mangrove Acanthus ilicifolius L. and its endophytic fungi.

Key words： mangroves Acanthus ilicifolius L.; endophytic fungi; Aspergillus terreus; secondary metabolites; antibacterial activities

Cite this article

AN Fan , JIANG Yue , WANG Yu , CAO Guangping , GAO Chenghai , LIU Yonghong , YI Xiangxi , BAI Meng . Studies on secondary metabolites of endophytic fungus Aspergillus terreus GXIMD 03158 isolated from mangroves Acanthus ilicifolius L.[J]. Journal of Tropical Oceanography, 2024 , 43(5) : 41 -48 . DOI: 10.11978/2023158

红树林主要分布在热带和亚热带之间, 兼具海洋和陆生植物的特性, 红树林微生物为了适应高盐、寡营养、周期性海水浸淹等特殊环境(Chen et al, 2017; Liao et al, 2019), 导致红树来源微生物自发地进化出与陆地微生物不同的代谢途径, 使得其代谢产物具有结构新颖、生物活性显著等特点(Duperron et al, 2019)。截至2023年共有1565个新颖结构化合物从红树内生真菌中分离获得, 主要包括聚酮类(63%)、萜类(19%)、生物碱类(12%)和肽类(6%); 其中613个化合物具有广谱生物活性, 为新药研发提供了导向(Carroll et al, 2020, 2021, 2022, 2023; Chen et al, 2022)。红树老鼠簕(Acanthus ilicifolius L.)是爵床科老鼠簕属的直立灌木植物。随着对海洋资源的开发利用, 如建造码头、围塘养殖等, 导致其红树资源逐渐减少(刘镜法, 2005; 李海生 等, 2019), 但是红树林微生物资源丰富, 从中分离得到的红树林真菌构成了海洋真菌的第二大类群(梁寒峭 等, 2023), 因而不少学者把研究对象转向药用红树内生真菌。据文献报道, 各国科学家从药用红树老鼠簕内生真菌的次级代谢产物中分离得到了多种类型化合物, 如生物碱类、甾醇类和简单芳香类化合物等, 部分化合物表现出良好的生物活性(李元跃 等, 2021; 高亚欣 等, 2023; 梁寒峭 等, 2023)。以上数据研究结果显示药用红树老鼠簕内生真菌的次级代谢产物具有良好的开发前景。
本课题组一直致力于药用红树内生真菌活性次级代谢产物研究(Bai et al, 2019; 白猛 等, 2020; 谭玉莹 等, 2022), 为了深入挖掘药用红树老鼠簕内生真菌活性次级代谢产物, 课题组前期从红树老鼠簕来源的内生真菌中发现1株具有显著抗菌活性的真菌Aspergillus terreus GXIMD 03158。本研究对该真菌进行了大米培养基固体发酵, 从发酵产物提取物中分离得到了10个化合物, 分别鉴定为dankasterones A (1)、(14α, 22E)-14-hydroxyergosta-4, 7, 22-triene-3, 6-dione (2)、steresterone B (3)、ergosta-4, 6, 8(14), 22-tetraen-3-one (4)、herbarulide (5)、demethylincisterol A3 (6)、isochaetochromin B1 (7)、isochaetochromin B2 (8)、(E)-7, 9-二烯-11-羰基硬脂酸 (9)和aspergilfuranone A (10) (图1)。其中6个高度氧化的甾体类化合物(1~6), 两个轴手性化合物(7和8), 且是一对阻转异构体, 通过圆二色谱仪(Circular Dichroism, CD)加以证实。
图1 化合物 1~10

Fig. 1 Chemical structures of compounds 1-10

1 材料和方法

1.1 主要仪器与试剂

Bruker AV-500MHz型核磁共振仪(Bruker公司, 瑞士), 岛津LC 2030C 3D Plus系列半制备型高效液相色谱(株式会社岛津制作所, 日本), C18半制备液相柱(250mm × 10mm, 5µm)(北京赛谱锐思科技有限公司, 中国), EYELAN1100V-W型旋转蒸发仪(东京理化株式会社, 日本), GC-MS QP 2010 Ultra质谱仪(株式会社岛津制作所, 日本), J-1500-150圆二色谱仪(分光株式会社, 日本), IP-digi300/3自动旋光仪(上海仪迈仪器科技有限公司, 中国), 真空控制器(北京爱科仪器设备科技有限公司, 中国), 中压制备色谱仪(上海沃珑仪器有限公司, 中国), 千分之一电子天平(上海菁海仪器有限公司, 中国), 超声波清洗器(昆山市超声仪器有限公司, 中国), HZ300L恒温摇床(武汉瑞华仪器设公司, 中国), 高压灭菌锅(重庆雅马拓科技有限公司, 中国), 恒温水浴锅(上海爱朗仪器有限公司, 中国), 柱色谱硅胶(青岛海洋化工有限公司, 中国), 分析纯石油醚、乙酸乙酯、甲醇(成都科隆化学品有限公司, 中国), 色谱纯甲醇、乙腈(上海星可高纯溶剂有限公司, 中国), 马铃薯葡萄糖水(广东环凯微生物科技有限公司, 中国), 测定核磁共振谱所用的氘代试剂(上海泰坦科技股份有限公司, 中国)。

1.2 试验方法

1.2.1 菌株的来源与发酵培养

内生真菌GXIMD 03158 (图2)分离自红树植物老鼠簕根部, 样品采集于中国广西山口红树林自然保护区, 老鼠簕根部由广西防城港市海洋局刘熊高级工程师鉴定, 菌种保存于广西中医药大学海洋药物研究院。对目标菌株GXIMD 03158的rDNA的ITS序列进行PCR扩增, 纯化后测序。在GenBank上与已知菌株进行BLAST序列对比, 发现该菌与Aspergillus terreus的相似度为99%, 因此鉴定该菌株为土曲霉Aspergillus terreus, GenBank号为OK036729。
图2 菌株GXIMD 03158菌落形态

Fig. 2 Colony morphology of strain GXIMD 03158

菌株Aspergillus terreus GXIMD 03158经复苏后, 转接到装有200mL马铃薯葡萄糖水培养基(含马铃薯浸出粉300.0g·L-1、葡萄糖20.0g·L-1和海盐3.3g·L-1)的500mL培养瓶中, 于28℃、180r·min-1的恒温摇床中振荡培养72h后得到种子液。菌株用大米培养基进行大规模发酵, 共发酵100瓶(每1000mL的培养瓶中加入80g大米、海盐5g·L-1), 在高压灭菌锅中121℃灭菌30min, 待培养基冷却后接种(每瓶加入8mL种子液), 在25℃条件下静置培养30d。

1.2.2 提取与分离

发酵结束后将大米捣碎, 使用等体积乙酸乙酯进行萃取, 重复3次, 合并萃取液, 减压浓缩后得到乙酸乙酯部位浸膏150.21g。浸膏经正相硅胶柱进行粗分, 用石油醚-乙酸乙酯 (100:0~0:100, v/v)、乙酸乙酯-甲醇 (3:1~0:100, v/v)依次洗脱, 根据薄层色谱法追踪目标分子, 分析各流份化合物成分合并相同流份, 浓缩, 得到15个组分Fr.1~Fr.15。
Fr.5经硅胶柱色谱分离, 用石油醚-乙酸乙酯 (100:0~0:100, v/v)、乙酸乙酯-甲醇(3:1~0:100, v/v)依次洗脱, 根据薄层色谱分析结果合并相同流份得到8个组分Fr.5-1~Fr.5-8。对Fr.5-5采用C18半制备高效液相色谱分离(流动相为乙腈-水=95:5, v/v, 流速为3mL·min-1, λ为230nm、290nm和410nm)得到化合物7 (tR=36.7min, 4.6mg)和8 (tR=37.4min, 8.2mg)。
Fr.2经反相硅胶柱色谱分离, 以水-甲醇(90:10~ 0:100, v/v)为流动相进行梯度洗脱, 根据TLC薄层色谱分析结果合并相同流份得到11个组分Fr.2-1~Fr.2-14。对Fr.2-4采用C18半制备高效液相色谱分离(流动相为乙腈-水=95:5, v/v, 流速为2mL·min-1, λ为200nm和272nm)得到化合物9 (tR=12.7min, 5.0mg)。对Fr.2-11采用C18半制备高效液相色谱分离(流动相为甲醇-水=95:5, v/v, 流速为2mL·min-1, λ为210nm和252nm)得到化合物1 (tR=13.6min, 79.2mg)和6 (tR=14.8min, 3.1mg)。对Fr.2-12采用C18半制备高效液相色谱分离(流动相为MeOH-H2O=80:20, v/v, 流速为2mL·min-1, λ为205nm和275nm)得到化合物2 (tR=19.2min, 8.5mg)、5 (tR=22.5min, 10.1mg)和3 (tR=18.4min, 2.5mg)。对Fr.2-2进行反相硅胶柱色谱分离, 以水-甲醇(90:10~0:100, v/v)为流动相进行梯度洗脱, 根据TLC薄层色谱分析结果合并相同流份得到14个组分Fr.2-2-1~Fr.2-2-14。对Fr.2-2-12经C18半制备高效液相色谱分离(流动相为MeOH-H2O=85:15, v/v, 流速为2mL·min-1, λ为203nm和350nm)得到化合物4 (tR=25.7min, 8.2mg)。对Fr.2-2-7经C18半制备高效液相色谱分离(流动相为甲醇-水=70:30, v/v, 流速为2mL·min-1, λ为211nm和257nm)得到化合物10 (tR=8.2min, 35.9mg)。

1.2.3 单体化合物MIC值测定方法

测定所有单体化合物对白色葡萄球菌(Staphylococcus albus)、金黄色葡萄球菌(Staphylococcus aureus)、表皮葡萄球菌(Staphylococcus epidermidis)、耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus, MRSA)和副溶血弧菌(Vibrio parahaemolyticus)的抗菌活性: 滤纸片扩散法对所有单体化合物(浓度为1mg·mL-1)进行抑菌活性初筛, 对有明显抑菌活性的单体化合物采用96孔微量肉汤稀释法检测其最小抑菌浓度(minimal inhibitory concentrations, MIC), 具体试验方法参考文献(Pierce et al, 2008)。滤纸片琼脂扩散法活性初筛具体方法如下: 配制致病菌生长所需的LB培养基, 121℃高压灭菌30min放置至冷却。在超净工作台中将活化好的致病菌转接至LB培养基中, 放入37℃、180r·min-1振荡器中培养至OD值为0.5, 即培养完成, 作为种子液。在超净工作台中, 采用灌注法用移液枪吸取1%的种子液固体LB培养基中, 充分摇匀, 再进行倒板。待平板冷却后, 对单体化合物采用滤纸片琼脂扩散法进行指示致病菌的活性初筛。

2 结果与分析

2.1 化合物波谱数据

化合物 1: 白色晶体, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$+44.4° (c 0.1, MeOH), HR-ESI-MS m/z 447.2874, [M+Na]+ (计算值C28H40O3Na, 447.2875)。1H NMR (500MHz, DMSO-d6) δH: 5.96 (1H, m, H-4), 5.29 (1H, dd, J=15.1, 6.8Hz, H-23), 5.27 (1H, dd, J=15.1, 6.8Hz, H-22), 2.77 (1H, td, J=9.0, 1.3Hz, H-9), 2.53 (2H, dt, J=17.6, 5.1Hz, H-2), 2.45 (1H, m, H-20), 2.42 (2H, dd, J=16.8, 1.3Hz, H-7), 2.04 (2H, m, H-11), 2.00 (2H, m, H-1), 1.87 (2H, m, H-15), 1.87 (1H, m, H-24), 1.69 (1H, dd, J=13.2, 4.2Hz, H-17), 1.63 (2H, m, H-12), 1.48 (1H, m, H-25), 1.20 (2H, m, H-16), 1.19 (3H, d, J=6.8Hz, H-21), 1.06 (3H, s, H-19), 0.88 (3H, d, J=6.8Hz, H-28), 0.79 (3H, s, H-18), 0.79 (3H, d, J=6.8Hz, H-26), 0.79 (3H, d, J=6.8Hz, H-27); 13C NMR (125MHz, DMSO-d6) δC: 215.8 (C, C-14), 200.5 (C, C-6), 199.6 (C, C-3), 157.8 (C, C-5), 134.0 (CH, C-23), 133.1 (CH, C-22), 124.8 (CH, C-4), 62.4 (C, C-8), 54.5 (CH, C-13), 49.1 (CH, C-9), 46.6 (CH, C-17), 42.7 (CH, C-24), 40.1 (CH2, C-7), 38.4 (CH2, C-1), 37.8 (CH2, C-15), 36.8 (CH2, C-12), 36.6 (CH, C-20), 36.1 (C, C-10), 34.2 (CH2, C-2), 32.8 (CH, C-25), 24.7 (CH2, C-11), 23.7 (CH3, C-19), 23.5 (CH3, C-21), 23.4 (CH2, C-16), 20.1 (CH3, C-27), 19.7 (CH3, C-26), 17.6 (CH3, C-28), 16.6 (CH3, C-18)。以上数据与文献(Amagata et al, 2007)报道基本一致, 故鉴定该化合物为dankasterones A。
化合物 2: 淡黄色粉末, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$+23.6° (c 0.05, MeOH), HR-ESI-MS m/z 447.2874, [M+Na]+ (计算值C28H40O3Na, 447.2875)。1H NMR (500MHz, DMSO-d6) δH: 6.29 (1H, s, H-7), 6.17 (1H, s, H-4), 5.24 (1H, d, J=11.7Hz, H-22), 5.18 (1H, dd, J=8.0, 4.1Hz, H-23), 2.91 (1H, t, J=10.5, 7.5Hz, H-9), 1.22 (3H, s, H-19), 0.99 (3H, d, J=6.5Hz, H-21), 0.90 (3H, d, J=6.8Hz, H-28), 0.82 (3H, d, J=6.7Hz, H-26), 0.81 (3H, d, J=6.7Hz, H-27), 0.67 (3H, s, H-18); 13C NMR (125MHz, DMSO-d6) δC: 199.0 (C, C-3), 187.3 (C, C-6), 169.2 (C, C-8), 158.2 (C, C-5), 135.5 (CH, C-22), 131.6 (CH, C-23), 124.9 (CH, C-7), 122.2 (CH, C-4), 83.1 (C, C-14), 49.9 (CH, C-17), 45.9 (C, C-13), 42.7 (CH, C-9), 42.0 (CH, C-24), 39.0 (C, C-10), 38.8 (CH, C-20), 34.4 (CH2, C-2), 33.9 (CH2, C-1), 32.5 (CH, C-25), 30.4 (CH2, C-15), 30.0 (CH2, C-12), 26.5 (CH2, C-16), 21.1 (CH3, C-21), 20.9 (CH2, C-11), 19.8 (CH3, C-26), 19.5 (CH3, C-27), 19.5 (CH3, C-19), 17.4 (CH3, C-28), 15.6 (CH3, C-18)。以上数据与文献(解双双 等, 2019)报道基本一致, 故鉴定该化合物为(14α, 22E)-14-hydroxyergosta-4, 7, 22-triene-3, 6-dione。
化合物 3: 黄色粉末, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$-33.7° (c 0.05, MeOH), HR-ESI-MS m/z 463.2793, [M+Na]+ (计算值C28H40O4Na, 463.2824)。1H NMR (500MHz, DMSO-d6) δH: 6.17 (1H, s, H-4), 5.99 (1H, s, H-7), 5.29 (1H, dd, J=15.3, 7.5Hz, H-23), 5.19 (1H, dd, J=15.3, 8.4Hz, H-22), 2.89 (1H, s, H-7), 2.51 (2H, m, H-2), 1.24 (3H, s, H-19), 0.97 (3H, d, J=6.6Hz, H-21), 0.90 (3H, d, J=6.8Hz, H-28), 0, 83 (3H, d, J=6.8Hz, H-27), 0.81 (3H, s, H-18), 0.77 (3H, d, J=6.8Hz, H-26); 13C NMR (125MHz, DMSO-d6) δC: 199.1 (C, C-3), 186.9 (C, C-6), 163.8 (C, C-8), 158.1 (C, C-5), 135.3 (CH, C-22), 131.8 (CH, C-23), 126.2 (CH, C-7), 124.9 (CH, C-4), 94.9 (C, C-14), 50.5 (CH, C-17), 47.2 (C, C-13), 42.4 (CH, C-9), 42.0 (CH, C-24), 40.1 (CH, C-20), 38.8 (C, C-10), 34.4 (CH2, C-1), 33.9 (CH2, C-2), 32.5 (CH, C-25), 29.6 (CH2, C-12), 26.5 (CH2, C-16), 24.2 (CH2, C-15), 21.0 (CH3, C-21), 20.1 (CH2, C-11), 19.8 (CH3, C-27), 19.5 (CH3, C-26), 18.8 (CH3, C-19), 17.3 (CH3, C-28), 16.4 (CH3, C-18)。以上数据与文献(Zhao et al, 2019)报道基本一致, 故鉴定该化合物为steresterone B。
化合物 4: 白色固体, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$+360.6° (c 0.05, MeOH), HR-ESI-MS m/z 393.3161, [M+H]+ (计算值C28H41O, 393.3157)。1H NMR (500MHz, DMSO-d6) δH: 6.68 (1H, d, J=9.5Hz, H-7), 6.14 (1H, d, J=9.5Hz, H-6), 5.70 (1H, s, H-4), 5.27 (1H, m, H-22), 5.27 (1H, m, H-23), 1.05 (3H, d, J=6.6Hz, H-21), 0.96 (3H, s, H-18), 0.95 (3H, s, H-18), 0.93 (3H, d, J=6.9Hz, H-28), 0.85 (3H, d, J=7.2Hz, H-26), 0.83 (3H, d, J=7.2Hz, H-27); 13C NMR (125MHz, DMSO-d6) δC: 197.9 (C, C-3), 163.5 (C, C-8), 155.4 (C, C-14), 135.0 (CH, C-22), 133.7 (CH, C-7), 131.8 (CH, C-23), 124.4 (C, C-5), 124.1 (CH, C-6), 122.6 (CH, C-4), 55.2 (CH, C-17), 43.8 (CH, C-9), 43.5 (C, C-13), 42.1 (CH, C-24), 38.8 (CH, C-20), 36.2 (C, C-10), 35.1 (CH2, C-12), 33.8 (CH2, C-1), 33.4 (CH2, C-2), 32.5 (CH, C-25), 27.4 (CH2, C-16), 24.8 (CH2, C-15), 21.1 (CH3, C-21), 19.9 (CH3, C-26), 19.5 (CH3, C-27), 18.7 (CH3, C-18), 18.5 (CH2, C-11), 17.4 (CH3, C-28), 16.3 (CH3, C-19)。以上数据与文献(Fujimoto et al, 2004)报道基本一致, 故鉴定该化合物为aspergilfuranone A。
化合物 5: 黄色固体, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$+169.8° (c 0.05, MeOH), HR-ESI-MS m/z 425.3038, [M+H]+ (计算值C28H41O3, 425.3056)。1H NMR (500MHz, DMSO-d6) δH: 5.66 (1H, s, H-4), 5.58 (1H, s, H-7), 5.26 (1H, dd, J=15.3, 7.5Hz, H-23), 5.17 (1H, dd, J=15.3, 8.3Hz, H-22), 1.10 (3H, s, H-19), 1.00 (3H, d, J=6.6Hz, H-21), 0.89 (3H, d, J=6.8Hz, H-28), 0.81 (3H, d, J=7.0Hz, H-26), 0.80 (3H, d, J=6.9Hz, H-27), 0.57 (3H, s, H-18); 13C NMR (125MHz, DMSO-d6) δC: 198.4 (C, C-3), 173.6 (C, C-5), 162.0 (C, C-6), 161.1 (C, C-8), 135.0 (CH, C-22), 131.8 (CH, C-23), 113.8 (CH, C-4), 111.8 (CH, C-7), 56.7 (CH, C-14), 55.6 (CH, C-17), 46.7 (CH, C-9), 46.0 (C, C-13), 42.1 (CH, C-24), 40.0 (CH, C-20), 39.0 (CH2, C-12), 38.0 (C, C-10), 33.4 (CH2, C-1), 33.0 (CH2, C-2), 32.5 (CH, C-25), 27.5 (CH2, C-16), 24.9 (CH2, C-11), 22.0 (CH2, C-15), 20.9 (CH3, C-21), 19.8 (CH3, C-19), 19.5 (CH3, C-26), 19.4 (CH3, C-27), 17.3 (CH3, C-28), 12.1 (CH3, C-18)。以上数据与文献(马新玥 等, 2018)报道基本一致, 故鉴定该化合物为herbarulide。
化合物 6: 黄色固体, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$+38.2° (c 0.1, MeOH), HR-ESI-MS m/z 355.2242, [M+Na]+ (计算值C21H32O3Na, 355.2249)。1H NMR (500MHz, DMSO-d6) δH: 5.77 (1H, d, J=1.8Hz, H-2), 5.26 (1H, dd, J=15.3, 7.4Hz, H-16), 5.19 (1H, dd, J=15.4, 8.3Hz, H-15), 1.00 (3H, d, J=6.6Hz, H-14), 0.89 (3H, d, J=6.8Hz, H-21), 0.81 (6H, d, J=7.0Hz, H-19, 20), 0.54 (3H, s, H-12); 13C NMR (125MHz, DMSO-d6) δC: 170.8 (C, C-1), 170.6 (C, C-3), 135.0 (CH, C-15), 131.8 (CH, C-16), 111.5 (CH, C-2), 105.0 (C, C-4), 54.6 (CH, C-11), 49.9 (CH, C-8), 48.3 (C, C-7), 42.1 (CH, C-17), 40.0 (CH, C-13), 35.0 (CH2, C-6), 34.8 (CH2, C-5), 32.5 (CH, C-18), 28.6 (CH2, C-10), 20.9 (CH3, C-14), 20.8 (CH2, C-9), 19.8 (CH3, C-19), 19.5 (CH3, C-20), 17.4 (CH3, C-21), 11.8 (CH3, C-12)。以上数据与文献(胡晓峰 等, 2021)报道基本一致, 故鉴定该化合物为demethylincisterol A3。
化合物 7: 黄绿色固体, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$-108.1° (c 0.05, MeOH), HR-ESI-MS m/z 547.1603, [M+H]+ (计算值C30H27O10, 547.1640)。1H NMR (400MHz, DMSO-d6) δH: 15.20 (1H, s, 5-OH), 15.11 (1H, s, 5′-OH), 10.01 (1H, s, 6-OH), 9.82 (1H, s, 6′-OH)), 6.58 (1H, s, H-10′), 6.51 (1H, s, H-7), 6.39 (1H, s, H-7′), 5.85 (1H, s, H-10), 4.68 (1H, dd, J=6.3, 3.0Hz, H-2′), 4.21 (1H, dd, J=11.0, 6.3Hz, H-2), 2.85 (1H, m, H-3), 2.78 (1H, m, H-3′), 1.33 (3H, d, J=6.2Hz, 2-CH3), 1.28 (3H, d, J=6.2Hz, 2′-CH3), 1.13 (3H, d, J=5.9Hz, 3-CH3), 1.09 (3H, d, J=6.7Hz, 3′-CH3); 13C NMR (100MHz, DMSO-d6) δC: 202.5 (C, C-4), 200.4 (C, C-4′), 165.3 (C, C-5), 165.3 (C, C-5′), 162.1 (C, C-8), 160.2 (C, C-8′), 159.0 (C, C-6), 157.3 (C, C-6′), 155.4 (C, C-10a), 154.1 (C, C-10a′), 141.2 (C, C-9a), 140.1 (C, C-9a′), 104.5 (C, C-5a), 104.1 (C, C-5a′), 101.7 (C, C-9), 101.7 (C, C-4a), 101.4 (C, C-4a′), 101.3 (C, C-9′), 100.4 (CH, C-7), 99.2 (CH, C-7′), 98.9 (CH, C-10), 98.9 (CH, C-10′), 78.3 (CH, C-2), 75.3 (CH, C-2′), 45.8 (CH, C-3), 44.2 (CH, C-3′), 19.8 (2-CH3), 16.6 (2′-CH3), 10.3 (3-CH3), 10.0 (3′-CH3)。以上数据与文献(Ugaki et al, 2012)报道基本一致, 故鉴定该化合物为isochaetochromin B1。
化合物 8: 黄绿色固体, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$+135.8° (c 0.05, MeOH), HR-ESI-MS m/z 547.1592, [M+H]+ (计算值C30H27O10, 547.1604)。1H NMR (400MHz, DMSO-d6) δH: 15.20 (1H, s, 5-OH), 15.18 (1H, s, 5′-OH), 9.96 (1H, s, 6-OH), 9.63 (1H, s, 6′-OH)), 6.53 (1H, s, H-10′), 6.46 (1H, s, H-7), 6.34 (1H, s, H-7′), 5.78 (1H, s, H-10), 4.62 (1H, dt, J=13.0, 6.2Hz, H-2′), 4.14 (1H, dt, J=13.0, 6.2Hz, H-2), 2.81 (1H, d, J=17.6Hz, H-3), 2.71 (1H, m, H-3′), 1.39 (3H, d, J=6.2Hz, 2-CH3), 1.27 (3H, d, J=6.2Hz, 2′-CH3), 1.18 (3H, d, J=5.9Hz, 3-CH3), 1.07 (3H, d, J=6.7Hz, 3′-CH3); 13C NMR (100MHz, DMSO-d6) δC: 202.4 (C, C-4), 200.5 (C, C-4′), 165.6 (C, C-5), 165.0 (C, C-5′), 161.9 (C, C-8), 160.1 (C, C-8′), 158.9 (C, C-6), 157.3 (C, C-6′), 154.8 (C, C-10a), 154.6 (C, C-10a′), 141.6 (C, C-9a), 141.3 (C, C-9a′), 104.6 (C, C-5a), 104.1 (C, C-5a′), 101.7 (C, C-9), 101.3 (C, C-9′), 101.0 (C, C-4a), 101.0 (C, C-4a′), 100.4 (CH, C-7), 99.0 (CH, C-7′), 99.0 (CH, C-10), 98.9 (CH, C-10′), 77.9 (CH, C-2), 75.5 (CH, C-2′), 45.9 (CH, C-3), 44.0 (CH, C-3′), 19.8 (2-CH3), 16.6 (2′-CH3), 10.2 (3-CH3), 10.0 (3′-CH3)。以上数据与文献(Ugaki et al, 2012)报道基本一致, 故鉴定该化合物为isochaetochromin B2。
化合物7和8在结构解析过程中发现是一对阻转异构体, 为了确定其阻转异构手性中心, 我们对化合物7和8进行了CD测试(图3)。结果发现化合物7和8的CD曲线在265nm和295nm处的卡滕效应呈镜像关系, 这与文献报道基本一致(Ugaki et al, 2012; Xu et al, 2014), 因此我们确定了化合物7和8是一对轴手性中心完全对称的阻转异构体, 轴手性分别为aR和aS。
图3 化合物7和8的圆二色光谱

Fig. 3 CD Spectra of compounds 7

化合物 9: 白色固体, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$+17.6° (c 0.1, MeOH), HR-ESI-MS m/z 317.2094, [M+Na]+ (计算值C18H30O3Na, 317.2093)。1H NMR (500MHz, DMSO-d6) δH: 7.18 (1H, dd, J=15.7, 9.5Hz, H-9), 6.28 (1H, dd, J=15.3, 8.6Hz, H-7), 6.23 (1H, dd, J=15.3, 9.6Hz, H-8), 6.09 (1H, d, J=15.6Hz, H-10), 2.17 (4H, q, J=6.9, 5.7Hz, H-2, 6), 1.45~1.51 (4H, m, H-3, 15), 1.39 (2H, t, J=7.1Hz, H-14), 1.21~1.31 (12H, m, H-4, 5, 12, 13, 16, 17), 0.86 (3H, t, J=6.8Hz, H-18); 13C NMR (125MHz, DMSO-d6) δC: 200.2 (C, C-11), 174.6 (C, C-1), 145.4 (CH, C-7), 142.8 (CH, C-9), 129.0 (CH, C-8), 128.1 (CH, C-10), 33.7 (CH2, C-2), 32.4 (CH2, C-6), 30.8 (CH2, C-12), 28.8 (CH2, C-13), 28.6 (CH2, C-5), 28.5 (CH2, C-4), 28.4 (CH2, C-14), 27.9 (CH2, C-15), 24.5 (CH2, C-3), 23.8 (CH2, C-16), 21.9 (CH2, C-17), 13.9 (CH3, C-18)。以上数据与文献(Zhang et al, 2021)报道的基本一致, 故鉴定该化合物为(E)-7, 9-二烯-11-羰基硬脂酸。
化合物 10: 黄色油状, $\left[ \alpha \right]_{\text{D}}^{\text{25}}$+20.0° (c 0.07, MeOH), HR-ESI-MS m/z 275.1054, [M+Na]+ (计算值C17H16O2Na, 275.1048)。1H NMR (500MHz, DMSO-d6) δH: 7.37 (1H, m, H-4), 7.36 (1H, m, H-2′), 7.36 (1H, m, H-6′), 7.23 (1H, m, H-2), 7.23 (1H, m, H-6), 7.22 (1H, m, H-3′), 7.22 (1H, m, H-5′), 7.14 (1H, m, H-4′), 7.13 (1H, m, H-3), 7.13 (1H, m, H-5), 4.18 (2H, m, H-9), 3.77 (1H, d, J=11.0Hz, H-7′), 3.01 (1H, m, H-8), 2.79 (2H, dd, J=7.3, 5.2Hz, H-7); 13C NMR (125MHz, DMSO-d6) δC: 177.0 (C, C-8′), 138.3 (C, C-1), 136.6 (C, C-1′), 128.8 (C, C-2′), 128.8 (C, C-6′), 128.6 (C, C-2), 128.6 (C, C-3), 128.6 (C, C-5), 128.6 (C, C-6), 128.5 (C, C-3′), 128.5 (C, C-5′), 127.3 (C, C-4), 126.4 (C, C-4′), 70.8 (CH2, C-9), 51.5 (CH, C-7′), 45.1 (CH, C-8), 36.7 (CH2, C-7)。以上数据与文献(Wang et al, 2021)报道基本一致, 故鉴定该化合物为ergosta-4, 6, 8(14), 22-tetraen-3-one。

2.2 化合物抑菌活性筛选数据

采用滤纸片扩散法测定所有单体化合物对S. albus、S. aureus、S. epidermidis、MRSA和V. parahaemolyticus的抑菌活性, 结果显示化合物6对S. albus、S. aureus和S. epidermidis均有抑菌活性, 化合物9对S. albus和S. aureus有抑菌活性; 采用微量肉汤稀释法测试它们的MIC值, 结果显示化合物6对S. albus、S. aureus和S. epidermidis均有抑菌活性, 其MIC值分别为3.12、6.25和3.12μg·mL-1; 化合物9对S. albus和S. aureus有显著抑菌活性, 其MIC值分别为1.56和3.12μg·mL-1。阳性药氯霉素的MIC值均为0.39μg·mL-1。

3 结论

本文综合运用了色谱分离-波谱解析等技术, 从红树老鼠簕来源的曲霉属内生真菌A. terreus GXIMD 03158大米培养基发酵产物中共分离鉴定了10个化合物, 包括6个甾醇类化合物(化合物1~6), 2个蒽醌类化合物(化合物7和8)和2个其他类化合物(化合物9和10)。其中化合物7和8是一对阻转异构体, 通过CD测试最终确定了化合物7和8的阻转异构轴手性中心。对所有化合物进行抗菌活性评价, 结果显示化合物9对S. albus和S. aureus有显著抑菌活性, 其MIC值分别为1.56和3.12μg·mL-1, 化合物6对S. albus、S. aureus和S. epidermidis均有抑菌活性, 其MIC值分别为3.12、6.25和3.12μg·mL-1, 对比化合物1~6的结构及抑菌活性结果, 推测化合物6结构中五元内酯环可能增强了甾体类化合物的抑菌活性, 该部分结构可能是活性基团。

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