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Journal of Tropical Oceanography >

2025 , Vol. 44 >Issue 1: 154 - 161

DOI: https://doi.org/10.11978/2024038

Exploitation of Marine Resources

Studies on secondary metabolites of Sargassum fusiforme derived fungus Aspergillus sp. GXIMD 02045

  • KONG Weiqi ,
  • JIN Xin ,
  • LIU Yonghong ,
  • GAO Chenghai ,
  • CHEN Xianqiang
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  • Guangxi Key Laboratory of Marine Drugs, Institute of Marine Drugs, Guangxi University of Chinese Medicine, Nanning 530200, China
CHEN Xianqiang. email: email:

Copy editor: SUN Cuici

Received date: 2024-02-07

  Revised date: 2024-02-24

  Online published: 2024-02-29

Supported by

National Natural Science Foundation of China(U20A20101)

Guangxi Natural Science Foundation of Guangxi Province(2020GXNSFGA297002)

Special Fund for Bagui Scholars of Guangxi(05019055)

GuiPai Traditional Chinese Medicine Inheritance and Innovation Team Project of Guangxi University of Chinese Medicine(2022A007)

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Abstract

To explore the diversity of secondary metabolites of fungus Aspergillus sp. GXIMD 02045 from Sargassum fusiforme, the secondary metabolites were separated with various chromatographic techniques. Chemical structures were identified using mass spectroscopy, nuclear magnetic resonance spectroscopy, and optical rotation data with comparison of literature reported ones. 15 compounds were isolated from Aspergillus sp. GXIMD 02045, and identified as (3S)-6-hydroxy-8-methoxy-3, 5-dimethylisochroman (1), (3S)-6-hydroxy-8-methoxy-3-methylisochroman (2), (3S, 4R)-6-hydroxy-8-methoxy-3, 5-dimethylisochroman (3), (1S, 3R)-3, 7-dimethyl-1, 8-dihydroxy-6-methoxyisochroman (4), (S)-6-hydroxy-7-methoxy-3, 5-dimethylisochroman-1-one (5), anserinone A (6), anserinone B (7), (+)-formylanserinone B (8), 4-hydroxyphenylacetic acid (9), p-hydroxyphenylacetic acid methyl ester (10), Waol A (11), (S)-7-methoxy-2, 5-dimethyl-2, 3-dihydrobenzofuran-6-ol (12), 6-methylcurvulinic acid methyl ester (13), 4-hydroxy-3, 6-dimethyl-2-pyrone (14), trans-ferulic acid (15). Antibacterial activity of secondary metabolites from A. sp. GXIMD02045 was tested, which showed that compounds 6−8 displayed inhibitory activity against methicillin-resistant Staphylococcus aureus, and the minimum inhibitory concentration (MIC) values were ranging from 3.91 μg·mL-1 to 7.81 μg·mL-1. Compounds 6−8 and 11 showed inhibitory activity against Bacillus subtilis, and the MIC values were ranging from 7.81 μg·mL-1 to 15.62 μg·mL-1. Compounds 4, 6−8, 10 and 11 showed inhibitory activity against Pseudomona aeruginosa, and the MIC values were ranging from 1.95 μg·mL-1 to 125.00 μg·mL-1. This study provides a theoretical basis for the research on development and utilization of mangrove Sargassum fusiforme and its symbiotic fungus.

Key words： Sargassum fusiforme; Aspergillus fungus; secondary metabolites; antibacterial activity

Cite this article

KONG Weiqi , JIN Xin , LIU Yonghong , GAO Chenghai , CHEN Xianqiang . Studies on secondary metabolites of Sargassum fusiforme derived fungus Aspergillus sp. GXIMD 02045[J]. Journal of Tropical Oceanography, 2025 , 44(1) : 154 -161 . DOI: 10.11978/2024038

真菌作为海洋微生物的重要类群, 富含多种结构新颖、活性显著的次级代谢产物, 其中部分化合物结构为新型药物的研发提供了重要支撑 (Xu et al, 2018), 随着对海洋来源微生物样品采集能力的提高以及化合物分离纯化、结构解析技术的进步, 研究人员发掘出越来越多的新颖化学结构 (Li et al, 2015), 具有巨大的药物研究潜力 (Nicoletti et al, 2016)。
羊栖菜 (Sargassum fusiforme) 是一种马尾藻科马尾藻属海藻, 在我国南方沿海大量养殖 (何维彬 等, 2023)。海藻共附生真菌会参与宿主自身的生理活动, 通过具有独特生物活性的次级代谢产物来帮助宿主抵御外部侵害 (叶禹秀 等, 2022), 这些化合物的结构类型包括萜类、生物碱、多肽、聚酮和复杂脂肪酸酯等 (冯婷 等, 2024)。近二十年来, 基于羊栖菜来源曲霉属(Aspergillus)真菌次级代谢产物的报道, 主要涉及异色满类化合物和降二萜类化合物。 这些报道指出异色满类化合物对胃癌细胞(NUGC-3)、宫颈癌细胞(HeLa-S3)和人早幼粒白血病细胞(HL-60)表现出选择性细胞毒性, 降二萜类化合物对部分海洋浮游动物(Artemia salina)和海洋浮游植物(Chattonella marina、 Heterosigma akashiwo和Alexandrium sp.)具有生物毒性(Ogawa et al, 2004; Miao et al, 2014)。课题组此前从涠洲岛海边红树林采集的羊栖菜中分离得到一株真菌, 通过对其DNA的提取、Intertek Testing Services(ITS)序列的扩增和数据库 (GenBank database) 对比, 鉴定为曲霉属真菌, 命名为Aspergillus sp. GXIMD 02045。为探索该菌株次级代谢产物的多样性, 先利用单菌多次级代谢产物(one-strain-many-compounds, OSMAC)策略, 发现该菌株在大米培养基中发酵获得的代谢产物较为丰富, 所以选择大米培养基进行大规模发酵。采用各种色谱技术分离该菌株的次级代谢产物, 利用质谱、核磁共振波谱等技术鉴定代谢产物的结构, 从其发酵产物中获得化合物15个 (结构见图1), 主要为异色满类(1—5)、对苯醌类(6—8)、对羟基苯乙酸及其甲酯(9、10)、呋喃类(11)、香豆酮类(12)、邻羧基苯乙酸类(13)、吡喃酮类(14)和肉桂酸类(15)化合物。这些化合物此前均有文献报道发现, 但生物活性研究多以它们的细胞毒性为主, 文献中较少提及它们的抑菌活性。本实验先利用滤纸片琼脂法对所有化合物进行抑菌活性粗筛, 再使用改良肉汤稀释法测定粗筛结果有活性的化合物对耐甲氧西林金黄葡萄球菌 (methicillin-resistant Staphylococcus aureus)、枯草芽孢杆菌 (Bacillus subtilis)和铜绿假单胞菌 (Pseudomona aeruginosa)的抑菌活性, 确定化合物对上述致病菌的最小抑菌浓度(minimum inhibitory concentration, MIC)。结果显示多个化合物对三种致病菌都具有一定的抑菌活性。实验结果扩充了对海藻共附生真菌的次级代谢产物的认知, 为海藻共附生真菌来源的次级代谢产物在抑制细菌领域的药物开发利用提供一定的科学依据, 推进对海洋真菌天然产物的深度研发。
图1 化合物1—15的化学结构

Fig. 1 Chemical structures of compounds 1−15

1 材料与方法

1.1 仪器与主要试剂

Avance Ⅲ HD 500M超导核磁共振波谱仪(Bruker公司); Waters Xevo G2-S 型高分辨 ESI 质谱(Waters 公司); MCP 100/150旋光仪(安东帕公司); LC-2030C 3D Plus型高效液相色谱仪(日本岛津公司); Sepacore型中压制备色谱仪(瑞士Buchi公司); EYELAN-1300D型旋转蒸发仪(东京理化器械株式会社); SHZ-CB型循环水真空泵(巩义市予华仪器有限公司); KQ-250DB型超声仪(巩义市予华仪器有限公司); Victor Nivo多功能酶标仪(珀金埃尔默); 培养箱(上海精宏实验设备有限公司); 100 ~200 目硅胶、200 ~300 目硅胶和薄层硅胶板均购于烟台江友硅胶开发有限公司; 反相硅胶购于YMC公司; 葡聚糖凝胶Sephadex LH-20购于GE公司; 甲醇、二氯甲烷、氯仿、乙酸乙酯、二甲基亚砜(dimethyl sulfoxide, DMSO)等分析纯试剂均购于广东光华科技股份有限公司。

1.2 指示菌

耐甲氧西林金黄葡萄球菌(methicillin-resistant Staphylococcus aureus ATCC 43300)、枯草芽孢杆菌(Bacillus subtilis ATCC 6051)和铜绿假单胞菌(Pseudomona aeruginosa ATCC 10145), 菌种由广西中医药大学海洋药物研究院保藏。

1.3 实验所用培养基与菌株分离鉴定

菌种活化培养基为MB固体培养基(麦芽提取物15g、海盐30g、蒸馏水1L和15~20g琼脂), 不加琼脂则为液体培养基。微生物分离所用培养基包括MB固体培养基(额外添加氨苄西林和氯霉素各100mg)、察氏培养基和孟加拉红琼脂培养基。抑菌活性测试所用培养基为 LB 固体培养基 (胰蛋白胨10g、NaCl10g、酵母提取粉5g、琼脂13g、蒸馏水1L, 调整pH为 7.2~7.4), 不加琼脂为液体培养基。真菌扩大发酵所用培养基为大米培养基(每1L的锥形瓶加入大米110g、海盐2g和蒸馏水110mL, 在高压灭菌锅中121℃灭菌30min后冷却)。
菌株分离自广西涠洲岛来源的羊栖菜, 分离操作方法参考本团队前期报道 (夏辰曦 等, 2023), 记录菌落数及菌落的形态特征。取新鲜菌体, 液氮研磨, 遵照DNA提取试剂盒的说明书, 按操作要求来提取真菌基因组。鉴定方法参考本团队前期报道(罗志宏 等, 2023), 采用真菌通用引物ITS1 (5'-TCCGTAGGTGAACCTGCGG-3')和ITS4 (5-'TCCTCCGCTTATTGATATGC-3')进行 PCR 扩增。扩增样品经琼脂糖凝胶电泳验证后, 委托生工生物工程(上海)股份有限公司进行测序分析, 在GenBank数据库上提交测序结果, 与已知菌株进行BLAST序列相似性比对, 确定与其相似程度高的种属信息, 初步将其鉴定为曲霉属, ITS序列相似度为96.58%, 编号为GXIMD 02045。

1.4 方法

1.4.1 真菌的发酵以及发酵产物的提取分离

将保存于4℃的菌株接种到事前准备好的MB固体培养基, 置于28℃下培养5d活化。参照本团队之前的报道 (邢楠楠 等, 2023)取适量的孢子接种至MB液体培养基中, 在25℃, 180r·min-1条件下振荡培养4d, 即获得种子液。将种子液接种到大米固体培养基上, 每瓶接种10mL, 共100瓶, 在28℃下静置培养30d。发酵结束后, 捣碎大米, 将其浸泡在等体积乙酸乙酯中, 超声提取代谢产物重复3次, 合并萃取液。使用旋转蒸发仪减压浓缩得到乙酸乙酯部位浸膏250g, 浸膏经正相硅胶(200~300目)柱层析, 使用二氯甲烷−甲醇(100: 0~0: 100, 体积比)来进行梯度洗脱, 根据薄层色谱板的显示结果进行合并, 得到4个组分(Fr.1—Fr.4)。
Fr.1(56.8g)经中压制备ODS色谱柱分离, 甲醇-水(上海宸乔生物科技有限公司中压玻璃柱TX36310, 36mm×310mm, 30: 70~70: 30, 体积比)梯度洗脱得到Fr.1.1、Fr.1.2组分。Fr.1.1(12.8g)运用HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 20mm×250mm, 乙腈-水体积比45: 55, 流速为6mL·min−1, λ为210nm和254nm)得到化合物1 (tR=21.04min, 142.8mg)。Fr.1.2(10.3g)运用HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 20mm×250mm, 乙腈-水45: 55(体积比), 流速为6mL·min−1, λ为210nm和254nm)得到化合物2 (tR=18.96min, 83.2mg)。
Fr.2(52.2g)经中压制备ODS色谱柱分离, 甲醇-水(上海宸乔生物科技有限公司中压玻璃柱TX36310, 36mm×310mm, 30: 70~70: 30, 体积比)梯度洗脱得到组分Fr.2.1—Fr.2.3。Fr.2.1(4.3g)经正相硅胶(200目~300目)柱层析, 石油醚-乙酸乙酯(50: 50, 体积比)等度洗脱后再HPLC 制备(YMC COSMOSIL 5C18-MS-Ⅱ, 10mm×250mm, 乙腈-水体积比10: 90,流速为3mL·min−1, λ为210nm和272nm)得到化合物14 (tR=11.62min, 25.8mg)。Fr.2.2(3.2g)经凝胶柱纯化后得到组分Fr.2.2A、Fr.2.2B、Fr.2.2C和Fr.2.2D。Fr.2.2A(0.8g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 20mm× 250mm, 乙腈-水体积比 22: 78, 流速为6mL·min−1, λ为210nm和254nm)得到化合物3 (tR=14.54min, 92.8mg)。Fr.2.2B(1.2g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 10mm×250mm, 乙腈−水32: 68, 体积比, 流速为3mL·min−1, λ为200nm和230nm)得到化合物12 (tR= 19.20min, 14.5mg)。Fr.2.2C(0.5g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 10mm×250mm, 乙腈-水体积比20: 80,流速为3mL·min−1, λ为205nm和230nm)得到化合物6 (tR=15.48min, 14.3mg)。Fr.2.2D (0.4g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 20mm×250mm, 乙腈-水体积比22: 78, 流速为6mL·min−1, λ为205nm和254nm)得到化合物7 (tR=14.51min, 75.7mg)。Fr.2.3(10.5g)经正相硅胶(200~300目)柱层析, 石油醚-乙酸乙酯(20: 80~80: 20, 体积比)梯度洗脱得到组分Fr.2.3A和Fr.2.3B。Fr.2.3A(6.8g)经反相色谱柱分离, 甲醇-水体积比 (40: 60~85: 15)梯度洗脱得到组分Fr.2.3A1和Fr.2.3A2。Fr.2.3A1(3.2g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 20mm×250mm, 乙腈-水体积比33: 67, 流速为3mL·min−1, λ为210nm和254nm)得到化合物8 (tR= 19.65min, 8.7mg)。Fr.2.3A2(2.2g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 10mm×250mm, 乙腈-水体积比33: 67, 流速为3mL·min−1, λ为210nm和254nm)得到化合物4 (tR=15.27min, 27.2mg)。Fr.2.3B(3.2g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 20mm× 250mm, 乙腈-水体积比33: 67, 流速为6mL·min−1, λ为210nm和254nm)得到化合物5 (tR=16.49min, 70.5mg)。
Fr.3(56.5g)经中压制备ODS色谱柱分离, 甲醇-水(上海宸乔生物科技有限公司中压玻璃柱TX36310, 36mm×310mm, 20: 80~70: 30, 体积比)梯度洗脱得到组分Fr.3.1—Fr.3.2。Fr.3.1(20.6g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 10mm×250mm, 乙腈-水体积比13: 87, 流速为3mL·min-1, λ为210nm和254nm)得到化合物9 (tR=12.07min, 41.1mg)。Fr.3.2(18.2g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 10mm× 250mm, 乙腈-水体积比20: 80, 流速为3mL·min-1, λ为200nm和254nm)得到化合物10 (tR=16.52min, 11.4mg)。
Fr.4(52.3g)经中压制备ODS色谱柱分离, 甲醇-水(上海宸乔生物科技有限公司中压玻璃柱TX36310, 36mm×310mm, 20: 80~70: 30, 体积比)梯度洗脱得到组分Fr.4.1—Fr.4.2。Fr.4.1(17.9g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 10mm×250mm, 乙腈-水体积比13: 87, 流速为3mL·min-1, λ为200nm和230nm)得到化合物13 (tR=18.37min, 8.3mg)。Fr.4.2(2.9g)经凝胶柱纯化后得到组分Fr.4.2A和Fr.4.2B。Fr.4.2A(1.2g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 20mm×250mm, 乙腈-水体积比28: 72, 流速为6mL·min-1, λ为210nm和254nm)得到化合物11 (tR=20.08min, 28.0mg)。Fr.4.2B(1.4g)经过HPLC制备(YMC COSMOSIL 5C18-MS-Ⅱ, 20mm×250mm, 乙腈-水体积比28: 72, 流速为6mL·min-1, λ为210nm和254nm)得到化合物15 (tR=14.40min, 18.5mg)。

1.4.2 共附生真菌的次级代谢产物的抑菌活性测试

测定所有单体化合物对耐甲氧西林金黄葡萄球菌、枯草芽孢杆菌和铜绿假单胞菌的抑菌活性, 预先采用滤纸片琼脂扩散法对单体化合物(每片滤纸片含有125μg化合物)进行抑菌活性初步筛选, 针对有明显抑菌圈(抑菌圈直径> 8mm)的活性化合物, 采用改良肉汤稀释法测定分离得到化合物的MIC值, 将待测化合物配成浓度为5mg·mL-1的DMSO溶液, 阳性对照药选择环丙沙星, 配成浓度为1mg·mL-1的DMSO溶液, 操作方法参考本团队前期报道(夏辰曦 等, 2023), 配置好的96孔板放入37℃恒温培养箱培养24h, 观察试验结果, 以孔内澄清对应最小质量浓度为抑制致病菌的MIC值。

2 试验结果

2.1 结构鉴定

化合物1: 白色粉末, 分子式为C12H16O3, HRESIMS m/z 209.1188 [M+H]+ (calcd for C12H17O3, 209.1178), [α]20 D+40.2° (c 0.1, MeOH)。 1H NMR (500 MHz, DMSO-d6) δH: 6.30 (s, 1H, H-7), 4.64 (d, J = 14.8 Hz, 1H, H-1), 4.39 (d, J = 14.9 Hz, 1H, H-1), 3.65 (s, 3H, H-8-OCH3), 3.61 (m, 1H, H-3), 2.56 (dd, J = 16.6, 2.3 Hz, 1H, H-4), 2.24 (dd, J = 16.6, 10.7 Hz, 1H, H-4), 1.91 (s, 3H, H-10), 1.24 (d, J = 6.1 Hz, 3H, H-9);13C NMR (125 MHz, DMSO-d6) δC: 153.9 (C-6), 153.1 (C-8), 133.7 (C-10), 113.2 (C-9), 112.8 (C-5), 96.0 (C-7), 70.0 (C-3), 63.8 (C-1), 54.9 (8-OCH3), 33.6 (C-4), 21.7 (C-9), 10.1 (C-10)。以上数据与文献报道(Orfali et al, 2015)基本一致, 故鉴定为(3S)-6-羟基-8-甲氧基-3, 5-二甲基异色满。
化合物2: 白色粉末, 分子式为 C11H14O3, HRESIMS m/z 195.1017 [M+H]+ (calcd for C11H15O3, 195.1021), [α]20 D+43.1° (c 0.1, MeOH)。1H NMR (500 MHz, CD3OD) δH: 6.20 (d, J = 2.1 Hz, 1H, H-7), 6.11 (d, J = 2.0 Hz, 1H, H-5), 4.74 (d, J = 14.9 Hz, 1H, H-1), 4.46 (d, J = 14.9 Hz, 1H, H-1), 3.69 (s, 3H, H-8-OCH3), 3.64 (m, 1H, H-3), 2.50 (dd, J = 16.2, 3.9 Hz, 1H, H-4), 2.46 (dd, J = 8.3, 7.9 Hz, 1H, H-4), 1.24 (d, J = 6.2 Hz, 3H, H-11);13C NMR (125 MHz, CD3OD) δC: 157.8 (C-6), 157.6 (C-8), 136.3 (C-10), 114.8 (C-9), 107.6 (C-5), 97.2 (C-7), 71.8 (C-3), 65.5 (C-1), 55.4 (8-OCH3), 36.7 (C-4), 21.7 (C-11)。以上数据与文献报道(Orfali et al, 2015)基本一致, 故鉴定为(3S)-6-羟基-8-甲氧基-3-甲基异色满。
化合物3: 白色粉末, 分子式为C12H16O4, HRESIMS m/z 247.0940 [M+Na]+ (calcd for C12H16O4Na, 247.0946), [α]20 D+41.5° (c 0.1, MeOH)。1H NMR (500 MHz, CD3OD) δH: 6.37 (s, 1H, H-7), 4.63 (d, J = 15.2 Hz, 1H, H-1), 4.52 (d, J = 15.2 Hz, 1H, H-1), 4.33 (d, J = 5.2 Hz, 1H, H-4), 3.78 (qd, J = 6.5, 3.2 Hz, 1H, H-3), 3.71 (s, 3H, H-8-OCH3), 2.18 (s, 3H, H-12), 1.25 (d, J = 6.5 Hz, 3H, H-11);13C NMR (125 MHz, CD3OD) δC: 155.9 (C-6), 154.5 (C-8), 135.4 (C-10), 116.8 (C-5), 115.8 (C-9), 98.5 (C-7), 77.0 (C-3), 69.1 (C-4), 62.3 (C-1), 55.6 (8-OCH3), 17.2 (C-11), 11.2 (C-12)。以上数据与文献报道(Orfali et al, 2015)基本一致, 故鉴定为(3S, 4R)-4, 6-二羟基-8-甲氧基-3, 5-二甲基异色满。
化合物4: 白色粉末, 分子式为C12H16O4, HRESIMS m/z 207.1106 [M+H-H2O]+ (calcd for C12H15O3, 207.1099), [α]20 D−18.5° (c 0.1, MeOH)。1H NMR (500 MHz, CD3OD) δH: 6.31 (s, 1H, H-5), 5.46 (s, 1H, H-1), 4.27 (m, 1H, H-3), 3.73 (s, 3H, H-6-OCH3), 2.64 (dd, J = 16.8, 3.3 Hz, 1H, H-4), 2.25 (dd, J = 16.8, 11.5 Hz, 1H, H-4), 1.98 (s, 3H, H-13), 1.33 (d, J = 6.2 Hz, 3H, H-11);13C NMR (125 MHz, CD3OD) δC: 157.0 (C-8), 157.0 (C-6), 136.0 (C-9), 114.8 (C-10), 114.2 (C-7), 97.4 (C-5), 97.1 (C-1), 63.8 (C-3), 55.8 (6-OCH3), 34.6 (C-4), 21.7 (C-11), 10.2 (C-13)。以上数据与文献报道(Quang et al, 2018)基本一致, 故鉴定为(1S, 3R)-3, 7-二甲基-1, 8-二羟基-6-甲基异色满。
化合物5: 白色粉末, 分子式为C12H14O4, HRESIMS m/z 245.0882 [M+Na]+ (calcd for C12H14O4Na, 245.0892), [α]20 D+171.1° (c 0.1, MeOH)。 1H NMR (500 MHz, DMSO-d6) δH: 10.38 (s, 1H, 6-OH), 6.46 (s, 1H, H-8), 4.35 (dqd, J = 12.4, 6.2, 2.8 Hz, 1H, H-3), 3.71 (s, 3H, H-7-OCH3), 2.92 (dd, J = 16.5, 2.8 Hz, 1H, H-4), 2.53 (dd, J = 14.0, 9.0 Hz, 1H, H-4), 1.97 (s, 3H, H-10), 1.34 (d, J = 6.2 Hz, 3H, H-9);13C NMR (125 MHz, DMSO-d6) δC: 161.8 (C-1), 160.7 (C-6), 160.5 (C-7), 142.0 (C-5), 112.6 (C-10), 104.8 (C-9), 97.8 (C-8), 72.4 (C-3), 55.4 (7-OCH3), 32.8 (C-4), 20.5 (C-11), 10.6 (C-12)。以上数据与文献报道(Zhang et al, 2023)基本一致, 故鉴定为(S)-6-羟基-7-甲氧基-3, 5-二甲基异色满-1-酮。
化合物6: 黄色粉末, 分子式为C11H12O4, HRESIMS m/z 209.0730 [M+H]+ (calcd for C11H13O4, 209.0814)。1H NMR (500 MHz, CD3OD) δH: 6.03 (s, 1H, H-6), 3.81 (s, 3H, H-5-OCH3), 3.74 (d, J = 10.9 Hz, 2H, H-8), 2.26 (s, 3H, H-10), 1.95 (s, 3H, H-7);13C NMR (125 MHz, CD3OD) δC: 206.4 (C-9), 188.5 (C-1), 182.9 (C-4), 156.0 (C-5), 145.0 (C-2), 137.9 (C-3), 108.3 (C-6), 56.9 (5-OCH3), 41.6 (C-8), 30.0 (C-10), 12.6 (C-7)。以上数据与文献报道(Wang et al, 1997)基本一致, 故鉴定为anserinone A。
化合物7: 黄色粉末, 分子式为C11H14O4, HRESIMS m/z 211.0894 [M+H]+ (calcd for C11H15O4, 211.0970), [α]20 D+45.2° (c 0.1, MeOH)。 1H NMR (500 MHz, CDCl3) δH: 5.86 (d, J = 18.3 Hz, 1H, H-6), 4.02 (m, 1H, H-9), 3.75 (s, 3H, H-5-OCH3), 2.63 (d, J = 6.4 Hz, 2H, H-8), 2.03 (s, 3H, H-7), 1.28 (m, 3H, H-10);13C NMR (125 MHz, CDCl3) δC: 187.8 (C-1), 183.1 (C-4), 158.5 (C-5), 143.7 (C-2), 139.9 (C-3), 107.5 (C-6), 67.7 (C-9), 56.5 (5-OCH3), 36.2 (C-8), 24.1 (C-10), 13.1 (C-7)。以上数据与文献报道(Smetanina et al, 2017)基本一致, 故鉴定为anserinone B。
化合物8: 黄色粉末, 分子式为 C12H14O5, HRESIMS m/z 261.0850 [M+Na]+ (calcd for C12H14O5Na, 261.0841), [α]20 D+20.0°(c 0.1, MeOH)。 1H NMR (500 MHz, CD3OD) δH: 7.99 (s, 1H, H-12), 6.00 (s, 1H, H-6), 5.25 (m, 1H, H-9), 3.81 (s, 3H, H-5-OCH3), 2.85 (dd, J = 13.5, 4.9 Hz, 1H, H-8), 2.80 (dd, J = 13.5, 8.5 Hz, 1H, H-8), 2.07 (s, 3H, H-7), 1.32 (d, J = 6.3 Hz, 3H, H-10);13C NMR (125 MHz, CD3OD) δC: 188.7 (C-1), 183.2 (C-4), 162.4 (C-12), 160.0 (C-5), 144.7 (C-2), 139.6 (C-3), 108.2 (C-6), 70.8 (C-9), 56.8 (5-OCH3), 33.8 (C-8), 20.7 (C-10), 12.8 (C-7)。以上数据与文献报道(Smetanina et al, 2016)基本一致, 故鉴定为(+)-formylanserinone B。
化合物9: 白色粉末, 分子式为C8H8O3, HRESIMS m/z 165.0460 [M+Na]+ (calcd for C8H8O3Na, 165.0473)。1H NMR (500 MHz, CD3OD) δH: 7.13 (m, 2H, H-2, H-6), 6.79 (m, 2H, H-3, H-5), 3.48 (s, 2H, H-7); 13C NMR (125 MHz, CD3OD) δC: 176.3 (C-8), 157.3 (C-4), 131.3 (C-2, C-6), 126.8 (C-1), 116.2 (C-3, C-5), 41.1 (C-7). 以上数据与文献报道(王艳颖 等, 2011)基本一致, 故鉴定为对羟基苯乙酸。
化合物10: 透明油状物, 分子式为C9H10O3, HRESIMS m/z 165.1750 [M-H]− (calcd for C9H9O4, 165.1760)。1H NMR (500 MHz, CD3OD) δH: 7.07 (d, J = 8.5 Hz, 2H, H-3, H-5), 6.75 (m, 2H, H-2, H-6), 3.66 (s, 3H, H-9), 3.58 (m, 2H, H-7);13C NMR (125 MHz, CD3OD) δC: 174.6 (C-8), 157.6 (C-4), 131.3 (CH2, C-3, C-5), 126.3 (C-1), 116.2 (CH, C-2, C-6), 52.4 (CH3, C-9), 40.9 (CH2, C-7)。以上数据与文献报道(刘媛媛 等, 2022)基本一致, 故鉴定为对羟基苯乙酸甲酯。
化合物11: 黄色油状物, 分子式为C13H16O4, HRESIMS m/z 237.1050 [M+H]+ (calcd for C13H17O4, 237.1127), [α]20 D−156.1° (c 0.1, MeOH)。1H NMR (500 MHz, CD3OD) δH: 6.77 (dd, J = 3.8, 2.4 Hz, 1H, H-4), 6.02 (dq, J = 15.4, 6.5 Hz, 1H, H-1′), 5.85 (dq, J = 15.3, 6.5 Hz, 1H, H-2′), 5.71 (ddq, J = 15.4, 7.5, 1.6 Hz, 1H, H-2′′), 5.64 (ddq, J = 15.4, 7.5, 1.6 Hz, 1H, H-1′), 4.59 (dd, J = 7.7, 7.5 Hz, 1H, H-7a), 4.44 (m, 1H, H-7), 4.41 (ddd, J = 7.7, 2.4, 2.4 Hz, 1H, H-3), 4.05 (bd, 2H, H-2), 1.81 (d, J = 6.5 Hz, 3H, H-3′), 1.75 (d, J = 6.5 Hz, 3H, H-3′′);13C NMR (125 MHz, CD3OD) δC: 168.8 (C-5), 135.2 (C-4), 134.6 (C-2′′), 133.7 (C-2′), 130.8 (C-4a), 128.2 (C-1′), 127.8 (C-1′′), 84.7 (C-2), 81.3 (C-7), 79.4 (C-7a), 64.9 (C-3), 18.2 (C-3′′), 18.0 (C-3′)。以上数据与文献报道(Nozawa et al, 2000)基本一致, 故鉴定为Waol A。
化合物12: 白色粉末, 分子式为C11H14O3, HRESIMS m/z 195.0973 [M+H]+ (calcd for C11H15O3, 195.0976), [α]20 D−16.5° (c 0.1, MeOH)。1H NMR (500 MHz, CD3OD) δH: 6.28 (s, 1H, H-8), 4.89 (m, 1H, H-2), 3.74 (s, 3H, H-7-OCH3), 3.19 (dd, J = 15.3, 8.6 Hz, 1H, H-3), 2.67 (dd, J = 15.3, 7.7 Hz, 1H, H-3), 2.01 (s, 3H, H-11), 1.40 (d, J = 6.2 Hz, 3H, H-1);13C NMR (125 MHz, CD3OD) δC: 150.1 (C-6), 142.9 (C-7), 141.6 (C-9), 129.1 (C-5), 114.1 (C-4), 101.2 (C-8), 81.1 (C-2), 56.7 (7-OCH3), 37.9 (C-3), 22.0 (C-1), 12.2 (C-5)。以上数据与文献报道(Dramae et al, 2022)基本一致, 故鉴定为(S)-7-甲氧基-2, 5-二甲基-2, 3-二氢苯并呋喃-6-醇。
化合物13: 白色固体, 分子式为C12H14O5, HRESIMS m/z 237.0750 [M-H]- (calcd for C12H13O5, 237.0763)。1H NMR (500 MHz, CD3OD) δH: 6.26 (s, 1H, H-4), 3.77 (s, 2H, H-2), 3.67 (s, 3H, 1-OCH3), 2.56 (m, 3H, H-10), 2.04 (s, 3H, H-11);13C NMR (125 MHz, CD3OD) δC: 206.1 (C-9), 173.9 (C-1), 161.3 (C-7), 160.6 (C-5), 134.4 (C-3), 118.9 (C-8), 112.3 (C-4), 111.9 (C-6), 52.5 (1-OCH3), 41.2 (C-2), 32.2 (C-10), 8.2 (C-11)。以上数据与文献报道(Smetanina et al, 2017)基本一致, 故鉴定为6-甲基-2-乙酰基-3, 5-二羟基苯乙酸甲酯。
化合物14: 淡黄色粉末, 分子式为 C7H8O3, HRESIMS m/z 141.0548 [M+H]+ (calcd for C7H9O3, 141.0552)。1H NMR (500 MHz, CD3OD) δH: 6.01 (s, 1H, H-5), 2.21 (s, 3H, H-7), 1.86 (s, 3H, H-1);13C NMR (125 MHz, CD3OD) δC: 169.1 (C-2), 168.0 (C-4), 161.4 (C-6), 101.5 (C-5), 98.7 (C-3), 19.5 (C-7), 8.2 (C-1)。以上数据与文献报道(Smetanina et al, 2017)基本一致, 故鉴定为4-羟基-3, 6-二甲基-2-吡喃酮。
化合物15:白色粉末, 分子式为C10H10O4, HRESIMS m/z 193.0508 [M-H]- (calcd for C10H9O4, 193.0501)。1H NMR (500 MHz, CD3OD) δH: 7.59 (d, J = 15.9 Hz, 1H, H-7), 7.18 (d, J = 1.8 Hz, 1H, H-2), 7.06 (dd, J = 8.2, 1.9 Hz, 1H, H-6), 6.81 (d, J = 8.2 Hz, 1H, H-5), 6.32 (d, J = 15.9 Hz, 1H, H-8), 3.89 (s, 3H, 3-OCH3);13C NMR (125 MHz, CD3OD) δC: 171.2 (C-9), 150.5 (C-3), 149.4 (C-4), 146.8 (C-7), 127.8 (C-1), 124.0 (C-6), 116.4 (C-5), 116.1 (C-8), 111.6 (C-2), 56.4 (3-OCH3)。以上数据与文献报道(黄正 等, 2017)基本一致, 故鉴定为反式阿魏酸。

2.2 抗菌活性测试结果

抑菌结果发现化合物6—8对耐甲氧西林金黄葡萄球菌有抑制活性, 具体详细结果可见表1, 其MIC值分别为7.81、3.91和7.81μg·mL-1, 阳性药的MIC值为0.78μg·mL-1。化合物6—8和11对枯草芽孢杆菌有抑制活性, 其MIC值分别为15.62、15.62、7.81和7.81μg·mL-1, 阳性药的MIC值为0.05μg·mL-1。化合物4、6—8、10和11对铜绿假单胞菌有抑制活性, 其MIC值分别为62.50、3.91、3.91、3.91、125.00和1.95μg·mL-1, 阳性药的MIC值为0.10μg·mL-1。
表1 化合物对致病菌的最小抑菌浓度(MIC, 单位μg·mL-1)

Tab. 1 MIC of the compounds against pathogenic bacteria

化合物 MIC-耐甲氧西林金黄葡萄球菌 MIC-枯草芽孢杆菌 MIC-铜绿假单胞菌
4 - - 62.50
6 7.81 15.62 3.91
7 3.91 15.62 3.91
8 7.81 7.81 3.91
10 - - 125.00
11 - 7.81 1.95
阳性药(环丙沙星) 0.78 0.05 0.10

注: “-”表示未能测出抑菌效果

3 结果和讨论

本文实验从羊栖菜共附生真菌 Aspergillus sp. GXIMD 02045中分离得到15个化合物, 化合物1—15均首次从涠洲岛来源的羊栖菜共附生曲霉属真菌中报道。
化合物1—5的结构类似, 都是以苯并二氢异吡喃为母核的异色满类化合物, 母核中氧原子的间位和邻位碳均不连有羰基氧原子, 导致结构上与香豆素和色原酮类似但有差异。多年前有报道, 从羊栖菜来源的曲霉属真菌代谢物中发现这类化合物(Ogawa et al, 2004), 本研究首次报道化合物4对铜绿假单胞菌的抑菌活性。初步构效关系分析发现4的1位羟基有助于对铜绿假单胞菌的抑菌活性。化合物6—8的母核相同, 是一类能抑制真菌和细菌生长的对苯醌类化合物, 8为7的9位羟基被取代得到的甲酸酯, 据文献报道其对小鼠艾氏腹水癌细胞(Ehrlich ascite carcinoma)和人乳腺癌高转移细胞(MDA-MB435)有细胞毒性(Gautschi et al, 2004; Smetanina et al, 2016), 本研究首次报道该化合物对于耐甲氧西林金黄葡萄球菌、枯草芽孢杆菌和铜绿假单胞菌的抑菌活性。初步构效关系分析发现8的 9位羟基被取代可能导致对枯草芽孢杆菌的抑菌活性加强。化合物10为对羟基苯乙酸甲酯, 初步构效关系分析发现10的结构类似于防腐剂尼伯金酯类, 推测结构上的相似可能是其对铜绿假单胞菌具有一定抑菌活性的原因。化合物11为呋喃类化合物, 文献报道其具有细胞毒活性(El-Elimat et al, 2013), 对金黄色葡萄球菌无明显抑菌活性(Hammerschmidt et al, 2014), 本研究首次报道该化合物对枯草芽孢杆菌和铜绿假单胞菌的抑菌活性。该化合物的抑菌活性相关研究报道较少, 有待进一步实验探究其对多种致病菌的抑菌活性。
耐甲氧西林金黄葡萄球菌、枯草芽孢杆菌和铜绿假单胞菌是常见医源性感染菌, 通常与严重医院交叉感染发生有关。对这些致病菌具有抑菌效果的次级代谢产物在医药领域有一定的潜在应用价值。目前, 目前在羊栖菜来源的共附生曲霉属真菌次级代谢产物领域, 缺少对抑菌活性成分的研究(Ogawa et al, 2004; Miao et al, 2014), 本研究结果可为充分开发羊栖菜及其共附生真菌的次级代谢产物提供理论基础, 为涠洲岛来源的生物活性成分挖掘提供科学依据。

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