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Journal of Tropical Oceanography >

2024 , Vol. 43 >Issue 1: 154 - 166

DOI: https://doi.org/10.11978/2023045

Exploitation of Marine Resources

Study on the chemical constituents and pharmacological activity of the marine-derived fungus Aspergillus fumigatus DL-p0m-g2 in the Beibu Gulf

  • FENG Ting ,
  • SUN Jian ,
  • WANG Yufei ,
  • PAN Weibin ,
  • QIN Xucan ,
  • QIN Bingyun ,
  • ZHOU Liman ,
  • WANG Cong ,
  • WANG Pei ,
  • KONG Fandong
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  • School of Chemistry and Chemical Engineering, Key Laboratory of Chemistry and Engineering of Forest Products, State Ethnic Affairs Commission, Guangxi Key Laboratory of Chemistry and Engineering of Forest Products, Guangxi Collaborative Innovation Center for Chemistry and Engineering of Forest Products, Key Laboratory of Universities in Guangxi for Excavation and Development of Ancient Ethnomedicinal Recipes, Guangxi Minzu University, Nanning 530006, China
KONG Fandong. email:

Editor: SUN Cuici

Received date: 2023-04-08

  Revised date: 2023-04-18

  Online published: 2023-05-18

Supported by

National Natural Science Foundation of Guangxi Province(2021GXNSFBA075036)

Specific Research Project of Guangxi for Research Bases and Talents(AD22035018)

National Natural Science Foundation of China(82104034)

Xiangsi Lake Youth Innovation Team Project of Guangxi Minzu University(2021RSCXSHQN01)

2021 University-Level Scientific Research Projects of Guangxi Minzu University(2021 MDKJ003)

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Abstract

The chemical constituents and their bioactivities of Aspergillus fumigatus DL-p0m-g2 from the Beibu Gulf were studied in order to obtain bioactive compounds. The compounds were isolated and purified by reversed-phase ODS column chromatography, semi-preparative liquid chromatography and other chromatographic methods. Their structures were identified by physicochemical properties, mass spectrometry and nuclear magnetic resonance spectroscopy. The cytotoxic, antibacterial and cholesterol transporter NPC1L1 protein binding activities of the isolated compounds were evaluated. As a result, a total of 21 alkaloids and one steroid were isolated and identified as 6-methoxyspirotryprostatin B (1), spirotryprostatin A (2), fumitremorgin C (3), cyclotryprostatin A (4), fumitremorgin B (5), pseurotin A (6), azaspirofuran A (7), azaspirofuran B (8), cephalimysin C (9), cephalimysin B (10), fumiquinazoline C (11), fumiquinazoline B (12), fumiquinazoline A (13), fumiquinazoline D (14), fumiquinazoline F (15), tryprostatin B (16), verruculogen (17), chaetominine (18), bisdethiobis(methylthio)glitoxin (19), helvolic acid (20), 7-deacetylpyripyropene A (21), terezine D (22). Compound 6 showed moderate cytotoxic activity against human hepatoma cells (HepG2), human lung cancer cells (A549) and human rectal cancer cells (HCT116). Compounds 1, 3 and 20 showed antibacterial activity against Staphylococcus aureus. Compound 14 showed a good binding with NPC1L1 protein, indicating its potential in the development of lipid-lowering drugs.

Key words： Aspergillus fumigatus; chemical constituents; cytotoxic activity; anti-bacteria activity; NPC1L1 protein binding

Cite this article

FENG Ting , SUN Jian , WANG Yufei , PAN Weibin , QIN Xucan , QIN Bingyun , ZHOU Liman , WANG Cong , WANG Pei , KONG Fandong . Study on the chemical constituents and pharmacological activity of the marine-derived fungus Aspergillus fumigatus DL-p0m-g2 in the Beibu Gulf[J]. Journal of Tropical Oceanography, 2024 , 43(1) : 154 -166 . DOI: 10.11978/2023045

海洋是生命起源的地方, 占据地球表面积的70%, 有80%的生物栖息于此, 拥有着丰富多彩的生物资源。海洋微生物资源丰富, 优势独特, 高压、高盐、低光照的特殊环境使得生存于此的微生物能够产生更多有别于陆地微生物的结构新颖、活性独特的天然产物, 是新的活性次生代谢产物的主要来源(Liu et al, 2018; Wang et al, 2020)。尤其是海洋真菌, 因其代谢产物高于海洋放线菌且种类更为丰富, 也成为国内外近十几年来研究的焦点(罗明和 等, 2015)。近年来, 从海洋真菌中发现了很多活性次级代谢产物, 包括生物碱类、萜类、大环内酯类以及聚酮类等多种天然活性物质, 并且发现这些物质具有抗肿瘤、抗癌、抗病毒、抗菌等多种活性(Newman et al, 2020)。自Cephalosporin C从海洋真菌中分离出来后, 越来越多的活性代谢产物被分离鉴定(张金新 等, 2023)。我国南海海域广阔, 生物种类资源繁多, 生态系统类型多样。全世界发现的超过3.5万种海洋天然产物中有20%来自中国南海(王庆琳 等, 2020)。Ye等(2018)从南海软珊瑚Sarcophyton infundibuliforme中分离得到两个新的骨架为三环[6.3.1.01,5]十二烷含氮二萜化合物sarinfacetamides A和B, sarinfacetamides A对ConA诱导的T淋巴细胞增殖具有明显的促进作用。Zhang等(2021)从海绵岩藻分离的Nocardiopsis dassonvillei SCSIO 40065中得到两个多环硫代生物碱dassonmycins A和B, 两种化合物均表现出中等的抗菌和细胞毒性活性。Yao等(2021)从南海来源的曲霉属真菌中分离得到三个新的环戊烯酮衍生物, 和五个新的环己烯酮衍生物。Cai等(2019)从红树林内生真菌Aspergillus sp. SK-28中分离得到具有6/5/4/5/6五环骨架的吲哚二酮哌嗪生物碱二聚体(+)-Asperginulin A对藤壶Balanus reticulatus具有防污活性。Jiao等(2020)从中国南海永兴岛分离的Aspergillus flavipes 164013菌株中得到三种氯化PKS-NRPS杂合代谢物flavipesides A-C均具有有效的胰脂肪酶抑制活性, IC50值(half maximal inhibitory concentration)为0.07~0.23μmol·L-1,。
北部湾位于我国南海西北部海域, 拥有丰富的海洋生物资源。至2020年, 从北部湾发现197种海洋真菌(徐新亚 等, 2020); 对北部湾的477种新报道的海洋天然产物进行来源归属发现微生物来源占52%, 其中从真菌中分离的海洋天然产物占微生物的91%(Wang et al, 2023)。从北部湾海洋真菌中分离得到的生物碱、萜类等化合物都表现出一定的抗肿瘤、抑菌等生物活性, 具有被应用开发的强大潜在价值(徐新亚 等, 2020)。Wang等(2021)从北部湾分离得到的珊瑚共生真菌Cladosporium halotolerans GXIMD 02502中发现Coniochaetone K对在10μmol·L-1的浓度下显示出显著的细胞毒性活性。Lin等(2023)从Trichoderma sp. GXIMD 01001中分离得到的7个新的18-residue peptaibols均显示出显著的细胞毒性, 对四种人类癌细胞系的最低IC50值为0.46~4.7μmol·L-1。高谕康等(2020)从一株海洋曲霉Aspergillus fumigatus MDCW-15的次级代谢产物中分离出一种新的烟曲霉酸对白色念珠菌有抑制活性, 最小抑菌浓度(minimal inhibition concentration, MIC)为32.0μg·mL-1。本次在对我国北部湾来源真菌Aspergillus fumigatus DL-p0m-g2的活性次生代谢产物的研究中, 获得了一系列生物碱类次生代谢产物(图1), 部分代谢产物对多种肿瘤细胞和金黄色葡萄球菌具有抑制活性, 且发现一个化合物与胆固醇转运蛋白NPC1L1具有良好的结合活性。
图1 化合物1—22的化学结构

Fig. 1 The chemical structures of compounds 1—22

1 仪器与材料

1.1 仪器与试剂

HCB-1300V洁净工作台(青岛海尔特种电器有限公司)、LRH-500A型生化培养箱(广东泰宏君科学仪器股份有限公司)、YXQ-LS-75SII型高压灭菌锅(上海博迅实业有限公司医疗设备厂)、CR-080R型春霖超声波清洗机(深圳市春霖清洗设备有限公司); Agilent 1260高效液相色谱仪(美国 Agilent 公司)、AL104型分析天平(瑞士梅特勒公司)、Brucker AVANCE 400MHz核磁共振仪(德国Brucker公司)、Agilent InfinityLab LC/MSD质谱仪(Agilent公司)、赛默飞高分辨质谱仪, 日立分析高效液相色谱, 兰博series Ⅲ制备型高效液相色谱, Nicolet is 10傅里叶变换红外光谱仪(美国赛默飞世尔科技公司), Agilent Cary60紫外-可见分光光度计(美国Agilent Technology公司)。分析高效液相色谱柱为5C18-MS-Ⅱ (C18, 250mm×4.6mm, 5μm, Nacalai tesque, 日本), 5PFP (250mm×4.6mm, 5μm, Nacalai tesque, 日本); 制备型高效液相色谱柱5C18-MS-Ⅱ (C18, 250mm×10mm, 5μm, Nacalai tesque, 日本), 5PFP (250mm×10mm, 5μm, Nacalai tesque, 日本), 反相硅胶YMC*GEL ODS-A-HG (日本YMC Group)。提取分离用乙酸乙酯、甲醇、二氯甲烷等均为工业用化学纯产品。

1.2 菌株来源与发酵培养

海洋菌株分离于北海钉螺(Oncomelania hupensis), 经对比生长形态与基因序列, 此菌株鉴定为Aspergillus fumigatus, 其系统发育树见图2。
图2 菌株Aspergillus fumigatus DL-p0m-g2的系统发育树

Fig. 2 Phylogenetic tree of strain Aspergillus fumigatus DL-p0m-g2

实验使用培养基包括真菌二号培养基: 葡萄糖1%, 甘露醇2%, 酵母膏0.3%, 麦芽糖2%, 味精1%, 磷酸二氢钾0.05%, 海水素0.33‰; 马铃薯土豆培养基: 马铃薯20.0%, 葡萄糖2%, 琼脂2.0%, 海水素0.33‰。
将保存于斜面中的菌株接种于马铃薯培养基, 置于28℃培养3d。挑取长有菌株的培养基于真菌二号培养基中, 室温培养30d。

1.3 化合物的提取与分离

对菌株Aspergillus fumigatus DL-p0m-g2进行真菌2号培养基发酵(33L, 室温, 30d), 用乙酸乙酯 (EtOAc)对发酵物浸提3次, 过滤和减压浓缩后得到20.84g粗提物(图3)。采用石油醚、90%甲醇-水对粗提物进行萃取, 得到石油醚层萃取物7.76g, 甲醇层萃取物14.49g。取甲醇萃取部位, 经反相ODS色谱柱, 依次用20%、 40%、 60%、 80%、 100%甲醇-水梯度洗脱, 每个梯度分3次收集。得Fr.1.1~ Fr.5.3共15个组分。Fr.3.2 (3.6g)经中压制备液相得Fr.3.2.1~ Fr.3.2.12共12个组分。Fr.3.2.5 (346.5mg)经半制备型液相色谱(HPLC) [50%甲醇-水] 纯化得到化合物1 (5.5mg, tR=34.2min)、化合物2 (2.0mg, tR=25.6min)和化合物18 (5.1mg, tR=28.4min)。Fr.3.2.7 (376.9mg)经半制备型液相色谱 [45%甲醇-水] 纯化得到化合物6 (17.3mg, tR=18.5min)与化合物19 (47.8mg, tR=25.2min)。Fr.4.1 (654.9mg)经半制备型液相色谱 [55%乙腈-水] 纯化得到化合物3 (17.7mg, tR = 10.7min)、化合物7(10.6mg, tR=18.7min) 和化合物8 (2.0mg, tR=19.5min)。Fr.4.3 (600.9mg)经半制备型液相色谱 [55%乙腈-水] 纯化得到化合物5 (3.3mg, tR=29.7min)、化合物17 (6.8mg, tR=18.8min)和化合物20 (3.7mg, tR=27.8min)。Fr.3.3 (1111.9mg)经反相ODS色谱柱, 依次用40%, 50%, 60%, 70%, 100%甲醇-水梯度洗脱, 得Fr.3.3.1~ Fr. 3.3.9共9个组分。Fr.3.3.6 (78.9mg)经半制备型液相色谱 [45%乙腈-水] 纯化得到化合物11 (3mg, tR=11.8min)和化合物14 (9.1mg, tR=15.9min)。Fr.3.3.5 (347.7mg)经半制备型液相色谱 [60%甲醇-水] 得到组分Fr.3.3.5.1~ Fr.3.3.5.5。Fr.3.3.5.3 (70mg) 经半制备型液相色谱 [40%乙腈-水] 纯化得到化合物9 (5.2mg, tR=24.1min) 、化合物10 (7mg, tR=26.0min)和化合物21 (4.4mg, tR=9.5min)。Fr.3.3.5.4 (95.6mg) 经半制备型液相色谱 [46%乙腈-水] 纯化得到组分Fr.3.3.5.4.2、化合物4 (5.7mg, tR=18.6min)、化合物12 (3.5mg, tR=11.7min)、化合物15 (1.7mg, tR=16.4min) 和化合物16 (1.9mg, tR=20.5min)。组分Fr.3.3.5.4.2 (6.0mg) 经半制备型液相色谱 [55%甲醇-水] 纯化得到化合物13 (2.5mg, tR=19.8min)和化合物22 (1.7mg, tR=18.7min)。
图3 菌株Aspergillus fumigatus DL-p0m-g2化合物1—22分离流程图

Fig. 3 The flowing chart of compounds 1—22

1.4 活性试验

1.4.1 细胞增殖抑制试验

实验参照CCK-8 (cell counting kit-8)法(王聪 等, 2017), 样品用DMSO (dimethyl sulfoxide)溶解, 母液浓度为20mmol·L-1。取对数生长期的肿瘤细胞, 稀释成8×103ind.·mL-1的细胞悬液, 96 孔板中每孔接种 100μL后于二氧化碳培养箱中培养24h, 去除原有培养基并加入100μL化合物稀释液后, 培养箱中培养48h, 再次去除培养基, 加入含有10% CCK-8增强型溶液的培养基培养4h, 在 450nm 处检测其吸光度并测量其抑制率。若化合物在50μmol·L-1浓度时, 抑制率大于 50%, 则说明该化合物对肿瘤细胞有较好抑制活性, 将对其进行半抑制浓度(IC50)测定。实验过程中, 每个测量浓度均设 3 个平行。

1.4.2 最小抑菌浓度测定

实验使用二倍稀释法(吴萌萌 等, 2021), 用96孔板对22个化合物进行细菌活性试验研究, 在96 孔板中加入100μL金黄色葡萄球菌液, 药物浓度为0.5μg·mL-1~256μg·mL-1。同时设置阳性药组, 阴性对照组, 每个药物浓度设置 3 个复孔。在 18h 后观察记录细菌生长情况, 测定最小抑菌浓度。

1.4.3 硫氧还蛋白还原酶活性测试实验

实验参照硫氧还蛋白还原酶活性检测试剂盒说明书, 样品用DMSO溶解, 母液浓度为20mmol·L-1。将样品与试剂混合配置成500μL待测样品, 先在96孔板中加入200μL待测样品在405nm处测定10s时吸光度; 然后将200μL待测样品放入37℃水浴5min后混匀测定10s吸光度。阳性药为姜黄素, 实验过程设置3个复孔。

1.4.4 NPC1L1蛋白活性测试实验

实验方法参考文献(Zhang et al, 2021)完成, 使用Biacore.T200 T200(Cytia)进行表面等离子共振(surface plasmon resonance, SPR)实验。用Amine Coupling Kit将NPC1L1蛋白偶联到CM5芯片表面, 首先将CM5芯片按照标准仪器程序进行安装和启动后, 将N-羟基琥珀酰亚胺和1-乙基（3-二甲基氨基丙基）碳酰氯)以1:1的比例混合并运行混合物, 总体积为200µL, 流速为10µL·min-1, 时间为600s。然后将NPC1L1蛋白在pH值为4.6的醋酸钠溶液中稀释至20µg·mL-1, 蛋白溶液总体积800µL, 流速10µL·min-1, 时间为3000s。最后, 乙醇胺溶液总体积200µL, 流速10µL·min-1, 时间为420s。然后, 使用含1% DMSO的磷酸盐缓冲液(phosphate buffer solution, PBS, pH 7.4)对CM5芯片进行预处理。用含DMSO的PBS-P将化合物稀释至0.1µmol·L-1, 确保DMSO终浓度为1%, 流速30µL·min-1, 结合时间60s, 解离时间120s。最终结果采用Biacore.T200进行分析。

2 实验结果

2.1 结构鉴定

化合物1: 淡黄色粉末状物, 分子式为C22H23N3O4, HRESIMS m/z 394.1761 [M + H]+。1H-NMR (400 MHz, CD3OD) δ: 7.02 (1H, d, J = 8.3 Hz, H-4), 6.56 (1H, dd, J = 2.3, 8.3 Hz, H-5), 6.50 (1H, d, J = 2.3 Hz, H-7), 5.74 (1H, s, H-8), 5.28 (1H, overlap, H-19), 5.28 (1H, overlap, H-18), 4.45 (1H, dd, J = 6.2, 11.3 Hz, H-12), 3.79 (3H, s, OMe-6), 3.79 (1H, m, H-15), 3.75 (1H, m, H-15), 1.97, 2.41 (2H, m, H-13), 1.97, 2.11 (2H, m, H-14), 1.59 (3H, s, H-21), 1.27 (3H, s, H-22); 13C-NMR (100 MHz, CD3OD) δ: 181.1 (C-2), 164.9 (C-11), 162.4 (C-6), 156.9 (C-17), 144.2 (C-7a), 139.0 (C-20), 138.9 (C-9), 129.5 (C-4), 122.2 (C-19), 120.3 (C-3a), 118.6 (C-8), 108.1 (C-5), 98.2 (C-7), 65.3 (C-18), 63.0 (C-12), 62.9 (C-3), 55.9 (OMe-6), 46.0 (C-15), 30.0 (C-13), 25.3 (C-14), 22.9 (C-21), 18.3 (C-22)。以上数据与文献(王亚楠 等, 2019)报道对照基本一致, 化合物1鉴定为6-methoxyspirotryprostatin B。
化合物2: 淡黄色油状物, 分子式为C22H25N3O4, HRESIMS m/z 418.1736 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 7.02 (1H, d, J = 8.4 Hz, H-4), 6.53 (1H, dd, J = 2.4, 8.4 Hz, H-5), 6.48 (1H, d, J = 2.4 Hz, H-7), 5.08 (1H, m, H-19), 5.02 (1H, m, H-9), 4.71 (1H, d, J = 9.2 Hz, H-18), 4.43 (1H, t, J = 7.8 Hz, H-12), 3.78 (3H, s, OMe-6), 3.55 (2H, m, H-15), 2.58 (1H, dd, J = 10.7, 13.3 Hz, H-8), 2.34 (1H, dd, J = 7.2, 13.3 Hz, H-8), 2.32 (1H, m, H-13), 2.11 (1H, m, H-13), 1.65 (3H, d, J = 0.9 Hz, H-22), 1.18 (3H, d, J = 1.1 Hz, H-21); 13C-NMR (100 MHz, CD3OD) δ: 183.2 (C-2), 169.4 (C-11), 168.9 (C-17), 162.2 (C-6), 144.4 (C-7a), 139.1 (C-20), 128.2 (C-4), 122.7 (C-19), 120.1 (C-3a), 107.7 (C-5), 97.7 (C-7), 62.3 (C-9), 61.8 (C-12), 59.8 (C-18), 57.1 (C-3), 55.9 (OMe-6), 46.2 (C-15), 35.3 (C-8), 28.5 (C-13), 25.6 (C-22), 24.5 (C-14), 18.1 (C-21)。以上数据与文献(陈雷 等, 2017)报道对照基本一致, 化合物2鉴定为spirotryprostatin A。
化合物3: 白色粉末状物, 分子式为C22H25N3O3, HRESIMS m/z 402.1794 [M + Na]+。1H-NMR (400 MHz, CDCl3) δ: 7.94 (1H, s, H-1), 7.43 (1H, d, J = 8.6 Hz, H-16), 6.85 (1H, d, J = 1.9 Hz, H-19), 6.81 (1H, dd, J = 2.1, 8.6 Hz, H-17), 5.98 (1H, d, J = 9.6 Hz, H-3), 4.90 (1H, d, J = 9.5 Hz, H-21), 4.17 (1H, dd, J = 4.8, 11.4 Hz, H-12), 4.10 (1H, dd, J = 8.3, 8.3 Hz, H-6), 3.82 (3H, s, OMe-18), 3.63 (2H, overlap, H-9), 3.51 (1H, dd, J = 5.1, 16.0 Hz, H-13a), 3.09 (1H, dd, J = 11.7, 15.9 Hz, H-13b), 2.40 (1H, m, H-7a), 2.23 (1H, m, H-7b), 2.05 (1H, m, H-8a), 1.98 (3H, s, H-24), 1.91 (1H, m, H-8b), 1.64 (3H, s, H-23); 13C-NMR (100 MHz, CDCl3) δ: 169.6 (C-5), 165.9 (C-11), 156.6 (C-18), 137.1 (C-20), 134.1 (C-22), 132.3 (C-2), 124.3 (C-21), 120.8 (C-15), 119.0 (C-16), 109.6 (C-17), 106.3 (C-14), 95.4 (C-19), 59.3 (C-6), 56.9 (C-12), 55.9 (OMe-18), 51.1 (C-3), 45.5 (C-9), 28.7 (C-7), 25.8 (C-23), 23.2 (C-8), 22.1 (C-13), 18.2 (C-24)。以上数据与文献(任虹 等, 2011)报道对照基本一致, 化合物3鉴定为fumitremorgin C。
化合物4: 白色粉末状物, 分子式为C22H25N3O5, HRESIMS m/z 434.1695 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 7.73 (1H, d, J = 8.7 Hz, H-16), 6.86 (1H, d, J = 2.2 Hz, H-19), 6.66 (1H, dd, J = 2.3, 8.7 Hz, H-17), 5.93 (1H, d, J = 9.6 Hz, H-3), 5.67 (1H, s, H-13), 4.78 (1H, dt, J = 1.2, 9.6 Hz, H-21), 4.49 (1H, d, J = 9.6 Hz, H-21), 3.80 (3H, s, OMe-18), 3.59 (2H, overlap, H-9), 2.00 (3H, d, J = 1.0 Hz, H-24), 1.65 (1H, d, J = 1.0 Hz, H-23); 13C-NMR (100 MHz, CD3OD) δ: 173.2 (C-11), 168.1 (C-5), 157.5 (C-18), 139.5 (C-20), 135.7 (C-22), 131.2 (C-2), 124.9 (C-21), 122.2 (C-15), 121.9 (C-16), 110.0 (C-17), 106.6 (C-14), 95.7 (C-19), 84.9 (C-12), 69.7 (C-13), 60.1 (C-6), 55.9 (OMe-18), 51.4 (C-3), 46.5 (C-9), 30.2 (C-7), 25.9 (C-23), 23.5 (C-8), 18.4 (C-24)。以上数据与文献(赵欢 等, 2018)报道对照基本一致, 化合物4鉴定为cyclotryprostatin A。
化合物5: 白色粉末状物, 分子式为C27H33N3O5, HRESIMS m/z 502.2311 [M + Na]+。1H-NMR (400 MHz, CDCl3) δ: 7.84 (1H, d, J = 8.7 Hz, H-16), 6.80 (1H, dd, J = 2.3, 8.7 Hz, H-17), 6.69 (1H, d, J = 2.3 Hz, H-19), 5.97 (1H, d, J = 10.0 Hz, H-26), 5.77 (1H, s, H-13), 5.03 (1H, t, J = 5.2 Hz, H-22), 4.70 (1H, m, H-3), 4.54 (2H, d, J = 5.6 Hz, H-21), 4.45 (1H, d, J = 7.0, 9.8 Hz, H-6), 3.84 (3H, s, OMe-18), 3.64 (2H, overlap, H-9), 2.47 (1H, m, H-7a), 2.09 (2H, m, H-8), 1.99 (3H, d, J = 1.0 Hz, H-29), 1.95 (1H, m, H-7b), 1.85 (3H, s, H-24), 1.70 (3H, d, J = 0.9 Hz, H-25), 1.63 (3H, d, J = 1.1 Hz, H-28); 13C-NMR (100 MHz, CDCl3) δ: 170.6 (C-5), 166.4 (C-11), 156.4 (C-18), 138.1 (C-20), 135.4 (C-27), 134.8 (C-23), 131.3 (C-2), 123.1 (C-26), 121.5 (C-16), 120.7 (C-15), 120.4 (C-22), 109.5 (C-17), 104.5 (C-14), 94.0 (C-19), 83.1 (C-12), 69.1 (C-13), 58.9 (C-6), 55.9 (OMe-18), 49.2 (C-3), 45.4 (C-9), 41.9 (C-21), 29.1 (C-7), 25.9 (C-25), 25.7 (C-29), 22.8 (C-8), 18.5 (C-24), 18.4 (C-28)。以上数据与文献(任虹 等, 2011)报道对照基本一致, 化合物5鉴定为fumitremorgin B。
化合物6: 白色晶状结晶, 分子式为C22H25NO8, HRESIMS m/z 454.1472 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 8.35 (2H, d, J = 8.5 Hz, H-19, H-23), 7.65 (1H, t, J = 7.4 Hz, H-21), 7.50 (2H, t, J = 7.9 Hz, H-20, H-22), 5.62 (1H, dt, J = 11.1, 7.4 Hz, H-13), 5.46 (1H, dd, J = 11.1, 9.0 Hz, H-12), 4.67 (1H, dd, J = 8.9, 7.0 Hz, H-10), 4.60 (1H, brs, H-7), 4.54 (1H, s, H-9), 4.50 (1H, d, J = 6.6 Hz, H-11), 3.34 (3H, s, OMe-8), 2.14 (2H, m, H-14), 1.76 (3H, s, H-16), 0.98 (3H, t, J = 7.52 Hz, H-15); 13C-NMR (100 MHz, CD3OD) δ: 199.2 (C-4), 197.1 (C-17), 188.7 (C-6), 169.2 (C-2), 137.3 (C-13), 135.1 (C-21), 134.9 (C-18), 131.7 (C-19, 23), 129.5 (C-20, 22), 128.8 (C-12), 114.4 (C-3), 93.9 (C-5), 93.6 (C-8), 76.3 (C-9), 72.9 (C-10), 69.5 (C-11), 52.5 (OMe-8), 22.2 (C-14), 14.5 (C-15), 5.8 (C-16)。以上数据与文献(陈雷 等, 2017)报道对照基本一致, 化合物6鉴定为pseurotin A。
化合物7: 黄色油状物, 分子式为C22H21NO7, HRESIMS m/z 434.1212 [M + Na]+。1H-NMR (400 MHz, CDCl3) δ: 8.32 (2H, d, J = 7.9 Hz, H-19, H-23), 7.61 (2H, dd, J = 7.3, 14.7 Hz, 7-NH, H-21), 7.48 (2H, t, J = 7.8 Hz, H-20, H-22), 7.03 (1H, d, J = 3.4 Hz, H-11), 6.22 (1H, d, J = 3.4 Hz, H-12), 4.69 (1H, d, J = 12.0 Hz, H-9), 4.19 (1H, d, J = 8.6 Hz, OH-9), 3.40 (3H, s, OMe-8), 2.74 (2H, q, J = 7.6 Hz, H-14), 2.00 (3H, s, H-16), 1.27 (3H, d, J = 7.6 Hz, H-15); 13C-NMR (100 MHz, CDCl3) δ: 195.8 (C-4), 194.8 (C-17), 172.7 (C-2), 166.4 (C-6), 163.9 (C-13), 143.5 (C-10), 134.6 (C-21), 132.6 (C-18), 130.7 (C-19, 23), 128.8 (C-20, 22), 118.4 (C-11), 108.1 (C-3), 107.9 (C-12), 91.7 (C-5), 89.9 (C-8), 74.4 (C-9), 51.8 (OMe-8), 21.9 (C-14), 11.9 (C-15), 6.3 (C-16)。以上数据与文献(Ren et al, 2010)报道对照基本一致, 化合物7鉴定为azaspirofuran A。
化合物8: 黄色油状物, 分子式为C21H19NO7, HRESIMS m/z 420.1054 [M + Na]+。1H-NMR (400 MHz, CDCl3) δ: 8.29 (2H, qd, J = 1.3, 9.8 Hz, H-19, H-23), 7.63 (1H, t, J = 7.3 Hz, H-21), 7.50 (2H, t, J = 7.7, 8.0 Hz, H-20, H-22), 7.20 (1H, d, J = 3.6 Hz, H-11), 7.09 (1H, s, NH-7), 6.30 (1H, d, J = 3.6 Hz, H-12), 5.01 (1H, s, H-9), 2.79 (1H, q, J = 7.6 Hz, OMe-8), 2.05 (3H, s, H-16), 1.30 (3H, t, J = 7.6 Hz, H-15); 13C-NMR (100 MHz, CDCl3) δ: 197.8 (C-4), 192.4 (C-17), 174.8 (C-2), 165.1 (C-13), 164.5 (C-6), 143.3 (C-10), 134.4 (C-21), 133.2 (C-18), 130.7 (C-19, C-23), 128.9 (C-20, C-22), 120.2 (C-11), 108.4 (C-12), 108.0 (C-3), 93.2 (C-5), 87.8 (C-8), 72.7 (C-9), 51.8, (OMe-8), 22.0 (C-14), 11.9 (C-15), 6.4 (C-16)。以上数据与文献(Ren et al, 2010)报道对照基本一致, 化合物8鉴定为azaspirofuran B。
化合物9: 黄色油状物, 分子式为C22H21NO7, HRESIMS m/z 434.1214 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 8.26 (2H, d, J = 8.0 Hz, H-19, H-23), 7.61 (1H, t, J = 7.41 Hz, H-21), 7.48 (2H, t, J = 7.8 Hz, H-20, H-22), 7.01 (1H, d, J = 3.6 Hz, H-11), 6.17 (1H, d, J = 3.5 Hz, H-12), 4.66 (1H, d, J = 4.2 Hz, H-9), 3.35 (3H, s, OMe-8), 3.15 (1H, d, J = 4.5 Hz, 9-OH), 2.74 (2H, q, J = 7.6 Hz, H-14), 2.02 (3H, s, H-16), 1.27 (3H, t, J = 7.6 Hz, H-15); 13C-NMR (100 MHz, CD3OD) δ: 196.0 (C-4), 193.6 (C-17), 170.3 (C-2), 168.3 (C-6), 163.9 (C-13), 143.3 (C-10), 134.2 (C-21), 134.0 (C-18), 129.9 (C-19, 23), 128.8 (C-20, 22), 118.1 (C-11), 108.2 (C-3), 107.8 (C-12), 95.2 (C-8), 88.0 (C-5), 76.9 (C-9), 51.6 (OMe-8), 21.9 (C-14), 11.9 (C-15), 6.3 (C-16)。以上数据与文献 (Yamada et al, 2010)报道对照基本一致, 化合物9鉴定为cephalimysin B。
化合物10: 黄色油状物, 分子式为C22H21NO7, HRESIMS m/z 434.1217 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 8.32 (2H, d, J = 8.4 Hz, H-19, H-23), 7.63 (1H, t, J = 7.5 Hz, H-21), 7.45 (3H, t, J = 7.9 Hz, H-20, H-22, H-7), 7.10 (1H, d, J = 3.5 Hz, H-11), 6.26 (1H, d, J = 3.5 Hz, H-12), 4.59 (1H, d, J = 12.4 Hz, H-9), 3.40 (1H, d, J = 12.4 Hz, 9-OH), 3.36 (3H, s, OMe-8), 2.78 (2H, q, J = 7.6 Hz, H-14), 2.02 (3H, s, H-16), 1.31 (3H, t, J = 7.6 Hz, H-15); 13C-NMR (100 MHz, CD3OD) δ: 198.3 (C-4), 194.0 (C-17), 171.4 (C-2), 167.3 (C-6), 163.9 (C-13), 143.6 (C-10), 134.7 (C-21), 133.1 (C-18), 130.8 (C-19, 23), 128.7 (C-20, 22), 117.9 (C-11), 109.5 (C-3), 107.8 (C-12), 92.6 (C-8), 86.8 (C-5), 73.6 (C-9), 51.8 (OMe-8), 21.9 (C-14), 11.9 (C-15), 6.4 (C-16)。以上数据与文献 (Yamada et al, 2010)报道对照基本一致, 化合物10鉴定为cephalimysin C。
化合物11: 淡黄色油状物, 分子式为C25H25N5O5, HRESIMS m/z 498.1753 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 8.19 (1H, dd, J = 1.4, 8.0 Hz, H-10), 7.83 (1H, m, H-8), 7.75 (1H, d, J = 7.8 Hz, H-7), 7.58 (1H, d, J = 7.7 Hz, H-27), 7.54 (1H, m, H-24), 7.49 (1H, d, J = 8.1 Hz, H-9), 7.34 (1H, td, J = 1.2, 7.7 Hz, H-25), 7.17 (1H, td, J = 1.2, 7.7 Hz, H-26), 5.95 (1H, dd, J = 6.9, 7.8 Hz, H-14), 5.40 (1H, d, J = 1.8 Hz, H-18), 4.20 (1H, q, J = 6.6 Hz, H-20), 3.40 (3H, s, OMe-3), 2.61 (1H, dd, J = 7.9, 14.3 Hz, H-15b), 2.41 (1H, dd, J = 6.8, 14.3 Hz, H-15a), 1.93 (3H, s, H-16), 1.30 (3H, d, J = 6.7 Hz, H-29); 13C-NMR (100 MHz, CD3OD) δ: 173.8 (C-1), 173.7 (C-21), 162.9 (C-12), 151.0 (C-4), 147.9 (C-6), 141.0 (C-28), 137.9 (C-23), 135.8 (C-8), 130.4 (C-25), 128.8 (C-7), 128.7 (C-9), 127.6 (C-10), 126.4 (C-26), 126.3 (C-27), 121.9 (C-11), 115.8 (C-24), 87.8 (C-18), 86.2 (C-3), 81.1 (C-17), 60.4 (C-20), 54.8 (C-14), 50.7 (OMe-3), 39.5 (C-15), 20.7 (C-16), 18.3 (C-29)。以上数据与文献 (Takahashi et al, 1995)报道对照基本一致, 化合物11鉴定为fumiquinazoline C。
化合物12: 淡黄色油状物, 分子式为C24H25N5O4, HRESIMS m/z 468.1649 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 8.19 (1H, dd, J = 1.1, 7.9 Hz, H-10), 7.81 (1H, td, J = 1.5, 7.8 Hz, H-8), 7.64 (1H, d, J = 8.5 Hz, H-7), 7.62 (1H, dd, J = 8.5 Hz, H-27), 7.51 (1H, td, J = 0.9, 7.6 Hz, H-24), 7.48 (1H, d, J = 8.5 Hz, H-9), 7.34 (1H, m, H-25), 7.20 (1H, m, H-26), 5.78 (1H, dd, J = 5.6, 10.0 Hz, H-14), 5.39 (1H, d, J = 1.7 Hz, H-18), 4.21 (1H, q, J = 6.7 Hz, H-20), 2.57 (1H, dd, J = 10.0, 13.9 Hz, H-15b), 2.35 (1H, dd, J = 5.6, 13.9 Hz, H-15a), 1.82 (3H, d, J = 7.2 Hz, H-16), 1.28 (3H, d, J = 6.7 Hz, H-29); 13C-NMR (100 MHz, CD3OD) δ: 173.5 (C-1), 171.4 (C-21), 162.3 (C-12), 153.8 (C-4), 148.4 (C-6), 140.7 (C-28), 137.9 (C-23), 136.1 (C-8), 130.5 (C-25), 128.2 (C-7), 127.7 (C-9), 127.6 (C-10), 126.5 (C-26), 126.4 (C-27), 121.3 (C-11), 115.8 (C-24), 87.8 (C-18), 81.2 (C-17), 60.3 (C-20), 53.5 (C-14), 53.5 (C-3), 39.2 (C-15), 24.7 (C-16), 18.4 (C-29)。以上数据与文献 (Takahashi et al, 1995)报道对照基本一致, 化合物12鉴定为fumiquinazoline B。
化合物13: 淡黄色油状物, 分子式为C24H23N5O4, HRESIMS m/z 468.1646 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 8.20 (1H, dd, J = 1.3, 8.0 Hz, H-10), 7.81 (1H, ddd, J = 1.5, 7.2, 8.2 Hz, H-8), 7.71 (1H, d, J = 7.9 Hz, H-27), 7.58 (1H, d, J = 7.5 Hz, H-7), 7.52 (1H, ddd, J = 1.1, 7.0, 8.0 Hz, H-24), 7.45 (1H, d, J = 7.8 Hz, H-9), 7.32 (1H, td, J = 1.2, 7.7 Hz, H-25), 7.18 (1H, td, J = 1.0, 7.6 Hz, H-26), 5.98 (1H, t, J = 7.9 Hz, H-14), 5.41 (1H, d, J = 1.7 Hz, H-18), 4.99 (1H, q, J = 6.6 Hz, H-3), 4.21 (1H, q, J = 6.6 Hz, H-20), 2.52 (1H, dd, J = 8.3, 14.1 Hz, H-15b), 2.23 (1H, dd, J = 7.5, 14.1 Hz, H-15a), 1.74 (3H, d, J = 6.6 Hz, H-16), 1.29 (3H, d, J = 6.6 Hz, H-29); 13C-NMR (100 MHz, CD3OD) δ: 173.5 (C-1), 172.4 (C-21), 162.7 (C-12), 153.8 (C-4), 148.7 (C-6), 141.0 (C-28), 137.7 (C-23), 135.8 (C-8), 130.5 (C-25), 128.5 (C-7), 128.2 (C-9), 127.6 (C-10), 126.5 (C-26), 126.3 (C-27), 121.6 (C-11), 115.8 (C-24), 87.8 (C-18), 81.2 (C-17), 60.2 (C-20), 54.7 (C-14), 50.3 (C-3), 37.0 (C-15), 18.5 (C-29), 16.8 (C-16)。以上数据与文献(Takahashi et al, 1995)报道对照基本一致, 化合物13鉴定为fumiquinazoline A。
化合物14: 淡黄色粉末状物, 分子式为C24H21N5O4, HRESIMS m/z 466.1486 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 8.20 (1H, d, J = 7.9 Hz, H-10), 7.82 (1H, t, J = 7.8 Hz, H-8), 7.72 (1H, d, J = 8.1 Hz, H-7), 7.54 (1H, t, J = 7.5 Hz, H-9), 7.43 (2H, overlap, H-27, H-24), 7.31 (1H, t, J = 7.6 Hz, H-25), 7.18 (1H, t, J = 7.6 Hz, H-26), 5.67 (1H, d, J = 1.6 Hz, H-14), 5.46 (1H, d, J = 9.7 Hz, H-18), 4.25 (1H, q, J = 6.4 Hz, H-20), 3.18 (1H, dd, J = 10.2, 15.2 Hz, H-15b), 2.23 (1H, d, J = 15.1 Hz, H-15a), 2.09 (3H, s, H-16), 1.09 (3H, d, J = 6.5 Hz, H-29); 13C-NMR (100 MHz, CD3OD) δ: 174.0 (C-1), 172.0 (C-21), 162.4 (C-12), 154.3 (C-4), 148.0 (C-5), 139.8 (C-22), 139.0 (C-28), 136.0 (C-8), 130.7 (C-25), 128.8 (C-7), 128.7 (C-9), 127.6 (C-10), 126.7 (C-26), 125.3 (C-27), 121.6 (C-11), 116.3 (C-24), 87.2 (C-18), 84.5 (C-17), 72.1 (C-3), 60.3 (C-20), 54.6 (C-14), 44.5 (C-15), 19.0 (C-16), 18.0 (C-29)。以上数据与文献(朱伟明等, 2015)报道对照基本一致, 化合物14鉴定为fumiquinazoline D。
化合物15: 淡黄色油状物, 分子式为C24H21N5O4, HRESIMS m/z 466.1486 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 8.30 (1H, dd, J = 1.1, 8.0 Hz, H-10), 7.91 (1H, td, J = 1.5, 8.3 Hz, H-2), 7.85 (1H, dd, J = 0.9, 8.3 Hz, H-8), 7.66 (1H, ddd, J = 1.3, 7.5, 7.5 Hz, H-9), 7.36 (3H, m, H-24, H-25, H-27), 7.24 (1H, ddd, J = 1.9, 7.2, 7.2 Hz, H-26), 5.57 (1H, dd, J = 1.0, 7.3 Hz, H-14), 5.29 (1H, s, H-18), 3.70 (1H, q, J = 7.0 Hz, H-20), 2.97 (1H, dd, J = 7.3, 15.1 Hz, H-15b), 2.09 (1H, dd, J = 0.9, 15.1 Hz, H-15a), 2.02 (3H, s, H-16), 1.02 (3H, d, J = 6.9 Hz, H-29); 13C-NMR (100 MHz, CD3OD) δ: 173.7 (C-1), 172.7 (C-21), 161.7 (C-12), 151.9 (C-4), 148.2 (C-6), 140.6 (C-28), 136.0 (C-8), 131.0 (C-25), 129.7 (C-9), 129.4 (C-7), 127.6 (C-10), 127.4 (C-26), 126.1 (C-27), 122.6 (C-11), 116.2 (C-24), 89.0 (C-17), 87.8 (C-18), 85.4 (C-3), 60.1 (C-20), 53.0 (C-14), 32.3 (C-15), 24.4 (C-16), 18.9 (C-29)。以上数据与文献 (Takahashi et al, 1995)报道对照基本一致, 化合物15鉴定为fumiquinazoline F。
化合物16: 白色粉末状物, 分子式为C21H25N3O2, HRESIMS m/z 374.1845 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 7.48 (1H, d, J = 7.8 Hz, H-4), 7.31 (1H, d, J = 7.9 Hz, H-7), 7.16 (1H, td, J = 1.1, 7.2 Hz, H-6), 7.09 (1H, td, J = 0.9, 7.9 Hz, H-5), 5.64 (1H, brs, H-10), 5.32 (1H, tt, J = 1.3, 7.2 Hz, H-19), 4.37 (1H, dd, J = 3.3, 11.5 Hz, H-12), 4.06 (1H, t, J = 7.9 Hz, H-9), 3.68 (2H, m, H-8, H-15a), 3.59 (1H, ddd, J = 3.2, 8.7, 11.7 Hz, H-15b), 3.48 (2H, t, J = 7.2 Hz, H-18), 2.96 (1H, dd, J = 11.4, 15.1 Hz, H-8), 2.33 (1H, m, H-13a), 2.06 (1H, m, H-13b), 1.91 (2H, m, H-14), 1.79 (3H, s, H-19), 1.75 (3H, s, H-20); 13C-NMR (100 MHz, CD3OD) δ: 170.3 (C-11), 167.5 (C-17), 138.8 (C-7a), 137.3 (C-20), 134.7 (C-2), 129.5 (C-3a), 122.1 (C-6), 121.9 (C-19), 119.8 (C-5), 119.1 (C-4), 111.7 (C-7), 104.4 (C-3), 60.1 (C-12), 57.2 (C-9), 46.0 (C-15), 29.0 (C-13), 28.6 (C-21), 26.2 (C-8), 25.9 (C-18), 22.5 (C-14), 18.0 (C-22)。以上数据与文献(赵文英 等, 2006)报道对照基本一致, 化合物16鉴定为tryprostatin B。
化合物17: 淡黄色粉末, 分子式为C28H35N3O7, HRESIMS m/z 534.2253 [M + Na]+。1H-NMR (400 MHz, CDCl3) δ: 7.90 (1H, d, J = 8.7 Hz, H-16), 6.83 (1H, dd, J = 2.2, 8.7 Hz, H-17), 6.64 (1H, d, J = 8.2 Hz, H-25), 6.59 (1H, d, J = 2.2 Hz, H-19), 6.05 (1H, d, J = 10.2 Hz, H-3), 5.66 (1H, s, H-13), 5.04 (1H, dt, J = 1.3, 8.1 Hz, H-26), 4.48 (1H, dd, J = 7.1, 9.6 Hz, H-6), 3.84 (3H, s, OMe-18), 3.64 (2H, overlap, H-9), 2.50 (1H, m, H-7), 2.00 (3H, s, H-29), 1.74 (3H, s, H-28), 1.72 (3H, s, H-24), 1.01 (3H, s, H-23); 13C-NMR (100 MHz, CDCl3) δ: 170.8 (C-11), 166.3 (C-5), 156.5 (C-18), 143.3 (C-27), 136.4 (C-20), 131.7 (C-2), 121.8 (C-16), 121.1 (C-15), 118.6 (C-26), 109.5 (C-17), 105.7 (C-14), 94.1 (C-19), 86.0 (C-25), 82.7 (C-12), 82.3 (C-22), 68.8 (C-13), 58.9 (C-6), 55.9 (OMe-18), 51.3 (C-9), 49.0 (C-3), 45.5 (C-21), 29.2 (C-7), 27.2 (C-24), 25.8 (C-23), 24.3 (C-29), 22.8 (C-8), 19.0 (C-28)。以上数据与文献(王聪 等, 2019)报道对照基本一致, 化合物17鉴定为verruculogen。
化合物18: 白色粉末状, 分子式为C22H18N4O4, HRESIMS m/z 403.1399 [M + H]+。1H-NMR (400 MHz, CD3OD) δ: 8.23 (1H, brs, H-25), 7.85 (1H, td, J = 1.5, 7.6 Hz, H-21), 7.71 (1H, d, J = 8.1 Hz, H-22), 7.58 (2H, overlap, H-20, H-5), 7.48 (2H, d, J = 7.5 Hz, H-8), 7.42 (1H, td, J = 1.2, 7.6 Hz, H-7), 7.25 (1H, td, J = 1.0, 7.6 Hz, H-6), 5.6 (1H, brs, H-2), 4.59 (1H, q, J = 6.9 Hz, H-11), 3.28 (1H, overlap, H-13), 2.61 (1H, dd, J = 3.1, 13.0 Hz, H-13), 1.71 (3H, d, J = 7.0 Hz, H-12); 13C-NMR (100 MHz, CD3OD) δ: 173.6 (C-10), 162.4 (C-17), 148.7 (C-23), 140.4 (C-9), 137.4 (C-21), 136.0 (C-4), 131.3 (C-7), 128.7 (C-20), 128.0 (C-22), 127.7 (C-19), 127.0 (C-6), 125.7 (C-5), 123.7 (C-18), 116.2 (C-8), 84.6 (C-2), 78.0 (C-3), 61.4 (C-11), 40.3 (C-13), 14.7 (C-12)。以上数据与文献(王宇 等, 2017)报道对照基本一致, 化合物18鉴定为chaetominine。
化合物19: 棕黄色油状物, 分子式为C22H25NO8, HRESIMS m/z 379.0767 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 5.99 (1H, m, H-9), 5.93 (1H, m, H-8), 5.68 (1H, d, J = 9.5 Hz, H-7), 4.94 (1H, d, J = 13.7 Hz, H-5a), 4.88 (1H, m, H-6), 4.26 (1H, d, J = 11.5 Hz, H-12), 3.87 (1H, d, J = 11.5 Hz, H-12), 3.12 (3H, s, H-11), 3.12 (1H, d, J = 15.8 Hz, H-10), 2.94 (1H, d, J = 15.8 Hz, H-10), 2.28 (3H, s, H-15), 2.25 (3H, s, H-13); 13C-NMR (100 MHz, CD3OD) δ: 168.4 (C-4), 167.8 (C-1), 134.0 (C-9a), 130.7 (C-7), 124.8 (C-8), 120.8 (C-9), 75.7 (C-6), 74.3 (C-3), 73.1 (C-10a), 70.5 (C-5a), 64.6 (C-12), 39.7 (C-10), 29.1 (C-11), 15.2 (C-13), 13.5 (C-15)。以上数据与文献 (Xu et al, 2017)报道对照基本一致, 化合物19鉴定为bisdethiobis(methylthio)glitoxin。
化合物20: 无色针状物, 分子式为C33H44O8, HRESIMS m/z 591.2935 [M + Na]+。1H-NMR (400 MHz, CDCl3) δ: 7.31 (1H, d, J = 10.1 Hz, H-1), 5.92 (1H, d, J = 8.4 Hz, H-16), 5.86 (1H, d, J = 10.0 Hz, H-2), 5.23 (1H, brs, H-6), 5.11 (1H, t, J = 6.5 Hz, H-24), 2.77 (1H, m, H-4), 2.61 (1H, dd, J = 2.2, 12.9 Hz, H-9), 2.56 (1H, d, J = 12.3 Hz, H-13), 2.45 (3H, m, H-12, H-22), 2.26 (1H, d, J = 12.4 Hz, H-5), 2.21 (1H, dd, J = 9.1, 15.0 Hz, H-15), 2.11 (3H, s, OCOCH3-6), 1.96 (3H, m, OCOCH3-16), 1.90 (1H, d, J = 15.0 Hz, H-15), 1.69 (3H, m, H-27), 1.60 (3H, m, H-26), 1.44 (3H, m, H-19), 1.28 (3H, d, J = 6.9 Hz, H-28), 1.17 (3H, m, H-29), 0.93 (3H, m, H-18); 13C-NMR (100 MHz, CDCl3) δ: 209.0 (C-7), 201.5 (C-3), 174.0 (C-21), 170.9 (OCOCH3-16), 169.0 (OCOCH3-6), 157.4 (C-1), 147.8 (C-17), 132.8 (C-25), 130.4 (C-20), 128.0 (C-2), 123.1 (C-24), 73.9 (C-6), 73.5 (C-16), 52.8 (C-8), 49.4 (C-13), 47.4 (C-5), 46.8 (C-14), 41.9 (C-9), 40.8 (C-15), 40.5 (C-4), 38.3 (C-10), 28.9 (C-22), 28.4 (C-23), 27.7 (C-19), 26.1 (C-12), 25.9 (C-27), 24.1 (C-11), 20.9 (OCOCH3-6), 20.8 (OCOCH3-16), 18.5 (C-29), 18.1 (C-18), 17.9 (C-26), 13.2 (C-28)。以上数据与文献 (Fujimoto et al, 1996)报道对照基本一致, 化合物20鉴定为helvolic acid。
化合物21: 淡黄色油状物, 分子式为C29H35NO9, HRESIMS m/z 542.2370 [M + H]+。1H-NMR (400 MHz, CD3OD) δ: 9.03 (1H, d, J = 1.6 Hz, H-2′), 8.64 (1H, dd, J = 1.2, 4.8 Hz, H-6"), 8.28 (1H, dt, J = 1.8, 8.1 Hz, H-4"), 7.57 (1H, dd, J = 4.9, 8.1 Hz, H-5"), 6.83 (1H, s, H-5'), 4.96 (1H, d, J = 3.6 Hz, H-13), 4.78 (1H, dd, J = 5.1, 11.4 Hz, H-1), 3.84 (2H, overlap, H-11), 3.73 (1H, dd, J = 4.8, 11.3 Hz, H-7), 2.14 (1H, dt, J = 3.2, 13.1 Hz, H-3), 2.04 (3H, s, OCOCH3-1), 2.03 (3H, s, OCOCH3-11), 1.64 (3H, s, H-14), 1.45 (3H, s, H-12), 0.93 (3H, s, H-15); 13C-NMR (100 MHz, CD3OD) δ: 172.5 (OCOCH3-1, OCOCH3-11), 165.3 (C-2'), 164.6 (C-2''), 158.1 (C-4'), 151.9 (C-6''), 147.3 (C-5''), 134.9 (C-4''), 129.3 (C-6'), 125.5 (C-3''), 104.4 (C-3'), 101.1 (C-5'), 86.7 (C-6), 78.3 (C-7), 75.2 (C-1), 66.1 (C-11), 60.4 (C-13), 55.3 (C-5), 46.8 (C-9), 41.8 (C-10), 39.2 (C-4), 37.1 (C-3), 28.8 (C-8), 23.8 (C-2), 21.0 (OCOCH3-1), 20.7 (OCOCH3-11), 18.0 (C-12), 15.9 (C-14), 13.4 (C-15)。以上数据与文献(Lan et al, 2016)报道对照基本一致, 化合物21鉴定为7-deacetylpyripyropene A。
化合物22: 白色粉末状物, 分子式为C19H23N3O2, HRESIMS m/z 348.1682 [M + Na]+。1H-NMR (400 MHz, CD3OD) δ: 7.43 (1H, d, J = 7.9 Hz, H-15), 7.07 (1H, s, H-9), 6.93 (1H, t, J = 7.5 Hz, H-14), 6.86 (1H, d, J = 7.0 Hz, H-13), 5.39 (1H, m, H-18), 4.26 (1H, td, J = 1.1, 4.2 Hz, H-2), 3.69 (1H, dq, J = 1.2, 7.0 Hz, H-5), 3.52 (2H, d, J = 7.2 Hz, H-17), 3.44 (1H, dd, J = 4.0, 14.6 Hz, H-7), 3.14 (1H, dd, J = 4.4, 14.6 Hz, H-7), 1.74 (3H, s, H-20), 1.73 (3H, s, H-21), 0.35 (3H, d, J = 7.1 Hz, H-22); 13C-NMR (100 MHz, CD3OD) δ: 170.7 (C-6), 169.5 (C-3), 136.5 (C-11), 133.7 (C-19), 129.2 (C-16), 125.7 (C-12), 125.7 (C-9), 123.4 (C-18), 121.7 (C-13), 120.5 (C-15), 117.8 (C-14), 109.7 (C-8), 57.5 (C-2), 51.8 (C-5), 30.9 (C-7), 30.5 (C-17), 25.9 (C-21), 19.9 (C-22), 17.9 (C-20)。以上数据与文献(Wang et al, 1995)报道对照基本一致, 化合物22鉴定为terezine D。

2.2 活性测试结果

2.2.1 细胞毒活性测试结果

对化合物进行抗肿瘤活性筛选发现化合物6对人肝癌细胞(HepG2)、人肺癌细胞(A549)、人直肠癌细胞(HCT116)都具有一定的细胞毒活性, IC50值如表1所示。
表1 化合物6细胞毒活性结果

Tab. 1 Cytotoxic activity results of compound 6

编号 名称 IC50 /(μmol·L-1)
HepG2 A549 HCT116 L02
1 6 35.86 ± 1.43 8.26 ± 1.89 17.04 ± 0.68 27.51 ± 2.84
P1 苏尼替尼 12.6 ± 3.14 9.4 ± 0.76 3.79 ± 0.2 20.6 ± 0.48
P2 盐酸阿霉素 0.66 ± 0.03 1.46 ± 0.72 0.03 ± 0.01 15.23 ± 1.24

2.2.2 抑菌活性测试结果

对22个单体化合进行抑菌活性筛选后, 发现除化合物1、3、20外其他单体化合物对金黄色葡萄球菌均没有抑菌活性, 且所有化合物对耐甲氧西林的金黄色葡萄球菌均无明显抑制活性。化合物1、3、20的最小抑菌浓度(minimal inhibitory concentration, MIC)如表2所示。
表2 化合物1、3、20抑制金黄色葡萄球菌活性结果

Tab. 2 Inhibition of Staphylococcus aureus activity results of compound 1, 3 and 20

名称 MIC/ (μg·mL-1)
1 64.0
3 8.0
20 1.0
环丙沙星 0.4

2.2.3 NPC1L1蛋白结合活性测试结果

NPC1L1作为胆固醇的特异性转运蛋白, 越来越受到人们的关注。对化合物1—20和化合物22进行NPC1L1蛋白结合活性测试, 使用阳性药为依折麦布, 结果显示化合物14与NPC1L1蛋白具有相互作用
(图4)。
图4 化合物14和依折麦布(ezetimibe)与NPC1L1相互作用的结合传感图

Fig. 4 Binding sensorgram for compound 14 and ezetimibe interaction with immobilized NPC1L1

3 讨论

目前, 从Aspergillus fumigatus中分离得到的化合物种类多为生物碱类、喹啉类衍生物和烟曲霉素类等。除此之外, 还从此菌株中首次分离得到吡喃酮类化合物(郭思雨 等, 2020)、酚酸类(钱诗筠 等, 2020)和肽类化合物(李基兴 等, 2020)。本文从海洋动物内生菌Aspergillus fumigatus DL-p0m-g2中分离得到22个化合物, 其中主要为生物碱类化合物, 丰富了烟曲霉代谢产物的种类。
据文献报道, 化合物19对P388小鼠白血病细胞有细胞毒活性, IC50 值为 0.11μmol·L-1 (Sun et al, 2012); 化合物18对人类白血病K562和结肠癌SW1116细胞系的细胞毒性大于联合检测的阳性药-氟尿嘧啶 (Jiao et al, 2006)。除此外, 还发现化合物3、17和20有抑制植物致病菌的活性等 (Li et al, 2012)。本文对分离得到的22个单体化合物进行抑菌活性测试发现所有化合物对耐甲氧基西林金黄色葡萄球菌均无明显活性, 但化合物1、3和20对金黄色葡萄球菌均有抑菌活性, 其中化合物20已经作为抑菌药物被开发利用, 化合物1和化合物3的最小抑菌浓度(MIC)分别为64μg·mL-1和8μg·mL-1。对所有化合物进行硫氧还蛋白还原酶活性测试发现所有化合物均无相应活性。化合物6在本次研究中对其进行细胞毒活性实验中首次发现其具有一定的人肝癌细胞 (HepG2)、人肺癌细胞(A549)、人直肠癌细胞(HCT116)抑制活性, IC50值分别为(35.86±1.43)μmol·L-1、(8.26±1.89)μmol·L-1和(17.04± 0.68)μmol·L-1, 其对A549的抑制活性与阳性药苏尼替尼相当。同时测试发现化合物6对于人正常肝细胞的IC50值要高于苏尼替尼和盐酸阿霉素, 这表明其安全性可能要优于这两种阳性药, 这使得其为后续药用价值的开发提供一定的参考。此外, 表面等离子共振测试显示化合物14和NPC1L1之间的相互作用, 这表明化合物14可能是潜在的NPC1L1抑制剂, 显示出其在抑制胆固醇吸收方面的潜力。

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