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热带海洋学报 >

2025 , Vol. 44 >Issue 3: 157 - 166

DOI: https://doi.org/10.11978/2024182

海洋生物学

石花菜共附生烟曲霉Aspergillus fumigatus 9-1次级代谢产物抗菌、抗氧化研究

  • 何鸣凤 ,
  • 黄佳翔 ,
  • 田永奇
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  • 福州大学生物科学与工程学院, 福建 福州 350108
田永奇。email:

何鸣凤(2001—), 女, 江西省赣州市人, 硕士研究生。email: ;

黄佳翔(1998—), 男, 福建省泉州市人, 硕士研究生。email:

Copy editor: 林强

收稿日期: 2024-09-24

  修回日期: 2024-11-01

  网络出版日期: 2024-11-25

基金资助

国家自然科学基金(42006094)

福建省自然科学基金(2022J01561)

福建省-印尼海洋食品联合研发中心开放课题(Y1-KF2201)

收起

Research on the antioxidant and antibacterial activities of secondary metabolites from Gelidium-derived fungus Aspergillus fumigatus 9-1

  • HE Mingfeng ,
  • HUANG Jiaxiang ,
  • TIAN Yongqi
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  • College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China
TIAN Yongqi. email:

Copy editor: LIN Qiang

Received date: 2024-09-24

  Revised date: 2024-11-01

  Online published: 2024-11-25

Supported by

National Natural Science Foundation of China(42006094)

Fujian Natural Science Foundation(2022J01561)

Fujian Province-Indonesia Marine Food Joint Research and Development Center Open-Ended Foundation(Y1-KF2201)

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摘要

文章对石花菜共附生烟曲霉Aspergillus fumigatus 9-1次级代谢产物的结构和活性进行了研究。通过薄层色谱、硅胶柱色谱、高效液相色谱、凝胶色谱等对石花菜共附生烟曲霉A. fumigatus 9-1次级代谢产物进行分离纯化, 从中得到了16个化合物。通过核磁共振波谱等波谱分析方法、结合文献对照, 鉴定它们的结构依次为: prenylcyclotryprostatin A (1)、verruculogen TR-2 (2)、cyclotryprostatin E (3)、cyclotryprostatin B (4)、asperfumigatin (5)、12R, 13S-dihydroxyfumitremorgin C (6)、spirocyclic diketopiperazine alkaloid (7)、fumigaclavine C (8)、fumigaclavine A (9)、chaetominine (10)、fumiquinazoline J (11)、thymidine (12)、pyripyropene A (13)、monomethylsulochrin (14)、questin (15)、helvolic acid (16)。其中化合物1首次从Aspergillus中发现。抗菌活性研究表明: 16对Listeria monocytogenes具有较强的抗菌活性, 最小抗菌浓度为4.03μg·mL−1。13—15对 L. monocytogenes具有中等抗菌活性, 最小抗菌浓度为分别为66.25、78.63、64.54μg·mL−1。同时体外活性抗氧化活性结果表明, 化合物6具有较强的DPPH(1, 1-diphenyl-2-picryl-hydrazyl)自由基清除活性, 半数抑制浓度为9.82μg·mL−1; 化合物6、8、9、14具有较强的ABTS[2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)]自由基清除活性, 半数抑制浓度分别为2.72、0.43、3.23和1.96μg·mL−1。

关键词： 石花菜共附生烟曲霉; 次级代谢产物; 抗菌活性; 抗氧化活性

本文引用格式

何鸣凤 , 黄佳翔 , 田永奇 . 石花菜共附生烟曲霉Aspergillus fumigatus 9-1次级代谢产物抗菌、抗氧化研究[J]. 热带海洋学报, 2025 , 44(3) : 157 -166 . DOI: 10.11978/2024182

Abstract

This paper examines the structure and activity of the secondary metabolites of Aspergillus fumigatus 9-1, a symbiotic epiphytic fungus of rockweed. The secondary metabolites produced by A. fumigatus 9-1 were systematically separated and purified using a combination of thin layer chromatography, silica gel column chromatography, high performance liquid chromatography, and gel column chromatography, resulting in the isolation of 16 compounds. Spectral analysis, including nuclear magnetic resonance (NMR) spectroscopy and comparison with literature data, led to the structural elucidation of these compounds as: prenylcyclotryprostatin A (1), verruculogen TR-2 (2), cyclotryprostatin E (3), cyclotryprostatin B (4), asperfumigatin (5), 12R, 13S-dihydroxyfumitremorgin C (6), spirocyclic diketopiperazine alkaloid (7), fumigaclavine C (8), fumigaclavine A (9), chaetominine (10), fumiquinazoline J (11), thymidine (12), pyripyropene A (13), monomethylsulochrin (14), questin (15), and helvolic acid (16). Compound 1 was first reported from Aspergillus. The antimicrobial activity showed that compound 16 had strong antibacterial activity against Listeria monocytogenes, with a minimum inhibitory concentration of 4.03 μg·mL-1. 13-15 had moderate antibacterial activity against L. monocytogenes, with MICs of 66.25, 78.63 and 64.54 μg·mL−1, respectively. The in vitro antioxidant activity results demonstrated that compound 6 exhibited robust DPPH (1, 1-diphenyl-2-picryl-hydrazyl radical) radical scavenging activity, with a half inhibitory concentration of 9.82μg·mL-1. Additionally, compounds 6, 8, 9, 14 displayed notable ABTS (2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)) radical scavenging activity, with a half inhibitory concentration of 2.72, 0.43, 3.23 and 1.96 μg·mL-1, respectively.

Key words： Aspergillus fumigatus; secondary metabolites; antibacterial activity; antioxidant activity

自由基由机体正常的代谢活动产生。自由基过量生成时, 会导致脱氧核糖核酸(deoxyribonucleic acid, DNA)、蛋白质等的破坏, 从而进一步导致机体的衰老和疾病产生, 因此对体内多余的自由基的清除显得尤为重要(Wu et al, 2018)。近年来, 全球食品安全问题愈发严重, 尤其是因病原性微生物污染导致的食品中毒(Guo et al, 2023)。因此, 对于病原性微生物的防治显得尤为重要。
海藻共附生真菌是指一类以内生、附生和共生形式存在于海藻组织中的丝状真菌。为了适应海洋环境, 海藻共附生真菌可产生大量具有生物活性的次级代谢产物, 帮助宿主抵抗外界的侵害(杨曦亮 等, 2020)。Li等(2014a)从福建平潭红藻来源的青霉菌Penicillium echinulatum pt-4发酵产物中分离得到1个新的混源萜Arisugacin K, 该化合物对大肠杆菌具有抑制作用(抑菌圈直径为8mm)。此外, Li等(2014b)还对马尾藻共附生真菌Aspergillus wentii EN-48进行培养发酵, 从其发酵产物中分离得到1个显著清除DPPH (1, 1-diphenyl-2-picryl-hydrazyl radical)自由基能力的新的蒽醌衍生物(半抑制浓度IC50= 5.2μg·mL−1)。还有研究人员(孔维琦 等, 2025)在羊栖菜(Sargassum fusiforme)共附生真菌Aspergillus sp. GXIMD 02045中获得了3个耐甲氧西林金黄色葡萄球菌、枯草芽孢杆菌、铜绿假单胞菌都有抑制活性的对苯醌类化合物。
石花菜属于石花菜科石花菜属(Gelidium), 分布在我国福建、浙江、山东和台湾等沿海地区, 一般生长于低潮带下2~4m 处的珊瑚礁上, 是温带重要的经济性红藻。石花菜中含有丰富的蛋白质、多糖和脂肪酸等, 在食品、生物技术和美容等领域都得到广泛应用(张英格 等, 2021)。本研究通过对石花菜共附生烟曲霉Aspergillus fumigatus 9-1进行培养以及分离纯化, 得到了16个化合物(图1)。并对分离的16个化合物进行了抗单增李斯特菌(Listeria monocytogenes)及铜绿假单胞菌(Pseudomonas aeruginosa)活性检测。同时评价了所有化合物的DPPH及ABTS [2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)]自由基清除活性。
图1 石花菜共附生烟曲霉9-1中化合物1—16的结构式

Fig. 1 Structures of compounds 1-16 from Aspergillus fumigatus 9-1

1 材料与方法

1.1 材料与试剂

研究材料来源于福建福州连江海域, 经福州大学生物科学与工程学院林向阳教授鉴定为石花菜(保藏于福州大学生物科学与工程学院食品与海洋生物资源研究所)。石花菜共附生烟曲霉9-1由生工生物工程(上海)股份有限公司测定其ITS rDNA序列, 并提交NCBI-BLAST网站中并比对, 确定其种属为Aspergillus fumigatus。
1, 1-二苯基-2-三硝基苯肼(1, 1-diphenyl-2-picrylhydrazyl)、2, 2-联氨-双-(3-乙基苯并噻唑啉-6-磺酸)二铵盐[2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)]、维生素C(vitamin C, VC)、四环素、酶标仪板(96孔)。
柱层析硅胶(山东烟台江友硅胶开发有限公司), 葡聚糖凝胶(Sephadex LH-20, GE Healthcare Bio-Sciences AB), 薄层层析硅胶板(山东烟台江友硅胶开发有限公司), 显色剂(10%硫酸香兰素溶液), 氘代试剂主要有氘代二甲基亚砜(dimethyl sulfoxide, DMSO)、氘代氯仿、氘代甲醇(美国CIL公司), 有机化学试剂主要有石油醚、二氯甲烷、乙酸乙酯、二甲基亚砜、甲醇等(分析纯, 广州化学试剂有限公司)。
单核细胞增生李斯特氏菌(Listeria monocytogene) ATCC 19115, 铜绿假单胞菌(Pseudomonas aeruginosa) PAO1(保藏于福州大学生物科学与工程学院)。BigDye测序反应盒(Thermo Fisher公司)、SanPrep柱式PCR产物纯化试剂盒(生工生物工程公司)。

1.2 仪器与设备

高效液相色谱仪(Agilent 1260), ODS(Daiso, Japan), 全数字化核磁共振仪(Bruker Avance II 400MHz / 600MHz), 高分辨质谱仪(Waters Xevo G2Q-TOF), ZF-C 型三用紫外分析仪(上海沪西分析仪器有限公司), 中压反相色谱(BUCHI), 旋转蒸发仪(R-300, BUCHI), 旋光仪(Anton Paar MCP5500)。

1.3 实验方法

1.3.1 培养基的配制

试验用培养基包括马铃薯葡萄糖琼脂培养基(potato dextrose agar, PDA): 200g土豆, 12g琼脂, 12g葡萄糖, 10g海盐, 1000mL超纯水, 溴化钠30g; 马铃薯葡萄糖汤培养基(potato dextrose broth, PDB): 200g土豆, 12g葡萄糖, 10g海盐, 1000mL超纯水, 溴化钠30g; 固体大米培养基: 在500mL的锥形瓶中加入大米100g, 海盐1g, 溴化钠3g, 超纯水100mL, 121°C灭菌30min备用; 溶原性培养基(lysogeny broth, LB): 胰蛋白胨10g, 酵母提取物5g, 氯化钠10g, 蒸馏水1000mL, pH 7.0。

1.3.2 菌株的鉴定和培养

将菌株培养在PDB培养基中, 设置摇床温度为28℃, 培养1周。将活化好的菌株进行测序分析。使用PCR扩增BigDye测序反应盒(Thermo Fisher公司)进行扩增, 引物为ITS1, 按照试剂盒所提供的方法将反应体系(25μL)加好后至于PCR扩增仪中扩增, 首先在98℃条件下变性2min, 随后进行PCR循环, 96℃条件下10s后50℃持续5s, 最后60℃维持4min, 进行25个循环, 结束后在4℃温度下保温。随后将得到的PCR产物利用SanPrep柱式PCR产物纯化试剂盒纯化, 并进行电泳随之得到菌株序列。将得到的菌株序列提交至NCBI-BLAST网站中并搜索, 从而得到菌株的种属为Aspergillus fumigatus, 命名为Aspergillus fumigatus(菌种编号: 9-1)。
石花菜共附生烟曲霉A. fumigatus 9-1在28℃的PDA培养基复苏培养7d, 后将其接种到30个50mL锥形瓶(含10mL PDB)种子培养基中摇床(28℃、180r·min-1)培养3d, 最后再转接到30个500mL的锥形瓶固体(含100g大米培养基)28℃静置培养30d。

1.3.3 提取与分离

对菌株A. fumigatus 9-1进行发酵后得到的发酵物用甲醇提取, 减压浓缩后采用乙酸乙酯萃取3次, 合并乙酸乙酯经过减压浓缩得到粗提物(34g)。粗提物用硅胶柱、中压反相色谱、LH-20(Sephadex LH-20)凝胶柱、高效液相色谱纯化得到化合物1—16。具体过程为: 粗提物(34g)经硅胶柱(100~200目、V二氯甲烷:V甲醇=10:0~9:1、t总=600min、v=30mL·min-1)分离, 洗脱下来的组分通过TLC(thin layer chromatography)后合并成3个组分(Fr.1—Fr.3)。Fr.1经中压反相色谱(40%MeOH/H2O-100%MeOH/H2O、5mL·min-1、3min) 得到化合物6 (tR=16.49min, 3.5mg), 15 (tR=12.76min, 1.5mg), 16 (tR=18.42min, 8.2mg) 以及5个组分(Fr.1.1—Fr.1.5)。Fr.1.1、Fr.1.2、Fr.1.4和Fr.1.5经LH-20凝胶柱(MeOH)后, 再经HPLC(high performance liquid chromatography)(5%-100%MeOH/H2O、2mL·min-1)得到化合物1 (tR=21.42min, 0.8mg)、4 (tR=15.44min, 6mg)、5 (tR=14.40min, 2.1mg)、7 (tR=20.02min, 3.7mg)、11 (tR=18.42min, 1mg)、13 (tR=12.78min, 3.5mg)和14 (tR=11.62min, 2.2mg)。组分Fr.1.3经LH-20凝胶柱(MeOH)后, 得到化合物2 (tR=22.72min, 49.6mg)和组分Fr.1.3.1。Fr.1.3.1经HPLC纯化后得到化合物3 (tR=15.85min, 4.2mg)。Fr.2 (5.5g) 通过中压反相色谱(10%MeOH/H2O~100%MeOH/H2O)得到2个组分(Fr.2.1、Fr.2.2)。Fr.2.1通过LH-20凝胶柱, 得到化合物10 (tR=13.66min, 7.4mg)。Fr.2.2经LH-20凝胶柱和HPLC纯化, 得到化合物8 (tR=19.20min, 4.2mg)。Fr.3 (1.4g)经过LH-20凝胶柱和HPLC分离, 得到化合物12 (tR=14.62min, 2.4mg)和9 (tR=17.52min, 6.3mg)。

1.3.4 体外抗菌活性的测定

参照文献(王小敏 等, 2015)的方法, 首先在无菌的96孔板中1—10列加入50μL的LB肉汤培养基, 11列加入100μL培养基作为空白对照, 12列加入50μL培养基作为阴性对照; 后在第1列加入50μL的一定浓度的样品, 样品用二甲基亚砜(dimethyl sulfoxide, DMSO)溶解为浓度5mg·mL-1的母液, 混匀, 依次倍比稀释到第10列; 后将OD600=0.35~0.40的细菌菌液稀释1000倍后在每个孔中加入50μL (除第11列外)。最后, 将处理好的96孔板在37℃黑暗中培养12h后, 肉眼观察是否有菌的产生。四环素为阳性对照, 用DMSO配制成1mg·mL-1。

1.3.5 体外抗氧化活性测定

1.3.5.1 DPPH自由基清除活力测定

参考文献 (Chen et al, 2006) 的方法, 首先配制一定浓度的样品。其次, 用甲醇将待测样品配制成浓度为1mg·mL-1的母液, 按二倍稀释法配成一系列不同样品浓度, 取不同浓度的样品溶液50μL和DPPH自由基溶液(0.1mmol·L-1, 无水甲醇配制)50μL于96孔板中, 每个浓度设定3个重复, 25℃避光反应30min, 用酶标仪在517nm处测定吸光度A1。同时用50μL甲醇代替50μL DPPH自由基溶液, 作为样品参比组, 其测定所得吸光度值A2; 用样品50μL甲醇代替50μL不同浓度样液, 作为空白组, 其测定所得吸光度值为A0。用甲醇进行校正凋零, 阳性对照为抗坏血酸(vitamin C, VC)。DPPH自由基清除率XDPPH(%)按公式(1)计算:
${{X}_{\text{DPPH}}}=\frac{{{A}_{\text{0}}}-({{A}_{1}}-{{A}_{2}})}{{{A}_{0}}}\times 100\text{ }\%$

1.3.5.2 清除ABTS自由基活力的测定

参考文献(Re et al, 1999)的方法, 配制过硫酸钾水溶液(2.45mmol·L-1) 和ABTS母液(7mmol·L-1, 蒸馏水配制), 临用前按1∶1比例进行混合, 室温避光静置反应16h后, 用甲醇进行稀释, 使得734nm 处所测的吸光值为0.70, 即ABTS自由基溶液。用甲醇溶剂将待测样品配制成浓度为1mg·mL-1的母液, 按二倍稀释法配成一系列不同样品浓度。取不同浓度样液50μL和ABTS自由基溶液50μL于96孔板中, 每个浓度设定3个重复, 25℃避光反应10min, 734nm处测定吸光度值Ap。同时用50μL甲醇代替50μL ABTS自由基溶液, 作为样品参比组, 其测定所得吸光度值为Ac; 用样品50μL甲醇代替100μL不同浓度样液, 作为空白组, 其测定所得吸光度值为Amax。用甲醇进行校正凋零, 阳性对照为VC。ABTS自由基清除率XABTS(%)按公式(2)计算:
${{X}_{\text{ABTS}}}=\frac{{{A}_{\max }}-({{A}_{\text{p}}}-A_{\text{c}}^{{}})}{{{A}_{\max }}}\times 100\text{ }\%$

2 实验结果

2.1 结构鉴定

化合物1: 白色无定形粉末, 分子式为C27H33N3O5, ESIMS 502.2 m/z [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ + 180 (c 0.1, MeOH)。1H NMR (400 MHz, DMSO-d6) δH 7.37 (1H, d, J = 8.5 Hz, H-16), 6.81 (1H, dd, J = 8.6, 2.1 Hz, H-17), 6.73 (H, m, H-3), 6.72 (H, s, H-19), 5.56 (1H, d, J = 7.0 Hz, H-21), 5.47 (1H, d, J = 9.6 Hz, H-27), 5.00 (1H, s, H-20), 4.57 (2H, dd, J = 16.4, 5.8 Hz, H2-26), 4.18 (1H, dd, J = 11.0, 6.1 Hz, H-6), 3.76 (3H, s, H3-25), 3.60 (1H, m, H-9a), 3.57 (1H, m, H-19b), 2.26 (1H, m, H-7a), 1.96 (1H, m, H-8a), 1.92 (3H, s, H3-23), 1.76 (1H, m, H-8b), 1.87 (1H, m, H-7b), 1.82 (3H, s, H3-29), 1.68 (3H, s, H3-24), 1.64 (3H, s, H3-27). 13C NMR (100 MHz, DMSO-d6) δC 165.9 (C, C-5), 165.3 (C, C-11), 155.6 (C, C-18), 136.9 (C, C-22), 135.2 (CH, C-19), 134.1 (C, C-28), 133 (C, C-2), 125.2 (CH, C-21), 120.5 (CH, C-27), 120.5 (C, C-15), 118.7 (CH, C-16), 108.8 (CH, C-17), 108 (C, C-14), 93.6 (CH, C-19), 86.8 (C, C-12), 70.5 (CH, C-13), 59.2 (CH, C-6), 55.4 (CH3, C-25), 46.9 (CH, C-3), 44.8 (CH2, C-9), 41.3 (CH2, C-26), 29.6 (CH2, C-7), 25.7 (CH3, C-24), 25.3 (CH3, C-30), 21.4 (CH2, C-8), 18.1 (CH3, C-23), 18.0 (CH3, C-29). 以上核磁数据都与文献(Zhou et al, 2021)数据基本一致, 因此将化合物1定为prenylcyclotryprostatin A。
化合物2: 黄色粉末, 分子式为C22H27N3O6, ESIMS 452.2 m/z [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ − 45 (c 0.55, CH2Cl2)。 1H NMR (500 MHz, DMSO-d6) δH 8.98 (1H, br s, H-1), 7.63 (1H, d, J = 8.7 Hz, H-16), 6.88 (1H, d, J = 2.3 Hz, H-19), 6.62 (1H, dd, J = 8.7, 2.3 Hz, H-17), 6.21 (1H, s, H-13), 5.37 (1H, dd, J = 8.1, 3.9 Hz, H-3), 4.40 (1H, t, J = 7.7 Hz, H-6), 3.74 (3H, s, H3-25), 3.66 (1H, m, H-9a), 3.61 (1H, m, H-9b), 2.52 (1H, m, H-7a), 2.07 (1H, m, H-8a), 1.95 (1H, m, H-7b), 1.96 (1H, m, H-8b), 2.02 (2H, m, H2-21), 1.07 (3H, s, H3-23), 0.98 (3H, s, H3-24). 13C NMR (125 MHz, DMSO-d6) δC 170.6 (C, C-11), 166.4 (C, C-5), 155.2 (C, C-18), 136.8 (C, C-20), 131.3 (C, C-2), 120.8 (CH, C-16), 120.7 (C, C-15), 108.5 (CH, C-17), 106.6 (C, C-14), 94.8 (CH, C-19), 83.5 (CH, C-12), 68.7 (C, C-22), 67.9 (CH, C-13), 58.7 (CH, C-3), 55.2 (CH3, C-25), 49.4 (CH2, C-9), 47.9 (CH, C-3), 45.0 (CH2, C-21), 30.3 (CH3, C-24), 28.9 (CH3, C-23), 28.9 (CH2, C-7), 22.4 (CH2, C-8). 以上核磁数据都与文献(姚佳晓 等, 2019)数据基本一致, 因此将化合物2定为verruculogen TR-2。
化合物3: 淡黄色粉末, 分子式为C23H29N3O6, ESIMS 466.2 m/z [M + Na]+, 比旋光值为[$[\alpha]_{\mathrm{D}}^{25}$ + 28.35 (c 0.23, CH3OH)。1H NMR (500 MHz, CD3OD-d4) δH 7.43 (1H, d, J = 8.7 Hz, H-16), 6.92 (1H, d, J = 2.1 Hz, H-19), 6.73 (1H, dd, J = 8.7, 2.23 Hz, H-17), 4.79 (1H, s, H-13), 4.29 (1H, dd, J = 11.3, 6.1 Hz, H-6), 3.83 (3H, s, 18-CH3O), 3.80 (3H, s, H-6), 3.74 (1H, m, H-21b), 3.59 (1H, m, H-21a), 3.38 (3H, s, 13-CH3O), 2.43 (1H, m, H-7a), 2.20 (1H, d, J = 6.1 Hz, H-9a), 2.16 (1H, d, J = 5.1 Hz, H-9b), 2.08 (1H, m, H-8a), 2.00 (1H, m, H-8b), 1.88 (1H, m, H-7b), 1.44 (3H, m, H3-24), 1.31 (3H, s, H3-23). 13C NMR (125 MHz, CD3OD-d4) δC 169.2 (C, C-11), 167.9 (C, C-5), 157.8 (C, C-18), 138.3 (C, C-15), 136.4 (C, C-2), 123.8 (C, C-20), 119.5 (CH, C-16), 110.8 (CH, C-17), 105.2 (C, C-14), 96.2 (CH, C-19), 87.5 (C, C-12), 71.4 (C, C-22), 77.7 (CH, C-13), 60.9 (CH, C-6), 57.5 (CH3, 13-CH3O), 56.2 (CH3, 18-CH3O), 51.0 (CH2, C-21), 49.0 (CH, C-3), 46.6 (CH2, C-9), 31.6 (CH3, C-23), 31.1 (CH2, C-7), 29.3 (CH3, C-24), 22.8 (CH2, C-8). 以上核磁数据都与文献(He et al, 2012)数据基本一致, 因此将化合物3定为cyclotryprostatin E。
化合物4: 淡黄色固体, 分子式为C23H27N3O5, ESIMS 448.2 m/z [M + Na] +, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ + 95.7 (c 0.36, CHCl3)。1H NMR (500 MHz, CD3OD-d4) δH 7.45 (1H, d, J = 8.6 Hz, H-16), 6.92 (1H, d, J = 2.2 Hz, H-19), 6.74 (1H, dd, J = 8.8, 2.3 Hz, H-17), 6.52 (1H, d, J = 9.9 Hz, H-3), 5.51 (1H, dm, J = 9.9 Hz, H-21), 4.84 (1H, s, H-13), 4.61 (1H, br s, 12-OH), 4.28 (1H, dd, J = 11.3, 6.3 Hz, H-6), 3.81 (3H, s, 18-OCH3), 3.75 (1H, m, H-9a), 3.56 (1H, m, H-9b), 3.39 (3H, s, 13-OCH3), 2.49 (2H, m, H2-7a), 2.10 (2H, m, H2-8b), 2.04 (3H, s, H3-24), 1.99 (2H, m, H2-7b), 1.99 (2H, m, H2-8a), 1.78 (3H, s, H3-23). 13C NMR (125 MHz, CD3OD-d4) δC 168.6 (C, C-5), 167.9 (C, C-11), 157.8 (C, C-18), 138.9 (C, C-22), 138.4 (C, C-20), 135.1 (C, C-2), 125.4 (CH, C-21), 123.9 (C, C-15), 119.4 (CH, C-16), 110.7 (CH, C-17), 105.4 (C, C-14), 96.2 (CH, C-19), 87.4 (C, C-12), 77.5 (CH, C-13), 60.8 (CH, C-6), 57.4 (CH3, 13-OCH3), 56.2 (CH3, 18-OCH3), 50.7 (CH, C-3), 46.6 (CH2, C-9), 31 (CH2, C-7), 26.4 (CH3, C-23), 22.8 (CH2, C-8), 18.5 (CH3, C-24). 以上核磁数据都与文献(Cui et al, 1997)数据基本一致, 因此将化合物4定为cyclotryprostatin B。
化合物5: 无定形黄色固体, 分子式为C27H33N3O7, ESIMS 534.2 m/z [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ −45.5 (c 0.2, MeOH)。1H NMR (600 MHz, CD3OD-d4) δH 7.93 (1H, d, J = 8.7 Hz, H-16), 7.42 (1H, d, J = 2.21 Hz, H-19), 6.87 (1H, dd, J = 8.8, 2.3 Hz, H-17), 6.41 (1H, s, H-26), 6.36 (1H, t, J = 6.06 Hz, H-3), 5.70 (1H, s, H-13), 4.56 (1H, m, H-6), 3.83 (3H, s, H3-30), 3.58 (1H, m, H-9b), 3.57 (1H, m, H-9a), 2.43 (1H, m, H-7a), 2.17 (3H, s, H3-29), 2.10 (3H, s, H3-28), 2.06 (1H, m, H-7b), 2.06 (1H, m, H-8a), 1.97 (1H, m, H-8b), 1.85 (1H, m, H-21a), 1.70 (1H, m, H-21b), 1.13 (3H, s, H3-23), 1.11 (3H, s, H3-24). 13C NMR (150 MHz, CD3OD-d4) δC 173.3 (C, C-11), 168.2 (C, C-5), 167.8 (C, C-25), 159.1 (C, C-27), 158.9 (C, C-18), 138.7 (C, C-20), 134.1 (C, C-2), 123.7 (CH, C-16), 123.3 (C, C-15), 121.9 (CH, C-26), 116.9 (C, C-14), 112.5 (CH, C-17), 100.6 (CH, C-19), 85.5 (C, C-12), 71.0 (C, C-22), 69.4 (CH, C-13), 61.0 (CH, C-6), 56.2 (CH3, C-30), 50.5 (CH2, C-21), 46.6 (CH, C-3), 45.3 (CH2, C-9), 30.3 (CH3, C-23), 30.1 (CH3, C-24), 29.6 (CH2, C-7), 27.4 (CH3, C-28), 24.0 (CH2, C-8), 21.3 (CH3, C-29). 以上核磁数据都与文献(Xie et al, 2015)数据基本一致, 因此将化合物5定为asperfumigatin。
化合物6: 淡黄色固体, 分子式为C22H25N3O5, ESI-MS m/z 434.2 [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$+ 18.1 (c 0.3, CHCl3)。1H NMR (400 MHz, CDCl3) δH 7.90 (1H, s, H-1), 7.79 (1H, d, J = 8.7 Hz, H-16), 6.83 (1H, d, J = 0.4 Hz, H-19), 6.80 (1H, dd, J = 8.7, 2.0 Hz, H-17), 5.87 (1H, d, J = 9.7 Hz, H-3), 4.79 (1H, d, J = 9.2 Hz, H-21), 4.42 (1H, dd, J = 8.5, 6.9 Hz, H-6), 4.20 (1H, br dd, J = 11.0, 5.5 Hz, H-12), 3.82 (3H, s, H3-18), 3.64 (1H, m, H-9a), 3.62 (1H, m, H-9b), 3.50 (1H, dd, J = 15.5, 5.5 Hz, H-13b), 3.10 (1H, dd, J = 15.5, 11.0 Hz, H-13a), 2.08 (1H, m, H-7a), 1.99 (3H, s, H3-23), 1.96 (1H, m, H-7b), 1.96 (2H, m, H2-8), 1.66 (3H, s, H3-24).13C NMR (100 MHz, CDCl3) δC 171.0 (C, C-11), 166.1 (C, C-5), 156.6 (C, C-18), 137.5 (C, C-20), 134.6 (C, C-22), 130.1 (C, C-2), 123.9 (CH, C-21), 121.2 (CH, C-15), 120.7 (C, C-16), 109.8 (CH, C-17), 105.3 (C, C-14), 95.0 (CH, C-19), 83.0 (C, C-12), 68.6 (CH, C-13), 58.7 (CH, C-6), 55.7 (CH3, C-18), 50.1 (CH, C-3), 45.3 (CH2, C-9), 29.1 (CH2, C-7), 22.5 (CH2, C-8), 25.7 (CH3, C-23), 18.3 (CH3, C-24). 以上核磁数据都与文献(Zhang et al, 2007)数据基本一致, 因此将化合物6定为12R, 13S-dihydroxyfumitremorgin C。
化合物7: 黄色油状物, 分子式为C22H25N3O6, ESIMS 450.2 m/z [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ + 147.2 (c 0.1, CHCl3)。1H NMR (600 MHz, DMSO-d6) δH 7.54 (1H, d, J = 8.7 Hz, H-4), 7.42 (1H, br s, 9-OH), 6.30 (1H, dd, J = 8.7, 1.9 Hz H-5), 6.07 (1H, d, J = 1.9 Hz, H-7), 5.38 (1H, br s, 8-OH), 4.80 (1H, s, H-18), 4.80 (1H, s, H-19), 4.75 (1H, s, H-8), 4.60 (1H, dd, J = 9.6, 7.6 Hz, H-12), 3.78 (3H, s, 6-OCH3), 3.56 (2H, m, H2-15), 2.37 (2H, m, H2-13b), 2.06 (2H, m, H2-13a), 2.06 (2H, m, H2-14b), 1.94 (2H, m, H2-14a), 1.63 (3H, s, H3-22), 1.38 (3H, s, H3-21).13C NMR (150 MHz, DMSO-d6) δC 202.2 (C, C-3), 170.0 (C, C-6), 169.3 (C, C-11), 164.9 (C, C-7a), 166.1 (C, C-17), 138.9 (C, C-20), 126.7 (CH, C-4), 121.1 (CH, C-19), 111 (C, C-3a), 109.5 (CH, C-5), 93.7 (CH, C-7), 86.5 (C, C-9), 76.6 (C, C-2), 72.8 (CH, C-8), 60.6 (CH, C-12), 56.2 (CH3, 6-OCH3), 54.7 (CH, C-18), 45.2 (CH2, C-15), 28.0 (CH2, C-15), 25.8 (CH3, C-22), 23.0 (CH2, C-14), 18.6 (CH3, C-21)。以上核磁数据都与文献(Zhang et al, 2019)数据基本一致, 因此将化合物7定为spirocyclic diketopiperazine alkaloid。
化合物8: 淡黄色固体, 分子式为C23H30N2O2, ESIMS 367.2 m/z [M + H]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ − 92.5 (c 0.20, CHCl3)。1H NMR (600 MHz, CD3OD-d4) δH 7.07 (1H, d, J = 8.0 Hz, H-14), 6.94 (1H, t, J = 7.3 Hz, H-13), 6.59 (1H, d, J = 7.2 Hz, H-12), 6.14 (1H, dd, J = 17.3, 10.7 Hz, H-22), 5.6 (1H, s, H-9), 5.06 (2H, d, J = 6.6 Hz, H2-23a), 5.04 (2H, s, H2-23b), 3.21(1H, d, H-10), 2.72 (2H, m, H2-7a), 2.67 (H, m, H-5), 2.65 (2H, m, H2-7b), 2.65 (2H, m, H2-4), 2.30 (3H, s, H3-17), 2.12 (H, m, H-8), 1.89 (3H, s, H3-25), 1.52 (3H, s, H3-20), 1.51 (3H, s, H3-21), 1.31 (3H, d, J = 7.3 Hz, H3-18). 13C NMR (150 MHz, CD3OD-d4) δC 171.2 (C, C-24), 146.2 (CH, C-22), 137.1 (C, C-2), 133 (C, C-15), 127.9 (C, C-16), 127.6 (C, C-11), 121.1 (CH, C-13), 111.4 (CH, C-12), 110.1 (CH2, C-23), 107.7 (CH, C-14), 104.6 (C, C-3), 71.4 (CH, C-9), 61.9 (CH, C-5), 57.5 (CH2, C-7), 42.5 (CH3, C-17), 39.0 (C, C-19), 38.9 (CH, C-10), 33.1 (CH, C-8), 27.4 (CH2, C-4), 26.7 (CH3, C-21), 26.6 (CH3, C-20), 19.6 (CH3, C-25), 15.5 (CH3, C-18). 以上核磁数据都与文献(Liu et al, 2020)数据基本一致, 因此将化合物8定为fumigaclavine C。
化合物9: 白色无定形粉末, 分子式为C18H22N2O2, ESIMS 299.2 m/z [M + H]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ − 91.3 (c 0.20, CHCl3)。1H NMR (600 MHz, CD3OD-d4) δH 7.21 (1H, d, J = 8.0 Hz, H-14), 7.09 (1H, dd, J = 8.1, 7.5 Hz, H-13), 7.07 (1H, d, J = 1.16 Hz, H-2), 6.75 (1H, d, J = 7.2 Hz, H-12), 5.77 (1H, br s, H-9), 3.50 (1H, d, J = 12.76 Hz, H-10), 3.43 (2H, dd, J = 20.4, 10.3 Hz, H2-4), 2.75 (2H, br d, J = 11.9 Hz, H2-7a), 2.66 (1H, br d, J = 10.6 Hz, H-5), 2.45 (H, s, H-17), 2.09 (1H, m, H-8), 1.88 (3H, s, H3-25), 1.97 (2H, br d, J = 11.9 Hz, H2-7b), 1.43 (3H, d, J = 7.8 Hz, H3-18). 13C NMR (150 MHz, CD3OD-d4) δC 172 (C, C-24), 135.6 (C, C-15), 127.5 (C, C-11), 126.8 (C, C-16), 124.0 (CH, C-13), 120.8 (CH, C-2), 113.8 (CH, C-12), 111 (C, C-3), 107.8 (CH, C-14), 70.1 (CH, C-9), 64.3 (CH, C-5), 58.3 (CH2, C-7), 42.7 (CH3, C-17), 39.5 (CH, C-10), 33.4 (CH, C-8), 25.3 (CH2, C-4), 21.1 (C, C-25), 15.8 (CH3, C-18). 以上核磁数据都与文献(Zhang et al, 2021)数据基本一致, 因此将化合物9定为fumigaclavine A。
化合物10: 无色晶体, 分子式为C22H18N4O4, ESIMS 425.1 m/z [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ − 70 (c 0.48, MeOH)。1H NMR (600 MHz, DMSO-d6) δH 8.28 (1H, br s, H-24), 8.18 (1H, br d, J = 7.8 Hz, H-19), 7.86 (1H, td, J = 7.5 Hz, H-21), 7.69 (1H, br d, J = 8.0 Hz, H-22), 7.58 (1H, br t, J = 7.5 Hz, H-20), 7.50 (1H, br d, J = 7.8 Hz, H-8), 7.49 (1H, br d, J = 7.8 Hz, H-5), 7.43 (1H, td, J = 7.9, 1.0 Hz, H-7), 7.25 (1H, td, J = 7.6, 1.0 Hz, H-6), 6.70 (1H, s, H-25), 5.92 (1H, br s, H-14), 5.60 (1H, s, H-2), 4.61 (1H, q, J = 6.8 Hz, H-11), 2.93 (1H, t, J = 12.5Hz, H-13b), 2.53 (1H, dd, J = 12.5, 2.5 Hz, H-13a), 1.59 (3H, d, J = 6.8 Hz, H3-12). 13C NMR (DMSO-d6, 150 MHz) δC 172.5 (C, C-10), 166.2 (C, C-17), 160.5 (C, C-15), 147.8 (CH, C-24), 147.2 (C, C-23), 139.2 (C, C-9), 137.2 (C, C-4), 135.2 (CH, C-21), 130.4 (CH, C-7), 127.8 (CH, C-20), 127.7 (CH, C-22), 126.9 (CH, C-19), 126.0 (CH, C-6), 125.3 (CH, C-5), 121.6 (C, C-18), 115.0 (CH, C-8), 83.0 (CH, C-2), 76.8 (C, C-3), 60.0 (CH, C-11), 49.1 (CH, C-14), 38.6 (CH2, C-13), 14.5 (CH3, C-12). 以上核磁数据都与文献(Jiao et al, 2006; 冯婷 等, 2024)数据基本一致, 因此将化合物10定为chaetominine。
化合物11: 白色固体, 分子式为C21H16N4O2, ESIMS 379.1 m/z [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ − 258 (c 0.243, EtOAc)。1H NMR (600 MHz, CD3OD-d4) δH 8.23 (1H, dd, J = 8.0, 1.2 Hz, H-10), 8.13 (1H, br s, H-19), 7.80 (1H, dd, J = 7.7, 1.4 Hz, H-8), 7.70 (1H, d, J = 8.0 Hz, H-7), 7.52 (1H, dt, J = 7.5, 0.9 Hz, H-9), 7.41 (1H, br d, J = 8.0 Hz, H-24), 7.37 (1H, br s, H-2), 7.13 (1H, t, J = 7.7 Hz, H-21), 7.13 (1H, t, J = 7.7 Hz, H-22), 7.01 (1H, t, J = 7.5 Hz, H-23), 5.93 (1H, dd, J = 4.7, 2.7 Hz, H-14), 3.50 (1H, dd, J = 17.3, 2.7 Hz, H-15a), 3.35 (1H, dd, J = 7.2, 4.6 Hz, H-15b), 2.23 (3H, s, H3-16). 13C NMR (150 MHz, CD3OD-d4) δC 172.5 (C, C-1), 162.1 (C, C-12), 155.8 (C, C-4), 148.9 (C, C-6), 137 (C, C-20), 136.2 (CH, C-8), 134.7 (C, C-18), 129.0 (C, C-25), 128.7 (CH, C-9), 127.8 (CH, C-7), 123.9 (CH, C-10), 121.8 (CH, C-22), 121.0 (C, C-11), 119.1 (CH, C-23), 117.5 (CH, C-24), 112.8 (CH, C-21), 107.2 (C, C-17), 56.6 (CH, C-14), 56.4 (C, C-3), 27.2 (CH2, C-15), 18.8 (CH3, C-16). 以上核磁数据都与文献(Sun et al, 2019)数据基本一致, 因此将化合物11定为fumiquinazoline J。
化合物12: 白色无定形固体, 分子式为C10H14N2O5, ESIMS 265.1 m/z [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$+ 78.0 (c 0.61, H2O)。1H NMR (600 MHz, DMSO-d6) δH 7.64 (1H, s, H-4), 6.11 (1H, t, J = 6.9 Hz, H-1'), 4.38 (1H, br d, H-3'), 3.89 (1H, br d, H-4'), 3.55 (2H, m, H2-5'), 2.07 (2H, m, H2-2'), 1.74 (3H, s, H3-5).13C NMR (150 MHz, DMSO-d6) δC 165.2 (C, C-2), 151.4 (C, C-1), 137.3 (CH, C-4), 110.6 (C, C-3), 87.7 (CH, C-4'), 84.8 (CH, C-1'), 71.2 (CH2, C-3'), 62.1 (CH2, C-5'), 40.9(CH2, C-2'), 12.9 (CH3, C-5). 以上核磁数据都与文献数据(Hannoda et al, 2007)基本一致, 因此将化合物12定为thymidine。
化合物13: 黄色无定形粉末, 分子式为C31H37NO10, ESIMS 584.3 m/z [M + H]+, 比旋光值为$\text{ }\!\![\!\!\text{ }\!\!\alpha\!\!\text{ }\!\!]\!\!\text{ }_{\text{D}}^{\text{18}}$ + 65.8(c 1.0, CHCl3)。1H NMR (400 MHz, CD3OD-d4) δH 9.03 (1H, d, J =1.5 Hz, H-2''), 8.63 (1H, dd, J = 4.7, 1.5 Hz, H-6''), 8.29 (1H, ddd, J = 8.2, 2.0, 1.3 Hz, H-4''), 7.55 (1H, dd, J = 8.3, 5.0 Hz, H-5''), 6.82 (1H, s, H-5'), 4.99 (1H, dd, J = 6.8, 4.9 Hz, H-1), 4.99 (1H, d, J = 3.67 Hz, H-13), 4.81 (1H, m, H-7), 3.78 (2H, dd, J = 21.5, 12.0 Hz, H2-11), 2.16 (2H, m, H2-3b), 2.13 (3H, s, 7-OCOCH3), 2.06 (3H, s, 1-OCOCH3), 2.03 (3H, s, 11-OCOCH3), 1.91 (2H, m, H2-2b), 1.86 (2H, m, H2-2a), 1.82 (2H, m, H2-8b), 1.63 (2H, m, H2-8a), 1.75 (3H, s, H3-14), 1.62 (1H, m, H-5), 1.49 (3H, s, H3-12), 1.38 (2H, m, H2-3b), 0.92 (3H, s, H3-15). 13C NMR (CD3OD-d4, 100 MHz) δC 172.6 (C, OCOCH3-11), 172.5 (C, OCOCH3-7), 172.0 (C, OCOCH3-1), 165.1 (C, C-2'), 164.1 (C, C-4'), 158.2 (C, C-6'), 151.9 (CH, C-6''), 147.4 (CH, C-2''), 135 (CH, C-4''), 129.2 (C, C-3''), 125.5 (CH, C-5''), 104.5 (C, C-3'), 101.0 (CH, C-5'), 84.5 (CH, C-6), 79.8 (CH, C-7), 75.3 (CH, C-1), 66.0 (CH2, C-11), 60.3 (CH, C-13), 55.5 (CH, C-5), 46.6 (CH, C-9), 41.7 (C, C-10), 39.1 (C, C-4), 37.1 (CH2, C-3), 26.1 (CH2, C-8), 23.8 (CH2, C-2), 21.1 (CH3, OCOCH3-7), 21.0 (CH3, OCOCH3-1), 20.7 (CH3, OCOCH3-11), 17.9 (CH3, C-12), 16.8 (CH3, C-14), 13.5 (CH3, C-15). 以上核磁数据都与文献(Zou et al, 2021)数据基本一致, 因此将化合物13定为pyripyropene A。
化合物14: 白色针状固体, 分子式为C18H18O7, ESIMS 369.1 m/z [M + Na]+。1H NMR (600 MHz, CDCl3) δH 7.03 (1H, d, J = 2.2 Hz, H-5), 6.63 (1H, d, J = 2.2 Hz, H-3), 6.47 (1H, br s, H-5'), 6.07 (1H, br s, H-3'), 3.69 (3H, s, H3-9), 3.69 (3H, s, H3-8), 3.38 (3H, s, H3-7'), 2.30 (3H, s, H3-8'), 3.71 (3H, s, H3-3').13C NMR (150 MHz, CDCl3) δC 199.6 (C, C-10), 166.3 (C, C-7), 164.5 (C, C-6'), 161.1 (C, C-2'), 157.3 (C, C-2), 156.3 (C, C-4), 148.1 (C, C-4'), 128.6 (C, C-6), 128.4 (C, C-1), 111.4 (C, C-1'), 111.2 (CH, C-5'), 108.0 (CH, C-5), 103.3 (CH, C-3'), 103.1 (CH, C-3), 56.4 (CH3, C-9), 55.9 (CH3, C-7'), 52.4 (CH3, C-8), 22.7 (CH3, C-8'). 以上核磁数据都与文献数据(Liu et al, 2006)基本一致, 因此将化合物14定为monomethylsulochrin。
化合物15: 黄色针状固体, 分子式为C16H12O5, ESIMS 285.1 m/z [M + H]+。1H NMR (400 MHz, DMSO-d6), δH 13.24 (1H, s, 1-OH), 11.25 (1H, s, 6-OH ), 7.43 (1H, d, J = 2.3 Hz, H-4), 7.20 (1H, d, J = 2.3 Hz, H-5), 7.12 (1H, d, J = 0.6 Hz, H-2), 6.83 (1H, d, J = 2.16 Hz, H-7), 3.90 (3H, s, 8-OCH3), 2.39 (3H, s, H3-11). 13C NMR (100 MHz, DMSO-d6) δC 186.4 (C, C-9), 182.4 (C, C-10), 164.6 (C, C-6), 163.6 (C, C-8), 161.8 (C, C-1), 146.8 (C, C-3), 136.9 (C, C-10a), 132.1 (C, C-4a), 124.3 (CH, C-2), 119.2 (CH, C-4), 114.5 (C, C-9a), 112.7 (C, C-8a), 107.1 (CH, C-5), 105.1 (CH, C-7), 56.4 (CH3, 8-OCH3), 21.5 (CH3, C-11). 以上核磁数据都与文献(Zhao et al, 2018)数据基本一致, 因此将化合物15定为questin。
化合物16: 白色无定形粉末, 分子式为C33H44O8, ESI-MS m/z 591.3 [M + Na]+, 比旋光值为$[\alpha]_{\mathrm{D}}^{25}$ − 41 (c 0.1, CHCl3)。1H NMR (400 MHz, CDCl3) δH 7.31 (1H, d, J = 10.1 Hz, H-1), 6.44 (1H, d, J =14.4 Hz, H-16), 6.01 (1H, d, J = 10.1 Hz, H-2), 5.38 (1H, m, H-25), 5.24 (1H, br s, H-6), 5.11 (1H, t, J = 7.3 Hz, H-24), 2.93 (1H, m, H-22b), 2.83 (1H, m, H-22a), 2.77 (1H, dq, J = 12.5, 6.8 Hz, H-4), 2.68 (1H, br d, J = 11.0 Hz, H-13), 2.68 (1H, dd, J = 13.0, 2.6 Hz, H-9), 2.57 (1H, m, H-23b), 2.54 (1H, m, H-5), 2.47 (1H, m, H-12b), 2.42 (1H, m, H-15b), 2.42 (1H, m, H-23a), 2.20 (1H, d, J = 14.4 Hz, H-15a), 2.12 (3H, s, H3-6-OCOCH3), 1.95 (3H, s, H3-16-OCOCH3), 1.87 (1H, m, H-11b), 1.85 (1H, m, H-12a), 1.70 (3H, s, H3-27), 1.62 (3H, s, H3-26), 1.55 (1H, m, H-11a), 1.45 (3H, s, H3-19), 1.29 (3H, d, J = 6.9 Hz, H3-28), 1.19 (3H, s, H3-18), 0.93 (3H, s, H3-29). 13C NMR (100 MHz, CDCl3) δC 208.7 (C, C-8), 201.3 (C, C-3), 173.9 (C, C-21), 170.1 (C, 16-OCOCH3), 168.9 (C, 6-OCOCH3), 157.2 (CH, C-1), 147.8 (C, C-17), 132.9 (C, C-25), 130.3 (C, C-20), 127.8 (CH, C-2), 122.7 (CH, C-24), 73.8 (CH, C-6), 73.4 (CH, C-16), 52.6 (C, C-8), 46.6 (C, C-14), 49.4 (CH, C-13), 47.2 (CH, C-5), 41.7 (CH, C-9), 40.6 (CH2, C-15), 40.4 (CH, C-4), 38.1 (CH, C-10), 28.6 (CH2, C-22), 28.3 (CH2, C-23), 27.5 (CH3, C-19), 25.9 (CH, C-12), 25.7 (CH3, C-27), 23.9 (CH2, C-11), 20.7 (CH3, 6-OCOCH3), 20.5 (CH3, 16-OCOCH3), 18.3 (CH3, C-26), 17.9 (CH3, C-18), 17.7 (CH3, C-29), 13.1 (CH3, C-28). 以上核磁数据都与文献(Sawadsitang et al, 2015)数据基本一致, 因此将化合物16定为helvolic acid。

2.2 化合物抗菌活性

本文测定了化合物对L. monocytogenes和P. aeruginosa的抗菌效果。结果表明, 阳性药对L. monocytogene的最小抑菌浓度(minimum inhibitory concentration, MIC)为0.78μg·mL−1, 将化合物抗菌效果与其对比。化合物16对L. monocytogenes具有较强的抗菌活性, MIC为4.03μg·mL−1。化合物13—15对 L. monocytogenes具有中等抗菌活性, MIC为分别为66.25、78.63、64.54μg·mL−1。本文得到的化合物对P. aeruginosa均无抑制效果。

2.3 化合物自由基清除活性

本文采用了DPPH自由基清除能力和ABTS自由清除能力的方法对化合物的抗氧化活性进行测试, VC作为阳性对照(图2)。结果表明, 化合物6具有较强的DPPH自由基清除活性, IC50为9.82μg·mL−1; 化合物6、8、9、14具有较强的ABTS自由基清除活性, IC50分别为2.72、0.43、3.23和1.96μg·mL−1。
图2 化合物DPPH(a)和ABTS(b)自由基清除能力(IC50)

Fig. 2 Radical scavenging ability (IC50) of the compounds. (a) DPPH; (b) ABTS

3 结果和讨论

本文对石花菜共附生烟曲霉Aspergillus fumigatus 9-1次级代谢产物进行化学研究, 通过传统分离纯化(TLC、硅胶柱、凝胶柱、HPLC等)手段, 系统研究了它们的次级代谢产物, 并评估了单体化合物的抗菌活性和体外抗氧化活性, 结果从中分离到7个色胺-脯氨酸二酮哌嗪(1—7)、2个麦角素型生物碱(8、9)、2个吲哚喹唑林酮类生物碱 (10、11)、1个嘧啶苷类(12), 1个混源倍半萜类(13)、1个苄基联苯类(14)、1个蒽醌(15)、1个四环三萜类(16)。其中化合物1首次从Aspergillus中发现。抗菌活性测定结果表明化合物16对L. monocytogenes具有较强的抗菌活性, MIC为4.03μg·mL−1, 化合物13—15对 L. monocytogenes具有中等抗菌活性, MIC分别为66.25、78.63、64.54μg·mL−1。虽然16的MIC与四环素(MIC, 0.78μg·mL−1)对比有不足, 但其MIC值仍远低于其他测试化合物。现有结论认为16对革兰氏阳性菌都有抗菌效果, 包括无乳链球菌(MIC, 8μg·mL−1)和金黄色葡萄球菌(MIC, 16μg·mL−1), 并且已经被开发利用(Kong et al, 2018; 冯婷 等, 2024)。本研究得到的所有化合物对P. aeruginosa均无抗菌效果, 这说明了不同化合物抗菌谱的特异性。体外活性抗氧化活性结果表明, 化合物6具有较强的DPPH自由基清除活性, IC50为9.82μg·mL-1; 化合物6、8、9、14具有较强的ABTS自由基清除活性, IC50分别为2.72、0.43、3.23和1.96μg·mL-1。化合物6及多个其他化合物在DPPH和ABTS自由基清除实验中结果都比较理想, 表明了它作为天然抗氧化剂的潜力。
此外, 这些化合物中的部分生物碱已被报道有较强的细胞毒活性, 如化合物5对人前列腺癌细胞细胞系显示出细胞毒活性, 还有研究证明化合物14对K562细胞也有一定的细胞毒活性(Liu et al, 2006; 丛梦静 等, 2022)。研究发现化合物10对人类白血病K562和结肠癌SW1116细胞系的细胞毒性大于抗癌药物氟尿嘧啶, 然而Zou 等人对化合物10经活性评估, 在10μg·mL-1浓度下, 化合物对人肝癌 HepG2 细胞没有明显的抑制毒性作用, 这表明化合物10可能对人类白血病癌细胞具有选择性生长抑制作用(Jiao et al, 2006; Xie et al, 2015; Zou et al, 2021)。这些结果都为上述化合物后续药用价值的开发提供了一定的参考, 以推动其从实验室走向实际应用。本论文研究成果为抗氧化剂、防腐剂和抗菌药物的研发提供先导分子, 同时也为食品工业中真菌毒素的分析、检测奠定基础。

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