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Journal of Tropical Oceanography >

2025 , Vol. 44 >Issue 1: 146 - 153

DOI: https://doi.org/10.11978/2024051

Exploitation of Marine Resources

γ-Aromatic butenolides lignans from soft coral-associated symbiotic and epiphytic fungi Aspergillus terreus EGF7-0-1(I)

  • HE Yueming , 1 ,
  • ZHAO Lining , 2 ,
  • CHEN Xinqi 1 ,
  • HE Jiahong 1 ,
  • FAN Hao 1 ,
  • CHEN Leyi 1 ,
  • ZHANG Cuixian , 1 ,
  • HE Xixin , 1
Expand
  • 1. School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
  • 2. Library, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
ZHANG Cuixian, email: ;
HE Xixin, email:

Copy editor: SUN Cuici

Received date: 2024-03-05

  Revised date: 2024-03-17

  Online published: 2024-04-02

Supported by

National Natural Science Foundation of China(82273845)

Special Fund of Guangdong Provincial Department of Natural Resources(GDNRC[2023]37)

Special Fund of Guangdong Provincial Department of Natural Resources(GDNRC[2020]039)

Special Fund of Guangdong Provincial Department of Natural Resources(GDNRC[2021]048)

Innovation and Entrepreneurship Program for College Students in Guangdong Province(202310572224)

Innovation and Entrepreneurship Program for College Students in Guangdong Province(202310572291)

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Abstract

In order to discover the novel and bioactive secondary metabolites, the soft coral-associated symbiotic and epiphytic fungi Aspergillus terreus EGF7-0-1 were fermented in rice media. The secondary metabolites were isolated using various chromatographic technologies, yielding eight γ-aromatic butenolide lignans. Based on nuclear magnetic resonance (NMR), high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) and comparison with the literature, their structures were determined to be butyrolactones I−V (1−5), aspernolide E (6), asperteretal D (7) and terrusnolide A (8). In this study, the inhibitory activity of these compounds against TDP1 (Tyrosyl-DNA phosphodiesterase 1) in vitro was studied for the first time, but it was unfortunate that compounds 1−8 did not show any inhibitory abilities against TDP1. This study provides theoretical data for the discovery of TDP1 inhibitors.

Key words： traditional marine Chinese medicine soft coral; Aspergillus terreus; γ-aromatic butenolides lignans; structural identification; TDP1 inhibitors

Cite this article

HE Yueming , ZHAO Lining , CHEN Xinqi , HE Jiahong , FAN Hao , CHEN Leyi , ZHANG Cuixian , HE Xixin . γ-Aromatic butenolides lignans from soft coral-associated symbiotic and epiphytic fungi Aspergillus terreus EGF7-0-1(I)[J]. Journal of Tropical Oceanography, 2025 , 44(1) : 146 -153 . DOI: 10.11978/2024051

海洋本草是中医药的重要组成部分, 由于海洋生境的特异性, 决定了其能产生迥异于陆生生物源的结构新颖、活性显著的天然产物。γ-丁烯内酯(γ-butenolides)是天然产物中一类重要骨架类型。γ-丁烯内酯类化合物来自多种极端环境(如盐土、植物、动物、海洋生物和湿地)的生物源, 是真菌重要的代谢产物(San-Martín et al, 2011; Bao et al, 2020; Peng et al, 2022)。由于其骨架类型的多样性, 该类化合物具有广泛的生理活性, 如抗氧化、抗病毒、抗菌、抗炎、α-葡萄糖苷酶抑制等活性(Davis, 1991; Hosoe et al, 2005; Chatterjee et al, 2021; Chen et al, 2022; Fan et al, 2022a)。海洋来源真菌土曲霉的次级代谢产物是γ-芳基丁烯内酯类化合物重要的天然来源, 目前共报道了178个此类化合物(Fan et al, 2022b), 主要结构为内酯环取代位置不同的三个类型(3, 4-、4, 5-或3, 5-二取代)和内酯环开环特征型。TDP1(tyrosyl-DNA phosphodiesterase 1)是近年来发现的DNA修复酶, 被认为是潜在的肿瘤治疗靶点(Hu et al, 2021)。至今为止, 虽然已报道50多个TDP1小分子抑制剂(胡翥 等, 2016; Laev et al, 2016), 结构类型上包括过渡金属钒酸盐和钨酸盐、氨基糖苷类、呋喃脒二胺型、磷酸酪氨酸模拟物、吲哚异喹啉类等, 但TDP1抑制剂的研发仍处于起步阶段, 尚未见关于此类药物上市或进入临床的相关报道。因此, 发现具有高活性及选择性的TDP1新型抑制剂对癌症治疗具有重要意义。
课题组长期致力于海洋微生物来源结构新颖的活性次生代谢物发现研究, 前期从Aspergillus terreus (A. terreus)中分离得到甾体类、酚酸类、杂萜类等化合物并对其活性进行研究(Cheng et al, 2021; 司徒美霞 等, 2023)。本研究聚焦A. terreus EGF7-0-1代谢物中的γ-芳环丁烯内酯类化学成分研究。采用多种分离手段, 从大米培养基的乙酸乙酯提取物中共分离得到8个γ-芳环丁烯内酯类化合物(图1): butyrolactones I—V(1—5)、aspernolide E(6)、asperteretal D(7)和terrusnolide A(8)。采用噻唑蓝(MTT)法首次对其进行TDP1体外抑制活性测试, 化合物1—8均无抑制作用。本研究为TDP1抑制剂的发现提供借鉴。
图1 来源于A. terreus EGF7-0-1中γ-芳环丁烯内酯类化合物1—8

Fig. 1 Compounds 1−8 from A. terreus EGF7-0-1

1 实验部分

1.1 仪器、试剂与材料

论文中菌种、微生物发酵用培养基、次生代谢产物提取、分离和化合物结构鉴定所用的仪器、试剂和材料与文献相同(陈传兵 等, 2022; 黎咏怡 等, 2022)。

1.2 菌株来源

菌株 EGF7-0-1是从中国南海三亚海域鳞指形软珊瑚Sinularia scabra中分离得到(由广西中医药大学海洋药物研究所刘昕明副研究员鉴定), 对菌株EGF7-0-1进行DNA提取, 通过 PCR(polymerase chain reaction) 扩增真菌核糖体ITS基因区段, 将扩序结果与NCBI(National Center for Biotechnology Information)网站的GenBank数据库中已知序列进行比对, 鉴定为Aspergillus terreus (Fan et al, 2023)。菌株保存于广州中医药大学中药学院海洋天然药物实验室(编号EGF7-0-1)。

1.3 菌株规模发酵

用无菌接种环沾取菌株EGF7-0-1, 接种至无菌PDA(potato dextrose agar)固体培养基, 待培养基上长出单菌落, 挑取单菌落接种到PDB(potato dextrose broth)液体培养基(1L培养瓶, 每瓶400mL)。恒温摇床上(28℃, 165r·min−1)培养2~3天, 即可获得菌株种子液。取10mL种子培养液接种于装有50g大米培养基的1L培养瓶中, 25℃静置培养 30天。共培养100L。

1.4 提取分离

菌株培养结束后, 向培养瓶中加入250mL乙酸乙酯灭活; 并置于摇床上振摇提取3次(165r·min−1, 每次8h), 过滤、合并乙酸乙酯浸提液, 经过减压浓缩后共获得浸膏(500g)。浸膏经硅胶柱色谱进行粗分, 用石油醚-乙酸乙酯体系(100: 0~0: 100, 体积比)梯度洗脱, 经TLC(thin-layer chromatography)薄层色谱追踪合并得到11个组分Fr.1—Fr.11。Fr.5(200g)经硅胶柱色谱分离, 用石油醚-乙酸乙酯(100: 0~0: 100, 体积比)梯度洗脱, 根据TLC薄层色谱分析结果合并相同流份得到8个亚组分Fr.5-1—Fr.5-6。组分Fr.5-2(6g)经Sephadex LH-20葡聚糖凝胶柱分离, 用二氯甲烷-甲醇(3: 7, 体积比)进行洗脱, 得到组分Fr.5-2-12(1.4g)。Fr.5-2-12经半制备HPLC(high performance liquid chromatography)分离(流动相MeOH: H2O=7: 23, 体积比, 流速2mL·min-1), 得到15个组份Fr.5-2-12-1—Fr.5-2-12-15。取Fr.5-2-12-1经半制备HPLC纯化(流动相MeOH: H2O=66: 34, 体积比, 流速2mL·min-1), 得化合物1 (27.7mg, tR=20.0min)、2 (11.0mg, tR=36.6min)。Fr.5-2-12-2经半制备HPLC纯化(流动相MeOH: H2O=65: 35, 体积比, 流速2mL·min-1), 得到化合物3 (18.5mg, tR=33.6min)。Fr.5-2-12-6经半制备HPLC纯化(流动相MeOH: H2O=65: 35, 体积比, 流速2mL·min-1), 得到化合物4 (12.7mg, tR=18.5min)。Fr.5-2-12-9经半制备HPLC纯化(流动相MeOH: H2O=60: 40, 体积比, 流速2mL·min-1), 得到化合物5 (13.7mg, tR=25.5min)。Fr.5-2-12-15经半制备HPLC纯化(流动相MeOH: H2O=53: 47, 体积比, 流速2mL·min-1), 得到化合物6 (130.3mg, tR=22.0min)、7 (13.7mg, tR=25.5min)、8 (9.3mg, tR=36.6min)。

1.5 物理常数和波谱数据

化合物1, 淡黄色油胶状物, $[\alpha ]_{\mathrm{D}}^{25}$ = +84.3(c 0.1, MeOH)。HR-ESI-MS m/z 425.1578 [M+H]+(计算值为C24H24O7, 425.1600), 1H NMR(CD3OD, 400 MHz)δH 和13C NMR CD3OD, 100 MHz)δC见表1。
表1 化合物1—4的NMR数据表(CD3OD)

Tab. 1 NMR data of 1−4 in CD3OD

No. 1 2 3 4
δH, mult, (J in Hz) δC, type δH, mult, (J in Hz) δC, type δH, mult, (J in Hz) δC, type δH, mult, (J in Hz) δC, type
2 / 170.3, C / 171.9, C / 171.5, C / 171.1, C
3 / 139.4, C / 140.3, C / 139.8, C / 139.8, C
4 / 129.2, C / 130.0, C / 129.3, C / 128.6, C
5 / 86.7, C / 86.9, C / 86.7, C / 86.9, C
6 a 3.45, d, 15.0
b 3.55, d, 15.0		 39.4, CH2 3.45, s 39.4, CH2 3.44, s 39.5, CH2 a 3.09, d, 14.7
b 3.43, d, 14.7		 39.6, CH2
7 / 171.3, C / 171.9, C / 170.3, C / 170.5, C
8 3.70, s 53.9, CH3 3.77, s 53.8, CH3 3.78, s 53.9, CH3 4.26, q, 7.1 63.6, CH2
9 / / / / / / 1.22, t, 7.1 14.3, CH3
1' / 122.8, C / 123.9, C / 123.1, C / 123.2, C
2' 7.58, d, 8.4 130.2, CH 7.61, d, 8.8 130.0, CH 7.57, d, 8.8 130.3, CH 7.60, d, 8.9 130.4, CH
3' 6.68, d, 8.4 116.5, CH 6.86, d, 8.8 116.5, CH 6.88, d, 8.8 116.6, CH 6.87, d, 8.9 116.6, CH
4' / 158.8, C / 158.8, C / 159.3, C / 159.3, C
5' 6.68, d, 8.4 116.5, CH 6.86, d, 8.8 116.5, CH 6.88, d, 8.8 116.6, CH 6.87, d, 8.9 116.6, CH
6' 7.58, d, 8.4 130.2, CH 7.61, d, 8.8 130.0, CH 7.57, d, 8.8 130.3, CH 7.60, d, 8.9 130.4, CH
1'' / 124.9, C / 123.9, C / 123.1, C / 123.6, C
2'' 6.45, s 132.1, CH 6.65, d, 8.8 132.6, CH 6.46, d, 2.1 132.8, CH 6.50, d, 2.1 132.4, CH
3'' / 128.2, C 6.51, d, 8.8 125.5, CH / 129.5, C / 129.8, C
4'' / 154.6, C / 157.4, C / 153.4, C / 155.1, C
5'' 6.48, d, 8.4 115.0, CH 6.51, d, 8.8 125.5, CH 6.55, d, 8.5 117.2, CH 6.56, d, 8.1 115.1, CH
6'' 6.52, d, 8.4 129.6, CH 6.65, d, 8.8 132.6, CH 6.52, dd, 8.5, 2.1 130.4, CH 6.54, dd, 8.1, 2.1 129.3, CH
7'' 3.05, br d, 6.2 28.5, CH2 / / 2.81, dd, 16.6, 5.20 31.9, CH2 3.07, br d, 7.4 28.7, CH2
8'' 5.02, t, 6.2 123.1, CH / / 3.66, m 70.4, CH 5.07, m 123.6, CH
9'' / 133.0, C / / / 77.9, C / 132.9, C
10'' 1.52, s 25.9, CH3 / / 1.26, s 25.8, CH3 1.57, s 25.9, CH3
11'' 1.61, s 17.7, CH3 / / 1.17, s 20.9, CH3 1.67, s 17.8, CH3

注: “/”表示无数据

化合物2, 淡黄色油胶状物, $[\alpha ]_{\mathrm{D}}^{25}$= +55.3(c 0.1, MeOH)。HR-ESI-MS m/z 356.0867 [M+H]+(计算值为C19H16O7, 356.0896), 1H NMR(CD3OD, 400 MHz)δH和13C NMR(CD3OD, 100 MHz)δC见表1。
化合物3, 淡黄色油胶状物, $[\alpha ]_{\mathrm{D}}^{25}$= +69.8(c 0.1, MeOH)。HR-ESI-MS m/z 440.1459 [M+H]+(计算值为C24H24O8, 440.1471), 1H NMR(CD3OD, 400 MHz)δH和13C NMR(CD3OD, 100 MHz)δC见表1。
化合物4, 淡黄色油胶状物, $[\alpha ]_{\mathrm{D}}^{25}$= +98.7(c 0.1, MeOH)。HR-ESI-MS m/z 438.1670 [M+H]+(计算值为C25H26O7, 438.1679), 1H NMR(CD3OD, 400 MHz)δH和13C NMR(CD3OD, 100 MHz)δC见表1。
化合物5, 淡黄色油胶状物, $[\alpha ]_{\mathrm{D}}^{25}$= +82.6(c 0.1, MeOH)。HR-ESI-MS m/z 440.1477 [M+H]+(计算值为C24H24O8, 440.1471), 1H NMR(CD3OD, 400 MHz)δH和13C NMR(CD3OD, 100 MHz)δC见表2。
表2 化合物5—8的NMR数据表(CD3OD)

Tab. 2 NMR data of 5−8 in CD3OD

No. 5 6 7 8
δH, mult, (J in Hz) δC, type δH, mult, (J in Hz) δC, type δH, mult, (J in Hz) δC, type δH, mult, (J in Hz) δC, type
1 / / / / / / 3.61, s 49.0, CH2
2 / 170.2, C / 171.5, C / 173.0, C / 210.0, C
3 / 139.4, C / 140.1, C / 139.7, C 3.59, s 49.0, CH2
4 / 129.4, C / 128.9, C / 158.2, C / /
5 / 86.7, C / 86.7, C 5.50, s 104.1, CH / /
6 3.40, s 39.4, CH2 a 3.45, d, 15.0
b 3.55, d, 15.0		 39.5, CH2 a 3.57, d, 15.2
b 3.91, d, 15.2		 32.8, CH2 / /
7 / 171.2, C / 170.5, C 3.53, s 57.4, CH3 / /
8 3.72, s 53.9, CH3 3.79, s 53.9, CH3 / / / /
1' / 122.9, C / 123.2, C / 121.5, C / 126.4, C
2' 7.55, d, 8.4 130.2, CH 7.57, d, 8.5 130.3, CH 7.35, d, 8.7 131.6, CH 6.93, d, 8.8 131.6, CH
3' 6.86, d, 8.4 116.6, CH 6.87, d, 8.5 116.6, CH 6.87, d, 8.7 116.1, CH 6.72, d, 8.8 116.3, CH
4' / 159.1, C / 159.3, C / 159.4, C / 157.3, C
5' 6.86, d, 8.4 116.6, CH 6.87, d, 8.5 116.6, CH 6.87, d, 8.7 116.1, CH 6.72, d, 8.8 116.3, CH
6' 7.55, d, 8.4 130.2, CH 7.57, d, 8.5 130.3, CH 7.35, d, 8.7 131.6, CH 6.93, d, 8.8 131.6, CH
1'' / 120.3, C / 123.2, C / 129.7, C / 128.8, C
2'' 6.45, s 132.8, CH 6.44, d, 2.0 132.0, CH 6.78, m 130.9, CH 6.79, m 129.5, CH
3'' / 125.9, C / 129.5, C / 128.0, C / 155.1, C
4'' / 159.1, C / 153.3, C / 155.1, C / 128.8, C
5'' 6.50, d, 8.4 117.1, CH 6.56, d, 8.0 116.5, CH 6.70, d, 8.6 116.4, CH 6.76, m 131.6, CH
6'' 6.44, d, 8.4 130.3, CH 6.54, dd, 8.0, 2.0 129.4, CH 6.83, m 128.2, CH 6.67, m 123.8, CH
7'' a 2.50, dd, 16.8, 6.4
b 2.72, dd, 16.8, 6.4		 39.4, CH2 6.10, d, 9.8 123.2, CH 3.25, d, 7.1 29.0, CH2 3.26, d, 7.2 29.1, CH2
8'' 3.64, m 70.2, CH 5.78, d, 9.8 132.0, CH 5.27, t, 7.1 123.7, CH 5.28, t, 7.2 126.2, CH
9'' / 77.8, C / 77.2, C / 133.3, C / 133.0, C
10'' 1.14, s 25.7, CH3 1.33, s 28.1, CH3 1.71, s 25.9, CH3 1.73, s 25.9, CH3
11'' 1.21, s 21.0, CH3 1.33, s 28.1, CH3 1.66, s 17.8, CH3 1.69, s 17.8, CH3

注: “/”表示无数据

化合物6, 淡黄色油胶状物, [α]25 D= +70.3(c 0.1, MeOH)。HR-ESI-MS m/z 422.1358 [M+H]+(计算值为C24H22O7, 422.1366), 1H NMR(CD3OD, 400 MHz)δH和13C NMR(CD3OD, 100 MHz)δC见表2。
化合物7, 淡黄色油胶状物, $[\alpha ]_{\mathrm{D}}^{25}$= +15.2(c 0.1, MeOH)。HR-ESI-MS m/z 380.1607 [M+H]+(计算值为C23H24O5, 380.1624), 1H NMR(CDCl3, 400 MHz)δH和13C NMR(CD3OD, 100 MHz)δC见表2。
化合物 8, 淡黄色油胶状物, HR-ESI-MS m/z 310.1556 [M+H]+(计算值为C20H22O3, 310.1569), 1H NMR(CD3OD, 400 MHz)δH和13C NMR(CD3OD, 100 MHz)δC见表2。

1.6 化合物活性测试

采用文献方法(Wei et al, 2022)测试化合物1—8对TDP1抑制活性测试, 试药浓度为100μmol·L−1。

2 结果与分析

化合物1, 结合HR-ESI-MS(m/z 425.1578 [M+H]+, Cal. 425.1600)、13C NMR及DEPT(24个C: 3个CH3、2个CH2、8个CH、11个C)确定其分子式C24H24O7, 不饱和度13。NMR显示该化合物含有2个羰基(δC 171.3, C, C-7; 170.3, C, C-2)、1个对位取代苯环(δH 7.58, 2H, d, J = 8.4 Hz, H-2'/H-6', δC 130.2, CH, C-2'/C-6'; δH 6.68, 2H, d, J = 8.4 Hz, H-3'/H-5', δC 116.5, CH, C-3'/C-5')、1个1, 3, 4-三取代苯环(δH 6.45, 1H, s, H-2'', δC 132.1, CH, C-2''; δH 6.48, 1H, d, J = 8.4 Hz, H-5'', δC 115.0, CH, C-5''; δH 6.52, 1H, d, J = 8.4 Hz, H-6'', δC 129.6, CH, C-6'')、一个全取代五元α, β-不饱和内酯环(δC 170.3, C, C-2; 139.4, C, C-3; 129.2, C, C-4; 86.7, C, C-5)、1个甲酯基(δH 3.70, 3H, s, H3-8, δC 53.9, CH3, C-8; δC 171.3, C, C-7)、一个含三取代双键的异戊烯基(δH 3.05, 2H, d, J = 6.2 Hz, H-7'', δC 28.5, CH2, C-7''; δH 5.02, 1H, t, J = 6.2 Hz, H-8'', δC 123.1, CH, C-8''; δH 1.52, 3H, s, H3-10'', δC 25.9, CH3, C-10''; δH 1.61, 3H, s, H3-11'', δC 17.7, CH3, C-11''; δC 133.0, C, C-9'')和一个饱和亚甲基信号(δH 3.45, 1H, d, J = 15.0 Hz, Ha-6, δH 3.55, 1H, d, J = 15.0 Hz, Hb-6, δC 39.4, CH2, C-6)。根据以上官能团信息推断化合物1为4, 5芳基取代的γ-丁烯内酯类化合物(Pal et al, 2020; Fan et al, 2022b)。将1的NMR数据和[α]25 D= +84.3(c 0.1, MeOH)与butyrolactone I的文献数据(Liu et al, 2018)对比, 二者数据基本一致, 确定1为butyrolactone I。
化合物2, 结合HR-ESI-MS(m/z 356.0867 [M+H]+, Cal. 356.0896)、NMR及DEPT(19个C: 1个CH3、1个CH2、8个CH、9个C)确定其分子式为C19H16O7, 不饱和度12。将2的NMR数据与1对比, 发现二者十分相似, 只是比1少了一组三取代异戊烯基信号; 同时2的NMR显示其含有两个对位取代芳信号: (δH 7.61, 2H, d, J = 8.8 Hz, H-2'/H-6', δC 130.0, CH, C-2'/C-6'; δH 6.86, 2H, d, J = 8.8 Hz, H-3'/H-5', δC 116.5, CH, C-3'/C-5'; δH 6.65, 2H, d, J = 8.8 Hz, H-2''/H-6'', δC 132.6, CH, C-2''/C-6''; δH 6.51, 2H, d, J = 8.8 Hz, H-3''/H-5'', δC 125.5, CH, C-3''/C-5'')。将2的NMR数据及[α]25 D= +55.3(c 0.1, MeOH)与butyrolactone II的文献数据(Sun et al, 2018)对比, 二者数据基本一致, 确定2为butyrolactone II。
化合物3, 结合HR-ESI-MS(m/z 440.1459 [M+H]+, Cal. 440.1471)、NMR及DEPT(24个C: 3个CH3、2个CH2、8个CH、11个C)确定其分子式为C24H24O8, 不饱和度13。NMR数据与化合物1相比, 二者十分相似, 只是比1缺少了一组三取代双键信号(δH 5.02, 1H, t, J = 6.2 Hz, H-8'', δC 123.1, CH, C-8''; δC 133.0, C, C-9'')而增加了两个连氧碳信号(δH 3.66, 1H, m, H-8'', δC 70.4, CH, C-8''; δC 77.9, C, C-9''), 同时甲基质子信号(1: δH 1.52, 3H, s, H3-10''; 1.61, 3H, s, H3-11''; 3: δH 1.26, 3H, s, H3-10''; 1.17, 3H, s, H3-11'')明显向高场移动; 且根据分子量和不饱和度信号推测其为环氧结构。将3的NMR数据和[α]25 D=+69.8(c 0.1, MeOH)与butyrolactone III的文献数据(Bao et al, 2021)对比, 二者数据基本一致, 确定3为butyrolactone III。
化合物4, 结合HR-ESI-MS(m/z 438.1670 [M+H]+, Cal. 438.1679)、NMR及DEPT(25个C: 3个CH3、3个CH2、8个CH、11个C)确定其分子式为C25H26O7, 不饱和度13。将4的NMR数据与1对照, 发现二者十分相似, 只是将1中的甲酯基(δH 3.70, 3H, s, H3-8, δC 53.9, CH3, C-8; δC 171.3, C, C-7)变成了乙酯基(δH 4.26, 2H, q, J = 7.1 Hz, H-8, δC 63.6, CH2, C-8; δH 1.22, 3H, t, J = 7.1 Hz, H3-9, δC 14.3, CH3, C-9; δC 170.5, C, C-7)。将4的NMR数据和[α]25 D=+98.7(c 0.1, MeOH)与butyrolactone VII的文献数据(巩婷 等, 2014)对比, 二者数据基本一致, 确定4为butyrolactone VII。
化合物5, 结合HR-ESI-MS(m/z 440.1477 [M+H]+, Cal. 440.1471)、NMR及DEPT(24个C: 3个CH3、2个CH2、8个CH、11个C)确定其分子式为C24H24O8, 不饱和度13。将5的NMR数据与1对比, 发现二者十分相似。只比1缺少了一组双键信号(δH 5.02, 1H, t, J = 6.2 Hz, H-8'', δC 123.1, CH, C-8''; δC 133.0, C, C-9''), 而增加了两个饱和连氧碳信号(δH 3.64, 1H, m, H-8'', δC 70.2, CH, C-8''; δC 77.8, C, C-9''), 同时甲基质子信号明显向高场移动(1: δH 1.52, 3H, s, H3-10''; 1.61, 3H, s, H3-11''; 5: δH 1.14, 3H, s, H3-10''; 1.21, 3H, s, H3-11'')。将5的NMR数据和[α]25 D=+82.6(c 0.1, MeOH)与butyrolactone V的文献数据(Wang et al, 2012)对比, 二者数据基本一致, 确定5为butyrolactone V。
化合物6, 结合HR-ESI-MS(m/z 422.1358 [M+H]+, Cal. 422.1366)、NMR及DEPT(24个C: 3个CH3、1个CH2、9个CH、11个C)确定其分子式为C24H22O7, 不饱和度14。将6的NMR数据与5对比发现二者十分相似, 只比5少了一个连氧次甲基信号(δH 3.64, 1H, m, H-8'', δC 70.2, CH, C-8''), 而多了一个二取代双键信号(δH 6.10, 1H, d, J = 9.8 Hz, H-7'', δC 123.2, CH, C-7''; δH 5.78, 1H, d, J = 9.8 Hz, H-8'', δC 132.0, CH, C-8'')。将6的NMR数据和[α]25 D= +70.3(c 0.1, MeOH)与aspernolide E的文献数据(Ye et al, 2014)对比, 二者数据基本一致, 确定6为aspernolide E。
化合物7结合HR-ESI-MS(m/z 380.1607 [M+H]+, Cal. 380.1624)、NMR及DEPT(23个C: 3个CH3、2个CH2、9个CH、9个C)确定其分子式为C23H24O5, 不饱和度12。对比化合物7和1的NMR数据发现, 二者信号十分相似, 只缺少了一个甲酯基信号(δH 3.70, 3H, s, H3-8, δC 53.9, CH3, C-8; δC 171.3, C, C-7), 而增加一个异头碳相连(δH 5.50, 1H, s, H-5, δC 104.1, CH, C-5)的甲氧基信号(δH 3.53, 3H, s, H-7, δC 57.4, CH3, C-7), 同时一个亚甲基(1: δH 3.45, 1H, d, J = 15.0 Hz, Ha-6, δH 3.55, 1H, d, J = 15.0 Hz, Hb-6, δC 39.4, CH2, C-6)的信号明显向低场移动(7: δH 3.57, 1H, d, J = 15.2 Hz, Ha-6, δH 3.91, 1H, d, J = 15.2 Hz, Hb-6, δC 32.8, CH2, C-6)。暗示7可能是3, 4芳基取代的γ-丁烯内酯类化合物(Fan et al, 2022b)。将7的NMR数据和[α]25 D= +15.2(c=0.1, MeOH)与asperteretal D的文献数据(Sun et al, 2018)对比, 二者数据基本一致, 确定7为asperteretal D。
化合物8, 结合HR-ESI-MS(m/z 310.1556 [M+H]+, Cal. 310.1569)、NMR及DEPT(20个C: 2个CH3、3个CH2、8个CH、7个C)确定其分子式为C20H22O3, 不饱和度10。NMR显示8同样存在一个对位取代的苯基(δH 6.93, 2H, d, J = 8.8 Hz, H-2'/H-6', δC 131.6, CH, C-2'/C-6'; δH 6.72, 2H, d, J = 8.8 Hz, H-3'/H-5', δC 116.3, CH, C-3'/C-5')、一个1, 3, 4-三取代苯基(δH 6.79, 1H, m, H-2'', δC 129.5, CH, C-2''; δH 6.76, 1H, m, H-5'', δC 131.6, CH, C-5''; δH 6.67, 1H, m, H-6'', δC 123.8, CH, C-6'')、一个酮羰基(δC 210.0, C, C-2)、一个含三取代双键的异戊烯基(δH 3.26, 2H, d, J = 7.2 Hz, H-7'', δC 29.1, CH2, C-7''; δH 5.28, 1H, t, J = 7.2 Hz, H-8'', δC 126.2, CH, C-8''; δH 1.73, 3H, s, H3-10'', δC 25.9, CH3, C-10''; δH 1.69, 3H, s, H3-11'', δC 17.8, CH3, C-11''; δC 133.0, C, C-9'')和两个明显处于低场的亚甲基(δH 3.61, 2H, s, H-1, δC 49.0, CH2, C-1; δH 3.59, 2H, s, H-3, δC 49.0, CH2, C-3), 暗示着8与上述γ-丁烯内酯类结构均不同, 可能为裂环类型的芳基取代的γ-丁烯内酯类化合物, 目前文献共有4个此类结构报道(Fan et al, 2022b)。将8的NMR数据与terrusnolide A的文献数据(Qi et al, 2018)对比, 二者数据基本一致, 确定8为terrusnolide A。

3 结论

本文从海洋软珊瑚共附生真菌A. terreus EGF7-0-1的大米培养基中共分离得到8个γ-芳环丁烯内酯类化合物, 结构类型包括4, 5-二取代、3, 4-二取代、裂环三类骨架类型。γ-芳环丁烯内酯类化合物与中药中C8-C8′位相连的木脂素类成分(Shen et al, 2022)相比, 其结构特征为两分子苯丙素首先通过C7-C8'连接, 然后“C9与C8′-OH”(4, 5取代)或“C8-OH与C9′-COOH”(3, 4取代)形成丁烯酸内酯杂环(Fan et al, 2022b)。因此, 从结构上看, γ-芳环丁烯内酯类化合物属于木脂素类化合物, 海洋微生物来源的γ-芳环丁烯内酯类化合物丰富了陆生木脂素的骨架结构。体外药理活性研究表明, 化合物1—8在100 μmol·L−1下均无抑制TDP1作用。而文献调研表明化合物1可以抑制BV-2细胞NF-κB通路来减轻神经炎症(Zhang et al, 2018); 化合物2—5和8具有清除自由基的能力(巩婷 等, 2014; Ye et al, 2014; Liu et al, 2018; Sun et al, 2018; Bao et al, 2020; Shen et al, 2022); 化合物7具有α-葡萄糖苷酶抑制活性(半抑制浓度IC50为8.6μmol·L−1)(Liu et al, 2018)。本研究表明, 菌株EGF7-0-1对γ-芳环丁烯内酯类化合物的产生具有较强的代谢能力, 为寻找TDP1抑制剂提供丰富的骨架信息, 也为后续此菌株次生代谢产物的深入挖掘和活性研究提供参考。

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