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Journal of Tropical Oceanography >

2025 , Vol. 44 >Issue 2: 56 - 63

DOI: https://doi.org/10.11978/2024104

Marine Biology

Secondary metabolites from the fungal-bacterial symbiont Aspergillus spelaeus GXIMD 04541 / Sphingomonas echinoides GXIMD 04532 derived from Mauritia arabica

  • YANG Jie , 1, 2 ,
  • YAO Feihua 1, 2 ,
  • LI Xiaoyan 1, 2 ,
  • SHI Jieyu 2, 3 ,
  • YI Xiangxi 2, 3 ,
  • GAO Chenghai , 1, 2
Expand
  • 1. Institute of Marine Drugs, Guangxi University of Chinese Medicine, Nanning 530200, China
  • 2. Guangxi Key Laboratory of Marine Drugs, Nanning 530200, China
  • 3. Faculty of Pharmacy, Guangxi University of Chinese Medicine, Nanning 530200, China
GAO Chenghai. email:

Copy editor: YIN Bo

Received date: 2024-05-17

  Revised date: 2024-06-19

  Online published: 2024-07-03

Supported by

National Natural Science Foundation of China(U20A20101)

Guangxi Key Research and Development Programme(AB24010109)

Guangxi Key Laboratory of Marine Drugs(2302603)

High-level Talent Inheritance, Innovation Team of Guangxi Traditional Chinese Medicine(2022A007)

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Abstract

In order to investigate the secondary metabolites of the fungal-bacterial symbiont Aspergillus spelaeus GXIMD 04541 / Sphingomonas echinoides GXIMD 04532 derived from Mauritia arabica, a series of isolation and purification techniques, including silica gel chromatography, gel column chromatography, and semi-preparative high performance liquid chromatography (HPLC), were employed. The structures of compounds were identified by spectroscopic analysis and literature comparison. Cytotoxic activity evaluation of compounds was conducted using the MTT (Methylthiazolyldiphenyl tetrazolium bromide) assay. Consequently, seven compounds were obtained from rice fermentation extracts of symbiont GXIMD 04541 / 04532, which were identified as trichalasins C (1), aspochalasin E (2), aspochalasin H (3), aspochalasin I (4), aspochalasin K (5), citreofuran (6), and cyclothiocurvularin B methyl ester (7). Activity assays demonstrated that compounds 2, 4, and 5 exhibited cytotoxic activity against PC3 cells, with IC50 (half maximal inhibitory concentration) values of 17.23, 15.18 and 8.71 μmol·L-1, respectively. However, compounds 1-5 showed no cytotoxicity against 22Rv1 cells.

Key words： Mauritia arabica; fungal-bacterial symbiont; secondary metabolites; cytotoxicity

Cite this article

YANG Jie , YAO Feihua , LI Xiaoyan , SHI Jieyu , YI Xiangxi , GAO Chenghai . Secondary metabolites from the fungal-bacterial symbiont Aspergillus spelaeus GXIMD 04541 / Sphingomonas echinoides GXIMD 04532 derived from Mauritia arabica[J]. Journal of Tropical Oceanography, 2025 , 44(2) : 56 -63 . DOI: 10.11978/2024104

“共生”是指两种不同生物之间所形成的紧密关系, 通常至少有益于一方(Xu et al, 2020)。生存于内生细菌与宿主真菌共生而形成的结合体称为真菌-细菌共生体, 内共生细菌在宿主中扮演着重要的角色, 通过调控真菌宿主的生长、分布和次级代谢过程深刻影响宿主细胞的功能, 有助于真菌宿主的适应、发育和进化(Hoffman et al, 2010)。陆生动植物中的致病真菌(Cheng et al, 2022)和菌根真菌(Sharmin et al, 2018 )一直是真菌-细菌共生体的主要研究对象。内共生细菌伯克霍尔德菌Burkholderia endofungorum与菌根真菌Rhizopus microsporus建立了互利关系, 产生抗肿瘤化合物rhizoxin (Partida-Martinez et al, 2005)。毛霉菌门真菌含有内共生细菌, 这些内细菌可以产生有毒的次生代谢物, 保护真菌宿主免受捕食性线虫的侵害(Okrasińska et al, 2021)。由于海洋生物的生存环境与陆地生物不同, 它们的新陈代谢途径也极为特殊, 逐渐研究人员开始关注海洋生境对真菌-细菌共生体的影响(张涵 等, 2021)。为了适应海洋中高压、低温、缺氧和低营养的恶劣环境, 海洋真菌-细菌共生体会进化出独特的代谢途径和防御机制(马丽丽 等, 2021)。这一过程极可能产生结构新颖、活性显著的次生代谢物, 成为海洋天然产物挖掘的重要来源。鞠建华课题组从南海北部大亚湾海底沉积物中得到一对真菌-细菌共生体Spiromastix sp. SCSIO F190 / Alcaligenes faecalis SCSIO B001, 并从中分离到活性化合物spiromarmycins (Shao et al, 2020)。Spiromarmycins对多种细菌耐药菌和真菌均具有很好的抑制活性, 有望开发成新型抗菌剂(鞠建华 等, 2022)。该研究证实了海洋中存在着真菌-细菌共生现象, 并且真菌-细菌共生体能够产生活性显著的次生代谢产物。本课题前期从广东雷州珍稀海洋生物国家级自然保护区采集的阿拉伯宝螺中分离获得真菌细菌共生体Aspergillus spelaeus GXIMD 04541 / Sphingomonas echinoides GXIMD 04532, 进一步从该菌株中分离鉴定出7个化合物, 并测试了部分化合物的细胞毒活性。

1 材料和方法

1.1 主要仪器与试剂

Bruker 500 MHz超导核磁共振仪(Bruker, 德国), Waters e2695高效液相色谱仪(high performance liquid chromatography, HPLC)(WATERS, 美国), 步琦C-620中压制备色谱仪(上海沃珑仪器有限公司, 中国), EYEL4 N-1400旋转蒸发仪(上海爱朗仪器有限公司, 中国), CP3102十万分之一电子天平(上海志荣电子科技有限公司, 中国), 优普UPH-11-20TN超纯水机(ELGA, 英国), COSMOSIL分析色谱柱(Nacalai tesque, 日本), WFH-2038暗箱式紫外分析仪(杭州齐威仪器有限公司, 中国), 200~300目正相硅胶(青岛海洋化工有限公司, 中国), HSGF 254薄层层析硅胶板(thin-layer chromatography silica gel plate, TLC)(烟台江友硅胶开发有限公司, 中国), 十八烷基硅烷键合硅胶(上海麦克林生化科技股份有限公司, 中国), Sephadex LH-20 (上海麦克林生化科技股份有限公司, 中国), 石油醚、乙酸乙酯、甲醇、乙醇(分析纯)(成都科隆化学品有限公司, 中国), 甲醇、乙腈(色谱纯)(上海星可高纯溶剂有限公司, 中国), 氯仿(分析纯)(昆山金城试剂有限公司, 中国)。

1.2 菌株来源及鉴定

生物样品采自广东雷州珍稀海洋生物国家级自然保护区, 由广西中医药大学刘昕明副研究员鉴定为阿拉伯宝螺(Mauritia arabica)。将螺肉消毒后, 用研钵和研杵研磨, 获得1.0g样品。然后将样品与10mL无菌海水混合均匀, 吸取0.1mL混合溶液在马铃薯葡萄糖琼脂(potato dextrose agar, PDA)平板上涂布, 置于25℃下培养7d。并经过多次纯化培养, 筛选出真菌纯培养物。采用真菌18S rRNA引物ITS1和ITS4扩增真菌ITS靶区(凌娟 等, 2023), 扩增产物通过1%琼脂糖凝胶电泳检验, 对18S rRNA扩增结果为阳性的真菌进行16S rRNA 测序鉴定。使用细菌16S rRNA通用引物27F和1492R进行PCR扩增(Frank et al, 2008)。PCR反应程序为: 95℃预变性10min, 94℃变性30s, 55℃退火30s, 72℃延伸60s, 34个循环结束后, 72℃延伸5min。通过1%琼脂糖凝胶电泳和Bio-RAD凝胶成像仪检验, 阳性结果委托生工生物工程(上海)股份有限公司进行测序。对菌株进行绿色荧光核酸染料(green fluorescent nucleic acid stain, SYTO9)特异性染色(Stiefel et al, 2015), 利用激光共聚焦显微镜在波长488nm处观察真菌菌丝内部特征。

1.3 菌株培养与发酵

菌株接种至500mL的锥形瓶(内含200mL马铃薯葡萄糖液体培养基, 已灭菌)中, 28℃、180r·min-1恒温振荡培养3d作为种子液。配制150瓶大米培养基(1L锥形瓶中含有大米 80.0g, 酵母粉 0.4g, 葡萄糖 0.4g, 海盐 1.6g, 加纯水至120mL), 经高压灭菌锅121℃灭菌20min, 待冷却后置于超净台中紫外灭菌30min, 用一次性无菌吸管取15mL种子液接种, 室温静置培养20d。挑取发酵第10天和第20天的共生体菌丝进行16S rRNA扩增和SYTO9特异性染色对发酵过程进行共生验证。发酵结束后加入等体积的乙酸乙酯进行多次提取, 直至提取液接近无色, 经旋转蒸发仪减压浓缩, 得到总浸膏190.3g。

1.4 分离与纯化

将总浸膏用100~200目的硅胶拌样, 经正相硅胶柱色谱(200~300目硅胶)进行等梯度洗脱分离, 用氯仿-甲醇(体积比为100/0、98/2、95/5、85/15、80/20、70/30、0/100)体系进行梯度洗脱, 每个梯度洗脱4个有效柱体积约为18L。根据TLC的结果, 将所接馏分最终合并得到5个组分, 分别标记为Fr.1 (68.4g)、Fr.2 (12.6g)、Fr.3 (30.4g)、Fr.4 (16.8g)和Fr.5 (9.7g)。Fr.3为黑棕色油状浸膏, 经中压反相柱色谱分离, 以甲醇/水体系(10%、20%、25%、30%、35%、40%、45%、50%、55%、60%、70%、90%、100%)梯度洗脱, 得到6个中压组分(Fr.3.1—Fr.3.6)。Fr.3.4经半制备高效液相色谱(CH3CN/H2O = 43/57, 流速2mL·min-1)得到化合物1 (7.0mg, tR = 30.0min)、化合物6 (10.6mg, tR = 29.2min)、化合物7 (4.8mg, tR = 29.4min)。Fr.3.6经半制备高效液相色谱(CH3CN/H2O = 56/44, 流速2mL·min-1)得到化合物2 (14.3mg, tR = 36.1min)、化合物3 (7.1mg, tR = 38.4min)。Fr.3.5经半制备高效液相色谱(CH3OH/H2O = 85/15, 流速2mL·min-1)得到化合物4 (4.0mg, tR = 34.1min)、化合物5 (8.9mg, tR = 34.2min)。

1.5 前列腺癌细胞毒活性测定方法

选用前列腺癌细胞PC3和22Rv1为试验对象, 通过溴化噻唑蓝四氮唑(methylthiazolyldiphenyl tetrazolium bromide, MTT)(高亚欣 等, 2023)对部分化合物进行细胞毒活性测定。将细胞以4×103个·孔-1的密度接种到96孔板中, 培养24h后, 化合物以每个20μmol·L-1的浓度处理细胞72h。然后每孔加入20μLMTT溶液(5.0mg·mL-1), 在37℃下培养4h后检测。通过GraphPad Prism 8 (Sandiego, 加拿大)分析软件计算化合物的生长抑制能力, 并用半数最大抑制浓度(half maximal inhibitory concentration, IC50)值表示, 阳性对照为多柔比星。

2 结果与分析

2.1 共生体GXIMD 04541 / 04532的鉴定

16S rRNA和18S rRNA扩增产物的1%琼脂糖凝胶电泳结果显示, 菌株中存在真菌条带(500bp), 也有细菌的条带(1500bp), 见图1。16S rRNA和18S rRNA基因测序与GenBank网站比对, 真菌GXIMD 04541鉴定为Aspergillus spelaeus, 细菌GXIMD 04532鉴定为Sphingomonas echinoides。SYTO9特异性染色结果(图2)显示, 真菌菌丝内有较明显的绿色荧光, 绿色荧光呈较规则的圆点, 表明该真菌菌丝内存在内共生细菌, 将其命名为Aspergillus spelaeus GXIMD 04541 / Sphingomonas echinoides GXIMD 04532, 保藏在广西中医药大学海洋药物研究院。
图1 GXIMD 04541 / 04532的琼脂糖凝胶电泳条带

a. 真菌GXIMD 04541的电泳条带; b. 细菌GXIMD 04532的电泳条带。M为100bp的片段marker

Fig. 1 Agarose gel electrophoresis strips of GXIMD 04541/04532. (a) Electrophoretic strip of fungus GXIMD 04541; (b) Electrophoretic strip of bacteria GXIMD 04532. M is a fragment marker of 100 bp

图2 绿色荧光核酸染料染色的激光扫描共聚焦荧光显微图

a. 共生菌菌丝SYTO9染色的显微图; b. 共生菌分生孢子SYTO9染色的显微图 。图中绿色荧光为内共生细菌, 存在于共生菌的菌丝和分子孢子中

Fig. 2 Laser scanning confocal micrographs of SYTO9 staining. (a) micrograph of SYTO9 staining of symbiotic mycelium; (b) micrograph of SYTO9 staining of symbiotic conidia. The green fluorescence in the figure is endosymbiotic bacteria, present in the hyphae and conidia of symbiot

2.2 共生体GXIMD 04541 / 04532的发酵

利用1%琼脂糖凝胶电泳和SYTO9特异性染色对共生体发酵过程进行了共生验证(图3、图4), 16S rRNA扩增产物的1%琼脂糖凝胶电泳结果显示, 第10天和第20天的共生体菌丝内都存在细菌条带(1500bp); SYTO9染色结果显示第10天和第20天的共生体菌丝内有绿色荧光, 表明了共生体GXIMD 04541 / 04532在发酵过程中真菌与细菌处于共生状态。
图3 GXIMD 04541 / 04532发酵第10天菌丝的琼脂糖凝胶电泳(a)和SYTO9染色结果(b)

M为100bp的片段marker, 绿色荧光为内共生细菌

Fig. 3 Agarose gel electrophoresis (a) and SYTO9 staining (b) of mycelium of GXIMD 04541 / 04532 at the 10th day of fermentation. M is a fragment marker of 100 bp, and green fluorescence is endosymbiotic bacteria

图4 GXIMD 04541 / 04532发酵第20天菌丝的琼脂糖凝胶电泳(a)和SYTO9特异性染色结果(b)

M为100bp的片段marker, 绿色荧光为内共生细菌

Fig. 4 Agarose gel electrophoresis (a) and SYTO9 staining (b) of mycelium of GXIMD 04541 / 04532 at the 20th day of fermentation. M is a fragment marker of 100 bp, and green fluorescence is endosymbiotic bacteria

2.3 化合物的结构解析

从共生体GXIMD 04541 / 04532的大米发酵提取物中分离7个单体化合物, 分别鉴定为trichalasins C (1)、aspochalasin E (2)、aspochalasin H (3)、aspochalasin I (4)、aspochalasin K (5)、citreofuran (6) 和cyclothiocurvularin B methyl ester (7), 如下图5所示。
图5 化合物1—7的化学结构

Fig. 5 Chemical structures of compounds 1—7

化合物 1: 白色粉末, UV(MeOH) λmax (log ε) 202 (1.82)、216 (1.71)、234 (0.53)nm; HR-ESI-MS m/z: 456.2369 [M+Na]+。1H NMR (500MHz, Methanol-d4) δH 7.05 (1H, dd, J=15.3, 2.2Hz, H-19), 6.06 (1H, d, J= 10.6Hz, H-13), 5.91 (1H, dd, J=15.3, 2.2Hz, H-20), 4.48 (1H, s, H-18), 3.89 (1H, d, J=9.8Hz, H-7), 3.78 (1H, s, H-4), 3.73 (1H, dd, J=7.6, 2.6Hz, H-17), 3.44 (1H, dd, J=8.2, 6.4Hz, H-3), 3.27 (1H, d, J=9.8Hz, H-8), 2.11 (1H, m, H-15a), 2.40 (1H, m, H-15b), 1.35 (1H, dd, J= 10.4, 15.2Hz, H-16a), 2.05 (1H, m, H-16b), 1.77 (3H, s, H-11), 1.70 (3H, s, H-12), 1.67 (1H, m, H-22), 1.46 (1H, m, H-10a), 1.58 (1H, m, H-10b), 1.39 (3H, s, H-25), 0.95 (3H, d, J=6.7Hz, H-23), 0.93 (3H, d, J=6.7Hz, H-24)。 13C NMR (125MHz, Methanol-d4) δC 175.0 (C-1), 57.0 (C-3), 53.0 (C-4), 127.6 (C-5), 135.1 (C-6), 72.6 (C-7), 47.4 (C-8), 86.7 (C-9), 46.3 (C-10), 18.3 (C-11), 14.9 (C-12), 123.2 (C-13), 142.5 (C-14), 41.5 (C-15), 28.3 (C-16), 79.4 (C-17), 74.0 (C-18), 154.2 (C-19), 120.4 (C-20), 168.6 (C-21), 26.2 (C-22), 23.5 (C-23), 22.5 (C-24), 15.6 (C-25)。以上数据与文献(Ding et al, 2012)报道基本一致, 确定化合物 1 为 trichalasins C。
化合物 2: 白色粉末, UV(MeOH) λmax (log ε) 202 (3.05)nm; HR-ESI-MS m/z: 442.2550 [M+Na]+。1H NMR (500MHz, CDCl3) δH 6.93 (1H, s, NH), 6.02 (1H, d, J=11.1Hz, H-13), 5.39 (1H, m, H-7), 3.90 (1H, d, J= 5.3Hz, H-20a), 2.00 (1H, m, H-20b), 3.62 (1H, m, H-19), 3.59 (1H, m, H-18), 3.44 (1H, m, H-17), 3.20 (1H, d, J= 11.2Hz, H-8), 3.14 (1H, dt, J=8.3, 3.7Hz, H-3), 2.56 (1H, m, H-5), 2.50 (1H, d, J=3.5Hz, H-4), 2.12 (2H, m, H-15), 1.73 (3H, d, J=2.5Hz, H-12), 1.57 (1H, m, H-22), 1.56 (1H, m, H-16a), 1.31 (1H, m, H-16b), 1.48 (3H, s, H-25), 1.20 (1H, m, H-10b), 1.11 (1H, m, H-10a), 1.18 (3H, s, H-11), 0.89 (3H, d, J=6.0Hz, H-23), 0.87 (3H, d, J= 6.0Hz, H-24)。13C NMR (125MHz, CDCl3) δC 176.0 (C-1), 50.8(C-3), 51.2 (C-4), 35.4 (C-5), 139.8 (C-6), 125.8 (C-7), 43.4 (C-8), 68.3 (C-9), 48.8 (C-10), 13.5 (C-11),20.0 (C-12), 124.9 (C-13), 137.0 (C-14), 37.8 (C-15), 29.5 (C-16), 72.6 (C-17), 78.8 (C-18), 69.0 (C-19), 44.2 (C-20), 212.1 (C-21), 25.0 (C-22), 21.6 (C-23), 23.7 (C-24), 15.9 (C-25)。以上数据与文献(Si et al, 2018)报道基本一致, 确定化合物 2 为 aspochalasin E。
化合物 3: 白色粉末, UV(MeOH) λmax (log ε) 200 (2.90)nm; HR-ESI-MS m/z: 440.2415 [M+Na]+。1H NMR (500MHz, CDCl3) δH 6.55 (1H, s, NH), 5.97 (1H, d, J=11.1Hz, H-13), 5.39 (1H, m, H-7), 4.19 (1H, d, J= 2.0Hz, H-20), 4.05 (1H, s, H-17), 3.38 (1H, d, J=8.1Hz, H-18), 3.09 (1H, dt, J=10.2, 3.5Hz, H-3), 3.03 (1H, d, J= 11.2Hz, H-8), 2.64 (1H, d, J=3.5Hz, H-4), 2.62 (1H, dd, J=7.0, 1.5Hz, H-19), 2.58 (1H, m, H-5), 2.40 (1H, m, H-15a), 2.11 (1H, m, H-15b), 2.25 (1H, t, J=13.0Hz, H-16a), 2.10 (1H, m, H-16b), 1.77 (3H, s, H-12), 1.54 (1H, m, H-22), 1.43 (3H, s, H-25), 1.32 (1H, m, H-10a), 1.19 (1H, m, H-10b), 1.21 (3H, d, J=6.7Hz, H-11), 0.91 (3H, d, J=6.0Hz, H-23), 0.91 (3H, d, J=6.0Hz, H-24)。13C NMR (125MHz, CDCl3) δC 174.9 (C-1), 52.1 (C-3), 53.0 (C-4), 35.6 (C-5), 140.9 (C-6), 125.6 (C-7), 44.2 (C-8), 67.7 (C-9), 48.7 (C-10), 13.8 (C-11), 20.3 (C-12), 124.6 (C-13), 136.1 (C-14), 35.0 (C-15), 29.2 (C-16), 72.8 (C-17), 78.4 (C-18), 62.1 (C-19), 55.0 (C-20), 205.9 (C-21), 25.2 (C-22), 23.8 (C-23), 21.3 (C-24), 15.6 (C-25)。以上数据与文献(Tomikawa et al, 2002)报道基本一致, 确定化合物 3 为 aspochalasin H。
化合物 4: 白色粉末, UV(MeOH) λmax (log ε) 201 (3.10)nm; HR-ESI-MS m/z: 440.2394 [M+Na]+。1H NMR (500MHz, CDCl3) δH 7.15 (1H, dd, J=15.3, 2.3Hz, H-19), 6.16 (1H, d, J=10.8Hz, H-13), 6.02 (1H, dd, J= 15.2, 2.3Hz, H-20), 5.28 (1H, s, H-7), 4.56 (1H, s, H-18), 3.86 (1H, dd, J=8.0, 2.6Hz, H-17), 3.80 (1H, d, J= 11.0Hz, H-8), 3.16 (1H, dt, J=10.2, 3.6Hz, H-3), 3.07 (1H, t, J=6.9Hz, H-5), 2.90 (1H, dd, J=5.0, 3.0Hz, H-4), 2.35 (1H, m, H-15a), 2.08 (1H, m, H-15b), 2.15 (1H, m, H-16a), 1.36 (1H, m, H-16b), 1.71 (3H, s, H-12), 1.62 (1H, m, H-22), 1.48 (1H, m, H-10a), 1.29 (1H, m, 10b), 1.37 (3H, s, H-25), 1.20 (3H, d, J=7.3Hz, H-11), 0.92 (3H, d, J=6.6Hz, H-23), 0.92 (3H, d, J=6.6Hz, H-24)。13C NMR (125MHz, CDCl3) δC 173.8 (C-1), 52.2 (C-3), 51.7 (C-4), 34.3 (C-5), 140.3 (C-6), 124.2 (C-7), 39.4 (C-8), 88.5 (C-9), 48.5 (C-10), 13.9 (C-11), 19.9 (C-12), 122.7 (C-13), 139.3 (C-14), 39.6 (C-15), 27.9 (C-16), 78.2 (C-17), 73.2 (C-18), 150.9 (C-19), 120.0 (C-20), 167.7 (C-21), 25.2 (C-22), 21.5 (C-23), 23.8 (C-24), 15.3 (C-25)。以上数据与文献(Zhou et al, 2004)报道基本一致, 确定化合物 4 为 aspochalasin I。
化合物 5: 白色粉末, UV(MeOH) λmax (log ε) 201 (3.15)nm; HR-ESI-MS m/z: 456.2711 [M+Na]+。1H NMR (500MHz, CDCl3) δH 6.26 (1H, s, NH), 6.02 (1H, d, J=11.1Hz, H-13), 5.43 (1H, m, H-7), 3.95 (1H, m, H-19), 3.88 (m, H-17), 3.88 (1H, d, J=5.3Hz, H-20a), 2.03 (1H, d, J=5.3Hz, H-20b), 3.29 (1H, d, J=11.2Hz, H-8), 3.15 (1H, d, J=3.5Hz, H-3), 3.12 (1H, m, H-18), 2.60 (1H, m, H-5), 2.47 (1H, d, J=3.5Hz, H-4), 2.11 (2H, m, H-15), 1.74 (3H, s, H-12), 1.61 (1H, m, H-22), 1.55 (1H, m, H-16a), 1.26 (1H, m, H-16b), 1.51 (3H, s, H-25), 1.19 (3H, d, J=7.1Hz, H-11), 1.15 (2H, m, H-10), 0.91 (3H, d, J=6.0Hz, H-23), 0.89 (3H, d, J=6.0Hz, H-24), 3.47 (3H, s, OCH3)。13C NMR (125MHz, CDCl3) δC 176.0 (C-1), 51.5(C-3), 53.6 (C-4), 35.6 (C-5), 139.6 (C-6), 125.9 (C-7), 43.7 (C-8), 67.8 (C-9), 48.9 (C-10), 13.5 (C-11), 20.3 (C-12), 124.5 (C-13), 136.8 (C-14), 37.3 (C-15), 29.9 (C-16), 72.8 (C-17), 78.7 (C-18), 61.8 (C-19), 42.0 (C-20), 212.0 (C-21), 25.0 (C-22), 21.7 (C-23), 23.5 (C-24), 15.8 (C-25), 57.1 (OCH3)。以上数据与文献(Hao et al, 2017)报道基本一致, 确定化合物 5 为 aspochalasin K。
化合物 6: 黄色结晶, UV(MeOH) λmax (log ε) 220 (3.20)、254 (3.04)、286 (2.95)nm; HR-ESI-MS m/z: 311.0890 [M+Na]+。1H NMR (500MHz, DMSO-d6) δH 6.34 (1H, d, J=2.4Hz, H-4), 6.30 (1H, d, J=2.2Hz, H-6), 6.21 (1H, d, J=3.1Hz, H-10), 6.09 (1H, d, J=3.1Hz, H-11), 5.15 (1H, m, H-15), 3.21 (1H, d, J=14.3Hz, H-2a), 3.11 (1H, d, J=14.3Hz, H-2b), 2.84 (1H, ddd, J=15.3, 6.2, 2.8Hz, H-13a), 2.67 (1H, ddd, J=15.3, 6.2, 2.8Hz, H-13b), 2.04 (1H, m, H-14a), 1.77 (1H, m, H-14b), 1.23 (3H, d, J=6.4Hz, H-16)。13C NMR (125MHz, DMSO-d6) δC 174.2 (C-1), 42.6 (C-2), 139.8 (C-3), 112.2 (C-4), 159.7 (C-5), 102.5 (C-6), 157.5 (C-7), 111.9 (C-8), 156.1 (C-9), 107.6 (C-10), 111.0 (C-11), 148.8 (C-12), 37.2 (C-13), 26.6 (C-14), 74.0 (C-15), 21.2 (C-16)。以上数据与文献(Fürstner et al, 2003)报道基本一致, 确定化合物 6 为 citreofuran。
化合物 7: 黄色油状物, UV(MeOH) λmax (log ε) 204 (4.20)、276 (3.70)、316 (3.60)nm; HR-ESI-MS m/z: 447.1092 [M+Na]+。1H NMR (500MHz, Methanol-d4) δH 6.25 (1H, d, J=2.3Hz, H-6), 6.20 (1H, d, J=2.2Hz, H-4), 5.01 (1H, m, H-15), 4.39 (1H, d, J=3.9Hz, H-10), 4.37 (1H, d, J=9.2Hz, H-2a), 3.27 (1H, d, J=9.2Hz, H-2b), 3.35 (1H, d, J=7.3Hz, H-17a), 2.91 (1H, d, J=7.3Hz, H-17b), 1.96 (1H, m, H-12a), 1.50 (1H, m, H-12b), 1.81 (1H, m, H-14a), 1.34 (1H, m, H-14b), 1.67 (1H, m, H-13a), 1.38 (1H, m, H-13b), 1.04 (3H, d, J=6.5Hz, H-16), 3.56 (3H, s, OCH3)。13C NMR (125MHz, CD3OD) δC 171.1 (C-1), 41.0 (C-2), 138.5 (C-3), 112.5 (C-4), 163.1 (C-5), 102.8 (C-6), 161.3 (C-7), 120.3 (C-8), 208.1 (C-9), 64.2 (C-10), 50.1 (C-11), 36.0 (C-12), 23.4 (C-13), 30.3 (C-14), 73.5 (C-15), 17.7 (C-16), 41.2 (C-17), 87.6 (C-18), 174.2 (C-19), 53.0 (OCH3)。以上数据与文献(De Castro et al, 2016)报道基本一致, 确定化合物 7 为 cyclothiocurvularin B methyl ester。

2.4 细胞毒活性测试

用MTT法测试化合物对前列腺癌细胞PC3和22Rv1的细胞毒活性。结果显示化合物2、4和5表现出PC3细胞毒活性(图6、表1), IC50分别为17.23、15.18和8.71μmol·L-1, 阳性对照多柔比星IC50为5.09μmol·L-1, 化合物1—5未表现出22Rv1细胞毒性。
图6 化合物1—5在20μmol·L-1浓度下进行细胞毒活性筛选

虚线表示在20μmol·L-1浓度下的50%细胞存活率

Fig. 6 Cytotoxicity screening of compounds 1—5 at 20 μmol·L-1 concentration. The dashed line indicates 50% cell viability at a concentration of 20 μmol·L-1

表1 化合物1—5对前列腺癌PC3细胞的生长抑制作用

Tab. 1 Growth inhibition of compounds 1—5 against prostate cancer PC3 cell

化合物 IC50/(μmol·L-1)
1 > 20
2 17.23
3 > 20
4 15.18
5 8.71
多柔比星 5.09

注: 多柔比星为阳性对照

3 结论与讨论

本研究从阿拉伯宝螺(Mauritia arabica)中分离鉴定出一对真菌-细菌共生体Aspergillus spelaeus GXIMD 04541 / Sphingomonas echinoides GXIMD 04532, 细菌特异性染色发现其宿主真菌菌丝内存在内共生细菌。对该共生体的大米发酵提取物进行化学成分研究, 从中分离出7个单体化合物, 包括5个细胞松弛素(化合物1—5)和2个苯内酯(化合物6—7)。对化合物1—5进行细胞毒活性筛选, 结果显示化合物2、4和5表现出PC3细胞毒活性, 其中化合物5对PC3细胞的抑制作用最强。内共生细菌通过调控宿主真菌的生长、分布和次级代谢过程, 有助于宿主真菌增强环境适应性和产生免受捕食者侵害的活性物质(Okrasińska et al, 2021)。该共生体在发酵过程中真菌细菌可能处于共生状态, 尝试用分离破碎法和原生质体法(Shao et al, 2020)去破坏共生体从而获得单独的内生细菌和宿主真菌, 但目前还是无法成功获得独立的细菌和真菌。因此推测内生细菌GXIMD 04532与宿主真菌GXIMD 04541共生, 能促进宿主真菌的生长发育, 从而帮助产生抑制前列腺癌PC3细胞的活性物质, 让两者在恶劣环境中更好地生存。细胞松弛素是一类结构复杂的化合物, 在结构上具有典型三环体系, 由高度取代的过氢异吲哚酮核心和9~15个碳的大环组成。该类化合物因能干预重要的细胞内生化过程, 从而对宫颈癌细胞Hela, 乳腺癌细胞MCF-7和结直肠癌细胞HCT116均具有细胞毒活性, 其IC50在6.3~17.6μmol·L-1之间(Guo et al, 2021)。此外, 细胞松弛素还因具有较强的抗藤壶污损(Zheng et al, 2013)、抗HIV (Pang et al, 2017)等生物活性而备受关注。化合物6和7属于苯二酚内酯类化合物, 其中7为含硫内酯化合物, 该类型中含硫化合物较为罕见, 该化合物具有抗炎(Yu et al, 2025)、抑菌(Ye et al, 2016)等生物活性。

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