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Journal of Tropical Oceanography >

2024 , Vol. 43 >Issue 6: 196 - 200

DOI: https://doi.org/10.11978/2024011

Marine Biology

Secondary metabolites from the sponge-derived fungus Aspergillus sp. SCSIO 41018 and their free radical scavenging activity

  • YUAN Yuan , 1, 2 ,
  • ZHANG Yuanyuan 3 ,
  • XU Fuquan 3 ,
  • LIU Qingchao , 1 ,
  • WANG Junfeng , 2, 4
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  • 1. School of Chemical Engineering, Northwest University, Xi’an 710069, China
  • 2. Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
  • 3. School of Marine Science and Fisheries, Jiangsu Ocean University, Lianyungang 222005, China
  • 4. Sanya Institute of Ocean Eco-Environmental Engineering, Sanya 572000, China
LIU Qingchao, email: ;
WANG Junfeng, email:

Editor: LIN Qiang

Received date: 2024-01-10

  Revised date: 2024-02-22

  Online published: 2024-02-27

Supported by

Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City(2021JJLH0097)

Guangdong Basic and Applied Basic Research Foundation, China(2022B1515120075)

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Abstract

This paper reported the identification of seven compounds from the rice ferment of Aspergillus sp. fungus SCSIO 41018 isolated from sponge Callyspongia sp. in the Xuwen County, Guangdong Province, China. These compounds were purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography and semi-preparative high performance liquid chromatography. The seven compounds were identified by nuclear magnetic resonance spectroscopy and mass spectrometry to determine their structures, in combination with the reported data, as: cycloechinulin (1), 4-methyl-1H-pyrazole-3-carboxylic acid (2), diorcinol (3), methyl-4-hydroxy-2-(3-hydroxy-5-methylphenoxy)-6-methylbenzoate (4), benzeneacetic acid (5), protocatechuic acid (6), indole-3-carboxaldehyde (7), wherein compound 2 was first isolated from Aspergillus sp. fungi. The free radical scavenging activity was evaluated, and the results showed that compounds 5 and 6 had stronger free radical scavenging ability and antioxidants potential than the positive control ascorbic acid (Vc, vitamin C).

Key words： Aspergillussp.; fungus; secondary metabolites; free radical scavenging activity

Cite this article

YUAN Yuan , ZHANG Yuanyuan , XU Fuquan , LIU Qingchao , WANG Junfeng . Secondary metabolites from the sponge-derived fungus Aspergillus sp. SCSIO 41018 and their free radical scavenging activity[J]. Journal of Tropical Oceanography, 2024 , 43(6) : 196 -200 . DOI: 10.11978/2024011

海绵是一种简单而古老的生物, 它的存在历史可以追溯到寒武纪(刘永宏 等, 2008)。人们对于海绵的研究已经持续六十年之久, 研究方向也随着海洋微生物热度的提高和研究的深入, 逐渐从海绵体内转移到与海绵共附生的微生物上。大量的共附生微生物存在于海绵独特的滤食性和腔状管道结构中, 这些生存在特殊生态环境下的微生物同时又是海绵化学防御体系的重要组成部分, 二者密不可分。海绵盛产活性天然产物, 迄今发现的新颖海洋天然产物约有1/3都来自于海绵(孙彩霞 等, 2022), 而真菌的次级代谢产物是海洋微生物天然产物最大的来源(Carroll et al, 2023)。从目前的研究成果来看, 海绵来源真菌产生的活性化合物主要有生物碱类, 聚酮类, 多肽类等, 这些化合物具有抗氧化, 抗炎, 抗病毒, 细胞毒, 抗菌等一种或多种活性, 为新药的开发提供了宝贵的资源(杨君泽 等, 2017; 金嘉悦 等, 2023)。
为进一步研究分离自美丽海绵属海绵(Callyspongia sp.)的曲霉属真菌(Aspergillus sp. SCSIO 41018)次级代谢产物多样性, 本文从这株曲霉属真菌的大米发酵物中分离并鉴定了7个已知的化合物(图1), 其中化合物2为首次从曲霉属真菌中分离获得。据文献报道, 化合物1具有较强的自由基清除活性(Dao et al, 2020), 为了探究化合物1以及其他所分离化合物的自由基清除活性, 本文对分离到的化合物1—6进行了自由基清除活性的体外试验。试验结果表明, 化合物5和6的自由基清除活性均强于阳性对照抗坏血酸(Vc), 是强效的抗氧化剂。
图1 化合物1—7的化学结构

Fig. 1 Chemical structures of compounds 1-7

1 材料与方法

1.1 仪器与试剂

BL-3000型电子天平(厦门佰伦斯电子科技); MVS-83型立式压力蒸汽灭菌器[冰山松洋生物科技(大连)]; HYG-C2型多功能摇床(苏州培英实验设备); ZQZY-CF8型振荡培养箱(上海知楚仪器); 613HTU型数控超声波清洗机(广州市星烁仪器); SB-1300型旋转蒸发仪(上海爱朗仪器); SHZ-95B型循环水真空泵(广州市星烁仪器); SepaBean machine—标准型中压快速液相制备系统(常州三泰科技); Infinity1260型高效液相色谱仪(Aglient); Primaide1430型高效液相色谱仪(HITACHI); AV500型/AVANCE Ⅲ HD 700MHz超导核磁共振仪; amaZon SL型低分辨离子阱质谱仪; HR-ESI-MS maXis impact型高分辨质谱仪(均为德国BRUKER); Master Touch-S15UV型低有机物型超纯水机(上海和泰仪器); 分离柱为C18填料反相色谱柱(250mm×4.6mm, 5µm, YMC); 分析纯甲醇、乙酸乙酯等(广州化学试剂厂), 色谱纯甲醇、乙腈(上海星可高), 氘代二甲基亚砜(CIL)。

1.2 试验方法

1.2.1 菌种来源与发酵

菌株Aspergillus sp. SCSIO 41018分离自广东省徐闻县的美丽海绵属海绵(Callyspongia sp. XWS01), 通过PCR扩增和DNA测序得到该菌株的ITS1-5.8S-ITS2序列与GenBank中的序列进行比对, 有585个碱基对与Aspergillus ochraceopetaliformis strain A180326相似度100%, 同时对比基于rDNA-ITS序列的邻接法构建的系统发育树(图2), 显示该株菌株与曲霉属的基因序列相似性聚为一簇, 因此确定该菌株为曲霉属真菌, 并将此菌株重新命名为Aspergillus sp. SCSIO 41018 (GenBank number: MH109740.1)。样品标本(编号: SCSIO 41018)保存于中国科学院南海海洋研究所(SCSIO)热带海洋生物资源与生态重点实验室。发酵时的操作是将新生的菌株在无菌环境下接种至大米培养基(配方为: 大米180g, 32‰海盐水180mL), 于28℃下静置培养60d。
图2 基于ITS基因序列构建的菌株SCSIO 41018系统发育树

Fig. 2 Phylogenetic tree based on the ITS gene sequence of strain SCSIO 41018

1.2.2 提取与分离

将乙酸乙酯与发酵液2:1充分混合, 萃取乙酸乙酯层, 反复三次, 得到粗提浸膏34.60g。 粗浸膏用正相硅胶柱层析分离, 经流动相乙酸乙酯:甲醇(100:0~0:100, V/V)洗脱分离, 根据薄层色谱结果合并得到5个组分Fr.1—Fr.5。
Fr.4经Sephadex LH-20柱层析得到3个组分Fr.4.1—Fr.4.3, 其中组分Fr.4.2再经Sephadex LH-20柱层析分离得到5个组分Fr.4.2.1~Fr.4.2.5, 组分Fr.4.2.4经半制备高效液相色谱进一步纯化(甲醇:水=65:35)得到化合物1 (3.30mg, tR=9.5min)和3 (5.72mg, tR= 11.6min)。组分Fr.5经Sephadex LH-20柱层析得到6个组分Fr.5.1—Fr.5.6, Fr.5.1利用半制备高效液相色谱继续纯化(甲醇:水=68:32)得到化合物2 (1.60mg, tR=10.8min); 组分Fr.5.2利用半制备高效液相色谱纯化(甲醇:水=70:30)得到化合物5 (7.50mg, tR=13.4min); 组分Fr.5.4利用半制备高效液相色谱进一步纯化(甲醇:水=82:18)得到化合物6 (44.15mg, tR=5.7min)。组分Fr.2经Sephadex LH-20柱层析分离得到6个组分Fr.2.1—Fr.2.6, 组分Fr.2.3经半制备高效液相色谱(甲醇:水=45:55)纯化, 得到化合物4 (3.43mg, tR=11.5min)。Fr.3经Sephadex LH-20柱层析分离得到4个组分Fr.3.1—Fr.3.4, 其中组分Fr.3.3经半制备高效液相色谱(甲醇:水=51:49)进一步分离纯化, 得到化合物7 (1.03mg, tR=12.5min)。

1.2.3 自由基清除活性试验

样品用甲醇根据不同化合物的抑制强度配制成浓度梯度为1000~4μg·mL-1的工作液, 以100μg·mL-1的DPPH• (1, 1-diphenyl-2-picryl-hydrazyl radical)溶液为底物, 50μg·mL-1的Vc溶液为阳性对照, 甲醇为空白对照。测试于96孔板上进行, 每组设3个复孔, 避光置于常温下反应30min, 反应结束后用微孔板酶标仪测试517nm下的吸光数值, 计算抑制率。

2 试验结果

2.1 结构鉴定

化合物1: 褐色固体, HRESIMS m/z 350.7 [M+H]+, 分子式为C20H21N3O3。1H-NMR (500MHz, DMSO-d6) δH 11.24 (1H, s, H-1), 8.34 (1H, d, J = 2.4 Hz, H-13), 7.42 (1H, d, J = 8.7 Hz, H-4), 7.29 (1H, s, H-10), 6.90 (1H, d, J = 2.3 Hz, H-7), 6.77 (1H, dd, J = 8.7, 2.3 Hz, H-5), 5.96 (1H, d, J = 8.2 Hz, H-18), 5.77 (1H, d, J = 8.2 Hz, H-17), 4.05 (1H, dq, J = 6.9, 2.2 Hz, H-14), 3.76 (3H, s, H-23), 1.60 (6H, s, H-20, 21), 1.31 (3H, d, J = 6.9 Hz, H-22); 13C-NMR (126MHz, DMSO-d6) δC 167.8 (C-15), 163.9 (C-12), 156.2 (C-6), 146.8 (C-2), 140.0 (C-18), 135.1 (C-8), 126.0 (C-11), 124.3 (C-9), 122.9 (C-17), 117.7 (C-4), 113.1 (C-10), 110.7 (C-5), 104.6 (C-3), 95.7 (C-7), 55.7 (C-23), 50.4 (C-14), 36.3 (C-19), 27.8 (C-20), 27.6 (C-21), 18.3 (C-22)。以上数据与文献de Guzman 等(1992)报道数据比对一致, 鉴定化合物为cycloechinulin。
化合物2: 褐色固体, HRESIMS m/z 127.1 [M+H]+, 分子式为C5H6N2O2。1H-NMR (700MHz, DMSO-d6) δH 11.00 (1H, s, H-NH), 7.25 (1H, s, H-5), 1.72 (3H, s, H-7); 13C-NMR (176MHz, DMSO-d6) δC 165.4 (C-6), 152.0 (C-3), 138.2 (C-5), 108.1 (C-4), 12.3 (C-7)。以上数据与文献Parameswaran等(1997)报道数据比对一致, 鉴定化合物为4-methyl-1H-pyrazole-3-carboxylic acid。
化合物3: 褐色固体, HRESIMS m/z 231.5 [M+H]+, 分子式为C14H14O3。1H-NMR (500MHz, DMSO-d6) δH 9.45 (2H, s, 3, 3’-OH), 6.34 (2H, s, J = 1.8 Hz, H-2, 2’), 6.24 (2H, s, J = 1.8 Hz, H-6, 6’), 6.15 (2H, s, J = 2.2 Hz, H-4, 4’), 2.18 (6H, s, H-7, 7’); 13C-NMR (126MHz, DMSO-d6) δC 158.9, 158.1 (C-1, 1’, 3, 3’), 140.5 (C-5), 111.6, 110.5 (C-4, 4’, 6, 6’), 103.4 (C-2, 2’), 21.6 (C-7, 7’)。以上数据与文献Yurchenko等(2010)报道数据比对一致, 鉴定化合物为diorcinol。
化合物4: 褐色固体, HRESIMS m/z 289.7 [M+H]+, 分子式为C16H16O5。1H-NMR (500MHz, DMSO-d6) δH 10.26 (1H, s, 5-OH), 9.56 (1H, s, 5’-OH), 6.41 (1H, s, H-4’), 6.35 (1H, s, H-4), 6.30 (1H, s, H-2), 6.27 (1H, s, H-6), 6.22 (1H, s, H-6’), 3.79 (3H, s, H-9), 2.21 (6H, s, H-7, 7’); 13C-NMR (126MHz, DMSO-d6) δC 169.0 (C-8), 159.5 (C-5’), 159.1 (C-5), 158.1 (C-1), 156.9 (C-1’), 140.9 (C-3), 139.5 (C-3’), 115.3 (C-2’), 112.5 (C-4’), 111.3 (C-4), 111.2 (C-2), 104.2 (C-6’), 103.2 (C-6), 52.4 (C-9), 21.6 (C-7), 20.7 (C-7’)。以上数据与文献Tian等(2018)报道数据比对一致, 鉴定该化合物为methyl-4-hydroxy-2-(3-hydroxy-5-methylphenoxy)-6-methylbenzoate。
化合物5: 褐色油状, HRESIMS m/z 167.8 [M - H]-, 分子式为C8H8O4。1H-NMR (500MHz, DMSO-d6) δH 6.64 (2H, m, H-2, 6), 6.47 (1H, dd, J = 8.0, 2.1 Hz, H-5), 3.32 (2H, s, H-7); 13C-NMR (126MHz, DMSO-d6) δC 173.8 (C-8), 145.5 (C-3), 144.5 (C-4), 126.3 (C-1), 120.5 (C-6), 117.2 (C-5), 115.8 (C-2), 40.9 (C-7)。以上数据与文献李骅轩 等(2019)报道数据比对一致, 鉴定化合物为benzeneacetic acid。
化合物6: 褐色油状, HRESIMS m/z 153.0 [M - H]-, 分子式为C7H6O4。1H-NMR (500MHz, DMSO-d6) δH 7.37 (1H, s, H-2), 7.32 (1H, dd, J = 8.2, 2.1 Hz, H-6), 6.82 (1H, d, J = 8.2 Hz, H-5); 13C-NMR (126MHz, DMSO-d6) δC 167.9 (C-7), 150.5 (C-3), 145.3 (C-4), 122.4 (C-1), 122.2 (C-6), 117.0 (C-2), 115.6 (C-5)。以上数据与文献Ramadan等(2009)报道数据比对一致, 鉴定化合物为protocatechuic acid。
化合物7: 褐色固体, HRESIMS m/z 146.2 [M+H]+, 分子式为C9H7NO。1H-NMR (700MHz, DMSO-d6) δH 12.17 (1H, s, H-NH), 9.93 (1H, s, H-CHO), 8.28 (1H, s, H-2), 8.09 (1H, dt, J = 7.7, 1.1 Hz, H-4), 7.51 (1H, d, J = 8.0 Hz, H-7), 7.26 (1H, ddd, J = 8.2, 7.0, 1.3 Hz, H-5), 7.22 (1H, td, J = 7.5, 1.1 Hz, H-6); 13C-NMR (176MHz, DMSO-d6) δC 185.4 (C-10), 139.0 (C-8), 137.5 (C-2), 124.6 (C-9), 123.9 (C-4), 122.6 (C-5), 121.3 (C-6), 118.6 (C-3), 112.9 (C-7)。以上数据与文献张浩 等(2022)报道数据比对一致, 鉴定化合物为indole-3-carboxaldehyde。

2.2 自由基清除活性测试结果

本试验采用96孔板法测试了化合物1—6对DPPH• 的自由基清除能力, 表1和图3中的数据表明, 化合物5 (IC50=4.69μg·mL-1)和6 (IC50=10.36μg·mL-1)的自由基清除能力均强于阳性对照Vc (IC50=12.75μg·mL-1), 化合物3、4也具有较弱的自由基清除活性, 而化合物1、2基本无活性。
表1 化合物1—6的自由基清除活性试验IC50

Tab. 1 Free radical scavenging activity assay IC50 of compounds 1-6

化合物 IC50 (μg·mL-1)
1 1 272.00
2 -
3 108.40
4 448.20
5 4.69
6 10.36
Vc 12.75
图3 化合物3、5、6和Vc的抑制曲线

Fig. 3 Inhibition curves of compounds 3, 5, 6

3 讨论

本文从美丽海绵属海绵来源曲霉属(Aspergillus sp.)真菌的大米发酵次级代谢产物中分离并鉴定了7个化合物, 对化合物1—6的DPPH•自由基清除能力进行体外活性测试, 结果表明化合物5 (IC50=4.69μg·mL-1)和6 (IC50=10.36μg·mL-1)的自由基清除能力均强于阳性对照Vc (IC50=12.75μg·mL-1), 是强效的抗氧化剂, 推测是由于化合物5和6的结构中多羟基与羧基, 这两种基团上的活泼氢极易脱出, 从而与游离的羟基自由基(•OH)反应, 使其具有显著的抗氧化性。羟基自由基(•OH)性质非常活泼, 可以氧化氨基酸、糖类、核酸、蛋白质和脂类等物质, 如果不及时清除, 会使机体的器官、组织和细胞受到损伤和破坏, 加快机体的衰老, 并在一定程度上促进肿瘤等疾病的发展(李艳 等, 2015)。因此化合物5和6在减少细胞损伤、抗衰和抗肿瘤等方面具有重要的研究潜力。海洋微生物来源的次级代谢产物一直以来都是药物先导化合物的重要来源, 这些天然产物的潜在活性与其生存环境和宿主的生长调节等生命活动密切相关, 更多结构新颖且活性显著的海洋微生物次级代谢产物有待挖掘与发现。

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