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桐花树土壤内生真菌Aspergillus fumigatus 06T03次生代谢产物的分离及其生物活性研究

花匠小妙招
2025-05-06 23:58

摘要

目的

研究红树植物桐花树土壤内生真菌Aspergillus fumigatus 06T03的次生代谢产物的分离纯化及其潜在的生物活性。

方法

菌株采用大米培养基经规模发酵，乙酸乙酯浸泡提取后经减压浓缩得到乙酸乙酯粗提物。采用多种现代色谱技术如硅胶柱色谱、Sephadex LH-20凝胶柱色谱、高效液相色谱等对粗提物进行化学成分分离。通过高分辨质谱、核磁共振波谱等综合谱学技术确定单体化合物结构。采用CCK-8法和Griess法初步评估单体化合物的抗肿瘤活性和抗炎活性。

结果

从Aspergillus fumigatus 06T03大米发酵产物中分离鉴定出9个化合物，分别为Monomethylsulochrin（1）、Questin（2）、Demethylsulochrin（3）、Fumiquinazoline C（4）、Fumiquinazoline J（5）、Azaspirofurans A（6）、Pseurotin A（7）、N-[2-(4-hydroxyphenyl) ethenyl] formamide（8）、Pyripyropene A（9）。其中，化合物1~3属于蒽醌类化合物，4~9属于生物碱类化合物。化合物1~9对肝癌细胞（Huh7）、卵巢癌细胞（A2780）、乳腺癌细胞（MDA-MB-231）无明显抗肿瘤活性，化合物1、3、6、7、8和9具有一定抗炎活性。

结论

从桐花树土壤内生真菌Aspergillus fumigatus 06T03中分离得到9个单体化合物，除化合物2，其余化合物均为首次从桐花树内生真菌中分离得到，化合物1、3、6、7、8和9具有一定的抗炎活性。本研究丰富了桐花树土壤内生真菌次生代谢产物的化学多样性，为后续深入开发提供了基础数据。

Abstract

Objective

To investigate the isolation and purification of the secondary metabolites and their potential biological activity in the endophytic fungus Aspergillus fumigatus 06T03 from the soil of Aegiceras corniculatum.

Methods

The strain was fermented on a large scale using rice medium, and then extracted by soaking ethyl acetate and concentrated under reduced pressure to obtain a crude extract of ethyl acetate. A variety of modern chromatographic techniques such as silica gel column chromatography, Sephadex LH-20 gel column chromatography, high -performance liquid chromatography were used to separate the chemical components of the crude extract. Their structures were determined by using comprehensive spectral techniques such as high-resolution mass spectra (MS) and nuclear magnetic resonance (NMR) spectroscopy. The antineoplastic and anti-inflammatory activities of the monomers were evaluated using cell counting kit-8 (CCK-8) assay and Griess assays.

Results

Nine compounds were isolated from the rice fermentation products of Aspergillus fumigatus 06T03 and identified as Monomethylsulochrin (1), Questin (2), Demethylsulochrin (3), Fumiquinazoline C (4), Fumiquinazoline J (5), Azaspirofurans A (6), Pseurotin A (7), N-[2-(4-hydroxyphenyl) ethenyl]formamide (8), and Pyripyropene A (9). Among them, compounds 1-3 belonged to anthraquinones, and 4-9 belonged to alkaloids. The results showed that compounds 1-9 failed to exhibit obvious antineoplastic activity against Huh7 cells, ovarian carcinoma cells (A2780), and breast cancer cells (MDA-MB-231), while compounds 1, 3, 6, 7, 8, and 9 had certain antiinflammatory activities.

Conclusion

Nine monomeric compounds, except compound 2 which has been isolated before, have been isolated for the first time from the endophytic fungus Aspergillus fumigatus 06T03 from the soil of Aegiceras corniculatum. Compounds 1, 3, 6, 7, 8, and 9 have certain anti-inflammatory activities. This study enriches the chemical diversity of the secondary metabolites of endophytic fungus from the soil of Aegiceras corniculatum, and provides basic data for subsequent further development.

图 1 桐花树内生真菌Aspergillus fumigatus分离得到化合物1~9的化学结构

图 2 化合物1~9对3株肿瘤细胞的抗增殖作用

A：化合物1~9（40 mmol/L）对肝癌细胞Huh7的细胞存活率影响；B：化合物1~9（40 mmol/L）对卵巢癌细胞A2780的细胞存活率影响；C：化合物1~9（40 mmol/L）对乳腺癌细胞MDA-MB-231的细胞存活率影响；1~9：化合物1~9实验组；n=3；与对照组比较，**P＜0.01。

图 3 化合物1~9对RAW264.7细胞存活率和对LPS诱导细胞产生NO含量的影响

A：化合物1~9（40 mmol/L）对小鼠巨噬细胞RAW264.7的细胞存活率的影响；1~9：化合物1~9实验组；n=3；与对照组比较，**P＜0.01，***P＜0.001；B：化合物1~3、6~9对LPS诱导的RAW264.7细胞生成的NO含量的影响；1~3、6~9：化合物1~3、6~9实验组；n=3；与对照组比较，###P＜0.001；与LPS组比较，*P＜0.05，***P＜0.001。

[1]

WU M J, XU B F, GUO Y W. Unusual secondary metabolites from the mangrove ecosystems: structures, bioactivities, chemical, and bio-syntheses[J]. Marine drugs, 2022, 20(8): 535. doi: 10.3390/md20080535

[2]

CADAMURO R D, DA SILVEIRA BASTOS I M A, SILVA I T, et al. Bioactive compounds from mangrove endophytic fungus and their uses for microorganism control[J]. Journal of fungi, 2021, 7(6): 455. doi: 10.3390/jof7060455

[3]

DING L, DAHSE H M, HERTWECK C. Cytotoxic alkaloids from Fusarium incarnatum associated with the mangrove tree Aegiceras corniculatum[J]. Journal of natural products, 2012, 75(4): 617-621. doi: 10.1021/np2008544

[4] 田晓萌, 蔡晓婧, 郭庆梅, 等. 桐花树药用研究进展[J]. 辽宁中医药大学学报, 2017, 19(11): 114-117. [5]

NUGRAHA A P, SIBERO M T, NUGRAHA A P, et al. Anti-periodontopathogenic ability of mangrove leaves (Aegiceras corniculatum) ethanol extract: in silico and in vitro study[J]. European journal of dentistry, 2023, 17(1): 46-56. doi: 10.1055/s-0041-1741374

[6]

RAHMAN M A, PAUL R R, BISWAS C, et al. Pesticidal activity of sundarban mangrove plant extracts against Sitophilus pests and identification of active constituents using LC-MS[J]. Advances in pharmacological and pharmaceutical sciences, 2021, 2021: 1540336.

[7]

HUANG X K, LIU Y H, QIAN C Y, et al. CHSY3 promotes proliferation and migration in gastric cancer and is associated with immune infiltration[J]. Journal of translational medicine, 2023, 21(1): 474. doi: 10.1186/s12967-023-04333-x

[8]

OUYANG P, LI K, XU W, et al. METTL3 recruiting M2-type immunosuppressed macrophages by targeting m6A-SNAIL-CXCL2 axis to promote colorectal cancer pulmonary metastasis[J]. Journal of experimental & clinical cancer research, 2024, 43(1): 111.

[9]

UNTERHOLZNER A, KUCK K, WEINZIERL A, et al. An unprecedented 4, 8-cycloeudesmane, further new sesquiterpenoids, a triterpene, steroids, and a lignan from the resin of Commiphora myrrha and their anti-inflammatory activity in vitro[J]. Molecules, 2024, 29(18): 4315. doi: 10.3390/molecules29184315

[10] 丛梦静, 胡怡伟, 赵凯, 等. 深海冷泉来源真菌Talaromyces helicus SCSIO41311中聚酮类化学成分研究[J]. 热带海洋学报, 2022, 41(5): 117-120. [11]

FUJIMOTO H, FUJIMAKI T, OKUYAMA E, et al. Immunomodulatory constituents from an ascomycete, Microascus tardifaciens[J]. Chemical & pharmaceutical bulletin, 1999, 47(10): 1426-1432.

[12]

HUANG R F, MA S Q, WU J L, et al. A new tetrahydroxy-benzophenone from a marine-associated fungus of Penicillium sp. and its potential antibacterial activity[J]. Natural product research, 2024: 1-8.

[13] 周彦伶, 许言超, 龚倩玉, 等. 贵州洞穴真菌Aspergillus fumigatus GZWMJZ-152的抗菌活性产物[J]. 中国抗生素杂志, 2018, 43(6): 675-682. [14] 韩小贤, 许晓妍, 崔承彬, 等. 海洋真菌烟曲霉H1-04生产的生物碱类化合物及其抗肿瘤活性[J]. 中国药物化学杂志, 2007, 17(4): 232-237. [15]

REN H, LIU R, CHEN L, et al. Two new hetero-spirocyclic gamma-lactam derivatives from marine sediment-derived fungus Aspergillus sydowi D2-6[J]. Archives of pharmacal research, 2010, 33(4): 499-502. doi: 10.1007/s12272-010-0401-4

[16] 易文娟, 陈波, 殷颖, 等. 绿僵菌破坏素合成缺失株中分离鉴定pseurotin A[J]. 菌物学报, 2019, 38(7): 1082-1089. [17]

KHAN I, ZHANG H B, LIU W, et al. Identification and bioactivity evaluation of secondary metabolites from Antarctic-derived Penicillium chrysogenum CCTCC M 2020019[J]. RSC advances, 2020, 10(35): 20738-20744. doi: 10.1039/D0RA03529G

[18]

LIANG W L, LE X, LI H J, et al. Exploring the chemodiversity and biological activities of the secondary metabolites from the marine fungus Neosartorya pseudofischeri [J]. Marine drugs, 2014, 12(11): 5657-5676. doi: 10.3390/md12115657

[19] 王诗怡, 王玉妃, 刘江晔, 等. 北部湾植物内生真菌的分离及次级代谢产物鉴定[J]. 中国抗生素杂志, 2024, 49(5): 510-518. [20] 殷娜, 宋娜丽, 普晓佳, 等. 民族药马利筋内生真菌生物活性研究[J]. 广西植物, 2022, 42(5): 781-789. [21]

GUPTA V K, MUKHERJEE K, ROY A. Two novel antifungals, acornine 1 and acornine 2, from the bark of mangrove plant Aegiceras corniculatum (Linn. ) Blanco from Sundarban Estuary[J]. Pharmacognosy magazine, 2014, 10(Suppl 2): S342-S349.

[22]

LIN Z J, LU Z Y, ZHU T J, et al. Penicillenols from Penicillium sp. GQ-7, an endophytic fungus associated with Aegiceras corniculatum[J]. Chemical & pharmaceutical bulletin, 2008, 56(2): 217-221.

[23]

SUNDARAM R, GANESAN R, MURUGESAN G. In vitro antiplasmodial activity of spiro benzofuran compound from mangrove plant of Southern India[J]. Asian Pacific journal of tropical medicine, 2012, 5(5): 358-361.