黄河三角洲盐碱地来源真菌Penicillium camembertiRD-37次级代谢产物的分离鉴定及活性评估

Isolation, identification, and activity evaluation of secondary metabolites from a fungus Penicillium camemberti RD-37 derived from saline-alkaline soil of the Yellow River Delta

摘要

目的 研究黄河三角洲盐碱地来源的真菌Penicillium camemberti RD-37的次级代谢产物及其生物活性。 方法 综合运用化学萃取、薄层色谱(thin layer chromatography, TLC)、ODS柱色谱(octadecylsilyl, ODS)和半制备型高效液相色谱(high performance liquid chromatography, HPLC)等分离技术对该株真菌发酵产物进行分离纯化;通过核磁共振(nuclear magnetic resonance, NMR)、质谱等波谱技术并结合文献数据比对,确定化合物结构。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 从真菌Penicillium camemberti RD-37大米发酵产物的乙酸乙酯提取物中共分离鉴定出11个单体化合物,分别为6-(methyl 3-methylbutanoate)-7-hydroxy-5-methoxy-4-methylphthalan-1-one(1)、7-hydroxy-5-methoxy-4-methyl-6-(3-methyl-4-oxopentyl)-phthalan-l-one(2)、F01-1358B(3)、Methyl mycophenolic acid(4)、6-((2E, 6E)-3, 7-dimethyldeca-2, 6-dienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one(5)、5,7-dihydroxy-4-methylisobenzofuran-1(3H)-one(6)、(2-hydroxypropan-2-yl)-6-methyl-2, 3-dihydrobenzofuran-4-ol(7)、Radstrictin K(8)、2, 3-dihydro-2-hydroxy-2, 4-dimethyl-5-trans-propenylfuran-3-one(9)、苯甲酸(10)、对甲氧基苯甲酸(11)。活性检测结果表明,化合物4(methyl mycophenolic acid)对人结肠腺癌细胞RKO和人卵巢癌细胞A2780细胞均表现出明显的细胞毒活性,IC₅₀值分别为7.7 μmol/L和8.2 μmol/L。在浓度为30 μmol/L时,脂多糖(lipopolysaccharide, LPS)诱导的炎症模型中化合物1、6、8、9、10和11对小鼠单核巨噬细胞RAW264.7表现出一定的抗炎活性,NO抑制率分别为45.9%、54.0%、62.3%、96.0%、59.5%和55.8%。 结论 真菌Penicillium camemberti RD-37具有产抗肿瘤抗炎活性成分的潜力,化合物4对肿瘤细胞具有显著的抑制活性。

Abstract

Objective To analyze the secondary metabolites and biological activities of the fungus Penicillium camemberti RD-37, which was isolated from the saline-alkaline soil of the Yellow River Delta. Methods The fermentation extract of this particular fungal strain was isolated and purified through integrated application of separation techniques, including chemical extraction, thin layer chromatography(TLC), octadecylsilyl column chromatography(ODS), and semi-preparative high performance liquid chromatography(HPLC). The structures of the compounds were determined using spectroscopic techniques such as nuclear magnetic resonance(NMR)and mass spectrometry, combined with a comparison of the literature data. The anti-tumor and anti-inflammatory activities of pure compounds were evaluated using CCK-8 and Griess assays. Results Eleven compounds were isolated and identified from the ethyl acetate extract of the rice fermentation of the fungus Penicillium camemberti RD-37, including 6-(methyl 3-methylbutanoate)-7-hydroxy-5-methoxy-4-methylphthalan-1-one(1), 7-hydroxy-5-methoxy-4-methyl-6-(3-methyl-4-oxopentyl)-phthalan-l-one(2), F01-1358B(3), Methyl mycophenolic acid(4), 6-((2E, 6E)-3,7-dimethyldeca-2, 6-dienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one(5), 5,7-dihydroxy-4-methylisobenzofuran-1(3H)-one(6),(2-hydroxypropan-2-yl)-6-methyl-2,3-dihydrobenzofuran-4-ol(7), Radstrictin K(8), 2,3-dihydro-2-hydroxy-2, 4-dimethyl-5-trans-propenylfuran-3-one(9), Benzoic acid(10), p-methoxybenzoic acid(11). The activity results indicated that compound 4(Methyl mycophenolic acid)exhibited significant cytotoxic activities against both human colorectal adenocarcinoma cells RKO and human ovarian cancer cells A2780 cells, with IC₅₀ values of 7.7 μmol/L and 8.2 μmol/L, respectively.In the LPS-induced inflammation model, at a concentration of 30 μmol/L, compounds 1, 6, 8, 9, 10 and 11 exhibited anti-inflammatory activities in mouse mononuclear macrophages cells RAW264.7, with NO inhibition rates of 45.9%, 54.0%, 62.3%, 96.0%, 59.5%, and 55.8%, respectively. Conclusion The fungus Penicillium camemberti RD-37 has the potential to produce compounds with antitumor and anti-inflammatory activities. Compound 4 exhibits significant inhibitory activity against tumor cells.

关键词

黄河三角洲 / 真菌 / Penicillium camemberti RD-37 / 次级代谢产物 / 细胞毒活性 / 抗炎活性

Key words

Yellow River Delta / Fungi / Penicillium camemberti RD-37 / Secondary metabolites / Cytotoxic activity / Anti-inflammatory activity

参考文献

[1] Aly AH, Debbab A, Proksch P. Fifty years of drug discovery from fungi[J]. Fungal Divers, 2011, 50(1): 3-19.

[2] Khan AA, Bacha N, Ahmad B, et al. Fungi as chemical industries and genetic engineering for the production of biologically active secondary metabolites[J]. Asian Pac J Trop Biomed, 2014, 4(11): 859-870.

[3] 刘富强, 王延平, 杨阳, 等. 黄河三角洲柽柳种群空间分布格局研究[J]. 西北林学院学报, 2009, 24(3): 7-11, 16.

[4] LIU Fuqiang, WANG Yanping, YANG Yang, et al. Spatial distribution pattern of Tamarix chinensis in Yellow River Delta[J]. Journal of Northwest Forestry University, 2009, 24(3): 7-11

[5] Agrawal S, Chavan P, Dufosse L. Hidden Treasure: halophilic Fungi as a repository of bioactive lead compounds[J]. J fungi(Basel), 2024, 10(4): 290-303.

[6] Méjanelle L, Lòpez JF, Gunde-Cimerman N, et al. Ergosterol biosynthesis in novel melanized fungi from hypersaline environments[J]. J Lipid Res, 2001, 42(3): 352-358.

[7] Ma LY, Zhang HB, Kang HH, et al. New butenolides and cyclopentenones from saline soil-derived fungus Aspergillus sclerotiorum[J]. Molecules, 2019, 24(14): 2642. doi: 10.3390/molecules24142642

[8] 徐广进, 温青云, 卜淑甜, 等. 海洋链霉菌OUCMDZ-2582产生的吲哚咔唑生物碱及其抗菌活性[J]. 中国抗生素杂志, 2025, 50(7): 759-769.

[9] XU Guangjin, WEN Qingyun, BU Shutian, et al. Indolocarbazole alkaloids from marine-derived Streptomyces sp. OUCMDZ-2582 and their antibacterial activity[J]. Chinese Journal of Antibiotics, 2025, 50(7): 759-769.

[10] Fu P, Liu PP, Qu HJ, et al. Α-pyrones and diketopiperazine derivatives from the marine-derived actinomycete Nocardiopsis dassonvillei HR10-5[J]. J Nat Prod, 2011, 74(10): 2219-2223.

[11] Kang HH, Zhong MJ, Ma LY, et al. Iizukines C-E from a saline soil fungus Aspergillus iizukae[J]. Bioorg Chem, 2019, 91: 103167. doi: 10.1016/j.bioorg.2019.103167

[12] Xu XY, Zhang XY, Nong XH, et al. Brevianamides and mycophenolic acid derivatives from the deep-sea-derived fungus Penicillium brevicompactum DFFSCS025[J]. Mar Drugs, 2017, 15(2): 43. doi: 10.3390/md15020043

[13] 王聪, 王玉妃, 徐辉, 等. 海洋真菌Penicillium sp. HD-1-1次级代谢产物研究[J]. 中草药, 2024, 55(7): 2142-2151.

[14] WANG Cong, WANG Yufei, XU Hui, et al. Study on secondary metabolites of marine-derived Penicillium sp. HD-1-1[J]. Chinese Traditional and Herbal Drugs, 2024, 55(7): 2142-2151.

[15] Lu XH, Zheng ZH, Zhang H, et al. Two new members of mycophenolic acid family from Penicillium brevicompactum Dierckx[J]. J Antibiot(Tokyo), 2009, 62(9): 527-529.

[16] Fan QY, Yin X, Li ZH, et al. Mycophenolic acid derivatives from cultures of the mushroom Laetiporus sulphureu[J]. Chin J Nat Med, 2014, 12(9): 685-688.

[17] 陈玲玲, 朱统汉, 朱国良, 等. 海洋来源真菌Penicillium brevicompactum OUCMDZ-4920的活性产物研究[J]. 有机化学, 2017, 37(10): 2752-2762.

[18] CHEN Lingling, ZHU Tonghan, ZHU Guoliang, et al. Studies on the bioactive constituents from a marine-derived fungus Aspergillus flavus OUCMDZ-2205[J]. Chinese Journal of Organic Chemistry, 2017, 37(10): 2752-2762.

[19] 李明琼, 黄惠彬, 陈玉婵, 等. 深海来源篮状菌Talaromyces indigoticus FS688中聚酮化合物及其细胞毒性[J]. 有机化学, 2022, 42(9): 2975-2980.

[20] LI Mingqiong, HUANG Huibin, CHEN Yuchan, et al. Polyketides from the deep-sea-derived fungus Talaromyces indigoticus FS688 and their cytotoxicites[J]. Chinese Journal of Organic Chemistry, 2022, 42(9): 2975-2980.

[21] Astakala RV, Preet G, Ebel R, et al. Methyl-4-hydroxy-2-(2-hydroxypropan-2-yl)-6-methyl-2,3-dihydrobenzofuran-5-carboxylate[J]. Molbank. 2023, 2023(3): M1697.

[22] Ibrahim A, Sorensen D, Jenkins HA, et al. Epoxynemanione A, nemanifuranones A-F, and nemanilactones A-C, from Nemania serpens, an endophytic fungus isolated from Riesling grapevines[J]. Phytochemistry, 2017, 140: 16-26.

[23] Karthikeyan N, Prince JJ, Ramalingam S, et al. Electronic [UV-Visible] and vibrational [FT-IR, FT-Raman] investigation and NMR-mass spectroscopic analysis of terephthalic acid using quantum Gaussian calculations[J]. Spectrochim Acta A Mol Biomol Spectrosc, 2015, 139: 229-242.

[24] 刘俊, 邹慧敏, 曾岗, 等. 土曲霉抗耐药菌活性成分及其逆转耐药性作用研究[J]. 中国药业, 2024, 33(21): 39-43.

[25] LIU Jun, ZOU Huimin, ZENG Gang, et al. Active ingredients of Aspergillus terrestris against multi-drug resistance and its reversal effect on bacterial resistance[J]. China Pharmaceuticals, 2024, 33(21): 39-43.

[26] 张玉芳, 李希来, 高志香, 等. 不同施肥组合对木里矿区人工植被和土壤微生物特征的影响[J]. 西北农业学报, 2022, 31(6): 741-754.

[27] ZHANG Yufang, LI Xilai, GAO Zhixiang, et al. Effects of different fertilization treatments on artificial vegetation and soil microbial characteristics in Muli mining area of Qinghai Province[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2022, 31(6): 741-754.

[28] Rodríguez-Blanco A, Sicardi M, Frioni L. Plant genotype and nitrogen fertilization effects on abundance and diversity of diazotrophic bacteria associated with maize(Zea mays L.)[J]. Biol Fertil Soils, 2015, 51(3): 391-402.

[29] Yao LH, Wang DJ, Kang L, et al. Effects of fertilizations on soil bacteria and fungi communities in a degraded arid steppe revealed by high through-put sequencing[J]. Peer J, 2018, 6: e4623. doi: 10.7717/peerj.4623

[30] Parmar DK, Thakur DR. Improvement in soil physical, chemical and microbiological properties during cropping cycles under different nutrient managements in western Himalayas[J]. Int J Curr Microbiol App Sci, 2017, 6(6): 487-496.

[31] Bentley R. Mycophenolic Acid: a one hundred year odyssey from antibiotic to immunosuppressant[J]. Chem Rev, 2000, 100(10): 3801-3826.

[32] Dahlke P, Jordan PM, Klepsch LC, et al. Enhanced efficiency of the microsomal prostaglandin E(2)synthase-1 inhibitor AGU661 in human whole blood by encapsulation into PLGA-based nanoparticles[J]. Mol Pharm, 2025, 22(11): 6803-6815.

[33] Silalai P, Pruksakorn D, Chairoungdua A, et al. Synthesis of propargylamine mycophenolate analogues and their selective cytotoxic activity towards neuroblastoma SH-SY5Y cell line[J]. Bioorg Med Chem Lett, 2021, 45: 128135. doi: 10.1016/j.bmcl.2021.128135

[34] de Mariz E, Miranda LS. The synergy between nucleotide biosynthesis inhibitors and antiviral nucleosides: new opportunities against viral infections?[J]. Arch Pharm(Weinheim), 2023, 356(1): e2200217. doi: 10.1002/ardp.202200217

[35] You C, Pan Y, Liu R, et al. Structural basis for substrate flexibility of the O-methyltransferase Mpa G' involved in mycophenolic acid biosynthesis[J]. Protein Sci, 2024, 33(9): e5144. doi: 10.1002/pro.5144.

[36] Liu XX, Sato N, Yabushita T, et al. IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia[J]. EMBO Mol Med, 2023, 15(1): e15631. doi: 10.15252/emmm.202115631

[37] Azar N, Liarzi O, Zavitan M, et al. Endophytic Penicillium species secretes mycophenolic acid that inhibits the growth of phytopathogenic fungi[J]. Microb Biotechnol, 2023, 16(8): 1629-1638.

基金资助

国家自然科学基金(81903537)

山东省医药卫生发展计划(202313050516)

山东省中医药科技发展计划(Z20244106)

“滨州市揭榜挂帅”技术攻关计划(2025JBGS007)