Abstract
To discover antimicrobial agents from higher fungi, mannonerolidol (3), a new nerolidol mannoside, together with known schizostatin (1) and nerolidol (2) were isolated from an antimicrobial fraction of the culture broth of Schizophyllum commune. Structures of these compounds were determined through spectroscopic methods. Compounds 1 and 3 exhibited antimicrobial activities against plant pathogenic fungi Rhizoctonia solani, Diaporthe sp., Botrytis cinerea, and Alternaria solani and bacteria Bacillus subtilis and Staphylococcus aureus.
The higher fungi produce various antibiotics to protect themselves from external environment and pathogens. Many antifungal and antibacterial compounds such as strobilurins, ganodermin, hypsin, and xylarinic acids have been isolated from mushrooms [1,2,3,4]. In our ongoing effort to search for antimicrobial compounds from fungal metabolites, we found that the culture broth of a higher fungus Schizophyllum commune exhibited significant antimicrobial activity. S. commune belonging to family Schizophyllacea is known to produce β-glucans such as schizophyllan, an immunostimulatory anticancer agent, schizines A and B that can inhibit cancer cell growth, schizostatin known to be a squalene synthase inhibitor, and schizocommunin that exhibits cytotoxicity [5,6,7,8]. In this study, we isolated a new nerolidol mannoside, designated as mannonerolidol (3), together with known schizostatin (1) and nerolidol (2) from an antimicrobial fraction of the culture broth of S. commune (Fig. 1).
Fungal strain S. commune NAAS 04688 was obtained from Rural Development Administration, Korea. To determine the phylogenetic position of NAAS 04688 strain (GenBank accession number: MH279792), ribosomal DNA internal transcribed spacer (ITS) region sequences containing the 5.8s rDNA gene previously reported in Genbank were aligned with NAAS 04688 strain using MEGA 7.0 software. Phylogenetic analysis showed that NAAS 04688 strain was closely related to S. commune strains, sharing sequence similarities of ≥99.3% with S. commune strains (see Supporting Information).
We cultured a fungal strain of S. commune NAAS 04688 in ten 4 l flasks (each containing 2 l of potato dextrose broth) at 27 °C in a stationary culture incubator for two weeks. The culture broth (20 l) was extracted with acetone at room temperature. After removing acetone under reduced pressure, the resulting extract was partitioned between chloroform and water. The chloroform-soluble layer showed significant antibiotic activity. The chloroform-soluble layer was concentrated and subjected to silica gel column chromatography eluted with CHCl3:MeOH (25:1, 5:1, MeOH only, v/v, stepwise). CHCl3:MeOH (5:1) fraction and MeOH fraction were combined, concentrated under reduced pressure, and subjected to reversed-phase medium pressure liquid chromatography eluted with 50% aq. MeOH to afford compound 1 (50 mg) and an active fraction. The active fraction was subjected to a reversed-phase Sep-pak cartridge eluted with 50-100% aq. MeOH followed by silica gel column chromatography eluted with CHCl3:MeOH (10:1, 5:1, v/v) to give two fractions. Each fraction was further separated by Sephadex LH-20 column chromatography eluted with MeOH to afford compounds 2 (8.3 mg) and 3 (4.6 mg) (see Supporting Information).
Compounds 1 and 2 were identified as schizostatin and (+)-trans-nerolidol, respectively, by comparing spectroscopic data with previously published data [9,10,11,12].
Compound 3 was obtained as a brown oil with specific rotation value of -83.0° ([α]D; 25 °C, c = 0.1, MeOH). It exhibited UV maxima (log ε) at 203 (3.9) nm. Its molecular formula was determined to be C21H36O6 through a high-resolution ESI-mass measurement (m/z 407.2407 [M + Na]+, Δ −0.3 mmu). The 1H NMR spectrum of compound 3 in CD3OD showed signals due to three olefinic methines at δ 6.03, 5.11, and 5.09, one terminal methylene at δ 5.23/5.16, five oxygenated methines at δ 4.59, 3.72, 3.57, 3.43, and 3.11, one oxygenated methylene at δ 3.80/3.69, four methylenes at δ 2.07, 2.04, 1.97, and 1.64, and four methyls at δ 1.67, 1.60, 1.60, and 1.33. Its 13C NMR spectrum showed 21 carbons, including two sp2 quaternary carbons at δ 136.1 and 132.2, three sp2 methine carbons at δ 144.4, 125.9, and 125.6, one sp2 terminal methylene carbon at δ 115.1, one anomeric methine carbon at δ 96.6, four sp3 oxygenated methine carbons at δ 78.0, 75.7, 74.2, and 68.5, one sp3 oxygenated quaternary carbon at δ 81.3, one oxygenated methylene carbon at δ 63.0, four sp3 methylene carbons at δ 41.4, 40.9, 27.9, and 23.7, and four methyl carbons at δ 26.0, 23.9, 17.9, and 16.3 (Table 1). All proton-bearing carbons were assigned by HMQC spectrum. Interpretation of 1H-1H COSY spectrum established three partial structures and a mannose moiety as shown in Fig. 2. The anomeric configuration of the mannose was considered as β because of the heteronuclear coupling constant (1JCH) of 154.0 Hz between the anomeric carbon and the respective proton [13]. Three partial structures were connected by the HMBC spectrum which showed long-range correlations from the methyl protons at δ 1.33 (3-CH3) to C-2, C-3, and C-4, from the methyl protons at δ 1.60 (7-CH3) to C-6, C-7, and C-8, and from two methyl protons at δ 1.60 (11-CH3) and δ 1.67 (C-12) to C-10 and C-11. These HMBC correlations established a sesquiterpene nerolidol moiety. In addition to HMBC correlations, chemical shift values of 1H and 13C NMR peaks were consistent with those of trans-nerolidol [10,11,12]. Finally, the chemical structure of compound 3 was confirmed by its HMBC spectrum which showed long-range correlations from anomeric proton of mannose at δ 4.59 (H-1‘) to a quaternary carbon at δ 81.3 (C-3) (Fig. 2). Therefore, compound 3 was determined to be nerolidol mannoside. The stereochemistry of C-6 was determined as S by specific rotation value (+36.1° ([α]D; 23°C, c = 0.05, MeOH) of nerolidol isolated from acid hydrolysate of 3 [11, 12].
Antimicrobial activities of compounds 1–3 were determined with conventional paper disk (6 mm in diameter) diffusion method. Test microorganisms used in this study included four fungi (Rhizoctonia solani, Diaporthe sp., Botrytis cinerea, and Alternaria solani) and two bacteria (Bacillus subtilis and Staphylococcus aureus). These fungi were incubated at 27°C for 4 days while these bacteria were incubated at 37°C for 30 h. Compound 1 at 50 μg disk-1 showed significant antifungal activities against R. solani, Diaporthe sp., B. cinerea, and A. solani, showing clear zone diameters of 15.6, 19.5, 12.7, and 20.2 mm, respectively. Compound 3 displayed moderate antifungal activities against R. solani and Diaporthe sp. with clear zone diameters of 11.4 and 10.9 mm, respectively. It has been reported that schizostatin possesses no antibacterial activity [7]. In this study, schizostatin at 50 μg disk-1 displayed antibacterial activities against B. subtilis and S. aureus, showing clear zone diameters of 11.2 and 21.2 mm, respectively. Nerolidol exhibited no antimicrobial activity.
References
Anke T, Oberwinkler F, Steglich W, Schramm G. The strobilurins-new antifungal antibiotics from the basidiomycete Strobilurus tenacellus. J Antibiot. 1977;30:806–10.
Wang H, Ng TB. Ganodermin, an antifungal protein from fruiting bodies of the medicinal mushroom Ganoderma lucidum. Peptides. 2006;27:27–30.
Lam SK, Ng TB. Hypsin, a novel thermostable ribosome-inactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochem Biophys Res Commun. 2001;285:1071–5.
Jang YW, Lee IK, Kim YS, Lee S, Lee HJ, Yu SH, Yun BS. Xylarinic acids A and B, new antifungal polypropionates from the fruiting body of Xylaria polymorpha. J Antibiot. 2007;60:696–9.
Komatsu N, Okubo S, Kikumoto S, Kimura K, Saito G, Sakai S. Host-mediated antitumor action of schizophyllan, a glucan produced by Schizophyllum commune. Gann. 1969;60:137–44.
Liu X, Frydenvang K, Liu H, Zhai L, Chen M, Olsen CE, Christensen SB. Iminolactones from Schizophyllum commune. J Nat Prod. 2015;78:1165–8.
Tanimoto T, Onodera K, Hosoya T, Takamatsu Y, Kinoshita T, Tago K, Kogen H, Fujioka T, Tsujita Y. Schizostatin, a novel squalene synthase inhibitor produced by the mushroom, Schizophyllum commune. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities. J Antibiot. 1996;49:617–23.
Hosoe T, Nozawa K, Kawahara N, Fukushima K, Nishimura K, Miyaji M, Kawai K. Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune. Mycopathologia. 1999;146:9–12.
Kogen H, Tago K, Kaneko S, Hamano K, Onodera K, Haruyama H, Minagawa K, Kinoshita T, Ishikawa T, Tsujita Y. Schizostatin, a novel squalene synthase inhibitor produced by the mushroom, Schizophyllum commune. J Antibiot. 1996;49:624–30.
Degenhardt J, Gershenzon J. Demonstration and characterization of (E)-nerolidol synthase from maize: a herbivore-inducible terpene synthase participating in (3E)-4,8-dimethyl-1,3,7-nonatriene biosynthesis. Planta . 2000;210:815–22.
Yanagisawa H, Ohshima Y, Okada Y, Takahashi K, Shibata S. A sesquiterpene glycoside, loquatifolin A, from the leaves of Eriobotrya japonica. Chem Pharm Bull. 1988;36:1270–4.
Schubert V, Dietrich A, Ulrich T, Mosandl A. The stereoisomers of nerolidol: separation, analysis and olfactoric properties. Z Naturforsch. 1992;47c:304–7.
Harms H, Rempel V, Kehraus S, Kaiser M, Hufendiek P, Müller CE, König GM. Indoloditerpenes from a marine-derived fungal strain of Dichotomomyces cejpii with antagonistic activity at GPR18 and cannabinoid receptors. J Nat Prod. 2014;77:673–7.
Acknowledgements
This work was supported by a grant (grant No. PJ012633) from the Agenda Project of the Rural Development Administration (RDA), Republic of Korea. Authors thank Ms. Ji-Young Oh, Center for University Research Facility (CURF) at Chonbuk National University, for contributing to NMR measurement.
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Woo, EE., Kim, JY., Kim, JS. et al. Mannonerolidol, a new nerolidol mannoside from culture broth of Schizophyllum commune. J Antibiot 72, 178–180 (2019). https://doi.org/10.1038/s41429-018-0130-3
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DOI: https://doi.org/10.1038/s41429-018-0130-3
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