Abstract
In our ongoing search for new secondary metabolites from fungi, a basidiomycete fungus Irpex consors was selected for mycochemical investigation, and three new zwitterionic alkaloids (1-3) and five known compounds (4-8) were isolated from the culture broth (16 l) of I. consors. The culture filtrate was fractionated by a series of column chromatography including Diaion HP-20, silica gel, and Sephadex LH-20, Sep-Pak C18 cartridge, medium pressure liquid chromatography (MPLC), and high pressure liquid chromatography (HPLC) to yield eight compounds (1-8). The structures of the isolated compounds were elucidated by the interpretation of nuclear magnetic resonance (NMR) spectra and high-resolution mass spectrometry (HR-MS). Their antioxidant and antibacterial activities were examined. The zwitterionic structures of three new sesquiterpene alkaloids (1-3) were determined together with five known compounds identified as stereumamide E (4), stereumamide G (5), stereumamide H (6), stereumamide D (7), and sterostrein H (8). This is the first report of the zwitterionic alkaloids in the culture broth of I. consors. Three new zwitterionic alkaloids were named as consoramides A–C (1-3).
Mushrooms are a good source of functional foods and traditional therapeutic agents [Citation1]. They produce a wide range of biologically active compounds with unique chemical structures [Citation2–4]. The mushroom Irpex consors, belonging to the family Meruliacease, is distributed in India and East Asian countries such as Korea and Japan [Citation5]. Previous investigations of I. consors have reported that it possesses tricyclic sesquiterpene derivatives with anti-bacterial and anti-tumor activities [Citation6–8]. In our ongoing search for new secondary metabolites from fungal strains, three new zwitterionic alkaloids (1-3) together with five known compounds (4-8) were isolated from the culture broth of the fungus I. consors. Herein, we describe the isolation and structure determination of these compounds ().
Figure 1. Structures of compounds 1-8.
Fungal strain Irpex consors was obtained from Rural Development Administration, Korea. The fungal strain I. consors was cultured on potato dextrose agar at 27 °C for two weeks. Small pieces of fresh mycelium were inoculated into 40 1-l flasks containing 400 ml of potato dextrose broth and cultured on a rotary shaker of 120 rpm at 27 °C for four weeks. The culture broth (about 16 l) was filtered to remove mycelia. The culture filtrate was fractionated by Diaion HP-20 column chromatography eluted with a mixture of methanol-water (30:70–100:0, v/v, stepwise), followed by silica gel column chromatography with stepwise chloroform-methanol (30:1–0:100, v/v) to afford four fractions (Fractions A-D). Fraction A was subjected to Sephadex LH-20 column chromatography, followed by medium pressure liquid chromatography (MPLC) to give two fractions A1 and A2. Fraction A1 was purified by Sep-Pak C18 cartridge eluted with 20% aqueous methanol to obtain two compounds 1 (4.3 mg) and 2 (12.9 mg). Fraction A2 was further separated by a Sep-Pak C18 cartridge eluted with 15% aqueous methanol to obtain compound 7 (4.5 mg). Fraction B was fractionated by Sephadex LH-20 column chromatography, followed by preparative reversed-phase high pressure liquid chromatography (HPLC) eluted with 18% aqueous methanol to yield two compounds 4 (9.3 mg) and 6 (9.5 mg). Fraction C was subjected to MPLC, followed by preparative reversed-phase HPLC eluted with 18% aqueous methanol to provide two compounds 5 (12.0 mg) and 8 (9.0 mg). Fraction D was separated by MPLC eluted with a gradient of increasing methanol (20–100%) in water, followed by preparative reversed-phase HPLC eluted with 27% aqueous methanol to obtain a compound 3 (4.8 mg).
Compound 1 was obtained as a brown powder with a specific rotation value of −92.8° (c = 0.1, 24.5 °C, methanol). Its molecular formula was established as C24H25NO4 by a high-resolution fast atom bombardment (FAB)-mass measurement (m/z 392.1849 [M + H]+, Δ −1.2 mmu). The 1H NMR spectrum of 1 revealed the presence of a substituted benzene moiety at δ 7.18 (× 2), 7.14, and 7.09 (× 2), a 3,4-disubstituted pyridine moiety at δ 9.05, 8.99, and 8.27, a olefinic methine at δ 6.93, two methines at δ 5.59 and 3.69, two methylenes at δ 3.88/3.47 and 2.10/1.97, and three methyls at δ 1.28, 1.24, and 1.19 (). In the 13C NMR spectrum, twenty-four carbons including one ketone carbon at δ 181.1, one carbonyl carbon at δ 171.0, nine sp2 methine carbons at δ 155.7, 148.6, 145.7, 130.1 (× 2), 129.9 (× 2), 128.6, and 125.0, four sp2 quaternary carbons at δ 171.4, 137.4, 137.3, and 131.6, one oxygenated quaternary carbon at δ 74.1, two methine carbons at δ 78.8 and 53.8, one quaternary carbon at δ 47.0, two methylene carbons at δ 40.7 and 40.6, and three methyl carbons at δ 28.6, 27.6, and 25.2 were evident (). In the 1H-1H correlated spectroscopy (COSY) spectrum, correlations between H-3 and H-4 and between H-13 and H-14 were observed, and the long-range correlations from H-1 to C-8 and C-9, from H-3 to C-5 and C-9, from H-10 and H-11 to C-1, C-2, and C-3, from H-12 to C-4, C-5, and C-6, from H-13 to C-5 and C-7, from H-14 to C-6 and C-15, and from H-15 to C-6 and C-8 established the presence of sterostrein Q moiety. The long-range correlations from H-2′ to C-1′, C-3′ and C-4′, from H-8′ to C-4′, and from H-9′ to C-3′ and C-7′ as well as 1H-1H COSY correlations of H-5′/H-6′/H-7′/H-8′/H-9′ and H-2′/H-3′ revealed the presence of a phenylalanine moiety. Finally, the long-range correlations from H-14 and H-15 to C-2′ and from H-2′ to C-14 and C-15 indicated that the phenylalanine moiety was connected to sterostrein Q via carbon-nitrogen bond (). The partial relative stereochemistry of 1 was established by the NOESY correlations. The cross peaks of H-4/H-3a and H-4/H-11 indicated the same face, while those of H-10/3b and H-3b/H-12 confirmed the other face. Therefore, the structure of 1 was determined as a new zwitterionic alkaloid and named consoramide A.
Figure 2. 1H-1H COSY and HMBC correlations of compounds 1-3.
Table 1. 1H and 13C NMR data of compounds 1-3 in methanol-d4.
Compound 2 was purified as a yellow oil with specific rotation of −107.2° (c = 1.0, 25.0 °C, methanol) and exhibited UV maxima (log ε) at 203 (3.34) and 238 (3.61) nm. Its molecular formula was determined to be C18H21NO4 by the high-resolution FAB-mass measurement (m/z 316.1531 [M + H]+, Δ −1.8 mmu). The 1 D NMR spectra of 2 revealed that the hydroxymethyl group in 5 was replaced by a methyl group (). The long-range correlations from H-3′ to C-1′ and C-2′ as well as 1H-1H COSY correlations between H-2´and H-3′ supported the presence of an alanine moiety in 2 (). Therefore, compound 2 was determined to be a new zwitterionic alkaloid and named consoramide B.
Compound 3 was obtained as a yellow powder with the specific rotation of −89.2° (c = 1.0, 25.0 °C, methanol) and showed UV maxima (log ε) at 202.0 (3.37) nm. Its molecular formula was established as C20H24N2O5 by the high-resolution FAB-mass measurement (m/z 373.1745 [M + H]+, Δ −1.9 mmu). The NMR spectra revealed that 3 was consisted of sterostrein Q and glutamine (). The glutamine moiety was determined by the 1H-1H COSY correlations and the long-range correlations from H-2′ to C-1′ and C-3′ and from H-3′ and H-4′ to C-5′ (). Thus, compound 3 was determined to be a new zwitterionic alkaloid and named consoramide C. The configuration of all amino acid moieties in 1-3 was tentatively deduced as L-form, because the L-amino acids are abundant in nature literature [Citation9,Citation11].
Compounds 4-8 were identified as stereumamide E (4), stereumamide G (5), stereumamide H (6), stereumamide D (7), and sterostrein H (8), respectively, by the comparison of their spectroscopic data with the literatures previously reported [Citation9–11].
The antioxidant activities of these compounds (1-8) were evaluated by the ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulonate]) and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging assays [Citation12]. All compounds (1-8) displayed no radical scavenging activity up to 200 µM. In the present study, all compounds exhibited no antibacterial activity up to 50 µg/disk against Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, and Escherichia coli.
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The online version of this article (https://doi.org/10.1038/s41429-017-00XX-X) contains supplementary material, which is available to authorized users.
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The authors thank Ms. Ji-Young Oh, Center for University-wide Research Facilities (CURF) at Jeonbuk National University, for NMR measurement.
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