Novel bioactive compounds have been intensively screened to identify their potential benefit in many fields of human life, including medicine, agriculture and industry. Natural compounds have been a continuous and important source of bioactive compounds, and have led to the discovery of not less than 200 000 bioactive compounds so far,1accounting for more than 50% of new medicines registered as anticancer, antibacterial, antifungal and antiviral agents during the period of 1981–2006.2

Fungal endophytes are defined as filamentous fungi that reside in the tissues of living plants without exerting any pathogenic effects. Judging from the fact more than one endophyte often inhabit a single plant, new and interesting endophytic microorganisms are likely to be found from the nearly 300 000 plant species, which inhabit the diverse environments and ecosystems of the earth.3 Moreover, considering that a great number of secondary metabolites with diverse chemical structures and various biological activities6, 7, 8 have been discovered from endophytes, endophytes can be regarded as a rich source of bioactive natural products.4, 5

Thailand is located in a tropical zone with abundant biodiversity and bioresources, suggesting that Thai endophytic fungi may be a rich source of bioactive compounds. A series of recent discoveries of novel bioactive substances, such as xylariaquinone A, scopararanes A and B, 11-hydroxymonocerin, phomoenamide, phomonitroester and deacetylphomoxanthone B, have confirmed the usefulness of fungal endophytes from Thai medicinal plants as promising bioresources.9, 10, 11, 12

In this study, novel compounds were screened from the endophytic fungus Xylaria feejeensis MU18, isolated from Eryngium foetidum Linn., a medicinal plant in Thailand. From the crude extract, one novel compound was isolated and its chemical structure was determined. The compound possesses a novel chemical structure comprising a γ-pyrone with a hydroxymethyl group and a methylpentyl group at C-2 and C-5, respectively. This is the first report of a natural or even a synthetic compound possessing a γ-pyrone moiety having these two side chains.

Materials and methods

General experimental procedures

The UV spectrum was recorded on a Hitachi U-3200 spectrophotometer (Hitachi Ltd., Tokyo, Japan). NMR spectra were recorded on a on a JEOL JNM-ECS400 (JEOL, Tokyo, Japan) at 400 MHz. The 1H and 13C chemical shifts were referenced to the solvent signal (δH 7.26 and δC 77.0 in CDCl3). HRFABMS was recorded on a JEOL JMS-700 spectrometer. Optical rotation was measured on a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan). IR spectra were recorded on a FTIR-8400S (Shimadzu, Kyoto, Japan).

Microorganism

The endophytic fungus MU18 was isolated from leaves of Eryngium foetidum Linn., obtained from Mahidol University, Bangkok, Thailand. The fungus was identified as X. feejeensis based on the DNA sequences of the internal transcribed spacer (ITS) ribosomal RNA region (DNA data bank of Japan (DDBJ) accession number AB569622). A Genbank search for similar ITS sequences confirmed that the fungus was X. feejeensis, with 99% sequence identity.13 The fungus was deposited as X. feejeensis MU18 at the culture collection of International Center for Biotechnology (ICBiotech; Osaka University, Osaka, Japan).

Fermentation and isolation

All chemicals, media and reagents were purchased from Wako (Osaka, Japan) unless stated otherwise. For seed culture preparation, the mycelia of X. feejeensis MU18 grown on a potato dextrose agar slant was inoculated into 5 ml of medium two (soluble starch 5%, Pharmamedia 2%, oatmeal 0.5%, KH2PO4 0.35%, Na2HPO4 0.25% and (NH4)2SO4 0.6%) in test tubes (Ø12.5 mm × 10.5 cm), and incubated for 3 days at 28 °C on a reciprocal shaker at 120 r.p.m. The seed culture (2 ml) was inoculated into 100 ml of medium two in 500-ml baffled flasks and cultivated for 21 days at 28 °C under a static condition.

After cultivation, culture broth (100 ml × 10 flasks) was mixed with an equal amount of EtOAc and left to stir for 1 h at room temperature. Mycelia were removed by filtration with Miracloth (Calbiochem, La Jolla, CA, USA), and the EtOAc layer was recovered from a separation funnel, dried over anhydrous Na2SO4 and evaporated to afford a crude extract (brown gum, 650 mg).

The crude extract (200 mg each) was at first separated on a C18 cartridge (Sep-Pak Vac 35 cc; Waters, Milford, MA, USA) by stepwise elution with increasing MeOH concentrations (MeOH/H2O=2:8, 4:6, 6:4, 8:2 and 1:0 v/v). The 60% MeOH fractions containing compound 1 were combined and evaporated (50 mg from two repeats). The compound 1 was further purified by preparative reversed-phase C18 HPLC (PU-1570; Jasco, equipped with a UVIDEC-100-V detector) using a CAPCELL PAK C18 column (UG80S5; Shiseido, Tokyo, Japan) with a shallow MeOH gradient in 0.1% TFA (a 60–75% MeOH gradient over a period of 20 min) to yield 10 mg of pure compound 1.

Antimicrobial assay

The minimum inhibitory concentration of xylaropyrone (1) was determined by a twofold broth microdilution method in three individual experiments according to the procedures of the Clinical and Laboratory Standards Institute for antimicrobial, anti-yeast and antifungal activity.14 Kojic acid was used as a reference for the γ-pyrone compound. The minimum inhibitory concentration is defined as the lowest concentration of the compound at which there is no visible growth of the indicator strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29923, Enterococcus faecalis ATCC 29212, Saccharomyces cerevisiae ATCC 6275, Candida albicans OUT 6266, Aspergillus niger ATCC 6275, Rhizopus oryzae ATCC10404 and Candida candidum IFO4598.

Xylaropyrone (1) was obtained as a yellow oil. The molecular formula was determined to be C12H18O3 on the basis of HRFABMS (obs. m/z 211.1335 [M+H]+, calcd. 211.1334 for C12H19O3), 1H and 13C NMR spectra data (Table 1). The IR spectrum showed a broadened OH absorption band at 3390 cm−1.

Table 1 NMR spectroscopic data of xylaropyrone (1) in CDCl3
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1H NMR data of 1 showed two methyl signals at δ 0.82 (t, J=6.9 Hz, 3H) and 0.85 (d, J=6.9 Hz, 3H), eight methylene protons at δ 1.14 (ddq, J=7.6, 15.0, 11.0 Hz, 1H), 1.31 (m, 1H), 1.41 (m, 1H), 1.61(dddd, J=6.7, 7.6, 13.4, 16.6 Hz, 1H), 2.48 (m, 2H) and 4.42 (s, 2H), one methine proton at δ 1.33 (m, 1H) and two aromatic protons at δ 7.74 (s, 1H) and 6.20 (s, 1H) (Supplementary Figure S1). The 13C NMR spectrum of 1 indicated one carbonyl carbon at δ 180.4, two quaternary carbons at δ 126.8 and 171.4, two methyl carbons at δ 11.2 and 18.3, four sp3 methylene carbons at δ 29.1, 31.3, 33.4 and 58.3, one sp3 methine carbon at δ 33.8 and two sp2 methine carbons at δ 113.4 and 152.4 (Supplementary Figure S2).

Three partial structures of 1, namely, a hydroxymethyl group, a methylpentyl group and a 2,5-disubstituted γ-pyrone, were deduced by comprehensive interpretation of its 1H, 13C NMR, COSY, heteronuclear single quantum correlation (HSQC) and HMBC spectra and other spectroscopic data (Figure 1). The observed carbon signals at δ 113.4, 126.8, 152.3, 171.4 and 180.4 in the 13C NMR spectrum suggested the presence of a γ-pyrone moiety.15 This was further supported by the maximum UV absorption at 252 nm and strong absorption band at 1660 cm−1 in the IR spectrum.15

Figure 1
figure 1

COSY and HMBC correlation of xylaropyrone (1).

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The key long range connections of H-8 (δ 2.48) with C-2 (δ 171.4) and C-3 (δ 113.4) and of H-7 (δ 4.42) with C-4 (δ 180.4), C-5 (δ 126.8) and C-6 (δ 152.4) indicated that the hydroxylmethyl and methylpentyl groups were connected to C-5 and C-2 of the γ-pyrone nucleus, respectively. Regarding the absolute configuration at C-10, it was deduced to be R, from the comparison of optical rotations on compounds having similar aliphatic chain with 3-methyl or 3-hydroxymethyl group: those of R-configuration were all minus ((R)-2-(3-(hydroxymethyl)pentyl)-4H-pyran-4-one ([α]20D −0.567), (R)-(3-methylpentyl)benzene ([α]25D −5.52), (R)-4-methylhexan-1-ol ([α]20D −8.1),16, 17, 18 whereas those of S-configuration were all plus.19, 20, 21 Thus, the structure of 1 was elucidated as (R)-5-(hydroxymethyl)-2-(3-methylpentyl)-4H-pyran-4-one (Figure 2).

Figure 2
figure 2

Structure of xylaropyrone (1).

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To the best of our knowledge, xylaropyrone, which consists of a γ-pyrone moiety, a hydroxymethyl group and a methylpentyl group, is a novel compound that has not previously been identified in natural resources or derived from chemical synthesis.

Xylaropyrone (1): a yellow oil; [α]26D −4.1 (c 0.10, MeOH); UV (MeOH) λmax (log ɛ) 215 (3.48), 252 (3.85); HRFABMS m/z [M+H]+ 211.1335 (calcd. for C12H19O3, 211.1334). IR νmax (film) 3161–3502 (OH), 2958, 2523, 2858, 1654 (C=O), 1596, 1419, 1338, 1184, 1128 and 1029 cm−1. 1H (CDCl3, 400 MHz), 13C (CDCl3, 100 MHz), H–H COSY and HMBC see Table 1.

As no antimicrobial data is available on a γ-pyrone compound possessing two side chains, especially to evaluate the effect of two side chains on a γ-pyrone on biological activities, the antimicrobial activities against typical prokaryotes and eukaryotes were measured using kojic acid as a reference. Xylaropyrone showed moderate activity against S. cerevisiae (minimum inhibitory concentration=32 μg ml−1), whereas kojic acid did not show any inhibition even at a concentration of 128 μg ml−1. Neither xylaropyrone nor kojic acid showed any inhibitory activity against E. coli, P. aeruginosa, S. aureus or E. faecalis when used at a concentration of 512 μg ml−1, or against A. niger, R. oryzae or C. candidum when administered at 128 μg ml−1.