Introduction

There are various ecosystems in the vast marine environment including hitherto unexploited and geographically and taxonomically undescribed organisms. Marine microorganisms, especially marine fungi, are well known to be rich sources of structurally interesting and biologically active compounds.1, 2 Chemical investigations of mangrove-derived endophytic fungi, especially those from the subtropical island of Hainan, China, have shown a sharp increase in recent years.3, 4, 5 As a result of adaptation to some special environments, mangrove endophytic fungi have formed unique genetic backgrounds and metabolic pathways.6, 7, 8 It is encouraging that bioactive compounds have been obtained from mangrove-derived fungi.9, 10, 11, 12 Endophytic fungi have also proven to be promising sources of new natural products with promising biological and pharmacological activities.13, 14, 15

In our search for new antibacterial natural products from marine fungi in the South China Sea, a fungus Stemphylium sp. 33231 obtained from the leaves of Brguiera sexangula var. rhynchopetala attracted our attention. The EtOAc extract of a fermentation broth of the fungus exhibited antimicrobial activities against tested bacterial strains. Bioassay-guided fractionation of the bioactive extract led to the isolation of two new α-pyrone derivatives, infectopyrone A (1) and infectopyrone B (2) (Figure 1). Compounds 1 and 2 were evaluated for their antibacterial activities. Herein, we report the isolation, structure elucidation and biological activities of these compounds.

Figure 1
figure 1

The structure of compounds 1, 2 and infectopyrone.

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Results and Discussion

Isolation and identification of compounds

The EtOAc extract of the fungal culture was treated with a combination of chromatographic materials, silica gel, octadecyl silane (ODS) column chromatography and Sephadex LH-20 column chromatography. The structures were elucidated by NMR and HRESI-MS spectroscopic data.

Compound 1 was isolated as a light yellow powder and had the molecular formula C14H16O6 (7 degrees of unsaturation) based on the prominent signal at m/z 303.0840 [M+Na]+ in the HRESI-MS, and combined with 1H and 13C NMR spectroscopic data (Table 1). The 1H NMR spectrum of 1 showed the presence of three olefinic protons in the downfield at δH 6.88 (1H, br s), 6.70 (1H, br s) and 5.79 (1H, br s). In the upfield, one oxygenated methylene group at δH 4.22 (2H, s), one methoxyl group at δH 3.98 (3H, s), two olefinic methyl groups at δH 2.09 (3H, s) and 2.25 (3H, s) were also observed. In addition, 14 carbon signals were detected in the 13C NMR spectrum (Table 1) including signals of two methyl groups, one methoxy group, one oxygenated methylene group, eight olefinic carbons, one ester carbonyl group and one carboxy1 group. These 1H and 13C NMR spectroscopic features suggested that 1 was very similar to infectopyrone. Infectopyrone is an α-pyrone derivative, which has been isolated from the fungus Alternaria infectoria,16 and was not isolated in this study (Figure 1), except for the disappearance of a methyl signal at δC 8.8 and δH 1.93 (3H, s) for 3, s) in infectopyrone16 and the presence of an oxygenated methylene signal at δC 52.1 and δH 4.22 (2H, s) for 3, CH2OH in 1. These characteristics implied that the methyl group in infectopyrone16 was replaced by an oxygenated methylene in 1. These were further supported by the HMBC correlations of the H-7-xygenated methylene, T Figure 2). Detailed analysis of 2D NMR (HSQC, 1H–1H COSY, HMBC and NOESY) spectra confirmed that the other parts of the molecule were the same as those of infectopyrone.16 Thus, the structure of compound 1 was identified as the Figure 1. We name compound 1 as infectopyrone A.

Table 1 NMR Data for compounds 1 and 2
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Figure 2
figure 2

Key partial structure of Compounds 1 and 2 from HMBC data, 1H–1H COSY and NOESY correlations.

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Compound 2 was isolated as a fine light yellow powder and had the molecular formula C15H18O6 (7 degrees of unsaturation) based on the prominent signal at m/z 317.0992 [M+Na]+ in the HRESI-MS. The 1H and 13C NMR data of 2 (Table 1) resembled with those of 1, except for the existence of one additional methyl group at δH 3.43 and δC 58.4 for 7′-OMe, which was in accordance with the increase in MW of 2 by 14 a.m.u. compared with 1. Further confirmation was achieved by the observed HMBC correlations of H-7′ to 7′-OMe, 2′, 3′ and 4′ (Figure 2). Detailed analysis of 2D NMR (HSQC, 1H–1H COSY, HMBC and NOESY) spectra confirmed that the other parts of the molecule were the same as those of 1. Thus, the structure of compound 2 was identified as infectopyrone B (Figure 1).

Biological properties of infectopyrones A and B

Preliminary antibacterial assay results showed that at a concentration of 20 μg ml−1, 1 and 2 exhibited a broad spectrum of antibacterial activity against five terrestrial pathogenic bacteria. The MIC values of 1 and 2 were further tested by the microplate assay method. The results in (Table 2) showed that 1 had moderate antibacterial activity against Bacillus subtilis (ATCC 6633), Micrococcus tetragenus (ATCC 13623) and Micrococcus luteus (ATCC 9341) with MIC values of 10.0 μg ml−1 for each. While compound 1 showed significant activity against S. albus (ATCC 8799) and Escherichia coli (ATCC 25922) with MIC values of 5.0 μg ml−1 and 2.5 μg ml−1, respectively. Compounds 1 and 2 had similar antibacterial activity, but 2 showed weaker activity against S. albus (ATCC 8799) with an MIC value of 10.0 μg ml−1. These results indicated that the 7′-OH group was important for antibacterial activity. Both compounds were tested for the microwell cytotoxicity assay using Artemia salina (brine shrimp).17 No significant cytotoxic activities against A. salina were observed at 50 μg ml−1.

Table 2 Antibacterial activity for compounds 1 and 2
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Methods

Fungal materials

The fungal strain Stemphylium sp. 33231 was isolated from the mangrove Brguiera sexangula var. rhynchopetala collected in the South China Sea in August, 2012. The strain was deposited in the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University of China, Hainan. The fungal strain was cultivated in 30 l potato glucose liquid medium (15 g of glucose and 30 g of sea salt in 1 l of potato infusion, in 1-l Erlenmeyer flasks each containing 300 ml of culture broth) at 25 °C without shaking for 4 weeks.

Identification of the fungus

The Fungus was identified according to its morphological characteristics and a molecular biological protocol by 18S rRNA amplification and sequencing of the ITS region. The sequence data have been submitted to GenBank, with an accession number KF479349, and the fungal strain was identified as Stemphylium sp.

General experimental procedures

Silica gel (Qing Dao Hai Yang Chemical Group Co., Qingdao, China; 200–300 mesh), octadecylsilyl silica gel (YMC, Kyoto, Japan; 12 nm–50 μm) and Sephadex LH-20 (GE Healthcare, Shanghai, China) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co., Yantai, China; G60, F-254) were used for TLC. 1H and 13C NMR spectra were recorded on a Bruker AV spectrometer (Bruker, Zurich, Switzerland) at 400 MHz in CDCl3 or DMSO-d6. Chemical shifts δ are reported in p.p.m., using tetramethylsilane (TMS) as an internal standard, and coupling constants (J) are in Hz. ESI-MS and HRESI-MS spectra were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. IR spectra were recorded on a Nicolet 6700 spectrophotometer (Thermo Fisher Scientific Co., Shanghai, China).

Extraction and isolation

The fungal cultures were filtered through cheesecloth and the filtrate was extracted with EtOAc (3 × 30 l, 10 h each). The EtOAc extracts were concentrated in vacuo to yield an oily residue (25.2 g). that was subjected to silica gel CC (petroleum ether/EtOAc v/v, 100:0–0:100) to generate five fractions (Fr. 1–Fr. 5). Fr. 4 was isolated by CC on silica gel eluted with petroleum ether—EtOAc (1:1), and then subjected to Sephadex LH-20 CC eluting with mixtures of petroleum CHCl3—MeOH (2:3) and further purified by using octadecylsilyl silica gel eluted with 50% MeOH/H2O to obtain compound 2 (6.2 mg). Fr. 5 was isolated by CC on silica gel eluted with petroleum ether—EtOAc (1:2), and then subjected to Sephadex LH-20 CC eluting with mixtures of petroleum CHCl3—MeOH (2:3) and further purified by using octadecylsilyl silica gel eluted with 40% MeOH/H2O to obtain compound 1 (11.2 mg).

Physical properties of compounds 1 and 2

Infectopyrone A (1): light yellow powder; UV (MeOH) λmax (log ɛ) 334 (1.12), 213 (2.29) nm; IR (KBr) νmax 3429, 1572, 1408 cm−1; 1H and 13C NMR: see Table 1; HRESI-MS m/z 303.0840 [M+Na]+ (calcd. for C14H16O6Na, 303.0839).

Infectopyrone B (2): light yellow powder; UV (MeOH) λmax (log ɛ) 309 (0.95), 211 (2.20) nm; IR (KBr) νmax 3429, 1570, 1413 cm−1; 1H and 13C NMR: see Table 1; HRESI-MS m/z 317.0992 [M+Na]+ (calcd for C15H18O6Na, 317.0995).

Antibacterial assays

Antibacterial activity was determined against five terrestrial pathogenic bacteria, including Staphylococcus albus (ATCC 8799), E. coli (ATCC 25922), B. subtilis (ATCC 6633), M. tetragenus (ATCC 13623) and M. luteus (ATCC 9341), by the microplate assay method.18