A new α-pyrone from the mangrove endophytic fungus Phomopsis sp. HNY29-2B

Abstract A new α-pyrone derivative, phomopyrone A (1), together with two known compounds (2–3), was isolated from the culture of the mangrove endophytic fungus Phomopsis sp. HNY29-2B. Their structures were determined by detailed analysis of spectroscopic data. The configuration of 1 was further confirmed by X-ray diffraction. All isolated compounds were evaluated for antibacterial and antioxidative activities. Compound 2 exhibited antibacterial activities with minimal inhibition concentration (MIC) values of 25 and 50 μM against Bacillus subtilis and Pseudomonas aeruginosa, and compound 3 showed activities against Staphylococcus aureus and B. subtilis with MIC values of 25 and 50 μM, respectively.


Introduction
Endophytic fungi have been known to produce important bioactive agents that inhibit or kill a wide variety of harmful micro-organisms including phytopathogens, bacteria, fungi, viruses and protozoans that affect humans and animals (Strobel et al. 2004;Aly et al. 2010). The Phomopsis as endophytes are a creative genus of which secondary metabolites have attracted a great deal of attention for various structures and diverse bioactivities (Gunatilaka 2006;Udayanga et al. 2011;Ma et al. 2016), such as antimigratory cytochalasans (Yan et al. 2016), cytotoxic xanthones (Ding et al. 2013), antibacterial pyrenocines J-M (Hussain et al. 2012), and antifungal lactones (Wu et al. 2008).
As part of our ongoing search for bioactive metabolites from mangrove endophytic fungi (Ding et al. 2013(Ding et al. , 2016Liu et al. 2015Liu et al. , 2016Chen et al. 2016), the crude extract of the fungus Phomopsis sp. HNY29-2B, isolated from a branch of mangrove plant Acanthus ilicifolius, displayed significant antibacterial activities against Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Further chemical investigation of the culture afforded one new α-pyrone, named phomopyrone A (1), together with two known compounds, acropyrone (2) and ampelanol (3) (Figure 1). All isolated compounds were evaluated for antibacterial and antioxidative activities. Herein, details of the isolation, structure elucidation and bioactivities of these compounds are reported.
The known compounds were identified as acropyrone (2) (Hammerschmidt et al. 2014) and ampelanol (3) (Aly et al. 2008) by comparing their detailed spectroscopic data with those reported in literature.
Previously, the secondary metabolites containing pyran-2-one moiety produced by fungi belonging to several genera and their antibiotic, antifungal, cytotoxic, neurotoxic, antiviral and phytotoxic activities were reported (Dickinson 1993;Evidente et al. 2003;Liu et al. 2011;Wang et al. 2016;Zheng et al. 2016). All isolated compounds were evaluated for their antibacterial and antioxidative activities. Compound 2 exhibited antibacterial activities against B. subtilis and P. aeruginosa with minimal inhibition concentration (MIC) values of 25 and 50 μM (Table S2) In addition, none of compounds displayed significant antioxidative activities (IC 50 > 100 μM).

General experimental procedures
Melting point was determined on a Fisher Johns hot-stage apparatus and was uncorrected. IR spectrum was recorded on a Nicolet Nexus 670 in KBr discs and UV spectrum was recorded in MeoH solution on a PERSEE TU-1900 spectrophotometer. The NMR spectra were recorded on Bruker Avance 500 spectrometers (500 and 125 MHz). All chemical shifts (δ) were given in ppm with reference to the solvent signal (δ C 49.0/δ H 3.31 for CD 3 oD, δ C 39.5/δ H 2.50 for DMSo and δ C 77.1/δ H 7.26 for CDCl 3 ). EIMS experiments were measured on a Thermo DSQ EIMS spectrometer and HREIMS were performed on a DMAT95XP high-resolution mass spectrometer. Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao Maine Chemical Factory) and Sephadex LH-20 (Amersham Pharmacia). Thin-layer chromatography (TLC) was performed on silica gel plates (Qingdao Huang Hai Chemical Group Co, G60, F-254). The fractions were monitored by TLC and all compounds were visualised by spraying first with 10% H 2 So 4 in EtoH followed by heating at 120 °C. X-ray diffraction intensity data were collected using an Agilent Gemini Ultra diffractometer with Cu Kα radiation.

Fungal material and culture conditions
Phomopsis sp. HNY29-2B was isolated from a branch of mangrove plant A. ilicifolius which was collected from South China Sea in Hainan Province, China. The strain was identified according to morphologic traits and molecular identification and the sequence data have submitted to GenBank (accession No. KF387574). It was grown at 25 °C for 28 days on a solid autoclaved rice substrate medium (60 1 L Erlenmeyer flasks each containing 50 g of rice and 50 mL of 3% of saline water).

Extraction and isolation
The fermentation mixture was extracted with MeoH three times. The organic extract was evaporated under reduced pressure to yield a brown crude 19.8 g, which was subjected by silica gel column (90 × 8 cm) using petroleum ether and ethyl acetate to afford eight fractions (Fr.

X-ray crystallographic analysis of phomopyrone A (1)
White crystals of 1 were obtained from MeoH-EtoAc. The X-ray diffraction data were collected at 293 K on an Agilent Gemini Ultra diffractometer with Cu Kα radiation (λ = 1.54178 Å). The crystal structure was solved by direct methods and refined using full-matrix least squares difference Fourier techniques. Then, all non-hydrogen atoms were refined and all hydrogen atoms were placed in idealised positions and refined as riding atoms with the relative isotropic parameters. Crystallographic data of 1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under the reference number CCDC 1469660. Copy of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge,CB21EZ, UK (e-mail: deposit@ccdc.cam.ac.uk).

Antibacterial assay
The antibacterial activities against Gram-positive B. subtilis (ATCC6633), S. aureus ATCC (25923) and Gram-negative P. aeruginosa (ATCC 9027) were performed by continuous dilution method in the 96-well plates (Appendino et al. 2008;Wang et al. 2014). All compounds were dissolved in DMSo and diluted in Mueller Hinton broth (MHB) to give a starting concentration (DMSo < 1%) of 1 mmol/mL. Each well containing 80 μL of MHB and 20 μL of sample was inoculated with 100 μL of bacteria suspension (10 6 CFU/mL). After incubation (37 °C, 20 h), the antibacterial potential was evaluated by optical density (oD) measurement. For further determination of the MIC, bacteria suspension (100 μL) were added to the solutions in 96-well to achieve a final volume of 200 μL and final sample concentrations from 50 to 0.39 μmol/L. Blank well was also incubated with only medium under the same condition.
Following incubation for 20 h at 37 °C, oD measurement was recorded at 595 nm. MIC was determined as the drug concentration at which 80% of bacterial growth was inhibited. All experiments were performed in three replicates and ciprofloxacin was the positive control.

Conclusion
In this study, chemical investigation of the mangrove endophytic fungus Phomopsis sp. HNY29-2B afforded one new α-pyrone derivative, phomopyrone A (1) and two known compounds (2-3). The α-pyrone moiety with a hydroxymethyl at C-3 in phomopyrone A (1) was rarely occurring in natural source. Compound 2 exhibited antibacterial activities against B. subtilis (25 μM) and P. aeruginosa (50 μM), and compound 3 showed activities against S. aureus (25 μM) and B. subtilis (50 μM). Considering the significant antibacterial activities of the crude extract, a synergistic or additive effect of all the weakly active secondary metabolites could be assumed. our study would expand the class of α-pyrone and enrich the chemical and biological diversities from mangrove endophytic fungus Phomopsis sp.

Supplementary material
Supplementary material relating to this article is available online, alongside Tables S1-S2 and Figures S1-S9.

Disclosure statement
No potential conflict of interest was reported by the authors.