A new pyrone derivative from an endophytic Aspergillus tubingensis of Lycium ruthenicum

Abstract A new pyrone named 6-isovaleryl-4-methoxy-pyran-2-one (1), along with three known pyrone compounds, rubrofusarin B (2), asperpyrones A (3) and campyrone A (4), was isolated from fermentation of Aspergillus tubingensis in Lycium ruthenicum. Their structures were confirmed by spectroscopic techniques, such as IR, NMR and HRESI-MS. Compound 2 indicated strong inhibitory activity against Escherichia coli, with MIC value of 1.95 μg/mL.


Introduction
Endophytic fungi, isolated from medicinal plants in adversity, have been recognised as a rich source of novel structure and potent bioactivity substances (Strobel 2003;Hu et al. 2011). Endophytic Aspergillus sp. can produce important secondary metabolites with interesting bioactivities (Deng et al. 2013;Xiao et al. 2014;Siriwardane et al. 2015). Lycium ruthenicum is medicinal plant, widely distributed throughout north-west barren soil in China. The plant is used in Chinese traditional medicine for the treatment of hypertension, diabetes, heart disease, etc. (Gan et al. 1997). Thus, the endophytic fungi from L. ruthenicum are considered a promising reservoir of biologically active natural products. Our group has conducted a preliminary study of the endophytic fungi of L. ruthenicum (Wang et al. 2013). In our continuing research for bioactive secondary metabolites from an endophytic Aspergillus tubingensis of L. ruthenicum, a new pyrone (1), together with three known compounds, rubrofusarin B (2), asperpyrones A(3) and campyrone A(4), was obtained. Details of the isolation, structure elucidation and biological activities of these compounds are reported.

Fungus identification
Colony of the fungus on potato dextrose agar (PDA) was smooth and white to black surface. It produced conidiophores and easy-scattered conidia which are typical characteristics of the genus Aspergillus. The strain was identified as A. tubingensis by morphological characters and ITS sequence. The sequence is available in GenBank with accession number KJ476743.

Structure elucidation
Compound 1 was obtained as a white needle crystal in MeOH; m.p. 101.8-102.9 °C. The molecular formula was established as C 11 H 14 O 4 (five degrees of unsaturation) based on the observed pseudomolecular ion [M + Na] + at m/z 233.0782 (calcd for 233.0790) by HR-ESI-MS. The 13 C NMR and DEPT spectra revealed 11 carbon signals, which were classified by chemical shifts as δ C : 192.6, 169.5, 161.6, 153.9, 104.8, 93.5, 56.9, 45.6, 23.8 and 22.2 (Table S1), of them, a methoxy at δ C 56.9,two methyl groups at δ C 22.2. The IR absorptions at 1719 and 1635 cm −1 indicated the presence of ester carbonyl and ketone carbonyl groups, respectively. The structure of compound 1 was supported by a comparison of its NMR data with those of a known compound named 6-pentyl-4-methoxy-pyran-2-one (Evidente et al. 2012) (Figure 1), which showed signals of 4-methoxy-pyran-2-one residue. The HMBC correlations from H-10 and H-11(δ H 0.91) to C-8 (δ C 45.6) suggested that compound 1 contained isobutyl fragment, of which fragment was also supported by the 1 H− 1 H COSY correlations of H-8/H-9, H-9/H-10 and H-9/H-11. What's more, the HMBC cross-peaks from H-9 (δ H 2.14-2.04) and H-5 (δ H 6.94) to C-7 (δ C 192.6) revealed that isovaleryl residue can be determined, and this residue confirmed the attachment of pyrone ring on C-6. Thus, from above mentioned evidences and by comparing the NMR spectral data of 6-pentyl-4-methoxy-pyran-2-one in the literature, compound 1 was unambiguously confirmed to be 6-isovaleryl-4-methoxy-pyran-2-one. The structure and main correlations in the HMBC of compound 1 are indicated in Figure 2.

Biological activities
The result of antibacterial activity indicated that compounds 1, 3 and 4 were weakly active, but compound 2 exhibited strong antibacterial activity against Escherichia coli with MIC value of 1.95 μg/mL. All compounds showed weak antioxygenation activities. Above results are displayed in Table 1.

General
All the solvents were purchased from Tianjin Hongyan Chemical Reagents Factory (China). The silica gel for column chromatography (200-300 mesh) was purchased from Tsingtao Marine Chemical Factory (China). Melting points were measured on an X-6 micro-melting point apparatus and were uncorrected. HR-ESI-MS were obtained in the positive ion mode with Bruker maXis 4G uHR-TOF. IR spectra were recorded with a Bruker VECTOR-22 FT-IR Spectrometer. 1D and 2D nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCEIII-400 MHz spectrometer with tetramethylsilane (TMS) as internal standard.

Fungal material
The fungus was isolated from the root of L. ruthenicum. The fungus was initially identified using morphological characteristics. And the fungus was further identified using a molecular biological protocol by DNA amplification and sequencing of the ITS region (Tao et al. 2008). The sequenced

Antimicrobial assay
Antimicrobial evaluation against two Gram-positive bacteria (S. aureus and S. lactis) and two Gram-negative bacteria (E. coli and P. aeruginosa) was carried out by the continuous dilution method in the 96-well plates (Appendino et al. 2008). Solutions of all compounds were prepared series of concentration from 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, to 0.98 μg/mL in DMSO. Each well containing 100 μL of the solution of sample was inoculated with 100 μL of bacteria suspension containing 10 6 CFu/mL, and plates were incubated at 37 °C for 24 h. The result of assay was recorded by the values of MIC (minimal inhibition concentration). The MIC was defined as the lowest concentration that produces complete growth inhibition of the tested. The mediums of bacteria were made up of beef extract 3.0 g/L, peptone 10.0 g/L, NaCl 5.0 g/L, pH 7.0-8.0. Penicillin sodium was positive control against Gram-positive bacteria; streptomycin sulphate was positive control against Gramnegative bacteria.

Antioxygenation assay
2,2′-Diphenylpicrylhydrazyl (DPPH) radical is widely used as a model system to test the scavenging activities of several natural products, Vitamin C always was regarded as standard to judge the ability of scavenging activities. Successive sample dilutions (100 μL/well, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, 0.98, 0.49 μg/mL) were mixed with 0.2 mg/mL DPPH solution in MeOH (100 μL/well), and absorbance was measured at 517 nm with a microplate reader in triplicate (Predes et al. 2011). The result of antioxygenation assay was indicated by the values of IC 50 . The IC 50 was the half maximal inhibitory concentration.

Conclusion
A new pyrone, 6-isovaleryl-4-methoxy-pyran-2-one, together with three known pyrone compounds were isolated from an endophytic A. tubingensis of L. ruthenicum. Compound 2 is the major constituents in the fermentation, which indicated strong inhibitory activity against E. coli. A. tubingensis deserved increasing attention as a source of antibacterial drug.

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

Funding
This work was co-financed by the Specialized Research Fund