A new furan derivative from an endophytic Aspergillus flavus of Cephalotaxus fortunei

A new furan derivative named 5-acetoxymethylfuran-3-carboxylic acid (2), together with a known furan compound, 5-hydroxymethylfuran-3-carboxylic acid (1), were isolated from the fermentation of Aspergillus flavus, endophytic fungi in Cephalotaxus fortunei. The structures of 1 and 2 were elucidated by NMR, IR, UV and MS data, as well as compared with literature data. The compounds 1 and 2 exhibited potent antibacterial activity against Staphylococcus aureus with MIC values of 31.3 and 15.6 μg/mL, respectively. The compound 2 showed moderate antioxidant activity.


Introduction
Endophytic fungi that colonise internal tissues of healthy plants represent one of the largest and relatively under-explored resources of biologically active small molecule natural products (Schulz et al. 2002;Strobel 2003;Zhang et al. 2006;Arnold 2007;Rodriguez et al. 2009). Medicinal plants are known to be a promising reservoir of bioactive natural products for drug discovery (Alonso-Castro et al. 2011;Chon 2012). There has been an increasing number of reports of biologically active metabolites from endophytic fungi of medicinal plants (Liu et al. 2011;Luo et al. 2013;Ortega et al. 2013). In the course of our ongoing effort to find valuable compounds from endophytic fungi Aspergillus sp. S19 of Cephalotaxus fortunei which is a traditional medicinal plant, a new furan derivative 5-acetoxymethylfuran-3-carboxylic acid (2) together with a known compound (1) were found. This article described the isolation and structural elucidation as well as antimicrobial and antioxidant activities evaluation of two compounds.

Results and discussion 2.1. Fungus identification
The strain of S19 was identified as Aspergillus flavus (GenBank accession number KJ473711), based on the internal transcribed spacer (ITS) sequence.
The NMR spectral (Table 1) data of compound 2 were similar to those of compound 1. The differences were in the 1 H NMR of compound 2 in contrast to those of compound 1 disappeared a hydroxyl hydrogen signal at d H 5.70 and appeared a methyl singlet at d H 2.11. Meanwhile, the 13 C NMR spectrum displayed the addition of one carbonyl signal (d C 169.54) and one methyl signal (d C 20.73) than 1. Furthermore, the methyl group was considered to be bound to carboxyl carbon by HMBC correlation between the methyl proton signal (d H 2.11) and carboxyl (d C 169.54). The above evidence suggested that it was an acetyl group. Moreover, the acetyl group was bound to alcoholic hydroxyl group by the HMBC correlation from the d H 4.94 to d C 169.54. According to the IR and NMR spectra, compound 2 was identified as acylation of the OH at C-7 in compound 1. Thus, compound 2 was unambiguously confirmed to be 5-acetoxymethylfuran-3-carboxylic acid. The main correlation in the HMBC of compound 2 was indicated in Figure 2. The model of conformation of compounds 1 and 2 is displayed in Figure 3.

Biological Activities
The screening data of the antibacterial activity showed that compounds 1 and 2 exhibited potent in vitro antibacteric activity, especially against Staphylococcus aureus with MIC 31.3 and 15.6 mg/mL, respectively. The compounds exhibited moderate antifungal activity. These results compared with known antibiotics as standards are shown in Table 2. In addition, 5acetoxymethylfuran-3-carboxylic acid (2) showed moderate activity for the scavenging 2,2diphenyl-1-picrylhydrazyl (DPPH) free radicals with an IC 50 value of 237 mg/mL in Table 2. The antioxidant activity of the compound 1 displayed with IC 50 value of 435 mg/ml. Melting points were determined on an X-6 micro-melting point apparatus and were uncorrected. HRESI-MS was obtained in the negative ion mode with Bruker maXis 4G UHR-TOF. UV spectra were obtained on a DR 5000. IR spectra were recorded with a Bruker VECTOR-22 FT-IR Spectrometer. 1D and 2D NMR spectra were recorded on Bruker AVANCE III-400 MHz spectrometer with tetramethylsilane as internal standard. Deuterated dimethylsulphoxide was purchased from Beijing Boya Dabei Technological Development (Beijing, China). All the solvents were purchased from Tianjin Hongyan Chemical Reagents Factory (Tianjin, China). The silica gel for column chromatography (200 -300 mesh) was purchased from Tsingtao Marine Chemical Factory (Tsingtao, Shandong Province, China). The bacteria (S. aureus, Streptococcus lactis, Escherichia coli and Pseudomonas aeruginosa) and the fungi (Botrytis cinerea, Valsa mali, Alternaria alternata and Alternaria brassicae) were provided from the College of Life Science & Engineering, Shaanxi University of Science & Technology (Xi'an, China).

Material
C. fortunei was collected from Taibai Mountains, Shaanxi Province, China, on July 2011. The endopytic fungus (No. S19) isolated from the stem of C. fortunei was deposited with the culture collection of the Laboratory of Natural Product Research, College of Chemistry & Chemical Engineering, Shaanxi University of Science & Technology. It was identified according to a molecular biological protocol by DNA amplification and sequencing of the ITS region.

Solid-state fermentation
S19 was grown on potato dextrose agar at 288C for 4 days. Five or six pieces (diameter 0.6 cm) of mycelial agar plugs were inoculated into Erlenmeyer flasks (10 £ 1000 mL) containing 600 mL Czapek's medium (sucrose 30 g/L, KH 2 PO 4 1.0 g/L, MgSO 4 z7H 2 O 0.5 g/L, NaNO 3 3.0 g/L, KCl 0.5 g/L and FeSO 4 0.01 g/L). The flasks were incubated for 10 days at 288C on a rotary shaker  (120 r/min) to obtain the fungus seed. Then, the seed liquid was added to 350 flasks (500 mL) each containing sterile culture medium consisted of 50 g rice and 60 mL of Czapek's medium without sugar. Then, the flasks were carried out statically at room temperature for 30 d (Lagemaat & Pyle 2011).

Extraction and isolation
The air-dried solid culture (3.7 kg) was extracted repeatedly with ethyl acetate (25 L £ 3) at room temperature for 24 h. The extract afford a dark brown residue (230 g), which was subjected to silica gel column chromatography eluting gradually with petroleum ether/ethyl acetate gradients (1:0, 1:1, 0:1, V:V) to give three fractions (A-C). The fraction C (125 g) was separated by silica gel column chromatography to yield pure compound 1 (120 g). Fraction B (80 g) was separated by silica gel column chromatography with a gradient eluent of ethyl acetate in petroleum ether to give twelve subfractions (B1 -12), B3 (5 g) was separated by repeated silica gel column chromatography and Sephadex LH-20 (methanol) to yield compound 2 (50 mg).

Antimicrobial assay
The antimicrobial activities were carried out by the continuous dilution method (Bharate et al. 2007;Zhang et al. 2008). Each compound was set at 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.98 mg/mL in DMSO, while the tested strains were incubated in the liquid mediums. For bacteria, the medium was made up of beef extract 3.0 g/L, peptone 10.0 g/L, NaCl 5.0 g/L, pH 7.0 -8.0, the culture temperature was 378C. For fungi, the potato -glucose medium was made up of percolate of 200 g potato under boiling for 30 min, glucose 20 g, and constant volume to be 1 L by water, the culture temperature was 288C. The in vitro antimicrobial activities were tested against two Gram-positive (G þ ) bacteria (S. aureus and S. lactis), two Gram-negative (G 2) bacteria (E. coli and P. aeruginosa) and four plant pathogenic fungi (B. cinerea, V. mali, A. alternata and A. brassicae). The activities were compared with the same concentrations of known antibiotics including penicillin sodium against G þ bacteria, streptomycin sulphate against G-bacteria and ketoconazole against fungi.

Antioxidation assay
Free radical scavenging activities of compounds were measured by DPPH (Predes et al. 2011) in 96 wells microtitre plates. The activities of the compounds were compared with that of Vc. In brief, 100 mL different concentration solutions (a two-fold dilution from 1.95 mg/mL to 1000 mg/ mL in methanol) were mixed with 100 mL of DPPH solution (0.2 mg/mL) and the miscible liquid incubated for 30 min at room temperature. The control was maintained by adding 100 mL of DPPH to 100 mL methanol. The absorbance (A) was measured at 492 nm in triplicate. The antioxidation activities were expressed as the half maximal inhibitory concentration (IC 50 ).   Note: -, the test has not been set.

Conclusion
In conclusion, in this study, a new furan derivative together with a known furan compound were isolated and elucidated from A. flavus, endophytic fungi in C. fortunei. Compounds 1 and 2 exhibited potent antibacterial activity against S. aureus. Compound 2 showed moderate antioxidant. Thus, the compound 2 is potential to be a antibacterial drug.

Supplementary material
Supplementary material relating to this article is available online.