A Novel Antioxidant Isobenzofuranone Derivative from Fungus Cephalosporium sp.AL031

Bioassay-guided fractionation of metabolites from the fungus Cephalosporium sp.AL031 isolated from Sinarundinaria nitida led to the discovery of a new isobenzofuranone derivative, 4,6-dihydroxy-5-methoxy-7-methylphthalide (1), together with three known compounds: 4,5,6-trihydroxy-7-methyl-1,3-dihydroisobenzofuran (2), 4,6-dihydroxy-5-methoxy-7-methyl-1,3-dihydroisobenzofuran (3) and 4,5,6-trihydroxy-7-methylphthalide (4). The structure of the new compound 1 was determined based on MS, 1D and 2D NMR spectral data. Compounds 1–4 showed potent antioxidant activity with EC50 values of 10, 7, 22 and 5 μM by 1,1-diphenyl-2-picryhydrazyl (DPPH) radical-scavenging assay.


Results and Discussion
Compound 1 was obtained as colorless needle-like crystals that gave a positive reaction in the 10% H 2 SO 4 -EtOH test. The molecular formula C 10 H 10 O 5 was deduced from a pseudomolecular ion [M + H] + at m/z 211.0628 in the HR-ESI-MS (calcd. for C 10 H 11 O 5 , 211.0607), which was in agreement with the 1 H-and 13 C-NMR spectra. The IR spectrum showed characteristic absorption bonds for hydroxyl groups (3365 cm −1 ), saturated methylene (2930 cm −1 ) and a carbonyl group (1685 cm −1 ).
The 1 H-NMR spectrum of 1 (Table 1) exhibited signals attributable to a methoxyl group at δ 3.87 (3H, s), an oxymethylene moiety at δ 5.12 (2H, brs) and a methyl group at δ 2.42 (3H, s). The 13 C-NMR and DEPT spectra of 1 (Table 1) also showed the occurrence of three sp 3 -carbon signals related to a methoxyl group (δ 59.6), an oxymethylene moiety (δ 66.8) and a methyl group (δ 8.2). Including one quaternary sp 2 -carbon signal which was attributed to a carbonyl moiety at δ 172.7, compound 1 also indicated six quaternary sp 2 -carbon signals at δ 125.8, 142.1, 140.8, 149.6, 116.5 and 117.7. The 1 H-NMR and 13 C-NMR data was assigned to an isobenzofuranone skeleton.  The 1 H-NMR and 13 C-NMR data of 1 are very similar to those of 4,5,6-trihydroxy-7methylphthalide [1] that has been isolated from the fungus Epicoccum sp. The only difference between these two compounds is the methoxyl group present in compound 1. HSQC and HMBC experiments revealed all significant connectivities. The correlation of the methyl group (δ 2.42) with C-6 (δ 149.6) and C-7a (δ 116.5) in the HMBC spectrum indicated the methyl unit connected to C-7. Similarly, the linkage of the methoxyl moiety at C-5 was deduced from the correlation between the methoxyl group (δ 3.87) and C-5 (δ 140.8). Furthermore, no correlations were observed between the methyl group (δ 2.42) or oxymethylene moiety (δ 5.12) and C-5, which unambiguously established the linkage of methoxyl moiety. All NMR data of compound 1 were assigned on the basis of DEPT, HSQC and HMBC experiments. Taken together, compound 1 was determined to be a new isobenzofuranone and named as 4,6-dihydroxy-5-methoxy-7-methylphthalide with the structure shown in Figure 1.
Together with the new compound 1, three known compounds 2-4 were identified by comparison of their spectroscopic data [1,13,14] with published values. We evaluated the antioxidant activity of isolated compounds 1-4 by DPPH radical-scavenging assay, which showed potent activity with the EC 50 values of 10, 7, 22 and 5 μM, respectively.

General
TLC was preformed with silica gel GF 254 (Marine Chemical Industry Factory, Qingdao, China), and the spots were visualized by spraying with 10% H 2 SO 4 -EtOH reagent, followed by heating. Column chromatography was performed using silica gel (Marine Chemical Industry Factory, Qingdao, China), reverse-phase C 18 silica gel (Merck, Germany) and Sephadex LH-20 (Sigma). All reagents were analytical grade and water was distilled-twice. Melting points were measured with an X-4 melting point apparatus and are uncorrected. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter. IR spectra were obtained on a Perkin-Elmer 577 spectrometer with KBr pellets. UV spectra were obtained on a Perkin-Elmer Lambda 900 UV/VIS/NIR spectrophotometer. NMR Spectra were recorded on a Brüker AV-400 spectrometer at 400 MHz 1 H and 100 MHz for 13 C in MeOD, and chemical shifts are presented as values relative to tetramethylsilane as an internal standard.
Low-resolution electrospray-ionization mass spectrometry (ESI-MS) and HR-ESI-MS were recorded on a Finnigan LCQ-Advantage mass spectrometer and a VG Auto-Spec-3000 mass spectrometer.

Fungal Material
The fungus Cephalosporium sp. AL031 was isolated from Sinarundinaria nitida grown at Ailao Mountain, Yunnan, China. It was identified as Cephalosporium sp. (strain number AL031), a member of Moniliaceae, by Professor Fa-Rong Yang in the Department of Biology, Yunnan University, and deposited in Key Laboratory of Industrial Microbial Fermentation Engineering of Yunnan Province, China. The isolated fungus was cultured on slants of potato dextrose agar (PDA) at 27 °C for 5 days, and then the fungus was grown on a medium consisting of rice, wheat bran, cornmeal, sugar cane bagasse with a ratio of 12:2:3:3, and incubated for 7 days at 28 °C.

Bioassay
The antioxidative properties of compounds 1-4 were assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. DPPH is a stable free radical with a strong UV/VIS absorption band at 517 nm. Addition of radical scavenging compounds to a solution of DPPH results in a decrease of absorption. The inhibitory percentage of DPPH was calculated according to the equation as follows: % scavenging activity = [1 − (As/Ac)] × 100; where As and Ac were the absorbance values at 517 nm of the DPPH solutions with sample and without sample, respectively. According to the previous method [15], we evaluated the antioxidant activity of isolated compounds 1-4 and the positive control gallic acid by DPPH radical-scavenging assay with the EC 50 values of 10, 7, 22, 5 and 2 μM, respectively.

Conclusions
Repeated column chromatography (including normal-phase silica gel and Sephadex LH-20) of the EtOAc extract of the fermentation of the fungus Cephalosporium sp. AL031 has led to the isolation of a novel isobenzofuranone derivative, namely 4,6-dihydroxy-5-methoxy-7-methylphthalide (1), together with three known ones: 4,5,6-trihydroxy-7-methyl-1,3-dihydroisobenzofuran (2), 4,6-dihydroxy-5methoxy-7-methyl-1,3-dihydroisobenzofuran (3), and 4,5,6-trihydroxy-7-methylphthalide (4). The structure of the new compound 1 was determined by spectroscopic methods, including 1D NMR, 2D NMR and MS experiments, and the structures of the known compounds including 2~4 were identified by comparing their NMR data with those of ones in the literature. All four compounds showed potent anti-oxidative activity by DPPH radical-scavenging assay, which are potential anti-oxidative agents. Comparing the antioxidant activity of compounds 1 and 4, 3 and 2, the latter containing three phenolic hydroxyl groups showed potent activity, which indicated the antioxidant activity correlates with the number of hydroxyl groups in the structure.