Chemical Constituents of the Entomopathogenic Fungus of Ophiocordyceps sobolifera

Abstract

The entomogenous fungi are intimately or even obligately associated with insects, other arthropods, and even nonarthropod microinvertebrates, mainly as pathogens or parasites [1]. Due to their capability of producing various secondary metabolites with diverse structural features and interesting biological activities, entomogenous fungi have been increasingly studied in recent years [2–6]. In our ongoing search for novel bioactive compounds from special ecological environmental fungi of Taiwan, an entomogenous fungus Ophiocordyceps sobolifera (Ophiocordycipitaceae) BCRC38180 was isolated from the cadaver of an unidentified insect collected from Ruisui Township, Hualien County, Taiwan, in May 2013. Bioassay involved fractionation of the EtOAc extract prepared from the liquid-substrate fermentation products, from which five metabolites, including two known isocoumarins, one benzenoid and two known steroids, were finally isolated. The structures of the isolated metabolites were determined by extensive analysis of their spectroscopic data as well as by comparison with literature data. The chemistry and bioactivity of O. sobolifera 38180 have never been studied previously. Among them, all compounds 1–5 were isolated for the first time from the genus Ophiocordyceps. The structures of the known compounds 1–5 were established by comparison of their 1H NMR and ESI-MS or EI-MS data with literature data: (R)-(–)-5-carbomethoxymellein (1) [7], (R)-(–)-7-hydroxymellein (2) [8], 4-(2′-hydroxyethyl)-phenol (3) [9], ergosterol (4) [10], and β-sitostenone (5) [11]. General. TLC: silica gel 60 F254 precoated plates (Merck). Column chromatography (CC): silica gel 60 (70–230 or 230–400 mesh, Merck) and spherical C18 100A reversed phase silica gel (RP-18) (particle size: 20–40 μm) (Silicycle). UV spectra: Jasco UV-240 spectrophotometer; λmax (log ε) in nm. Optical rotation: Jasco DIP-370 polarimeter, in CHCl3. IR spectra: PerkinElmer-2000 FT-IR spectrophotometer, νmax in cm –1. 1H, 13C, and 2D NMR spectra: Varian-VNMRS-600 and Varian-Mercury-400 spectrometers, δ in ppm rel. to Me4Si, J in Hz. EI-MS: VG-Biotech Quatro-5022 mass spectrometer, m/z (rel. %). HR-EI-MS spectra were recorded on a Finnigan/Thermo Quest NAT mass spectrometer. ESIand HR-ESI-MS: Bruker APEX-II mass spectrometer, in m/z. Microorganism, Cultivation, and Preparation of the Fungal Strain. The fungus used in this study was collected in May 2013 in Ruisui Township, Hualien County, Taiwan, and identified by one of the authors, Dr. Sung-Yuan Hsieh. The strain is preserved with the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) under ID No. 38180. Five-day-old colonies of the O. sobolifera strain on malt extract agar (MEA) medium in 9-cm Petri dish were cut into the bottle and blended for 30 s with 100 mL of distilled H2O to prepare the fungal inoculum for solid fermentation. To each 500-mL flask containing 150 mL of liquid cultural media (ingredients: 1.5% soluble starch, 0.06%