Stereochemical determination of a new and cytotoxic euphane triterpenoid from the plant endophytic fungus Phomopsis chimonanthi

The culture of P. chimonanthi was isolated from branches of the salt-tolerant wild medicinal plant Tamarix chinensis in the yellow river delta, Dongying, in May 2011. The isolate was identified as P. chimonanthi by one of the authors (AJ) based on the sequence (GenBank Accession number JQ234963) analysis of the internal transcribed spacer (ITS) region of the recombinant DNA, and deposited in the Biology Institute, Shandong Academy of Sciences (Registration No. YG-1), Jinan. The fungal strain was cultured on slants of potato dextrose agar at 28 °C for 10 days. The agar plugs were inoculated in 250 ml Erlenmeyer flasks, each containing 50 ml of media (0.4% glucose, 1% malt extract and 0.4% yeast extract), and the final pH of the media was adjusted to 6.5 before sterilization. Flask cultures were incubated at 28 °C on a rotary shaker at 170 r.p.m. for 5 days. Fermentation was carried out in four 500 ml Fernbach flasks each containing 80 g of rice. Spore inoculum was prepared by suspension in sterile, distilled H₂O to give a final spore/cell suspension of 1 × 10⁶ ml⁻¹. Distilled H₂O (100 ml) was added to each flask and the contents were soaked overnight before autoclaving at 15 lb in⁻¹ for 30 min.² After cooling to room temperature, each flask was inoculated with 5.0 ml of the spore inoculum and incubated at 28 °C for 40 days.

The fermented rice substrate was extracted repeatedly with ethyl acetate (EtOAc) (4 × 500 ml), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (5.2 g). The extract was fractionated by Silica gel vacuum liquid chromatography using petroleum ether–EtOAc gradient elution. The fraction (125 mg) eluted with 80% EtOAc was separated by Sephadex LH-20 column chromatography and repeatedly eluted with 1 : 1 CHCl₃–CH₃OH to give compound 1 (5 mg). The fraction (236 mg) eluted with 8% EtOAc was separated by Sephadex LH-20 column chromatography eluted with 1 : 1 CHCl₃–CH₃OH to obtain compound 2 (10 mg). The fraction (180 mg) eluted with 20% EtOAc was separated by Sephadex LH-20 column chromatography eluted with 1 : 1 CHCl₃–CH₃OH to produce compound 3 (7 mg).

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