Cytochalasans with different amino-acid origin from the plant endophytic fungus Trichoderma gamsii

The culture of T. gamsii was isolated from traditional Chinese medicinal plant P. notoginseng (BurK.) F.H.Chen. The isolate was identified based on the sequence (Genbank Accession No. JF964996) obtained the by analysis of ITS region of the rDNA. The fungal strain was cultured on the slants of potato dextrose agar at 25 °C for 10 days. The agar plugs were used to inoculate 250-ml Erlenmeyer flasks, each containing 40 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 25 °C on a rotary shaker at 170 r.p.m. for 5 days. Fermentation was carried out in Fernbach flasks (500 ml) 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 per ml. Distilled H₂O (100 ml) was added to each flask, and the contents were soaked overnight before autoclaving at 15 lb in⁻² for 30 min. After cooling to room temperature, each flask was inoculated with 5 ml of the spore inoculum and incubated at 25 °C for 40 days.

The fermented material was extracted with ethyl acetate (20 l), and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (100 g), which was fractionated by silica gel column chromatography (10 × 100 cm) using CH₂Cl₂–CH₃OH gradient elution. The fraction (0.8 g) which eluted with 100:2 CH₂Cl₂–CH₃OH was separated by column chromatography, and further purification by reversed-phase HPLC (Lumtech, Berlin, Germany; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 ml min⁻¹, 75% MeOH in H₂O for 5 min, and followed by 75–90% for 60 min) afforded trichalasin A (1; 1.5 mg, t_R 21.5 min) and aspochalasin J (4; 5 mg, t_R 29.3 min). Other fraction (0.3 g) eluted with 100:2 CH₂Cl₂–CH₃OH was separated by Sephadex LH-20 CC repeatedly to obtain trichalasin B (2; 2 mg). One fraction eluted with 100:8 CH₂Cl₂–CH₃OH was separated by Sephadex LH-20 and reversed-phase HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 ml min⁻¹, 70% MeOH in H₂O for 5 min, and followed by 70–85% for 60 min) to give aspochalasin I (3; 5 mg, t_R 35.9 min). UV data were recorded on a Shimadzu UV-2550 UV-vis spectrophotometer (Shimadzu, Kyoto, Japan). IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer. ¹H and ¹³C NMR data were acquired with a Bruker 600 (Bruker, Billerica, MA, USA) and Varian Inova 600 spectrometer (Varian Inova, Salt Lake City, UT, USA) using solvent signals (CDCl₃; δ_H 7.26/δ_C 77.6, DMSO-d₆; δ_H 2.49/δ_C 39.5) as references. The HMQC and HMBC experiments were optimized for 145 and 8 Hz, respectively. HRESIMS data were acquired using a LTQ Orbitrap XL mass spectrometer (Thermo, Waltham, MA, USA).

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