Secondary Metabolites from an Algicolous Aspergillus versicolor Strain

Two new compounds, asperversin A (1) and 9ξ-O-2(2,3-dimethylbut-3-enyl)brevianamide Q (2), and nine known compounds, brevianamide K (3), brevianamide M (4), aversin (5), 6,8-di-O-methylnidurufin (6), 6,8-di-O-methylaverufin (7), 6-O-methylaverufin (8), 5α,8α-epidioxyergosta-6,22-dien-3β-ol (9), ergosta-7,22-diene-3β,5α,6β-triol (10), and 6β-methoxyergosta-7,22-diene-3β,5α-diol (11), were obtained from the culture of Aspergillus versicolor, an endophytic fungus isolated from the marine brown alga Sargassum thunbergii. The structures of these compounds were established by spectroscopic techniques. Compounds 4, 7 and 8 exhibited antibacterial activities against Escherichia coli and Staphyloccocus aureus, and 7 also showed lethality against brine shrimp (Artemia salina) with an LC50 value of 0.5 μg/mL.


General
NMR spectra were recorded in CDCl 3 at 500 and 125 MHz for 1 H and 13 C, respectively, on a Bruker Avance III 500 NMR spectrometer using TMS as internal standard.High resolution mass data were acquired on Autospec Premier P776 mass spectrometer with an EI source.IR spectra were obtained on a JASCO FT/IR-4100 Fourier Transform InfraRed spectrometer.UV spectrum was measured on a TU-1810 Spectrophotometer.HPLC separation was carried out on an Elite HPLC system (P270 pump, UV230+ detector, Dalian Elite Analytical Instruments Co., Ltd., Dalian, China) using an Eclipse XDB-C18 (5 μm, 9.4 × 250 mm) column.Column chromatography was performed with silica gel (100-200 and 200-300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China) and Sephadex LH-20 (Pharmacia).Precoated silica gel plates (GF-254, Qingdao Haiyang Chemical Co., Qingdao, China) were used for preparative TLC purification.All solvents were of analytical grade.

Microorganism and Fermentation
The endophytic fungus A. versicolor pt20 was isolated from a fresh, surface-sterilized tissue sample of the marine brown alga S. thunbergii, which was collected from Pingtan Island, China.The fungus was identified based on morphological and molecular taxonomic methods by one of the authors (F.-P.M.).A voucher sample has been preserved in Bio-Resource Laboratory of Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences.The initial cultures were maintained on the potato dextrose agar plates.Pieces of mycelia were cut into small segments and aseptically inoculated into 1000 mL Erlenmeyer flasks containing 300 mL potato dextrose broth (PDB) culture media.The static fermentation was carried out for 30 days at room temperature (25 °C).

Antimicrobial Assay
Antibacterial and antifungal activities were assayed as described previously [11].

Brine Shrimp Lethality Assay
Brine shrimp (Artemia salina) lethality assay procedure followed the micro-well plate method described by Solis et al with some modifications [12].Briefly, brine shrimp eggs were left to hatch in sea water for 48 hours at 28 °C under natural light.For brine shrimp lethality testing, compounds were dissolved in DMSO prior to preparing serial dilutions in 200 µL volume of sea water prepared in 96 well microplates.A well containing DMSO without compounds added was used as a positive control.Approximately, 10 brine shrimp were placed in a well with a volume of 200 µL sea water for lethality testing.Brine shrimp lethality was observed after 24 hours of cultivation under continuous light.Dead shrimp were identified with the aid of a handheld magnifying lens.

Table 1 .
Cont.Compound 2 was obtained as colorless crystals from CHCl 3 .The molecular formula was established to be C 27 H 33 N 3 O 3 based on HREIMS (m/z 447.2507 [M] + , calcd.for C 27 H 33 N 3 O 3 , 447.2522), requiring thirteen degrees of unsaturation.The 1 H NMR spectrum (Table