Nonpolar Compounds and Free Fatty Acids from Several Marine Isolates of Fungus Aspergillus ustus and Actinobacterium Nocardiopsis umidischolae

In continuation of research on secondary metabolites of marine fungi and actinobacteria [1, 2], hexane fractions and fractions of free fatty acids and the sterol fraction of actinobacterium were obtained and analyzed from cultures of marine isolates of the fungus Aspergillus ustus KMM 4642 and KMM 4664 isolated from sediment (Okhotsk Shelf, Sakhalin Island, 26.5 m depth) and the actinobacterium Nocardiopsis umidischolae KMM 7036 isolated from Mycale sp. of sponge (Deryugin Basin, Okhotsk Sea). Strain KMM 4642 was cultivated in malt-agar medium prepared with sea water for 14 d [3] and on rice medium prepared with seawater for 21 d [4]. Strain KMM 4664 was cultivated only on rice medium prepared with seawater. Actinobacterium was cultivated in a special medium containing peptone (5.0 g/L), meat extract (3.0), starch (20.0), agar (16.0) and seawater at pH 7.5 and 23°C for 23 d. Cultures were extracted with EtOAc. The extracts were evaporated to dryness. The resulting residues were dissolved in EtOH (10%) and extracted successively with hexane, EtOAc, and BuOH. The hexane fractions were evaporated at reduced pressure and analyzed by GC-MS. The data were compared with the mass spectrometric fragmentation of standards using the NIST98 database. The hexane fraction of A. ustus KMM 4642 consisted of 96.88% diethylhexylphthalate. The remainder consisted of fatty acid ethyl esters and pentadecane. The hexane fraction of A. ustus KMM 4664 contained fatty acid ethyl esters, squalene, octadecane, and sterols such as 24-cholest-4-en-3-one, 24-ethylcholest-7-en-3 -ol, 24-ethylcholesta-3,5-diene, and 24-ethylcholest-5-en-3 -ol at concentrations of 1–2% of the studied mixture. These results differed from those obtained earlier for marine isolates of A. ustus [1]. The hexane fraction of actinobacterium N. umidischolae contained hydrocarbons at concentrations of 1.5–3% that included linear C15, C16, C17, C18, C20, and C22; C18, C19, C20, C22, and C24 with an iso-carbon chain; C18, C19, and C22 with a terminal double bond; and dibutylphthalate (5%) and diethylhexylphthalate (45%). The EtOAc fraction of each culture was chromatographed over a column of silica gel using a hexane–EtOAc gradient (100:0 90:10) to afford fractions of free fatty acids. The obtained total acids were analyzed as the methyl esters (methylated by diazomethane in Et2O) and pyrrolides [5] using GC-MS. Derivatives were identified by comparing their mass spectra with those of standards using the NIST98 database. The composition and content of A. ustus acid esters are given below (mass%):