N. N. Kirichuk

Biologically active metabolites of the facultative marine fungus Penicillium citrinum

Four sesquiterpenoid metabolites, JBIR-27, petasol, sporogen AO-1, and dihydrosporogen AO-1, that were previously unknown for Penicillium citrinum were isolated from the marine isolate of this fungus. The structures of the compounds were established using NMR spectral data and high-resolution mass spectrometry. It was shown that sporogen AO-1 exhibited weak antimicrobial and cytotoxic properties. Cytotoxic action was observed for dihydrosporogen AO-1 against Ehrlich carcinoma cells (ED50 0.4 mM).

NEW 3-(2(R)-HYDROXYBUTYL)-7-HYDROXYPHTHALIDE FROM MARINE ISOLATE OF THE FUNGUS Penicillium claviforme

The new phthalide derivative 3-[2′(R)-hydroxybutyl]-7-hydroxyphthalide (2) and the five known compounds (−)-3-butyl-7-hydroxyphthalide (1), isopatulin (3), m-hydroxybenzyl alcohol (4), cyclopenin (5), and cyclopeptine (6) were isolated from the marine isolate of the fungus Penicillium claviforme associated with the seagrass Zostera marina. The structures of the compounds were established using NMR spectroscopy and mass spectrometry. The absolute configuration of the C-2′ asymmetric center was determined using a modified Mosher method.

New Diorcinol J Produced by Co-Cultivation of Marine Fungi Aspergillus sulphureus and Isaria felina

New diorcinol J (1) and four known diorcinols B-E (2-5) were isolated from the EtOAc extract of a co-culture of marine isolates of the fungi Aspergillus sulphureus KMM 4640 and Isaria felina KMM 4639. The structure of diorcinol J was established using NMR spectroscopy and high-resolution mass spectrometry. The cytotoxicity of the isolated compounds and their ability to enhance expression of heat shock protein Hsp70 in Ehrlich ascites carcinoma cells were investigated.

Metabolites of the Marine Fungus Penicillium citrinum Associated with a Brown Alga Padina sp.

Fungi associated with marine algae are some of the most promising marine fungal producers. Greater than 10% of all new marine fungal metabolites were recently isolated from this ecological fungal class [1, 2]. We isolated the fungus Penicillium citrinum, which was identified using morphological signatures, from the surface of a brown alga Padina sp. (South China Sea, Vietnam coast) during a search for producers of new biologically active compounds among marine fungi associated with algae. The fungus was cultivated for 21 d at 22°C in 20 1-L Ehrlenmeyer flasks, each of which contained medium consisting of rice (10 g), sodium tartrate (0.005 g), yeast extract (0.01 g), KH2PO4 (0.005 g), and natural seawater (20 mL). Mycelium together with medium was extracted (2 ) with EtOAc. The extract was evaporated. The residue was dissolved in EtOH–H2O (1:4). The resulting solution was extracted sequentially with hexane, EtOAc, and BuOH. The EtOAc fraction developed a crystalline precipitate of 5 (500 mg). The mother liquor was evaporated at a reduced pressure. The obtained residue (2 g) was chromatographed over a column (25 2 cm) with silica gel using hexane–EtOAc with a stepwise gradient to 5%. The resulting fractions were rechromatographed over Sephadex LH-20 using EtOH–CHCl3 (1:1) to afford 1 (7.7 mg), 2 (2.2 mg), 3 (5.0 mg), and 4 (450.0 mg).

Spiroketals from Marine Isolates of the Fungi Penicillium thomii KMM 4645 and P. lividum KMM 4663

Recent research showed that micromycete fungi isolated from marine sources are interesting as producers of biologically active compounds [1–3]. In continuation of work on the discovery of biologically active compounds in extracts of marine isolates of micromycete fungi, we isolated the two strains Penicillium lividum and P. thomii from the surface of Sargassum miyabei (collected in Lazurnaya Bay, Peter the Great Gulf, Sea of Japan). Work with the fungi was carried out in parallel. Each fungus was cultivated on standard solid medium. The biomass was worked up with EtOAc followed by separation of the dry residue over silica gel using eluent of increasing polarity. The resulting fractions were separated by reversed-phase HPLC to afford from P. lividum compounds 1 (5 mg) and 2 (38 mg); from P. thomii, 1 (2 mg) and 2 (30 mg).

Asperpentyn from the Facultative Marine Fungus Curvularia inaequalis

Marine micromycete fungi are rich sources of new compounds that often exhibit strong biological activity. Fungi of the genus Curvularia were also described several times as producers of such compounds [1, 2]. The isolate of C. inaequalis, which was reported earlier by us as a producer of several known polyketides, was isolated during research on fungal metabolites from strains in the oceans of the Russian Far East [3]. Fungi were cultivated for 21 d in five 1-L Pyrex flasks, each of which contained medium consisting of malt extract (50 mL), agar (5 g), and seawater (200 mL). Mycelium together with medium was extracted twice with EtOAc. The extract was evaporated. The residue (0.4 g) was chromatographed over a column of silica gel (2 10 cm) with elution successively by hexane and hexane–EtOAc (stepwise gradient, 25:1 10:1). The fraction eluted by the 10:1 system (24 mg) was separated using HPLC over a ChiraDex chiral column and MeOH–H2O (40:60) to afford pure (–)-asperpentyn (1.5 mg).

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%):

Non-polar compounds and free fatty acids from several marine isolates of fungi of the genus Aspergillus

Marine mycelial fungi of the genus Aspergillus are known producers of biologically active compounds with broad spectra of action and unusual chemical structures [1, 2]. The biosynthesis of such compounds may be due to the need to adapt to specific conditions of the marine habitat. One of the consequences of such adaptation can be an unusual composition of non-polar compounds in addition to extracellular fatty acids (FAs) produced by fungal marine isolates. It was shown that they are producers of such important acids as 16:0, 18:0, 18:1n9, 18:2n6, 18:3n3, and 20:4n6 [3]. An unusually high percent content of branched and unsaturated FAs was noted in them [4]. Extracts of fungal marine isolates contained also phthalates, which inhibited the protein catabolism enzyme catepsin B [5]. According to our data, several types of marine isolates of A. ustus produced significant quantities of various hydrocarbons [6]. In continuation of research on metabolites of marine fungi, we investigated fractions of marine isolates of the fungi A. sulfureus KMM 4640, A. versicolor KMM 4644, A. carneus KMM 4646, and A. versicolor KMM 4647 that were isolated from sediment on the Sea of Okhotsk shelf, Sakhalin Island, at a depth of 26-28 m in addition to the fungus A. carneus KMM4638 that was isolated from the marine alga Laminaria sachalinensis (Miyabe). Fungal strains were cultivated in modified rice medium at a constant 25°C for 21 d [7]. Cultures were extracted with EtOH. The extracts were concentrated at reduced pressure. The resulting residuals 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. Compounds were identified by comparing their mass spectra with those of standards using the NIST98 database. The hexane extract of A. versicolor KMM 4644 contained hydrocarbons with linear carbon chains from C20 to C32 at concentrations from 2 to 18%. Non-polar compounds were not observed in hexane extracts of the other cultures. The EtOAc fractions from each culture were chromatographed over a column of silica gel with gradient elution by hexane:EtOAc (100:0 90:10) to afford fractions of free FAs. The resulting total acids were analyzed as methyl esters (methylated by Et2O solution of diazomethane) and pyrrolidides [8] using GC-MS. Derivatives were identified by comparing their mass spectra with those of standards using the NIST98 database. All cultures produced significant amounts of heptadecanoic, octadecadienoic, octadecaenoic, and octadecanoic acids. The strain A. carneus that was isolated from laminaria produced insignificant amounts of linear acids with C22–C24 C atoms whereas that isolated from sediment had a broader set of FAs. All fractions contained insignificant amounts of phthalates. The isolate of A. carneus KMM4646 produced small amounts of squalene and 1,4,6-trimethylnaphthalene. The qualitative compositions of the FA fractions agreed in general with those published earlier [9]. Table 1 presents the results.

Non-polar compounds and free fatty acids from marine isolates of mycelial fungi

Marine mycelia fungi are known to be producers of biologically active compounds with broad spectra of activity and unusual chemical structures [1, 2]. Such compounds are biosynthesized by marine organisms in order to adapt to specific marine habitats. One of the consequences of such adaptation may be the qualitative and quantitative composition of non-polar compounds and extracellular fatty acids produced by isolates of marine fungi. Previous studies showed that several isolates of marine fungi produced important acids such as 16:0, 18:0, 18:1n9, 18:2n6, 18:3n3, and 20:4n6 and those with normal and branched chains [3]. Extracts of several isolates of marine fungi contained phthalates, which inhibit the protein-catabolism enzyme catepsin B [4]. In continuation of research on metabolites of marine fungi, we studied extracts of marine isolates of 10 strains of the mycelial fungi Penicillium implicatum KMM 4648 (1), P. citrinum KMM 4649 (2) and KMM 4650 (3), P. glabrum KMM 4651 (4), P. restrictum KMM 4652 (5), P. expansum KMM 4653 (6), Isaria felina KMM 4659 (7), Curvularia inaequalis KMM 4660 (8), Scopulariopsis brumptii KMM 4661 (9), and Acremonium roseum KMM 4662 (10). Cultures of facultative marine fungi were isolated from various sources, i.e., 1, 4, 9, from seaweed Zostera sp.; 3, from soft coral Zoantharia sp.; 2, 5, 6, 7, 8, 10, from marine soil. Strains of fungi were cultivated on malt-extract agar [5] (strains 3, 4, 7, 8, 10) and rice [6] (strains 1, 2, 4, 5, 6, 7, 8, 9) using stationary seawater at 25°C for 14 and 21 d, respectively. Fungal cultures were extracted with EtOAc. The extracts were concentrated at reduced pressure. 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. Compounds of the hexane fraction and derivatives of the EtOAc eluate were identified by comparison of their mass spectra with those of standard compounds using the NIST98 database. Strain P. implicatum (1) produced an insignificant amount of linear hydrocarbon C16 (0.2%); strain P. expansum (6), hydrocarbons with a terminal double bond C18 (3%), C20 (4%), C22 (2.4%), and C24 (0.8%) in addition to saturated C20 (43%) and fatty acids proportional to the content in the fatty-acid fraction (Table 1). The hexane fraction of I. felina (7), which was cultivated on marine malt agar, contained linear hydrocarbons C16 (5.3%), C18 (12.2%), C20 (9.4%), and C22 (5.0%); linear hydrocarbons with a terminal double bond C16 (9.2%), C18 (19.7%), C20 (17.7%), C22 (8.6%), and C24 (1.4%); and hydrocarbons with a terminal cyclohexane group C16 (3.4%), C18 (4.4%), and C20 (3.6%). Non-polar compounds were not detected in extracts of the other cultures. The EtOAc fractions of each culture were chromatographed over a column of silica gel using a gradient of hexane:EtOAc (100:0 90:10) to afford fractions of fatty acids. The resulting total acids were analyzed as methyl esters (methylation by diazomethane in Et2O) and pyrrolidides using GC-MS [7]. All cultures produced 16:0, 18:2, 18:1, and 18:0 fatty acids. Strain P. expansum (6) and I. felina (7) produced trace amounts of 14:0 acid; strain P. citrinum (3), which was isolated from soft coral, 17:0 acid. Table 1 presents the results. All culture samples contained phthalates in small (1–3%) quantities. However, their content in extracts of P. glabrum (4) and P. restrictum (5) exceeded 50% of the fraction contents. The qualitative composition of fatty acid fractions of the studied strains agreed in general with previously published data [8]. The results supplemented our previous investigations of non-polar compounds and fatty acids from marine isolates of fungi [9, 10].

New Kipukasin from Marine Isolate of the Fungus Aspergillus flavus

A new aromatic nucleoside kipukasin J (1) and two known compounds decumbenone B (2) and cyclopenol (3) were separated from marine isolate of the fungus Aspergillus flavus. The structure of kipukasin J was elucidated using NMR spectroscopy and high-resolution mass spectrometry. The antimicrobial activity of the separated compounds was investigated.