Yu. V. Khudyakova

Metabolites from the marine fungus Eurotium repens

Abstract1,8-Dihydroxy-6-methoxy-3-methyl-9,10-anthracenedione (physcion, 1), 3,4-dihydro-3,6,9-trihydroxy-8-methoxy-3-methyl-1(2H)-anthraceneone (asperflavin, 2), and 2,5-dihydroxy-3-(3-methyl-2-butenyl)-6-[(1E)-1-heptenyl]-benzaldehyde (tetrahydroauroglaucin, 3) were shown to be the main pigments of the marine isolate of the fungus Eurotium repens. In addition to the pigments, the fungal metabolites included the diketopiperazine alkaloid echinulin (4). The structures of the compounds were identified using NMR spectroscopy and mass spectrometry. The cytotoxic activity of 1–3 toward sex cells of the sea urchin Strongylocentrotus intermedius was determined.

Fungi in sediments of the sea of Japan and their biologically active metabolites

The most abundant marine fungi encountered in various regions of the Sea of Japan belong to the generaPenicillium, Aspergillus, Wardomyces, Trichoderma, Chrysosporium, andChaetomium. Facultative marine fungi of the generaScytalidium, Verticillium, andOidiodendron and obligate marine fungi of the genusDendryphiella are much less abundant. The composition of marine sediments and the anthropogenic load on them were found to influence the abundance and species diversity of fungi, as well as the occurrence of fungal strains producing hemolytically active substances. The biodiversity of mycobiota and the abundance of hemotoxin-producing fungi in marine sediments may be used to evaluate the anthropogenic load on marine biocenoses. Hemolytic compounds were produced by 57% of the fungi isolated from marine sediments. The hemolytic activity ofChaetomium spiculipilium was revealed in the fraction of the culture liquid containing extracellular fatty acids and pigments. The fatty acid composition of this marine fungus was determined.

Fungal assemblages associated with sponges of the southern coast of Sakhalin Island

This paper examines the biodiversity of fungi associated with six species of sponges from the southern coast of Sakhalin Island. The species richness of fungal assemblages is represented by 78 species, mainly Anamorphic fungi. The diversity and numbers of fungi depend on the species of sponge, which is due to the rigidity of the skeleton and the morphological characteristics of the sponges.

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.

3β-Methoxyolean-18-ene (miliacin) from the marine fungus Chaetomium olivaceum

A pentacyclic tritepenoid, 3β-methoxyolean-18-ene (miliacin), was isolated for the first time from the marine fungus Chaetomium olivaceum. Its structure was determined on the basis of 2D NMR spectroscopy and X-ray diffraction data.

Marine fungus Stilbella aciculosa as a potential producer of prostaglandins

The amount and composition of fatty acids in the fungus Stilbella aciculosa associated with the marine macroorganism Apostichopus japonica (trepang) were determined by gas-liquid chromatography and gas chromatography-mass spectrometry. In the culture liquid of S. aciculosa, prostaglandins (PG) of groups E and F were revealed by UV spectroscopy. This finding was confirmed by the presence of direct precursors of PG, polyunsaturated eicosapentaenoic and docosahexaenoic acids, in the culture liquid. The biomass of this fungus contained PG of group B.

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).

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].