Sh. Sh. Afiyatullov

A comparative study in vitro of physiological activity of triterpene glycosides of marine invertebrates of echinoderm type

1. The cytostatic and antimicrobial activity of triterpene glycosides of 19 holothurian species of the Pacific tropical zone has been studied. 2. It has been demonstrated that yeast and tumor cells display a comparable sensibility to the action of triterpene oligosides of sea cucumbers. 3. Gram-positive and Gram-negative bacteria are not sensible to the action of glycosides in doses to 500 mkg/ml. 4. Triterpene glycosides-stichoposides, thelothurins and oligosides of Holothuria of genus Bohadschia are the most active in relation to fungal, yeast microflora and tumor cells.

Structures of cucumariosides C1 and C2 — Two new triterpene glycosides from the holothurian Eupentacta fraudatrix

Two new triterpene glycosides — cucmariosides C1 and C2 — have been isolated from the Far Eastern holothurianEupentacta (=Cucumaria)fraudatrix Djakonov et Baranova. Their structures have been established with the aid of13C NMR and PMR spectroscopy, partial acid hydrolysis, periodate oxidation, and methylation as 16β-acetoxy-3-{[3-O-methyl-β-D-xylopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)] [β-D-xylopyranosyl-(1→2)]-β-D-quinovopyranosyl-(1→2)-β-D-xylopyranosyloxy}holosta-7,23,24(cis)-triene and 16β-acetoxy-3-{[3-O-methyl-β-D-xylopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)] [β-D-xylopyranosyl-(1→2)]-β-D-quinovopyranosyl-(1→2)-β-D-xylopyranosyloxy}holosta-7,22,24(trans)-triene, respectively.

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).

Diketopiperazines from Marine Isolate of Actinobacterium Nocardiopsis umidischolae KMM 7036

In continuation of research on secondary metabolites of marine isolates of microorganisms [1, 2], we investigated actinobacterium Nocardiopsis umidischolae KMM 7036, which was isolated from Mycale sp. fungi, collected in Deryugin Basin, Sea of Okhotsk. The actinobacterium was cultivated and the culture was processed as described earlier [3]. Multiple column chromatography over silica gel using a gradient of hexane EtOAc (100% hexane 100% EtOAc) of the culture extract isolated 1 (11.4 mg), 2 (0.9 mg), and 3 (2.2 mg).

Metabolites from the Marine Isolate of the Fungus Aspergillus versicolor KMM 4644

In continuation of the search for biologically active compounds in extracts of marine isolates of mycelial fungi, we isolated the strain Aspergillus versicolor KMM 4644 from sediment of Sakhalin Bay, Sea of Okhotsk. The fungus was cultivated on solid medium for 21 d at room temperature in 20 Ehrlenmeyer flasks (500 cm3), each of which contained medium of the following composition: rice, 15.0 g; yeast extract, 0.02 g; KH2PO4, 0.01 g; sodium tartrate, 0.01 g; seawater, 20 mL. Mycelium with medium was extracted with EtOAc. The extract was evaporated in vacuo. The dry residue (3.15 g) was chromatographed over a column of silica gel using hexane:EtOAc mixtures of increasing polarity. A fraction (hexane:EtOAc, 70:30) was separated by HPLC over a Diaspher-110-C18 column using H2O:MeOH (50:50) to isolate 1 (12 mg), 2 (2.5 mg), and 3 (7 mg). Separation of another fraction (hexane:EtOAc, 95:5) using the same column and H2O:MeOH (85:15) afforded 4 (2 mg) and 5 (6.5 mg).

Triterpene glycosides of the holothurian Eupentacta pseudoquinquisemita

The holothurianEupentacta pseudoquinquisemita Deichmann collected in Kraternaya Bay, Ushishir Islands has yielded two triterpene pentaosides — the previously known cucumarioside C2, and cucumarioside H, which is a new glycoside. With the aid of13C NMR spectroscopy and solvolytic desulfation its structure has been determined as 6β-acetoxy-3β-([3-O-methyl-β-D-xylopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 4)] [β-D-xylopyranosyl-(1 → 4)] [β-D-xylopyranosyl-(1 → 2)]-β-D-quinovopy-ranosyl-(1 → 2)-(4-O-sulfato-β-D-xylopyranosyloxy)holosta-7,22,24(trans)-triene. Cucumarioside H was also identified inEupentacta (=Cucumaria)fraudatrix from Posyet Bay, Sea of Japan.

Nonpolar Compounds and Free Fatty Acids from Several Isolates of Marine Fungi of the Genus Penicillium

In continuation of research on biologically active compounds in extracts of marine isolates of micromycete fungi [1, 2], nonpolar compounds and fatty acids in hexane and EtOAc fractions of marine isolates of Penicillium fungi isolated from the surface of brown algae and sediment collected in the Sea of Okhotsk, South China Sea, and the Sea of Japan were analyzed (Table 1). The fungi strains were cultivated for 14 d in rice medium prepared from seawater [3]. Mycelium together with medium was extracted with EtOAc. The extracts were concentrated at reduced pressure. The obtained dry residues were dissolved in EtOH (10%) and extracted successively with hexane, EtOAc, and BuOH. The hexane fractions of the EtOAc extract 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. Strain P. citrinum (5) produced a linear olefinic hydrocarbon with a terminal docosene double bond. The hexane fraction of Penicillium sp. (6) that was isolated from the surface of Sargassum pallidum contained linear paraffinic hydrocarbons C21 (4.45%), C22 (6.64), C23 (8.34), C24 (10.85), C25 (11.94), C26 (10.94), C27 (11.74), C28 (10.34), and C29 (7.34) in addition to dibutylphthalate (10.44%). Cultures of Penicillium spp. (2 and 4) that were isolated from sediment produced linear olefinic hydrocarbons with a terminal double bond and their isomers: 2, C16 (4.54%), C18 (13.65), iso-C20 (10.45), iso-C22 (9.94), C22 (11.54), C24 (7.74), C26 (6.94); 4, C18 (11.25%), C20 (19.34), C22 (16.34), C24 (13.54), C26 (9.34), and C28 (5.25).

Nonpolar Compounds and Free Fatty Acids from Marine Isolates of Several Fungi and Bacteria

In continuation of research on biologically active compounds in extracts of microorganism marine isolates [1, 2], nonpolar compounds and fatty acids in hexane and EtOAc fractions of isolates from marine fungi and bacteria that were obtained from various marine species and sediments and collected in various regions were analyzed (Table 1). Fungal strains 1–5, 6, and 6a were cultivated on modified rice medium for 14 d [3]. Culture 6 was treated with KBr (3% of medium composition); 6a, with Aspergillus sulfureus KMM 4640 [4] culture (data for nonpolar compounds and fatty acids from KMM 4640 were published earlier [5]) after cultivation for 7 d. Bacteria were cultivated in liquid medium as described previously [6]. The obtained cultures were extracted (3 ) with EtOAc (1:1). The extracts were concentrated at reduced pressure. The obtained dry residues were dissolved in EtOH (10%) and extracted sequentially 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 and by using the NIST98 database. Strain A. candidus (5) produced a linear C22H44 hydrocarbon with a terminal double bond, i.e., docosene. Hydrocarbons were not detected in extracts of other fungal cultures. The hexane fraction of bacterial culture KMM 457 contained linear C21 (4.04%), C22 (5.35), C23 (9.27), C24 (11.29), C25 (19.11), C26 (14.05), C27 (11.81), C28 (8.93), and C29 (9.22). Bacterial strain KMM 1922 produced a broader spectrum of linear hydrocarbons, i.e., C18 (2.07%), C19 (4.84), C20 (9.96), C21 (12.72), C22 (11.34), C23 (9.27), C24 (9.82), C25 (9.41), C26 (9.68), C27 (8.02), C28 (5.26), C29 (4.15), C30 (2.21), and C31 (1.24). EtOAc fractions of each culture were chromatographed over a column of silica gel using a hexane–EtOAc gradient (100:0 90:10) in order to isolate free fatty-acid fractions. The obtained total acids were analyzed by GC-MS as methyl esters (methylation by diazomethane in Et2O) and pyrrolidides [7]. Derivatives were identified by comparing their mass spectra with those of standards and by using the NIST98 database. Table 2 presents the results. It is noteworthy that A. carneus (4) produced dodecanoic acid in significant quantities (24.42%). Extracts of Aspergillus fungi could contain 3% C12 acid [5]; Chaetomium spiculipilium, 1–2% [8]. Culture extracts of E. nigrum (2) isolated from Ulva linza and of B. subtilis (7) isolated from soft coral contained diethylhexylphthalate (11.81% calculated for fatty-acid fraction and 6.92%, for hexane fraction). Culture extract of B. subtilis (8) isolated from sponge did not contain fatty acids but did contain dibutylphthalate (8.22%) and diethylhexylphthalate (91.77%). The results agreed in general with those published previously [1, 9] and supplemented our data for the contents of hydrocarbons and free fatty acids in marine isolates of microorganisms. The changes of medium composition that were made for culture 6 did not affect the fatty-acid composition.

Structure of cucumarioside G1 — A new triterpene glycoside from the holothurianCucumaria fraudatrix

A new triterpene glycoside — cucamarioside G1 — has been isolated from the Pacific Ocean holothurianCucumaria fraudatrix. On the basis of physicochemical characteristics and the results of chemical transformations, its structure has been established as 16β-acetoxy-3β-[O-(3-O-methyl-β-D-xylopyranosyl)-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-D-quinovopyranosyl-(1 → 2)-(4-O-sulfato-β-D-xylopyranosyloxy)]-holosta-7,24-diene.