I. I. Kapustina

Biosynthetic studies of marine lipids. 42. Biosynthesis of steroid and triterpenoid metabolites in the sea cucumber Eupentacta fraudatrix.

The saponins, conjugated sterols, and free sterols of the sea cucumber Eupentacta fraudatrix were examined. A total of 85 steroids, twelve of them new, were identified in the free sterol, sulfated sterol, and sterol-xyloside fractions. The free sterol fraction contained 4 alpha,14 alpha-dimethylcholest-9(11)-en-3 beta-ol(6) and 14 alpha-methylcholest-9(11)-en-3 beta-ol(7) together with 18 minor sterols. Examination of the aglycone moieties of the sterol-beta-xyloside fraction afforded 31 different sterols. Cholestan-3 beta-ol (15) and 24-methylcholesta-7,22-dien-3 beta-ol (20) were the major sterols in this group. Cholestanol sulfate (74) and cholesterol sulfate (64) were identified as the major components among the 34 different sterol sulfates present. Finally, cucumariosides G1 (1), C1 (2), C2 (3), H (4), and G2 (5) were isolated from the saponin fraction. Radiolabeling experiments indicated that there are two pathways of sterol biosynthesis in E. fraudratix. The first involves transformation of squalene to produce lanosta-9(11),24-dien-3 beta-ol(parkeol) which is subsequently demethylated to form 4 alpha,14 alpha-dimethylcholest-9(11)-en-3 beta-ol (6) and 14 alpha-methylcholest-9(11)-en-3 beta-ol (7). The second proceeds through squalene to lanosterol which is further metabolized to produce the triterpene saponins, 5 alpha-cholest-7-en-3 beta-ol (19) and its xyloside (49).

New diterpenoids from the far-eastern gorgonian coral Plumarella sp.

Abstract Two new cytotoxic diterpenoids, plumarellide ( 1 ) and the ethyl ester of plumarellic acid ( 2 ), were isolated from the alcoholic extract of the gorgonian coral Plumarella sp. and their structures were established by NMR, EIMS, MALDI TOF MS, IR and UV spectroscopy.

Sesquiterpenoids from the Far-Eastern Gorgonaria Stenogorgia SP.

In continuation of the search for biologically active natural compounds in marine coelenterates, we investigated the composition of the sesquiterpenoid fraction from the Far-Eastern Gorgonaria Stenogorgia sp. [1, 2]. The Gorgonaria was collected in August 2008 near Urup Island (Kuril Islands) from gravelly ground by a bottom trawler at a depth of 350 m. Ground animal was extracted with EtOH. The evaporated extract was dissolved in water and extracted with hexane. Compounds 1 and 2 were isolated from the hexane extract by column chromatography over Sephadex LH-20 (EtOH) and silica gel (KSK, 50-160 μm) using a hexane:EtOAc gradient (100:0–90:10) with subsequent HPLC on an Altex UltrasphereTM Si column (5 μm, 4.6 × 250 mm, hexane). Compound 3 was obtained by a series of chromatographic procedures including an LH-20 column (EtOH), silica gel (hexane:EtOAc, 92:8), and monocratic HPLC on UltrasphereTM Si (5 μm, 4.6 × 250 mm, hexane:EtOAc, 3.5:1) and Agilent Hypersil ODS (5 μm, 4.0 × 250 mm, EtOH:H2O, 7:3) columns.

STIMULATORY EFFECTS OF STARFISH SAPOGENINS (ASTERIAS AMURENSIS AND LETHASTERIAS NANIMENSIS CHELIFERA) ON MOLLUSCAN HEART (SPISULA SACHALINENSIS)

Sapogenins from the starfish Asterias amurensis and Lethasterias nanimensis chelifera, 5 alpha-pregn-9(11)-ene-3 beta,6 alpha-diol-20-one, 5 alpha-cholest-9(11)-ene-3 beta,6 alpha-diol-23-one, 5 alpha-cholesta-9(11),24(25)-diene-3 beta,6 alpha-diol-23-one, (20E)-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one and 24 zeta-methyl-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one, stimulated the contractile force of the heart of the mollusk Spisula sachalinensis at concentration of 5 x 10(-5) M. Ouabain, a specific inhibitor of Na+,K(+)-ATPase, at concentration of 5 x 10(-5) M had no effect on this physiological model. Starfish sapogenins of the cholestane series moderately inhibited rat brain cortex Na+,K(+)-ATPase and decreased Ca2+ influx into Ehrlich carcinoma cells. In contrast, pregnane asterogenin asterone did not inhibit Na+,K(+)-ATPase and increased the influx of Ca2+ into cells. These effects were not the result of cell membrane damage, because none of the compounds tested have hemolytic activity.

Sterols from the toxin-containing far-east sponge Monanchora pulchra

It was shown earlier that toxin-containing marine invertebrates, in particular sponges (phylum Porifera) and sea cucumbers (phylum Echinodermata, class Holothurioidea), often contain unusual sterols instead of cholesterol and other 5-sterols, in contrast with other animals [1]. They differ from cholesterol either by the position of the double bond in the steroid core (sometimes its absence) [2–4] or by the presence of highly alkylated side chains [5, 6]. According to a hypothesis proposed by us, the unusual sterols protect the biomembranes of toxin-producing marine invertebrates from the lytic action of their own toxins as a result of sterol–toxin biochemical coordination occurring in such instances [7]. In continuation of the search for new sources of biologically active compounds among invertebrates of Far-East seas, we recently found that EtOH extracts of the sponge Monanchora pulchra exhibited high cytotoxic activity. It was shown that this sponge contained a new cytotoxic polycyclic guanidine alkaloid that we called monanchocidin [8]. We investigated the sterol fraction from this sponge collected near the shores of Urup Is. (Kuril Islands, 46°07.0 N, 150°02 E) in August 2008 in order to test further this hypothesis. The sponge for chemical investigation was stored at –20°C. Thawed ground biological material (18.9 g dry wt.) was extracted (3 ) with EtOH. The combined extracts were concentrated to dryness in vacuo and distributed between H2O and BuOH. The evaporated BuOH layer (2.14 g) was dissolved in EtOH (90%) and extracted (3 ) with hexane. The combined hexane layers were concentrated to afford total nonpolar substances (0.69 g). A part (0.215 g) of this total was chromatographed over a column of silica gel (KSK, 50–160 m) using a hexane EtOAc gradient (4:1) to afford the total sterol fraction (57 mg, 0.92% of dry wt) that was separated using HPLC into three subfractions consisting of 5(1.3 mg) and 7-sterols (24.3 mg), and stanols (21 mg). These were analyzed using PMR as described before [2, 3, 9, 10]. The structures of the free sterols as the acetylated derivatives were identified using GC and comparison with the relative retention times (RRT) of standard sterols and using GC–MS as before [2, 3, 9, 10]. Table 1 presents the results. Table 1 shows that the sterol fraction from the studied sponge contained 29 previously known C26, C27, C28, C29, and C30 compounds that had double bonds in the 7, 7,22, 7,24(28), 0, 22, 5, 5,22, and 5,24(28) positions. Sterols of the 7series were basic and made up 52.1% of the total sterol fraction. The stanol content was 45.1% whereas 5-sterols were a minor fraction with a content of 2.8%. The principal components of the fraction were identified as 5-cholestan-3 -ol and 5 -cholest-7-en-3 -ol. Thus, yet another instance of toxin—(unusual sterols) biochemical coordination was found.

Diterpenoids and Other Metabolites from the Vietnamese Gorgonians Lophogorgia sp. and Junceella sp.

In continuation of research on isoprenoids from gorgonian coral [1, 2], we isolated seven known compounds (1–7) [3–8] from the Vietnamese gorgonians Lophogorgia sp. and Junceella sp. Compounds 1, 5, 6, and 7 were not previously isolated from gorgonian species studied by us. The structures of 1–7 were established using mass and high-resolution NMR spectroscopic data and comparisons with the literature [3–7, 9, 10].

New asterosaponins from the starfishDismolasterias nipon

Three new glycosides, D1, D2, and D3, have been isolated from the Far Eastern starfishDistolasterias nipon. They have been identified by chemical and physicochemical methods as 5α-cholestane÷3β,6α,8β,15β,24ξ-pentaol 3,24-di-O-β-D-xylopyranoside, tα-cholest-22-ene-3β,6α,8β,15β,24ξ-pentaol 3,24-di-O-β-D-xylopyranoside (II), and 5α-cholestane-3β,6α,8β,15β,24ξ-pentaol 24-O-β-D-glucopyranoside 3-O-β-D-xylopyranoside (III).

Sterols and Fatty Acids from Attheya Planktonic Diatoms

Sterols and fatty-acids isolated from laboratory cultures of the diatoms Attheya decora, A. arenicola, and A. longicornis were studied. The studied cultures of these species were substantially different with respect to the quantitative contents of polyunsaturated fatty acids and sterols. Rare sterols were found in several strains. It was shown that A. arenicola contained primarily 24-ethylcholesterol; A. longicornis and A. decora, 24-ethylcholesterol, 24-methylcholesterol, fucosterol, and 24-methylenecholesterol. Such differences indicate that the genus comprises various groups of species, the phylogenetic positions of which need to be defined.

Sterols and Fatty Acids Isolated from Laboratory Culture of the Toxic Marine Diatom Pseudo-nitzschia pungens

Marine diatoms of the genus Pseudo-nitzschia H. Peragallo are widely distributed in the world’s oceans and are of interest because 16 of 44 known species of this genus contain amnesic toxin (domoic acid) [1–3] that accumulates in edible mollusks and fish through food chains and causes serious poisoning in people and marine fauna [4–7]. The chemical compositions of many Pseudo-nitzschia are still little studied despite the interest in this group of diatoms. Their non-toxic metabolites are insufficiently studied [8]. The exceptions are fatty acid derivatives bacillariolides, which were isolated earlier from Pseudonitzschia pungens (Grunow ex Cleve) G. R. Hasle [9]. Herein, we communicate data on the isolation and structural analysis of sterol and fatty-acid fractions from algologically pure non-axenic culture of P. pungens (clone PPR-12). Developed methods were used to isolate the diatom in the culture [10]. The stock culture was inoculated into ten 1000-mL Ehrlenmeyer flasks with culture medium f [11] (700 mL) and grown at 20 ± 2°C and 3500 lux illumination. The culture was illuminated with luminescent lamps (FAR 15 W/m2) with 12-h light and dark cycles. Cell mass (1040 mg dry wt., 84,400 cells/mL) was separated by filtration at the end of the logarithmic growth phase. The mass on the filter was rinsed with warm distilled H2O and extracted by CHCl3–MeOH (2:1). The extract was concentrated to afford a dark-green amorphous precipitate (36.5 mg) that represented total nonpolar constituents isolated from the biomass. The obtained compound (30 mg) was chromatographed over a column of KSK silica gel (50–160 m) that was thoroughly rinsed before use by hexane–EtOAc (4:1) and then hexane. The chromatography used a gradient of hexane hexane–EtOAc (4:1) to produce a sterol fraction (2.5 mg) that gave a spot on silica gel with the same TLC Rf value as standard cholesterol (0.24% of starting biomass). Sterols were acetylated by AcOH–Py (1:1) at room temperature for 24 h. The reagents were removed by heating to 40°C in vacuo. Total sterol acetates were purified by column chromatography over KSK silica gel using hexane hexane–EtOAc (40:1) to afford a colorless amorphous mass (2.8 mg) that was analyzed by NMR and GC-MS [Hewlett Packard HP 5973 MSD/HP instrument, HP-5ms column with 5% phenylmethylsiloxane (30.0 m 25 m 0.25 m), temperature program 100°C (1 min) 20°C/min 270°C (30 min), carrier gas He, 69.9 eV ionization potential]. GC-MS analysis showed that the obtained fraction contained only two sterol acetates, i.e., cholesta-5,22-dien-3 -ol and cholesterol acetates. Therefore, only the two corresponding sterols were biosynthesized in the studied culture (Table 1). Because one of the sterols predominated in the obtained fraction, it was purified by crystallization from EtOH. The structure of the main sterol was confirmed as cholesta-5,22-dienol by NMR spectroscopy. The PMR spectrum of the obtained compounds (Bruker Avance 500, CDCl3, TMS as internal standard) showed resonances characteristic of 5,22-sterol acetates [12] as singlets for CH3-18, CH3-19, and CH3CO groups at 0.69, 1.02, and 2.03 ppm; doublets for CH3-21, CH3-26, and CH3-27 at 0.92, 0.87, and 0.89; and multiplets for CH-3 at 4.6 ppm and CH-22, CH-23, and CH-6 at 5.22, 5.27, and 5.37, respectively. Fatty acids (FA) were analyzed using reproduced culture (1.3 L) (191 mg of diatom biomass). Total lipids were extracted by the literature method [13]. Methyl esters were prepared, analyzed, and identified as described before [14]. The FA contents were determined using an internal standard (C21:0 acid) that was added to the lipids before the methylation. The calculated content was 0.49 mg (0.26% calculated for initial biomass). Table 2 presents the analytical results.

Sesquiterpenoids from a Vietnamese Gorgonian Eunicea sp.

The class Anthozoa (corals, including gorgonian) is a group of marine invertebrates (Coelenterata) that is a renewable source of terpenoids with various skeletons that, as a rule, exhibit biological activity [1]. In continuation of research on isoprenoids from gorgonian corals [2, 3], we isolated two new compounds 1 and 2 from a Vietnam gorgonian Eunice sp. [4–7]. The gorgonian was collected in May 2010 in the South China Sea at 12 m depth (Vietnam, Van Phong Bay, 12°32.8 N, 109°23.7 E). Animals were ground and extracted with EtOH. The partially evaporated extract was distributed between aqueous EtOH and hexane. The non-polar fraction was evaporated to dryness and separated over columns of KSK silica gel (50–160 m) using a hexane hexane–EtOAc (99:1) gradient and then Sephadex LH-20 using CHCl3 and HPLC over an Altex UltrasphereTM-Si column (5 m, 4.6 250 mm) using hexane–EtOAc (4:1) to afford 1.