Timofey V. Malyarenko

Four new asterosaponins, hippasteriosides A - D, from the Far Eastern starfish Hippasteria kurilensis.

Four new sulfated steroidal glycosides (asterosaponins), hippasteriosides A–D (1–4, resp.), were isolated from the alcoholic extract of the Far Eastern starfish Hippasteria kurilensis, collected in the Sea of Okhotsk near Kuril Islands. Compounds 1–4 were determined to contain the same unprecedented hexasaccharide moiety, β‐D‐xylopyranosyl‐(1→3)‐β‐D‐fucopyranosyl‐(1→2)‐β‐D‐quinovopyranosyl‐(1→4)‐[β‐D‐quinovopyranosyl‐(1→2)]‐β‐D‐xylopyranosyl‐(1→3)‐β‐D‐quinovopyranosyl, linked to C(6) of 3‐O‐sulfonylated steroidal aglycons, which differ from each other in their side‐chain structures. Structures 1–4 were elucidated by extensive NMR and MS techniques, and chemical transformations. Not all the asterosaponins show a prominent cytotoxic activity against human colon tumor HT‐29 cells, but some of them, especially compound 4 demonstrate a remarkable inhibition of the HT‐29 colony formation in soft‐agar clonogenic assay, suggesting its anticancerogenic properties.

Cyclic Steroid Glycosides from the Starfish Echinaster luzonicus: Structures and Immunomodulatory Activities

Five new steroid glycosides, luzonicosides B-E (2-5), belonging to a rare structure group of marine glycosides, containing carbohydrate moieties incorporated into a macrocycle, and a related open carbohydrate chain steroid glycoside, luzonicoside F (6), were isolated from the starfish Echinaster luzonicus along with the previously known cyclic steroid glycoside luzonicoside A (1). The structures of compounds 2-6 were established by extensive NMR and ESIMS techniques as well as chemical transformations. Luzonicoside A (1) at concentrations of 0.01-0.1 μM was shown to be potent in lysosomal activity stimulation, intracellular ROS level elevation, and NO synthesis up-regulation in RAW 264.7 murine macrophages. Luzonicoside D (4) was less active in these biotests.

Asterosaponins from the Far Eastern starfish Leptasterias ochotensis and their anticancer activity.

Six new asterosaponins, leptasteriosides A-F (3-8), one new and one previously known asterogenins (1, 2) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of 1-8 were elucidated by extensive NMR and ESI-MS techniques. Compounds 2-8 showed slight or moderate cytotoxic activities against cancer cell lines RPMI-7951 and T-47D. The asterosaponins 3-5 demonstrated a significant inhibition of RPMI-7951 and T-47D cell colony formation in soft agar clonogenic assay in nontoxic doses.

Four New Sulfated Polar Steroids from the Far Eastern Starfish Leptasterias ochotensis: Structures and Activities

Three new sulfated steroid monoglycosides, leptaochotensosides A–C (1–3), and a new sulfated polyhydroxylated steroid (4) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of compounds 1–4 were established by extensive nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESIMS) analyses and chemical transformations. Although the isolated compounds did not show any apparent cytotoxicity against melanoma RPMI-7951 and breast cancer T-47D cell lines, leptaochotensoside A (1) demonstrated inhibition of T-47D cell colony formation in a soft agar clonogenic assay at nontoxic doses. In addition, this compound decreased the epidermal growth factor (EGF)-induced colony formation of mouse epidermal JB6 Cl41 cells. The cancer preventive action of 1 is realized through regulation of mitogen-activated protein kinase (MAPK) signaling pathway.

Cariniferosides A-F and other steroidal biglycosides from the starfish Asteropsis carinifera.

Six new steroidal biglycosides, cariniferosides A-F (1-6), were isolated along with six previously known glycosides, 7-12, from the alcoholic extract of the starfish Asteropsis carinifera. The structures of 1-6 were determined by extensive NMR and ESIMS techniques. The isolated compounds did not show any apparent cytotoxicity in cancer cell lines HCT-116, RPMI-7951, and T-47D, but sulfated compounds 6, 11, and 12 demonstrated a significant inhibition of RPMI-7951 and T-47D cell colony formation in a soft agar clonogenic assay.

Biosynthesis of polar steroids from the Far Eastern starfish Patiria (=Asterina) pectinifera. Cholesterol and cholesterol sulfate are converted into polyhydroxylated sterols and monoglycoside asterosaponin P1 in feeding experiments.

For the first time, it is experimentally established that the dietary cholesterol and cholesterol sulfate are biosynthetic precursors of polyhydroxysteroids and related low molecular weight glycosides in starfishes. These deuterium labeled precursors were converted into partly deuterated 5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol, 5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol, and steroid monoside asterosaponin P1 in result of feeding experiments on the Far Eastern starfish Patiria (=Asterina) pectinifera. The incorporations of deuterium were established by MS and NMR spectroscopy. Scheme of the first stages of biosynthesis of polar steroids in these animals was suggested on the basis of inclusion of three from six deuterium atoms and determination of their positions in biosynthetic products, when [2,2,3,4,4,6-(2)H6]cholesterol 3-sulfate was used as precursor. It was also shown that labeled cholesterol is transformed into Δ(7)-cholesterol (lathosterol) in digestive organs and gonads of the starfish.

Metabolite profiling of polar steroid constituents in the Far Eastern starfish Aphelasterias japonica using LC–ESI MS/MS

Numerous polar steroids are characteristic metabolites of starfish which determine physiological activities of their extracts. The Far Eastern starfish Aphelasterias japonica is a rich source of different steroid glycosides and polyhydroxysteroids. For detailed analysis of complicated mixture of steroids from this species, isolated by solid-phase extraction, a liquid chromatography–electrospray tandem mass spectrometry (LC–ESI MS/MS) approach was selected and applied. The characteristic fragmentations in ESI MS/MS spectra of steroid glycosides allowed determining types of aglycones, presence of sulfate groups, sugar sequences as well as branching. In addition, main structural features of polyhydroxylated polar steroids including position of hydroxylation and level of sulfation were also established. Totally, 68 metabolites, comprising of 33 asterosaponins, 28 sulfated polyhydroxysteroid mono- and biosides and 7 sulfated polyhydroxysteroid compounds were found by this method. In addition to 15 previously isolated compounds from A. japonica, many new steroid glycosides including asterosaponins with unusual carbohydrate chains were discovered and characterized by their ESI product ion mass spectra. Some details of biosynthesis of polyhydroxylated steroids and their conjugated forms in the species studied such as a role of sulfation and order of introduction of hydroxyl group in A. japonica were proposed.

LC–MS-based metabolome analysis on steroid metabolites from the starfish Patiria (=Asterina) pectinifera in conditions of active feeding and stresses

IntroductionStarfish are recognized as interesting source of natural steroid products with pharmaceutical potential. Polar steroid metabolites of starfish have unique chemical structures and exhibit various biological activities but their biological functions are controversial.ObjectivesThe objective of this study was to investigate the response of polar steroid metabolome of the starfish Patiria (=Asterina) pectinifera on various environmental factors and stresses.MethodsHere we first have applied MS-based environmental metabolomics to elucidate the metabolic changes of polar steroid metabolome of starfish. Using HPLC–ESI–Q/TOF–MS approach followed by statistical analysis including principal component analysis and partial least squares discriminant analysis for data classification and potential biomarkers selection, we investigated the changes induced by feeding, injury, variations in water temperature and salinity, and oxygen deficiency.ResultsAccording to multivariate and univariate statistical analysis the responses to feeding, injury and water heating were better expressed than the others and have some similarity in their action on the steroid metabolome of the starfish P. pectinifera. Most constituents of asterosaponin pool were reduced and most constituents of polyhydroxysteroid and related glycoside pool were increased at that.ConclusionOur results indicate that various metabolic changes in polar steroid constituents of P. pectinifera are induced by feeding and stresses. We believe that these responses are connected with biological multifunctionality of these compounds.

Anthenosides L–U, Steroidal Glycosides with Unusual Structural Features from the Starfish Anthenea aspera

Ten new polyhydroxysteroidal glycosides, anthenosides L-U (1-10), with rare positions of carbohydrate fragment attachments, were isolated from the starfish Anthenea aspera. The structures of 1-10 were established by NMR and ESIMS techniques as well as by chemical transformations. The unoxidized Δ22-24-nor-cholestane (1), (24S)-Δ22-24-methylcholestane (2-5), and Δ22-cholestane (7) side chains of the steroidal aglycons, 3-O-methyl-β-d-galactofuranosyl residue (2, 8), and 5α-cholest-8(14)-ene-3α,7β,16α-trihydroxysteroidal nucleus (9, 10) have not been found previously in starfish polar steroidal compounds. The mixture of glycosides 9 and 10 showed hemolytic activity with an EC50 = 8 μM. Compound 4 at a dose of 10 μM exhibited a potential immunomodulatory action, decreasing by 24% the intracellular ROS content in RAW 264.7 murine macrophages, induced by pro-inflammatory endotoxic lipopolysaccharide from E. coli.

Asteropsiside A and other asterosaponins from the starfish Asteropsis carinifera

Three asterosaponins were isolated from the tropical starfish Asteropsis carinifera: a new one, asteropsiside A, and two known ones, regularoside A and thornasteroside A. The structure of the new compound was established using 2D NMR spectroscopy and ESI mass spectrometry as the sodium salt of 3-O-sulfonato-(20E)-6-O-{β-d-fucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→4)-[β-d-quinovopyranosyl-(1→2)]-β-d-xylopyranosyl-(1→3)-β-d-quinovopyranosyl}-3β,6α-dihydroxy-5α-cholesta-9(11),20(22)-dien-23-one. Regularoside A and thornasteroside A were shown to display the ability to inhibit the growth of the T-47D and RPMI-7951 tumor cell colonies in vitro.