Vladimir I. Kalinin

Sea Cucumbers Triterpene Glycosides, the Recent Progress in Structural Elucidation and Chemotaxonomy

Triterpene glycosides are characteristic metabolites of sea cucumbers (Holothurioidea, Echinodermata). Majority of the glycosides belong to holostane type (lanostane derivatives with 18(20)-lactone). Carbohydrate chains of these glycosides contain xylose, glucose, quinovose, 3-O-methylglucose and 3-O-methyl sylose. During the last 5 years, main investigations were focused on holothurians belonging to the order Dendrochirotida collected in the North Pacific, North Atlantic, Antarctic and in subtropical waters. The glycosides of holothurians belonging to the order Aspidochirotida have also been studied. The most uncommon structural features of carbohydrate chains of new glycosides were: (1) the presence of quinovose as fifth terminal monosaccharide unit and the presence of two quinovose residues; (2) the presence of glucose instead of common xylose as fifth terminal monosaccharide unit; (3) trisaccharide carbohydrate chain; (4) the presence of two 3-O-methylxylose terminal monosaccharide units; (5) the presence of sulfate group at C-3 of quinovose residue. New glycosides without lactone or with 18(16)-lactone and having shortened side chains have also been isolated. The presence of 17α and 12α-hydroxyls, which are characteristic for glycosides from holothurians belonging to the family Holothuriidae (Aspidochirotida) in glycosides of dendrochirotids confirms parallel and relatively independent character of evolution of glycosides. All three families belonging to the order Aspidochirotida: Holothuriidae, Stichopodidae and Synallactidae have similar and parallel trends in evolution of the glycosides carbohydrate chains, namely from non-sulfated hexaosides to sulfated tetraosides. Sets of aglycones in glycosides from holothurians belonging to the genus Cucumaria (Cucumariidae, Dendrochirotida) are specific for each species. The carbohydrate chains are similar in all representatives of the genus Cucumaria.

Hemolytic activities of triterpene glycosides from the holothurian order dendrochirotida: Some trends in the evolution of this group of toxins

Hemolysis and K+ loss from mouse erythrocytes, induced by triterpene glycosides and their derivatives from this order of sea cucumbers were studied. Sulfate groups, attached to position 4 of the first xylose residue and to position 6 of the third glucose residue of the branched pentaosides, having 3-O-methyl-groups in terminal monosaccharide moieties increase K+ loss. A sulfate group at C-4 of the first xylose residue increases the hemolytic activity while a sulfate at C-6 of the third monosaccharide unit decreases it. A sulfate group at C-6 of terminal 3-O-methylglucose drastically decreases the hemolytic activity and rate of K+ loss. The presence of a sulfate group at the first xylose residue in glycosides having no 3-O-methyl group at the terminal monosaccharide decreases hemolytic activity and rate of K+ loss. The presence of the 16-ketone group in aglycones having the 7(8)-double bond significantly decreases activity. These results correlate with the previously proposed trends in evolution of sea cucumber glycosides from substances having sulfate groups at C-6 of glucose and 3-O-methylglucose units to substances sulfated at C-4 of the first xylose or having no sulfate groups, and from substances with aglycone 16-ketone to substances having no oxygen functions in this position.

Constituents of the sea cucumber Cucumaria okhotensis. Structures of okhotosides B1-B3 and cytotoxic activities of some glycosides from this species

Three new triterpene oligoglycosides, okhotosides B 1 ( 1), B 2 ( 2), and B 3 ( 3), have been isolated from the sea cucumber Cucumaria okhotensis along with the known compounds frondoside A ( 4), frondoside A 1, cucumarioside A 2-5, and koreoside A. The structures of 1- 3 were elucidated on the basis of their spectroscopic data (2D NMR and MS). Compounds 1- 3 were moderately toxic against HeLa tumor cells. Frondoside A ( 4) showed more potent cytotoxicity against THP-1 and HeLa tumor cell lines (with IC 50 values of 4.5 and 2.1 microg/mL, respectively) and decreased both the AP-1-dependent trascriptional activities induced by UVB, EGF, or TPA in JB6-LucAP-1 cells and the EGF-induced NF-kappaB-dependent transcriptional activity in JB6-LucNF-kB cells at doses of about 1 microg/mL. At the same doses, it increased the p53-dependent transcriptional activity in nonactivated JB6-Lucp53 cells and inhibited the colony formation of JB6 P (+) Cl 41 cells activated with EGF (INCC 50 = 0.8 microg/mL).

Triterpene Glycosides from Sea Cucucmbers (Holothurioidea, Echinodermata). Biological Activities and Functions

Abstract The holothuroid triterpene glycosides have strong membranolytic action against cellular and model membranes containing Δ5-sterols as result of the formation of single-ion channels and more large pores that is the basis of hemolytic, antifungal, antitumor cytotoxic activities of these compounds. The binding presence of an 18(20)-lactone, and at any rate of one oxygen functional group near it for compounds with 9(11)-double bonds in lanostane aglycon moiety, is very important for the membranotropic action of these substances. A linear tetrasaccharide fragment in the carbohydrate chain is also essential for membranolytic action. A sulfate group at C-4 of the first xylose residue increases the effect against membranes. The absence of a sulfate group at C-4 of the xylose residue in biosides decreases the activities more than one order of magnitude. The presence of a sulfate at C-4 of the first xylose of branched pentaosides having 3-O-methyl group at a terminal monosaccharide increases activity but the same sulfate decreases the activity of branched pentaosides having terminal glucose residues. Sulfate groups attached to a C-6 position of terminal glucose and 3-O-methylglucose residues greatly decrease activity. Some glycosides may inhibit chemokine receptor subtype 5 (CCR 5). This activity is correlated with toxicity. Some glycosides possess immunostimulatory action in subtoxic doses. The most effective immunostimulants are monosulfated glycosides but di- and trisulfated are immunosuppressors. The network diagrams illustrating the relationships between glycoside structural elements and functional components (partial activities) as well as with general glycoside activity are shown to be useful for non-quantative predication of biological activity. The glycosides regulate of oocyte maturation in the sea cucumbers to synchronize maturation processes. This role is caused by the modifying action of the glycosides on the membranes of holothuroid eggs. The absence or very low concentration of Δ5-sterols in oocyte membranes suggests another nature of the modifying action in comparison with that against membranes containing Δ5-sterols. Holothuroid glycosides have defensive function against predators.

Anticancer Activity of Sea Cucumber Triterpene Glycosides

Triterpene glycosides are characteristic secondary metabolites of sea cucumbers (Holothurioidea, Echinodermata). They have hemolytic, cytotoxic, antifungal, and other biological activities caused by membranotropic action. These natural products suppress the proliferation of various human tumor cell lines in vitro and, more importantly, intraperitoneal administration in rodents of solutions of some sea cucumber triterpene glycosides significantly reduces both tumor burden and metastasis. The anticancer molecular mechanisms include the induction of tumor cell apoptosis through the activation of intracellular caspase cell death pathways, arrest of the cell cycle at S or G2/M phases, influence on nuclear factors, NF-κB, and up-down regulation of certain cellular receptors and enzymes participating in cancerogenesis, such as EGFR (epidermal growth factor receptor), Akt (protein kinase B), ERK (extracellular signal-regulated kinases), FAK (focal adhesion kinase), MMP-9 (matrix metalloproteinase-9) and others. Administration of some glycosides leads to a reduction of cancer cell adhesion, suppression of cell migration and tube formation in those cells, suppression of angiogenesis, inhibition of cell proliferation, colony formation and tumor invasion. As a result, marked growth inhibition of tumors occurs in vitro and in vivo. Some holothurian triterpene glycosides have the potential to be used as P-gp mediated MDR reversal agents in combined therapy with standard cytostatics.

Antitumor activity of the immunomodulatory lead Cumaside

A new immunomodulatory lead Cumaside that is a complex of monosulfated triterpene glycosides from the sea cucumber Cucumaria japonica and cholesterol possesses significantly less cytotoxic activity against sea urchin embryos and Ehrlich carcinoma cells than the corresponding glycosides. Nevertheless Cumaside has an antitumor activity against different forms of experimental mouse Ehrlich carcinoma in vivo both independently and in combination with cytostatics. The highest effect occurs at a treatment once a day for 7 days before the tumor inoculation followed by Cumaside treatment once a day for 7 days. Prophylactic treatment with Cumaside and subsequent therapeutic application of 5-fluorouracil suppressed the tumor growth by 43%.

Immunomodulatory Properties of Frondoside A, a Major Triterpene Glycoside from the North Atlantic Commercially Harvested Sea Cucumber Cucumaria frondosa

Frondoside A, a major triterpene glycoside from North Atlantic commercially harvested sea cucumber Cucumaria frondosa, possesses strong immunomodulatory properties in subtoxic doses. Frondoside A stimulates lysosomal activity of mouse macrophages in vivo at a maximal effective stimulatory dose of 0.2 microg per mouse and is maintained over 10 days. This glycoside also shows a 30% stimulation of lysosomal activity in mouse macrophages in vitro at concentrations of 0.1-0.38 microg/mL. Frondoside A enhances macrophage phagocytosis of the bacterium Staphylococcus aureus in vitro at a maximal effective concentration of 0.001 microg/mL. Frondoside A stimulates reactive oxygen species formation in macrophages in vitro at a maximal effective concentration of 0.001 microg/mL. Frondoside A stimulates an increase in the number of antibody plaque-forming cells (normally B-cells in spleen) in vivo with a maximal stimulatory effect at a concentration of 0.2 microg per mouse (stimulatory index, 1.86). Frondoside A has a weak effect upon immunoglobulin (Ig) M production after immunization with sheep erythrocytes in mice. Frondoside A does not stimulate Ig production in mice and does not significantly enhance the ovalbumin-stimulated IgM and IgG antibody levels in ovalbumin-immunized mice. Hence frondoside A is an immunostimulant of cell-based immunity including phagocytosis without a significant effect on amplification of humoral immune activity or adjuvant properties. Therefore, frondoside A may provide curative and/or preventive treatment options against diseases wherein a depleted immune status contributes to the pathological processes.

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).

Immunomodulatory effects of holothurian triterpene glycosides on mammalian splenocytes determined by mass spectrometric proteome analysis

Spleen is a prime organ in which immuno-stimulation takes place in mammalians. Proteome analysis was used to investigate the elicited effects on mouse splenocytes upon exposure to holothurian triterpene glycosides. Cucumarioside A(2)-2, and Frondoside A, respectively, have been used to in-vitro stimulate primary splenocyte cultures. Differential protein expression was monitored by 2D gel analysis and proteins in spots of interest were identified by MALDI ToF MS and nano LC-ESI Q-ToF MS/MS, respectively. Differential image analysis of gels from control vs. gels from stimulated primary splenocyte cultures showed that approximately thirty protein spots were differentially expressed. Prime examples of differentially expressed proteins are NSFL1 cofactor p47 and hnRNP K (down-regulated), as well as Septin-2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, and GRB2-related adaptor protein 2 (up-regulated). Immuno-analytical assays confirmed differential protein expression. Together with results from proliferation and cell adhesion assays, our results show that cellular proliferation is stimulated by holothurian triterpene glycosides. In conclusion, holothurian triterpene glycosides are thought to express their immuno-stimulatory effects by enhancing the natural cellular defense barrier that is necessary to fight pathogens and for which lymphocytes and splenocytes have to be recruited constantly due to limited lifetimes of B-cells and T-cells in the body.

Triterpene Glycosides from Antarctic Sea Cucumbers. 1. Structure of Liouvillosides A1, A2, A3, B1, and B2 from the Sea Cucumber Staurocucumis liouvillei: New Procedure for Separation of Highly Polar Glycoside Fractions and Taxonomic Revision

Five new triterpene glycosides, liouvillosides A1 (1), A2 (2), A3 (3), B1 (4), and B2 (5), have been isolated from the Antarctic sea cucumber Staurocucumis liouviellei along with the known liouvilloside A(6), isolated earlier from the same species, and hemoiedemosides A (7) and B (8), isolated earlier from the Patagonian sea cucumber Hemioedema spectabilis. The isolation was carried out using a new chromatographic procedure including application of ion-pair reversed-phase chromatography followed by chiral chromatography on a cyclodextrin ChiraDex column. The structures of the new glycosides were elucidated using extensive NMR spectroscopy (1H and 13C NMR spectrometry, DEPT, 1H-(1)H COSY, HMBC, HMQC, and NOESY), ESI-FTMS, and CID MS/MS, and chemical transformations. Glycosides 1-3 are disulfated tetraosides and glycosides 4 and 5 are trisulfated tetraosides. Glycosides 2 and 3 contain 3-O-methylquinovose, found for the first time as a natural monosaccharide in sea cucumber glycosides. On the basis of analyses of glycoside structures a taxonomic revision is proposed.