In vitro characterization of bioactive compounds extracted from sea urchin (Stomopneustes variolaris) using green and conventional techniques.

This study investigated the in vitro bioactivities of extracts obtained from viscera, spines, shells, and gonads of Stomopneustes variolaris using subcritical water extraction (SWE) at 110 °C, 150 °C, 190 °C, and 230 °C and Soxhlet extraction. The highest amounts of phenolics (22.68 ± 0.05 mg GAE/g), flavonoids (27.11 ± 0.10 mg RE/g), and proteins (40.25 ± 0.84 mg BSA/g) were recorded from gonads at 230 °C, whereas maximum sugar content (23.38 ± 1.30 mg glucose/g) was in viscera at 150 °C. Gonads at 230 °C exhibited the highest DPPH activity (78.68 ± 0.18%), whereas viscera at 150 °C exhibited the highest ABTS+ (98.92 ± 1.27%) and protein denaturation inhibition activity (37.13 ± 9.94%). Viscera at 110 °C claimed the highest amylase inhibition (42.46 ± 0.83%), and spines at 150 °C had the highest anticancer activity (IC50 = 767.47 µg/mL). SWE achieved superior results in bioactive compound recovery and detected higher levels of bioactivities (p < 0.05). Results suggest processing sea urchin extracts via SWE has potential application to the food and pharmaceutical industries.

PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line.

Polyhydroxylated naphthoquinones (PHNQs), known as spinochromes that can be extracted from sea urchins, are bioactive compounds reported to have medicinal properties and antioxidant activity. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay showed that pure echinochrome A exhibited a cytotoxic effect on Saos-2 cells in a dose-dependent manner within the test concentration range (15.625-65.5 µg/mL). The PHNQ extract from New Zealand sea urchin Evechinus chloroticus did not induce any cytotoxicity within the same concentration range after 21 days of incubation. Adding calcium chloride (CaCl2) with echinochrome A increased the number of viable cells, but when CaCl2 was added with the PHNQs, cell viability decreased. The effect of PHNQs extracted on mineralized nodule formation in Saos-2 cells was investigated using xylenol orange and von Kossa staining methods. Echinochrome A decreased the mineralized nodule formation significantly (p < 0.05), while nodule formation was not affected in the PHNQ treatment group. A significant (p < 0.05) increase in mineralization was observed in the presence of PHNQs (62.5 µg/mL) supplemented with 1.5 mM CaCl2. In conclusion, the results indicate that PHNQs have the potential to improve the formation of bone mineral phase in vitro, and future research in an animal model is warranted.

Anticoagulant and Antithrombotic Properties of Three Structurally Correlated Sea Urchin Sulfated Glycans and Their Low-Molecular-Weight Derivatives.

The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from Strongylocentrotus franciscanus was observed to be poorly active in most assays, the 4-sulfated fucan from Lytechinus variegatus, the 2-sulfated galactan from Echinometra lucunter and their derivatives showed multiple effects. All marine compounds showed no capacity to activate factor XII and similar low bleeding tendencies regardless of the dose concentrations used to achieve the highest antithrombotic effect observed. The 2-sulfated galactan showed the best combination of results. Our work improves the background about the structure-function relationship of the marine sulfated glycans in anticoagulation and antithrombosis. Besides confirming the negative effect of the 2-sulfated fucose and the positive effect of the 2-sulfated galactose on anticoagulation in vitro, our results also demonstrate the importance of this set of structural requirements on antithrombosis in vivo, and further support the involvement of high-molecular weight and 4-sulfated fucose in both activities.

The Sea Urchin Arbacia lixula: A Novel Natural Source of Astaxanthin.

Several echinoderms, including sea urchins, are valuable sources of bioactive compounds but their nutraceutical potential is largely unexplored. In fact, the gonads of some sea urchin species contain antioxidants including carotenoids and polyhydroxylated naphthoquinones (PHNQ's), such as echinochrome A. Astaxanthin is known to have particular bioactivity for the prevention of neurodegenerative diseases. This carotenoid is produced by microalgae, while several marine invertebrates can bioaccumulate or synthetize it from metabolic precursors. We determined the carotenoid content and analyzed the bioactivity potential of non-harvested Atlantic-Mediterranean sea urchin Arbacia lixula. The comparison of methanol crude extracts obtained from eggs of farmed and wild specimens revealed a higher bioactivity in farmed individuals fed with a customized fodder. HPLC-analysis revealed a high concentration of astaxanthin (27.0 µg/mg), which was the only pigment observed. This study highlights the potential of farmed A. lixula as a new source of the active stereoisomer of astaxanthin.

New Aminonaphthoquinone from the Sea Urchins Strongylocentrotus pallidus and Mesocentrotus nudus.

The aminonaphthoquinone, spinamine E (5), was isolated for the first time from the sea urchins Strongylocentrotus pallidus (Sars G.O., 1872) and Mesocentrotus nudus (A. Agassiz, 1864). The structure of 5 was elucidated as 2-amino-3,5,6,7,8-pentahydroxy-1,4-naphthoquinone using ID 1H-, 13C- and 2D NMR procedures, and HR-ESI mass-spectrometric data of 5 and its trimethyl ether. Spinamine E, as well as two other aminonaphthoquinones of sea urchins, echinamines A (2) and B (3), along with their hydroxylated analogues, spinochrome E (4) and echinochrome A (1), were tested for their ability to scavenge the stable DPPH radical and to inhibit lipid peroxidation. All investigated naphthoquinones obtained from the sea urchins showed a high antiradical activity, which was up to 1.5 times higher than that of α-tocopherol. Echinamine B showed the highest scavenging effect (EC50 = 6.5-10(-6) M); this effect decreases in the series 3>5>1>2>4>α-tocopherol. In a lipid peroxidation inhibition testing model, echinamine B and spinamine E showed the highest inhibitory effect. The stability of compounds 1-5 in weakly alkaline solutions was evaluated.

Inhibitory Effects of Echinochrome A, Isolated from Shell of the Sea Urchin Anthocidaris crassispina, on Antigen-Stimulated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells through Suppression of Lyn Activation.

Echinochrome A (Echi-A) was isolated from the sea urchin Anthocidaris crassispina and its structure determined using ID and 2D-NMR. In the present study, we examined the inhibitory effect of Echi-A on antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells, which were suppressed in a dose dependent manner.. The antigens bind to the high affinity immunoglobulin E receptor, which is expressed on the surface of mast cells and basophils and activate intracellular signal transduction, resulting in the release of biologically active mediators such as histamine. In order to disclose the inhibitory mechanisms of degranulation by Echi-A, we examined the elevation in intracellular C²âº concentration ([Ca²âº]i), production levels of intracellular reactive oxygen species (ROS) and early intracellular signaling events. Both elevation of [Ca²âº] and intracellular ROS production were markedly suppressed in cells treated with Echi-A. Echi-A also suppressed the activation of Lyn, Syk, and PLCyl/2 in antigen-stimulated cells. These results indicated that inhibition of antigen-stimulated degranulation in RBL-2H3 cells by Echi-A is mainly due to the inactivation of Lyn/Syk/PLCy signaling pathways. Our findings suggest that Echi-A could be a beneficial agent for alleviating the symptoms of type I allergy.

Preparative Synthesis of Spinochrome D, a Pigment of Different Sea Urchin Species.

A simple and effective synthesis of spinochrome D (1) (2,3,5,6,8-pentahydroxy-1,4-naphthoquinone), a pigment of different sea urchin species, has been developed starting from 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (13), easily available by cycloacylation of 1,4-dimethoxybenzene (11) with dichloromaleic anhydride (12). Bromination of 13 with either bromine or dioxane dibromide to 6-bromo-2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (21), followed by nucleophilic substitution of the halogen atoms by methoxy groups in 21 and hydrolysis of trimethyl ether 10 produce the target compound in overall yield from 62 to 65%.

Synthesis, Cytotoxic and Contraceptive Activity of 6,8,9-Trihydroxy-2-methyl-2H-naphtho[2,3-b]pyran-5,10-dione, a Pigment of Echinothrix diadema, and its Analogs.

6,8,9-Trihydroxy-2-methyl-2H-naphtho[2,3-b]pyran-5,10-dion, a pigment of the sea urchin Echinothrix diadema, and six analogs were synthesized. The cytotoxic activity and contraceptive properties of the synthesized pyranonaphthazarins have been investigated using the sperm and eggs of the sea urchin Strongylocentrotus intermedius.

Steroidal constituents from the edible sea urchin Diadema savignyi Michelin induce apoptosis in human cancer cells.

Bioassay-directed fractionation and purification were used to isolate 12 steroids (1-12) from a CH(2)Cl(2) extract of the edible Vietnamese sea urchin Diadema savignyi Michelin. The cytotoxic activity of the CH(2)Cl(2) extract and 12 steroids was evaluated in three human cancer cell lines (HL-60, PC-3, and SNU-C5). Relative to the effects of the positive control, mitoxantrone, the CH(2)Cl(2) extract (with an inhibitory concentration of 50% [IC(50)] values ranging from 1.37±0.15 to 3.11±0.15 µg/mL) and compounds 2 (with IC(50) values ranging from 5.29±0.11 to 6.80±0.67 µM) and 11 (with IC(50) values ranging from 4.95±0.07 to 6.99±0.28 µM) exhibited potent cytotoxic effects against all three tested human cancer cell lines. In addition, the CH(2)Cl(2) extract and compounds 2 and 11 were found to induce apoptosis. The induction of apoptosis was accompanied by alterations of the apoptosis-related protein expression, inactivation of ERK1/2 mitogen-activated protein kinase signaling, and decreased c-Myc expression. These data suggest that compounds 2 and 11 from the edible sea urchin D. savignyi may have potential for the treatment of colon cancer, leukemia, and prostate cancer as complementary cancer remedies.

Preparative production of spinochrome E, a pigment of different sea urchin species.

A concise route to spinochrome E (1) (2,3,5,6,7,8-hexahydroxy-1,4-naphthoquinone), a pigment isolated from sea urchins of different species, has been developed starting from either commercially available 5,8-dihydroxy-1,4-naphthoquinone (11) or 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (12). The protocol involves 3 steps, the chlorination of either 11 or 12 to tetrachloronaphthazarin (13), the total nucleophilic substitution of the chlorine atoms in 13 by methoxy groups, and hydrolysis of tetramethyl ether14; this makes possible the preparation of the target compound in overall yields from 41 to 46%.

Traditional Chinese medicine--sea urchin.

The sea urchin is an ancient, common, seafloor-dwelling marine invertebrate that belongs to the phylum Echinodermata. There are multiple species of sea urchin with resources that are widely distributed in China, where they were used in ancient times as Traditional Chinese Medicine for treating a variety of diseases. At present, it is known that the shell, spine and gonad of the sea urchin have many medicinal values determined through modern research. In this paper, we summarized the major chemical constituents and medicinal value of the sea urchin.

[Pharmacological activity echinochrome A singly and consisting of BAA "Timarin"].

Pharmacological activity of echinochrome A (EchA) alone and in the biologically active additives (BAA) "Timarin", administered per os has been investigated on volunteers. EchA decreased serum glutatione (GSH) and increased catalase activity 1 h after treatment; catalase activity normalized, while GSH exceeded the initial level 3 h after the treatment. Changes in serum lipid spectrum, demonstrating reduction of the risk atherogenesis were determined.

Free radical scavenging activities of naturally occurring and synthetic analogues of sea urchin naphthazarin pigments.

Antioxidant activities of minor pigments of sea urchins (1-5) and synthetic naphthazarins (7-13) were evaluated and compared with echinochrome A (6) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) scavenging assays. Structure-activity relationships showed that the antioxidant activities of the tested compounds depended on the number and positions of hydroxyl groups. Compounds bearing 3 or 2 hydroxyl groups on a naphthazarin core (5,8-dihydroxy-1,4-naphthoquinone) were the most active in both assays. Echinochrome A (6) (IC50 7.0 microM) and its monomethyl ethers 7 (IC50 15.0 microM) and 8 (IC50 15.0 microM) displayed stronger activities than Trolox (IC50 16.0 microM) in the DPPH and ABTS assays (TE = 3.41, 2.35, and 2.35 mM, respectively). Compounds with either one or without hydroxyl groups on a naphthazarin core displayed activities significantly lower than Trolox in both assays. These results suggest that hydroxylated naphthazarin pigments of sea urchins have a potential use as natural antioxidants.

Nutraceutical functionalities of polysaccharides from marine invertebrates.

Many researchers are seeking functional materials from marine resources. These marine resources can be used as traditional food additives, and specifically, these are based on polysaccharides. To date, there is a big opportunity to develop new high-value added products with indispensable functional characteristics, which can be used in nutraceuticals either as additives or supplements. Also, a crossover in the pharmaceutical market may be established. Some glycosaminoglycans (GAGs) mimetic-type molecules are already being utilized in the field of nutrition as well as in the cosmetics industry. This chemical is used as a dietary supplement to maintain the structure and function of cartilages, for the relief of pain caused by osteoarthritic joints, and can also be used as an anti-inflammatory agent. Recently, in relation to the prevalence of mad cow disease and avian influenza, the production of GAGs from marine invertebrates offers new market opportunities as compared with that obtained from bovine or avian livestock.

Short and regiospecific synthesis of echinamine A--the pigment of sea urchin Scaphechinus mirabilis.

New convenient two-step synthesis of natural pigment echinamine A by the condensation of 2,3-dichloro-6-ethyl-7-hydroxy-1,4-naphthoquinone with sodium nitrite and subsequent reduction of intermediate 7-ethyl-2,5,6,8-tetrahydroxy-3-nitro-1,4-naphthoquinone has been developed.

[Novel compounds increasing chaperone Hsp70 expression and their biological activity].

Hsp70 possesses chaperonic activity, the property associated with the protective function that was demonstrated in experiments on a great number of cell and animal models. Therefore, it seems important to search for the substances able to innocuously elevate the chaperone concentration in an organism cells and tissues. In our work, we screened of more that 60 compounds and found two chemicals, derivatives of shikonin and echinochrome that able to increase the chaperone level in a variety of human cells. It was shown that in human erythroleukemia K562 cells treated with the both substances concomitantly with elevation of Hsp70 level the absolute chaperonic activity was also increased; this can indicate mobilization of the whole cellular chaperonic machinery by above mentioned compounds. Estimating biological activity of the two substances, we demonstrated that treatments of cells by them prior to hard heat stress, hydrogen peroxide or staurosporine reduced cell mortality by 20-50 % depending on a cytotoxic factor. The results show that after simple chemical modifications these compounds might be taken as a basis of pharmaceuticals for therapy of wide range of disorders.

The grinding tip of the sea urchin tooth exhibits exquisite control over calcite crystal orientation and Mg distribution.

The sea urchin tooth is a remarkable grinding tool. Even though the tooth is composed almost entirely of calcite, it is used to grind holes into a rocky substrate itself often composed of calcite. Here, we use 3 complementary high-resolution tools to probe aspects of the structure of the grinding tip: X-ray photoelectron emission spectromicroscopy (X-PEEM), X-ray microdiffraction, and NanoSIMS. We confirm that the needles and plates are aligned and show here that even the high Mg polycrystalline matrix constituents are aligned with the other 2 structural elements when imaged at 20-nm resolution. Furthermore, we show that the entire tooth is composed of 2 cooriented polycrystalline blocks that differ in their orientations by only a few degrees. A unique feature of the grinding tip is that the structural elements from each coaligned block interdigitate. This interdigitation may influence the fracture process by creating a corrugated grinding surface. We also show that the overall Mg content of the tooth structural elements increases toward the grinding tip. This probably contributes to the increasing hardness of the tooth from the periphery to the tip. Clearly the formation of the tooth, and the tooth tip in particular, is amazingly well controlled. The improved understanding of these structural features could lead to the design of better mechanical grinding and cutting tools.

Accumulation trends of petroleum hydrocarbons in commercial shellfish from the Galician coast (NW Spain) affected by the Prestige oil spill.

Aliphatic and aromatic hydrocarbons were determined in three species of commercial shellfish, namely razor shells (Ensis arcuatus and Ensis siliqua), goose barnacle (Pollicipes cornucopia) and sea urchin (Paracentrotus lividus), living in different habitats and exhibiting different feeding behaviors. The samples were collected monthly, from January 2003 to October 2004, in three stations of the Galicia coast (NW Spain), following the Prestige oil spill, with the aim of assessing their response to the spill and, therefore, their suitability for monitoring purposes. The aliphatic fractions were mostly dominated by biogenic hydrocarbons, reflecting the diet composition of the organisms and their low metabolic capacity. The presence of oil was assessed by the determination of chemical markers. The analysis of the aromatic fractions revealed the occurrence of 3-6 ring parent and alkylated PAHs, consistent with a mixed petrogenic-pyrolytic origin, with the common feature of the predominance of chrysene in all samples collected after the spill. However, the distributions exhibited both temporal and interspecies variations. The PAH concentrations (Sigma13) increased significantly after the spill and decreased 6-7 months later close to background levels for the region. One year after the accident, the median values were: 58 microg/kg for razor shells, 26 microg/kg for barnacles, and 25 microg/kg for sea urchins. The temporal evolution of the PAH concentrations along the survey period was used to estimate loss rates for bioavailable PAHs in barnacles and sea urchins after the spill. Half-life values were in the order of 30 and 60 d, respectively. The results of the study demonstrate that barnacles can be suitable species for oil spill monitoring.

Purification of novel kinesins from embryonic systems.

Several kinesin holoenzymes, including the heterotrimeric kinesin-II and bipolar KLP61F complexes described here, are being purified in our laboratory using microtubule affinity precipitation and conventional biochemical fractionation procedures. These protocols have been optimized by using pan-kinesin peptide antibodies and subunit-specific antibodies to monitor the enrichment of kinesin-related polypeptides in particular fractions by immunoblotting. Protein purification represents the most direct route available for determining the oligomeric state and subunit composition of a kinesin holoenzyme, for identifying tightly associated accessory subunits such as SpKAP115, and for determining the molecular architecture and functional properties of native kinesin motors. Protein purification methods therefore represent an important complementary approach to molecular genetic approaches that are being pursued in many other laboratories.

WEE1-like CDK tyrosine kinase mRNA level is regulated temporally and spatially in sea urchin embryos.

A cDNA from the sea urchin Strongylocentrotus purpuratus encodes a 624 amino acid polypeptide (WEE1S.purp) with a high degree of similarity to the Mik1 and Wee1 protein tyrosine kinases. These kinases act as negative regulators of mitosis by inactivating cyclin-dependent kinases (CDK). Wee1 activity varies during the cell-cycle, and is generated only when required. The pattern of WEE1S.purp mRNA expression was examined temporally and spatially in sea urchin embryos. Only a trace amount of WEE1S.purp mRNA is present in the egg and through the fifth cell cycle post-fertilization. During the next three cycles to the mid-blastula stage, its concentration rises transiently to 2.5 x 10(4) transcripts per embryo. Its developmental profile during this early period is the inverse of that reported for cyclin mRNAs, which are at a high level in the egg and through the fifth cell cycle, then decline upon further development. WEE1S.purp mRNA in the gastrula and pluteus stages becomes restricted to cells engaged in DNA replication, including the endoderm (gut), oral ectoderm, and arm rudiments. It is absent from the aboral ectoderm, which lacks cycling cells. In the pluteus larva of the species Lytechinus pictus, WEE1 mRNA was detected in the arm rudiments during cellular proliferation and arm elongation, but not after the completion of the arms. Putative regulatory motifs in the sea urchin Wee1-like cDNA suggest a capacity for rapid turnover of both its mRNA and protein: The WEE1S.purp mRNA 3' UTR contains 13 AUUUA pentamers, which have been characterized as determinants of mRNA lability; and the N-terminal domain of the predicted WEE1S.purp polypeptide is enriched in S/TP-containing, potential kinase-target sites, as well as high-value "PEST' sequences, associated with protein lability. The developmental appearance of WEE1S.purp mRNA may coincide with the introduction of a gap phase in the cell cycle. Its spatial pattern during embryogenesis appears to reflect distinct programs of regulated cell cycling in differentiating tissues.