Sulfated Fucoidan Research Articles

ASEXUAL GROWTH OF BABESIA BOVIS IS INHIBITED BY SPECIFIC SULFATED GLYCOCONJUGATES

In the present study, inhibitory effects of several sulfated and nonsulfated glycoconjugates were evaluated on the in vitro asexual growth of Babesia bovis. Among the selected sulfated glycoconjugates, dextran sulfate, heparin, heparan sulfate, fucoidan, and chondroitin sulfate B strongly inhibited the parasitic growth, and all but chondroitin sulfate B induced a significant accumulation of extracellular merozoites in culture. In contrast, chondroitin sulfate A, keratan sulfate, and protamine sulfate, as well as nonsulfated dextran and hyaluronic acid, did not influence the growth. These findings indicate that the asexual growth of B. bovis merozoites is inhibited by specific sulfated glycoconjugates, possibly providing us with an important insight into the molecular interaction(or interactions) during the process of the erythrocyte invasion by B. bovis merozoites.

ANALYSES OF EXPRESSED SEQUENCE TAGS FROM SARGASSUM BINDERI (PHAEOPHYTA)1

Sargassum is a source for many metabolites, including alginic acid, alginates, sulfated fucoidans, pigments, oils, sterols, and mannitols. The present study is the first attempt to examine Sargassum binderi (Sonder) J. Agardh, a tropical brown seaweed, using an expressed sequence tag (EST) approach. Single‐pass 5′ sequencing of 2304 cDNA clones generated 1876 ESTs of good quality, from which 1270 tentative unique genes (TUGs) consisting of 991 singletons and 279 contigs were obtained. Approximately 39.5% of the TUGs from S. binderi showed significant matches to the existing data bases and were further divided into nine subcategories: metabolism (24.9%); transcription and translation (22.5%); cellular processes (7.2%); protein folding, sorting, and degradation (5.6%); signaling (2.8%); membrane and transport (3%); receptor (1.1%); other functions (4.2%); and unknown proteins (28.7%). The sequences were compared with ESTs from Laminaria digitata (Hudson) J. V. Lamour.

Effect of Monosaccharide L-fucose and Polysaccharide Fucoidan on Sperm ??L-fucosidase Activity and Relation to Sperm-oocyte Interaction in Pig

Carbohydrate-protein interactions are known to be important in gamete interactions. Several evidence indicated that a fucose-containing sulfated polysaccharide fucoidan was potential inhibitor of fertilization in vitro and thus fucose seemed to be part of the recognition signal of gamete interaction in mammals. In recent investigation we found that α-L-fucosidase activity was present in boar spermatozoa and it was related to sperm binding to and penetration into zona pellucida (ZP) in vitro. The objective of this study was to determine the effects of monosaccharide L-fucose and polysaccharide fucoidan on sperm α-L-fucosidase activity and relation to sperm-oocyte interaction in pig. Results indicated that the activity of sperm α-L-fucosidase was largely inhibited (62%) when sperm suspension was treated with monosaccharide L-fucose. It also significantly inhibited the number of sperm binding to ZP (32%) and penetration into zona-intact oocytes (72%), but did not inhibit penetration into zona-free oocytes when fertilization medium contained L-fucose. The chlorotetracycline (CTC) assessment showed that L-fucose did not affect induction of sperm capacitation and acrosome reaction. In contrast, the activity of sperm α-L-fucosidase was not inhibited when sperm suspension was treated with polysaccharide fucoidan but sperm-ZP binding was greatly inhibited (85%) and completely blocked sperm penetration into zona-intact or zona-free oocytes. The CTC assessment showed that fucoidan increased the F pattern and decreased the AR pattern sperm. These results suggested that the different inhibitory mechanisms were present between monosaccharide L-fucose and polysaccharide fucoidan on sperm-oocyte interaction, the inhibition effect of α-L-fucose on sperm binding and penetrating into ZP caused sperm α-L-fucosidase inhibited by α-L-fucose.

Effect of native fucoidan, sulfated fucoidan, heparin and 6-aminohexanoic acid on the activation of glutamic-plasminogen by urokinase: role of NaCl

Addition of a physiological concentration of NaCl (0.9%) to 0.05 mol/l Tris buffer (pH 7.4) reversed the enhancement of the activation of glutamic-type plasminogen (Glu-Plg) by low molecular weight urokinase by fucoidan and heparin, while addition of aminohexanoic acid (6-AH) enhanced the activation. Native fucoidan (N-2), sulfated fucoidan (S-2) and heparin alone and in the presence of 6-AH were investigated to determine the effect of NaCl addition to 0.05 mol/l Tris (pH 7.4) on the activation of Glu-plg by high molecular weight urokinase (HMW u-Pa). Heparin alone and in conjunction with 6-AH enhanced 4.5 to 5.5-fold the initial rate of activation of Glu-plg, while N-2 alone or in conjunction with 6-AH gave 3 to 5.5-fold enhancement and S-2 gave no enhancement of activation using 0.05 mol/l Tris buffer (pH 7.4). Addition of 0.9% NaCl to the buffer reversed the enhancement by the cofactors but, in the presence of 6-AH, N-2 gave two-fold and S-2 gave three-fold enhancement of activation while heparin gave 25% enhancement. The mechanism of enhancement by S-2 in the presence of 6-AH was investigated by dilution and enzyme kinetic studies. The results show that the enhancement was due to interaction with HMW u-PA and not with Glu-plg.

Effect of oversulfation on the chemical and biological properties of fucoidan

Fucoidan was sulfated using chlorosulfonic acid–pyridine complex and isolated as the sodium salt. Infrared analysis of the native and sulfated fucoidans gave identical results in respect to the O–H stretching, hemiacetal stretching and S O stretching. Absorption around 840 cm −1 was also present in both the fucoidans representing the sulfate at the axial C-4 position except for a shoulder at 820 cm −1, which was present only in the sulfated compound indicating the presence of sulfate groups at the equatorial C-2 position. The sulfated compound showed four times higher anticoagulant activity in doubling prothrombin time of normal citrated human plasma in comparison with native fucoidan. Earlier studies using native fucoidan showed that the activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) or by urokinase (u-PA) was enhanced when the in vitro studies were conducted using 0.05 M Tris buffer (pH 7.4) and that the addition of physiological concentration of NaCl (0.9%) to the buffer reversed the activation. The results of the current studies showed that sulfated fucoidan gave higher stimulations of Glu-Plg activation by u-PA and by t-PA in comparison to native fucoidan when the in vitro studies are conducted using Tris buffer containing 0.9% NaCl.

Mechanism of the stimulatory effect of native fucoidan, highly sulfated fucoidan and heparin on plasminogen activation by tissue plasminogen activator: The role of chloride

Native Fucoidan and unfractionated heparin enhanced by 6-fold the in vitro activation of human glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) using 0.05M Tris buffer pH 7.4, while sulfated fucoidan inhibited the activation under these conditions. Double reciprocal plots of these interactions showed that sulfated fucoidan inhibited the activation in a noncompetitive manner while the enhancements by heparin or native fucoidan were due to an increase of Vmax without affecting Km. To determine whether the stimulatory effect of the individual cofactor was due to its interaction with Glu-Plg or with t-PA, experiments were performed at a fixed level of the cofactor and either varying in a serial fashion the concentration of Glu-Plg or of t-PA. The ratios of the initial rate of plasmin generation in the presence or absence of the cofactors were plotted against the inverse of the volume fraction of Glu-Plg or of t-PA. The results showed that heparin interacted with Glu-Plg while native fucoidan and sulfated fucoidan interacted with t-PA. Studies were also conducted on the effect of the two fucoidans and heparin on the activation of Glu-Plg by t-PA using 0.05M Tris buffer pH 7.4 containing 0.1 M NaCl. Under these conditions, sulfated fucoidan was most effective in enhancing the activation followed by native fucoidan and heparin respectively. The results of this study showed that in presence of the buffer containing 0.1 M NaCl, heparin was interacting with t-PA while the two fucoidans were interacting with both t-PA and Glu-Plg. A comparison of the double reciprocal plots of the rate of enhancement by the cofactors using 0.05M Tris buffer pH 7.4 containing 0.1M NaCl or in presence of buffer alone showed that the cofactors were more effective using 0.05M Tris buffer pH 7.4 alone and that addition of NaCl to the buffer slowed down the reactions by decreasing Vmax without affecting Km. Circular Dichroism (CD) studies of Glu-Plg in the far ultraviolet (UV) range showed that addition of NaCl destabilized the beta sheet structure which was reversed by addition of 6-aminohexanoic acid (6-AH) or one of the cofactors, while the near UV CD spectra of Glu-Plg in presence of 0.1 M NaCl was enhanced by the cofactors by increasing its ellipticity as reported earlier for 6-AH.

한국산 길조류에서 추출한 Fucoidan의 황산기에 따른 항암작용

Fucoidan은 갈조류에 다량 함유되어있는 다당류로서 항암작용, 항혈응고작용, 항혈전작용, 항염증작용, 항virus작용과 같은 생리활성작용력을 가지고 있어 최근 많은 연구가 수행되어지고 있다. 본 논문은 갈조류에서 fucoidan을 추출 하여 그 항암활성을 SV40 DNA replication assay, RPA-ssDNA binding assay, 그리고 MCF7 cell growth inhibition assay를 이용하여 알아보았으며 이 항암활성은 fucoidan의 황산기가 중요한 역할을 하고 있음을 알아내었다. 화학적으로 황산기의 함유량을 증가시킨 fucoidan이 황산기의 함유량을 제거시킨 fucoidan보다 항암활성이 뛰어났고, 이는 RPA의 ssDNA 결함력을 떨어 EM리기 때문이라 예측되어 진다. 본 연구는 한국산 갈조류에서 추출한 황산기를 함유한 fucoidan의 항암활성능력을 보여주었다. Fucoidan is a kind of polysaccharides in brown seaweeds. For the past years have been extensively studied due to their numerous biological activities : anticancer, anticoagulant, antithrombotic, anti-inflammatory and antiviral. In this study, we h ave extracted fucoidan from the Korean brown seaweeds and examined it's anticancer activities for employed SV40 DNA replication assay, RPA-ssDNA binding assay of replication protein A(RPA: known as human single-stranded DNA-binding protein essential for DNA rep-lication) and MCF7 cell growth inhibition assay. In addition to, we found that chemically sulfated fucoidan'santicancer activity is more higher than natural and desulfated fucoidan. It seem that fucoidan's sulfate group affect on DNA replication, cause of decrease RPA's DNA binding activity. These results suggests that sulfated fucoidan from Korean brown seaweeds have anticancer activity.

Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

Effect of fucoidan treatment on collagenase-induced intracerebral hemorrhage in rats

Inflammatory cells are postulated to mediate some of the brain damage following ischemic stroke. Intracerebral hemorrhage is associated with more inflammation than Ischemic stroke. We tested the sulfated polysaccharide fucoidan, which has been reported to reduce inflammatory brain damage, in a rat model of intracerebral hemorrhage induced by injection of bacterial collagenase into the caudate nucleus. Rats were treated with seven day intravenous infusion of fucoidan (30ugh''1) or vehicle. The hematoma was assessed in vivo by magnetic resonance imaging. Motor behavior, passive avoidance, and skilled forelimb function were tested repeatedly for six weeks. Fucoidan-treated rats exhibited evidence of impaired blood clotting and hemodilution, had larger hematomas, and tended to have less inflammation in the vicinity of the hematoma after three days. They showed significantly more rapid improvement of motor function in the first week following hemorrhage and better memory retention in the passive avoidance test. Acute white matter edema and eventual neuronal loss in the striatum adjacent to the hematoma did not differ between the two groups. Investigation of more specific anti-inflammatory agents and hemodiluting agents are warranted in intracerebral hemorrhage. [Neurol Res 1999; 21: 415-419]

ANTICOAGULANT FUCOIDAN FRACTIONS FROM FUCUS VESICULOSUS INDUCE PLATELET ACTIVATION IN VITRO

Anticoagulant fucoidan fractions of different molecular weight and sulfate content were prepared and investigated for their effects on platelet function in vitro. The fucoidan fractions were incubated with human platelet rich plasma (PRP) at concentrations of 5, 10 and 50 μg/ml. Platelet activation was subsequently studied by a standard aggregation assay and flow cytometric determination of the activation dependent platelet-surface markers CD62p (P-selectin, GMP-140) and CD63 (GP53). All fucoidan fractions induced irreversible platelet aggregation in a dose-dependent manner. Comparing fractions of identical molecular weight (100 kDa) the low sulfate content fucoidan FF5 (S=7,6%) exerted a significantly greater effect than the highly sulfated fucoidan FF7 (S= 10,2%) over the whole concentration range (n=5, P<0,05). Among fractions of identical sulfate content fucoidan-induced platelet aggregation was also found to depend on the molecular weight of the fucoidan. At concentrations of 10 and 50 μg/ml the high molecular weight fraction FF7/1 (150 kDa) showed a significantly greater effect than the 50 kDa fraction FF7/3 (24,8 ± 6,7 vs. 7,0 ± 3,5 and 54,6 ± 13,5 vs. 15,0 ± 9,0%, respectively; mean ± SD, n=5, P<0,05). The molecular weight dependence of the fucoidan effect was also reflected by the flow cytometric data. Coincubation of FF7/1 and FF7/3 (10μg/ml) with PRP increased the number of CD62p and CD63 positive platelets by 9,0 ± 3,3 vs. 2 ± 1,9 and 7,1 ± 2,4 vs. 3,2 ± 2,6% over control values, respectively (n=5, P< 0,05). In conclusion, our results show that the low molecular weight fucoidan FF7/3 combines potent anticoagulant and fibrinolytic properties with only minor platelet activating effects and is therefore a suitable substance for further pharmacological studies. © 1997 Elsevier Science Ltd

Inhibition of lactoferrin and vitronectin binding toStaphylococcus aureus by heparin

Cells ofStaphylococcus aureus strain ISP 546, previously shown to express high binding of vitronectin (Vn), were shown also to bind radioactive iodine-labeled human lactoferrin (Lf). The binding was specific, and heat treatment ofS. aureus cells abolished Lf binding. When cells were preincubated with heparin (2.5 μg/ml), binding of Vn and Lf was inhibited by more than 90%. Two other sulfated carbohydrate polymers, fucoidan and dextran sulfate (mol weights 5000 and 8000) inhibited binding of both iodine-labeled Vn and Lf by about 75% each. No synergistic inhibitory effect between heparin and fuocidan on Lf binding byS. aureus was detected. Mannose, mannoseamine and poly-α-sialic acid (colominic acid) inhibited Vn binding by about 40% but had no effect on Lf binding. A cell surface extract of ISP 546 inhibited Vn binding but did not affect Lf binding. Lf binding toS. aureus was inhibited by preincubation of the cells with Vn, and Vn binding toS. aureus was inhibited by preincubation with Lf. Our data indicate that Vn and Lf bind to different molecules on theS. aureus cell surface, involving heparin and other carbohydrate interactions.

Fibrinolytic and anticoagulant activities of highly sulfated fucoidan

A series of fucoidan [sulfated poly( l-fucopyranose)] derivatives were prepared by chemical sulfation and desulfation, and they were tested for their abilities to stimulate tissue plasminogen activator (t-PA)-catalyzed plasminogen activation, clot lysis, and the inhibition of fibrin polymer formation. The magnitude of their activities was dependent upon the degree of sulfation. A striking feature of the sulfated fucoidan was that, unlike heparin, it stimulated t-PA-induced plasma clot lysis by protecting plasmin activity from α 2-plasmin inhibitor and decreased the rate of fibrin polymer formation. The inhibition of hyaluronic acid-mediated enhancement of fibrin clot formation was also observed with the fucoidan derivative. We also showed that highly sulfated fucoidan prevents significantly endotoxin-induced hepatic vein thrombosis in the hyperlipemic rat model. The present results are the first to describe the fibrinolytic and anticoagulant activities of fucoidan, and thus may provide useful clues for the development of an ideal thrombolytic agent.

Sulfation of fucoidan in Fucus embryos : II. Separation from initiation of polar growth

Sulfation of the brown algal polysaccharide fucoidan at the time and location of rhizoid formation in zygotes of Fucus distichus L. Powell suggested a possible role of fucoidan sulfate in the initiation of polarity. However, when sulfation of fucoidan was prevented by growing zygotes in artificial sea water lacking sulfate but containing methionine (10 mM), normal rhizoids were observed and protein synthesis was unaffected. Hence, establishment of a polar axis is not dependent upon fucoidan sulfation. The net negative charge introduced on fucoidan by sulfation, however, may still be involved in localizing this polysaccharide into the rhizoid cell by a self-electrophoresis mechanism.