Sulfated polysaccharides with antioxidant and anticoagulant activity from the sea cucumber Holothuria fuscogliva

Sea cucumber is a traditional nutritional food and medicinal resource with many bioactive components in China.Holothuria fuscogliva is a big sea cucumber with a rich of bioactive polysaccharides.To investigate the bioactivities of the polysaccharides from sea cucumber H.fuscogliva,we prepared the sulfated polysaccharides(HfP) from sea cucumber H.fuscogliva using a protease hydrolysis method.Antioxidant activities of HfP were investigated,including hydroxyl radical scavenging activity and superoxide radical scavenging activity.And,the anticoagulant activities of HfP were studied,including the activated partial thromboplastin time(APTT),prothrombin time(PT) and thrombin time(TT).The average molecular weight was 1 867.1 Da,with a sulfate content of 20.7%.In addition,the molar ratio of monosaccharide composition of HfP was Man:Rha:Glc A:Glc:Gal:Xyl:Fuc=0.083 6:0.437:0.134:0:1.182:0.748:1.It had a strong antioxidant activity,the hydroxyl and superoxide radical scavenging activity EC_(50) of HfP was 3.74 and 0.037 mg/mL,respectively.It also showed a good anticoagulant activity in our study.The APTT of HfP was much higher than that of heparin sodium,and the PT and TT of HfP was close to that of heparin sodium at a low concentration.Therefore,HfP shows a good antioxidant and anticoagulant activity and it may become a potential candidate of the natural antioxidant and anticoagulant and will have a good application future in health product or medicine industry.     

Chemical characteristics and anticoagulant activities of two sulfated polysaccharides from Enteromorpha linza (Chlorophyta)

Two sulfated polysaccharides, designated MP and SP, were extracted from the marine green alga Enteromorpha linza using hot water and then purified using ion-exchange and size-exclusion chromatography. The anticoagulant activities of MP and SP were examined by determination of their activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using human plasma. Results showed that MP and SP were composed of abundant rhamnose with small amounts of xylose and glucuronic acid, whereas SP also contained a small amount of galactose. Approximate molecular weights of MP and SP were 535 and 502 kDa, respectively. As compared with SP, MP had higher contents of sulfate ester (19.0%) and uronic acid (14.9%). The MP mainly consisted of (1→4)-linked rhamnose residues with partially sulfated groups at the C-3 position, and small amounts of (1→3, 4)-linked rhamnose, (1→2, 4)-linked rhamnose, (1→4)-linked glucuronic acid and (1→4)-linked xylose residues. The SP contained abundant (1→4)-linked rhamnose with minor amounts of (1→3)-linked rhamnose, (1→3, 4)-linked rhamnose, (1→2, 4)-linked rhamnose, (1→4)-linked glucuronic acid, (1→4)-linked xylose, and (1→3)-linked galactose residues. The sulfate groups were mainly located at C-3 of (1→4)-linked rhamnose residues. Both MP and SP, in particular the former, effectively prolonged APTT and TT. This work demonstrates that MP and SP have unique structural characteristics distinct from those of other sulfated polysaccharides from Enteromorpha. The MP is a potential source of anticoagulant, and the difference in anticoagulant activities of the two sulfated polysaccharides is directly linked to the discrepancy of their chemical features.     

Preparation of low molecular weight Sargassum fusiforme polysaccharide and its anticoagulant activity

Heparin has been used as an anticoagulant drug for many years, but it has significant side ef fects. In the search for good substitutes, low molecular weight(MW) polysaccharides from S argassum fusiforme have been examined and confi rmed to possess biological activities. Here, S. fusiforme polysaccharides(SFP) were extracted and subjected to a hydrogen peroxide(H_2O_2) oxidation method for the preparation of low-MW SFP(LSFP). The effects of temperature, pH, and H_2O_2 concentration on the degradation process were also examined. Several LSFP of 36, 9, 5.7, and 2.7 kDa were obtained under different conditions, and their anticoagulant activities studied in vitro. The results showed that SFP and LSFP prolonged activated partial thromboplastin(APTT), prothrombin(PT) and thrombin times(TT) significantly, indicating that these low MW polysaccharides possessed anticoagulant activity in the intrinsic, extrinsic, and common coagulation pathways. As these ef fects were related to the MW of the polysaccharides in APTT and TT but not in PT, the contents of the monosaccharide fucose and sulfate and the polysaccharide MW could have exerted combined ef fects. The details of this mechanism require further verifi cation.     

Study on antithrombotic and antiplatelet activities of low molecular weight fucoidan from Laminaria japonica

The antithrombotic and antiplatelet effects of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica were compared in order to examine the influence of chemical character on their antithrombotic activity and the possible mechanism. Both LMW fucoidan fractions exhibited favorable antithrombotic activity in an Fecl3-induced arterial thrombosis. The antithrombotic activity of LMW fucoidan was related with decrease of TXB2 and whole blood viscosity and hematocrit. LMW fucoidan showed a correlation between anticoagulant, antiaggregant and antithrombotic effects in vivo. For LMW fucoidan, antithrombotic activity required high dose of 5-10 nmol kg-1, concomitantly with increase in anticoagulant activity and inhibition of platelet aggregation. Administration of LMW fucoidan significantly promoted the 6-keto-PGF1α content and decreased the TXB2 content, indicating its inhibition of tissue factor pathway and regulation of metabolism of arachidonic acid. By comparison, highly sulfated fucoidan LF2 with Mw 3900 seemed to be a more suitable choice for antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.     

Study on extraction of agaropectin from Gelidium amansii and its anticoagulant activity

Gelidium amansii agar was fractionated on DEAE-cellulose and four fractions were obtained sequentially. The yields of 1.0 mol/L NaCl fraction and 2.5 mol/L NaCl fraction were 2.80% and 2.03%. They are highly sulfated agar, and named as agaropectin with sulfate content being 22.8% and 32.5%, respectively. The anticoagulant experiment results show that agaropectin could effectively prolong the coagulation time in a dose-dependent manner in vitro. Agaropection could be absorbed and effectively prolong the plasma coagulation time in vivo. After intragastric administration at the doses of 100, 200, and 400 mg/kg-d in rats for 15 days, TT （thrombin time）, CT （coagulation time）, PT （prothrombin time）, and APTT （activated partial thromboplastin time） could be effectively prolonged and the plasma Fib level could be significantly lowered.     

Isolation and characterization of a fucoidan-degrading bacterium from Laminaria japonica

Fucoidan, a polysaccharide containing abundant fucose and sulfate ester group, was prepared from Laminaria japonica. In order to obtain fucoidan-degrading enzyme, bacteria capable of degrading fucoidan were screened from kelp. A bacterial strain named RC2-3 was obtained, which degraded fucoidan by the maximum extent of 54% ± 1.3%, the highest among all bacterial isolates. High-performance size exclusion chromatography(HPSEC) showed that the molecular weight of fucoidan was gradually reduced by RC2-3 with culturing time, suggesting the production of fucoidan-degrading enzyme by RC2-3. Phylogenetic analysis of partial 16S ribosomal RNA gene(16S rDNA) sequence showed that RC2-3 belonged to the family Flavobacteriaceae. However, it showed different physiological and biochemical characteristics from the known Flavobacteriaceae members producing fucoidan-degrading enzyme, thus RC2-3 was proposed to be a new member of this family.     

Polyguluronate Sulfate,Polymannuronate Sulfate,and Their Oligosaccharides Have Antithrombin Ⅲ-and Heparin Cofactor Ⅱ-independent Anticoagulant Activity

Cardiovascular disease is the leading causes of death.However,the complications can be treated with heparin and heparinoids,such as heparin pentasaccharide Fondaparinux,dermatan sulfate,and PSS made from alginate extracted from brown seaweeds by chemical sulfation.Alginate is composed of a linear backbone of polymannuronate(PM),polyguluronate(PG),and alternate residues of mannuronic acid and guluronic acid.It is unknown if heparin and sulfated PG(PGS)/PM(PMS) have the same or different anticoagulant molecular targets.In the current study,the anticoagulant activities of PGS,PMS,and their oligosaccharides were directly compared to that of heparin,Fondaparinux,and dermatan sulfate by the activated partial thrombinplastin time(aP TT) assay using normal,antithrombin III(ATIII)-deficient,heparin co-factor II(HCII)-deficient,and ATIII-and HCII-double deficient human plasmas.Our results showed that PGS,PMS,and their oligosaccharides had better anticoagulant activity than that of Fondaparinux in all four human plasmas tested.As expected,heparin was the best anticoagulant in normal plasma.Moreover,PGS,PGS6,PGS12,PGS25,PMS6,PMS12,and PMS25 were better anticoagulants than dermatan sulfate in HCII-deficient plasma.Most strikingly,PGS,PGS12,PGS25,PMS6,PMS12,and PMS25 were better anticoagulants than that of heparin in ATIII-and HCII-double deficient human plasma.The results revealed for the first time that sulfated alginate had ATIII-and HCII-independent anticoagulant activities.Therefore,developing PGS and PMS-based anticoagulants might require to discover their major molecular targets and to develop target-specific anticoagulant assays.     

Characterization of Polysaccharides Extracted from a Cultivated Brown Alga Costaria costata During the Harvest Period

In this study,variations in composition and properties of polysaccharides isolated from brown algae Costaria costata were analyzed.The algae were collected from May to July of the harvest period.The carbohydrates alginate and fucoidan were extracted with selected solvents.1 H Nuclear magnetic resonance and rheology were used to investigate the monomer composition and rheological characteristics of alginate.Gas chromatography and Fourier transform-infrared spectroscopy were employed to investigate the compositional properties of purified fucoidan.The results indicated that the composition and properties of alginate and fucoidan varied during the life of this alga.The alginate from the alga harvested in May and June had a higher molecular weight,viscosity,and proportion of mannuronic acid,whereas that harvested in July had a lower molecular weight and viscosity but a higher proportion of guluronic acid.The alginate from C.costata had a higher molecular weight and a different mannuronic acid:guluronic acid ratios compared with other algae;thus,it could be used in the chemical,food,cosmetics,and pharmaceutical industries.Fucoidan content reached the maximum in June.Substantial changes in the molecular weight distribution,monosaccharide composition,and sulfate content occurred simultaneously.The fraction of fucose in the polysaccharides decreased significantly from June to July,whereas that of mannose increased.This alga can be harvested during different growth periods to obtain fucoidans and alginates with different compositions and,therefore,with different biological properties.     

The Hydrolyzation of Collagen by Fucoidan Olisosaccharide's Complex with Ce~(IV)

Fucoidan is such a polysaccharide that its hydroxies are easy to combine with lanthanons ion (CeIV) to form complex. This work obtained the complexes of three fucoidan oligosaccharides with different molecular weights F1(>5000), F2 (1000-5000) and F3(<1000) by hydrolyzing Oligosaccharide collagen with sulfuric acid. It is found that the fucoidan oligosaccharide F3 can form complex with more CeIV than F1 and F2. Hydrolyzing collagen with the complex was carried out to produce amino acid and peptides. All the three fucoidan oligosaccharide complexes with CeIV( F1, F2, F3) can catalyze by the artificial hydrolytic enzyme, and the activity of the complex of F3 is the highest.     

The Hydrolyzation of Collagen by Fucoidan Oligosaccharide''s Complex with CeⅣ

Fucoidan is such a polysaccharide that its hydroxies are easy to combine with lanthanons ion (CeⅣ) to form complex. This work obtained the complexes of three fucoidan oligosaccharides with different molecular weights F1( ＞5 000), F2(1 000-5 000) and F3( ＜ 1 000) by hydrolyzing Oligosaccharide collagen with sulfuric acid. It is found that the fucoidan oligosaccharide F3 can form complex with more CeⅣ than F1 and F2. Hydrolyzing collagen with the complex was carried out to produce amino acid and peptides. All the three fucoidan oligosaccharide complexes with CeⅣ( F1, F2, F3) can catalyze by the artificial hydrolytic enzyme, and the activity of the complex of F3 is the highest.     

Structural analysis and anti-complement activity of polysaccharides extracted from Grateloupia livida (Harv.) Yamada

Polysaccharides were extracted from Grateloupia livida(Harv.) Yamada using hot water(extracted product denoted WGW) and then degraded in dilute sulfuric acid(degraded product denoted WGWD). The degraded mixture was then separated into four fractions through anion exchange chromatography on 2-diethylaminoethanol(DEAE)-Bio-Gel Agarose FF gel. Electrospray ionization collision-induced dissociation tandem mass spectrometry(ESI-CID-MS/MS) was performed to elucidate the structural features of all fractions. In combination with nuclear magnetic resonance spectroscopy(NMR)and infrared spectroscopy(IR) data, the major polysaccharide structures were concluded to be μ-carrageenan and κ-carrageenan. μ-Carrageenan usually has a backbone of alternating 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-galactopyranose residues sulfated at C-6, while κ-carrageenan consists of alternating 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-3,6-anhydrogalactopyranose residues. Trace v-carrageenan, composed of 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-galactopyranose residues sulfated at C-2 and C-6, was also detected. Furthermore, the polysaccharide had a backbone comprising 1,3-linked β-D-galactopyranose and1,4-linked α-L-galactopyranose sulfated at C-6, which is the agarose precursor. The hydroxy groups in the galactopyranose were partially substituted by methyl and pyruvic acid acetal(PA) groups. The anticomplementary activities of WGW and its derivatives against classical pathways were measured. The native polysaccharides in WGW had higher activities, while the derivatives had much weaker activities. This indicated that the molecular weight and sulfate content were important factors affecting the anti-complement activity.     

The Hydrolyzation of Collagen by Fucoidan Oligosaccharide＇s Complex with CeⅣ

Fucoidan is such a polysaccharide that its hydroxies are easy to combine with lanthanons ion （Ce^IV） to form complex. This work obtained the complexes of three fucoidan oligosaccharides with different molecular weights F1 （〉5 000）, F2 （1 000-5 000） and F3 （〈 1 000） by hydrolyzing Oligosaccharide collagen with sulfuric acid. It is found that the fucoidan oligosaccharide F3 can form complex with more Ce^IV than F1 and F2. Hydrolyzing collagen with the complex was carried out to produce amino acid and peptides. All the three fucoidan oligosaccharide complexes with CeIV（ F1, F2, F3） can catalyze by the artificial hydrolytic enzyme, and the activity of the complex of F3 is the highest.     

Mechanism Study of Endothelial Protection and Inhibits Platelet Activation of Low Molecular Weight Fucoidan from Laminaria japonica

Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline(0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. v WF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce v WF level in vascular endothelial injury rats and also significantly reduce v WF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and v WF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.     

Studies on Antiviral and Immuno-Regulation Activity of Low Molecular Weight Fucoidan from Laminaria japonica

The antiviral activity in vitro and in vivo and the effect of the immune system of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica(LMW fucoidans) were investigated in order to examine the possible mechanism. In vitro, I-type influenza virus, adenovirus and Parainfluenza virus I were used to infect Hep-2, Hela and MDCK cells, respectively. And 50% tissue culture infective dose was calculated to detect the antiviral activity of two LMW fucoidans. The results indicated that compared with the control group, 2 kinds of LMW fucoidans had remarkable antiviral activity in vitro in middle and high doses, while at low doses, the antiviral activity of 2 kinds of LMW fucoidans was not statistically different from that in the blank control group. And there was no statistically difference between two LMW fucoidans in antiviral activity. In vivo, LMW fucoidans could prolong the survival time of virus-infected mice, and could improve the lung index of virus-infected mice significantly, which have statistical differences with the control group significantly(p 0.01). However, the survival time of the two LMW fucoidans was not statistically significant(p 0.05). In this study, it was shown that both of two LMW fucoidans(LF1, LF2) could increase the thymus index, spleen index, phagocytic index, phagocytosis coefficient and half hemolysin value in middle and high doses, which suggested that LMW fucoidans could play an antiviral role by improving the quality of immune organs, improving immune cell phagocytosis and humoral immunity.     

Enzyme-like activities of algal polysaccharide - cerium complexes

Water-soluble algal polysaccharides (APS) (alginic acid, fucoidan and laminaran) possess many pharmacological activities. The results of this study showed that the APS-Ce^4 complexes have some enzyme-like activities. Fucoidan and its complex with Ce^4 have activities similar to those of SOD. The activities of laminaran, alginic acid and their complexes are not measurable. The APS do not show measurable activities in the digestion of plasmid DNA. In contrast, the APS - Ce^4 complexes show these measurable activities under the comparable condition when APS bind Ce^4 and form homogenous solutions. The laminaran-Ce^4 complex shows the most obvious activity in the digestion of plasmid DNA, pNPP and chloropyrifos under neutral conditions.     

Isolation and characterization of fucoidans from five brown algae and evaluation of their antioxidant activity

In this study, we evaluated the chemical property and antioxidant activity of fucoidans isolated from brown algae, Laminaria japonica(LJF), Lessonia nigrescens(LNF), Lessonia trabeculata(LTF), Ascophyllum mackaii(AMF), and Ecklonia maxima(EMF). LJF was less in sulfate content(14.16%) and more in galactose and mannose content(1.08 and 0.68) than the documented early. EMF contained 20%–30% of sulfate and fucose, 0.97 in molar ratio which was lower than that of sulfate to other four fucoidans(1.21–1.41). AMF(162 kDa) and EMF(150 kDa) were the first two largest in molecular weight, which were followed by LJP(126 kDa), LNF(113 kDa) and LTF(105 kDa). The fucoidans isolated these algae showed a wide range of antioxidant activity in vitro. It was found that the reducing power of the isolated fucoidans was positively correlated with their sulfate content and molecular weight. In addition, LNF and LTF at low concentrations exhibited high superoxide and hydroxyl radical scavenging activity. This demonstrated that low molecular weight fucoidans may perform a high antioxidant activity.     

Extraction,isolation and structural characterization of polysaccharides from a red alga <Emphasis Type="Italic">Gloiopeltis furcata</Emphasis>

Three kinds of polysaccharides:GFW,GFH and GFA,were sequentially extracted from a red alga Gloiopeltis furcatawith 25℃and 85℃water,and 60℃ 4%NaOHwater solution.Based on the defatted alga,the yields of the polysaccharide were57.9%,2.5%and 2.6%,respectively.Their monosaccharide compositions,average molecular weights and structural characters weredetermined by gas chromatography(GC),high performance liquid chromatography(HPLC),fourier transform infrared spectroscopy(FTIR)or13C-NMR spectroscopy.The results show...     

Purification and Characterization of An Alkaline Protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates （azocasein and casein）. The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55℃ and the optimal pH value was 5.5. Ion Ca^2＋ could enhance the proteolytic activity of the protease, while Cu^2＋, EDTA and PMSF could inhibit its activity.     

Purification and characterization of an alkaline protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca²⁺ could enhance the proteolytic activity of the protease, while Cu²⁺, EDTA and PMSF could inhibit its activity.     

Analysis of the monosaccharide composition of fucoidan by precolumn derivation HPLC

We developed an HPLC method for analysis of the monosaccharide composition of fucoidans.The fucoidan was hydrolyzed into monosaccharides with 2 mol/L trifluoroacetic acid.Using ribose as the internal standard,the monosaccharide derivatives,obtained with 1-Phenyl-3-methyl-5-pyrazolone(PMP),were separated by reverse-phase HPLC using a gradient elution process,and monitored by ultraviolet detection at 245 nm.In the concentration range of 0.1-2.0 mmol/L,the peak area of each monosaccharide had a good linear rel...