Biological activities of sulfated polysaccharides from tropical seaweeds.

Sulfated polysaccharides from 11 species of tropical marine algae (one edible specie of Rhodophyta, six species of Phaeophyta and four species of Chlorophyta) collected from Natal city coast (Northeast of Brazil) were evaluated for their anticoagulant, antioxidant and antiproliverative in vitro activities. In the activated partial thromboplastin time (APTT) test, which evaluates the intrinsic coagulation pathway, seven seaweeds presented anticoagulant activity. Dictyota cervicornis showed the highest activity, prolonging the coagulation time to double the baseline value in the APTT with only 0.01 mg/100 microl of plasma, 1.4-fold lesser than Clexane, a low molecular weight heparin. In the protrombin time (PT) test, which evaluates the extrinsic coagulation pathway, only Caulerpa cupresoides showed anticoagulant activity. All species collected showed antioxidant activities. This screening emphasized the great antioxidant potential (total capacity antioxidant, power reducing and ferrous chelating) of four species: C. sertularioide; Dictyota cervicornis; Sargassum filipendula and Dictyopteris delicatula. After 72 h incubation, HeLa cell proliferation was inhibited (p<0.05) between 33.0 and 67.5% by S. filipendula; 31.4 and 65.7% by D. delicatula; 36.3 and 58.4% by Caulerpa prolifera and 40.2 and 61.0% by Dictyota menstrualis at 0.01-2mg/mL algal polysaccharides. The antiproliferative efficacy of these algal polysaccharides were positively correlated with the sulfate content (r=0.934). Several polysaccharides demonstrated promising antioxidant, antiproliferative an/or anticoagulant potential and have been selected for further studies on bioguided fractionation, isolation and characterization of pure polysaccharides from these species as well as in vivo experiments are needed and are already in progress.

Sulfated galactofucan from Lobophora variegata: anticoagulant and anti-inflammatory properties.

Sulfated polysaccharides (fucans and fucoidans) from brown algae show several biological activities, including anticoagulant and anti-inflammatory activities. We have extracted a sulfated heterofucan from the brown seaweed Lobophora variegata by proteolytic digestion, followed by acetone fractionation, molecular sieving, and ion-exchange chromatography. Chemical analyses and 13C-NMR and IR spectroscopy showed that this fucoidan is composed of fucose, galactose, and sulfate at molar ratios of 1 : 3 : 2. We compared the anticoagulant activity of L. variegata fucoidan with those of a commercial sulfated polysaccharide (also named fucoidan) from Fucus vesiculosus and heparin. The experimental inflammation models utilized in this work revealed that fucoidan from L. variegata inhibits leukocyte migration to the inflammation site. Ear swelling caused by croton oil was also inhibited when sulfated polysaccharides from F. vesiculosus and L. variegata were used. The precise mechanism of different action between homo- and heterofucans is not clear; nevertheless, the polysaccharides studied here may have therapeutic potential in inflammatory disorders.

Inhibition of reverse transcriptase activity of HIV by polysaccharides of brown algae.

Brown algae have two kinds of acid polysaccharides present in the extracellular matrix: sulfated fucan and alginic acid. We have previously isolated and characterized fucans from several species of brown seaweed. The characterized fucans from Dictyotaceae are heterofucans containing mainly fucose, galactose, glucose, xylose, and/or uronic acid. The fucan from Fucus vesiculosus is a homofucan containing only sulfated fucose. We assessed the activity of these fucans as inhibitors of HIV from reverse transcriptase (RT). Using activated DNA and template primers poly(rA)-oligo(dT), we found that fucans at a concentration of 0.5-1.0 microg/mL had a pronounced inhibitory effect in vitro on the avian reverse transcriptase, with the exception of xylogalactofucan isolated from Spatoglossum schröederi, which had no inhibitory activity. The alginic acid (1.0 microg/mL) inhibited the reverse transcriptase activity by 51.1% using activated DNA. The inhibitory effect of fucans was eliminated by their desulfation. Furthermore, only xylofucoglucuronan from S. schröederi lost its activity after carboxyreduction. We suggest that fucan activity is not only dependent on the ionic changes but also on the sugar rings that act to spatially orientate the charges in a configuration that recognizes the enzyme, thus determining the specificity of the binding.

Cytotoxicity effect of algal polysaccharides on HL60 cells.

The present study shows the cytotoxic effect of three different classes of algal polysaccharides on HL60 cells. Three galactofucans, fucoidan, and glucan were the polysaccharides utilized in this analysis. The parameters used for evaluating cell viability were [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) reduction, protein content, and phosphatase activity. We demonstrated stimulation of phosphatase activity, MTT reduction, and protein content in relation to three types of galactofucans (1, 2, and 3) with different molecular weights (1600, 1200, and 360 kD). However, when HL60 cells were treated with galactofucan type 3, the total protein remained unchanged. Under the same experimental conditions, an expressed increase in the phosphatase activity was detected when galactofucan 3 was utilized. In relation to the mitochondrial function, the stimulation was higher in cells treated with galactofucan type 1. Fucoidan did not have a significant effect on MTT reduction, but protein content was decreased (IC50 around 30 microg/ml). Glucan also activated all the parameters that were analyzed, and this effect was more expressed in the phosphatase activity and in the protein content. This study provides new insights into the cytotoxic action of polysaccharides on HL60 cells and suggests for the first time the possible involvement of phosphatases in this process.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora.

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50% acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-beta-D-glucuronic acid 1-> or 4-beta-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or beta-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50 percent acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

Sulfated fucan as support for antibiotic immobilization.

Xylofucoglucuronan from Spatoglossum schröederi algae was tested as a support for antibiotic immobilization. The polysaccharide (20 mg in 6 ml) was first activated using carbodiimide, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide methiodide (20 mg in 2 ml), under stirring for 1 h at 25 masculine C and pH from 4.5 to 5.0. After adjusting the pH to 8.0, either gentamicin or amikacin (62.5 mg in 1.25 ml) was then immobilized on this chemically modified polysaccharide with shaking for 24 h in a cold room. Infrared spectra of the activated carbodiimide xylofucoglucuronan showed two bands to carbonyl (C=O at 1647.9 and 1700.7 cm(-1)) and to amide (C-NH2) groups (1662.8 and 1714.0 cm(-1)). Microbial characterization of the derivatives was carried out by the disk diffusion method using Staphylococcus aureus or Klebsiella pneumoniae incorporated in Müller Hinton medium. Inhibition halos of bacterial growth were observed for the antibiotics immobilized on this sulfated heteropolysaccharide before and after dialysis. However, the halos resulting from the samples after dialysis were much smaller, suggesting that dialysis removed either non-covalently bound antibiotic or other small molecules. In contrast, bacterial growth was not inhibited by either xylofucoglucuronan or its activated form or by gentamicin or amikacin after dialysis. An additional experiment was carried out which demonstrated that the sulfated heteropolysaccharide was hydrolyzed by the microorganism. Therefore, the antibiotic immobilized on xylofucoglucuronan can be proposed as a controlled drug delivery system. Furthermore, this sulfated heteropolysaccharide can be extracted easily from sea algae Spatoglossum schröederi.

Sulfated fucan as support for antibiotic immobilization

Xylofucoglucuronan from Spatoglossum schrõederi algae was tested as a support for antibiotic immobilization. The polysaccharide (20 mg in 6 ml) was first activated using carbodiimide, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide methiodide (20 mg in 2 ml), under stirring for 1 h at 25ºC and pH from 4.5 to 5.0. After adjusting the pH to 8.0, either gentamicin or amikacin (62.5 mg in 1.25 ml) was then immobilized on this chemically modified polysaccharide with shaking for 24 h in a cold room. Infrared spectra of the activated carbodiimide xylofucoglucuronan showed two bands to carbonyl (C = O at 1647.9 and 1700.7 cm-1) and to amide (Cpsi-NH2) groups (1662.8 and 1714.0 cm-1). Microbial characterization of the derivatives was carried out by the disk diffusion method using Staphylococcus aureus or Klebsiella pneumoniae incorporated in Müller Hinton medium. Inhibition halos of bacterial growth were observed for the antibiotics immobilized on this sulfated heteropolysaccharide before and after dialysis. However, the halos resulting from the samples after dialysis were much smaller, suggesting that dialysis removed either non-covalently bound antibiotic or other small molecules. In contrast, bacterial growth was not inhibited by either xylofucoglucuronan or its activated form or by gentamicin or amikacin after dialysis. An additional experiment was carried out which demonstrated that the sulfated heteropolysaccharide was hydrolyzed by the microorganism. Therefore, the antibiotic immobilized on xylofucoglucuronan can be proposed as a controlled drug delivery system. Furthermore, this sulfated heteropolysaccharide can be extracted easily from sea algae Spatoglossum schrõederi.

Heterofucans from Dictyota menstrualis have anticoagulant activity.

Fucan is a term used to denote a family of sulfated L-fucose-rich polysaccharides which are present in the extracellular matrix of brown seaweed and in the egg jelly coat of sea urchins. Plant fucans have several biological activities, including anticoagulant and antithrombotic, related to the structural and chemical composition of polysaccharides. We have extracted sulfated polysaccharides from the brown seaweed Dictyota menstrualis by proteolytic digestion, followed by separation into 5 fractions by sequential acetone precipitation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9.0, stained with 0.1% toluidine blue, showed the presence of sulfated polysaccharides in all fractions. The chemical analyses demonstrated that all fractions are composed mainly of fucose, xylose, galactose, uronic acid, and sulfate. The anticoagulant activity of these heterofucans was determined by activated partial thromboplastin time (APTT) using citrate normal human plasma. Only the fucans F1.0v and F1.5v showed anticoagulant activity. To prolong the coagulation time to double the baseline value in the APTT, the required concentration of fucan F1.0v (20 g/ml) was only 4.88-fold higher than that of the low molecular weight heparin Clexane (4.1 g/ml), whereas 80 g/ml fucan 1.5 was needed to obtain the same effect. For both fucans this effect was abolished by desulfation. These polymers are composed of fucose, xylose, uronic acid, galactose, and sulfate at molar ratios of 1.0:0.8:0.7:0.8:0.4 and 1.0:0.3:0.4:1.5:1.3, respectively. This is the fist report indicating the presence of a heterofucan with higher anticoagulant activity from brown seaweed.

Heterofucans from Dictyota menstrualis have anticoagulant activity

Fucan is a term used to denote a family of sulfated L-fucose-rich polysaccharides which are present in the extracellular matrix of brown seaweed and in the egg jelly coat of sea urchins. Plant fucans have several biological activities, including anticoagulant and antithrombotic, related to the structural and chemical composition of polysaccharides. We have extracted sulfated polysaccharides from the brown seaweed Dictyota menstrualis by proteolytic digestion, followed by separation into 5 fractions by sequential acetone precipitation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9.0, stained with 0.1 percent toluidine blue, showed the presence of sulfated polysaccharides in all fractions. The chemical analyses demonstrated that all fractions are composed mainly of fucose, xylose, galactose, uronic acid, and sulfate. The anticoagulant activity of these heterofucans was determined by activated partial thromboplastin time (APTT) using citrate normal human plasma. Only the fucans F1.0v and F1.5v showed anticoagulant activity. To prolong the coagulation time to double the baseline value in the APTT, the required concentration of fucan F1.0v (20 æg/ml) was only 4.88-fold higher than that of the low molecular weight heparin Clexane® (4.1 æg/ml), whereas 80 æg/ml fucan 1.5 was needed to obtain the same effect. For both fucans this effect was abolished by desulfation. These polymers are composed of fucose, xylose, uronic acid, galactose, and sulfate at molar ratios of 1.0:0.8:0.7:0.8:0.4 and 1.0:0.3:0.4:1.5:1.3, respectively. This is the fist report indicating the presence of a heterofucan with higher anticoagulant activity from brown seaweed.

Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase

We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase

Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase.

We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells.

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5))()and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins.

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5)) and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?

The distribution and structure of heparan sulfate and heparin are briefly reviewed. Heparan sulfate is a ubiquitous compound of animal cells whose structure has been maintained throughout evolution, showing an enormous variability regarding the relative amounts of its disaccharide units. Heparin, on the other hand, is present only in a few tissues and species of the animal kingdom and in the form of granules inside organelles in the cytoplasm of special cells. Thus, the distribution as well as the main structural features of the molecule, including its main disaccharide unit, have been maintained through evolution. These and other studies led to the proposal that heparan sulfate may be involved in the cell-cell recognition phenomena and control of cell growth, whereas heparin may be involved in defense mechanisms against bacteria and other foreign materials. All indications obtained thus far suggest that these molecules perform the same functions in vertebrates and invertebrates.

Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?

The distribution and structure of heparan sulfate and heparin are briefly reviewed. Heparan sulfate is a ubiquitous compound of animal cells whose structure has been maintained throughout evolution, showing an enormous variability regarding the relative amounts of its disaccharide units. Heparin, on the other hand, is present only in a few tissues and species of the animal kingdom and in the form of granules inside organelles in the cytoplasm of special cells. Thus, the distribution as well as the main structural features of the molecule, including its main disaccharide unit, have been maintained through evolution. These and other studies led to the proposal that heparan sulfate may be involved in the cell-cell recognition phenomena and control of cell growth, whereas heparin may be involved in defense mechanisms against bacteria and other foreign materials. All indications obtained thus far suggest that these molecules perform the same functions in vertebrates and invertebrates

New pathway of heparan sulphate degradation. Iduronate sulphatase and N-sulphoglucosamine 6-sulphatase act on the polymer chain prior to depolymerisation by a N-sulpho-glucosaminidase and glycuronidases in the mollusc Tagelus gibbus.

It has previously been shown that in the mollusc Anomalocardia brasiliana the desulphation of chondroitin sulphate precedes its depolymerisation by beta-glucuronidase and beta-N-acetylgalactosaminidase (Sousa Jr. et al. J. Biol. Chem. 1990;265:20150-20155). This led us to investigate whether in molluscs, sulphatases also act on heparan sulphate before its depolymerisation by glycosidases. Radioactively labelled [35S]heparan sulphate was extensively degraded by enzyme extracts prepared from the mollusc Tagelus gibbus. Several enzymes acting in concert degrade the compound to inorganic sulphate, glucosamine N-sulphate, N-acetylglucosamine-6 sulphate and other oligosaccharide products. These results indicate the presence of iduronate sulphatase, N-sulphoglucosamine 6-sulphatase alpha-N-sulphoglucosaminidase, beta-glucuronidase and alpha-L-iduronidase. The di- and mono-saccharide composition of the oligosaccharides were analysed with the aid of heparitinase II from Flavobacterium heparinum. These analyses led to the characterisation of two sulphatases that act on the polymer chain removing sulphates from the C-2 position of iduronic acid residues and the C-6 position of the glucosamine moieties, respectively. The different enzymes were partially fractionated by ion exchange chromatography and molecular sieving. These results led to the proposition of a new pathway of degradation of heparan sulphate where sulphatases act directly on the polymer chain which is then depolymerised by several glycosidases.