Hugo Alexandre Oliveira Rocha

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.

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.

Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent

The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked β-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked α-fucose and one or two nonsulfated, 4-linked β-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several “in vitro” assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the “in vivo” antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.

A sulfated polysaccharide, fucans, isolated from brown algae Sargassum vulgare with anticoagulant, antithrombotic, antioxidant and anti-inflammatory effects.

Fucan (SV1) sulfated polysaccharides from the brown algae Sargassum vulgare were extracted, fractionated in acetone and examined with respect to chemical composition, anticoagulant, anti-inflammatory, antithrombotic effects and cellular proliferation. These polysaccharides contain low levels of protein, high level of carbohydrate and sulfate. Monosaccharides analysis revealed that SV1 was composed of fucose, galactose, xylose, glucuronic acid and mannose. SV1 polysaccharide prolonged activated partial thromboplastin time (aPTT) and exhibited high antithrombotic action in vivo, with a concentration ten times higher than heparin activity. PSV1, a purified form in gel filtration showed very low biological activities. SV1 stimulated the enzymatic activity of FXa. Its action on DPPH radical scavenging activity was 22%. This polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups. It displays strong anti-inflammatory action at all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration.

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.

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.

BC nanofibres: In vitro study of genotoxicity and cell proliferation

Nanomaterials have unusual properties not found in the bulk materials, which can be exploited in numerous applications such as biosensing, electronics, scaffolds for tissue engineering, diagnostics and drug delivery. However, research in the past few years has turned up a range of potential health hazards, which has given birth to the new discipline of nanotoxicology. Bacterial cellulose (BC) is a promising material for biomedical applications, namely due its biocompatibility. Although BC has been shown not to be cytotoxic or genotoxic, the properties of isolated BC nanofibres (NFs) on cells and tissues has never been analysed. Considering the toxicity associated to other fibre-shaped nanoparticles, it seems crucial to evaluate the toxicity associated to the BC-NFs. In this work, nanofibres were produced from bacterial cellulose by a combination of acid and ultrasonic treatment. The genotoxicity of nanofibres from bacterial cellulose was analysed in vitro, using techniques previously demonstrated to detect the genotoxicity of fibrous nanoparticles. The results from single cell gel electrophoresis (also known as comet assay) and the Salmonella reversion assays showed that NFs are not genotoxicity under the conditions tested. A proliferation assay using fibroblasts and CHO cells reveals a slight reduction in the proliferation rate, although no modification in the cell morphology is observed.

Antioxidant and Antiproliferative Activities of Heterofucans from the Seaweed Sargassum filipendula

Fucan is a term used to denominate a type of polysaccharide which contains substantial percentages of l-fucose and sulfate ester groups. We obtained five heterofucans from Sargassum filipendula by proteolytic digestion followed by sequential acetone precipitation. These heterofucans are composed mainly of fucose, glucose, glucuronic acid, galactose and sulfate. These fucans did not show anticoagulant activity in PT and aPTT tests. Their antioxidant activity was evaluated using the follow tests; total antioxidant capacity, scavenging hydroxyl and superoxide radicals, reducing power and ferrous ion [Fe(II)] chelating. All heterofucans displayed considerable activity, especially SF-1.0v which showed the most significant antioxidant potential with 90.7 ascorbic acid equivalents in a total antioxidant capacity test and similar activity when compared with vitamin C in a reducing power assay. The fucan antiproliferative activity was performed with HeLa, PC3 and HepG2 cells using MTT test. In all tested conditions the heterofucans exhibited a dose-dependent effect. The strongest inhibition was observed in HeLa cells, where SF-1.0 and SF-1.5 exhibited considerable activity with an IC50 value of 15.69 and 13.83 μM, respectively. These results clearly indicate the beneficial effect of S. filipendula polysaccharides as antiproliferative and antioxidant. Further purification steps and additional studies on structural features as well as in vivo experiments are needed to test the viability of their use as therapeutic agents.

Heterofucans from the brown seaweed Canistrocarpus cervicornis with anticoagulant and antioxidant activities.

Fucan is a term used to denominate a family of sulfated polysaccharides rich in sulfated l-fucose. We extracted six fucans from Canistrocarpus cervicornis by proteolytic digestion followed by sequential acetone precipitation. These heterofucans are composed mainly of fucose, glucuronic acid, galactose and sulfate. No polysaccharide was capable of prolonging prothrombin time (PT) at the concentration assayed. However, all polysaccharides prolonged activated partial thromboplastin time (aPTT). Four sulfated polysaccharides (CC-0.3/CC-0.5/CC-0.7/CC-1.0) doubled aPTT with only 0.1 mg/mL of plasma, only 1.25-fold less than Clexane®, a commercial low molecular weight heparin. Heterofucans exhibited total antioxidant capacity, low hydroxyl radical scavenging activity, good superoxide radical scavenging efficiency (except CC-1.0), and excellent ferrous chelating ability (except CC-0.3). These results clearly indicate the beneficial effect of C. cervicornis polysaccharides as anticoagulants and antioxidants. Further purification steps and additional studies on structural features as well as in vivo experiments are needed to test the viability of their use as therapeutic agents.

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.