Anticoagulant activity of sulfated polysaccharides fractions from an aqueous extract obtained from the red seaweed Halymenia floresia

Heparin (HEP) is known due to their side effects and the red seaweed Halymenia floresia (Hf) sulfated polysaccharides (SP) are heparinoids. In this study we purified the Hf-SP obtained from an aqueous extract and evaluated their anticoagulant activities. Hf-SP1 (25°C), Hf-SP2 (80°C) and Hf-SP3 (80°C) were sequentially isolated. Hf-SP3 had the highest sulfate content (37.45%). Hf-SP3 was fractionated by ion exchange chromatography on a DEAEcellulose column using a NaCl gradient. Fractions were lyophilized and submitted to 0.5% agarose gel electrophoresis. The anticoagulant activity was evaluated by the activated partial thromboplastin time using rabbits plasma and expressed in international units per mg of SP using standard HEP (193 IU mg). The chromatographic procedure separated into four different SP fractions (F I, F II, F III and F IV) eluted at concentrations of 0.50, 0.75, 1.00 and 1.25 M of NaCl, respectively, reveling among them different marked on charge density, when compared by electrophoresis. F III had the highest anticoagulant activity (10.72 IU mg), suggesting that the sulfate is important in this process. In conclusion, our results suggest that sequential extractions of Hf-SP are an important biotechnological tool for identification of novel anticoagulants and studies of structural characterization are already in progress.

In this context, we also expanded our investigations to purification of SP from marine alga.In case of red seaweeds, we have observed that SP species of the Halymenia genus occur on distinct molecular characteristics into the algal tissue when obtained by successive extractions.These studies have also revealed as an important biotechnological tool for identification of novel anticoagulants (RODRIGUES et al., 2009(RODRIGUES et al., , 2010b)).More recently, we extended our studies to green alga Caulerpa cupressoides (RODRIGUES et al., 2011a).The C. cupressoides SP also occur on distinct molecular characteristics into the algal tissue.However, we observed macromolecules excessively polydisperses along of the technique.Thus, the studies suggest that the molecular characteristics of seaweed SP vary among different species, when obtained by papain digestion.
The Halymenia floresia SP (Hf-SP) have been studied (AMORIM et al., 2011).We report now the purification and anticoagulant activity of Hf-SP fractions from an aqueous extract obtained from this species.

Marine algae
The red seaweed H. floresia (Clemente) C. Agardh was collected in March, 2004 on the Northeast coast of Brazil (Pedra Rachada Beach, Ceará State).After collection, the material was washed with distilled water, and stored at -20°C at Carbohydrate and Lectins Laboratory (CarboLec), Department of Biochemistry and Molecular Biology, Federal University of Ceará, Brazil.Hf-SP were extracted as previously described (AMORIM et al., 2011).Briefly (Figure 1), the algae were submitted to mechanical stirring for 24h at room temperature in water at 1.5% (w v -1 ).The residue was removed by centrifugation (5.000 × g for 15 min.at 4 o C).The supernatant was precipitated with absolute EtOH (1:3, v v -1 ), centrifuged, re-dissolved in distilled water, dialyzed against water, freezedried and denominated Hf-SP 1 .The algal residue was reextracted but this time at 80°C for 4h, followed by centrifugation under the same conditions.The hot extraction was repeated once more, using the second extraction residue.The supernatants were precipitated with absolute EtOH (1:3, v v -1 ), and denominated Hf-SP 2 and Hf-SP 3 for the second and third extractions, respectively.Acta Scientiarum. Technology Maringá, v. 33, n. 4, p. 371-378, 2011 Chemical composition of Hf-SP The total sugars (TS) content was estimated by phenol-sulfuric acid analysis using D-galactose as the standard (DUBOIS et al., 1956) at 490 nm.After acid hydrolysis of the soluble polysaccharides (1 mL of HCl for 5h at 100°C), free sulfate (FS) was measured by the BaCl 2 /gelatin method (DODGSON; PRICE, 1962).The contaminant proteins (CP) content was measured by the method of Bradford (1976), using bovine serum albumin to construct the standard curve.

Total sugar (TS) content of metachromatic fractions obtained by ion-exchange chromatography (DEAEcellulose)
The TS content was estimated by phenolsulfuric acid analysis using D-galactose as the standard (DUBOIS et al., 1956) a plate (MASUKO et al., 2005), using an Elisa raider (AMERSHAM BIOSCIENCES, BIOTRAK II) at 492 nm.The presence of sulfate in the obtained fractions by ion exchange chromatography (DEAE-cellulose) was also estimated by the metachromatic integrated area using the 7.0 ORIGIN program.

Agarose gel electrophoresis
The fractions obtained by ion-exchange chromatography (DEAE-cellulose) were analyzed by 0.5% agarose gel electroforesis according to Dietrich and Dietrich (1976).Sample of 25 μg was applied to a gel and run for 1h at 110 V in 0.05 M 1,3 diaminopropane-acetate buffer (pH 9.0).SP on gel were fixed with 0.1% N-cetyl-N-N-Ntrimethylammonium bromide solution.After 12h, the gel was dried and stained with 0.1% toluidine blue and discolored with an acetic acid: absolute ethanol: distillated water solution (0.1:0.45:0.45).

Evaluation of SP fractions by the Activated Partial Thromboplastin Time (APTT) test
The assay was carried out using citrated rabbit plasma according to the manufacturers' specifications.50 μL of rabbit plasma was mixed with 10 μL of a solution of different amounts of polysaccharide before addition of 50 μL of APTT reagent.The mixture was then incubated at 37°C for 3 min.Then, 50 μL of 0.025 M of calcium chloride reagent was added to the mixture to trigger the coagulation cascade.The clotting time was recorded in a coagulometer (DRAKE QUICK TIMER).HEP (National Institute for Biological Standards and Control (Potters Bar, UK)) with 193 international units per mg of polysaccharide (IU mg -1 ) was used as the standard.All tests were performed in triplicate.

Results and discussion
We have previously reported that the red seaweed H. floresia is composed by three crude SP (Hf-SP 1 , Hf-SP 2 and Hf-SP 3 ).These polysaccharides showed in vitro anticoagulant activity (APTT test) dependent of the sulfate content.The Hf-SP accelerates thrombin inhibition by heparin cofactor II.The chemical composition also showed that the Hf-SP is composed of 6-O-metylgalactose and 3,6-anidrogalactose (AMORIM et al., 2011).
Here, to further evaluate other characteristics of Hf-SP, we extended our investigation to an aqueous extract (80°C) (denomined Hf-SP 3 ) obtained and fractionated by ion-exchange chromatography on a DEAE-cellulose column and analyzed by agarose gel electrophoresis procedure.Initially, the different obtained aqueous extracts (Hf-SP 1 (25°C), Hf-SP 2 (80°C) and Hf-SP 3 (80°C)) were obtained.Among them, the crude Hf-SP 3 had the highest FS content (37.45%),TS (85.88%) and the low CP content (1.63%), as shown in Table 1, and was used on subsequent studies.The high CP found in these crude SP may perhaps be the presence of amino acids (GHOSH et al., 2004) and/or polysaccharidesprotein complex forms (MELO et al., 2002;PUSHPAMALI et al., 2008).Thus, a more detailed study of these macromolecules is suggested.In this study, the obtaining of distinguish isolated SP at different temperatures from the H. floresia tissue support the hypothesis of Percival and McDowell (1967), suggesting that the use of consecutive extractions result in the obtaining of different macromolecules in chemical composition.This fact further justifies the occurrence of distinct SP into tissue of the studied species.Therefore, such disproportions in chemical composition also justify the complexity and heterogeneity of these polymers (FARIAS et al., 2000;GHOSH et al., 2004;SILVA et al., 2005;RODRIGUES et al., 2010aRODRIGUES et al., , 2011b)).From these data (Table 1), we chose the Hf-SP 3 (80°C) which was submitted to ion-exchange chromatography on a DEAE-cellulose column, an important technique for separation of these compounds.

Ion-exchange chromatography
The DEAE-cellulose chromatography profile is shown in Figure 1.The chromatographic profile indicated the separation into four different fractions of SP (F I, F II, F III, and F IV) eluted at concentrations of 0.50, 0.75, 1.00, and 1.25 M of NaCl, respectively.F II had the highest metachromatic peak compared to other obtained fractions.The highest yield of SP and TS and FS contents were also obtained in F II, eluted with 0.75 M of NaCl, compared to F I, F III and F IV (Table 2).The employment of DEAE-cellulose as a matrix has been widely reported for separation of SP, to reveal the characteristics of different algal species, such as on Gelidium crinale (PEREIRA et al., 2005), Ecklonia cava (ATHUKORALA et al., 2006), Champia feldmannii (ASSREUY et al., 2008), Lomentaria catenata (PUSHPAMALI et al., 2008), Halymenia pseudofloresia (RODRIGUES et al., 2009), C. cupressoides, C. racemosa (RODRIGUES et al., 2010a), Halymenia sp.(RODRIGUES et al., 2010b) and Hypnea musciformis (RODRIGUES et al., 2011b).

Agarose gel electrophoresis
The electrophoretic profile is shown in Figure 2. The agarose gel electrophoresis procedure showed marked differences in charge density among the isolated fractions.However, this was not corroborated by the higher presence of sulfate (Table 2).Thus, fractions F I, F II and F III were not observed on agarose gel, suggesting little sulfated groups in their chemical structures.On the other hand, F III, eluted with 1.00 M of salt, had a strong metachromatic band on gel, showing a similar migration to glycosaminoglycan condroitin sulfate (CS), while the Hf-SP 3 showed a similar charge density when compared to glycosaminoglycan dermatan sulfate (DS), both glycosaminoglycans obtained from animal tissues.Curiously, F III was also a homogeneous SP when compared to Hf-SP 3 .This suggests that the native crude compound (Hf-SP 3 ) is homogeneous molecule (Figure 2) and the ion-exchange chromatography procedure is efficient for separation of Hf-SP (Figure 1).Therefore, the isolation of these compounds could be a useful tool for posterior structural characterization studies (FARIAS et al., 2000;PEREIRA et al., 2005;RODRIGUES et al., 2009, 2010a andb;SILVA et al., 2005).In this study, it was observed more homogeneous molecules in third aqueous extraction (Figure 2) compared to first (25°C) and second (80°C) ones, respectively (data not shown).According some researchers involving the obtaining of SP by successive extractions from other Halymenia species (RODRIGUES et al., 2009(RODRIGUES et al., , 2010b)).It seems that their molecular characteristics are common on this genus, but not show the same profile when compared to polysaccharides isolated from green seaweed C. cupressoides (RODRIGUES et al., 2011a).Therefore, this technique could also be a valuable tool for identification of molecular characteristics among different algal species and biological agents.However, the chemical composition of these compounds can vary due to, for example, temperature, light and water nutrients, as well as the place and season of the year (MARINHO-SORIANO; BOURRET, 2003;PERCIVAL;McDOWELL, 1967).In this connection, a more detailed study of these macromolecules is indicated.Our study also involved to evaluate the potential anticoagulant from their isolated fractions.In this line, the homogeneous SP (F III) shown by the electrophoretic technique led us to conduct anticoagulant assays.

Anticoagulant activity
We have been recently described that Hf-SP are heparinoids.The anticoagulant activity determined by APTT test for crude fractions Hf-SP 1 , Hf-SP 2 and Hf-SP 3 about 37, 68 and 36 IU mg -1 , respectively.Sulfate content is important in this process (AMORIM et al., 2011).Although showing the lowest activity, Hf-SP 3 was explored because of its more homogeneous form (Figure 2).Thus, the anticoagulant assays (APTT) showed fractions of SP capable of modifying the normal coagulation time (Table 3).Fraction F III (1.00 M of NaCl), at a concentration of 0.25 mg mL -1 of SP, prolonged the APTT of normal rabbit plasma (20.45 s), whose activity was 10.72 IU mg -1 , comparing to standard HEP (193.00IU mg -1 ).Fractions F I, F II and F IV, eluted with 0.50, 0.75 and 1.25 M of NaCl, respectively, no practically extended the APTT, when 1.00 mg mL -1 of SP was measured.Therefore, Hf-SP 3 fractions had a low anticoagulant potential.
Based on the popular use of marine algae for disorders such as cardiovascular diseases and cancer, studies with SP from algae have intensified.In this context, the discovery of new anticoagulant compounds is needed (MOURÃO; PEREIRA, 1999), and seaweed SP are a potentially attractive source of macromolecules to investigate.These anticoagulant SP include fucoidans, sulfated galactans, ulvans and rhamnose (ASSREUY et al., 2008;ATHUKORALA et al., 2006;FONSECA et al., 2008;MOURÃO, 2004;PEREIRA et al., 2002;PUSHPAMALI et al., 2008;RODRIGUES et al., 2009RODRIGUES et al., , 2010aRODRIGUES et al., , 2011b;;SILVA et al., 2005;MEDEIROS et al., 2008;YOON et al., 2007;ZHANG et al., 2008).It has been believed that the sulfate is important in this process (AZEVEDO et al., 2009;FARIAS et al., 2000;NISHINO et al., 1991;RODRIGUES et al., 2011a).In the present study, we extended to explore of SP fractions from H. floresia (Hf-SP 3 ) obtained by ion-exchange chromatography (DEAE-cellulose).All the SP fractions were capable of prolonging the APTT (Table 3).Fraction F III had the highest activity when compared to F I, F II and F IV.As expected, the difference in the activities of these fractions was dependent of the charge density observed by electrophoresis procedure, showing clearly that the presence of sulfate groups is also important in this process.This point of view was observed in a previously investigation, using a dessulfated crude SP (AMORIM et al., 2011).On the another hand, the effect of SP on coagulation system do not occur merely as function of charge density, but also of chemical composition, position of sulfate groups and the occurrence of dessulfated units (MOURÃO, 2004).Each polysaccharide has a structural requirement for interaction with coagulation cofactors and their target proteases are stereospecific (FARIAS et al., 2000;PEREIRA et al., 2002PEREIRA et al., , 2005)).Therefore, the chemical characteristics are also prerequisites for understanding of these polymers as their structure/biological function relationships (AZEVEDO et al., 2009;ZHANG et al., 2008).Animals model of thrombosis have been important tools in these investigations (MOURÃO;PEREIRA, 1999;FARIAS et al., 2001;FONSECA et al., 2008).
Our study suggest the hypothesis of Farias et al. (2000) that an addition of sulfate ester in a single unit of α -galactose and the molecular weight of the galactan have an amplifying effect on the Acta Scientiarum. Technology Maringá, v. 33, n. 4, p. 371-378, 2011 prolongation of clotting time, a finding also reported for the algae Botryocladia occidentalis.The inhibitory mechanism of the anticoagulant activity previously reported for H. floresia showed that the crude polysaccharide (Hf-SP 3 ) is able to inhibit the action of thrombin by heparin cofactor II.The clotting time (APTT) is also considerably prolonged in the presence of cofactor VIII and IX deficient plasma.However, the results showed that these two factors are not important to the inhibitory effects of polysaccharide (AMORIM et al., 2011).Thus, the obtained polysaccharide (Figure 2) could also be very important to evaluate and compare not only the doses required to achieve thrombosis inhibition, but also the persistence of the effect, circulating plasma levels, the correlation between the anticoagulant action and antithrombotic effect, as well as bioavailability and absorption when administered by different routes (MOURÃO; PEREIRA, 1999).Overall, our study reported a homogeneous heparinoid from H. floresia.Although showing a low anticoagulant potential, its posterior investigation may help to determine a close relationship between the structure and anticoagulant activity of SP, as has already been reported for heparin, arousing thus a great interesting for our group.Structural analysis of this SP fraction by infrared and NMR spectroscopies can help to this end, including animal studies.

Conclusion
The anticoagulant activity of sulfated polysaccharides fractions obtained from an aqueous extracted from the red alga Halymenia floresia were fewer actives than heparin.However, the used technique for isolation of these molecules showed to be an important tool in the identification of more homogeneous polysaccharides.Structural analysis of polysaccharide by infrared and NMR spectroscopies and its mechanism of action in posterior studies can help to better understanding its particular biological action using animal models of thrombosis.
a -Fractions obtained on DEAE-cellulose column; b -Yields from a sample of SP applied on DEAE-cellulose column; c -Expressed by Dubois et al. (1956)' method in plate by Masuko et al. (2005)' method using D-galactose as standard; d -Sulfate expressed by metachromatic integrated area from the chromatographic profile (DEAE-cellulose).

Table 1 .
Chemical composition of aqueous extracts obtained from the red seaweed Halymenia floresia.

Table 2 .
Yield and chemical composition of sulfated polysaccharides fractions obtained by ion exchange chromatography (DEAEcellulose) from the red seaweed Halymenia floresia.