Extraction and anticoagulant activity of sulfated polysaccharides from Caulerpa cupressoides var . lycopodium ( Vahl ) C . Agardh ( Chlorophyceae )

The reportedly low standard quality of heparin (HEP) for use in cardiac surgeries has led to concern in the Brazilian and international markets. Sulfated polysaccharides (SPs) from seaweeds have been regarded as promising substitutes for HEP. The aim of this study was to sequentially extract total SPs (TSPs) from Caulerpa cupressoides (Chlorophyceae) with papain in 100 mM sodium acetate buffer (pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by fractionation by ion-exchange chromatography (DEAE-cellulose), and then evaluate the anticoagulant potential of SP fractions by activated partial thromboplastin time (APTT) using normal human plasma and compare it to standard HEP (193 IU mg). The obtained fractions were chemically characterized by chemical composition and agarose gel electrophoresis. The yield was 4.61%, and three fractions of SP (F I, F II and F III) eluted with 0.50, 0.75 and 1.00 M of NaCl, respectively, were observed on chromatography profiles; however, differences in charge densities patterns and degree of resolution among them were revealed by electrophoresis. SPs were capable of modifying APTT only in fractions eluted with 0.75 M of NaCl, whose activities were 23.37 and 25.76 IU mg, respectively, and the charge density was prerequisite to activity. Therefore, C. cupressoides is a source of SPs possessing low anticoagulant potential compared to HEP.


Introduction
Sedentary lifestyle, poor eating habits and stress are immunosuppressive agents that can potentially lead to thromboembolic disease.Heart attack, stroke, deep-vein thrombosis and pulmonary embolism are the most prevalent cardiovascular diseases worldwide, usually leading to patient death or else partial or total disability (NADER et al., 2001;RANG et al., 2007).
Heparin glycosaminoglycan (HEP) is a mucilage material obtained commercially from bovine lung and swine mucosa.It is administered therapeutically Acta Scientiarum.Biological Sciences Maringá, v. 33, n. 2, p. 133-140, 2011 as an anticoagulant and antithrombotic agent in the prevention and treatment of patients suffering from thromboembolic disease and for heart surgery involving extracorporeal circulation, such as in hemodialysis (NADER et al., 2001).However, this sulfated polysaccharide (SP) is also known for its undesirable side effects, such as hemorrhage (main adverse effect) and thrombocytopenia (RANG et al., 2007;THOMAS, 1997).Moreover, it has become increasingly difficult to acquire porcine HEP in the international market as a result of the rising demand for low-molecular-weight HEPs (ALBAN, 2005).
There have also been reports of a reduction in quality standards for HEP preparations used routinely in heart surgeries under extracorporeal circulation.A significant change in molecular weight resulting from the presence of contaminants and low anticoagulant activity recorded by APTT test for some commercial HEP brands have been recently reported by Melo et al. (2008).SPs are structural components of the cell wall of marine algae, in which they are found in high concentrations (PAINTER, 1983;PEREIRA et al., 2005;RODRIGUES et al., 2009RODRIGUES et al., , 2010a)).The use of these molecules as alternative sources of new heparinoids is justified by the fact that algae are phylogenetically distant from mammals, significantly reducing contamination by viral particles (LEITE et al., 1998).Some seaweed SPs have already been described as heparinoids.For instance, Farias et al. (2000) and Pereira et al. (2005) isolated sulfated galactans with anticoagulant activity from marine red algae Botryocladia occidentalis and Gelidium crinale.When these galactans were evaluated in experimental models of thrombosis in rats, different biological effects were found between the two species; G. crinale featured procoagulant effects at low doses, and was regarded as a promising therapeutic source for patients with bleeding disorders (hemophilia) (FONSECA et al., 2008).They are thus suggested as biotechnology tools to better understand the physiopathology of thromboembolic diseases.
Marine green algae also contain SPs with anticoagulant activity; however, their biological potential has been little explored.The difficulty in characterizing the chemical structure of these compounds makes it harder to relate structure to biological function (ZHANG et al., 2008).Matsubara et al. (2001), while evaluating the anticoagulant potential of polysaccharides extracted in water medium from marine green alga Codium cylindricum, observed a different anticoagulant mechanism from that reported for HEP.SPs from chlorophycean Monostroma latissimum, when fragmented in several molecular weights, produce different effects on blood coagulation in vitro (ZHANG et al., 2008).
Genus Caulerpa Lamouroux is represented by benthonic marine green algae, macroscopically featuring creeping thallus formed by rhizomes that expand along the substrate, fixed by structures known as rhizoids (JOLY, 1965;BRAYNER et al., 2008).Several species of this genus can be found along the Brazilian coast (JOLY, 1965;BEZERRA-NETO et al., 2008;BRAYNER et al., 2008;VANDERLEI et al., 2010) and some studies report important biological properties of their SPs, such as antiviral, anticoagulant and antitumor activities (GHOSH et al., 2004;JI et al., 2008).Studies have shown that by using sequential extraction it is possible to identify new SPs with anticoagulant activity in marine algae (RODRIGUES et al., 2009(RODRIGUES et al., , 2010a)).
Our group has contributed with studies on the chemical characterization and identification of new SPs with anticoagulant activity present in marine algae native to the coast of the state of Ceará, Brazil (PEREIRA et al., 2005;FONSECA et al., 2008;RODRIGUES et al., 2010b).Among them is chlorophycean Caulerpa cupressoides (RODRIGUES et al., 2010c).Thus, continuing with these investigations, the present study had as objective to evaluate, using sequential extraction, the anticoagulant potential of SPs from this species, also contributing to the characterization of these molecules isolated from representatives of the same genus.Total SPs (TSPs) extraction was carried out as previously described by Rodrigues et al. (2010c).To that end, algae (5 g) dehydrated at room temperature and macerated with liquid N 2 were hydrated in 250 mL of 100 mM sodium acetate buffer (pH 5.0) (Vetec Química) containing 5 mM EDTA (QEEL) and 5 mM cysteine (Sigma Chemical), and Acta Scientiarum.Biological Sciences Maringá, v. 33, n. 2, p. 133-140, 2011 subjected to digestion with 17 mL of crude papain solution (30 mg mL -1 ) during 6 hours at 60°C in water bath (MARCONI,model MA 159).Next, the material was filtered, and SPs in the mixture were concentrated by adding 16 mL of cetylpyridinium chloride (CPC) (Sigma Chemical) at 10% (24h, 25°C).Following precipitation, the polysaccharide extract was rinsed (CPC 0.05%; 200 mL), dissolved in 174 mL of 2 M NaCl: commercial-grade ethanol (100:15, v v -1 ) and precipitated again by adding 200 mL of commercial-grade ethanol (24h, 4°C).Right after the second precipitation, the obtained material was rinsed with 200 mL of 80% commercial-grade ethanol (2 ×), commercial-grade ethanol (200 mL, 1 ×) and lyophilized in order to obtain TSPs.For optimum TSPs yield, the residues obtained from the extractions were re-digested with papain (RODRIGUES et al., 2009(RODRIGUES et al., , 2010a)).

Agarose gel electrophoresis
Due to the lack of standardization for algae, the study used animal glycosaminoglycans -chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and heparin (HEP) -to characterize SPs obtained from algae with regard to standards and charge density.Thus, TSPs and SP fractions (25 g) were analyzed by 0.5% agarose gel electrophoresis (Bioagency) in 50 mM 1,3-diaminopropane acetate buffer (Aldrich) (pH 9.0).SPs were applied on the gel and run under constant voltage (110 V) during 60 min.After the procedure, the SPs present in the gel were fixed 0.1% N-cetyl-N,N,Ntrimethylammonium bromide solution (Vetec Química) for 24 hours.The gel was next stained with 0.1% toluidine blue and dyed with a solution containing absolute ethanol, distilled water and concentrated acetic acid (4.95:4.95:0.1,v v -1 v -1 ) as described by Dietrich and Dietrich (1976).

Chemical analyses
Total carbohydrates (TCs) content in the fractions was determined by the phenol-sulfuric acid method (DUBOIS et al., 1956) on a microplate format (MASUKO et al., 2005) using D-galactose to obtain the standard curve in an Elisa reader (Amersham Biosciences, model Biotrak II).Contaminating proteins (CPs) content was estimated using the method described by Bradford (1976), using bovine serum albumin as the standard.

Anticoagulant assay
For the test, normal citrated human blood was obtained from different donors at the Hematology and Hemotherapy Center of Ceará (HEMOCE) and Activated Partial Thromboplastin Time (APTT) was measured according to manufacturer specifications.First, the blood was centrifuged (73.75 × g, 15 min.)to obtain platelet-poor plasma.To perform the test, 50 L of human plasma were incubated at 37°C for 3 min.with 10 L of SP solution and 50 L of APTT reagent (CLOT, Bios Diagnóstica).After incubation, 50 L of 25 mM calcium chloride (CLOT, Bios Diagnóstica) were added to the mixture to trigger the coagulation cascade.Trials were performed in triplicate; coagulation time was recorded automatically with a coagulometer (DRAKE, QUICK-TIMER model) and anticoagulant activity was expressed in international units (IU) per mg of polysaccharide, using HEP with 193 IU mg -1 as reference.

Yield
The methodology of using enzymatic digestion of proteins by proteolytic enzymes (papain) (6h, 60°C) to extract TSPs resulted in three consecutive extractions for C. cupressoides.The largest quantity of these compounds was obtained in the first extraction (3.15%), whereas during the second and third extractions yields decreased sharply to 1.11% and 0.35%, respectively, totaling 4.61% of TSPs, from dehydrated (25°) and macerated algae.
In studies with marine algae B. occidentalis and G. crinale (Rhodophyceae), Farias et al. (2000) and Pereira et al. (2005)  Therefore, the variation in yield of these compounds among different species also involves the use of various techniques.In the present study, three consecutive TSP extractions resulted in a marked reduction in yield from C. cupressoides.This suggests an extraction of TSPs from different layers of algal tissue, thus favoring the identification of new bioactive compounds (RODRIGUES et al., 2009(RODRIGUES et al., , 2010a)), which can also be applied in other economic sectors, such as in agricultural technology (ARAÚJO et al., 2008).

Ion-exchange chromatography (DEAE-cellulose)
Given the low yield obtained in the third extraction (0.35%), only the first two TSPs extracts were used.Thus, the chromatography profiles obtained in ion-exchange column (DEAE-cellulose) showed three separate SP fractions (F I, F II and F III) eluted at concentrations of 0.50, 0.75 and 1.00 M of NaCl in both extractions, respectively (Figure 1).The fraction with highest yield was obtained in F II, eluted with 0.75 M of NaCl, in comparison to the other fractions obtained in boh extractions (Table 1).In the present studies we observed that the methodology resulted in similar chromatographic profiles for all TSP extractions from chlorophycean C. cupressoides, whereas marked differences during fractioning in DEAE-cellulose of sulfated galactans from read algae H. pseudofloresia and Halymenia sp. were reported by Rodrigues et al. (2009Rodrigues et al. ( , 2010a)).
Therefore, the studies demonstrate that the use of this technique makes it possible to identify SPs with different characteristics among macroalgae species.The efficiency in separating SPs demonstrated in Figure 1 can be advantageous, considering the complex and heterogeneous nature of these compounds, which encumbers studies on structural characterizations and its relationships with biological activities (FARIAS et al., 2000;PEREIRA et al., 2005;RODRIGUES et al., 2009RODRIGUES et al., , 2010a)).

Chemical analyses
The chemical composition of freeze-dried fractions obtained from DEAE-cellulose also revealed differences among the extractions (Table 1).Overall, the TCs content of the fractions was similar in the first extraction (F I and F II), while lower levels (F I and F III) were found in the second extraction.No soluble proteins were detected, denoting the efficacy of the method in the enzymatic digestion of proteins by proteolytic enzymes for the extraction of TSPs present in C. cupressoides, as also previously described to other studies (RODRIGUES et al., 2010b(RODRIGUES et al., , 2010c) )  Such disparities in the chemical composition of SP fractions from C. cupressoides are the result of complex and heterogeneous characteristics of these macromolecules, when compared to other studies (FARIAS et al., 2000;PEREIRA et al., 2005;ZHANG et al., 2008;AZEVEDO et al., 2009;RODRIGUES et al., 2010b).

Agarose gel electrophoresis
Agarose gel electrophoresis revealed differences between the resolution degree of fractions and TSPs, as well as in the charge densities of SP B A fractions obtained from the different extractions (Figure 2).The fractions eluted in the first extraction (F I, F II and F III) (Figure 2A) showed differences in charge density among them on gel, in which fraction F II, eluted with 0.75 M of NaCl, showed a higher resolution degree and similar mobility to glycosaminoglycan heparan sulfate (HS) (Figure 2A).Conversely, fractions F I and F III practically were not observed, suggesting a smaller presence of sulfate groupings.During the second extraction, practically the same electrophoretic profile was observed (Figure 2B).However, small differences were observed in negative charge densities between fractions F II and F III (eluted with 0.75 and 1.00 M of NaCl, respectively), when subjected to potential difference.When compared among them, (Figure 2C), fraction F II (Cc1 -first extraction and Cc2 -second extraction, respectively) also showed the same mobility pattern among extractions and unfractioned HEP, although with different resolutions on gel.The higher resolution degree observed for fraction F II of the first TSPs extraction (Cc1) compared to that of the second extraction (Cc2) (Figure 2C) suggests the occurrence of SPs with different negative charge patterns in different coenocyte portions of C. cupressoides.This suggests that the procedure of separating these SPs by ionexchange chromatography (DEAE-cellulose) combined with the electrophoresis technique proved to be a very efficient tool for partial characterization of these polymers, given that marine green algae have more complex components in their chemical structure (D-galactose, D-xylose, D-glucuronic acid, L-arabinose and L-rhamnose), thus making it more difficult to elucidate the native chemical structure of these compounds (PAINTER, 1983;ZHANG et al., 2008).Rodrigues et al. (2009Rodrigues et al. ( , 2010a)), using the TSPs re-extractions technique for Rhodophyceae, also observed marked differences in DEAE-cellulose fractioning and the electrophoresis procedure.The authors concluded that more homogenous SPs in negative charges occurred in the third extraction compared to the first and second extractions in species from genus Halymenia.This constitutes an important tool for future characterization of those macromolecules.
Considering our study, it is known that the cell wall of algae generally consists of two components: fibrillary (wall skeleton) and amorphous (mucilage matrix) (PAINTER, 1983).Extracellular-matrix polysaccharides are linked to mechanical, osmotic and ionic functions, favoring the survival of these organisms in marine environments (KLOAREG; QUATRANO, 1998).However, the absence of defined cells or cell organization that could allow a microscopic differentiation among the different species of the genus Caulerpa (structure known as coenocyte) (JOLY, 1965;BRAYNER et al., 2008) suggest that perhaps the detection of these macromolecules may in the future be regarded as an additional biotechnology tool for strategies aiming to identify species without microscopic identification.Alternatively, the application of the technique of enzymatic digestion of proteins by proteolytic enzymes (papain) in the consecutive extraction of TSPs from marine algae of genus Caulerpa followed by analysis of these compounds by ion-exchange chromatography on DEAE-cellulose column and agarose gel electrophoresis may be regarded as the first record using a marine green alga.Thus, further studies are recommended, including a larger number of species from this genus, in order to strengthen these observations, as well as list these compounds with regard to their possible use as taxonomic, phylogenetic and biogeographic molecular markers (TEIXEIRA, 2002;RODRIGUES et al., 2009).

Anticoagulant assay
The anticoagulant assay, performed according to the APTT test, revealed SP fractions capable of altering normal coagulation time (Table 2).The minimum SP concentration to prolong APTT was 0.10 mg mL -1 in fractions F II for both extractions; their activities were 23.37 and 25.76 IU mg -1 , respectively.No prolongations were detected at a high SP concentration (1.00 mg mL -1 ) in the SP fractions separation in 0.50 and 1.00 M elutions of Acta Scientiarum.Biological Sciences Maringá, v. 33, n. 2, p. 133-140, 2011 NaCl.Anticoagulant activity was also dosedependent, requiring a high concentration greater than 0.75 mg mL -1 of SP (F II -first extraction) to effectively prolong APTT, whereas for the fraction obtained at the same concentration (0.75 M of NaCl) in the second extraction from C. cupressoides, activity was significantly prolonged above 0.25 mg mL -1 of PS (F II -2nd).Thus, the anticoagulant activity of SPs isolated from that species was lower than that of unfractioned HEP, and the APTT test demonstrated that these SPs inhibited the intrinsic and/or common coagulation cascade, whereas prothrombin time (data not shown) was not altered, denoting that these compounds do not inhibit coagulation through the extrinsic pathway (ZHANG et al., 2008;AZEVEDO et al., 2009;RODRIGUES et al., 2009RODRIGUES et al., , 2010a, b), b).Side effects from HEP use have led to a search for alternative sources of new anticoagulant compounds (SPs).However, there is little scientific information on the biologic functions of the chemical structures of these compounds (FARIAS et al., 2000;GHOSH et al., 2004;MOURÃO, 2004;PEREIRA et al., 2005;BEZERRA-NETO et al., 2008;FONSECA et al., 2008;ZHANG et al., 2008;AZEVEDO et al., 2009;RODRIGUES et al., 2010b).
Studies indicate some action mechanisms of the polymers in blood coagulation.According to Farias et al. (2000), sulfated D-galactan isolated from B. occidentalis (Rhodophyceae) has anticoagulant activity by inhibiting thrombin via antithrombin and heparin cofactor II (endogenous blood coagulation regulators) from the presence of two sulfate esters in a single galactose residue.Matsubara et al. (2001) reported that the polysaccharide isolated from marine green alga C. cylindricum has a direct inhibitory mechanism on thrombin, regardless of antithrombin III and heparin cofactor II.More recently, Fonseca et al. (2008), while testing two sulfated galactans isolated from Rhodophyceae B. occidentalis and Gelidium crinale, observed that they dissociate in vivo systemic events in rats.Their actions involve naturally existing differences among the ratios and/or distributions of sulfated radical in the chemical structure.This results in different mechanisms of interaction with proteases, inhibitors and activators of the coagulation system, and expressing anti-and pro-coagulant activities, as well as anti-and pro-thrombotic actions.
The anticoagulant activity of C. cupressoides SPs was detected only in F II (Table 2), in both extractions, which reinforces the resolutions of these compounds when observed by electrophoresis (Figure 2) -activities were 23.37 and 25.76 IU mg -1 for the first and second extractions, respectively.Thus, the activity of these polysaccharides seems to be dependent on negative charge density, given that the other SP fractions from the same species were generally unable to alter normal APTT.On the other hand, the in vitro effects of these compounds recorded in coagulation did not occur merely as a function of charge densities (MOURÃO, 2004); further studies are necessary to elucidate their structural requirements of anticoagulation action of SPs from C. cupressoides in coagulation (MATSUBARA et al., 2001;PEREIRA et al., 2005;FONSECA et al., 2008;ZHANG et al., 2008), as well as relate them to other biologic activities of interest in biomedicine (GHOSH et al., 2004;MOURÃO, 2004;JI et al., 2008).
Evaluating the in vitro anticoagulant activity of SP fractions from red alga H. pseudofloresia using citrated rabbit plasma, Rodrigues et al. (2009) observed marked changes in APTT.The fractions obtained in the first (464.20,211.60, 103.50 and 101.70 IU mg -1 ) were more active compared to those from the third extraction (137.10,96.50 and 89.20 IU mg -1 ).Its actions were considered superior to the existing HEP standard (100 IU mg -1 ) and to SPs from the same-genus species Halymenia sp (RODRIGUES et al., 2010a).
As such, the use of these polysaccharides in several biotechnology processes justifies further studies by our research group, particularly with thrombosis models using animals.Those studies would aim to investigate the effect of these molecules in order to distinguish systemic events and delineate their use in medical practice, such as in the case of patients undergoing heparin therapy and/or thromboembolic disorders (FONSECA et al., 2008).Studies on these compounds are also recommended to characterize other species of the same genus.

Conclusion
Applying the methodology -which uses enzymatic digestion of proteins (papain) in the next extraction, followed by the chromatography procedure in ion-exchange column (DEAEcellulose), combined with the electrophoresis technique -it is possible to partially characterize sulfated polysaccharides from marine green alga Caulerpa cupressoides.These polysaccharides, however, show low anticoagulant potential in vitro compared to heparin.

Figure 1 .
Figure 1.Ion-exchange chromatography of TSPs obtained in the first (A) and second (B) extractions from Caulerpa cupressoides.The column was equilibrated and eluted with 50 mM sodium acetate buffer (pH 5.0) containing 5 mM cysteine and 5 mM EDTA.The SPs adsorbed in gel were eluted by adding NaCl (0.50, 0.75 and 1.00 M). () metachromasia; () NaCl concentration (M).

Table 1 .
Yield and chemical composition of SP fractions obtained by ion-exchange chromatography (DEAE-cellulose) from chlorophycean Caulerpa cupressoides.

Table 2 .
Anticoagulant activity of SP fractions obtained by ionexchange chromatography (DEAE-cellulose) from chlorophycean Caulerpa cupressoides in relation to HEP.