Elsevier

Carbohydrate Polymers

Volume 154, 10 December 2016, Pages 139-150
Carbohydrate Polymers

Novel sulfated xylogalactoarabinans from green seaweed Cladophora falklandica: Chemical structure and action on the fibrin network

https://doi.org/10.1016/j.carbpol.2016.07.088Get rights and content

Highlights

Abstract

The water-soluble sulfated xylogalactoarabinans from green seaweed Cladophora falklandica are constituted by a backbone of 4-linked β-l-arabinopyranose units partially sulfated mainly on C3 and also on C2. Besides, partial glycosylation mostly on C2 with single stubs of β-d-xylopyranose, or single stubs of β-d-galactofuranose or short chains comprising (1  5)- and/or (1  6)-linkages, was also found. These compounds showed anticoagulant activity, although much lower than that of heparin. The effect of a purified fraction (F1) on the fibrin network was studied in detail. It modifies the kinetics of fibrin formation, suggesting an impaired polymerization process. Scanning electron microscopy showed a laxer conformation, with larger interstitial pores than the control. Accordingly, this network was lysed more easily. These fibrin properties would reduce the time of permanence of the clot in the blood vessel, inducing a lesser thrombogenic state. One of the possible mechanisms of its anticoagulant effect is direct thrombin inhibition.

Introduction

Cladophora Kützing is one of the largest green algal genera and has a worldwide distribution. Within the class Cladophorophyceae, the genus Cladophora is characterized by its simple thallus architecture: branched, uniseriate filaments of multinucleate cells. Cladophora cell walls, which are several micrometers thick, were reported to consist largely of cellulose, which represents about 15% of the biomass (Wissel, Mayr, & Lücke, 2008).

In spite of its wide distribution, which comprises mostly marine, but also fresh water environments, and of its abundance, there is only scarce information about the structure of the sulfated polysaccharides biosynthesized by algae of this genus. Percival and coworkers studied the structural features of the water soluble polysaccharides from Cladophora rupestris (Percival & McDowell, 1981). These studies established the presence of arabinose, galactose, xylose, rhamnose, and glucose in the approximate molar proportions of 3.7:2.8:1.0:0.4:0.2 (+8% protein) and 19.6% half ester sulfate. Application of degradative studies provided evidence of a highly branched structure, with xylose and galactose units at the ends of the branches and galactose, arabinose, and rhamnose residues occurring in the inner part of the molecules. Evidence of 6-linked and/or 6-sulfated galactofuranose units was obtained. Partial hydrolysis experiments led to the separation and characterization of the following fragments: l-arabinose 3-sulfate, d-galactose 6-sulfate, 3-linked and 6-linked d-galactobioses, 4- or 5-linked l-arabinobiose 3-sulfate, 4-linked d-xylobiose, a mixture of trisaccharides containing sulfated galactose and arabinose, and a mixture of pentasaccharides in which the molar ratio of arabinose to galactose was 4:1.

Later, the structural characteristics of the polysaccharides obtained from Cladophora socialis by extraction with dilute acid were reported (Sri Ramana & Venkata Rao, 1991). A molar ratio galactose:arabinose:xylose of 4.5:3.0:1.0 and 16.9% of sulfate were determined. Similar conclusions were obtained regards the arabinose units, but no galactose in the furanose form was detected. In this case, galactose units were found to be 3-linked and sulfated on C4 or C4 and C6.

Between the sulfated polysaccharides, sulfated fucans (SFs) and sulfated galactans (SGs) are currently the marine non-glycosaminoglycan (GAG) sulfated compounds most studied in glycomics. These polysaccharides exhibit therapeutic effects in several pathophysiological systems such as blood coagulation, thrombosis, neovascularization, cancer, inflammation, and microbial infections. As analogs of the largely employed GAGs and due to some limitations of the GAG-based therapies (Anand, Yusuf, Pogue, Ginsberg, & Hirsh, 2003; Kelton & Hirsh, 1980; Kelton & Warkentin, 2008), SFs and SGs comprise new carbohydrate-based therapeutics available for clinical studies. However, the degree and mechanism of action varies greatly, not only with the degree of sulfation, but also with their chemical structure, and other sulfated polysaccharides with different carbohydrate backbones and sulfate distributions have been investigated and were found to have similar properties (Ciancia, Quinitana, & Cerezo, 2010, Fernández et al., 2013).

In a previous paper, the effect in the fibrin network formation of sulfated galactans from green seaweeds of the order Bryopsidales was studied by measuring optical density (OD) at 405 nm vs time. It was shown that these galactans caused an impaired assembly of fibrin monomers into the fibrin polymer (Arata et al., 2015).

Kinetics of fibrin formation gives a different insight into the study of the coagulation process, contributing to the understanding of the mechanism of anticoagulant activity. On the other hand, the study of fibrinolysis is also crucial, because currently approved thrombolytic drugs have side effects, such as bleeding, low specificity on account of being indirect plasminogen activators, and high cost. The ‘holy grail’ for antithrombotic therapy, a drug that prevents coagulation without promoting bleeding, has yet to be found.

In this paper, structure of the major water-soluble sulfated polysaccharides from Cladophora falklandica was determined, and their anticoagulant effect was investigated, not only by general coagulation tests, but also through studies of their influence in fibrin formation and fibrinolysis. Besides, characteristics of the fibrin clot were examined using scanning electron microscopy.

Section snippets

Algal sample

Specimens of Cladophora falklandica (J.D. Hooker & Harvey) J.D. Hooker & Harvey were collected in Punta Este − Puerto Madryn − Chubut − Argentina (42°46′48″S, 64°57′W), in September 2010. The samples used in this work were in the vegetative state. Thalli of the seaweeds were washed with sea water and analyzed for epiphytic and epizoic contaminants in a Nikon AFX-II macroscope (Nikon, Japan). Voucher specimens were deposited in the Museum Bernandino Rivadavia, Buenos Aires Argentina (collection

Extraction and characterization of the sulfated polysaccharides

Water-soluble polysaccharides from green seaweed Cladophora falklandica were obtained by sequential extraction at room temperature and at 90 °C from the alcohol insoluble residue, giving a total yield of 29.2% of the dry milled material.

All the extracts have important amounts of carbohydrates and sulfate (Supplementary Table 1). Colorimetric assays indicated only trace amounts of uronic acids and no pyruvic acid ketals in these extracts, which have the same monosaccharides, but in different

Conclusions

The major water-soluble polysaccharides from Cladophora falklandica are sulfated xylogalactoarabinans. Their backbone is constituted by 4-linked pyranosic β-l-arabinose units. 3-Linked sulfated pyranosic arabinans were previously isolated from Codium species (Bryopsidales). On the other hand, pyranosic arabinose units were found to be part of arabinogalactans from green seaweeds Caulerpa racemosa (Bryopsidales) and Cladophora rupestris; however, their structure was not fully determined. In

Acknowledgement

This work was supported by a grant from the University of Buenos Aires, UBACYT 2014–2017, 20020130100576BA.

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