The system of fucoidans from the brown seaweed Dictyota dichotoma: Chemical analysis and antiviral activity
Introduction
Brown seaweeds (Phaeophyta) produce different polysaccharides, namely alginates, laminarans and fucoidans (Painter, 1983). The latter polysaccharides, which contain mainly l-fucose and sulfate, but also minor amounts of other sugars (Bilan et al., 2010, Duarte et al., 2001, Ponce et al., 2003), have received preferential attention due to their different biological activities: anticoagulant (Athukorala et al., 2006, Chandia and Matsuhiro, 2008, Chevolot et al., 1999, Chevolot et al., 2001, Nardella et al., 1996, Nishino and Nagumo, 1992, Nishino et al., 1991a, Nishino et al., 1991b, Pereira et al., 1999, Wang et al., 2010), antithrombotic (Mauray et al., 1995); antiinflamatory (Blondin, Fischer, Boisson-Vidal, Kazatchkine, & Jozenfovicz, 1994), antitumoral (Athukorala et al., 2009, Croci et al., 2011, Rocha de Souza et al., 2007, Yang et al., 2008, Zhuang et al., 1995), contraceptive (Mahony et al., 1993, Mahony et al., 1991), apoptotic (Foley, Mulloy, & Tuohy, 2011), antioxidant (Rocha de Souza et al., 2007, Ruperez et al., 2002, Wang et al., 2008b, Wang et al., 2010) and antiviral (Bandyopadhyay et al., 2011, Feldman et al., 1999, Harden et al., 2009, Hayashi et al., 2008, Hidari et al., 2008, McClure et al., 1991, Ponce et al., 2003, Trinchero et al., 2009, Venkateswaran et al., 1989, Wang et al., 2008a). They have particularly been described as inhibitors of the replication of several enveloped viruses, as human immunodeficiency virus, herpes simplex virus and human cytomegalovirus (Beress et al., 1993, McClure et al., 1991). Its action against HIV is of particular importance (Queiroz et al., 2008, Trinchero et al., 2009). Attempts to systematize the fine structure of the fucoidans as is already established for red seaweed galactans have been unsuccessful, as only few examples of regularity were found: linkages, branching, sulfate position, other sugars appear to be variable and thus the relationship between structure and biological activity is not clearly established (Ale, Mikkelsen, & Meyer, 2011). However, about four different types of polysaccharides were found within fucoidan samples: one containing high proportions of fucose, galactose and sulfate groups (the “galactofucan” or “fucogalactan”), another one containing just fucose and sulfate (the “fucan”) and another one containing different monosaccharide moieties, less sulfate, and important proportions of uronic acids, known first as “uronofucoidans” (Duarte et al., 2001, Ponce et al., 2003) but later subdivided into “fucoglucuronans” and “fucomannoglucuronans” (Bilan et al., 2010, Croci et al., 2011). It has been shown that only the formers have strong biological activities (Croci et al., 2011, Ponce et al., 2003). For the fucan moieties, in the last years two main types of fucoidans were differentiated: one carrying alternating 3- and 4-linked α-l-fucopyranosyl units, recognized earlier in Ascophyllum nodosum (Chevolot et al., 2001), but also present in some Fucus species as F. vesiculosus (Chevolot et al., 2001), F. evanescens (Bilan et al., 2002), F. distichus (Bilan et al., 2004) and F. serratus (Bilan, Grachev, Shashkov, Nifantiev, & Usov, 2006), and another one carrying mainly 3-linked α-l-fucopyranosyl units, as found in Cladosiphon okamunarus (Hidari et al., 2008), Chordaria flagelliformis (Bilan et al., 2008), Lessonia vadosa (Chandia & Matsuhiro, 2008) and Saccharina latissima (formerly known as Laminaria saccharina, Bilan et al., 2010). Other authors indicate both 3- and 4-linkages, but not an alternating fashion; usually, 3-linked fucose units tend to predominate (Adhikari et al., 2006, Lee et al., 2011, Ponce et al., 2003). The presence of 2-linked fucose units has also been postulated (Karmakar et al., 2009) in Padina tetrastromatica. These backbones appear heavily sulfated, mainly on C-2 and/or C-4, and sometimes also branched. For the galactofucan, a core containing 6-linked β-d-galactopyranosyl units has been postulated (Bilan et al., 2010, Duarte et al., 2001), whereas for the glucuronic acid-containing polymers four different motifs were encountered: terminal GlcA units on C-2 of a 3-linked fucose backbone (Bilan et al., 2008, Hidari et al., 2008); a 3-linked β-d-GlcAp backbone carrying fucose units on C-4 (Bilan et al., 2010), a backbone containing both 4-linked β-d-GlcAp and 2-linked α-d-Man moieties, possibly alternating (Bilan et al., 2010, Li et al., 2006), and the linear structure →4)-β-d-GlcAp-(1→2)-α-l-Fucp-(1→3)-α-d-Manp-(1→, partially sulfated on O-3 of the fucose units, and on O-6 of the mannose units (Sakai et al., 2003). Xylose was found to appear mainly as branching points. Some terminal fucofuranose units have also been found (Bilan et al., 2008, Nishino et al., 1991b, Ponce et al., 2003).
Dictyota dichotoma is a cosmopolitan brown seaweed. Many reports, especially from Asian countries, have recently reported biological activities of its secondary metabolites (Ayrad et al., 2011, Ravikumar et al., 2011, Tabarsa et al., 2012). However, this seaweed also appears in Europe, South Africa and Bermuda, according to molecular markers (Tronholm et al., 2010), and all around the world. In the late seventies, it has been determined that the polysaccharides from an Egyptian sample of D. dichotoma contained residues of d-glucuronic acid, d-galactose, d-mannose, d-xylose, and l-fucose in substantial amounts (Abdel-Fattah et al., 1978, Magdel-Din Hussein et al., 1979). The antimicrobial activity of its non-characterized polysaccharides was reported (Kantachumpoo & Chirapart, 2010). In order to assess the potential of a cosmopolitan seaweed as a source of fucoidan, and to study its characteristics, we have studied the whole system of polysaccharides of D. dichotoma and screened their antiviral activity against HSV-1 and CVB3. Herein the extraction of the polysaccharides from this seaweed using diluted hydrochloric acid both at room temperature and at 70 °C is reported, together with their purification, analysis, fractionation and assessment of the antiviral activity of some selected fractions.
Section snippets
Materials
The brown seaweed Dictyota dichotoma (Hudson) Lamouroux was collected in summer at the shores near Bahía Bustamante (Chubut Province, Argentina). The thalli were air-dried and milled to a fine powder. All the reagents indicated below were of analytical grade.
Analytical methods
Total carbohydrates were estimated by the phenol-H2SO4 method using fucose as standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Uronic acids were determined using the method of Filisetti-Cozzi and Carpita (1991) using
Extraction using room-temperature acid solution
The milled seaweed was extracted with ethanol first in order to remove low-molecular weight material. About 40% of the original dry weight of the seaweed was extracted with ethanol; mannitol was an important constituent of that extract, although it also contained reducing carbohydrates, proteins, uronic acids and sulfate. The residue was extracted with aqueous hydrochloric acid (pH 2) for 7 h. This solvent usually maximizes the extraction of fucoidans while it minimizes the coextraction of
Conclusion
The brown seaweed Dictyota dichotoma produces large amounts of heterogeneous polysaccharides when submitted to the extraction procedures used to obtain fucoidans. By cetrimide fractionation of the two first extracts, i.e. that extracted with room-temperature acid (EAR), and that extracted in the first place by hot acid (EAH-1), typical heavily sulfated galactofucans/fucogalactans are obtained, with no major signs of heterogeneity. However, the proportion of subfractions from these two extracts
Acknowledgements
This work was supported by grants from University of Buenos Aires and CONICET. M.R. is a recipient of a Fellowship from CONICET, whereas N.M.A.P., D.A.N., R.M.G. and C.A.S. are Research Members of the same Institution. The authors are indebted to Dr. L. Cavallaro (UBA) for the gift of a sample of ribavirin.
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