Fucoidans from the brown seaweed Adenocystis utricularis: extraction methods, antiviral activity and structural studies

doi:10.1016/S0008-6215(02)00403-2

Carbohydrate Research

Volume 338, Issue 2, 20 January 2003, Pages 153-165

https://doi.org/10.1016/S0008-6215(02)00403-2 Get rights and content

Abstract

The brown seaweed Adenocystis utricularis (family Adenocystaceae, order Ectocarpales sensu lato) was extracted in parallel with three solvents usually utilized for obtaining fucoidans: distilled water, 2% calcium chloride solution and diluted hydrochloric acid (pH 2) solution. In each case, the extraction was effected at room temperature and then at 70 °C. The extraction yields and characteristics of the products were similar in the three cases, with only minor differences. The analytical features of the products indicate that two different types of fucoidans are present in this seaweed. One of them, mostly extracted at room temperature, is composed mainly of l-fucose, d-galactose and ester sulfate (the ‘galactofucan’). The other product (the ‘uronofucoidan’) is the major component of the extracts obtained at 70 °C. It is composed mainly of fucose, accompanied by other monosaccharides (mostly Man, but also Glc, Xyl, Rha and Gal), significant amounts of uronic acids and low proportions of sulfate ester. Fractionation with the cationic detergent cetrimide has allowed achieving a better separation of the galactofucan and uronofucoidan components. The galactofucans show a high inhibitory activity against herpes simplex virus 1 and 2, with no cytotoxicity, whereas the uronofucoidans carry no antiviral activity. Structural studies on the galactofucan fractions were carried out by methylation analysis, desulfation and NMR spectroscopy. The fucan constituent is mainly composed of 3-linked α-l-fucopyranosyl backbone, mostly sulfated at C-4, and branched at C-2 with non-sulfated fucofuranosyl and fucopyranosyl units, and 2-sulfated fucopyranosyl units. The galactan moiety is more heterogeneous, with predominant d-galactopyranose units linked on C-3 and C-6, and sulfation mostly on C-4, even in terminal non-reducing units. It may be inferred that at least some of these galactose units carry the α-configuration.

The brown seaweed Adenocystis utricularis biosynthesizes at least two different types of fucoidans: the galactofucans and the uronofucoidans. The galactofucans show high antiviral activities.

Introduction

Brown seaweeds (Phaeophycophyta) are known to produce different polysaccharides, namely alginates, laminarans and fucoidans.1., 2. The latter polysaccharides usually contain large proportions of l-fucose and sulfate, together with minor amounts of other sugars like xylose, galactose, mannose and glucuronic acid.1., 3. Several biological activities4., 5. have been attributed to the fucoidans: anticoagulant,6., 7., 8., 9., 10., 11., 12. antithrombotic,¹³ antiinflammatory,¹⁴ antitumoral,5., 15. contraceptive,16., 17. and antiviral.18., 19., 20. They have particularly been described as inhibitors of the replication of several enveloped viruses, as human immunodeficiency virus, herpes simplex virus and human cytomegalovirus.20., 21., 22. In spite of the many studies attempting to determine the fine structure of the fucoidans, 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 was not established. Different techniques for extracting the fucoidans free from contaminants have been used.23., 24. They included the action of calcium-containing solvents, acid media, or plain water.3., 24., 25., 26., 27., 28.

Adenocystis utricularis is a brown seaweed from the cold waters of the Southern Hemisphere. It is found close to the Antarctica, as well as on the coasts of Chile, Argentina, New Zealand and Australia.²⁹ Its classification was revised many times, although it is now considered within the family Adenocystaceae,³⁰ of the order Ectocarpales sensu lato.30., 31. Herein the extraction of the polysaccharides from this seaweed by different methods is reported, together with their purification, analysis, fractionation, assessment of their antiviral activity, and structural analysis of the fractions with antiviral properties.

Section snippets

Algal material

The brown, globular seaweed Adenocystis utricularis was collected in summer at the shores near Comodoro Rivadavia (Chubut Province). The thalli were air-dried and milled to a fine powder.

Analytical methods

Total carbohydrates were determined by the phenol–H₂SO₄ method using fucose as standard.³² Uronic acids were determined using the method of Filisetti-Cozzi and Carpita³³ using glucuronolactone as standard. The percentages of sulfate were measured by turbidimetry³⁴ after hydrolysis with 1 M HCl, while the

Extraction

The milled seaweed was extracted as depicted in Scheme 1. Yields and general analyses of the extracts are shown on Table 1, while the monosaccharide composition is shown on Table 2. Treatment of the seaweed with 80% aqueous ethanol at room temperature and 70 °C removed large amounts of materials. Their analysis revealed small proportions of carbohydrates and proteins, while the monosaccharide composition indicated that mannitol was nearly the only carbohydrate present. Hydrolysis yielded traces

Discussion

Several different procedures have been used for the extraction of fucoidans.24., 26., 27., 28. The main concern in their isolation procedures was to avoid contamination with other polysaccharides, like laminaran and especially alginic acid.⁴⁹ First extraction attempts were carried out by the use of plain water, often acidified, or other solvents.⁴⁹ The first attempt to carry out a systematic approach to extraction was effected by Mian and Percival.²⁴ They developed a sequential extraction that

Acknowledgements

N.M.A.P. was recipient of a fellowship from FOMEC-UBA. E.B.D. and C.A.S. are Research Members of the National Research Council of Argentina (CONICET). M.L.F. is member of RIPRONAMED (Iberoamerican Network on Medicinal Natural Products), Sub-Program X (Pharmaceutical Fine Chemistry) of CYTED. This work was supported by grants from UBA, CONICET, and UNPSJB.

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