Natalia M. Shevchenko

Structural analysis of a fucoidan from the brown alga Fucus evanescens by MALDI-TOF and tandem ESI mass spectrometry.

A fucoidan, a heterogeneous sulfated polysaccharide from the brown alga Fucus evanescens, was depolymerized under solvolytic conditions, and its ethanol-extracted low-molecular-weight fraction was analyzed by MALDI-TOFMS and ESIMS/MS. It was found that the mixture contained unsulfated oligosaccharides including some monosulfated components, which were shown to consist of mainly (1-->3)-linked 2-O-sulfonated fucose residues (from 1 to 4). Minor components of the mixture were shown to contain 2-O- and 4-O-sulfonated xylose and galactose residues. Among them, mixed monosulfonated fucooligosaccharides were detected and characterized: Xyl-(1-->4)-Fuc, Gal-(1-->4)-Fuc, Gal-(1-->4)-Gal-(1-->4)-Fuc, Gal-(1-->4)-Gal. Fucose, galactose, and xylose residues were shown to be mainly 2-O-sulfonated with traces of 4-O-sulfonation. Glucuronic acid was also found as a part of non-sulfated fucooligosaccharides: Fuc-(1-->3)-GlcA, Fuc-(1-->4)-Fuc-(1-->3)-GlcA, Fuc-(1-->3)-Fuc-(1-->4)-Fuc-(1-->3)-GlcA.

Structural analysis of a highly sulfated fucan from the brown alga Laminaria cichorioides by tandem MALDI and ESI mass spectrometry.

Water-soluble polysaccharide fractions were extracted from the brown alga Laminaria cichorioides. Samples were collected monthly from May to October in Troitsa Bay (Japan Sea, Russia). Analysis showed that the content and monosaccharide composition of the fractions changed with the collection season. Fucoidan was isolated and purified from the most fucose-rich fraction, collected in July, and subjected to autohydrolysis to obtain fucooligosaccharides, suitable for mass-spectrometric analysis. Both ESIMS and MALDI-TOFMS analyses show that multisulfated (up to 3) fucooligosaccharides with polymerization degree n from 2 to 5, including mono- and disulfated-fucose residues, were the major products of autohydrolysis. The structural features of the fucooligosaccharides and their alditol derivatives were elucidated by tandem MALDI-TOFMS and ESIMS. The results obtained allowed us to conclude that fragments of the fucoidan, collected in July, were predominantly linked with a (1→3)-type of linkage and that sulfate groups occupied mostly C-2 or C-2/C-4 of the α-l-fucose residues.

Polysaccharide and lipid composition of the brown seaweed Laminaria gurjanovae

Polysaccharide and lipid composition of the Pacific brown seaweed Laminaria gurjanovae is determined. Alginic acid is shown to be the main polysaccharide of its biomass (about 28%); it consists of mannuronic and guluronic acid residues at a ratio of 3: 1. The yield of water-soluble polymannuronic acid is low and does not exceed 1.1% of dry biomass. High laminaran content (about 22%) is found, whereas the yield of fucoidan is no more than 3.6%. Laminaran consists of two fractions, soluble and insoluble in cold water, their ratio is 2.5: 1. Insoluble laminaran is a practically linear 1,3-β-D-glucan, and the soluble fraction was shown to be 1,3;1,6-β-D-glucan. The oligosaccharide products of desulfation or partial acidic hydrolysis of fucoidan were studied by MALDI TOF MS; they were found to be fuco- and galactooligosaccharides. The fucoidan is suggested to be a highly sulfated partially acetylated galactofucan (Fuc/Gal is ∼1: 1). The main lipid components of the dried L. gurjanovae are neutral lipids and glyceroglycolipids, whereas phospholipids are found in minor amounts. The main fatty acid components of lipids are 14:0, 16:0, 16:1 ω-7, 18:1 ω-7 and 18:2 ω-6 acids.

ESIMS analysis of fucoidan preparations from Costaria costata, extracted from alga at different life-stages

Four fucoidan fractions from brown alga Costaria costata, collected at different life-stages: vegetative, May (5F2 and 5F3) and generative, July (7F1 and 7F2) collections were characterized. It was found that seaweed synthesizes different set of fucoidans - one with high fucose content and substantial percentage of hexoses and uronic acid and lower sulfate content (7F1, 5F2 and 5F3) and other - highly sulfated galactofucan (7F2). Structural features of fractions 7F2 and 5F3 were predominantly determined by mass spectrometric analysis of low-molecular-weight (LMW) oligosaccharide fragments, obtained by autohydrolysis of 7F2 and mild acid hydrolysis of 5F3 fucoidans. It was found that oligosaccharides from 7F2 fractions were mainly built up of sulfated at C-2 and/or at C-2/C-4 (1→3)-linked α-l-fucopyranose residues. d-Galactose residues, sulfated either at C-2 or C-6, were found as parts of mixed di- and trisaccharides at both termini and, probably, internal. Fucose residues in 5F3 fucoidan fragments were sulfated at C-2 and sometimes at C-4. Galactose residues were sulfated at C-4 and less frequently at C-2. Resistant to hydrolysis fraction was probably a core, built up with fucose, mannose and glucuronic acid. Presumably, oligosaccharide fragments were branches at C-4 of GlcA. They were sulfated at C-2 and sometimes at C-4 (1→3)- and/or (1→4)-linked fucooligosaccharides (sometimes terminated with (1→3)-linked galactose) and sulfated at C-4 or C-2 (1→4)- or, probably, (1→6)-linked galactooligosaccharides, probably, with own branches, formed by (1→2)-linked galactose residues. Unsulfated xylose residues were probably terminal in chains built up of fucose. It was confirmed, that monosaccharide content and structure of fucoidans of vegetative algae changed following its life stage. Generative alga in general produced highly sulfated galactofucan having lower MW along with less sulfated mannoglucuronofucan with higher MW, which was extensively synthesized by vegetative algae.

Further studies on structure of fucoidan from brown alga Saccharina gurjanovae

A sulfated galactofucan SgF (MW 123kDa) was purified from the brown alga Saccharina gurjanovae. Polysaccharide was depolymerized by autohydrolysis at 25 and 60°C, and products were studied by mass spectrometry and (13)C NMR spectroscopy. According to results of investigation, the main chain of this polysaccharide is built of a repeating units →3)-α-L-Fucp-(2,4-OSO3(-))-(1→. Fucose chains could be sometimes terminated by (1→3)-linked galactose residues. Shorter (1→4)- and/or (1→6)-linked sulfated galactose chains are attached at positions C-2, C-3 of fucose residues. Sulfate groups can occupy positions C-2 and/or sometimes C-3 of Gal residues, but a sulfation at C-4 of the galactofucan could not be excluded. The SgF-AH25-H preparation (71kDa) was obtained by autohydrolysis of SgF at 25°C, which leaded to a selective desulfation at C-2 and, probably, to a cleavage of galactose chains, since structure of SgF-AH25-H represented a repeating unit →3)-α-l-Fucp-(4-OSO3(-))-(1→, which was definitely established by (13)C NMR spectroscopy. Galactofucan SgF and its derivative SgF-AH25-H exhibited no cytotoxic activity and leaded to about the same colony formation inhibition in colon cancer DLD-1 cells. Hence, structural simplification of SgF by lowering its molecular weight, desulfation at C-2 and removing of galactose residues by autohydrolysis at 25°C did not decrease its anticancer activity. This procedure allows obtaining standardized products which can be used as medical.

Structure and anticancer activity in vitro of sulfated galactofucan from brown alga Alaria angusta.

Laminaran and three fractions of fucoidan were isolated from brown alga Alaria angusta. The laminaran AaL was characterized as a typical 1,3;1,6-β-D-glucan (ratio of bonds 1,3:1,6 = 10:1). Fucoidans AaF1 and AaF2 are sulfated heteropolysaccharides, containing fucose, galactose, mannose and xylose. The fraction AaF3 is sulfated and acetylated galactofucan with the main chain represented by a repeating unit → 3)-α-L-Fucp-(2,4-SO3(-))-(1 →. According the data of methylation analysis, AaF3 contains mainly 1,3-linked fucose, less 1,4-linked and 1,4,6-linked galactose residues. The autohydrolysis (37 °C) of fucoidan AaF3 allowed to obtain selectively 2-desulfaled polysaccharide fraction, built up of fucose only, and low molecular weight (LMW) fraction. The negative-ion tandem mass spectrometry of LMW fraction, further hydrolyzed by acid hydrolysis identified the following fragments: Gal-2-SO3(-)-(1 → 4)-Gal, Gal-4-SO3(-)-(1 → 4)-Gal, Gal-(1 → 2)-Gal-4-SO3(-), Fuc-2-SO3(-)-(1 → 4)-Gal, Gal-2-SO3(-)-(1 → 3)-Fuc-(1 → 3)-Fuc, Fuc-2-SO3(-)-(1 → 3)-Fuc-(1 → 4)-Gal. The laminaran AaL and the fucoidan AaF3 exhibited no cytotoxicity in vitro for HT 29, T-47D, and SK-MEL-28 cell lines. The AaF3 fraction suppressed colony formation of HT 29 and T-47D cells, AaL-only HT 29 cells.

Structural similarities of fucoidans from brown algae Silvetia babingtonii and Fucus evanescens, determined by tandem MALDI-TOF mass spectrometry

Rapid mass spectrometric investigation of oligosaccharides, obtained by autohydrolysis of fucoidans from brown algae Silvetia babingtonii and Fucus evanescens (Fucales, Phaeophyceae) has shown both similarities and differences in structural features/sulfation pattern of their fragments, obtained in the same conditions. Tandem MALDI-TOF MS of fucooligosaccharides with even DP (degree of polymerization) was close to that observed for fucoidan from F. evanescens. Slight differences in tandem mass spectra of fragments with odd DP indicated, probably, sulfation at C-3 (instead of C-2 in F. evanescens) of some (1→4)-linked α-L-Fucp residues and/or the presence of short blocks, built up of (1→3)-linked α-L-Fucp residues.

Polysaccharides of Diatoms Occurring in Lake Baikal

Polysaccharide composition of neutral, acid- and alkali-soluble fractions of the diatoms Stephanodiscus meyerii Genkal et Popovsk and Aulacoseira baicalensis (K. Meyer) Simonsen of Lake Baikal has been studied. Neutral polysaccharides were represented by chrysolaminarans (1→3;1→6-β-D-glucans). The chrysolaminaran from S. meyerii consists of the high- and low-molecular-weight fractions (40 and 2–5 kDa, respectively) and contains a large number of β-1→6-bound glucose residues. The chrysolaminaran from A. baicalensis is a low-molecular-weight 1→3;1→6-β-D-glucan containing a small number of β-1→6-bonds, with mannitol being attached to the reducing unit of its chain. Acid- and alkali-soluble polysaccharide fractions are practically absent in S. meyerii. The alkali-soluble fraction from A. baicalensis is a low-molecular-weight (2-kDa) glycoprotein, the carbohydrate moiety of which is represented by a heteropolysaccharide.

Structural features and anticancer activity in vitro of fucoidan derivatives from brown alga Saccharina cichorioides

A fucoidan ScF from brown alga Saccharina cichorioides was extracted, purified and partially depolymerized by autohydrolysis at 37°C for 24, 48 and 72h. Supernatant (SN) and pellet (PL) fractions were obtained by ethanol precipitation of each sample. Unlike spectral data of ScF, NMR of PL derivatives clearly suggested the structure: 1,3-linked α-l-Fucp-4-OSO3- repeating unit. Molecular weights (MWs) of PL fractions were 30, 26 and 18kDa for 24, 48 and 72h of autohydrolyis, respectively. MALDI-TOFMS, size-exclusion HPLC and carbohydrate polyacrylamide-gel electrophoresis (C-PAGE) indicated a similarity of SN mixtures. They consisted mainly of a polysaccharide part (MW 6kDa, C-PAGE data) with a structure similar to PL components (NMR data) and monosaccharides α-l-Fucp-4-OSO3-, α-l-Fucp-2,4-di-OSO3-. PL fractions exhibited almost identical antiproliferative activity in vitro as native fucoidan, while an SN sample for 72h of autohydrolysis was slightly more active against colony formation of colorectal carcinoma cells HT-29.

The comparison of structure and anticancer activity in vitro of polysaccharides from brown algae Alaria marginata and A. angusta

Laminaran and three fucoidan fractions were obtained from the brown alga Alaria marginata. Alaria angusta, studied earlier by us, has the same polysaccharide composition. Galactofucan AmF3 from A. marginata has a main chain of →3)-α-l-Fucp-(2,4-SO3(-))-(1→residues, similar to galactofucan from A. angusta. However, the structure of the branches in fucoidan AmF3 can differ from those in the fucoidan from A. angusta. The following fragments were identified in AmF3: HexA-(1→2)-Fuc, HexA-(1→2)-Gal, Gal-(1→4)-HexA, Fuc-(1→2)-Gal-6-SO3(-), Fuc-4-SO3(-)-(1→6)-Gal, Gal-(1→2)-Gal-2-SO3(-), Gal-4-SO3(-)-(1 →6)-Gal, Gal-4-SO3(-)-(1→3)-Fuc-(1→3)-Fuc, Fuc-4-SO3(-)-(1→6)-Gal-(1→4)-Gal, Gal-(1→4)-Gal-(1→3)-Fuc, Gal-2-SO3(-)-(1→4)-Gal-(1→4)-Gal, Gal-(1→4)-Gal-6-SO3(-)-(1→2)-Gal. Chains of galactose residues (DP up to 9) were found in AmF3 fucoidan. The laminarans, galactofucans and their derivatives from both algae exhibited no cytotoxicity in vitro. Polysaccharides from A. angusta were more effective against colony formation of HT-29 cells, while those from A. marginata had a greater effect on T-47D cells. Sulfated and desulfated fucoidans possessed weak antitumor activity using SK-MEL-28 cells.