A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]     

A fucoidan fraction from Ascophyllum nodosum.

A fucoidan fraction was purified from the brown alga Ascophyllum nodosum. The polysaccharide contained L-fucose and sulfate as the only constituents. Combination of methylation analysis, Smith degradation, FTIR and NMR spectroscopy on the native and the de-sulfated polymers demonstrated that the fucoidan consisted of a highly branched core region with primarily alpha-(1-->3)-linked fucosyl residues and a few alpha-(1-->4) linkages. Branch points were at position 2 of the -->3-linked internal residues. The side chains consisted of single and multi-unit fucosyl residues. The combined analytical data suggested also a complex sulfation pattern with substitution principally at position 2 and/or position 4. Such diversity in the structural features of this fucoidan may be of importance for its various biological properties.     

Effect of Enzyme Preparation from the Marine Mollusk Littorina kurila on Fucoidan from the Brown Alga Fucus distichus

A fucoidanase preparation from the marine mollusk Littorina kurila cleaved some glycosidic bonds in fucoidan from the brown alga Fucus distichus, but neither fucose nor lower oligosaccharides were produced. The main product isolated from the incubation mixture was a polysaccharide built up of disaccharide repeating units -->3)-alpha-L-Fucp-(2,4-di-SO3(-))-(1-->4)-alpha-L-Fucp-(2SO3(-))-(1-->, the structure coinciding with the idealized formula proposed for the initial substance. A polymer fraction with the same carbohydrate chain but sulfated only at positions 2 and nonstoichiometrically acetylated at positions 3 and 4 of fucose residues was isolated as a minor component. It is suggested that the native polysaccharide should contain small amounts of non-sulfated and non-acetylated fucose residues, and only their glycosidic bonds are cleaved by the enzyme. The enzymatic hydrolysis showed that irregular regions of the native polysaccharide containing acetylated and partially sulfated repeating units were assembled in blocks.     

Sulfation of fucoidan in Fucus embryos. I. Possible role in localization

Zygotes of the brown alga Fucus distichus L. Powell divide into two cells which are structurally and biochemically different from each other. Cytochemical staining and autoradiography indicate that a sulfated polysaccharide is localized in only one of the two cells. Up to 10 hr after fertilization, no localization of sulfated polysaccharides is detectable in zygotes, and little ³⁵S (Na₂³⁵SO₄) is incorporated into an acid-soluble carbohydrate fraction. Between 10 and 16 hr, during rhizoid initiation and several hours before the first cell division, there is a large increase in the amount of ³⁵S incorporated into this fraction. The label is found associated with the sulfated fucose polymer fucoidan. Various extraction techniques and labeling experiments demonstrate that fucoidan is unsulfated at fertilization and undergoes little metabolic activity or turnover during the first 24 hr. Thus, the incorporation of sulfate into this carbohydrate fraction appears to involve a sulfation of a preexisting, unsulfated fucan polymer. The degree of sulfation achieved at this time in vivo is sufficient for migration of fucoidan through an electric field in agarose or acrylamide gels. The possible role of sulfation as a mechanism for the localization of fucoidan in the rhizoid cell by means of an intracellular electrical gradient is discussed.     

Structure of a fucoidan from the brown seaweed Fucus serratus L

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1:0.1 and small amounts of xylose and galactose were isolated from the brown seaweed Fucus serratus L. The fucoidan structure was investigated by 1D and 2D 1H and 13C NMR spectroscopy of its desulfated and de-O-acetylated derivatives as well as by methylation analysis of the native and desulfated polysaccharides. A branched structure was suggested for the fucoidan with a backbone of alternating 3- and 4-linked alpha-L-fucopyranose residues, -->3)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->, about half of the 3-linked residues being substituted at C-4 by trifucoside units alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-alpha-L-Fucp-(1-->. Minor chains built up of 4-linked alpha-fucopyranose and beta-xylose residues were also detected, but their location, as well as the position of galactose residues, remained unknown. Sulfate groups were shown to occupy mainly C-2 and sometimes C-4, although 3,4-diglycosylated and some terminal fucose residues may be nonsulfated. Acetate was found to occupy C-4 of 3-linked Fuc and C-3 of 4-linked Fuc in a ratio of about 7:3.     

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of -->2)-alpha-D-Man(1--> and -->4)-beta-D-GlcA(1-->, with a minor portion of -->4)-beta-D-Gal(1--> units. The branch points were at C-3 of -->2)-Man-(1-->, C-2 of -->4)-Gal-(1--> and C-2 of -->6)-Gal-(1-->. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1-->4)-, (1-->3)- and (1-->2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1-->4)-linked. Most of the mannose units were (1-->2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1-->6)-linked. The absolute configurations of the sugar residues were alpha-D-Manp, alpha-L-Fucp, alpha-D-Xylp, beta-D-Galp and beta-D-GlcpA. Sulfate groups in F32 were at C-6 of -->2,3)-Man-(1-->, C-4 and C-6 of -->2)-Man-(1-->, C-3 of -->6)-Gal-(1-->, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.     

Characterization of a fucoidan from Lessonia vadosa (Phaeophyta) and its anticoagulant and elicitor properties

Blades of Lessonia vadosa (Phaeophyta) were extracted with 2% CaCl(2) solution, affording in 4.4% yield a polysaccharide which contained fucose and sulfate groups in the molar ratio 1.0:1.12. The high negative optical activity value ([alpha](D)(22)=-134.0 degrees ), FT-IR and NMR analysis suggest the presence of a fucoidan. (13)C NMR spectrum of the polysaccharide obtained by solvolytic desulfation of native fucoidan indicated the major presence of 1-->3 linked alpha-l-fucan. Depolymerization of the native fucoidan with H(2)O(2) in the presence of copper(II) acetate gave in 54.8% yield a fraction with 33.7% of sulfate content. The native fucoidan (MW 320,000) showed good anticoagulant activity whereas the radical depolymerized fraction (MW 32,000) presented a weak anticoagulant activity. These polysaccharides showed significant activation of phenylalanine-ammonia lyase (PAL), lipooxygenase (LOX) and glutathione-S-transferase (GST) defence enzyme activities in tobacco plants.     

Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1.23:0.36 was isolated from the Pacific brown seaweed Fucus evanescens. The structures of its desulfated and de-O-acetylated derivatives were investigated by 1D and 2D (1)H and (13)C NMR spectroscopy, and the data obtained were confirmed by methylation analysis of the native and desulfated polysaccharides. The fucoidan was shown to contain a linear backbone of alternating 3- and 4-linked alpha-L-fucopyranose 2-sulfate residues: -->3)-alpha-L-Fucp(2SO(3)(-))-(1-->4)-alpha-L-Fucp(2SO(3)(-))-(1-->. Additional sulfate occupies position 4 in a part of 3-linked fucose residues, whereas a part of the remaining hydroxyl groups is randomly acetylated.     

Differentiation of the fucoidan sulfated L-fucose isomers constituents by CE-ESIMS and molecular modeling

Alpha-L-fucose, the monosaccharide component of fucoidan, is found in the polysaccharide mainly as its sulfated form where sulfate groups are in position 2 and/or 4 and/or 3. The correlation between biological activities and structure of fucoidan requires the determination of the sulfation pattern of the fucose residues. Therefore, it is of importance to discriminate between the isobaric sulfated fucose isomers. For this purpose, the three isomers 2-O-, 3-O-, and 4-O-sulfated fucose have been analyzed using electrospray ion trap mass spectrometry and capillary electrophoresis. The results reported herein show that it is possible to differentiate between these three positional isomers of sulfated fucose based on their fragmentation pattern upon MS/MS experiments. 3-O-Sulfated fucose was characterized by the loss of the hydrogenosulfate anion HSO4- as the main fragmentation product, while the two other isomers 2-O-, and 4-O-sulfated fucose exhibited cross-ring fragmentation yielding to distinctive (0,2)X and (0,2)A daughter ion, respectively. A computational study of the conformation of the sulfated fucose isomers was carried out providing an understanding of the fragmentation pattern with respect to the position of the sulfate group.     

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.     

Optimization of citric acid treatment for the sequential extraction of fucoidan and alginate from Sargassum latifolium and their potential antioxidant and Fe(III) chelation properties

In this study, citric acid was applied as a safe organic acid for the treatment of the brown macroalga Sargassum latifolium to facilitate the sequential extraction of fucoidan and alginate without cross contamination. Box-Behnken experimental design was used to investigate the effects of citric acid concentration, temperature, and time on fucoidan yield, its fucose and sulfate contents, and molecular weight (MW), while the investigated responses of the sequentially extracted alginate were yield, mannuronic/guluronic acid ratio (M/G) and MW. Under the optimized conditions, the fucoidan yield, fucose content, sulfate content, and MW were 6.55%, 21.01%, 30.92% and 7.12 × 10³ Da, respectively, while alginate yield, M/G, and MW were 28.81%, 0.36, and 1.50 × 10⁵ Da, respectively. The optimized products showed good Fe(III) chelating properties and the iron contents were 509.82 mg g^?1 for fucoidan-Fe and 406.71 ± 5.18 mg g^?1 for alginate-Fe. Under simulated gastrointestinal conditions, fucoidan-Fe and alginate-Fe complexes released 46 and 57% iron, respectively, which are nutritionally relevant amounts. Both fucoidan and alginate showed potent antioxidant properties, but their Fe complexes exhibited a reduced activity. The results of the present study indicated that citric acid could support the basis for an economical biorefinery process for the sequential extraction of fucoidan and alginate. Additionally, these polysaccharides could be good candidates for the preparations of iron supplements.

     

Separation,purification, anticoagulant activity and preliminary structural characterization of two sulfated polysaccharides from sea cucumber Acaudina molpadioidea and Holothuria nobilis

In this study, we isolated two fucosylated polysaccharide sulfates (ACP and HOP) from sea cucumber Acaudina molpadioidea and Holothuria nobilis, with an average molecular weight of 90.8 and 135.8 kDa, respectively. We investigated and compared their anticoagulant activities through anticoagulant assay. Our data showed that both polysaccharides possessed good anticoagulant activity, but HOP's activity was higher than that of ACP. Due to the different anticoagulant activities of ACP and HOP, we compared the preliminary structural characterizations of these two sulfated polysaccharides, and found that both ACP and HOP consisted of β-d-glucuronic acid, β-d-N-acetyl-galactosamine, α-l-fucose and sulfate groups. ACP and HOP had almost identical ratios of glucuronic acid, N-acetyl-galactosamine and fucose. However, the sulfate contents and sulfation patterns of fucose residues of ACP and HOP were obviously different. There were 4-O-sulfated fucose, 3,4-O-disulfated fucose and 2,4-O-disulfated fucose in ACP, but only 3-O-sulfated fucose and 2,4-O-disulfated fucose were present in HOP. Therefore, their distinct anticoagulant activities might be due to the different sulfate contents and sulfation patterns of their fucose residues.     

Structural studies on fucoidans from the brown seaweed Sargassum stenophyllum.

The brown seaweed Sargassum stenophyllum biosynthesizes two different sets of fucoidans. One of them is characterized by higher percentages of glucuronic acid and fewer sulfate groups, which are situated on different sugar units. alpha-L-Fucose was the major component but other sugars like beta-D-galactose, beta-D-mannose, alpha-D-glucuronic acid, alpha-D-glucose and beta-D-xylose were also in substantial amounts. Fucoidans from the other set contain small amounts of alpha-D-glucuronic acid and high percentages of sulfate groups, which are concentrated on the fucose residues, with only fucose and galactose as major components. Structural studies of one fucoidan from each set suggest that these products have a general basic structure that has a formal resemblance to that of the fucosylated chondroitin sulfates from the body wall of sea cucumbers, namely, a linear core (formed mainly by (1-->6)-beta-D-galactose and/or (1-->2)-beta-D-mannose units) with branched chains of 'fucans' (formed by (1-->3) and/or (1-->4)-alpha-L-fucose, (1-->4)-alpha-D-glucuronic acid, terminal beta-D-xylose and, sometimes, (1-->4)-alpha-D-glucose). In fucoidans from the second set, the 'core' is reduced to short galactan chains.     

Regioselective desulfation of sulfated L-fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximus: application to the structural study of algal fucoidan (Ascophyllum nodosum).

The study of the structural bases of the biological properties of algal fucoidan (Ascophyllum nodosum) led us to look for enzymes able to modify this sulfated polysaccharide. In this context, we found a sulfoesterase activity in the digestive glands of the common marine mollusk Pecten maximus, which is active on fucoidan. This sulfoesterase activity was shown by capillary electrophoresis and 13C-1H NMR (500 MHz) analysis of the enzymatic hydrolysis of the fucoidan, of fucoidan oligosaccharides and of sulfated fucose isomers. We report the exhaustive list of all proton and carbon chemical shifts for each of the three isomers of sulfated-l-fucose (including of their alpha/beta anomers), i.e. the 2-O-, 3-O- and 4-O-sulfated fucose, which have been useful reference values for the assignments of NMR spectra of fucoidan oligosaccharides upon enzymatic desulfation. Our results demonstrated a high regioselectivity for this sulfoesterase, which hydrolyzes only the sulfate group at the 2-O position of the fucopyranoside. Therefore, this sulfoesterase is a helpful tool in the structure-activity study of the fucoidan, as the literature data suggest that the 2-O-sulfation level play a central role in the biological properties of the polysaccharide.     

Structural Characterization and α‐Glucosidase Inhibitory and Antioxidant Activities of Fucoidans Extracted from Saccharina japonica

Two sulfated fucoidan fractions (Lj3 and Lj5) were extracted from Saccharina japonica and then subjected to acid hydrolysis to obtain Lj3h and Lj5h. Lj3h and Lj5h were characterized using IR, methylation analysis, and mass spectrometry. It was found that Lj3h and Lj5h were homogeneous low molecular weight fucoidans. Specifically, Lj3h was composed of the main chain of 1,3‐linked α‐L‐fucopyranose residues with sulfate at C‐2 and/or C‐4 and three different monosaccharides (galactose, glucose, mannose) branched at C‐2 and/or C‐4 of fucose residue. Lj5h contained backbones of alternating galactopyranose residues and fucopyranose residues attached via a 1→3 linkage (galactofucan) and 1→6 linked galactan. The sulfation pattern was mainly located at C2/C4 fucose or galactose residues and more branches occupied at C‐4 of fucose residue and C‐2, C‐3 or/and C‐6 of galactose residue. In vitro assay indicated that, among the four fucoidans tested, only Lj5 showed potent α‐glucosidase inhibitory activity with IC₅₀ of 153.27±22.89 μg/mL, and the two parent fucoidans, Lj3 and Lj5, showed better antioxidant activity than their derivatives. These findings highlight the structure and bioactivity diversity of Saccharina japonica‐derived fucoidans.     

Polysaccharides of algae: 60. Fucoidan from the Pacific brown alga Analipus japonicus (Harv.) Winne (Ectocarpales, Scytosiphonaceae)

A fucoidan containing L-fucose, sulfate, and O-acetyl groups at a molar ratio of 3 : 2 : 1, as well as minor amounts of xylose, galactose, and uronic acids was isolated from the brown alga Analipus japonicus collected in the Sea of Japan. The structures of the native polysaccharide and the products of its desulfation and deacetylation were studied by the methods of methylation, periodate oxidation, and NMR spectroscopy. It was shown that the polysaccharide molecule mainly consists of a linear carbohydrate chain of (1-->3)-linked alpha-L-fucopyranose residues, which bear numerous branches in the form of single alpha-L-fucopyranose residues (three branches at position 4 and one branch at position 2 per each ten residues of the main chain). Sulfate groups occupy positions 2 and (to a lesser extent) 4, most of the terminal nonreducing fucose residues being sulfated twice. The acetyl groups are located predominantly at positions 4. The structural role of minor monosaccharides was not established.     

Structural characteristics and biological activity of Fucoidans from the brown algae Alaria sp. and Saccharina japonica of different reproductive status

Structural characteristics and the antitumor activity of fucoidans isolated from vegetative and reproductive tissue of the brown algae Alaria sp. and Saccharina japonica were studied. The reproductive status of the brown algae affected the yield of fucoidans and their structural characteristics. The fucoidan yield was 5.7% (w/w on the basis of the dried algae weight) for fertile and 3.8% for sterile Alaria sp. and 1.42 and 0.71% for fertile and sterile S. japonica, respectively. The fucoidans from fertile Alaria sp. and S. japonica had a slightly higher degree of sulfation and a somewhat more homogeneous monosaccharide composition, with predominate amounts of fucose and galactose, than those isolated from sterile algae tissue. The fucoidans from both the sterile and fertile brown algae tissue tested possessed selective cytotoxicity towards human breast cancer (T-47D) and melanoma (RPMI-7951) cell lines, but not to normal mouse epidermal cells (JB6 Cl41), and effectively inhibited the proliferation and colony formation of the breast cancer and melanoma cell lines. The fucoidans from reproductive tissue of brown algae possessed higher antitumor activity than those from vegetative plants.     

A highly regular fraction of a fucoidan from the brown seaweed Fucus distichus L

A fucoidan fraction consisting of L-fucose, sulfate, and acetate in a molar proportion of 1:1.21:0.08 was isolated from the brown seaweed Fucus distichus collected from the Barents Sea. The 13C NMR spectrum of the fraction was typical of regular polysaccharides containing disaccharide repeating units. According to 1D and 2D 1H and 13C NMR spectra, the fucoidan molecules are built up of alternating 3-linked alpha-L-fucopyranose 2,4-disulfate and 4-linked alpha-L-fucopyranose 2-sulfate residues: -->3)-alpha-L-Fucp-(2,4-di-SO3-)-(1-->4)-alpha-L-Fucp-(2SO3-)-(1-->. The regular structure may be only slightly masked by random acetylation and undersulfation of several disaccharide repeating units.     

Separation and structural analysis of some saponins from Quillaja saponaria Molina

A fraction of saponins from Quillaja saponaria Molina, QH-B, was fractionated by consecutive separations on three different reverse-phase HPLC systems. Eight compounds were isolated and the structures of these were elucidated mainly by sugar analysis and NMR spectroscopy. The structures consisted of a quillaic acid substituted with two different trisaccharides at C-3, beta-D-Galp-(1-->2)-[alpha-L-Rhap-(1-->3)]-beta-D-GlcpA and beta-D-Galp-(1-->2)-[beta-D-Xylp-(1-->3)]-beta-D-GlcpA, and a tetra- or pentasaccharide at C-28, beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3)]-alpha-L-Rhap-(1--> 2)-beta-D-Fucp and beta-D-Apif-(1-->3)-beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3) ]-alpha-L- Rhap-(1-->2)-beta-D-Fucp. These compounds were further substituted with an acyl group either at O-3 or O-4 of the fucose residue, which is the sugar linked to C-28 of the quillaic acid.     

羊栖菜褐藻糖胶抗凝血活性的研究

本文研究了羊栖菜褐藻糖胶的化学组成和抗凝血活性之间的关系。采用热水提取得羊栖菜粗多糖,CaCl2纯化得褐藻糖胶,DEAE Sepharose CL-6B柱层析与Sepharose CL-6B柱层析对褐藻糖胶进行分级,得到F1、F2、F31、F32和F33五个级分,均为岩藻糖、半乳糖和甘露糖等糖基组成的杂多糖,并含有硫酸酯和糖醛酸以及少量的蛋白质,相对分子质量范围2．5万～95万。采用活化部分凝血活酶时间(APTT)和凝血酶时间(TT)检测了这5个级分的抗凝血活性,结果显示,羊栖菜褐藻糖胶能显著延长APTT的凝血时间,而对TT的影响不明显。F1、F31和F32对APTT的影响比较显著,而F2、F33和羊栖菜粗多糖的影响较小。研究表明,羊栖菜褐藻糖胶主要是通过抑制内源凝血途径而达到抗凝血的效果,其抗凝血活性与褐藻糖胶的硫酸基含量成正相关,而与相对分子质量和糖醛酸含量无关。