Galactans from Cryptonemia species. Part II: Studies on the system of galactans of Cryptonemia seminervis (Halymeniales) and on the structure of major fractions

Cryptonemia seminervis biosynthesizes a family of d,l-hybrid galactans based on the classical 3-linked β-d-galactopyranosyl→4-linked α-d- and α-l-galactopyranosyl alternating sequence (A-units→B-units) with major amounts of α-d- and α-l-galactose and 3,6-anhydro-d- and l-galactose and lesser percentages of 3,6-anhydro-2-O-methyl-l-galactose, 2-O-methyl-, 4-O-methyl- and 6-O-methylgalactoses. The dispersion of structures in this family is based on five structural factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and glycosyl side-chain (4-O-methyl galactopyranosyl and/or xylosyl); (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d,l-galactoses and d,l-3,6-anhydrogalactoses also in the B-units, (d) the formation of diads and (e) the sequence of the diads in the linear backbone. Considering these variables it is not unexpected to find in the fractions studied at least 18 structural units producing highly complex structures. Structural studies carried out in two major fractions (S2S-3 and S2S-4) showed that these galactans were formed mainly by β-d-galactopyranosyl 2-sulfate (20 and 11.9 mol %), β-d-galactopyranosyl 2-sulfate 4,6-O-(1′-carboxyethylidene) (8.9 and 6.0 mol %) and β-d-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol %), together with 3,6-anhydro-α-l-galactopyranosyl (11.4 and 7.3 mol %) and 3,6-anhydro-α-l-galactopyranosyl 2-sulfate (4.9 and 15.4 mol %) and minor quantities of 12-15 other structural units.Preparative alkaline treatment carried out on fraction (S2S-3) produced a quantitative formation of 3,6-anhydro α-l-galactopyranosyl units from precursor units (α-l-galactose 6-sulfate and α-l-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 × 10⁴ s⁻¹ indicating diads of the type G→L6S/2,6S. Data from chemical, spectroscopic and kinetic studies suggest that, in S2S-3, the agaran block in the d,l-hybrid galactan is composed of the following diads: G(6R)→L6S/2,6S and G2S(P)(2,6S)→LA(2S)(2R)(2M) and the carrageenan block of G2S(P)→D(2S)(2,3S)(3S)(3,6S) in a molar ratio of agaran to carrageenan structures of ∼2:1.     

Chemical characteristics of a polysaccharide from Porphyra capensis (Rhodophyta)

The structure of a polysaccharide from the red seaweed, Porphyra capensis, growing along the coast of Namibia and South Africa was investigated. Algae growing at different sites and collected at different times gave a polysaccharide extract with similar chemical components. FTIR and NMR spectral analysis showed that the polysaccharide from P. capensis had a typical porphyran structure. It has the linear backbone of alternating 3-linked beta-D-galactose and 4-linked alpha-L-galactose-6-sulfate or 3,6-anhydro-alpha-L-galactose units. The ratio of alpha-L-galactose-6-sulfate and the 3,6-anhydrogalactose is 1.2:1, as reflected by a 1H NMR spectrum. A high degree of methylation occurred at the C-6 position of the D-galactose units. The degree of methylation was 0.64 for the D-galactose residues.     

Chemical structure of the complex pyruvylated and sulfated agaran from the red seaweed Palisada flagellifera (Ceramiales, Rhodophyta)

A homogeneous agaran fraction from Palisada flagellifera (Laurencia complex, Rhodomelaceae, Ceramiales) was obtained by aqueous room-temperature extraction, followed by ion-exchange chromatography. This galactan presents a highly complex structure with at least 18 different types of derivatives. The A units were found mostly pyruvylated, 2-sulfated (～34%), and 6-methylated (～34%), with the latter partially 2- and 2,4-sulfated. Minor amounts of β-D-galactopyranosyl units 2-, 6- and 2,6-sulfated, 6-glycosylated, and non-substituted are also present. The B-units are L-sugars composed predominantly of their cyclized derivatives, 3,6-anhydrogalactose and 3,6-anhydro-2-O-methylgalactose (～56%). The former are linked to β-D-galactosyl (6-methyl) (6-glycosylated) units, as well as to 4,6-O-(1-carboxyethylidene)-β-D-galactose 2-sulfate in the proportion of 3:1.8, respectively. A significant amount (～18%) of the α-L-galactopyranosyl units are linked to pyruvylated β-D-galactose 2-sulfate residues. An important part of the B-units (20%) is represented by α-L-galactose 6-sulfate substituted on C-3 by xylosyl, galactosyl and/or 2,3-di-O-methylgalactose units or sulfate groups that preclude their cyclization to 3,6-anhydrogalactosyl derivative. The precursor units are present in relatively low percentages. Kinetic studies suggest that in P. flagellifera agaran the cyclizable units are linked to 6-O-methyl-β-D-galactosyl and/or β-D-galactosyl units (6-glycosylated). The structural complexity of this polysaccharide is increased by the presence of 2- and 3,6-sulfated α-L-galactoses, with the latter additionally 2-O-methylated. Therefore, the major subfraction obtained from the cold extract contains structurally complex sulfated, methylated, and pyruvylated agaran.     

Immunostimulatory activity of sulfated galactans isolated from the red seaweed Gracilaria fisheri and development of resistance against white spot syndrome virus (WSSV) in shrimp

Sulfated galactans (SG) were isolated from the red seaweed Gracilaria fisheri (G. fisheri). Chemical analysis revealed SG contains sulfate (12.7%) and total carbohydrate (42.2%) with an estimated molecular mass of 100 kDa. Structure analysis by NMR and FT-IR spectroscopy revealed that SG is a complex structure with a linear backbone of alternating 3-linked β-d-galactopyranose and 4-linked 3,6-anhydrogalactose units with partial 6-O-methylate-β-d-galactopyranose and with sulfation occurring on C4 of d-galactopyranose and C6 of l-galactopyranose units. SG treatment enhanced immune parameters including total haemocytes, phenoloxidase activity, superoxide anions and superoxide dismutase in shrimp Penaeus monodon. Shrimp fed with Artemia salina enriched with SG (100 and 200 μg ml⁻¹) and inoculated with white spot syndrome virus (WSSV) showed a significantly lower mortality rate and lower viral VP 28 amplification and expression than control. The results suggest that SG from G. fisheri exhibits immune stimulatory and antiviral activities that could protect P. monodon from WSSV infection.     

The structure of a sulfated galactan from Porphyra haitanensis and its in vivo antioxidant activity

The sulfated galactan fraction F1 isolated from the red seaweed, Porphyra haitanensis, showed typical porphyran structure. It has a linear backbone of alternating 3-linked beta-D-galactosyl units and 4-linked alpha-L-galactosyl 6-sulfate and 3,6-anhydro-alpha-L-galactosyl units. The L-residues are mainly composed of alpha-L-galactosyl 6-sulfate units, and the 3,6-anhydrogalactosyl units are minor. Partial methylation occurred at the C-6 position of the D-galactosyl units and at the C-2 position of the 3,6-anhydro-alpha-L-galactosyl units. Intraperitoneal administration of F1 significantly decreased the lipid peroxidation in aging mice. F1 treatment increased the total antioxidant capacity and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in aging mice. The results indicated that F1 had significant in vivo antioxidant activity.     

A sulfated galactan from the mucilaginous sheath of the red filamentous alga Chroodactylon ornatum (Stylonematophyceae,Rhodophyta)

The pseudofilamentous red alga Chroodactylon ornatum was grown in f/2 culture medium with the addition of 10, 34, and 100 μM (nominal concentration) cupric sulfate. The bioassays were terminated at two selected end points (days 4 and 12). Growth inhibition, changes in pigment composition, and oxidative stress indicators such as phenolic compounds and lipid peroxidation (dosed as thiobarbituric reactive substances) were observed in cultures with 34 and 100-μM cupric sulfate. Quinacrine (Atebrin) and chlorotetracycline fluorochromes showed abundant vacuoles of acidic content, related with mucilage secretion. Structural analyses by methylation, desulfation-methylation, alkaline treatment, and NMR spectroscopy revealed that the mucilaginous sheath of C. ornatum contains a sulfated galactan with a backbone of alternating 3-linked β-d-galactopyranose and 4-linked α-l-galactopyranose moieties, i.e., an agaran. The absence of 3,6-anhydrogalactose and of its precursor unit (α-galactose 6-sulfate) were confirmed. The highly sulfated polysaccharide contained these ester groups on the C-2 and C-4 hydroxyl groups of the 3-linked unit and on C-3 of the 4-linked units. The large proportion of sulfate esterification in this polysaccharide can be related to the extracellular biosorption of copper divalent cation (2.5?±?0.4 mg per gram of dry weight) and to copper tolerance in bioassays.     

Unusual structures in the polysaccharides from the red seaweed Pterocladiella capillacea (Gelidiaceae,Gelidiales)

Sequential extraction of tetrasporic Pterocladiella capillacea with water at room temperature and then at 50 degrees C led to the isolation of two products that were each fractionated with cetrimide to give a soluble fraction and a precipitate. The precipitates were then subjected to fractional solubilization in solutions of increasing sodium chloride concentration. The whole treatment yielded two major fractions in each case, one soluble in the cetrimide medium and the other soluble in 0.5 M NaCl, which were further fractionated by anion-exchange chromatography. Structural analysis, carried out by methylation, desulfation-methylation, 13C NMR spectroscopy and determination of the absolute configuration of the 2,6-di-O-methylgalactose units in the permethylated products, indicated the presence of xylogalactans, with low content of 3,6-anhydrogalactose and low molecular weight. These polysaccharides varied in the level of xylopyranosyl and sulfate substitution, primarily on the 6-position of the 3-linked beta-D-galactopyranosyl and on the 3-position of the 4-linked alpha-galactopyranosyl units. Moreover, herein we report, for the first time, the presence of 3-substituted, 4-linked D-galactopyranosyl residues in an alga belonging to the Gelidiales.     

The system of sulfated galactans from the red seaweed Gymnogongrus torulosus (Phyllophoraceae, Rhodophyta): Location and structural analysis

Sulfated polysaccharides were localized in the cuticle, cortex and medulla of the gametophyte thallus, being more concentrated in the intercellular matrix than in the cell walls. During the water extraction sequence, a small percentage of galactan sulfates (5.1% of dry seaweed) with average low M_r (6–11.4 kDa) were extracted at room temperature without disturbing the cellular arrangement, while sulfated galactans of average medium M_r (18–45 kDa) were obtained by further hot-water extractions (52.4% of dry seaweed), with diorganization of the tissue. The residue (40.0% of dry seaweed) still contained carrageenan-type (major) and agaran-type (minor) galactans. Part of these galactans was extracted with 8.4% LiCl solution in DMSO, from which “pure” κ/ι-carrageenans were isolated.Carrageenans and agarans were extracted in a ratio 1:0.5, showing the highest amount of agaran-structures for a carrageenophyte. The galactans comprise alternating 4-sulfated (major) and non-sulfated (minor) 3-linked β-d-galactopyranose units, and 4-linked α-galactopyranose units with the following substitutions: (i) non-sulfated and 2-sulfated 3,6-anhydro-α-d-galactopyranose residues in the carrageenan-structures, which belong to the κ-family (κ/ι-carrageenans); (ii) 3-sulfated α-l-galactopyranose units and 2-sulfated 3,6-anhydro-α-l-galactopyranose residues in the agaran-structures.Alkaline treatment and alkaline dialysis of the main extracts gave “pure” κ/ι-carrageenans, showing that carrageenan molecules are extracted together with low M_r agarans or agaran-dl-hybrids.     

Structural analysis and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi (Gigartinales, Rhodophyta)

Aqueous extraction of gametophytic Schizymenia binderi afforded a polysaccharide composed of galactose and sulfate groups in a molar ratio of 1.0:0.89 together with uronic acids (6.8 wt%) and minor amounts of other neutral sugars. Alkali-treatment of the polysaccharide afforded a polysaccharide devoid of 3,6-anhydrogalactose. 13C NMR spectroscopy of the desulfated alkali-treated polysaccharide showed a backbone structure of alternating 3-linked beta-D-galactopyranosyl and 4-linked alpha-galactopyranosyl units that are predominantly of the D-configuration and partly of the L-configuration. Methylation, ethylation and NMR spectroscopic studies of the alkali-treated polysaccharide indicated that the sulfate groups are located mainly at positions O-2 of 3-linked beta-D-galactopyranosyl residue and at position O-3 of 4-linked-alpha-galactopyranosyl residues, the latter is partially glycosylated at position O-2. The sulfated galactan from S. binderi exhibited highly selective antiviral activity against Herpes simplex virus types 1 and 2, with selectivity indices (ratio cytotoxicity/antiviral activity) >1000 for all assayed virus strains. This compound was shown to interfere with the initial adsorption of viruses to cells.     

The structure of the agaran sulfate from Acanthophora spicifera (Rhodomelaceae, Ceramiales) and its antiviral activity. Relation between structure and antiviral activity in agarans

The sulfated agaran isolated by water extraction from the red seaweed, Acanthophora spicifera (Rhodomelaceae, Ceramiales), is made up of A-units highly substituted with sulfate groups on C-2 (28-30%), sulfates on C-2 and 4,6-O-(1'-carboxyethylidene) groups (9-15%), and only the C-2 sulfate groups (5-8%) with small amounts of C-6 sulfate, 6-O-methyl, and nonsubstituted residues. B-units are formed mainly by 3,6-anhydro-alpha-L-galactose (15-16%) and its precursor, alpha-L-galactose 6-sulfate (10-17%), together with lesser amounts of 3,6-anhydro-alpha-L-galactose 2-sulfate, alpha-L-galactose 2,6-disulfate, alpha-L-galactose 2,3,6-tri-sulfate, alpha-L-galactose 2,6-disulfate 3-xylose, 2-O-methyl-alpha-L-galactose, and unsubstituted alpha-L-galactose. Small, but significant quantities of beta-D-xylose were found in all the fractions, together with small amounts to traces of D-glucose. Some of the fractions have high antiviral activity. Attempts to correlate structure and antiviral activity in agarans are presented.     

Structural analysis of carrageenans from the red alga, Callophyllis hombroniana Mont. Kütz (Kallymeniaceae, Rhodophyta)

The use of a range of modern analytical techniques has facilitated the structural characterisation of the polysaccharide from the New Zealand endemic red alga, Callophyllis hombroniana. The native polysaccharide contains a number of structural units with the largest proportion consisting of 3-linked beta-D-galactopyranosyl 2-sulfate units, alternating with 4-linked 3,6-anhydro-alpha-D-galactopyranosyl 2-sulfate units, that is, theta-carrageenan (36 mol%). C. hombroniana is the first red seaweed reported to naturally contain such a large proportion of theta-carrageenan.     

The system of low-molecular-weight carrageenans and agaroids from the room-temperature-extracted fraction of Kappaphycus alvarezii

The room-temperature-extracted fraction from the red seaweed Kappaphycus alvarezii consists mainly of low-molecular-weight carrageenans, with structural dispersion around a basic kappa-pattern. This dispersion results from: (a) low percentages of 3,6-anhydrogalactose and the presence of precursor units; (b) important quantities of 6-O-methyl beta-D-galactose (4-sulfate) residues; (c) significant amounts of iota-repeating structure, and (d) small amounts of non-sulfated and disulfated beta-D-galactose residues. Significant quantities of alpha-L-galactose units suggest the presence of agaroids, as it has been reported in several other carrageenophytes.     

Carrageenan from Sarconema scinaioides (Gigartinales, Rhodophyta) of Indian waters

Carrageenan was extracted from the red seaweed Sarconema scinaioides of Indian waters and was characterized. The crude carrageenan as well as its alkali modified derivative was composed of 3,6-anhydro galactose, 6-O-methyl galactose as well as galactose moieties in various proportions. Linkage analysis exhibited that these two carrageenan samples consisted of 4-linked 3,6-anhydrogalactose residue sulphated at position 2, and 3-linked galactose residue sulphated at position 4. The physicochemical and rheological data along with molecular weight data, FT-IR, 1D and 2D NMR (¹H, ¹³C, COSY and HSQC) spectrometry suggested that the polysaccharide was composed predominantly of iota- along with a small amount of its precursor nu (ν)-carrageenan, unlike the hybrid carrageenans (iota-, pyruvated- and kappa-carrageenans) from this seaweed reported in the literature. This Indian seaweed species would be a potentially important source of iota-carrageenan.     

Comparative analysis of sulfated galactans from red algae by reductive hydrolysis and mild methanolysis coupled to two different HPLC techniques

The sugar determination of the sulfated galactans, agars and carrageenans of various red algae was performed using two different techniques of depolymerisation with subsequent HPLC analysis: 1) reductive hydrolysis/ HPAEC-PAD; 2) mild methanolysis/ RPLC-DR. Both techniques were optimized to release quantitatively the composite sugars (galactose, 6-O-methyl-galactose, the labile 3,6-anhydrogalactose and 2-O-methyl-3,6-anhydrogalactose residues) and precise relative response factors of authentic 3,6-anhydrogalactose were determined. The methanolysate neutralisation step, performed subsequently to methanolysis depolymerisation, was demonstrated as a key step for the quantitative recovery of the anhydrogalactose residues. The yield of the main sugars released by the two techniques were in good agreement for the commercial agarose and iota and kappa carrageenans studied. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antioxidant, cytotoxic and hemolytic effects of sulfated galactans from edible red alga Hypnea musciformis

Polysaccharides, galactans, obtained from edible red seaweed Hypnea musciformis were characterized by molecular weight and infrared spectroscopy analysis and were evaluated for antioxidant activity in vitro and for their effects on cell viability. The main components were galactose and sulfate presenting low protein contamination. These sulfated galactans (F1.0) showed a polydisperse profile, and signs in infrared analysis were attributed to a sulfate ester S?=?O bond, the presence of a 3,6-anhydrogalactose C–O bond, nonsulfated β-d-galactose, and a C–O–SO₄ bond in galactose C4. The NMR analysis showed signals at about 95 and 92 attributed to anomeric carbon of 4-linked 3,6-anhydro-α-d-galactopyranose residue of κ-carrageenans and 4-linked 3,6-anhydro-α-d-galactopyranose2-sulfate of ι-carrageenans. Sulfated galactan F1.0 showed strong antioxidant activity under lipid peroxidation assay where F1.0 at 8 mg mL^?1 promoted 57.92% peroxidation inhibition and displayed the scavenging activity on hydroxyl radicals in a dose-dependent manner leading to 32.5% scavenging of these radicals when 5 mg mL^?1 of sulfated galactan F1.0 was used. The sulfated galactan fraction also exhibited strong inhibition on the H₂O₂-induced hemolysis model. Sulfated galactan F1.0 displayed low cytotoxic action in 3 T3 cells and moderate antitumoral action in HeLa cells. These results suggest that sulfated galactan F1.0 from H. musciformis has antioxidant potential, which is a great effect for a compound used as food and in the food industry.     

Chemical structure analysis of water-soluble sulfated polysaccharide fromSchizymenia dubyi (Rhodophyta,Gigartinales)

Chemical and spectroscopic methods showed that the water-soluble polysaccharide extracted fromSchizymenia dubyi from Sicily was composed of 1/0.75/1.3 galactose, glucuronic acids and sulfate groups; 45% of total galactose was present as the L-form and no 3,6-anhydrogalactose was detected. The structural characteristics of this galactan of molecular weight 290 000 were close to sulfated polysaccharide with 1,3-, 1,4- and terminal-linked galactose units and secondary ramifications in 1,3,6; 1,4,6; 1,3,4 and 1,6. Permethylation analysis suggested the presence of sulfate groups on positions O-2 and/or O-3 of 1,4-linked galactose and on O-2 and/or O-4 of 1,3-linked residues.Author for correspondence     

Chemical characteristics and gelling properties of agar from two Philippine Gracilaria spp. (Gracilariales, Rhodophyta)

The chemical structure of agars extracted from Philippine Gracilaria arcuata and G. tenuistipitata were determined by NMR and infrared spectroscopy. Agar with alternating 3-linked 6-O-methyl-β-D-galactopyranosyl and 4-linked 3,6-anhydro-2- O-methyl-α-L-galactopyranosyl units was isolated from G. arcuata, while the agar from G. tenuistipitata possesses the regular agarobiose repeating unit with partial methylation at the 6-position of the D-galactosyl residues. Both agars exhibit sulphate substitution at varying positions in the polymer. Chemical analyses reveal higher 3,6-anhydrogalactose and lower sulphate contents in alkali-modified than in native agar from both samples. Also, alkali modification enhanced agar gel strength and syneresis. Native G. arcuata agar produces a viscous solution (2000 cP at 75 °C) with a high gelling point (>60 °C) that forms a soft gel even after alkali modification (gel strength: <300 g cm⁻²). On the other hand, the agar from G. tenuistipitata exhibits gel qualities typical of most Gracilaria agars. This revised version was published online in June 2006 with corrections to the Cover Date.     

A new chemical tool for characterization and partial depolymerization of red algal galactans

Complete acid hydrolysis of red algal galactans in the presence of borane - 4-methylmorpholine complex has been shown to prevent the acid degradation of 3,6-anhydrogalactose derivatives by their reduction to the corresponding 3,6-anhydro-galactitols, whereas all the other monosaccharides are liberated essentially in the non-reduced form; the reductive hydrolysis products may be determined quantitatively using gas-liquid chromatography (GLC). The method is recommended for preliminary characterization of the polysaccharide composition of red algal biomass. Partial acid hydrolysis of galactans in the presence of the same reducing agent gives rise to reduced oligosaccharides having terminal 3,6-anhydrogalactitol residues. Based on this reaction, the attribution of unknown galactans to the agar or carrageenan groups is possible by partial reductive hydrolysis of small samples of algal biomass with subsequent identification of agarobiitol or carrabiitol acetates by GLC. Sulfate groups are substantially retained under partial reductive hydrolysis conditions; the isolation by liquid chromatography and elucidation of structures of reduced sulfated oligosaccharides may be of great value for the structural analysis of complex red algal galactans.     

The structure of a galactan sulfate from the red seaweed Bostrychia montagnei

The sulfated, methylated galactan isolated from the red seaweed Bostrychia montagnei, showed an unusually narrow structural dispersion. This agaran has the defining linear backbone of alternating 3-linked beta-D-galactopyranosyl units and 4-linked alpha-L-galactopyranosyl and 3,6-anhydrogalactopyranosyl residues. The D-units have C-6 methylation, C-6 single stubs of xylopyranosyl and minor to trace amounts of (possible) C-6 linked single stubs of galactopyranosyl. These units are mainly sulfated on C-4 with lesser sulfation at C-6 and minor at C-2. The L-residues are mainly methylated on C-2 of the 3,6-anhydrogalactopyranosyl and sulfated on C-3 of the L-galactopyranosyl; minor amounts of 2,3- and 3,6-disulfated and 2-O-methyl or 2-O-glycosyl 3-sulfated L-galactopyranosyl were also found.     

NMR structural determination of dissolved O-acetylated galactoglucomannan isolated from spruce thermomechanical pulp

Water-soluble O-acetylated galactoglucomannan (GGM) isolated from spruce thermomechanical pulp (TMP) by hot-water extraction was characterized by 1D and 2D (homo- and heteronuclear) NMR analysis. The backbone was found to consist of (1-->4)-linked mannopyranosyl and glucopyranosyl units in a ratio of 10:1.9-2.6. The mannopyranosyl units were acetylated at C-2 and C-3 with a degree of acetylation around 0.28-0.37 as determined by NMR. A slightly larger amount of 2-O-acetylated mannopyranosyl was detected when compared to the 3-O-acetylated component. Approximately every 10th mannopyranosyl unit was substituted at C-6 by a single alpha-galactopyranosyl unit. Fine structure determination based on sequence-specific chemical shift variations showed that the distribution of glycosyl residues is random. Small amounts of other minor polysaccharide species including xylans and galactans could also be identified by NMR.