Complex sulfated galactans from hot water extracts of red seaweed Asparagopsis taxiformis comprise carrageenan and agaran structures.

Hot water extraction from the red seaweed Asparagopsis taxiformis yielded three extracts which showed sulfated galactans with a D:L-galactose ratio non consistent with carrageenan or agaran backbones. The major extract was fractionated by cetrimide precipitation and redissolution with increasing sodium chloride concentrations due to their low solubility. Seven fractions were obtained, and studied by methylation analysis, desulfation-methylation, and NMR spectroscopy of the partially hydrolyzed and the native samples. Fractions with the highest yield were those obtained at high concentrations of NaCl. They comprised both agaran and crageenan structures in considerable amounts. The main agaran structures were ß-D-galactose 4-sulfate and ß-D-galactose 2-sulfate units linked to α-L-galactose 2,3-disulfate residues, and ß-D-galactose linked to α-L-galactose 3-sulfate or 6-sulfate, or substituted with single stubs of ß-D-xylose on C3, while the carrageenan structures comprised ß-D-galactose (2-sulfate) linked to α-D-galactose 3-sulfate or 2,3-disulfate, and ß-D-galactose 2,4-disulfate linked to α-D-galactose 2,3-disulfate. Between the less sulfated fractions, the one obtained by solubilization in 0.5 M NaCl was mainly constituted by agarans, which included 3,6-anhydro-α-L-galactose units. Anticoagulant activity was assayed by general coagulation tests (aPTT and TT), showing a moderate action compared with heparin. This is the first detailed study of the sulfated galactans from the order Bonnemaisoniales.

NMR spectroscopy for structural elucidation of sulfated polysaccharides from red seaweeds.

Some sulfated polysaccharides from red seaweeds are used as hydrocolloids. In addition, it is well known that there are sulfated galactans (carrageenans and agarans) and sulfated mannans, with remarkable biological properties, as antiviral, antitumoral, immunomodulating, antiangiogenic, antioxidant, anticoagulant, and antithrombotic activities, and so on. Knowledge of the detailed structure of the active compound is essential and difficult to acquire. The substitution patterns of the polymer chain, as degree of sulfation and position of sulfate groups, as well as other substituents of the backbone, determine their biological behavior. NMR spectroscopy is a powerful and versatile tool for structural determination. It can be used for elucidation of structures of polysaccharides from new algal sources with novel substitutions or to detect the already known structures from different algal sources, and it could even help to monitor the quality of the active compound on a productive scale. In this review, the available information about NMR spectroscopy of sulfated polysaccharides from red seaweeds is revised and rationalized, to help other researchers working in different fields to study their structures. In addition, considerations about the effects of different structural features, as well as some recording conditions on the chemical shifts of the signals are analyzed.

Paramylon and synthesis of its ionic derivatives: Applications as pharmaceutical tablet disintegrants and as colloid flocculants.

Paramylon, a high molecular weight polysaccharide, is a linear and unbranched (1 → 3)-ß-d-glucan. Despite its numerous biological benefits, the poor aqueous solubility of crystalline paramylon is a drawback that has hampered some of its applications. In an effort to make this biomaterial amenable to practical uses, cationic and anionic paramylon derivatives were obtained. The degrees of substitution of both products were determined. The products were characterized by FT-IR spectrocopy, ESI mass spectrometry, 1H, 13C and 1H-13C NMR and SEM microscopy. These techniques confirmed the success of the substitution reactions. 1H NMR analysis was used to develop alternative methods for an approximate estimation of the degree of substitution. 1H-13C HSQC NMR spectra were assigned for both derivatives. New applications of native, cationic and anionic paramylon were found. Native paramylon showed similar performance as pharmaceutical tablet disintegrant than sodium croscarmellose. Cationic paramylon behavior as colloid flocculant was comparable with commercial cationic polyacrylamides. The anionic derivative could eventually be used in the formulation of matrix controlled release systems or as a suspending agent.

Gracilariopsis hommersandii, a red seaweed, source of agar and sulfated polysaccharides with unusual structures.

Red seaweed Gracilariopsis hommersandii produces important amounts of non-gelling galactans, which were extracted with hot water (GrC, yield, 37%, viscosity average molecular weight, Mv 109 kDa), comprising agarose and sulfated galactan structures. The alkali modified derivative, GrCTr (Mv 95 kDa), gave a galactose:3,6-anhydrogalactose molar ratio of 1.0:0.9, and a more regular structure, favouring gelation (melting and gelling temperatures 64 and 14 °C, respectively). The rheological properties of this product suggest possible applications as hydrocolloid. G. hommersandii also biosynthesizes non gelling sulfated galactan fractions with diads constituted by ß-d-galactose and partially cyclized α-l-galactose units or non-cyclized α-d-galactose residues. Sulfation was mainly detected on C6 or C4 of the ß-d-galactose units, and on C6 and, in minor amounts, on C3 of the α-l-galactose units. The presence of ß-apiuronic acid was demonstrated for these fractions as side chains of the galactan backbone. Carrageenan structures were found for the first time in an agarophyte of the Gracilariales.

Chiroptical characterization of homopolymeric block fractions in alginates.

Homopolymannuronic and homopolyguluronic fractions were obtained by partial hydrolysis of the alkaline extracts from the brown seaweeds Ascoseira mirabilis, Desmarestia menziessi, Desmarestia ligulata and Durvillaea sp. collected in southern Chile. Full characterization of the fractions was achieved by FT-IR and NMR spectroscopy. Total hydrolysis with 90% formic acid of the homopolymeric fractions allowed the preparation of mannuronic and guluronic acids. Both monomers and homopolymeric fractions as neutral salts were studied by CD and ORD. Chiroptical spectra were similar in shape and sign to those previously published in the literature, and permitted to assign D configuration to mannuronic acid and L configuration to guluronic acid in alginic acids. Specific optical rotation values at the sodium D light for the homopolymannuronic (∼-100°) and homopolyguluronic (∼-110°) acid fractions were obtained. These high negative values are proposed for the assignment of the absolute configuration of monomers in homopolymeric fractions.

Carrageenan and agaran structures from the red seaweed Gymnogongrus tenuis.

The galactan system biosynthesized by the red seaweed Gymnogongrus tenuis (Phyllophoraceae) is constituted by major amounts of κ/ι-carrageenans, with predominance of ι-structures, which were isolated by extraction with hot water in high yield (∼ 45%). A small amount of non-cyclized carrageenans mostly of the ν-type was also obtained. Besides, 12% of these galactans are agaran structures, which were present in major quantities in the room temperature water extracts, but they were also found in the hot water extract. They are constituted by 3-linked ß-D-galactose units partially substituted on C-6 with sulfate or single stubs of ß-D-xylose and 4-linked residues that comprise α-L-galactose units partially sulfated or methoxylated on C-3 or sulfated on C-3 and C-6 and 3,6-anhydro-α-L-galactose. Related structural patterns were previously found for agarans synthesized by other carrageenophytes. Results presented here show that these agarans are low molecular weight molecules independent of the carrageenan structures, with strong interactions between them.

Cationization of kappa- and iota-carrageenan--Characterization and properties of amphoteric polysaccharides.

Commercial kappa- and iota carrageenans were cationized with 3-chloro-2-hydroxypropyltrimethylammonium chloride in aqueous sodium hydroxide solution. For kappa-carrageenan three derivatives with different degrees of substitution were obtained. Native and amphoteric kappa-carrageenans were characterized by NMR and infrared spectroscopy, scanning electron and atomic force microscopy; methanolysis products were studied by electrospray ionization mass spectrometry. Young moduli and the strain at break of films, differential scanning calorimetry, rheological and flocculation behavior were also evaluated; the native and the amphoteric derivatives showed different and interesting properties. Cationization of iota-carrageenan was more difficult, indicating as it was previously observed for agarose, that substitution starts preferentially on the 2-position of 3,6-anhydrogalactose residues; in iota-carrageenan this latter unit is sulfated.

In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from red seaweed Nemalion helminthoides.

Water-soluble sulfated polysaccharides from the red seaweed Nemalion helminthoides: two xylomannan fractions (N3 and N4) and a mannan fraction (N6) were investigated to determine their in vitro and in vivo immunomodulatory activities. N3 and N4 induced in vitro proliferation of macrophages of the murine cell line RAW 264.7 and significantly stimulated the production of nitric oxide (NO) and cytokines (IL-6 and TNF-α) in the same cells, whereas this response was not observed with the mannan N6. The cytokine production was also stimulated by sulfated xylomannans in vivo in BALB/c mice inoculated intravenously with these polysaccharides. Remarkably, when mice were treated with N3 and N4 for 1 h before being infected with Herpes simplex virus type 2, they remained asymptomatic with no signs of disease. The in vitro and in vivo results suggest that sulfated xylomannans could be strong immunomodulators.

Structure of highly substituted agarans from the red seaweeds Laurencia obtusa and Laurencia filiformis.

The water extracts from red seaweeds Laurencia obtusa and Laurencia filiformis comprise complex sulfated agarans. Those from L. obtusa have 3-linked ß-d-galactose units in part sulfated on 2-position or methylated on 6-position, while the 4-linked units are mostly 3,6-anhydro-α-l-galactose and α-l-galactose 6-sulfate, some of the latter units are substituted with ß-d-xylose on 3-position, precluding alkaline cyclization. The 3-linked ß-d-galactose units of the agarans from L. filiformis are mostly sulfated on 2-position, but approximately half of these residues also carry the 4,6-O-(1-carboxyethylidene) group. The 4-linked 3,6-anhydro-α-l-galactose units are methylated or substituted in part with single stubs of ß-d-xylose on 2-position. This is the first time that substitution with xylose of 3,6-anhydro-α-l-galactose is reported. Besides, α-l-galactose 2-sulfate carrying single stubs of ß-d-xylose on 3-position was also detected. These galactans have some common structural characteristics with those of other species of this genus, but also others that are specific for these species.

Preparation and characterization of controlled release matrices based on novel seaweed interpolyelectrolyte complexes.

Novel interpolyelectrolyte complexes (IPECs) between naturally sulfated polysaccharides of the seaweed Polysiphonia nigrescens (PN) and cationized agaroses (CAG) and Eudragit E (EE) were prepared using an organic solvent free process, characterized, and explored for controlled drug release. Tablets containing model drug ibuprofen and IPECs were prepared by direct compression. Drug release in acid medium was low owing to the low solubility of ibuprofen in that condition and to the matrix action. Zero order drug release was determined in the buffer stage (pH=6.8), with Fickian diffusion predominating over relaxation during the initial phases. Relaxation appears to increase along the release process and even overcomes diffusion for some systems. Drug release profiles could be controlled by varying the content of IPECs in the tablets. Also, the change in molecular weight and the degree of substitution of the components allowed altering the release profiles.

Chemical modifications of algal mannans and xylomannans: effects on antiviral activity.

The structures of two sulfated xylomannans extracted from the red alga Nemalion helminthoides were determined. These two fractions plus a sulfated mannan, isolated from the same alga and whose structure was previously reported, were subjected to chemical modification. The mannan was oversulfated with SO(3)-pyridine in dimethyl sulfoxide at 60 °C during two and three hours and the xylomannans were subjected to Smith degradation in order to eliminate xylose side-chains. Structural analysis of all derivatives was carried out by methylation analysis and (13)C NMR spectroscopy. Antiviral activity against herpes simplex virus type 1 and 2, and dengue virus type 2 of native and modified mannans and xylomannans was estimated. Anticoagulant effect of the active fractions was also determined.

ß-D-(1→4), ß-D-(1→3) 'mixed linkage' xylans from red seaweeds of the order Nemaliales and Palmariales.

Xylans from five seaweeds belonging to the order Nemaliales (Galaxaura marginata, Galaxaura obtusata, Tricleocarpacylindrica, Tricleocarpa fragilis, and Scinaia halliae) and one of the order Palmariales (Palmaria palmata) collected on the Brazilian coasts were extracted with hot water and purified from acid xylomannans and/or xylogalactans through Cetavlon precipitation of the acid polysaccharides. The ß-D-(1→4), ß-D-(1→3) 'mixed linkage' structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of ß-(1→4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations.

Studies on the cationization of agarose.

Cationized agaroses with different degrees of substitution (0.04-0.77) were synthesized, employing 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC). The influence of different reaction parameters on the substitution degree and molecular weight was evaluated. The investigated parameters were concentration of reagents, temperature, time, and addition of NaBH(4). The products were characterized by means of scanning electronic microscopy, infrared spectroscopy, viscosimetry, and NMR spectroscopy. Methanolysis products were studied by electrospray ionization mass spectrometry. The higher the concentration of CHPTAC employed, a higher degree of substitution was obtained, if the optimum concentration of NaOH in each case was employed. Insufficient quantities of NaOH reduced epoxide formation and the reacting alkoxides of the polysaccharide, whereas an excess of NaOH favored degradation of the epoxide and decrease in the molecular weight of the product. A reaction time of 2h was sufficient to obtain products with the maximum degree of substitution for each case. The addition of NaBH(4) gave products with a slightly higher molecular weight, but the extra cost involved should not justify its use for large-scale application.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides.

MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid-n-butylamine and ferulic acid-n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M-Na](-) together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na](+)), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied.

Preparation and characterization of a novel starch-based interpolyelectrolyte complex as matrix for controlled drug release.

A novel cationized starch-based interpolyelectrolyte complex (IPEC) was formed using kappa-carrageenan as the counter polyion. Characterization of the product by turbidity measurements and elemental analyses indicated a 1:1 interaction of the repeating units. FT-IR spectra for the IPEC showed some differences in comparison with either IPEC constituents or physical mixture. The swelling of tablets obtained by direct compression was independent of pH, and a maximum value of 742% was attained after 24h. The performance of the IPEC as matrix for controlled release of ibuprofen indicates that drug delivery takes place in a zero-order manner. Experimental dissolution data in the buffer stage were properly represented by a model accounting for contributions of Fickian diffusion and relaxation phenomena; this model suggests that the former predominates over the latter, for the modeled range.

Sulfated mannans from the red seaweed Nemalion helminthoides of the South Atlantic.

Nemalion helminthoides, collected in the Argentine South Atlantic coast, was extracted with hot water and the crude product fractionated using cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration and seven fractions were separated and analyzed. Structural analysis of the main fractions, those soluble in 3.0 and 4.0 M NaCl (yields 21.0% and 13.8%, respectively) and those insoluble in 4.0 M NaCl (yield 20.0%), indicated that this seaweed biosynthesizes (1-->3)-linked alpha-D-mannans that are sulfated at positions 4 and 6. Three mannan fractions comprising considerable amounts of xylose were also isolated in very low total yield (2.0%). The fractions that were soluble in 3.0 and 4.0 M NaCl showed low antiherpetic activity whereas this activity was considerable for the fraction solubilized in 2.0 M NaCl (yield 0.5%) which contained single stubs of beta-D-xylose. A xylan, soluble in cetrimide solution, containing (1-->3, 1-->4)-linked beta-D-xylose residues, was also isolated in minor amount.

Agarans from the red seaweed Polysiphonia nigrescens (Rhodomelaceae, Ceramiales).

Polysiphonia nigrescens was sequentially extracted with water at room temperature, 70 and 90 degrees C. The extracts were analyzed and the major one, isolated at 70 degrees C, was fractionated by ion-exchange chromatography, eluting with water and solutions of increasing sodium chloride concentration; five main fractions were separated. Structural analysis, carried out by methylation analysis and NMR spectroscopy, showed that four of these were partially cyclized agarans that were highly substituted on C-6 mainly with sulfate, although methyl ether and single stubs of beta-D-xylose were found in minor proportions. A fifth fraction comprising 6-sulfated agarose was also isolated. The use of 2D NMR techniques allowed us to assign the 1H and 13C NMR resonances of the G6S-->L6S diad for the first time.

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight (UV-MALDI-TOF) mass spectra of N-acylated and N,O-acylated glycosylamines.

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments.

Galactans from cystocarpic plants of the red seaweed Callophyllis variegata (Kallymeniaceae, Gigartinales).

The polysaccharide extracted from cystocarpic Callophyllis variegata was fractionated with potassium chloride yielding three small fractions that precipitated in the ranges of 0-0.05 M KCl, 1.20-1.25 M KCl, and 1.80-2.00 M KCl, and a main product soluble in 2.00 M KCl. These fractions were analyzed, and as the first one contained very high amounts of protein, it was not studied further. Structural analysis of the rest of the fractions (F1-F3) was carried out by methylation, desulfation-methylation, IR, and 13C NMR spectroscopy. The results are consistent for F1 with a carrageenan-type backbone mainly constituted by beta-D-galactose 2-sulfate linked to alpha-D-galactose 2,3,6-trisulfate and beta-D-galactose 2,4-disulfate linked to 3,6-anhydro-D-galactose 2-sulfate as dominant diads. In F2 these diads are present together with low amounts of beta-D-galactose 2-sulfate linked to 3,6-anhydro-D-galactose 2-sulfate, whose contribution becomes higher in F3. In addition, minor but significant amounts of L-galactose were detected. F1-F3 showed potent antiviral activity against herpes simplex types 1 and 2 and dengue type 2.

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.