Water- and alkali-soluble glucans from oak lichen

A water-soluble glucan, [α]²_D +217° (water), and an alkali-soluble glucan,

+152° (sodium hydroxide), have been isolated from the oak lichen Evernia prunastri (L.) Ach. On the basis of methylation analysis, periodate oxidation, and partial acid hydrolysis, the water-soluble polysaccharide has been shown to be a neutral, slightly branched glucan with a main chain composed of (1→3)- and (1→4)- linked glucopyranose residues in the ratio 1?:1. Branching occurs most probably at position 2 of (1→4)-linked glucopyranose residues. On the basis of optical rotation and i.r. spectral data, and enzymic hydrolysis, the α-D configuration has been assigned to the glycosidic linkages. Likewise, the alkali-soluble polysaccharide was shown to be a neutral, branched glucan with a main chain composed of (1→3)- and (1→4)-linked α-D-glucopyranose residues in the ratio 6:1. Each of the (1→4)-linked units was a branch point involving position 6. The presence of some β-D linkages is not excluded since hydrolysis with β-D-glucosidase occurred to a small extent.     

Further studies on the composition and structure of a fucoidan preparation from the brown alga Saccharina latissima

The polysaccharide composition of a fucoidan preparation isolated from the brown alga Saccharina latissima (formerly Laminaria saccharina) was reinvestigated. The preparation was fractionated by anion-exchange chromatography, and the fractions obtained were analyzed by chemical methods combined with NMR spectroscopy. Several 2D procedures, including HSQC, HMQC-TOCSY, and HMQC-NOESY, were used to obtain reliable structural information from the complex spectra, and the signal assignments were additionally confirmed by comparison with the literature spectra of the related polysaccharides and synthetic oligosaccharides. In accordance with the previous data, the main polysaccharide component was shown to be a fucan sulfate containing a backbone of 3-linked α-l-fucopyranose residues sulfated at C-4 and/or at C-2 and branched at C-2 by single sulfated α-l-fucopyranose residues. In addition, three other types of sulfated polysaccharide molecules were detected in the total fucoidan preparation: (i) a fucogalactan having a backbone of 6-linked β-d-galactopyranose residues branched mainly at C-4 and containing both terminal galactose and fucose residues; (ii) a fucoglucuronomannan having a backbone of alternating 4-linked β-d-glucopyranosyluronic acid and 2-linked α-d-mannopyranose residues with α-l-fucopyranose residues as single branches at C-3 of α-d-Manp; and (iii) a fucoglucuronan having a backbone of 3-linked β-d-glucopyranosyluronic acid residues with α-l-fucopyranose residues as single branches at C-4. Hence, even a single algal species may contain, at least in minor amounts, several sulfated polysaccharides differing in molecular structure. Partial resolution of these polysaccharides has been accomplished, but unambiguous evidence on their presence as separate entities was not obtained.     

Mucilage from a fresh-water red alga of the genus Batrachospermum

The gelatinous polysaccharides of a Batrachospermum species have been extracted from the alga. The major polysaccharide is acidic and has been separated from neutral polysaccharides by chromatography on DEAE-cellulose. The constituent sugars of the acidic polysaccharide include d- and l-galactose, d-mannose, d-xylose, l-rhamnose, d-glucuronic acid, and two O-methyl sugars, which have been characterized as 3-O-methyl-l-rhamnose (l-acofriose and 3-O-methyl-d-galactose. Partial acid hydrolysis of this polysaccharide has given a complex mixture of neutral and acidic oligosaccharides. The two preponderant acidic oligosaccharides contained galactose and glucuronic acid in 1:1 ratio, suggesting the presence of a repeating sequence of these two residues as a major structural feature of the polysaccharide.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

Characterization of a neutral polysaccharide with antioxidant capacity from red wine

A neutral fraction (PS-SI) (0.3 g/L) with MW of 74 kDa, which contained galactose, arabinose, mannose, and glucose in the molar ratio of 1.0:0.6:0.4:0.2 was obtained by treatment of the whole polysaccharide extracted from red wine with cetrimide, followed by gel permeation chromatography. Spectroscopic and methylation analyses indicated that PS-SI is a mixture of neutral polysaccharides, consisting mainly of β (1→3)-linked galactopyranosyl residues, with side chains of galactopyranosyl residues at positions O-6. Arabinofuranosyl residues linked α (1→5), α-mannopyranosyl and glucosyl residues appear to be components of different polysaccharides. The in vitro antioxidant capacity of fractions of wine polysaccharide was studied by hydroxyl radical scavenging and ORAC assays. Fraction PS-SI presented the strongest effect on hydroxyl radicals (IC₅₀ = 0.21).     

Composition and properties of pectin polysaccharides of St. John’s wort Hypericum Perforatum L.

Three fractions of acidic water-soluble polysaccharides (concentration of glucuronic acid 10?C65%) were obtained from the above-ground part of St. Johns wort Hypericum perforatum L. by serial extraction with water and 0.7% aqueous solution of ammonium oxalate. Enzymatic hydrolysis of these polysaccharides using endo-polygalacturonase indicates that their carbohydrate chains contain the units of galacturone formed by 1,4-??-linked residues of non-substituted D-galacturonic acid. The extracted polysaccharides have been purified by means of gel filtration. It has been shown that water-soluble polysaccharides obtained by extraction with water manly contain the residues of galactose, mannose, glucose, and arabinose (the concentration of glucuronic acid being 10?C27%) while the polysaccharide fraction extracted using 0.7% aqueous solution of ammonium oxalate is presented by pectin polysaccharides. Only the residues of galacturonic acid (55?C72%) have been identified among glucuronic acids in its composition using chromatography/mass spectrometry of trimethylsilyl derivatives. In addition, this fraction contains the residues of the neutral monosaccharides which are typical for pectins: arabinoses, galactoses, rhamnoses, and glucose; there are also minor concentrations of residues of xylose and mannose. IR spectra of pectin polysaccharides of St. John??s wort have absorption bands in the ranges 1740, 1640?C1620, 1236?C1200, and 1200?C1000 cm^?1 which are typical for pectins. It has been demonstrated that aqueous solutions of pectin polysaccharides of St. John??s wort (2 mg/mL) have pronounced antioxidant activity (44% of the activity of trolox taken for 100%).     

Anionic polymers of the cell wall of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> subsp. <Emphasis Type="Italic">subtilis</Emphasis> VKM B-501<Superscript>T</Superscript>

Teichoic acid and disaccharide-1-phosphate polymer were identified in the cell walls of Bacillus subtilis subsp. subtilis VKM B-501^T. The teichoic acid represents 1,3-poly(glycerol phosphate) 80% substituted by α-D-glucopyranose residues at O-2 of glycerol. The linear repeating unit of disaccharide-1-phosphate polymer contains the residues of β-D-glucopyranose, N-acetyl-α-D-galactosamine, and phosphate and has the following structure: -6)-β-D-Glcp-(1→3)-α-D-GalpNAc-(1-P-. The structures of two anionic polymers were determined by chemical and NMR-spectroscopic methods. The ¹H- and ¹³C-NMR spectral data on disaccharide-1-phosphate polymer are presented for the first time.     

Structural and immunochemical characterization of the acidic arabinomannan of mycobacterium smegmatis

A serologically active, acidic arabinomannan has been isolated from Mycobacterium smegmatis. The polysaccharide contains approximately 56 arabinosyl and 11 mannosyl residues, and 2 phosphate, 6 monoesterified succinate, and 4 ether-linked lactate groups. After saponification to remove succinyl groups, the polysaccharide can be separated into phosphorylated (55%) and nonphosphorylated (45%) forms, the former containing a little more arabinose and a little less mannose than the latter. The structures of these polysaccharides were investigated by ¹H- and ¹³C-n.m.r. spectroscopy and methylation analysis, before and after selective cleavage of furanosyl linkages. The phosphorylated and nonphosphorylated forms of the polysaccharide were found to have similar, if not identical, structures. The main structural feature of the polysaccharides is the presence of chains of contiguous arabinofuranosyl residues linked α-(1→5). These chains are attached at O-4 of arabinopyranosyl residues that are present in a core region of the polysaccharide that also contains mannopyranosyl residues. Immunochemical studies demonstrated that the polysaccharide is an effective, precipitating antigen with antisera from rabbits immunized with cell walls or heat-killed cells of M. smegmatis. The polysaccharide is, however, more effective as a precipitating antigen after removal of the succinate groups, and completely ineffective after removal of arabinofuranosyl residues. The polysaccharide therefore contains an important antigen in common with the arabinogalactan lipopolysaccharide of the cell wall of the bacterium, i.e., chains of contiguous α-(1→5)-linked arabinofuranosyl residues.     

Structural characterization and biological activities of two α-glucans from <Emphasis Type="Italic">Radix Paeoniae Alba</Emphasis>

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, cancer, and other diseases. In this study, two polysaccharides RPAPW1 and RPAPW2 were isolated from Radix Paeoniae Alba by DEAE-52 cellulose chromatography and G-25 sephadex. According to physicochemical methods, NMR and methylation analysis, RPAPW1 and RPAPW2 were established to be α-glucans consisting of predominant 4-linked α- Glc residues branched at O-6 and contained trace amount of protein and uronic acid. Immunological tests indicated that RPAPW1, RPAPW2 and could promote splenocyte proliferation and RAW264.7 phagocytic activity. In vitro, RPAPW1 and RPAPW2 elicited a week reducing power, DPPH scavenging activity and could not protect the PC12 cells from H₂O₂ damage. These data implied polysaccharides RPAPW1 and RPAPW2 had the potential to be a natural immunopotentiating and antioxidant supplement for preparing functional foods and nutraceuticals.     

Conformational studies of -glucans

A study of the effect of linkage on the possible conformations of di-and polysaccharides of α-D -glucose and also the probable intramolecular hydrogen bonds has been made. The differences in the nature of linkage is shown to effect the energetically preferred conformations; (1 → 2) linkages lead only to righthanded helical conformations, (1 → 3) linkages lead to extended as well as both left and righthanded helical conformations; (1 → 4) linkages lead to both right-and lefthanded wide helical conformations. The possible hydrogen bonds between adjacent residues are also dependent on the nature of the linkage. A comparison of the conformational data of α-D -glucans with those of β-D -glucans has indicated that the favored conformations and hydrogen bonds between contiguous residues in the chain are influenced by the configuration at the anomeric carbon atom in all the three types of polysaccharides. From the energy calculations a probable conformation (?M = ?10°, ψM = ?30°, ?N = ?23°, ψN = ?19°) has also been proposed for crystalline mycodextran in conformity with x-ray data. This conformation contains two types of hydrogen bonds between contiguous residues one between 0–2 and 0–3 atoms at (1 → 4) linkage and the other between 0?2 and 0–4 atoms at (1 → 3) linkage in the chain. The conformation of maltose unit (?10°,?30°) that is likely to occur in the crystalline mycodextran coincides with the minimum energy conformation of maltose.     

Polysaccharides of basidiomycetes. Alkali-soluble polysaccharides from the mycelium of white rot fungus <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> (Curt.: Fr.) P. Karst

Two polysaccharides were isolated from submergedly cultured mycelium of the basidiomycete Ganoderma lucidum by extraction with alkali followed by fractionation with Fehling reagent. The polysaccharides were shown to be a linear (1→3)-α-D-glucan and a highly branched xylomannan containing a backbone built up of (1→3)-linked α-D-mannopyranose residues, the majority of which are substituted at O-4 by single β-D-xylopyranose residues or by disaccharide fragments β-D-Manp-(1→3)-β-D-Xylp-(1→. Polysaccharide structures were elucidated by NMR spectroscopy in combination with methylation analysis and periodate oxidation. An interesting feature of the xylomannan is the simultaneous presence of α-D-mannopyranose and β-D-mannopyranose residues, the first forming the backbone, and the second being the non-reducing terminal units of disaccharide side chains.     

Electron-impact mass spectrometry of methyl O-methylglucopyranosiduranamides

6-O-Acetyl-2,4-diazido-3-O-benzyl-2,4-dideoxy-β-D-glucopyranosyl chloride and 2,6-diazido-3,4-di-O-benzyl-2,6-dideoxy-β-D-glucopyranosyl chloride are two valuable building units suitable for the synthesis of α-linked disaccharides containing 2,4-diamino-2,4-dideoxy- or 2,6-diamino-2,6-dideoxy-D-glucose as nonreducing moieties. The glycoside synthesis is accomplished stereoselectively under mild conditions in the presence of silver perchlorate. The α-(1→3)-linked disaccharides 2,4-diacetamido-2,4-dideoxy-3-O-(2,4-diacetamido-2,4-dideoxy-α-D-glucopyranosyl)-D-glucopyranose and 2-acetamido-2-deoxy-3-O-(2,6-diacetamido-2,6-dideoxy-α-D-glucopyranosyl)-D-glucopyranose have been prepared.     

Structural characterization of feruloylated arabinoxylans and xylans released from water-unextractable cell walls of rye outer layers upon treatment with lichenase and cellulase

Destarched and deproteinated water-unextractable material (WUM) of rye outer layers was sequentially treated with lichenase and cellulase to digest β-glucans and a part of the cellulose. As a result, the polymeric cell-wall material (CWM) initially associated with these polysaccharides was released into solution (AXL and AXC for lichenase- and cellulase-extractable fractions, respectively). A portion of the material that self-aggregated during extractions was further solubilized with DMSO (XD and XD-P for the fraction left in the solution and that precipitated during dialysis, respectively). Arabinoxylans (AXs) recovered from these fractions were composed of populations with different degrees of substitution with α-l-arabinofuranosyl residues (Araf). Their counterparts present in the AXL and AXC fractions exhibited higher (0.60 and 0.75) arabinose-to-xylose ratios (Ara/Xyl) and represented 27% and 32% of the isolated AXs, respectively. The xylans of the XD and XD-P fractions had a very low Ara/Xyl ratio (0.16 and 0.09) and accounted for 23% and 18%, respectively. Based on the results of ammonium sulfate fractionation and sugar analysis, it has been shown that AXL consisted of AX subfractions having Ara/Xyl in a narrow range (0.50-0.66). By contrast, the cellulase-extractable AXs were characterized by the presence of the highly branched subfractions (Ara/Xyl of 1.00) as well. Quite unexpectedly, the higher amounts of ferulic acid (FA) were found in the cell-wall fractions enriched in xylans than in the AX-containing fractions. Furthermore, as demonstrated by ¹H NMR and Fourier transform infrared spectroscopy, xylans were substituted with α-d-glucuronopyranosyl residues (GlcpA).     

Étude complémentaire de la structure de trois galactoxyloglucanes (amyloïdes) de graines

D-Galacto-D-xylo-D-glucans (amyloids) from Balsamina, Tropaeolum, and Tamarindus seeds behave in a similar manner in the presence of various glycosidase preparations: slow depolymerization by enzymes from several germinated or non-germinated seeds, and hydrolysis into monosaccharides and oligosaccharides by commercial cellulase and hemicellulase preparations from fungi. A purified cellulase from Penicillium notatum gave a dialyzable fraction almost exclusively composed of α-D-xylopyranosyl-(1→6)-D-glucose residues and a nondialyzable fraction composed of chains of β-D-(1→4)[withsome (1→3)]-glucopyranosyl residues; β-D-galacto-pyranosyl-(1→2)-α-D-xylosyl groups are linked to some of the β-D-glucosyl residues at 0-6. The presence of (1→3)-linkages in the D-glucan chain of the Balsamina was verified by methylation and sequential periodate oxidation-borohydride reduction; the distribution of the substituents on the D-glucan chain is not regular. The main D-glucan backbone, where the β-D-glucosyl residues are partly linked at 0-6 to β-D-galactosyl-(1→2)-D-xylosyl groups, is linked to D-glucan chains where almost all the D-glucose units are linked at 0-6 by one α-D-xylosyl group. The presence of 3,6-di-O-methyl-D-glucose after permethylation and hydrolysis suggests that the xyloglucan chains are linked to 0-2 of the D-glucosyl units of the galactoxyloglucan backbone.     

Comparison of Structure and Antitumor Activity of Polysaccharides Isolated from Fukurotake,the Fruiting Body of Volvariella volvacea

An α-linked d-manno-d-galactan, glycogen, and three kinds of branched (1→3)-γ-d-glucans were isolated from Fukurotake, the fruiting body of Volvariella volvacea by successive extractions with cold and hot water, cold and hot alkali, and dimethyl sulfoxide. The mannogalactan, purified from the cold water extract, had a MW of 4x 10⁵, and consisted of an α-(1→6) linked d-galactose backbone, one out of every three d-galactose residues being substituted with a single α-d-mannosyl group. The glycogen, isolated from the hot water extract, had a MW of 12 × 10⁵, and 14 ~ 15 d-glucose residues as an average chain length, as revealed by methylation analysis. The α-(1→4)-linked unit chains of this glycogen were distributed from DP 6 to 13, approximately in equal numbers.

There were three kinds of branched (1→3)-γ-d-glucans, isolated from alkali and dimethyl sulfoxide extractions. They contain a backbone of (1→3)-linked d-glucose residues with side chains of single d-glucosyl groups, but having different degrees of branching. In addition, alkali-extracted glucans contain small but significant proportions of (1→6)-linked sugar units.

Among these polysaccharides, the cold alkali-extracted glucan (degree of branching, 1:5) showed a potent antitumor activity against Sarcoma 180 solid tumors implanted in mice, and chemical modifications changed its original activity, confirming our previous results. Other polysaccharides, such as the mannogalactan and other (1→3)-γ-d-glucans, showed no or lower antitumor activity.     

A New Fungal α-D-glucan,elsinan, elaborated by Elsinoe leucospila

A new α-D-glucan, designated elsinan, has been isolated from the culture filtrate of Elsinoe leucospila grown in potato extract-sucrose medium. Acid hydrolysis of the methylated polysaccharide gave 2,3,6- and 2,4,6-tri-O-methyl-D-glucose, in the ratio of 2.5:1.0, together with small proportions of 2,3,4,6-tetra- (0.7%) and 2,4-di-O-methyl-D-glucose (0.5%), indicating that the glucan is an essentially linear polymer containing (1→4)- and (1→3)-α-D-glucosidic linkages. Periodate oxidation, followed by borohydride reduction and mild hydrolysis with acid (mild Smith degradation) yielded 2-O-α-D-glucosyl-D-erythritol and erythritol, in the molar ratio of 1.0:1.4, and a trace of glycerol. Partial acid hydrolysis, and also acetolysis, of elsinan gave nigerose, maltose, O-α-D-glucopyranosyl-(1→3)-O-α-D-glucopyranosyl (1→4)-D-glucopyranose, O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→3)-D-glucopyranose, maltotriose, and a small proportion of maltotetraose. It is concluded that elsinan is composed mainly of maltotriose residues joined by α-(1→3)-linkages, in the sequence →3)-α-D-Glcp-(1→4)-α-D-Glcp-(1→.The unique structural features of elsinan are discussed in comparison with other glucans.     

Carbohydrate Specificity of Antibodies against Phytopathogenic Fungi of the <Emphasis Type="Italic">Aspergillus</Emphasis> Genus

Brush rabbits were immunized with injections prepared from the fungi Aspergillus fumigatus, Aspergillus niger, and Aspergillus repens. A library of synthetic biotinylated oligosaccharides containing the key fragments of antigenic polysaccharides of the fungal cell wall—galctomannan, α- and β-glucans, mannan, and chitin—was used to analyze carbohydrate specificity. The anticarbohydrate antibodies obtained from animals immunized with preparations from A. fumigatus and A. repens predominantly recognized epitopes containing galactofuranoside residues, while the majority of the antibodies against A. niger bound the chitooligosaccharide ligand. These results are the basis for the identification of specific markers required for the development of immunoenzyme test systems.     

Les polysaccharides des graines de quelques liliacees et iridacees

The alkali-soluble polysaccharides have been surveyed in the seeds of 7 species of the Liliaceae and 2 species of the Iridaceae. All appear to contain galactoglucomannans and/or glucomannans. The structure of the water-soluble galactoglucomannan from the endosperm of Asparagus officinalis has been studied in detail. It contains residues of glucose, mannose and galactose in the ratio 43:49:7. Hydrolysis of the fully methylated polysaccharide released 2,3,4,6-tetra-O-methyl-d-hexoses (mannose and glucose), 2,3,4,6-tetra-O-methyl-d-galactose, 2,3,6-tri-O-methyl-d-mannose, 2,3,6-tri-O-methyl-d-glucose, 2,3-di-O-methyl-d-mannose and 2,3-di-O-methyl-d-glucose in the molar proportions of 1:4.5:50:41:2:1·5. The following oligosaccharides were identified on partial hydrolysis of the galactoglucomannan: mannobiose, mannotriose, mannotetraose, cellobiose, glucopyranosylmannose, mannopyranosylglucose and a trisaccharide composed of two mannosyl residues and one glucosyl residue. The galactoglucomannan consists of a linear chain of β(1 → 4)-Iinked d-mannosyl and d-glucosyl residues, to which are attached single-unit galactosyl side chains. The galactose residues are linked 1 → 6, probably α. The terminal, non-reducing residues of the main chain may be either glucosyl or mannosyl units but the former predominate.     

Total hemicelluloses from Hordeum vulgare plants at different stages of maturity

The changes in the composition of the total hemicelluloses of leaf and stem tissues of field-grown barley plants have been examined at different stages of maturation. In each plant the proportion of xylose residues in the total hemicellulose increases with tissue maturity, that of galactose varies little, and the proportions of arabinose, glucose and uronic acid residues decrease. The ratio of β(1 → 3) to β(1 → 4) linkages in the β-glucans decreases with tissue maturity and there is a decrease in the $\text{DP}$_n of these β-glucans.     

Characterization of Laurencia arbuscula spore mucilage and cell walls with stains and FITC-labelled lectins

In most red algae, spores are liberated without a cell wall, within a sheath of mucilage that is responsible for its primary attachment. Utilizing fluorescent-labelled lectins, we identified carbohydrate residues and their location in the mucilage and cell walls of spores of Laurencia arbuscula. Cell wall formation and mucilage composition were studied with Calcofluor, Toluidine Blue (AT-O), Alcian Blue (AB) and periodic acid-Schiff (PAS). In the mucilage, we identified α-d-mannose, α-d-glucose, N-acetyl-glucosamine, N-acetyl-galactosamine and β-d-galactose. All sugar residues were found in the cell wall, in the spore body rather than in the rhizoid, which suggests that the residues may be related to initial substrate adhesion. A cell wall is produced soon after the spore's attachment, beginning with a deposition of cellulose around the spore, as indicated by Calcofluor. A polarization of the cell wall triggers the process of germination. The cell-wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while neutral polysaccharides were positive to PAS.