Fractionation and anti-tumor activity of the mycelia of liquid-cultured Phellinus linteus

All the fractions of Phellinus linteus mycelia showed anti-tumor activity toward solid tumors planted in mice. The highest anti-tumor activity of 81.2% was observed in the protein-glucan complex obtained by precipitating the 24% NaOH extract at pH 6.0. This protein-glucan complex consisted of 39.3% polysaccharide and 49.4% protein. Its (13)C- and (1)H-NMR data showed that the main glucan part of the complex was simple alpha-1,3-glucan chains.     

The isolation, preliminary study of structure and physiological activity of water-soluble polysaccharides from squeezed berries of Snowball tree Viburnum opulus

Water-soluble polysaccharide fractions VO1-VO4 were isolated from the squeezed berries of snowball tree (Viburnum opulus) by successive extraction with water at various temperatures and pH and with aqueous solutions of ammonium oxalate. These fractions were purified by ion-exchange chromatography on DEAE cellulose, and the homogeneity of the purified polysaccharides was determined by gel filtration on Sephacryl S-500. Acidic polysaccharides close to pectins in their sugar composition were found in all the extracts (fractions VO1-1, VO2-1, VO3-2, and VO4-2). Residues of galacturonic acid, galactose, arabinose, and (to a lesser extent) rhamnose are their main constituents. Neutral polysaccharides composed mainly of galactose and mannose residues were additionally found in fractions extracted with acidified water (pH 4.0) and with aqueous ammonium oxalate solutions. Partial acidic hydrolysis and digestion with pectinase of acidic polysaccharides indicated that their carbohydrate backbone consists of alpha-1,4-linked residues of D-galacturonic acid. NMR spectra of acidic polysaccharides (fractions VO3-2 and VO3-3) confirmed this and demonstrated that their side oligosaccharide chains are composed of beta-1,4-linked galactopyranose residues and of terminal and 2,5- and 3,5-substituted residues of alpha-arabinofuranose at a Gal: Ara ratio of 3:1. Some polysaccharides from V. opulus were found to possess an immunostimulating activity: they enhance phagocytosis, in particular, the phagocytic index and the secretion of lysosomal enzymes with peritoneal macrophages. Calcium ions were found to be necessary for the appearance of the stimulating effect of acidic polysaccharides from V. opulus.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

The isolation, preliminary structural studies, and physiological activity of water-soluble polysaccharides from the squeezed berries of the snowball treeViburnum opulus

Water-soluble polysaccharide fractions VO1–VO4 were isolated from the squeezed berries of the snowball tree (Viburnum opulus) by successive extraction with water at various temperatures and pH and with aqueous solutions of ammonium oxalate. These fractions were purified by ion-exchange chromatography on DEAE cellulose, and the homogeneity of the purified polysaccharides was determined by gel filtration on Sephacryl S-500. Acidic polysaccharides close to pectins in their sugar composition were found in all the extracts (fractions VO1-1, VO2-1, VO3-2, and VO4-2). Residues of galacturonic acid, galactose, arabinose, and (to a lesser extent) rhamnose are their main constituents. Neutral polysaccharides composed mainly of galactose and mannose residues were additionally found in fractions extracted with acidified water (pH 4.0) and with aqueous ammonium oxalate solutions. Partial acidic hydrolysis and digestion with pectinase of acidic polysaccharides indicated that their carbohydrate backbone consists of α-1,4-linked residues ofD-galacturonic acid. NMR spectra of acidic polysaccharides (fractions VO3-2 and VO3-3) confirmed this and demonstrated that their side oligosaccharide chains are composed of β-1,4-linked galactopyranose residues and of terminal and 2,5- and 3,5-substituted residues of α-arabinofuranose at a Gal : Ara ratio of 3 : 1. Some polysaccharides fromV. opulus were found to possess an immunostimulating activity: they enhance phagocytosis, in particular, the phagocytic index and the secretion of lysosomal enzymes with peritoneal macrophages. Calcium ions were found to be necessary for the appearance of the stimulating effect of acidic polysaccharides fromV. opulus.     

Structural data for the carbohydrate of ispaghula husk ex Plantago ovata forsk

The husk from the seeds of Plantago ovata Forsk yielded two fractions when exposed to mild aikali, namely, the mucilage polysaccharide (85%, apparently a single species) and the non-polysaccharide component (15%). Methylation analysis and partial hydrolysis with acid showed the mucilage polysaccharide to be a highly branched, acidic arabinoxylan, the xylan backbone having both (1→4) and (1→3) linkages. The majority of the residues in the xylan backbone are variously substituted at O-2 and O-3 with arabinose, xylose, and an aldobiouronic acid identified as 2-O-(galactopyranosyluronic acid)-rhamnose. A structure incorporating these features for the husk polysaccharide is proposed.     

Isolation and some characterization of an acidic polysaccharide with anti-calcification activity from coccoliths of a marine alga, Pleurochrysis carterae.

Coccolith is a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, and consists of calcium carbonate crystals and organic matrices. EDTA-soluble organic materials extracted from coccoliths of Pleurochrysis carterae showed anti-calcification activity. They were separated by anion-exchange HPLC, and two fractions, fractions A and B, were obtained. Fraction B, which was more active than fraction A, was further separated into six consecutive fractions, B1-B6, by second anion-exchange HPLC. 1H NMR spectral analyses of these fractions suggested that a novel acidic polysaccharide, designated CMAP, existed throughout B1-B6 and that the latter four fractions mainly contained another acidic polysaccharide, PS-2, characterized previously. Since PS-2 did not show anti-calcification activity, CMAP was found to be the active principle.     

ON ARABINOSE AS A CONSTITUENT OF HYALURONIC ACID FROM BOVINE BRAIN

(1) Wardi , Allen , Turner and Stary (1966) and Margolis (1967) have reported that arabinose is a component of hyaluronic acid from mammalian brain. (2) In the present study, total acidic polysaccharide and hyaluronic acid fractions were isolated from lipid-extracted and proteolysed bovine brain by precipitation with cetyltri-methylammonium bromide. These fractions were analysed for arabinose by paper chromatography of deionized hydrolysates and by gas-liquid chromatography of per(trimethylsilyl)ated methanolysates. (3) Two pentoses, xylose and ribose, were detected. Arabinose was analytically undetectable in both polysaccharide fractions, but was easily detected in a control polysaccharide containing 0-1% (w/w) arabinose. Arabinose, if present in hyaluronic acid from bovine brain, constitutes less than 0.1 mol per mol of hyaluronic acid (molecular weight 1.5 x 10⁶ daltons).     

Antigenic polysaccharides of bacteria of the genus Shigella. Determination of the structure of polysaccharide chains of Shigella boydii type 9 lipopolysaccharide and detection of unusually high molecular weight glycolipid

Specific acidic polysaccharide has been isolated from the Shigella boydii type 9 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and L-rhamnose. From the results of methylation analysis, partial acid hydrolysis and 13C NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [----4)DGlcp(alpha 1----4)DGlcAp(beta 1----3)DGlcNAcp(alpha 1----3)LRhap(alpha 1----]n. The lipopolysaccharide from Sh. boydii 9 was fractionated by gel chromatography on the Sephadex G-200 column in a buffer containing sodium deoxycholate into three fractions. PAGE-SDS of the fractions obtained, 13C NMR- and chromato-mass-spectrometry data indicated that the three fractions contained the O-specific polysaccharide as the only carbohydrate component. The substance from the most high-molecular weight fraction contained unusually long O-specific chains (60,000 dalton). In the fat acid composition this fraction differed from other lipopolysaccharides by absence of beta-hydroxymyristic acid.     

Characterization and antioxidant activity of Ginkgo biloba exocarp polysaccharides

Ginkgo biloba exocarp polysaccharide (GBEP) was obtained by hot water extraction, the crude polysaccharide was deproteinized by Sevag method and fractionized by a DEAE Sepharose fast flow anion-exchange column. Five fragments were obtained, including neutral polysaccharide (GBEP-N) and four acidic polysaccharides (GBEP-A1, GBEP-A2, GBEP-A3 and GBEP-A4). GBEP-N and GBEP-A3 were further purified by Superdex 200 gel column chromatography. The resulted two fractions GBEP-NN, and GBEP-AA were characterized by FT-IR, and HPGFC (high pressure gel filtration chromatography). Monosaccharide composition was determined by RP-HPLC method of precolumn derivatization with 1-phenyl-3-5-pyrazolone. GBEP-NN was mainly composed of rhamnose, arabinose, mannose, glucose and galactose, while GBEP-AA was mainly made up of mannose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose. The crude GBEP exhibited certain antioxidant activity. At the concentration of 5 mg/mL, the hydroxyl radical scavenging effect of GBEP was 90.52%, greater than 77.37% for the positive control ascorbic acid.     

The xylanase system of Coniophora cerebella

1. The culture filtrate of the fungus Coniophora cerebella grown on poplar 4-O-methylglucuronoxylan as carbon source and enzyme inducer contained an enzyme system that degraded the polysaccharide to xylose, acidic and neutral oligosaccharides and an enzyme-resistant polymer. Free uronic acid was not produced. 2. Cold ethanol fractionation of the culture filtrate yielded two active fractions, one of which had only xylanase (EC 3.2.1.8) and the other both xylanase and xylosidase (EC 3.2.1.37) activities. Further fractionation on DEAE-cellulose resolved the xylanase and xylosidase activities. 3. The xylanase degraded poplar 4-O-methylglucuronoxylan in an essentially random manner, producing oligosaccharides, but some xylose residues in the vicinity of uronic acid side groups were protected from hydrolysis, preventing a truly random attack. The xylosidase attacked the polysaccharide very slowly, releasing xylose, but the oligosaccharides produced by the action of the xylanase were much more susceptible to hydrolysis by the xylosidase. 4. The products of xylanase action were separated into neutral and acidic fractions. The neutral oligosaccharides were separated by chromatography on charcoal-Celite, and the major products were characterized as xylobiose, xylotriose, xylotetraose and xylopentaose. Some of the acidic sugars were branched, having the uronic acid residue attached to a xylose residue other than the terminal non-reducing one. 5. Gel filtration of various xylanase fractions gave values for the molecular weight of the enzyme from 34000 to 38000.     

Isolation and Some Characterization of an Acidic Polysaccharide with Anti-calcification Activity from Coccoliths of a Marine Alga,Pleurochrysis carterae

Coccolith is a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, and consists of calcium carbonate crystals and organic matrices. EDTA-soluble organic materials extracted from coccoliths of Pleurochrysis carterae showed anti-calcification activity. They were separated by anion-exchange HPLC, and two fractions, fractions A and B, were obtained. Fraction B, which was more active than fraction A, was further separated into six consecutive fractions, B1-B6, by second anion-exchange HPLC. ¹H NMR spectral analyses of these fractions suggested that a novel acidic polysaccharide, designated CMAP, existed throughout B1-B6 and that the latter four fractions mainly contained another acidic polysaccharide, PS-2, characterized previously. Since PS-2 did not show anti-calcification activity, CMAP was found to be the active principle.     

Extracellular polysaccharides of cowpea rhizobia: compositional and functional studies

Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharides and low molecular weight neutral glucans. These polymers were fractionated using gel filtration chromatography. Purified fractions of the acidic heteropolymer reacted with peanut agglutinin to give precipitin bands when subjected to Ouchterlony gel diffusion. The acidic exopolysaccharide was found to contain mainly glucose, galactose, glucuronic acid, mannose and fucose. The non-carbohydrate substituents of the acidic heteropolymer were pyruvate, acetate and uronate which were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.Abbreviations EPS Extracellular polysaccharide - YEM yeast extract mannitol - PNA peanut agglutination - ¹H-NMR proton nuclear magnetic resonance     

Structural characterization of the released polysaccharide of desiccation-tolerant Nostoc commune DRH-1

The structure of the viscous extracellular polysaccharide (glycan) of desiccation-tolerant Nostoc commune DRH-1 was determined through chromatographic and spectroscopic methods. The polysaccharide is novel in that it possesses a 1-4-linked xylogalactoglucan backbone with D-ribofuranose and 3-O-[(R)-1-carboxyethyl]-D-glucuronic acid (nosturonic acid) pendant groups. The presence of D-ribose and nosturonic acid as peripheral groups is unusual, and their potential roles in modulating the rheological properties of the glycan are discussed. Nosturonic acid was present in the glycans of N. commune from diverse geographic locations, suggesting that this uronic acid is an integral component of this cosmopolitan anhydrophile.     

Antioxidant activities potential of tea polysaccharide fractions obtained by ultra filtration

Three polysaccharide fractions (TPS1, TPS2 and TPS3) with different molecular weights were obtained using ultra filtration membranes from crude tea polysaccharide (CTPS) extracted from abandoned lower grade tea leaves. Each fraction contained different contents of neutral sugar, uronic acid, protein, and total polyphenols. These differences provided basis for the antioxidant and free radical scavenging activity of these polysaccharide fractions. The molecular weights of TPS1, TPS2, and TPS3 were around 2.40×10(5) Da, 2.14×10(4) Da, and 2.46×10(3) Da, respectively. In general, TPS1 and CTPS had stronger antioxidant activity, TPS2 and TPS3 had lower antioxidant activity. TPS1 had higher activity for DPPH and lipid per oxidation inhibition. But it had lower capacity for reducing power and metal chelating. This might be due to its higher content of hexuronic acid and larger molecular weight. The order of inhibition activity of lipid per oxidation of various polysaccharide fractions was the same as DPPH radical scavenging activity, as well as the order of metal chelating activity of various polysaccharide fractions similar to hydroxyl radical scavenging activity, which demonstrated that hydroxyl radical scavenging activity of polysaccharide relied heavily on the Fe(2+) metal chelating to decrease the generation of hydroxyl radical.     

Involvement of a highly polyvalent glycan in the cell-binding of the aggregation factor from the marine sponge Microciona prolifera

A proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) mediates cell-cell recognition via a cell-binding and a self-association domain. After repetitive and prolonged treatment of MAF with glycopeptide-N-glycosidase (PNGase) the specific binding of MAF to homotypic cells was decreased by 72%. Polyacrylamide gel electrophoresis and gel filtration analysis of such PNGase digests showed that: 1) the enzyme released a single glycan type of Mr = 6 X 10(3) (G-6) from MAF, 2) 1 mole of MAF contains at least 830 moles of N-linked chains of G-6 glycan. The correlation between the loss of the binding activity of MAF and the extent of the release of the repetitive G-6 polysaccharide strongly suggests its involvement in MAF-cell association via highly polyvalent interactions.     

Structure of an acidic glycan present in the lipopolysaccharide extract from the reference strain for Serratia marcescens serogroup O18.

The lipopolysaccharide extract from the cell wall of the reference strain for Serratia marcescens serogroup O18 contained, in addition to a neutral glycan characterised previously, an acidic glycan. Acidity was contributed both by D-glucuronic acid and by 4-O-[(R)-1-carboxyethyl]-D-glucose (4-O-Lac-D-Glc). By using n.m.r. spectroscopy, methylation analysis, and chemical degradations, the repeating unit of the acidic glycan was identified as a branched hexasaccharide having the structure shown; an O-acetyl group also present was not located. The glycan is believed to define the O18 serogroup, but is probably not an integral component of the lipopolysaccharide. [formula: see text].     

Chromatofocusing fails to separate hFSH isoforms on the basis of glycan structure

Follicle-stimulating hormone (FSH) glycosylation is regulated by feedback from the gonads, resulting in an array of glycans associated with FSH preparations derived from pools of pituitary or urine extracts. FSH glycosylation varies due to inhibition of FSHbeta N-glycosylation, elaboration of 1-4 branches possessed by mature N-glycans, and the number and linkage of terminal sialic acid residues. To characterize FSH glycosylation, FSH isoforms in pituitary gland extracts and a variety of physiological fluids are commonly separated by chromatofocusing. Variations in the ratios of immunological and biological activities in the resulting FSH isoform preparations are generally attributed to changes in glycosylation, which are most often defined in terms of sialic acid content. Using Western blotting to assess human FSHbeta glycosylation inhibition revealed 30-47% nonglycosylated hFSHbeta associated with four of six hFSH isoform preparations derived by chromatofocusing. Glycopeptide mass spectrometry assessment of glycan branching in these isoforms extensively characterized two N-glycosylation sites, one at alphaAsn52, the critical glycan for FSH function, and the other at betaAsn24. With two to four N-glycans per FSH molecule, many combinations of charges distributed over these sites can provide the same isoelectric point. Indeed, several glycans were common to all isoform fractions that were analyzed. There was no trend showing predominantly monoantennary glycans associated with the high-pI fractions, nor were predominantly tri- and tetra-antennary glycans associated with low-pI fractions. Thus, differences in receptor binding activity could not be associated with any specific glycan type or location in the hormone. FSH aggregation was associated with reduced receptor binding activity but did not affect immunological activity. However, as gel filtration indicated sufficient heterodimer was present in each isoform preparation to generate complete inhibition curves, the near total loss of receptor binding activity in several preparations could not be explained by aggregation alone, and the mechanism remains unknown.     

Isolation of two pure polysaccharides from the hemicellulose of slash pine (Pinus elliottii)

Two pure, acidic polysaccharides have been isolated from the hemicellulose of slash pine in yields of 1–2% and 4–5%. Their properties are compared, and the structure of one of them has been investigated by methylation analysis. The results indicate that the glycan is a β-D-(1→4)-linked xylan chain with many branch points. 4-O-Methyl-D-glucopyranosyluronic acid, L-arabinofuranose, and D-xylopyranose residues occur as non-reducing end groups. The uronic acid occurs as single-unit attachments to the main chain. Some of the D-xylose residues in the polysaccharide are doubly branched. The total hemicellulose components of the wood probably represent a complex mixture of chemical types, from which the two pure fractions described above may be separated fortuitously by careful, fractional precipitation.     

Bioactive polysaccharides from the stems of the Thai medicinal plant Acanthus ebracteatus: their chemical and physical features

Crude water-soluble polysaccharides were isolated from Acanthus ebracteatus by hot water extraction followed by ethanol precipitation after pre-treatment with 80% ethanol. The crude polysaccharides were separated into neutral and acidic polysaccharides by anion-exchange chromatography. The neutral polysaccharide (A1001) was rich in galactose, 3-O-methylgalactose and arabinose, whereas the acidic polysaccharide (A1002) consisted mainly of galacturonic acid along with rhamnose, arabinose and galactose as minor components indicating a pectin-type polysaccharide with rhamnogalacturonan type I (RG-1) backbone. 3-O-Methylgalactose is also present in the acidic fraction. Both neutral and acidic fractions showed potent effects on the complement system using pectic polysaccharide PM II from Plantago major as a positive control. A small amount of 3-O-methylgalactose present in the pectin seemed to be of importance for activity enhancement in addition to the amount of neutral sugar side chains attached to RG-1. The relationship between chemical structure and effect on the complement system of the isolated polysaccharides is considered in the light of these data. The presence of the rare monosaccharide 3-O-methylgalactose may indicate that this can be used as a chemotaxonomic marker. The traditional way of using this plant as a medical remedy appears to have a scientific basis.