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Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds.

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.

Animals↗

Recovery of polysaccharides from marc and pomace through sequential extractions assisted by ultrasound, enzymes and acid maceration.

This study evaluated the pilot-scale recovery of polysaccharides from Vitis vinifera pomace/marc using sequential extraction strategies combining high-power ultrasound (UAE), enzymes (EAE), and acid maceration (AAE). Laboratory-scale trials identified optimal conditions for enzyme dosage and liquid/solid ratio (L/S). Pilot-scale trials demonstrated that the extraction sequence and the processing byproducts influenced extraction efficiency, total soluble polysaccharide in the extract (TSP), and polysaccharide composition. Post-maceration at pH 3.2, with/without the maximum enzyme dose after UAE in a L/S of 1.3/1, improved structural polysaccharide extraction from Viura pomace, while Tempranillo marc showed better recovery of pectic families and TSP with UAE + EAE. Separating grape pomace extract (UAE) from the post-maceration stage at pH 3.2 produced two extracts: E1, with higher yield (19.9%), enriched in structural polysaccharides and oligosaccharides, and E2, enriched in high and medium molecular weight pectic polysaccharides (58.03%), a low degree of esterification (17.1%) and more complex rhamnogalacturan structures.

Polysaccharides↗

Water-soluble polysaccharides from Angelica sinensis (Oliv.) Diels: Preparation, characterization and bioactivity.

Crude water-soluble polysaccharides (ASP) were separated from Angelica sinensis (Oliv.) Diels by hot water extraction. They were fractionated into neutral and acidic polysaccharides by anion-exchange chromatography. The neutral polysaccharide (ASP1) was rich in glucose, galactose, and arabinose suggesting a mixture of glucan and arabinogalactan. The acidic polysaccharide (ASP2, ASP3) consisted mainly of galacturonic acid along with rhamnose, arabinose, and galactose indicating a pectic polysaccharide. The degree of esterification of ASP and ASP3 were 54.06% and 47.14% for the crude and purified sample, respectively. ASP3, with a molecular weight of 3.4x10(4)Da determined by high-performance size-exclusion chromatography (HPSEC), was the major constituent for the crude extracts. The radioprotective effect of the pectic polysaccharide ASP3 was studied in murine models. ASP3 pretreated mice exhibited a significant decrease of apoptosis (P<0.05, dosage of 200 mg/kgd body weight) in peripheral lymphocytes compared to the irradiated control. The results showed that ASP3 can protect leucocytes and lymphocytes of mice against radiation induced damage, which has potential radioprotective effect on acute radiation injured mice.

Angelica sinensis↗

Human milk antibodies with polysaccharide kinase activity.

It was shown for the first time that a small fraction of milk secretory IgA (sIgA) is tightly bound to oligosaccharides (oligoSACs) and polysaccharides (polySACs). The ability of sIgA to phosphorylate oligo- and polysaccharides was shown to be an intrinsic property of this antibody. In contrast to known kinases, sIgAs with polysaccharide kinase activity can transfer phosphoryl group to oligo- and polysaccharides not only from [gamma-(32)P]ATP but can also use [(32)P]orthophosphate as a substrate of phosphorylation reaction. An extremely unusual property of polysaccharide kinase Abs is their high affinity for orthophosphate (K(m) = 15-77 microM), and orthophosphate is a better substrate than ATP. Two first examples of natural abzymes (Abzs) with synthetic activity were milk sIgA with protein and lipid kinase activities. Polysaccharide kinase sIgA of human milk is the third example of natural antibodies (Abs) with synthetic activity.

Antibodies, Catalytic↗

Botanical polysaccharides: macrophage immunomodulation and therapeutic potential.

Botanical polysaccharides exhibit a number of beneficial therapeutic properties, and it is thought that the mechanisms involved in these effects are due to the modulation of innate immunity and, more specifically, macrophage function. In this review, we summarize our current state of understanding of the macrophage modulatory effects of botanical polysaccharides isolated from a wide array of different species of flora, including higher plants, mushrooms, lichens and algae. Overall, the primary effect of botanical polysaccharides is to enhance and/or activate macrophage immune responses, leading to immunomodulation, anti-tumor activity, wound-healing and other therapeutic effects. Furthermore, botanical and microbial polysaccharides bind to common surface receptors and induce similar immunomodulatory responses in macrophages, suggesting that evolutionarily conserved polysaccharide structural features are shared between these organisms. Thus, the evaluation of botanical polysaccharides provides a unique opportunity for the discovery of novel therapeutic agents and adjuvants that exhibit beneficial immunomodulatory properties.

Adjuvants, Immunologic↗

Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells.

The main purposes of this study were to investigate the regulation of polysaccharides isolated from A. cinnamomea on vascular endothelial growth factor (VEGF)-induced cyclin D1 expression and down stream signaling pathway that may correlate with their anti-angiogenc effects in endothelial cells (ECs). Crude and fractionated polysaccharides (Fra-1 to Fra-4) of A. cinnamomea showed slightly toxicity to ECs as compared with their inhibition concentration on angiogenic-related gene expression. The crude extract and fractionated fractions, except for Fra-2, of A. cinnamomea polysaccharides significantly decreased VEGFR2 phosphorylation on tyrosine 1054/1059, cyclin D1 promotor activity, and protein expression induced by VEGF. Crude extract of A. cinnamomea polysaccharides inhibited the binding of VEGF to KDR/flk-1 in a dose-dependent manner. These results indicated that inhibition of VEGF interaction with VEGF receptor 2 is the mechanism serves A. cinnamomea as a protective mechanism composing the anti-angiogenesis function. Furthermore, A. cinnamomea polysaccharides also blocked VEGF-induced migration and capillary-like tube formation of ECs on Matrigel. Taken together, these results indicate that A. cinnamomea polysaccharides inhibit cyclin D1 expression through inhibition of VEGF receptor signaling, leading to the suppression of angiogenesis.

Angiogenesis Inhibitors↗

Antibody response to pneumolysin and to pneumococcal capsular polysaccharide in healthy individuals and Streptococcus pneumoniae infected patients.

BACKGROUND: Animal experiments have shown that antibodies against capsular polysaccharide enhance phagocytosis of pneumococcal bacteria and that antibodies against pneumolysin are anti-inflammatory and prevent pneumococcal invasion. It is not known if an antibody response to pneumolysin can be acquired from natural exposure to pneumococcal bacteria or how the concentration of pneumolysin antibody at the mucosal surface compares with that of antibodies against pneumococcal capsular polysaccharide. This study used an equal potency method to measure specific antibody concentrations against pneumolysin and pneumococcal capsular polysaccharides in order to facilitate comparative estimates of concentrations in saliva and serum. The results may provide experimental information as a basis for an improved pneumococcal vaccine strategy. RESULTS: Healthy individuals had higher IgM and IgG antibody concentrations against capsular polysaccharide than against pneumolysin in both saliva and serum, but for IgA the converse was true. Patients with acute pneumococcal infection had significantly lower concentrations of specific IgG antibodies against both antigens than the healthy group. These patients also had significantly higher concentrations of IgM antibody against both antigens than the healthy control group. DISCUSSION: Healthy individuals acquire a comparatively lower concentration of antibody to pneumolysin than to pneumococcal capsular polysaccharides from natural exposure to pneumococcal bacteria. Patients infected by pneumococcal bacteria have lower specific IgG antibody concentrations to both antigens than healthy individuals. These findings support the view that pneumolysin could potentially be used as a vaccine. For enhanced effectiveness, it could be used as a supplement to Pneumovax((R))II rather than as a replacement. The two acquired antibodies, i.e. to pneumolysin and to capsular polysaccharide, could then play their protective roles at different stages in the course of pneumococcal infection, and together contribute to an effective immune defence against Streptococcus pneumoniae.

Adult↗

Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE).

Experimental carbohydrate-conjugate vaccines composed of the 13 amino acid universal Pan HLA-DR Epitope (PADRE) and Streptococcus pneumoniae capsular polysaccharides from serotypes 14, 6B and 9V were produced. Simple carbodiimide-mediated condensation chemistry was used to conjugate the PADRE synthetic peptide to the three chemically different capsular polysaccharides in a 1:1 molar ratio. The immunogenicity of the PADRE peptide component of the conjugate vaccines was confirmed by the induction of PADRE-specific CD4+ helper T cell (HTL) responses following immunization of C57BL/6 mice. High titer antibody responses specific for polysaccharides of S. pneumoniae serotypes 14, 6B and 9V were induced using Complete Freund's Adjuvant (CFA) and alhydrogel Al(OH)3 formulations. The HTL, or carrier, effect of the PADRE synthetic peptide was only evident using the PADRE-polysaccharide conjugates; simple mixtures of the PADRE peptide and polysaccharides were essentially nonimmunogenic. The functional or potential protective value of the polysaccharide-specific antibodies was measured as a function of opsonophagocytic activity for the 6B serotype. High titers of opsonophagocytic activity were measured in sera from mice immunized with formulations containing both adjuvants. These data demonstrate that the PADRE synthetic peptide can induce the HTL responses needed to support the development of antibodies specific for bacterial carbohydrates used in conjugate vaccines.

Adjuvants, Immunologic↗

Characterization and phase behaviour of phospholipid bilayers adsorbed on spherical polysaccharidic nanoparticles.

In this paper a new drug carrier, the Light-biovector, is described. These biovectors are composed of a neutral, anionic or cationic polysaccharidic core surrounded by phospholipids. They can be prepared with high yield and in a nearly pure form as determined by density analysis on sucrose gradients. These particles showed great stability with no sedimentation being observed after more than one year of storage. Physicochemical studies carried out with dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol mixtures showed that in Light-biovectors, the lipids are organized in bilayer surrounding the polysaccharidic core. In presence of a neutral polysaccharidic core, the gel to liquid phase transition temperature Tm of DPPC was only slightly affected as compared to liposomal dispersions of the lipid. In contrast, for cationic and anionic Light-biovectors, the Tm of the lipids was affected by the electric charge born by the polysaccharidic core, indicating that electrostatic interactions contribute to the organization of the lipid bilayer in these systems. It was also found that the association of anionic membrane to anionic polysaccharidic cores and the association of cationic membrane to cationic polysaccharidic cores was possible.

1,2-Dipalmitoylphosphatidylcholine↗

Selective cleavage of glycosidic linkages: studies with the O-specific polysaccharide from Shigella dysenteriae type 3.

Treatment of the O-specific polysaccharide from Shigella dysenteriae Type 3 with hydrazine in the presence of hydrazine sulphate resulted in quantitative N-deacetylation with the formation of a modified polysaccharide containing free amino groups. Oxidation of the modified polysaccharide with periodate did not destroy the 2-amino-2-deoxygalactose residues, thus indicating that they were substituted at position 3. Acid hydrolysis of the modified polysaccharide afforded 3-O-(2-amino-2-deoxy-beta-D-galactopyranosyl)-D-galactose, which was identified as the N-acetyl derivative. Deamination of the modified polysaccharide with nitrous acid cleaved the 2-amino-2-deoxy-D-galactopyranosyl linkages to give a pentasaccharide as the major product, which appeared to be the modified chemical repeating unit of the O-specific polysaccharide.

Acetylation↗

Structure of the capsular polysaccharide of Klebsiella type 73 (Enterobacter aerogenes).

The capsular polysaccharide from Klebsiella Type 73 (Enterobacter aerogenes) was found to contain equimolar amounts of D-galactose, D-glucose, L-rhamnose, and D-glucuronic acid. Acid hydrolysis of the polysaccharide gave one aldobio-and one aldotrio-uronic acid, whose structures were established by acid hydrolysis and by methylation analysis. The anomeric configurations of the different sugar residues were determined from the specific rotations of the polysaccharide and the aldobio-and aldotrio-uronic acids, and also by oxidation of the native and the carboxyl-reduced polysaccharide with chromium trioxide. Methylation analysis of the polysaccharide provided information about the linkages of the different sugar residues. Based on all of these results, the structure assigned to the repeating unit of the polysaccharide is as follows. (Formula: see text).

Carbohydrate Conformation↗

Structure of the Klebsiella type 10 capsular polysaccharide.

The capsular polysaccharide from Klebsiella Type 10 was found to contain D-galactose, D-glucose, D-mannose, and D-glucuronic acid in the ratios 3:1:1:1. Acid hydrolysis of the polysaccharide gave one aldobiouronic acid, one aldotriouronic acid, one aldotetraouronic acid, and two neutral disaccharides the structures of which were established. The native and carboxyl-reduced polysaccharide have been subjected as appropriate to methylation analysis and Smith degradation. Degradation of the methylated polysaccharide with base established the identity of the sugar unit preceding the glucosyluronic acid residue. The anomeric configurations of the sugar residues were determined by oxidation of the acetylated native and carboxyl-reduced polysaccharides with chromium trioxide. Based on these studies, the hexasaccharide structure 1 has been assigned to the repeating unit of the K-10 polysaccharide.

Bacterial Capsules↗

Location and identity of the acyl substituents on the extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum.

The basic structures of the extracellular polysaccharides of Rhizobium leguminosarum and Rhizobium trifolii were found to be identical, but their acylation patterns differ. Liquid hydrogen fluoride at -40 degrees degrades the two polysaccharides to a series of oligosaccharides representing the repeating units of the polysaccharides and their higher homologs. At -23 degrees, it degrades the polymers to a mixture of oligosaccharides from which a tetrasaccharide constituting a unit of the backbone of the polysaccharide, and a trisaccharide representing all but the non-reducing terminus of the side chain, could be readily purified. The location and identity of the acyl substituents were determined by 1H-n.m.r. spectroscopy, methylation analysis, and f.a.b. mass spectrometry. The unusual substituent D-3-hydroxybutanoate was found esterified to O-3 of a terminal 4,6-O-pyruvic acetylated D-galactose in both strains of R. leguminosarum, and in one of the three strains of R. trifolii tested. All of the strains tested contained a 3-O-acetyl substituent on the (1----4)-beta-D-glucopyranosyl residues in the backbone of the polysaccharide. Only the R. leguminosarum polysaccharides contained a combination of 2- and 3-O-acetyl groups on the branching sugar of the backbone of the polymer.

Acylation↗

Polysaccharides and food processing.

The rôle of polysaccharides during processing and for the quality of foods is discussed. Starch is the most important energy source for man. Most other polysaccharides are not metabolized for energy, but play an important rôle as dietary fibres. Pectins, alginates, carrageenans, and galactomannans are discussed as functional food additives in relation to their structure and their rheological behaviour, stability and interactions. Endogenous polysaccharides of fruits and vegetables and in products derived from them are responsible for such phenomena as texture (changes), press yields, ease of filtration and clarification, cloud stability, and mouth feel. To achieve desirable properties, the action of endogenous enzymes on polysaccharides must be inactivated and/or exogenous enzymes added as processing aids. This is also true for overcoming haze phenomena in clear juices or to break down undesirable microbial polysaccharides. Dough properties for bread baking can be improved by enzymic breakdown of a restrictive pentoglycan network. Network formation may come about by oxidative coupling of phenol rings of ferulic acid bound to hemicelluloses by ester links. Gels may be made by inducing oxidative coupling in natural or synthetic systems. Stagnation in development of new polysaccharide food additives is ascribed to difficulties in obtaining government approval for food use.

Carbohydrate Conformation↗

Structures of two polysaccharides of Campylobacter jejuni 81116.

Campylobacter jejuni 81116 has been extensively investigated in studies on genes associated with the synthesis of Campylobacter lipopoly/lipooligosaccharides (LPS/LOS). Despite these investigations, data on the chemical structure of polysaccharides from C. jejuni 81116 have been absent. The present study was undertaken to fill that void. Biomass was grown in large quantities on agar medium, harvested and extracted by hot phenol-water extraction. Subsequently, extracts were treated by DNase, RNase and proteinase K to remove contaminants. After mild acid treatment, followed by preparative gel-permeation and anion-exchange chromatography, fractions were isolated and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, 1H,(13)C HMQC and HMBC experiments. These advanced investigations revealed the occurrence of two different polysaccharides in the approximate ratio of 3:1, each having a tetrasaccharide repeating unit. Polysaccharide A contained glucose, glucuronic acid and mannose, and is O-acetylated. Polysaccharide B contained glucose, galactose and N-acetylglucosamine. Importantly, polysaccharide A is acidic, whereas polysaccharide B is neutral. [carbohydrate structure: see text]

Campylobacter jejuni↗

Ozonolytic depolymerization of polysaccharides in aqueous solution.

The selective oxidation of beta-D-glycosidic linkages of polysaccharides by ozone has great utility as a general method for depolymerization of polysaccharides. Here we describe a 'one-step' method whereby polysaccharides dissolved in water or basic solutions are depolymerized by ozonolysis. The oxidation of glycosidic linkages of unprotected carbohydrates by ozone is complicated by several side reactions. We describe here optimized conditions for carrying out ozonolysis degradation. We also characterize the major pathways for unwanted degradation by various side reactions. In the preferred oxidation pathway, the aldosidic linkage is oxidized to an aldonic ester function that hydrolyzes under the basic conditions employed to give a free aldonate, with cleavage of the polysaccharide chain. Nonselective degradation pathways include oxidative degradation by radical species that oxidize glycosyl residues to formic, acetic, and oxalic acids. The nonselective degradation caused by acids is minimized by basic buffers. The products of polysaccharide depolymerization form a size distribution around a nominal molecular weight, and the average molecular weight of the products can be controlled by the rate or amount of ozone passed through the reaction mixture. The ozonolysis method described herein provides a convenient, inexpensive, and controllable means for generating small polysaccharides or large oligosaccharide fragments.

Bacterial Capsules↗

Water-soluble polysaccharides from Ginkgo biloba leaves.

The water-soluble polysaccharides from dried Ginkgo biloba leaves were isolated after exhaustive extraction with organic solvents. The polysaccharide mixture could be separated into a neutral (GF1) and two acidic (GF2 and GF3) polysaccharide fractions by ion exchange chromatography. According to the Mr distribution GF1 and GF3 seemed to be homogenous, whereas GF2 could be further fractionated into two subfractions (GF2a and GF2b) by gel permeation chromatography. GF1 (Mr 23,000) showed the structural features of a branched arabinan. The main chain was composed of 1,5-linked arabinose residues and three in 12 arabinose molecules were branched via C-2 or C-3. GF2a (Mr 500,000) consisted mainly of 1,2,4-branched mannose (29%), 1,4-linked glucuronic (32%) and galacturonic (8%) acid as well as terminal rhamnose (25%). After removal of ca 70% of the terminal rhamnose the remaining polysaccharide showed a decrease in 1,2,4-branched mannose and an increase in 1,2-linked mannose indicating that at least half of the rhamnose residues were linked to mannose via C-4. GF3 (Mr 40,000) consisted of 1,4-linked galacturonic (30%) and glucuronic (16) acid, 1,3,6-branched galactose (15%), 1,2-linked (5%) and 1,2,4-branched (3.5%) rhamnose as well as 1,5-linked arabinose (11%). Rhamnose (5%) and arabinose (10%) were present as terminal groups. Mild acid hydrolysis selectively cleaved arabinose and the remaining polysaccharide showed an increased amount of 1,6-linked and terminal galactose and a decreased quantity of 1,3,6-branched galactose. These results indicated that the terminal as well as the 1,5-linked arabinose were mainly connected to galactose via C-3. The GF3 polysaccharide appeared to be a rhamnogalacturonan with arabinogalactan side chains.

Carbohydrate Sequence↗

Chemical modification, characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides.

A natural lacquer polysaccharide with complex branches was separated into two fractions, LPH (MW 16.9x10(4)) and LPL (MW 6.85x10(4)). Results of 13C NMR and FT-IR indicated they had the same structure. The treatment of LPL with sodium periodate led to a partial cut-off of side chains with 4-O-methyl-D-glucuronic acid in the terminal. These polysaccharides were sulfated in the presence of Py*SO3/DMSO. Depending on the reaction conditions, the products showed a different degree of sulfation (DS) ranging from 0.57 to 1.57 and different molecular weights ranging from 1.71x10(4) to 3.49x10(4). FT-IR analysis showed the equatorial primary OH at O-6 and the axial secondary OH at O-4 were sulfated. Activated partial thromboplastin time (APTT), prothrombin time and thrombin time (TT) assays showed the sulfated polysaccharides could prolong APTT and TT, but not TP. These activities strongly depended on the DS, the molecular weights (MW) and the branching structure of polysaccharides. DS of above 0.8 was essential for anticoagulant activity. The anticoagulant activity increased with the DS and the molecular weights. The molecular weights played a more important role. The branching structure of polysaccharides increased the activities. In our studies, the sulfated polysaccharides with the DS of 1.15 and the highest MW of 3.49x10(4) had the best blood anticoagulant activities.

Anticoagulants↗