Anti-oxidation activity of different types of natural Cordyceps sinensis and cultured Cordyceps mycelia.

Cordyceps, one of the well-known traditional Chinese medicines, consists of the dried fungus Cordyceps sinensis growing on the larva of the caterpillar. It is commonly used for the replenishment of body health. One of the known pharmacological effects is its anti-oxidation activity. However, there is a great variation of the quality in different sources of Cordyceps. Here, the water extracts of various sources of natural C. sinensis and cultured Cordyceps mycelia were analyzed for their anti-oxidation activity by using three different assay methods such as the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. The results showed that Cordyceps, in general, possesses a strong anti-oxidation activity in all assays tested. However, both natural and cultured Cordyceps showed the lowest inhibition in the lipid peroxidation when compared with the other two assay methods. The cultured Cordyceps mycelia had equally strong anti-oxidation activity as compared to the natural Cordyceps. Besides, the anti-oxidation activities were increased to 10-30 folds in the partially purified polysaccharide fractions from the cultured Cordyceps mycelia, which suggested that the activity could be derived partly from Cordyceps polysaccharides.     

Modification of polysaccharides containing uronic acid residues

Klebsiella Type 47 capsular polysaccharide has side chains attached to the main chain viaD-glucuronic acid residues. The side chains have been removed to yield an essentially linear polysaccharide by the following sequence of reactions: (1) substitution of hydroxyl and car?yl groups with methyl vinyl ether; (2) β-elimination by treatment with base; (3) removal of modified uronic acid residues and protecting groups by mild acid hydrolysis. The possibility of modifying other uronic acid-containing polysaccharides by this method is discussed.     

Antiviral Effects of Sulfated Exopolysaccharide from the Marine Microalga <Emphasis Type="Italic">Gyrodinium impudicum</Emphasis> Strain KG03

The sulfated exopolysaccharide p-KG03, which is produced by the marine microalga Gyrodinium impudicum strain KG03, exhibited impressive antiviral activity in vitro (EC₅₀ = 26.9 µg/ml) against the encephalomyocarditis virus (EMCV). Depending on the p-KG03 concentration, the development of cytopathic effects in EMCV-infected HeLa cells was either inhibited completely or slowed. Moreover, p-KG03 did not show any cytotoxic effects on HeLa cells, even at concentrations up to 1000 µg/ml. The polysaccharide was purified by repeated precipitation in ethanol, followed by gel filtration. The p-KG03 polysaccharide had a molecular weight of 1.87 × 10⁷, and was characterized as a homopolysaccharide of galactose with uronic acid (2.96% wt/wt) and sulfate groups (10.32% wt/wt). The biological activities of p-KG03 suggest that sulfated metabolites from marine organisms are a rich source of antiviral agents. This is the first reported marine source of antiviral sulfated polysaccharides against EMCV. The p-KG03 polysaccharide may be useful in the development of marine bioactive exopolysaccharide for biotechnological and pharmaceutical products.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-3H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-³H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.     

Characterization of uniformly and atom-specifically C-labeled heparin and heparan sulfate polysaccharide precursors using C NMR spectroscopy and ESI mass spectrometry

The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin and heparan sulfate oligosaccharide or disaccharide fragments using simple 1D ¹H NMR spectroscopy is often complicated by the extensive signal overlap. ¹³C NMR signals, on the other hand, overlap less and therefore, ¹³C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific ¹³C-labeled heparin and heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly ¹³C- and ¹⁵N-labeled precursors were also produced and characterized by ¹³C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of ¹³C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% ¹³C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome-proteome interactions.     

Precursor-Product Relationships during Sulfate Incorporation into Porphyridium Capsular Polysaccharide

This study describes the kinetics of ³⁵S-incorporation during in vivo sulfate esterification of Porphyridium aerugineum capsular polysaccharide. Techniques were developed to isolate the precursor pool (free sulfate), cell-associated product, and extracellular product. Specific radioactivities of these three fractions were monitored during pulse-chase sequences. Label rapidly appeared in the pool during the pulse, then declined asymptotically during the chase as equilibrium was approached. Efflux of small quantities of isotope from the cell during chase periods was not the result of backleakage, but the result of washing untransported isotope from the free-space. During the pulse, intracellular product was labeled at 25% of the rate at which the pool was labeled. Fully 50% of the label which left the pool was incorporated into the polysaccharide as ester sulfate, indicating that polysaccharide esterification is a major metabolic pathway for sulfate. The specific radioactivity of the extracellular product increased slowly throughout pulse and chase periods.     

Purification and Biological Activities of Enzymatically Degraded Sargassum fusiforme Polysaccharides

Enzymatic hydrolysate of the crude polysaccharide (SFP) extracted from Sargassum fusiforme was purified by column DEAE-52 and Sephadex G-100 to yield four components, namely, ESFP1, ESFP2, ESFP3 and ESFP4. These components were characterized by chemical composition assay, GC/MS, HPGPC, UV and FT-IR techniques. The in vitro antioxidant activities of the four purified fractions were investigated by measuring their radical scavenging activity and reducing power. The results suggested that all the four components possess good antioxidant activities. Among them, ESFP1 was found to possess the strongest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and hydroxyl radical-scavenging activity, and the greatest ferric reducing power. The immunomodulatory effect of these four polysaccharides was demonstrated by their ability to promote proliferation, and to enhance both phagocytic activity and NO release in a macrophage RAW264.7 model. The results revealed that the bioactivities of the polysaccharides are related to their molecular weight, and the uronic acid and sulfate contents.     

Profiling Carbohydrate-Receptor Interaction with Recombinant Innate Immunity Receptor-Fc Fusion Proteins

The recognition of bacteria, viruses, fungi, and other microbes is controlled by host immune cells, which are equipped with many innate immunity receptors, such as Toll-like receptors, C-type lectin receptors, and immunoglobulin-like receptors. Our studies indicate that the immune modulating properties of many herbal drugs, for instance, the medicinal fungus Reishi (Ganoderma lucidum) and Cordyceps sinensis, could be attributed to their polysaccharide components. These polysaccharides specifically interact with and activate surface receptors involved in innate immunity. However, due to the complexity of polysaccharides and their various sources from medicinal fungi, quantitative analysis of medicinal polysaccharide extracts with regard to their functions represents a major challenge. To profile carbohydrate-immune receptor interactions, the extracellular domains of 17 receptors were cloned as Fc-fusion proteins, such that their interactions with immobilized polysaccharides could be probed in an enzyme-linked immunosorbent assay. The results show that several innate immune receptors, including Dectin-1, DC-SIGN, Langerin, Kupffer cell receptor, macrophage mannose receptor, TLR2, and TLR4, interact with the polysaccharide extracts from G. lucidum (GLPS). This analysis revealed distinct polysaccharide profiles from different sources of medicinal fungi, and the innate immune receptor-based enzyme-linked immunosorbent assay described here can serve as a high-throughput profiling method for the characterization and quality control of medicinal polysaccharides. It also provides a means to dissect the molecular mechanism of medicinal polysaccharide-induced immunomodulation events.     

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° degrades the two polysaccharides to a series of oligosaccharides representing the repeating units of the polysaccharides and their higher homologs. At −23°, 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 acetalated 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)-β-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.     

Anti-Inflammatory Properties of the Medicinal Mushroom Cordyceps militaris Might Be Related to Its Linear (1→3)-β-D-Glucan

The Ascomycete Cordyceps militaris, an entomopathogenic fungus, is one of the most important traditional Chinese medicines. Studies related to its pharmacological properties suggest that this mushroom can exert interesting biological activities. Aqueous (CW and HW) and alkaline (K5) extracts containing polysaccharides were prepared from this mushroom, and a β-D-glucan was purified. This polymer was analysed by GC-MS and NMR spectrometry, showing a linear chain composed of β-D-Glcp (1→3)-linked. The six main signals in the ¹³C-NMR spectrum were assigned by comparison to reported data. The aqueous (CW, HW) extracts stimulated the expression of IL-1β, TNF-α, and COX-2 by THP-1 macrophages, while the alkaline (K5) extract did not show any effect. However, when the extracts were added to the cells in the presence of LPS, K5 showed the highest inhibition of the pro-inflammatory genes expression. This inhibitory effect was also observed for the purified β-(1→3)-D-glucan, that seems to be the most potent anti-inflammatory compound present in the polysaccharide extracts of C. militaris. In vivo, β-(1→3)-D-glucan also inhibited significantly the inflammatory phase of formalin-induced nociceptive response, and, in addition, it reduced the migration of total leukocytes but not the neutrophils induced by LPS. In conclusion, this study clearly demonstrates the anti-inflammatory effect of β-(1→3)-D-glucan.     

Effect of extraction temperature on the diffusion coefficient of polysaccharides from Spirulina and the optimal separation method

The extraction temperature had a significant impact on the concentration of polysaccharides derived from solid-liquid extraction of Spirulina. The polysaccharide concentration was significantly higher when the extraction was performed at 90°C than when it was performed at 80, 70, and 50°C. This result is related to the diffusion coefficients of the polysaccharides, which increased from 1.07 × 10^?12 at 50°C to 3.02 × 10^?12 m²/sec at 90°C. Using the Arrhenius equation, the pre-exponential factor (D ₀ ) and the activation energy (E _a ) for Spirulina polysaccharide extraction were calculated as 7.958 × 10^?9 m²/sec and 24.0 kJ/mol, respectively. Among the methods used for the separation of Spirulina polysaccharides, cetyltrimethylammonium bromide (CTAB, method I) and organic solvent (ethanol, in methods II and III) provided similar yields of polysaccharides. However, the separation of polysaccharides using an ultrafiltration (UF) process (method III) and ethanol precipitation was superior to separation via CTAB or vacuum rotary evaporation (method II). The use of a membrane with a molecular weight cut-off (MWCO) of 30 kDa and an area of 0.01 m² at a feed pressure of 103 kPa with a mean permeate flux of 39.3 L/m²/h and a retention rate of 95% was optimal for the UF process. The addition of two volumes (v/v) of ethanol, which gave a total polysaccharide content of approximately 4% dry weight, was found to be most suitable for polysaccharide precipitation. The results of a Sepharose 6B column separation showed that the molecular weights of the polysaccharides in fractions I and II were 212 and 12.6 kDa, respectively.     

A MODIFIED CELL WALL MUTANT OF THE RED MICROALGA RHODELLA RETICULATA RESISTANT TO THE HERBICIDE 2,6-DICHLOROBENZONITRILE1

The rigid component of the cell walls of red macroalgae, cellulose, is lacking in the red microalgae. Instead, the cells are encapsulated within an amorphous polysaccharide. These complex sul fated polysaccharides are composed of at least 10 different sugars, but their structure is not known, When the herbicide 2,6-dichlorobenzonitrile (DCB), a compound that specifically inhibits cellulose biosynthesis, was applied to cultures of the red microalga Rhodella reticulata upon inoculation, growth was inhibited. When added during the stationary phase of growth (after cell division had ceased), DCB did not affect cell number but it did inhibit polysaccharide production. A spontaneous mutant resistant to DCB was selected; it had physiological characteristics similar to those of the wild-type parent. The composition of the cell wall polysaccharide of the mutant was totally modified, being composed almost entirely (98% of its dry matter, as compared to 2.9% in the wild type) of methyl galactose, but retaining the same sulfate content. The molecular mass of the mutant polysaccharide was, however, similar to that of the wild-type parent (～6 × 10⁶ daltons), although its viscosity was significantly lower.     

Comparison of Cell Wall Polysaccharide Hydrolysis by a Dilute Acid/Enzymatic Saccharification Process and Rumen Microorganisms

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72?h conversion assay (CONV) with in vitro rumen microflora incubation for 72?h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r?=?0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.     

Comparative studies of mucopolysaccharides from marine animals. I. Raja eglanteria Bosc.

A mucopolysaccharide (MPS) was extracted from the pectoral fins of the skate, Raja eglanteria Bosc. The purified MPS had an anticoagulant activity of 28 IU/mg and an optical rotation value, [α]_D²⁵, of ?9°. The MPS was analyzed for uronic acid, hexosamine, N-sulfate, O-sulfate, O-sulfate, and neutral sugar. This analysis suggests that this MPS is an over-sulfated chondroitin sulfate, the electrophoretic pattern and infra-red spectrum of which are similar to chondroitin sulfate.     

Isolation of a chondroitin sulfate--dermatan sulfate proteoglycan from bovine aorta.

Material containing proteoglycans was extracted from bovine aorta by the dissociative solvent 3.0 m MgCl². The proteoglycan that remained in solution at low ionic strength was purified by isopycnic CsCl centrifugation (?, 1.75 – 1.89 g/ml). From the lower third of the gradient a proteoglycan was isolated which behaved as a homogeneous material when analyzed by the ultracentrifuge and by electrophoresis on cellulose acetate. The proteoglycan contained 12% protein, 21% uronic acid, and 28% hexosamine. Analyses by hyaluronidase digestion and gas-liquid chromatography of the polysaccharide moieties of the proteoglycan showed a composition of 56% chondroitin 6-sulfate, 20% chondroitin 4-sulfate, and 7% dermatan sulfate. A copolymeric structure for the polysaccharide of the proteoglycan is proposed.     

Polar Constituents of Brown Seaweed Colpomenia peregrina (Sauv.) Hamel from the Black Sea

GC-MS of trimethylsilyl derivatives of the compounds present in the butanolic extract of biomass of brown seaweed Colpomenia peregrina from the Black Sea aided in identification of 24 components, including aliphatic hydroxy and keto and aromatic acids, glycerol, mannitol, floridoside, and monosaccharides. The polysaccharide composition of the biomass was also studied, with high sodium alginate and laminaran contents and a comparatively low level of fucoidan being revealed. The polysaccharides were isolated from the biomass by fractional extraction and purified by precipitation or ion exchange chromatography. The structures of alginic acid and laminaran were deduced from ¹³C NMR spectra and confirmed, in the case of laminaran, by methylation analysis. The sodium alginate was shown to contain more guluronic (G) than mannuronic acid (M) residues, the M/G ratio being 0.48. Laminaran was demonstrated to be a -glucan with 1 3 linkages in its backbone and 1 6 linkages in its branching points, which is characteristic of brown algae. Fucoidan turned out to be a complex heteropolysaccharide containing, in addition to fucose and sulfate, other neutral monosaccharides and uronic acids.     

Glycosaminoglycans from earthworms (<Emphasis Type="Italic">Eisenia andrei</Emphasis>)

The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycan-degrading enzymes confirmed the presence of chondroitin sulfate/dermatan sulfate and heparan sulfate in the 2.0 M NaCl fraction. The content of GAGs (uronic acid containing polysaccharide) in the 2.0 M NaCl fraction determined by carbazole assay was 2%. Disaccharide compositional analysis using liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) analysis after chondroitinase digestion (ABC and ACII), showed that the chondroitin sulfate/dermatan sulfate contained a 4-O-sulfo (76%), 2,4-di-O-sulfo (15%), 6-O-sulfo (6%), and unsulfated (4%) uronic acid linked N-acetylgalactosamine residues. LC–ESI–MS analysis of heparin lyase I/II/III digests demonstrated the presence of N-sulfo (69%), N-sulfo-6-O-sulfo (25%) and 2-O-sulfo-N-sulfo-6-O-sulfo (5%) uronic acid linked N-acetylglucosamine residues.     

Crithidia spp.: Structural comparison of polysaccharides for taxonomic significance

The chemical structures of polysaccharide components of cells of several Crithidia species have been partially elucidated. The structures have been used as criteria to evaluate evolutionary lines previously proposed for species of Crithidia and Herpetomonas and for Trypanosoma cruzi. In accord with the suggestion that Crithidia and Herpetomonas are closely interrelated, all species investigated synthesize a linear (1→2)-linked β-d-mannopyranan and a heteropolysaccharide. These differ from T. cruzi polysaccharide, which contains α-d-mannopyranosyl structures and likely indicates a separate evolutionary route for this flagellate. Crithidia fasciculata, Crithidia harmosae, and Crithidia luciliae form a closely knit group since they form arabinogalactans with related structures. The similarity is particularly close between arabinogalactans of C. fasciculata and C. harmosae whose ¹³C nuclear magnetic resonance spectra show a high degree of resemblance. An unnamed Crithidia sp. contains polysaccharides with fucose and xylose units, intermediate between those of Crithidia deanei, which gave glucose and fucose on hydrolysis, and Herpetomnas samuelpessoai, which gave glucuronic acid and xylose.     

THE PRODUCTION OF EXTRACELLULAR POLYSACCHARIDE BY THE UNICELLULAR RED ALGA PORPHYRIDIUM AERUGINEUM1,2

The unicellular red alga Porphyridium aerugineum was shown to be encapsulated by an amorphous, water-soluble, polyanionic polysaccharide of high molecular weight. The encapsulating polysaccharide is qualitatively identical with polysaccharide found dissolved in large quantity in the culture medium. The kinetics of extracellular polysaccharide production as a function of cell age was studied. Rates of production (on a per cell basis) of both encapsulating and dissolved polysaccharides are greatest in stationary phase light-grown cultures. Dissolved polysaccharide was quantitatively isolated by precipitation with cetyl pyridinium chloride, conversion to the calcium salt, and reprecipitation with ethanol. The procedure yields a spectrally pure product, which is composed of glucose, galactose, xylose, and 2 undetermined, sugar components, and has a sulfate content of 7.6% by weight. Electron microscopy of Porphyridium revealed that Golgi vesicles transport, polymerized polysaccharides to and through the cell membrane. Similar vesicles were observed in the multicellular Pseudogloiophloea, indicating that the Golgi complex plays a crucial role in the production of extracellular polysaccharides by the red algae. H¹⁴CO₃^- pulse-label experiments resulted in labeled extracellular polysaccharide in which all the constituent components contained ¹⁴C. Rates of excretion of polysaccharide were found, to follow a cyclic pattern, correlated generally with the division cycle, of the cell.