Structural characteristic of pectin-glucuronoxylan complex from Dolichos lablab L. hull.

Polysaccharides are important constituents in Dolichos lablab hull. Herein, pectin-glucuronoxylan complex from D. lablab hull designated as DLHP-3 (D. lablab hull polysaccharide,) was prepared by ion exchange and gel permeation chromatography, and further characterized by acid degradation and enzymatic hydrolysis, methylation combined with GC-MS, NMR and MALDI-TOF-MS analysis. Both of pectin and glucuronoxylan regions were found in DLHP-3. The glucuronoxylan region consisted of a →4)-ß-Xylp-(1→ backbone with branches of α-GlcpA-(1→ substituted at O-2 site, and the ratio of xylose to glucuronic acid was about 5:1. Acetyl groups were mainly attached to O-3 site of →2,4)-ß-Xylp-(1→ residues. The main chain of pectin region could be represented by →4)-α-GalpA-(1→4)-α-GalpA-(1→ and →2)-α-Rhap-(1→4)-α-GalpA-(1→ with partial methyl-esterification. The side chains were deduced to embrace arabinan and arabinogalactan linked to rhamnogalacturonan-I region. Pectin was probably covalently bound to glucuronoxylan. Our findings uncovered the molecular structure of pectin-glucuronoxylan complex from D. lablab hull.

Revealing the architecture and solution properties of polysaccharide fractions from Macrolepiota albuminosa (Berk.) Pegler.

Macrolepiota albuminosa (Berk.) Pegler is abundant in active polysaccharides, but little is known about their structures and solution properties. In this study, water-extracted polysaccharides from M. albuminosa (MAWP) were purified into three fractions with structural heterogeneity, which was attributed to the diversity in molecular weight, monosaccharide composition and linkage patterns, further affecting their solution properties. Methylation and NMR analysis revealed MAWP-60p and MAWP-70 were a 3-O-methylated glucomannogalactan and a previously unreported glucomannogalactan, whereas MAWP-80 was elucidated as a branched galactoglucan. Besides, three fractions exhibited random coil conformation in aqueous solution, while MAWP-60p had the highest viscosity due to its highest molecular weight, mean square radius of gyration (Rg) and O-methyl group attached to the backbone. The molecular weight, monosaccharide composition and glycosidic linkages might be the major contributors to the flexibility, molecular size and stereochemistry of mushroom polysaccharide chains.

Isolation and structure characterization of a low methyl-esterified pectin from the tuber of Dioscorea opposita Thunb.

A low methyl-esterified pectin (33.2% methyl-esterification degree) was isolated from the tuber of Dioscorea opposita Thunb., which was an edible and medicinal material in China. This pectin (Mw of 1.3 × 104 g/mol) contained the ~59.1% homogalacturonan (HG) and ~38.1% highly branched rhamnogalacturonan I (RG-I) region with possible side chains embracing arabinogalactan II, arabinan or arabinogalactan I. The fragments including HG backbone consisting of â†’ 4)-α-GalpA-(1 â†’ and â†’ 4)-α-GalpA-6-O-methyl-(1 â†’ with molar ratio of ~2:1, and repeating unit of arabinogalactan II side chain composed of α-Araf-(1 â†’ and â†’ 3,6)-ß-Galp-(1→, were speculated through methylation analysis and NMR spectra. However, the linkage pattern for RG-I backbone and side chains were indiscernible due to limited resolution of NMR spectra. Besides, the pectin adopted a flexible chain conformation in 0.1 M NaNO3 solution. These results provided a structural basis for study on polysaccharide from D. opposite, which was benefit for development of functional food of yam.

A review of NMR analysis in polysaccharide structure and conformation: Progress, challenge and perspective.

Nuclear magnetic resonance (NMR) has been widely used as an analytical chemistry technique to investigate the molecular structure and conformation of polysaccharides. Combined with 1D spectra, chemical shifts and coupling constants in both homo- and heteronuclear 2D NMR spectra are able to infer the linkage and sequence of sugar residues. Besides, NMR has also been applied in conformation, quantitative analysis, cell wall in situ, degradation, polysaccharide mixture interaction analysis, as well as carbohydrates impurities profiling. This review summarizes the principle and development of NMR in polysaccharides analysis, and provides NMR spectra data collections of some common polysaccharides. It will help to promote the application of NMR in complex polysaccharides of biochemical interest, and provide valuable information on commercial polysaccharide products.

Sulfated polysaccharides from Cyclocarya paliurus reduce H2O2-induced oxidative stress in RAW264.7 cells.

In this study, two sulfated polysaccharides (S-CP1-4 and S-CP1-8) from Cyclocarya paliurus were produced by chlorosulfonic acid-pyridine method. Hydrogen peroxide (H2O2) was used to develop an oxidative stress model in the mouse macrophage cell line RAW264.7. Effects of the two sulfated polysaccharides on H2O2-induced oxidative stress were investigated. The results showed that S-CP(1-8) improved the viability of the H2O2-induced stressed RAW264.7 cells, as well as inhibited the lipid oxidation as determined by the level of malondialdehyde (MDA). Meanwhile, treatment with S-CP(1-4) increased superoxide dismutase (SOD) activity in these cells. The sulfated polysaccharides were found to have a better protective effect against H2O2-induced oxidative stress as compared to the native polysaccharide. Scanning electron microscopy also showed a significant change in the surface morphology of sulfated polysaccharides, but the degradation of main chain of polysaccharides was unconspicuous according to the results of monosaccharide composition. In addition, the sulfated polysaccharides had noticeable DPPH radical scavenging activity. In summary, our results demonstrated that H2O2 was able to induce oxidative stress in RAW264.7 cells, and sulfated group might play an important role in resistance to H2O2-induced oxidative damage.

Sulfated modification, characterization and property of a water-insoluble polysaccharide from Ganoderma atrum.

Sulfated modification was carried out to modify a water-insoluble polysaccharide from Ganoderma atrum (AGAP). The effects of sulfation on structure, physicochemical and functional properties of AGAP were investigated. Three sulfated derivatives were prepared, designated as S-1, S-2 and S-3 with degree of substitution (DS) of 0.35, 0.74 and 1.14, respectively. AGAP was elucidated as an α-(1→3)-glucan with few branches terminated by single mannose or xylose residues. The molecular weight (Mw) and radius of gyration (Rg) were estimated to be 1665 kDa and 65.49 nm, respectively. After sulfated modification, non-selective sulfation occurred preferably at O-6, partially at O-2 and O-4 positions of the glucosyl residues. The water-solubility of the derivatives was significantly improved in a DS-dependent manner. Mw of the derivatives showed a sharp decrease, and the chain conformation was estimated to be expanded stiff in phosphate buffer. In vitro tests showed that sulfated modification improved its antioxidant activities and anti-proliferative ability against S-180 tumor cells. This study suggested that sulfated modification was an effective approach to improve the water-solubility and functional properties of insoluble polysaccharides.

Signaling pathway involved in the immunomodulatory effect of Ganoderma atrum polysaccharide in spleen lymphocytes.

The aim of this study was to investigate the molecular mechanism underlying the immunomodulatory effect of Ganoderma atrum polysaccharide (PSG-1) in spleen lymphocytes. Our results showed that PSG-1 increased the intracellular Ca2+ concentration and calcineurin (CaN) activity. Moreover, PSG-1 was found to elevate nuclear factor of activated T cells (NFAT) activity, but this effect could be diminished by the treatment of CaN inhibitors (cyclosporin A and FK506). PSG-1-induced interleukin (IL)-2 production was also inhibited by cyclosporin A and FK506. In addition, PSG-1 was found to significantly enhance protein kinase C (PKC) activity. PKC was involved in induction of NFAT activity by PSG-1, as evidenced by abrogation of NFAT activity by PKC inhibitor calphostin C, which significantly decreased PSG-1-induced IL-2 production. On the basis of these results, we concluded that PSG-1 may induce activation of spleen lymphocytes at least in part via the Ca2+/CaN/NFAT/IL-2 signaling pathway and the PKC/NFAT/IL-2 signaling pathway cooperatively regulated PSG-1-induced activation of spleen lymphocytes.

Toll-like receptor 4 mediates the antitumor host response induced by Ganoderma atrum polysaccharide.

The aim of this study is to investigate the role of Toll-like receptor (TLR) 4 in Ganoderma atrum polysaccharide (PSG-1)-induced antitumor activity. In vitro, the apoptosis rate of S-180 cells was increased in PSG-1-induced peritoneal macrophage derived from C3H/HeN (wild-type) mice, but not from C3H/HeJ (TLR4-deficient) mice. In the S-180 tumor model, phagocytosis, NO and ROS release, phosphorylation of MAPKs and Akt, and expression of NF-κB were increased by PSG-1 in peritoneal macrophage derived from C3H/HeN mice. Furthermore, PSG-1 elevated Th1 cytokine production and enhanced the cytotoxic activity of CTL and NK cells in C3H/HeN mice. In addition, PSG-1 decreased the tumor weight and increased the apoptosis rate and caspase-3 and caspase-9 activities of tumor derived from the C3H/HeN mice. However, none of these activities were observed in C3H/HeJ mice. In summary, these findings demonstrated that the antitumor activity of PSG-1 is mediated by TLR4.

Toll-like receptor 4-mediated ROS signaling pathway involved in Ganoderma atrum polysaccharide-induced tumor necrosis factor-α secretion during macrophage activation.

Ganoderma atrum has been used as Chinese traditional medicine and healthful mushroom for thousands of years. The polysaccharide is regarded as the major bioactive substances in G. atrum. To delineate the underlying mechanism and signaling cascade involved in the immunomodulatory property of G. atrum polysaccharide (PSG-1). Specifically, this study is designed to examine the possibility of TLR4 as a candidate receptor interacted with G. atrum polysaccharide (PSG-1) and elucidate the role of reactive oxygen species (ROS) in PSG-1-induced tumor necrosis factor-α (TNF-α) production during macrophage activation. Flow cytometric and confocal laser-scanning microscopy analysis showed that fluorescence-labeled PSG-1 bind specifically to the macrophages. Moreover, PSG-1 stimulated TNF-α secretion of peritoneal macrophages from C3H/HeN mice, but not from C3H/HeJ mice. PSG-1-indcued TNF-α production was suppressed by anti-TLR4 mAb. Furthermore, ROS production was mediated by TLR4, and NADPH oxidase-derived ROS act as upstream of phosphoinositide 3-kinase(PI3K)/Akt/mitogen-activated protein kinases(MAPKs)/nuclear factor(NF)-κB signaling pathway in the regulation of PSG-1 stimulated TNF-α production. Taken together, we conclude that PSG-1 induces TNF-α secretion through TLR4/ROS/PI3K/Akt/MAPKs/NF-κB pathways during macrophage activation. Our findings provide a molecular basis for the potential of PSG-1 as a novel immunomodulatory agent.

Chemoprotective effects of Ganoderma atrum polysaccharide in cyclophosphamide-induced mice.

In this study, the chemoprotective effects of Ganoderma atrum polysaccharide (PSG-1) in cyclophosphamide (Cy) treated mice were investigated. In Cy-treated mice, PSG-1 treatment accelerated recovery dose-dependently of peripheral red blood cells, white blood cells and platelets, enhanced splenic natural killer cell activity and cytotoxic T lymphocyte activity. In addition, PSG-1 elevated CD4(+) T lymphocyte counts as well as the CD4(+)/CD8(+) ratio dose-dependently. Furthermore, PSG-1 restored the levels of IL-2, INF-γ, IL-10, IgA, IgM and IgG, as well as hemolysin in the sera. Finally, PSG-1 can also significantly increase the total antioxidant capacity, activities of superoxidase dismutase, catalase and glutathione peroxidase, and decrease the malondialdehyde level in vivo. These findings indicate that PSG-1 plays an important role in the protection against myelosuppression and immunosuppression and oxidative stress in Cy-treated mice and could be a potential immunomodulatory agent.

Polysaccharide from Ganoderma atrum induces tumor necrosis factor-α secretion via phosphoinositide 3-kinase/Akt, mitogen-activated protein kinase and nuclear factor-κB signaling pathways in RAW264.7 cells.

The aim of this study was to investigate the signaling pathways involved in the macrophage activation by Ganoderma atrum polysaccharide (PSG-1) and elucidate the molecular mechanism of PSG-1-induced signal transduction in the regulation of tumor necrosis factor (TNF)-α secretion. Our results illustrated that the mitogen-activated protein kinase (MAPK) pathways were simultaneously activated and involved in PSG-1-induced TNF-α secretion in RAW264.7 cells. Moreover, our results also demonstrated that the phosphoinositide 3-kinase (PI3K)/Akt pathway was stimulated and played an important role in the PSG-1 induced TNF-α secretion. Additionally, the present study showed that nuclear factor (NF)-κB activation by PSG-1 was triggered by PI3K/Akt/MAPK pathway and NF-κB participated in PSG-1 stimulated TNF-α production. In conclusion, we have elucidated the mechanism of PSG-1-mediated immunomodulatory activities, and provide a theoretical basis for the potential of PSG-1 as a novel immunomodulating agent.