Immunomodulatory Effect of Polysaccharides from the Mushroom-Forming Basidiomycete Gymnopilus imperialis (Agaricomycetes, Basidiomycota).

Gymnopilus consists of a widely distributed genus of basidiomycetes, especially in tropical regions of the world, such as Japan, Australia, Paraguay, and Brazil. This genus biosynthesizes interesting bioactive compounds, such as sesquiterpenoids, oligoisoprenoids, styrylpyrones, and lectins. In the present study, the aqueous extract of the basidiomata of Gymnopilus imperialis (Basidiomycota, Agaricomycetes, Agaricales, Hymenogastraceae) was obtained by using the accelerated solvent extraction (ASE) technique, followed by the precipitation of polysaccharide fraction with ethanol. Further purification by freeze-thawing processes, Fehling solution precipitation, and membrane dialysis with different pore sizes yield three main polysaccharide fractions (Gi-MRSW, Gi-PFME, and Gi-SFME). According to monosaccharide composition and 13C-NMR data, the Gi-MRSW and Gi-SFME fractions showed to be composed mainly of ß-glucans and Gi-PFME by a heterogalactan. Moreover, the immunomodulatory potential of Gi-MRSW was evaluated using RAW 264.7 murine macrophage as a study model. The nitric oxide production was significantly increased in treated samples, and the expression of inducible nitric oxide synthase (iNOS) showed that the fraction Gi-MRSW from G. imperialis induces the M1 polarization phenotype.

MG-Pe: A Novel Galectin-3 Ligand with Antimelanoma Properties and Adjuvant Effects to Dacarbazine.

Melanoma is a highly metastatic and rapidly progressing cancer, a leading cause of mortality among skin cancers. The melanoma microenvironment, formed from the activity of malignant cells on the extracellular matrix and the recruitment of immune cells, plays an active role in the development of drug resistance and tumor recurrence, which are clinical challenges in cancer treatment. These tumoral metabolic processes are affected by proteins, including Galectin-3 (Gal-3), which is extensively involved in cancer development. Previously, we characterized a partially methylated mannogalactan (MG-Pe) with antimelanoma activities. In vivo models of melanoma were used to observe MG-Pe effects in survival, spontaneous, and experimental metastases and in tissue oxidative stress. Analytical assays for the molecular interaction of MG-Pe and Gal-3 were performed using a quartz crystal microbalance, atomic force microscopy, and contact angle tensiometer. MG-Pe exhibits an additive effect when administered together with the chemotherapeutic agent dacarbazine, leading to increased survival of treated mice, metastases reduction, and the modulation of oxidative stress. MG-Pe binds to galectin-3. Furthermore, MG-Pe antitumor effects were substantially reduced in Gal-3/KO mice. Our results showed that the novel Gal-3 ligand, MG-Pe, has both antitumor and antimetastatic effects, alone or in combination with chemotherapy.

A galactose-rich heteropolysaccharide extracted from "jaboticaba" (Plinia cauliflora) peels.

The objective of this work was to extract, identify and characterize a galactose-rich heteropolysaccharide (GH) from "jaboticaba" peel. The best conditions to extract the GH according to a 23 full-factorial experimental design were 90 °C/30 min/pH 1.0, resulting in a 32.32 % yield using lyophilized sample. The chemical structure analyzed by GC/MS and NMR spectra (HSQC/HSQC-TOCSY) showed that the main chain of GH consists of a (1→4) galactoside branched at carbon 3, containing galactose (67.21 %), glucose (15.78 %), arabinose (9.78 %), rhamnose (2.26 %) and traces of esterified and non-esterified uronic acids. Rheological studies revealed that GH suspensions behave as a Newtonian fluid, with calculated molecular mass of 1.48 × 105 Da. The absolute viscosity of 1 % (w/v) aqueous suspension of GH decreased from 25 mPa s to 10 mPa s in NaCl and 7 mPa s in CaCl2, indicating the polyelectrolyte character of GH.

Structure elucidation of a bioactive fucomannogalactan from the edible mushroom Hypsizygus marmoreus.

A fucomannogalactan (FMG-Hm), with a molecular weight of 17.1 kDa, obtained from fruiting bodies of Hypsizygus marmoreus exhibited promising in vitro antimelanoma effects. FMG-Hm was not cytotoxic, nor did it alter the cell morphology and proliferation, but was able to inhibit colony-forming ability and cell migration in B16-F10 murine melanoma cells. An analysis of the monosaccharide composition indicated that FMG-Hm was composed of fucose, mannose, and galactose in a ratio of 1.00:1.08:3.17. The FMG-Hm was structurally characterized based on methylation analysis, partial acid hydrolysis, and NMR experiments. The results indicated that FMG-Hm contained a α-(1→6)-linked galactopyranosyl main chain, partially substituted at O-2 by non-reducing ends of α-L-fucopyranose and ß-D-mannopyranose. The predicted structure of the heteropolysaccharide was established as.

Inhibition of Leishmania amazonensis arginase by fucogalactan isolated from Agrocybe aegerita mushroom.

The inhibition of arginase from Leishmania spp. is considered a promising approach to the leishmaniasis treatment. In this study, the potential of a fucogalactan isolated from the medicinal mushroom Agrocybe aegerita was evaluated against arginase (ARG) from Leishmania amazonensis. The polysaccharide was obtained via aqueous extraction, and purified by freeze thawing and precipitation with Fehling solution. Its chemical structure was established by monosaccharide composition, methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The data indicated that it is a fucogalactan (FG-Aa; Mw = 13.8 kDa), having a (1→6)-linked α-D-Galp main-chain partially substituted in O-2 by non-reducing end-units of α-L-Fucp. FG-Aa showed significant inhibitory activity on ARG with IC50potency of 5.82 ± 0.57 µM. The mechanism of ARG inhibition by the heterogalactan was the competitive type, with Kiof 1.54 ± 0.15 µM. This is the first report of an inhibitory activity of arginase from L. amazonensis by biopolymers, which encourages us to investigate further polysaccharides as a new class of ARG inhibitors.

Chemical structure of a partially 3-O-methylated mannofucogalactan from edible mushroom Grifola frondosa.

An unusual heteropolysaccharide was isolated from the fruiting bodies of the medicinal mushroom Grifola frondosa, via successive cold aqueous extraction, followed by fractionation through freeze-thawing, precipitation with Fehling solution and dialysis using a membrane with a size exclusion cut-off of 500 kDa. Its chemical structure was determined based on total acid hydrolysis, methylation analysis and NMR studies. The mannofucogalactan had a molar mass of 15.9 × 103 g mol-1, which was determinate by HPSEC-MALLS. This heteropolymer showed to have a main chain of (1 → 6)-linked α-d-Galp partially substituted at O-2 by 3-O-α-d-mannopyranosyl-α-l-fucopyranosyl groups and in a minor proportion with α-l-Fucp single-unit side chains. Moreover, the presence of 3-O-Me-Galp units could also be observed in the main chain of the G. frondosa mannofucogalactan.

Partially methylated galactans containing different proportions of 3-O-methyl-galactose from Pleurotus citrinopileatus.

Pleurotus citrinopileatus, popularly known as "golden oyster mushroom" have medicinal properties, which are attributed mainly to the presence of bioactive polysaccharides. In this work, two partially 3-O-methylated galactans were isolated from the fruiting bodies of this fungus, via successive aqueous extraction, followed by fractionation by freeze-thawing, and precipitation of soluble material with Fehling solution. The structural assignments were carried out using mono- and bidimensional NMR spectroscopy, monosaccharide composition, and methylation analyses. The polysaccharides were characterized as linear, partially 3-O-methylated (1 → 6)-linked α-galactopyranans, containing only Gal and 3-O-Me-Gal, in 2:1 and 1:1 molar ratios, with molar masses of 37.6 × 103 g/mol and 28.5 × 103 g/mol, respectively. Similar structures have been described for other Pleurotus spp., but showing a lower content of 3-O-Me-Gal.

Water-soluble polysaccharides from Pleurotus ostreatus var. florida mycelial biomass.

Pleurotus ostreatus var. florida known as Hiratake has a high nutritional value, presents medicinal and nutraceutical properties and it is one of the consumed mushrooms in Brazil. Thus, the aim of this study was to characterize the chemical structure of polysaccharides found in mycelial biomass produced by submerged culture of P. ostreatus var. florida in order to compare with those found in P. ostreatus var. florida fruit bodies. Aqueous and alkali extracts obtained from mycelial biomass were purified, 13C NMR, GC-MS and chemical techniques were used to characterize three polysaccharide structures: a mannogalactan (MG-PfM) with α-D-Galp and 3-O-Me-α-D-Galp units, both (1→6)-linked, highly substituted at O-2 by D-Manp, a glycogen-like polymer (GLY-PfM) with α-D-Glp (1→4)-linked main chain, partially substituted at O-6 by α-D-Glcp side chains and a (1→3), (1→6) ß-D-glucan (ßGLC-PfM) with a main chain of ß-D-Glcp (1→3)-linked units, partially substituted at O-6 by side chains of 6-O-substituted ß-D-glucopyranosyl units, on an average of one to every two residues of the backbone. These results show the possibility to obtain similar and also different molecules from those found in the fruiting body of the same mushroom species, therefore the submerged culture of mushroom is a promising way to give raise molecules of interest.

Glucuronoarabinoxylan from coconut palm gum exudate: chemical structure and gastroprotective effect.

A glucuronoarabinoxylan (CNAL) was extracted with 1% aq. KOH (25°C) from Cocos nucifera gum exudate. It had a homogeneous profile on HPSEC-MALLS-RI (Mw 4.6 × 10(4)g/mol) and was composed of Fuc, Ara, Xyl, GlcpA (and 4-O-GlcpA) in a 7:28:62:3 molar ratio. Methylation data showed a branched structure with 39% of non-reducing end units, 3-O-substituted Araf (8%), 3,4-di-O- (15%), 2,4-di-O- (5%) and 2,3,4-tri-O-substituted Xylp units (17%). The anomeric region of CNAL (13)C NMR spectrum contained 9 signals, indicating a complex structure. The main chain of CNAL was characterized by analysis of a Smith-degraded polysaccharide. Its (13)C NMR spectrum showed 5 main signals at δ 101.6, δ 75.5, δ 73.9, δ 72.5, and δ 63.1 that were attributed to C-1, C-4, C-3, C-2 and C-5 of (1→4)-linked ß-Xylp-main chain units, respectively. CNAL exhibited gastroprotective effect, by reducing gastric hemorrhagic lesions, when orally administered (1 and 3mg/kg) to rats prior to ethanol administration.

Fucomannogalactan and glucan from mushroom Amanita muscaria: structure and inflammatory pain inhibition.

A fucomannogalactan (FMG-Am) and a (1→3), (1→6)-linked ß-D-glucan (ßGLC-Am) were isolated from Amanita muscaria fruiting bodies. These compounds' structures were determined using mono- and bi-dimensional NMR spectroscopy, methylation analysis, and controlled Smith degradation. FMG-Am was shown to be a heterogalactan formed by a (1→6)-linked α-D-galactopyranosyl main chain partially substituted at O-2 mainly by α-L-fucopyranose and a minor proportion of ß-D-mannopyranose non-reducing end units. ßGLC-Am was identified as a (1→3)-linked ß-D-glucan partially substituted at O-6 by mono- and a few oligosaccharide side chains, which was confirmed after controlled Smith degradation. Both the homo- and heteropolysaccharide were evaluated for their anti-inflammatory and antinociceptive potential, and they produced potent inhibition of inflammatory pain, specifically, 91±8% (30 mg kg(-1)) and 88±7% (10 mg kg(-1)), respectively.

Lactarius rufus (1→3),(1→6)-ß-D-glucans: structure, antinociceptive and anti-inflammatory effects.

Medicinal health benefits uses of edible as well as non-edible mushrooms have been long recognized. The pharmacological potential of mushrooms, especially antitumor, immunostimulatory and anti-inflammatory activities has been documented. Wild ectomycorrhizal mushroom, Lactarius rufus had the anti-inflammatory and antinociceptive potential of their polysaccharides evaluated using the formalin model. Two structurally different (1→3),(1→6)-linked ß-D-glucans were isolated from fruiting bodies. Soluble (FSHW) ß-D-glucan 1-30 mg kg(-1) produced potent inhibition of inflammatory pain caused by formalin when compared with the insoluble one (IHW), suggesting that solubility and/or branching degree could alter the activity of ß-glucans. Their structures were determined using mono- and bi-dimensional NMR spectroscopy, methylation analysis, and controlled Smith degradation. They were ß-D-glucans, with a main chain of (1→3)-linked Glcp residues, substituted at O-6 by single-unit Glcp side chains (IHW), on average to every fourth residue of the backbone, or by mono- and few oligosaccharide side chains for soluble ß-glucan.

Sulfonation and anticoagulant activity of fungal exocellular ß-(1→6)-D-glucan (lasiodiplodan).

An exocellular ß-(1→6)-D-glucan (lasiodiplodan) produced by a strain of Lasiodiplodia theobromae (MMLR) grown on sucrose was derivatized by sulfonation to promote anticoagulant activity. The structural features of the sulfonated ß-(1→6)-D-glucan were investigated by UV-vis, FT-IR and (13)C NMR spectroscopy, and the anticoagulant activity was investigated by the classical coagulation assays APTT, PT and TT using heparin as standard. The content of sulfur and degree of substitution of the sulfonated glucan was 11.73% and 0.95, respectively. UV spectroscopy showed a band at 261 nm due to the unsaturated bond formed in the sulfonation reaction. Results of FT-IR and (13)C NMR indicated that sulfonyl groups were inserted on the polysaccharide. The sulfonated ß-(1→6)-D-glucan presented anticoagulant activity as demonstrated by the increase in dose dependence of APTT and TT, and these actions most likely occurred because of the inserted sulfonate groups on the polysaccharide. The lasiodiplodan did not inhibit the coagulation tests.

Polysaccharide glucomannan isolated from Heterodermia obscurata attenuates acute and chronic pain in mice.

Glucomannan (GM) is a polysaccharide obtained from Heterodermia obscurata lichens. The present study was conducted to elucidate the antinociceptive effect of GM in behavioural models of acute and chronic pain in mice. GM reduced mechanical allodynia and the levels of interleukin 1-ß (IL-1ß) in spinal cord and nerve in the partial sciatic nerve ligation (PSNL) model. Systemic treatment with GM inhibited the nociception induced by intraplantar injection of glutamate and by intrathecal injection of N-methyl-d-aspartic acid (NMDA), (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), tumour necrosis factor α (TNF-α) and IL-1ß. Taken together, our data demonstrate that GM has significant antinociceptive effect in acute and chronic pain, suggesting a potential interest in the development of new clinically relevant drugs for the management of pain.

Agaricus bisporus fucogalactan: structural characterization and pharmacological approaches.

The fucogalactan from Agaricus bisporus (EFP-Ab) obtained on aqueous extraction followed by purification had M(w) 37.1 × 10(4)g mol(-1) relative to a (1→6)-linked α-D-Galp main-chain partially methylated at HO-3, and partially substituted at O-2 by nonreducing end-units of α-L-Fucp or ß-d-Galp. EFP-Ab also inhibited significantly the neurogenic and inflammatory phases of formalin-induced licking, however, the antinociceptive effect was more pronounced against the inflammatory phase with ID(50) of 36.0 (25.8-50.3)mg kg(-1). In addition, EFP-Ab decreased the lethality induced by CLP. Its administration reduced the late mortality rate by 40%, prevented neutrophil accumulation in lungs and markedly decreased iNOS and COX-2 protein expression by ileum cells. These data show for the first time that EFP-Ab has significant anti-sepsis, antinociceptive and anti-inflammatory actions, which seems to be related to the decreased iNOS and COX-2 expression. Collectively, the present results demonstrate that EFP-Ab could constitute an attractive molecule of interest for the development of new drugs.

Chemical and biological properties of a highly branched ß-glucan from edible mushroom Pleurotus sajor-caju.

Hot aqueous extraction of the basidiocarps of the mushroom Pleurotus sajor-caju provided a cold water-soluble, gel-like glucan, which was characterized chemically, and its effects on RAW 264.7 cell line (mouse leukaemic monocyte macrophage) activation were determined. NMR spectroscopy, HPSEC, methylation analysis, and a controlled Smith degradation showed it to have a branched structure with a (1→3)-linked ß-Glcp main-chain, substituted at O-6 by single-unit ß-Glcp side-chains, on the average of two to every third residues of the backbone, with a molar mass of 9.75 × 10(5) g mol(-1). In macrophage cell culture, the ß-glucan induced production of NO and the cytokines TNF-α, IL-1ß, these effects being very similar as those of Escherichia coli serotype 0111:B4 Sigma-Aldrich lipopolysaccharide (LPS), although not modifying the response of LPS-activated macrophages. The results suggest that the (1→3), (1→6)-linked ß-glucan from P. sajor-caju may have potential for immunological activities, although additional experiments are necessary for a better understanding of the mechanisms involved.

Chemical structure and selected biological properties of a glucomannan from the lichenized fungus Heterodermia obscurata.

Successive aqueous and alkaline extraction of the thallus of the lichenized fungus Heterodermia obscurata provided a highly branched glucomannan fraction (GM), whose chemical structure was determined. This was based on monosaccharide composition, methylation, partial acid hydrolysis, and NMR spectroscopic analysis. It consisted of a main chain of (1→6)-linked α-D-mannopyranosyl units, almost all being substituted at O-2 with α-D-glucopyranosyl, α-D-mannopyranosyl, and 4-O-substituted α-D-mannopyranosyl groups. Intra-peritoneal administration of this GM induced a marked and dose-dependent inhibition of acetic acid-induced visceral pain with an ID(50) of 0.6 (0.2-2.0) mg/kg and inhibition of 88±4%. It also reduced leukocyte migration by 58±4%, but did not alter plasmatic extravasation to the peritoneal cavity. The results suggest that the glucomannan from the H. obscurata might have potential for antinociceptive and anti-inflammatory utilization.

Antithrombin and heparin cofactor II-mediated inactivation of alpha-thrombin by a synthetic, sulfated mannogalactan.

INTRODUCTION: A mannogalactan from Pleurotus ostreatoroseus (MgPr) was chemically sulfated to give MgPr-S1, which was evaluated for its anticoagulant and antithrombotic activities, bleeding tendency, and platelet aggregation. MATERIALS AND METHODS: MgPr-S1 was partially characterized by HPSEC-MALLS, methylation analysis, and 13C NMR spectroscopy. Its anticoagulant activity was determined by assays of aPTT, TT, alpha-thrombin and factor Xa residual activity, heparin cofactor II (HCII)-, or antithrombin (AT)-mediated inhibition. The antithrombotic effect was evaluated in rats using a venous thrombosis model and the bleeding tendency was also tested in vivo. Platelet aggregation was investigated by an adaptation of the method of Born [1]. RESULTS: The hydroxyl groups of beta-D-Manp units and OH-2 and OH-4 of the (1-->6)-linked alpha-D-Galp units were preferentially substituted. The anticoagulant activity of MgPr-S1 was mainly by thrombin inhibition with antithrombin and HCII, and had an effect on platelet aggregation induced by ADP and alpha-thrombin. It almost completely inhibited thrombus formation in vivo at a dose of 6 mg/kg and heparin inhibited thrombus formation at a dose of 0.200 mg/kg. CONCLUSIONS: These results suggested that the chemically sulfated mannogalactan could act as an alternative to heparin as anticoagulant.

Structure of Agaricus spp. fucogalactans and their anti-inflammatory and antinociceptive properties.

Fucogalactans from Agaricus brasiliensis (EPF-Ab) and A. bisporus var. hortensis (EPF-Ah) were prepared via by aqueous extraction and a purification procedure. EPF-Ab had M(w) 19.4 x 10(3)g/mol and EPF-Ah M(w) 31.1 x 10(3)g/mol. EPF-Ab had a (1-->6)-linked alpha-D-Galp main-chain partially substituted in O-2 by non-reducing end-units of alpha-L-Fucp. EPF-Ah had a similar main-chain with O-2 substitution, but was partially methylated at HO-3, as well as having 2.5% non-reducing end-units of beta-D-Gal. In mice, EPF-Ab gave 39% antinociceptive inhibition (ID(50)>100mg/kg) and no anti-inflammatory activity. EPF-Ah also gave an inhibition of 39% at ID(50) 0.33 mg/kg and also inhibited by 61% (ID(50) 5.0mg/kg) total cell migration and by 32% peritoneal capillary permeability, which is related to the anti-inflammatory effect. The small differences in chemical structure in these polysaccharides thus modified their biological activities.

Anti-inflammatory and analgesic properties in a rodent model of a (1-->3),(1-->6)-linked beta-glucan isolated from Pleurotus pulmonarius.

A glucan was extracted with hot water from the basidiomycete Pleurotus pulmonarius and shown to have a (1-->3)-linked beta-D-glucopyranosyl main-chain substituted at O-6 of every third unit by single beta-D-glucopyranosyl non-reducing end units. This was shown by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and a controlled Smith degradation. The glucan was tested for its effects on the acetic acid-induced writhing reaction in mice, a typical model for quantifying inflammatory pain. It caused a marked and dose-dependent anti-inflammatory response, demonstrated by the inhibition of leukocyte migration to injured tissues (82 +/- 6%) with an ID50 of 1.19 (0.74-1.92) mg/kg. Furthermore, animals previously treated with the glucan (3 mg/kg i.p.), showed a reduction of 85 +/- 5% of writhes, after receiving the acetic acid injection. Furthermore, in the formalin test, the glucan (3-30 mg/kg, i.p.) also caused significant inhibition of both the early (neurogenic pain) and the late phases (inflammatory pain) of formalin-induced licking. However, it was more potent and effective in relation to the late phase of the formalin test, with mean ID(50) values for the neurogenic and the inflammatory phases of > 30 and 12.9 (6.7-24.6) mg/kg and the inhibitions observed were 43 +/- 5% and 96 +/- 4%, respectively. These data showed that the glucan had potent anti-inflammatory and analgesic (antinociceptive) activities, possibly by the inhibition of pro-inflammatory cytokines.

Three exopolysaccharides of the beta-(1-->6)-D-glucan type and a beta-(1-->3;1-->6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit.

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and (13)C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1-->3) linkages substituted at O-6 as shown in the putative structure below: [carbohydrate structure: see text]. The EPS of the other fungal isolates consisted of a linear chain of (1-->6)-linked glucopyranosyl residues of the following structure: [carbohydrate structure: see text]. FTIR spectra showed one band at 891 cm(-1), and (13)C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1-->6)-d-Glucans produced as exocellular polysaccharides by fungi are uncommon.