Andrea Caroline Ruthes

Polysaccharides from Agaricus bisporus and Agaricus brasiliensis show similarities in their structures and their immunomodulatory effects on human monocytic THP-1 cells

BackgroundMushroom polysaccharides have traditionally been used for the prevention and treatment of a multitude of disorders like infectious illnesses, cancers and various autoimmune diseases. Crude mushroom extracts have been tested without detailed chemical analyses of its polysaccharide content. For the present study we decided to chemically determine the carbohydrate composition of semi-purified extracts from 2 closely related and well known basidiomycete species, i.e. Agaricus bisporus and A. brasiliensis and to study their effects on the innate immune system, in particular on the in vitro induction of pro-inflammatory cytokines, using THP-1 cells.MethodsMushroom polysaccharide extracts were prepared by hot water extraction and precipitation with ethanol. Their composition was analyzed by GC-MS and NMR spectroscopy. PMA activated THP-1 cells were treated with the extracts under different conditions and the production of pro-inflammatory cytokines was evaluated by qPCR.ResultsSemi-purified polysaccharide extracts of A. bisporus and A. brasiliensis (= blazei) were found to contain (1→6),(1→4)-linked α-glucan, (1→6)-linked β-glucan, and mannogalactan. Their proportions were determined by integration of 1H-NMR signs, and were considerably different for the two species. A. brasiliensis showed a higher content of β-glucan, while A. bisporus presented mannogalactan as its main polysaccharide. The extracts induced a comparable increase of transcription of the pro-inflammatory cytokine genes IL-1β and TNF-α as well as of COX-2 in PMA differentiated THP-1 cells. Pro-inflammatory effects of bacterial LPS in this assay could be reduced significantly by the simultaneous addition of A. brasiliensis extract.ConclusionsThe polysaccharide preparations from the closely related species A. bisporus and A. brasiliensis show major differences in composition: A. bisporus shows high mannogalactan content whereas A. brasiliensis has mostly β-glucan. Semi-purified polysaccharide extracts from both Agaricus species stimulated the production of pro-inflammatory cytokines and enzymes, while the polysaccharide extract of A. brasiliensis reduced synthesis of these cytokines induced by LPS, suggesting programmable immunomodulation.

d-Glucans from edible mushrooms: A review on the extraction, purification and chemical characterization approaches

D-Glucans from edible mushrooms present diversified chemical structures. The most common type consists of a backbone of β-D-glucose (1→3)-linked frequently branched at O-6 by β-D-glucose residues as side chains. However it is possible to distinguish α-, β- and mixed D-glucans. Further discrimination could be made on the basis of glycosidic bond position in a pyranoid ring, distribution of specific glycosidic bonds along the chain, branching and molecular weight. The present manuscript reviews the processes of extraction, purification and chemical characterization of D-glucans, such as NMR studies, methylation analysis, Smith degradation, and some other methodologies employed in carbohydrate chemistry characterization. In addition, these polysaccharides are important because they can provide many therapeutic benefits related to their biological activity in animals and humans, either immunostimulatory activity, inhibiting tumor growth, as well as exerting antinociceptive and anti-inflammatory action, among others, which are usually attached to their structure, molecular weight and degree of branching.

Lactarius rufus (1→3),(1→6)-β-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 β-D-glucans were isolated from fruiting bodies. Soluble (FSHW) β-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 β-glucans. Their structures were determined using mono- and bi-dimensional NMR spectroscopy, methylation analysis, and controlled Smith degradation. They were β-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 β-glucan.

Chemical and biological properties of a highly branched β-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 β-Glcp main-chain, substituted at O-6 by single-unit β-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 β-glucan induced production of NO and the cytokines TNF-α, IL-1β, 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 β-glucan from P. sajor-caju may have potential for immunological activities, although additional experiments are necessary for a better understanding of the mechanisms involved.

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 β-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.

Structural characterization and anti-inflammatory activity of a linear β-d-glucan isolated from Pleurotus sajor-caju

Glucans comprise an important class of polysaccharides present in basidiomycetes with potential biological activities. A (1 → 3)-β-D-glucan was isolated from Pleurotus sajor-caju via extraction with hot water followed by fractionation by freeze-thawing and finally by dimethyl sulfoxide extraction. The purified polysaccharide showed a (13)C-NMR spectrum with six signals consisting of a linear glucan with a β-anomeric signal at 102.8 ppm and a signal at 86.1 ppm relative to O-3 substitution. The other signals at 76.2, 72.9, 68.3, and 60.8 ppm were attributed to C5, C2, C4, and C6, respectively. This structure was confirmed by methylation analysis, and HSQC studies. The β-d-glucan from P. sajor-caju presented an immunomodulatory activity on THP-1 macrophages, inhibited the inflammatory phase of nociception induced by formalin in mice, and reduced the number of total leukocytes and myeloperoxidase levels induced by LPS. Taken together, these results demonstrate that this β-d-glucan exhibits a significant anti-inflammatory activity.

Exopolysaccharide produced by Pleurotus sajor-caju: Its chemical structure and anti-inflammatory activity

Edible mushrooms are high nutritional value foods, which contain proteins, fibers, minerals, vitamins, and carbohydrates. Among their carbohydrates are some polysaccharides with recognized therapeutic effects. It was reported in this manuscript the structural characterization and antinociceptive and anti-inflammatory activities of an exopolysaccharide (EPS) produced by Pleurotus sajor-caju. The purified EPS was a mannogalactan (PEIsR), which was composed by mannose (37.0%), galactose (39.7%), and 3-O-methyl-galactose (23.3%). The polysaccharide was purified by freeze-thawing and dialysis, and it was characterized by GC-MS analysis and NMR spectroscopy. The mannogalactan is constituted by a main chain of (1 → 6)-linked α-D-Galp and 3-O-methyl-α-D-Galp units. Some of the α-D-Galp units were substituted at O-2 by non-reducing end units of β-D-Manp. According to the literature review conducted, this is the first time that a methylated polysaccharide was observed on EPS of P. sajor-caju. The mannogalactan was able to reduce the nociception, in vivo, in the writhing and formalin tests and also reduced the carrageenan-induced paw edema, which indicates that it could be an effective antinociceptive and anti-inflammatory agent.

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) β-D-glucan (βGLC-PfM) with a main chain of β-D-Glcp (1→3)-linked units, partially substituted at O-6 by side chains of 6-O-substituted β-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.

Cytotoxic effect of Agaricus bisporus and Lactarius rufus β-d-glucans on HepG2 cells

The cytotoxic activity of β-D-glucans isolated from Agaricus bisporus and Lactarius rufus fruiting bodies was evaluated on human hepatocellular carcinoma cells (HepG2). NMR and methylation analysis suggest that these β-d-glucans were composed of a linear (1→6)-linked and a branched (1→3), (1→6)-linked backbone, respectively. They both decreased cell viability at concentrations of up to 100 μg mL(-1), as shown by MTT assay. The amount of LDH released and the analysis of cell morphology corroborated these values and also showed that the β-D-glucan of L. rufus was more cytotoxic to HepG2 cells than that of A. bisporus. The treatment of HepG2 cells with L. rufus and A. bisporus β-D-glucans at a dose of 200 μg mL(-1) for 24h promoted an increase of cytochrome c release and a decrease of ATP content, suggesting that these polysaccharides could promote cell death by apoptosis. Both β-D-glucans were tested against murine primary hepatocytes at a dose of 200 μg mL(-1). The results suggest that the L. rufus β-d-glucan was as cytotoxic for hepatocytes as for HepG2 cells, whereas the A. bisporus β-D-glucan, under the same conditions, was cytotoxic only for HepG2 cells, suggesting cell selectivity. These results open new possibilities for use of mushroom β-D-glucans in cancer therapy.

Polysaccharides present in cultivated Teloschistes flavicans symbiosis: comparison with those of the thallus.

The chemical structures of polysaccharides present in aposymbiotically cultured myco- and photobionts of the lichen Teloschistes flavicans were determined, in order to compare them with those previously found in the intact thallus. The mycobiont was cultured on a solid Lilly and Barnett medium and the resulting colonies were freeze dried, defatted, and their polysaccharides were extracted successively with 2%, 10% and 30% aq. KOH, each at 100 degrees C. The extracts were neutralized (HOAc) and fractionated, giving rise to three homogeneous fractions, PFSK2 from 2% KOH, which contained a (1-->4),(1-->6)-linked alpha-glucan (1:1 ratio, pullulan), fraction PK10 from 10% KOH extraction, which was a linear (1-->3)-linked linear beta-glucan (laminaran), and fraction PK30 from 30% KOH extraction, being a branched (1-->3),(1-->6)-linked beta-glucan. The photobiont (Trebouxia sp. de Puymaly) was cultured in liquid nutrient medium, and after purification, a linear (1-->5)-linked beta-galactofuranan was characterized. The galactofuranan and the laminaran were not present in the symbiotic thallus, in contrast to the glucans, showing that the mycobiont alone produces them without participation of the photobiont.