Fhernanda R. Smiderle

Anti-inflammatory and analgesic properties in a rodent model of a (1→3),(1→6)-linked β-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.

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.

A 3-O-methylated mannogalactan from Pleurotus pulmonarius: structure and antinociceptive effect.

A polysaccharide (Mw 2.39x10(4)g/mol) was extracted with cold water from the basidiomycete Pleurotus pulmonarius, and its antinociceptive and anti-inflammatory properties were evaluated. It was a mannogalactan (MG), whose structure was characterized using mono- and two-dimensional NMR spectroscopy, methylation analysis, and a controlled Smith degradation. It had a main chain of (1-->6)-linked alpha-D-galactopyranosyl and 3-O-methyl-alpha-D-galactopyranosyl units, both of which are partially substituted at O-2 by beta-D-mannopyranosyl non-reducing ends. The MG was tested for its effects on the acetic acid-induced writhing reaction in mice, a typical model for inflammatory pain, causing a marked and dose-dependent inhibition of the nociceptive response, with ID50 of 16.2 (14.7-17.7)mg/kg and inhibition of 93+/-3% at a dose of 30mg/kg. An inflammatory response was not inhibited.

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.

Agaricus bisporus and Agaricus brasiliensis (1 → 6)-β-d-glucans show immunostimulatory activity on human THP-1 derived macrophages

The (1→6)-β-D-glucans from Agaricus bisporus and Agaricus brasiliensis were purified to evaluate their effects on the innate immune system. THP-1 macrophages were used to investigate the induction of the expression of TNF-α, IL1β, and COX-2 by RT-PCR. The purification of the polysaccharides gave rise to fractions containing 96-98% of glucose. The samples were analyzed by GC-MS, HPSEC and (13)C NMR, which confirmed the presence of homogeneous (1→6)-β-D-glucans. The β-glucans were incubated with THP-1 derived macrophages, for 3 h and 6 h to evaluate their effects on the expression of pro-inflammatory genes. Both β-glucans stimulated the expression of such genes as much as the pro-inflammatory control (LPS). When the cells were incubated with LPS+β-glucan, a significant inhibition of the expression of IL-1β and COX-2 was observed for both treatments after 3 h of incubation. By the results, we conclude that the (1→6)-β-D-glucans present an immunostimulatory activity when administered to THP-1 derived macrophages.

Isolation and chemical characterization of a glucogalactomannan of the medicinal mushroom Cordyceps militaris

Cordyceps militaris dried fruiting bodies were extracted with 5% KOH solution. The extract was purified by freeze-thawing treatment, and dialysis (100 kDa), giving rise to a homogeneous polysaccharide (Mw 23,000 Da). Its monosaccharide composition was mannose (56.7%), galactose (34.5%), and glucose (8.8%). The anomeric configurations were determined by their coupling constants. A complex polysaccharide was identified by NMR and methylation analysis. The HSQC spectrum showed signals at δ 107.7/5.06 and 106.1/5.14; 105.9/5.12 relative to β-d-Galf, and O-2-substituted β-d-Galf units, respectively. The sign at δ 104.4/5.21 corresponded to α-d-Galf. Other signals corresponded to α-d-Manp O-6- and O-2-substituted (δ 100.2/4.94; 100.5/5.27; 100.6/5.23; 100.7/5.16), and α-d-Manp 2,6-di-O-substituted (from δ 99.3 to 99.9). The main linkages, confirmed by methylation analysis, showed the derivatives: 2,3,4-Me3-Manp (11.9%) and 3,4,6-Me3-Manp (28.6%). The branches were (1→6)-linked-α-d-Manp or (1→2)-linked-β-d-Galf, terminating with β-d-Galf, α-d-Galf, α-d-Galp, or α-d-Manp. 42.7% of the partially hydrolyzed product consisted of 3,4,6-Me3-Manp, suggesting a (1→2)-linked backbone.

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.

Antinociception of β-d-glucan from Pleurotus pulmonarius is possibly related to protein kinase C inhibition

β-D-Glucan, a polysaccharide isolated from an edible mushroom Pleurotus pulmonarius (Fr.) Quel., presented antinociceptive activity in mice. This study evaluated the involvement of transient receptor potential (TRP) channels and protein kinase C (PKC) on antinociceptive effect of a (1→3),(1→6)-linked β-D-glucan (GL) in mice. Intraperitoneal administration of GL potently inhibited nociceptive responses induced by intraplantar injections of capsaicin, cinnamaldehyde, menthol, acidified saline and phorbol myristate acetate (PMA). Moreover, Western blot analysis revealed that GL treatment also prevented PMA-induced PKCɛ activation. Collectively, present results demonstrate that GL could constitute an attractive molecule of interest for the development of new analgesic drugs.

Antinociceptive Effects of (1→3),(1→6)-Linked β-Glucan Isolated From Pleurotus pulmonarius in Models of Acute and Neuropathic Pain in Mice: Evidence for a Role for Glutamatergic Receptors and Cytokine Pathways

UNLABELLED The present study evaluated the antinociceptive effect of (1→3),(1→6)-linked β-glucan (GL) isolated from Pleurotus pulmonarius (Fr.) Quel. in mice and its possible mechanism of action. Intraperitoneal administration of GL inhibited glutamate-induced licking with an ID(50) of 0.34 mg/kg and inhibition of 96% ± 3%. The treatment of animals with GL (1 mg/kg i.p.) inhibited nociception induced by intrathecal injection of N-methyl-D-aspartic acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate and interleukin -1β in 67% ± 13%, 89% ± 11%, 74% ± 9%, and 75% ± 7%, respectively, but not the nociceptive response induced by (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid, substance P, and tumor necrosis factor-α. Moreover, GL (30 mg/kg i.p.) also reduced mechanical allodynia caused by partial sciatic nerve ligation for 2 hours, with inhibition of 47% ± 10% observed 0.5 hours after treatment. When given chronically (twice a day) over 7 days, GL reversed the mechanical allodynia caused by partial sciatic nerve ligation (inhibition of 45% ± 13% to 60% ± 8%). Interestingly, GL did not affect the locomotor activity of mice in an open field test with doses that produce antinociceptive effects. Our findings show that GL inhibits acute and neuropathic pain in mice through mechanisms that involve the inhibition of ionotropic glutamate receptors and the interleukin -1β pathway. PERSPECTIVE This article presents the antinociceptive activity of GL in acute and neuropathic pain with participation of ionotropic glutamate receptors and pro-inflammatory cytokines (interleukin-1β). After further experiments, this compound may represent a new pharmacological agent for the treatment of clinical pain.