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.

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.

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.

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.

Unusual partially 3-O-methylated alpha-galactan from mushrooms of the genus Pleurotus.

Indistinguishable partially 3-O-methylated galactans were isolated from the edible basidiomycetes Pleurotus eryngii and Pleurotus ostreatoroseus. They were obtained via successive aqueous extraction, freeze-thawing, precipitation with Fehling solution of soluble material, and ultrafiltration. Mono- and bidimensional 13C and 1H-nuclear magnetic resonance spectroscopy (HMBC, HETEROTOCSY, COSY, and HMQC), and methylation analysis were used to determine their structures. They were linear, partially 3-O-methylated, (1-->6)-linked alpha-d-galactans containing Gal and 3-Me-Gal, in a 3:1 molar ratio (GC-MS of alditol acetates).

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.

A gel-forming beta-glucan isolated from the fruit bodies of the edible mushroom Pleurotus florida.

Glucans of basidiomycetes are considered to be an important class of polysaccharides, as they can act as biological response modifiers. We now isolate a gel-forming, water-soluble beta-glucan, with a molecular mass of 1.2 x 10(6)g/mol (HPSEC), from the fruit bodies of the edible mushroom Pleurotus florida, via alkaline extraction, followed by fractionation by freeze-thawing. Structural assignments were carried out using mono- and bi-dimensional nuclear magnetic resonance spectroscopy, monosaccharide composition, methylation analyses, and a controlled Smith degradation. It was a branched beta-glucan, with a main chain of (1-->3)-linked-Glcp residues, substituted at O-6 by single-unit Glcp side chains, on average to every fourth residue of the backbone.

Structural characterization of the cell wall D-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05.

Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chromatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and 13C NMR spectroscopy and, after methylation, by GC-MS. The hot-water extract produced a fraction designated Q1A that was a beta-(1-->6)-D-glucan with the following structure: [Formula: see text] The alkaline extract, when subjected to repeated freeze-thawing, yielded two fractions: K1P (insoluble) that comprised a beta-(1-->3)-D-glucan with beta-D-glucose branches at C-6 with the structure: [Formula: see text] and K1SA (soluble) consisting of a backbone chain of alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucopyranosyl residues: [Formula: see text]

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.

Chemical and immunological modifications of an arabinogalactan present in tea preparations of Phyllanthus niruri after treatment with gastric fluid.

An arabinogalactan (AG) obtained from tea preparations of Phyllanthus niruri was previously investigated and presented immunological properties when tested with peritoneal mice macrophages. AG was now submitted to acidic and neutral gastric conditions using human gastric fluids and aq. HCl solution. Since the acidic procedures gave rise to the same free monosaccharidic composition, the acid hydrolyzate of AG at pH 2.00 was treated with ethanol to form insoluble (AG-P) and soluble fractions (AG-S). These were analyzed using (13)C NMR, HPSEC, and GC-MS for monosaccharide composition and methylation analyses. The results showed an intense partial degradation, including cleavages of the main chain. AG-S presented the monosaccharides released from the native polymer and some oligosaccharides as shown by methylation data. AG-P contained larger molecular fragments comprising the internal units from AG, which were not attacked by the hydrolysis condition. Both fractions were tested in peritoneal mice macrophages and remained active, promoting an increase of superoxide anion production of 2.0 and 2.3-fold, at 250 microg/mL, for AG-S and AG-P, respectively. When compared to AG, a slight diminished response was observed, revealing a structure-activity relation. The significance of the results is that most plant extracts are orally ingested and will reach the gastrointestinal tract before performing a biological function, so checking these changes is crucial to propose future clinical therapies based on the rational use of phytomedicine.

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.

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.

Structural characterization of a polysaccharide and a beta-glucan isolated from the edible mushroom Flammulina velutipes.

Two polysaccharides were isolated from the basidiomycete Flammulina velutipes, via successive hot extraction with water, 2% and 25% aq. KOH, and then submitted to freeze-drying. The precipitate formed by repeated freeze-thawing from the 2% aq. KOH extraction PK2 was analyzed by determination of its monosaccharide composition, as well as by methylation analyses using GC-MS, mono- ((13)C, (1)H NMR) and bidimensional ((1)H (obs.), (13)C HMQC) spectroscopy, and controlled Smith degradations. It was established to be a branched beta-glucan, with a main chain of (1-->3)-linked-Glcp residues, substituted at O-6 by single-unit beta-Glcp side chains. The precipitate formed by repeated freeze-thawing from the 25% KOH extraction PK25 contained Xyl, Man, and Glc and was heterogeneous by HSPEC and extraction with DMSO gave a soluble xylomannan (XM). It was homogeneous with a molar mass 30.8 x 10(4)g/mol (dn/dc=0.186). Using the above chemical analyses, it was a xylomannan with Man and Xyl in a 3:2 molar ratio. Its main chain consisted of (1-->3)-linked alpha-Manp units, mainly substituted at O-4 by beta-Xylp units or with some beta-Xylp-(1-->3)-beta-Xylp groups.

A fungus-type beta-galactofuranan in the cultivated Trebouxia photobiont of the lichen Ramalina gracilis.

A structural characterization of polysaccharides extracted from the aposymbiotically cultured photobiont of the lichen Ramalina gracilis was carried out in order to compare them with those previously found in the symbiotic thallus. The photobiont was isolated from thallus fragments, following the method of Yamamoto, and cultivated in a liquid nutrient medium. Freeze-dried cells were defatted, and the polysaccharides extracted successively with water and aq. 10% KOH, each at 100 degrees C. After purification, the soluble fractions provided a polysaccharide containing a (1-->5)-linked beta-galactofuranosyl backbone, substituted in a small proportion at O-6 by beta-Galf units. Amylose was also found, as insoluble material obtained on freeze-thawing of the alkaline extract. These polysaccharides have not been found in the symbiotic thallus of Ramalina gracilis, which contained only water-soluble (isolichenan) and insoluble glucans (nigeran and laminaran), and galactomannan. Surprisingly, the galactofuranan has similarities with those found in some fungal cell walls.

Glucans of lichenized fungi: significance for taxonomy of the genera Parmotrema and Rimelia.

The glucans of lichenized fungi are an important class of polysaccharides with structural and chemotaxonomic roles. The water-insoluble glucans of the genus Parmotrema (P. austrosinense, P. delicatulum, P. mantiqueirense, P. schindleri, and P. tinctorum) and those of Rimelia (R. cetrata and R. reticulata), were investigated in order to evaluate the significance in chemotyping, with nigeran [(1-->3),(1-->4)-alpha-glucan] and lichenan [(1-->3),(1-->4)-beta-glucan] characterized using (1)H and (13)C NMR, methylation analysis, and controlled Smith degradations. Results from all species were similar, suggesting that glucan chemistry does not support separation of Rimelia from Parmotrema.

Galactomannans with novel structures from the lichen Roccella decipiens Darb.

Two homogeneous galactomannan fractions were isolated from the lichen, Roccella decipiens, one (FP) containing Man and Gal in an 81:19 molar ratio and the other (RFS), having Man, Gal, and Glc in a 43:56:1 molar ratio. FP consisted of a main chain with (1-->4)-linked alpha-D-Manp units, most of which were substituted at O-2 with side chains consisting of nonreducing end-, 2-O- and 6-O-substituted alpha-Manp units. The latter appeared to be substituted by single-unit beta-D-Galf nonreducing ends. RFS contained a similar alpha-D-Manp core structure, but with side chains containing nonreducing end, 5-O-, 6-O-, and 5,6-di-O-substituted beta-D-Galf units. Such polysaccharide structures have not been previously reported.

Polysaccharides from the fruit bodies of the basidiomycete Laetiporus sulphureus (Bull.: Fr.) Murr.

The two main polysaccharides from the basidiomycetous fungus Laetiporus sulphureus were isolated, purified and characterized. The structural assignments were carried out using (13)C, (1)H, and (1)H,(13) HSQC nuclear magnetic resonance spectroscopy, methylation analysis, and Smith degradation. One was a linear beta-glucan having a (1-->3)-linked main chain, namely laminaran. The other was a fucomannogalactan, which consisted of a main chain of (1-->6)-linked alpha-D-galactopyranosyl residues, a part of them being substituted at O-2 by 3-O-D-mannopyranosyl-L-fucopyranosyl, alpha-D-mannopyranosyl and in a minor proportion, alpha-L-fucopyranosyl groups. This heteropolysaccharide is related to those of other Basidiomycetes heterogalactans, although it differs distinctly in its side-chain structures. Whereas part of the single-unit L-fucopyranosyl and/or 3-O-alpha-mannopyranosyl-L-fucopyranosyl residues are present as side chains of the other heterogalactans, additional alpha-D-mannopyranosyl units are present in our fucomannogalactan of L. sulphureus.

The presence of partially 3-O-methylated mannogalactan from the fruit bodies of edible basidiomycetes Pleurotus ostreatus 'florida' Berk. and Pleurotus ostreatoroseus Sing.

The partially 3-O-methylated mannogalactans were isolated from the fruiting bodies of edible basidiomycetes Pleurotus ostreatus 'florida' Berk. and Pleurotus ostreatoroseus Sing. They were obtained via successive aqueous extraction, freeze thawing, and precipitation with Fehling solution and then investigated using (13)C- and (1)H-nuclear magnetic resonance spectroscopy (including COSY, TOCSY and HMQC techniques), methylation analysis and Smith degradation. The main chain consisted of (1-->6)-linked alpha-D-galactopyranosyl residues containing 3-O-Me-alpha-D-galactopyranoses, a part of these units being substituted in the position O-2 with beta-D-mannopyranose residues. The heteropolysaccharides found were similar with differences only in the levels of the 3-O-Me-alpha-D-galactopyranoses residues. The presence of partially 3-O-methylated mannogalactan appears to be typical of Pleurotus spp.