Estudo químico e farmacológico do extrato hidroalcoólico do cogumelo Agaricus blazei Murill e seu emprego no desenvolvimento de scaffolds para o reparo tecidual

Abstract

The present work aimed to characterize the chemical profile and pharmacological potential of the hydroalcoholic extract from Agaricus blazei Murill mushroom (EAb) and its bioactive compounds (mannitol and polysaccharides), as well as to employ it in scaffolds for tissue repair. The EAb was prepared by dynamic maceration from a hydroethanolic mixture (70%, v v-1) as solvent, while the polysaccharides (PAb) were isolated by hot water methodology. The chemical composition of EAb was studied by Two-Dimensional Nuclear Magnetic Resonance (2D NMR) and Gas ChromatographyMass Spectrometry (GC-MS). In addition, its antioxidant activity was determined by chemical methods, as well as the content of reducing sugars. The PAbs were characterized by ¹H and ¹³C NMR and High Performance Liquid Chromatography (HPLC). The safety and efficacy of EAb, mannitol and PAb were studied using red blood cells, platelets and human neutrophils. The polymer matrix of the scaffolds consisted of alginate and carboxymethylcellulose reinforced with starch nanocrystals (SNCs). SNCs were synthesized by acid hydrolysis and characterized based on their morphology and colloidal properties. The structural, thermal, morphological and mechanical properties of the scaffolds were also evaluated. Through the analysis of 2D NMR, GC-MS and quantification of reducing sugars, it was found that the chemical profile of the EAb consists mostly of sugars and amino acids that together represent more than 80% of the chemical variety of this complex matrix. In addition, EAb showed significant antioxidant activity, especially in the superoxide anion scavenging assay (IC50 < 1 µg mL-1). The analysis of the monosaccharide composition of PAb by HPLC indicated glucose as the major monosaccharide in a content of 76.36%. EAb, mannitol and PAb showed low cytotoxicity against red blood cells, platelets and human neutrophils in a concentration range that varied from 1 to 200 µg mL-1, parameters that were studied through the hemolysis index, evaluation of membrane integrity and cell viability. At the same time, EAb demonstrated potential in vitro anti-inflammatory activity against the reduction of myeloperoxidase release by PMA-stimulated neutrophils (Log CE50 = 1.35 µg mL-1) with better results than those obtained for indomethacin. Mannitol significantly reduced (p < 0.05) the release of MPO only at the highest concentration (80 µg mL-1), suggesting the existence of an additive effect between this compound and the others present in the EAb. On the other hand, PAbs showed a modulating effect on MPO release in a concentrationdependent manner, showing an anti-inflammatory effect at lower concentrations (1-10 µg mL-1) and pro-inflammatory at higher concentrations (100-200 µg mL-1). The SNCs had a hydrodynamic diameter lower than 200 nm and a zeta potential higher than the modulus of 20 mV. Furthermore, in the microscopic analysis, particles with even smaller diameter (~100 nm) and oblong morphology were observed. Scaffolds characterization showed that the use of EAb and SNCs increases the porosity, thermal stability and absorption capacity of the formulations, which may be due to intermolecular interactions between the chemical constituents of the EAb and the polymeric matrix, which was evidenced by spectroscopic techniques. Therefore, with these data, we can suggest that the incorporation of EAb in composite scaffolds based on alginate/carboxymethylcellulose/starch nanocrystals is a promising strategy for the development of new formulations with potential for tissue repair.