Atividade antifúngica in vitro e in vivo do extrato metanólico de mitracarpus frigidus na sua forma livre e incorporado a uma formulação a base de quitosana

Abstract

The emergence of multiresistant microorganisms can be attributed to the indiscriminate use of antimicrobials for disease control, which has been causing intense selective pressure on microorganisms. Responsible for most infections, we can highlight Candida albicans, an opportunistic pathogenic agent, associated with some important clinical infections, such as vulvovaginal candidiasis (CVV). Recent research has been investing in new formulations, several of these of natural origin. The species Mitracarpus frigidus (Willd. Ex Reem Schult.) K. Schum (MFM) presents biological activities already reported, with emphasis on antimicrobial activities. This work aims to evaluate the in vitro and in vivo antifungal activities of MFM in its live and incorporated into a chitosan gel against C. albicans (ATCC® 10231) resistant fluconazole, itraconazole, voriconazole and anidulafungin. In vitro assays were performed only with free MFM. For this, planktonic cell activity was analyzed using minimum inhibitory concentration (MIC), minimum fungicidal concentration (CFM), growth curve, cell density and action mechanism (permeability with violet crystal, membrane extravasation, sorbitol protection, exogenous ergosterol binding assay, hydrophobicity, ergosterol dosage and viability). Subsequently, biofilm assays were carried out by proliferation and adhesion analysis, Whole Slide Imaging (WSI), Scanning Electron Microscopy (SEM) and carbohydrate content analysis. The in vivo assays comprised experimental models of CVV, testing in addition to free MFM, its form incorporated into a chitosan gel. The evaluation of MIC and MFC determined a value of 500 μg/mL and fungistatic effect for C. albicans, respectively. Growth curve assay resulted in an inhibition of fungal growth against MFM. For cell density, MFM proved a 70% reduction in the number of fungal cells. Capability of violet crystal was increased. The membrane extravasation assay demonstrated that MFM significantly increased the release of nucleotides. For the analysis of sorbitol and ergosterol, a value of MIC 4000 μg/mL and MIC 8000 μg/mL, respectively, was obtained, indicating an action on the wall and cell membrane of the yeast. Lipid content assays indicated a greater release of these components in the samples tested. Cell viability indicated a decrease in fungal load and a reduction in the presence of hyphae, altering the virulence factor of samples treated with MFM. In relation to biofilm assays, MFM was able to reduce pre-established biofilm and interfere with its adhesion. The use of WSI and SEM suggested the reduction of formed biofilms. Carbohydrates analysis also indicated a decrease of this structure and its exopolysaccharide matrix. In vivo assays demonstrated that MFM in its two analyzed forms were able to reduce fungal load and inflammatory process. The results obtained further highlighted the scientific and therapeutic potential of this plant, opening new perspectives for the treatment of infectious diseases and possibly a new formulation based on chitosan with the capacity of dermatological applications, providing its future use for CVV treatment.