Caracterização química e avaliação in vitro da atividade antifúngica e mecanismos de ação de extratos de Caryocar coriaceum Wittm. frente a Candida spp.

Abstract

Species of Candida spp. are classified as priority fungal pathogens due to fungal resistance, mortality rates, treatment challenges, access to diagnosis, annual incidence, complications, and sequelae from infections. The antimicrobial potential of Caryocar coriaceum, known as pequi, has been evaluated. However, many studies are limited to verifying only antimicrobial activity, without understanding the interactions involved in the process, resulting in unexplored mechanisms of action. Thus, the aim of this study was to analyze the chemical composition and evaluate the activity of three extracts of C. coriaceum Wittm. against Candida spp. in both its planktonic and biofilm forms, as well as to analyze the possible mechanisms of action involved. The leaves of the pequi tree were collected on the campus of Ceará State University, Fortaleza, Brazil. Three extracts were produced: the pequi leaf acetate extract (EAFP), pequi leaf methanol extract (EMFP), and pequi leaf ethanol extract (EEFP). The composition was analyzed using high-performance liquid chromatography (HPLC). Seven strains of Candida spp. were used for microbiological analysis. The antifungal sensitivity test was performed according to the M27-A3 standard of the Clinical and Laboratory Standards Institute. The extracts were combined with the antifungals fluconazole, itraconazole, and amphotericin B (AMB) using the checkerboard method. The mechanism of action evaluation was performed by flow cytometry and electrophoresis. The activity of the extracts on mature C. albicans biofilms was analyzed in 96-well plates. Human lymphocytes were used to assess the cytotoxicity of the extracts. The leaf extracts revealed antifungal activity against Candida spp., with a minimum inhibitory concentration (MIC) ranging from 0.5 to 512 μg/mL, although with limited effect on biofilms. High-performance liquid chromatography characterization revealed the presence of gallic acid, ellagic acid, rutin, and quercetin, which were identified as responsible for the antifungal activity. Analysis of the mechanism of action indicated the production of reactive oxygen species, mitochondrial depolarization, DNA damage, and reduced cell density caused by the apoptotic process triggered. The extracts had low cytotoxicity with respect to human lymphocyte viability. Furthermore, synergism was observed when the extracts were combined with amphotericin B, revealing a potentially synergistic association against Candida infections. In cases where synergism occurred, compared to treatment with the actives alone, the interaction between EAFP and AMB resulted in a four to sixteen-fold decrease in the MIC, while the combinations of EMFP and EEFP with AMB reduced the MIC by approximately four-fold.