Análise antifúngica de compostos bioativos, da sua ação em fatores de virulência de Candida albicans e na modulação da interação Candida-hospedeiro

Abstract

OBJECTIVE: To analyze the in vitro antifungal activity against Candida spp of S. aromaticum essential oil (EO), and bioactive compounds (eugenol, β-caryophyllene, geraniol, citronellal and linalool), as well as its action on virulence factors of Candida albicans, and in the modulation of Candida-host interaction, considering the associated in vivo toxicity. MATERIAL AND METHOD: The antifungal activity was analyzed by Broth Microdilution Method on Candida species (C. albicans, C. glabrata, C. tropicalis and C. dubliniensis) and antibiofilm activity on C. albicans, for that it was established, respectively, the Minimal Inhibitory Concentration (MIC), and the counting of Colony Forming Units/mL (CFU/mL), normalized by the biofilm dry weight (CFU/mL/g). Cytotoxicity was analyzed in human oral epithelial cells (TR146 - ECACC 10032305) and in human monocytes (THP-1 - ATCC TIB-202) by the Cell Titer Blue viability assay. Based on the microbiological and cytotoxic results, geraniol (MIC) was selected for analysis of the capacity to modulate Candida-host interaction, through the dual chamber co-culture method. with THP-1 cells and TR146 cells (infected with C. albicans), RT-PCR were used to assess gene expression of C. albicans proteolytic enzymes (ACT-1 and PLB-1) and host inflammatory cytokines (IL-1β, IL-6, IL-17, IL-18, IL-10, and TNF). Fluorescence microscopy was performed with TR146 and C. albicans co- culture. An analysis of systemic toxicity was performed in a model of Galleria mellonella. RESULTS: All compounds, with the exception of β-caryophyllene (MIC > 8000 μg/mL), showed antifungal activity with MIC determined at: 500-1000 μg/mL for EO and eugenol, 1.25-5 mM/mL for geraniol, 25-100 mM/ml for linalool and 100-200 mM/ml for citronellal. All concentrations tested for EO, eugenol and geraniol reduced the viability of C. albicans during biofilm formation and in the mature biofilm. Linalool inhibited biofilm formation, but in the mature biofilm only 10xCIM was effective. Citronellal did not show antibiofilm activity at the tested concentration. For cell cytotoxicity, the LD50 were defined, respectively, for TR146 and THP-1: EO 59.37 and 79.54 µg/mL; eugenol 55.35 and 84.16 µg/mL; geraniol 5883 mM/ml and 8027 mM/ml; linalool 1432 mM/ml and 1709 mM/ml; and citronellal 0.3006 mM/mL and 0.1825 mM/mL. Geraniol 5 mM/mL (MIC) was able to modulate the Candida-host interaction by down-regulating the expression of Candida proteolytic enzymes and the host pro-inflammatory cytokines IL-1β, IL-6 and IL-18. The substantial reduction in growth of C. albicans by geraniol was also seen microscopically. Geraniol (up to 8000 mM/Kg) and EO (up to 100 mg/Kg) did not induce in vivo toxicity. Conclusion: The essential oil of S. aromaticum and the evaluated compounds, except for β-caryophyllene and citronellal, had anti-Candida activity, capable of inhibiting biofilm formation and reducing the viability of a mature C. albicans biofilm. Geraniol modulates the Candida-host interaction, reducing the expression of virulence factors and pro-inflammatory cytokines, without evidence of toxicity.