Cytotoxic and Anti-Candida spp. Activity of Essential Oils and Terpenes

Abstract

ABSTRACT: Objective: To determine the cytotoxicity and anti-Candida spp. activity of essential oils and terpenes. Materials & Methods: A total of twelve essential oils (EOs) from different plant species collected in Colombia and eight commercial terpenes, were evaluated against nine strains of clinically relevant Candida spp. (C.albicans ATCC 10231, C. albicans ATCC 64550, C. parapsilosis ATCC 22019, C. tropicalis ATCC 750, C. tropicalis ATCC 200956, C. glabrata LMDM 34, C.metapsilosis MUM 1512, C.orthopsilosis MUM 1713 and C.lusitaniae MUM 1708) with different antifungal susceptibility profiles. EOs were obtained from plant material by using hydro-distillation, and their components were analyzed by GC/MS. Antifungal activity was determined using the broth microdilution reference method CLSI M27-A3. Initially, a screening of all EOs and terpenes was carried out at 256 μg/mL, and for those in which >90% growth inhibition was observed, minimal inhibitory concentrations (MICs) were determined using concentrations from 256 to 16 μg/mL. The MICs corresponded to the lowest concentration of the EOs or terpenes where a >90% reduction of visible fungal growth was observed. In addition, the susceptibility profiles of all yeasts were evaluated with fluconazole(FLZ), amphotericine B(AMB)and itraconazole(ITZ). The results were expressed as ranges and geometric means (GM). The cytotoxicity of the compounds that showed the highest antifungal activity was tested by means of an MTT assay using the human immortalized keratinocyte cell line HaCat. Results: The strains of Candida spp. showed different antifungal susceptibility pro- files; GM MICs and ranges values were: 0.13 (0.03–2 μg/mL), 0.11 (0.03->16 μg/mL) and 4.66 (0.5–32 μg/mL) for AMB, ITZ and FLZ, respectively. The terpenes thymol and limonene showed the best antifungal activity, inhibiting all yeasts with GM-MIC ranges of 256–128 ug/mL and 128–8 μg/mL respectively. Candida tropicalis ATCC 200956, a yeast with known resistance to azoles and amphotericin B, was susceptible to 18 of 20 com-pounds evaluated. C. lusitaniae MUM 1708 and C. methapsilosis MUM 1512 presented sensitivity to eight EOs (GM-MIC range 256–128 μg/mL) and five terpenes (GM-MICrange 256–8 μg/mL), while ATCC 10231, C. albicans ATCC 64550, C. glabrata LMDM 34 and C. tropicalis ATCC 750, just were susceptible to terpenes, and none of the EOs showe any activity. The cytotoxic activity obtained from EOs and terpenes on the HaCat cell line showed IC50 values ranging from 354.7 to 903.6 μg/mL. Limonene displayed higher selectivity to C. methapsilosis MUM 1512, C. orthopsilosis MUM 1713, C. tropicalis ATCC 200956 and C. glabrata LMDM 34 with selectivity index (SI) of 50, 25, 12 and 6 respectively. Conclusions: The (SI) obtained allow one to identify compounds as possible alterna-tives for the design of products for the eradication of resistant yeasts. The broad sensitivity of the multiresistant C. tropicalis ATCC 200956 to terpenes and EOs suggests that their mechanism of action may be different from that of the main antifungals of clinical use.