Otimização de nanoemulsões contendo óleo essencial de tomilho: estabilidade, atividade antifúngica e liberação in vitro

Abstract

The main objective of this work was the optimization of nanoemulsions containing Thymus vulgaris essential oil (20 mg/mL) obtained from the high speed homogenization method, without the use of organic solvents and high temperatures, aiming to explore the antifungal activity due to the presence of thymol and carvacrol in its composition. Thyme essential oil was identified according to genus and species, characterized and quantified by gas chromatography. Two nanoemulsions were developed from an experimental design using the response surface methodology, differing among themselves by surfactant concentrations. Nanoemulsions were evaluated for their physicochemical properties, such as average droplet size, polydispersion index, zeta potential, pH and thymol content, which was determined by co-validation of analytical methodology by high performance liquid chromatography. The formulations had nanometer size droplets (<150 nm), low polydispersity index (0.21 - 0.26), negative zeta potential (around -8.0 mV), slightly acidic pH (about 5.0 ) and thymol content close to the theoretical. The same parameters were analyzed for the stability study (room temperature and 5 °C) up to 100 days of storage. The parameters were maintained for the highest surfactant nanoemulsion at both storage temperatures. In order to investigate the protection exerted by nanostructured systems against volatilization of essential oil components, a second stability study was performed from the extraction and concentration of nanoemulsion samples by the solid phase microextraction technique (HS-SPME) and later quantification by gas chromatography. For this assay, nanoemulsions were stored at 5, 20 and 40 °C, protected from light for 30 days. Due to the volume of data obtained and for a better understanding of the results, Principal Component Analysis (PCA) was applied. A distinct behavior was found for formulations containing different concentrations of emulsifying agents. The formulation with the highest amount of surfactant presented physicochemical characteristics more adequate to the nanostructured system. Thus, a significant difference (p <0.05) was observed for the formulations maintained at 40 °C, as they presented higher concentration of volatile compounds in the headspace at the end of the study, including thymol and carvacrol, which demonstrated greater system destabilization. Concomitant to this study, in vitro antifungal activity was evaluated by the agar diffusion method against Candida albicans yeast, with the same formulations stored at 5, 20 and 40 °C, at the beginning and end of the storage period. This study aimed to verify if nanoemulsion breakdown and consequent decrease of oil in the nanostructure influenced the antimicrobial activity of the formulations. After 30 days, a reduction in the inhibition zone was observed for all formulations when compared to the initial storage time. However, the lowest antifungal activity was observed for nanoemulsions stored at 40 °C, which is explained by the greater destabilization of nanoemulsion. when stored at this temperature. The in vitro release study of thymol from nanoemulsions showed the ability of nanostructured systems to control thymol release when compared to free (unassociated) oil.