Estudo de penetração cutânea e desenvolvimento de formulações tópicas contendo estigmasterol e beta-sitosterol presentes na partição hexânica de Pereskia aculeata

Abstract

Pereskia aculeata Muller, commonly known as ora-pro-nobis, is widely studied due to its therapeutic properties. The anti-inflammatory effect of the species is known in the literature and can be associated with the phytosterols beta-sitosterol (β-SIT) and stigmasterol (EST). Therefore, the objective of this study was to develop topical formulations containing EST and β-SIT and evaluate their skin penetration. For this, the hydromethanolic extract of P. aculeata leaves was produced followed by partitioning in hexane (PHEX). The isolated substances EST and β-SIT were quantified in the PHEX. Cytotoxicity was evaluated in L929 fibroblast cells, and PHEX did not show cytotoxicity at concentrations of up to 200 µg/mL. EST and β-SIT did not show cytotoxicity at concentrations of up to 25 µg/mL. Another in vitro test carried out was the inhibition of the nitric oxide radical using the Griess method. In this test, PHEX (200 µg/mL) inhibited 66% of the nitric oxide radical. EST and β-SIT (25 µg/mL) inhibited 33.73% and 34.94%, respectively. In vivo acute toxicity assays were performed using Zophobas morio larvae. The LD50 calculation was obtained using the dose-response curve, being 229.6 mg/kg for PHEX, 101.5 mg/kg for EST and 103.8 mg/kg for β-SIT. Two topical formulations, gel-cream and topical film, were developed to incorporate EST and β-SIT. Thereafter, the development and validation of the analytical method for quantification of EST and β-SIT was carried out using High-performance Liquid Chromatography coupled with the Ultraviolet Diode-Array Detector (HPLC-UV-DAD). The analytical method was selective, linear, precise, and accurate. Furthermore, it was able to recover EST and β-SIT from the pig ear skin. Ex vivo skin penetration was assessed in Vertical Franz Diffusion Cells, using pig ear skin. According to the skin penetration results, β-SIT penetrated into the viable epidermis and dermis 2.5 times more in the topical film than in the gel-cream (44.16 ± 19.69 vs 17.65 ± 15.68 µg/cm2 ). Thus, to date, this is the first study that evaluated the skin penetration of EST and βSIT in formulations.