Photochemoprotective evaluation of apple extract and rutin in in vitro and in vivo skin models

Author(s)

Silvia de Siqueira

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2014-10-24

Examining board members

Maria Jose Vieira Fonseca; Silvia Berlanga de Moraes Barros; Marcelo Dias Baruffi; Wanderley Pereira de Oliveira; Maria Beatriz Puzzi

• Advisor: Maria Jose Vieira Fonseca

Abstract

Several studies have shown that the ultraviolet radiation (UVR) - induced skin damage are related to DNA photolesions, generation of reactive oxygen species and activation of inflammatory mediators. Therefore, there is an increasing interest in the use of antioxidants with photochemoprotective potential, such as apple extract and rutin. The most used model for evaluation of photochemoprotective agents is the exposure of hairless mice to UVR. However, efforts to reduce or even avoid the use of animals in scientific trials has pursued for alternative methods to replace/reduce animal testing. The first step of this work aimed to optimize parameters for the extraction of apple powder and the characterization of the obtained extract and rutin. Thus, the optimized apple extraction conditions were extraction time of 22 h, ethanol content of 60% (w/w) and plant:solvent ratio of 18 (w/w). The apple extract concentration led to an enriched apple polyphenols extract (EEPM), which showed strong in vitro antioxidant activity and rutin content of 6.1 ± 0.3 ?g / g. The actives showed low or no toxicity against MRC5 fibroblasts, protected these cells against UVR - induced death and inhibited lipid peroxidation in irradiated cells (treatment with 4000 and 100 ?g/mL of EEPM and rutin, respectively). The second step of this study aimed to evaluate the photochemoprotective potential of EEPM and rutin added in a topical formulation and assayed in in vitro models of human skin biopsy and human reconstructed skin and in vivo hairless mouse. The EEPM (1.25%) and rutin (0.75%) formulations were evaluated for skin retention in vitro using the Franz diffusion cell and, although it was not possible to detect compounds of EEPM, rutin was retained in the human skin biopsy (2.04 ± 0.19 mg/cm2). As regard the The photochemoprotective efficacy in in vitro models of human skin, EEPM and rutin formulations were able to prevent/reduce the formation of sunburn cells, induction of caspase-3, cyclobutane pyrimidine dimers, matrix metalloproteinases and lipid peroxidation in skin exposed to UVR. As for in vivo functional activity, EEPM showed a slight inhibitory effect of UVR-induced inflammatory infiltrate, while both EEPM and rutin completely inhibited endogenous GSH levels depletion. These results are promising and suggest the use of EEPM and rutin in the prevention/treatment of ultraviolet radiationinduced damage to the skin. (AU)

• FAPESP's process: 10/11846-0 - Comparison of photochemopreventive effect of apple extract and rutin in "in vitro" skin explants culture and "in vivo" skin hairless mice models
• Grantee: Silvia de Siqueira
• Support Opportunities: Scholarships in Brazil - Doctorate