Identifying a Role of Red and White Wine Extracts in Counteracting Skin Aging: Effects of Antioxidants on Fibroblast Behavior

Abstract

Dermal fibroblasts are the main actor in many proteins' secretion, including collagen, preserving skin function. Free radicals are involved in skin aging and damages involving different cellular components. The imbalance between reactive oxygen species (ROS) amount and natural antioxidant enzymes negatively affects skin homeostasis. Natural compounds have recently emerged as a potential anti-aging tool in tissue regeneration. In the present paper we evaluated the antioxidant activity of white and red wines, considering their probable use, as raw materials, for the formulation of cosmetic products with anti-aging properties. We studied a method that would allow the removal of the alcoholic fraction of wines and determined their composition by LC-MS analysis. We then tested the possible cytotoxic effects of red and white wines on fibroblasts by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, and their antioxidant activity by the catalase activity test in stressing conditions. Finally, we evaluated their anti-aging potential through the β-galactosidase colorimetric assay. Our results showed that wine extracts exhibit a remarkable antioxidant and anti-aging activity, especially on cells exposed to a marked stressful event. These properties could suggest their possible application as cosmetical products for skin regeneration.

Keywords: antioxidants; bioactive molecules; cell proliferation; cell senescence; oxidative stress; skin aging.

Figure 1

Chemical LC-ESI-LTQ-Orbitrap mass spectrometry (MS). profiles of red wine samples before (A) and after evaporation (B).

Figure 2

Chemical LC-ESI-LTQ-Orbitrap MS profiles of white wine samples before (A) and after evaporation (B).

Figure 3

MTT vitality assay. The cytotoxicity of the extracts was evaluated in cultured cells in the presence of white and red wine extracts at concentrations of 100, 200, 300, 400, and 500 mg/mL for 24 h (A), 48 h (B), and 72 h (C). Cell viability was compared to untreated controls, cultured in the presence of the only basic growing medium, and expressed as absorbance at 570 nm. The experiments were performed two times with three technical replicates for each treatment. Data are expressed as mean ± SD referring to the control (∗ p ≤ 0.05) and evaluated with Kruskal–Wallis rank sum and Wilcoxon signed-rank tests.

Figure 4

Catalase activity. The activity of catalase was evaluated in cells pretreated with H₂O₂ and grown in the presence of white and red wine extracts at concentrations of 100, 200, 300, 400, and 500 mg/mL for 24 h (A), 48 h (B), and 72 h (C). The activity of wine extracts-treated cells was compared to H₂O₂-untreated controls, cultured in the presence of the only basic growing medium (Ctrl). Positive control of oxidative stress were cells pre-treated with H₂O₂ alone (H₂O₂). The absorbance of the various samples was measured at 520 nm. The experiments were performed two times with three technical replicates for each treatment. Data are expressed as mean± SD referring to the control (∗ p ≤ 0.05) and evaluated with Kruskal–Wallis rank sum and Wilcoxon signed-rank tests.

Figure 5

β-galactosidase activity. (A) The activity of β-galactosidase was evaluated in cells pretreated with H₂O₂ and grown in the presence of white and red wine extracts at a concentration of 500 mg/mL. Scale bar = 100 μm. (B) Senescent (blue) cells were evaluated by light microscopic observation. The percentage of SA-β-Gal-positive cells for each treatment was calculated as the number of positive cells divided by the total number of cells counted using an image software analysis (ImageJ). Data are expressed as mean ± SD referring to the control (∗ p ≤ 0.05).