Determination of the antioxidant capacity of an antioxidant combination using the fluoroscan assay in vitro and visualization of its effects using histological methods
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The effects of a well-defined combination of antioxidants on oxidative stress were investigated in vitro using classical techniques together and its protective effects against UV damage were investigated using a newly developed skin model. After determining the cytotoxic potential of the combination, its quenching effect on the oxidative stress induced by hydrogen peroxide was quantified by a nonfluorescent (C-H2DCF-DA/AM)/fluorescent (C-DCF) dye system using the fluoroscan assay. Two different skin models consisting of normal human skin fibroblasts and the keratinocyte cell line HaCaT were developed and subsequently used to visualize the protective effects of the combination against UVA damage. No evidence of any cytotoxic potential of the combination could be seen. Supplementation of human skin fibroblasts demonstrated a clear, dose-dependent enhancement of the antioxidative capacity of these cells. Histological findings confirmed the beneficial effects of the antioxidants present in the combination in the skin models used. Supplementation induced morphological changes leading to a thicker epidermal layer providing evidence of the positive effects of the treatment on the viability of the keratinocytes after UVA irradiation. This in vitro study provided convincing evidence of the combined antioxidative action of α-tocopherol, β-carotene, tomato extract, grape seed extract, ascorbic acid and selenium yeast, and indicated a potential beneficial action of the combination against oxidative stress caused by external oxidative stress factors such as UV irradiation.
KeywordsAntioxidants Skin equivalents HaCaT Fluoroscan assay Histology UVA
The authors wish to thank Pharmaton SA, Lugano, Switzerland, for kindly providing the active substances and for financial support.
- 15.Fuchs J, Packer L (1999) Antioxidant protection from solar-simulated radiation-induced suppression of contact hypersensitivity to the recall antigen nickel sulfate in human skin. Free Radic Biol Med 27:422–427Google Scholar
- 17.Gollnick HPM, Hopfenmüller W, Hemmes C, Chun SC, Schmid C, Sundermeier K, Biesalski HK (1996) Systemic beta carotene plus topical UV-sunscreen are an optimal protection against harmful effects of natural UV-sunlight: results of the Berlin-Eilath study. Eur J Dermatol 6:200–205Google Scholar
- 18.Greul AK, Grundmann JU, Heinrich F, Pfitzner I, Bernhardt J, Ambach A, Biesalski HK, Gollnick H (2002) Photoprotection of UV-irradiated human skin: an antioxidative combination of vitamins E and C, carotenoids, selenium and proanthocyanidins. Skin Pharmacol Appl Skin Physiol 15:307–315CrossRefPubMedGoogle Scholar
- 21.Jentzsch AM, Streicher H, Engelhart K (2001) The synergistic antioxidative effect of ascorbyl 2-phosphate and alpha-tocopheryl acetate. Cosmet Toiletries 116:55–64Google Scholar
- 22.Kagan V, Witt E, Goldman R, Scita G, Packer L (1992) Ultraviolet light-induced generation of vitamin E radicals and their recycling. A possible photosensitizing effect of vitamin E in skin. Free Radic Res Commun 16:51–64Google Scholar
- 30.Ponec M (2002) Skin constructs for replacement of skin tissues for in vitro testing. Adv Drug Deliv Rev 54 [Suppl 1]: S19–S30Google Scholar