Assessment of antioxidant activity of Phloretin and Neohesperidin Dihydrochalcone and their mixture using New Approach Methodologies (NAMs)

Ultraviolet (UV) radiation generates reactive oxygen species (ROS), triggering skin inflammation, activating AP-1, increasing matrix metalloproteinase (MMP) expression, and inhibiting collagen synthesis, ultimately promoting extracellular matrix degradation and photoaging. New Approach Methodologies (NAMs), including human cell-based in vitro assays, provide relevant non-animal tools for evaluating the biological activity of cosmetic ingredients. The aim of this study was to assess the antioxidant of two natural compounds (Phloretin, NF02; and Neohesperidin dihydrochalcone, NHDC), individually and in combination, and to investigate possible synergistic effects. UV-Vis absorption spectra were obtained according to OECD 101 (1981). Cytotoxicity was evaluated in HaCaT keratinocytes (Eucellbank, Celltec–Universitat de Barcelona) exposed to 10–100 µg/mL of each compound or their mixture for 24 h using the Neutral Red Uptake (NRU) and MTT assays. Intracellular ROS production was determined after treatment with 10 or 20 µg/mL of the compounds followed by UVA irradiation (10 J/cm²). DCFH-DA was used as the fluorescent probe, and fluorescence values were normalized to cellular protein content determined by the Bradford assay. N-acetyl-L-cysteine and untreated cells were included as positive and negative antioxidant controls, respectively. MTT results showed an IC50 of approximately 100 µg/mL, whereas no IC₅₀ could be determined with the NRU assay. All compounds exhibited high molar extinction coefficients. Both individual compounds and their combination significantly reduced UVA-induced ROS production, with the mixture producing the greatest inhibition, suggesting a synergistic antioxidant effect. These findings support the usefulness of NAMs for the characterization of bioactive cosmetic ingredients.