Upcycling of Citrus Waste by Natural Deep Eutectic Solvents: Green Extraction of Bioactive Compounds with Antioxidant and Regenerative Properties on Human Keratinocytes

<b>Background:</b> The citrus processing industry generates over 40 million tons of waste annually, representing a significant environmental challenge. Citrus by-products are rich in bioactive compounds with proven health benefits. This study aims to upcycle citrus waste by developing green extracts and evaluating their biological activities for cosmeceutical applications. <b>Methods:</b> Three NaDES formulations-choline chloride-urea (ChCl: U), choline chloride-citric acid (ChCl: CA), and betaine-urea (Bet: U)-were optimized to extract polyphenols from orange and lemon waste using roller agitation. Extracts were characterized by HPLC-ESI-MS/MS. Biological activities were assessed in human keratinocytes (HaCaT). Antioxidant activity was measured using a chemiluminescent assay that detects intracellular H₂O₂ production. The wound-healing potential was evaluated using scratch assays, and cytokine release (IL-6, IL-8, IL-1β, IL-10) was assessed by ELISA. DNA damage protection was evaluated by quantifying 53BP1 foci following genotoxic exposure (neocarzinostatin). <b>Results</b>: All NaDES extracts showed high polyphenol content, with hesperidin being the primary compound. Pretreatment with the extracts for 24 h significantly reduced intracellular H₂O₂ levels, confirming their antioxidant efficacy. In scratch assays, extracts enhanced wound closure; notably, the Bet: U-derived orange extract achieved complete closure within 48 h. All extracts increased IL-6 and IL-8 release, consistent with an early pro-regenerative response. Pretreatment with the Bet: U orange extract lowered the number of cells with high 53BP1 foci after genotoxic stress, indicating partial DNA damage protection. <b>Conclusions:</b> These findings highlight citrus by-product extracts as sustainable bioactive ingredients with great potential for skin repair and anti-aging formulations, promoting responsible cosmeceutical innovation.