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

  • Nutrients. 2025 Nov 25;17(23):3692. doi: 10.3390/nu17233692.
Alessia Silla  1  2 ,  Angela Punzo  1  2 ,  Rossana Comito  3 ,  Emanuele Porru  2  3 ,  Greta Gozzi  1 ,  Maria Cristina Barbalace  4 ,  Matteo Perillo  1 ,  Antonello Lorenzini  1 ,  Marco Malaguti  4 ,  Silvana Hrelia  4 ,  Cristiana Caliceti  1  2  5  6
Affiliations
  • 1. Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum, University of Bologna, 40126 Bologna, Italy.
  • 2. Biostructures and Biosystems National Institute (INBB), 00185 Rome, Italy.
  • 3. Occupational Medicine Unit, Department of Medical and Surgical Sciences, Alma Mater Studiorum, University of Bologna, 40138 Bologna, Italy.
  • 4. Department for Life Quality Studies, Alma Mater Studiorum, University of Bologna, 47921 Rimini, Italy.
  • 5. Interdepartmental Centre for Renewable Sources, Environment, Sea and Energy (CIRI FRAME), Alma Mater Studiorum, University of Bologna, 40126 Bologna, Italy.
  • 6. Interdepartmental Centre for Industrial Agrofood Research-CIRI Agrofood, University of Bologna, 47521 Cesena, Italy.
PMID: 41373983 DOI: 10.3390/nu17233692
Abstract

Background: 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. Methods: 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). Results: 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. Conclusions: 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.

Keywords
antioxidant activity; citrus waste; cosmeceuticals; keratinocytes; natural deep eutectic solvents (NaDES); upcycling; wound healing.
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