Recovery of Neohesperidin and Other Bioactive Flavonoids from Bitter Orange By-Products: Impact of Hydrogen Bond Donors Using NADES-UAE as a Sustainable Approach

Natural deep eutectic solvents (NADES) have been increasingly explored as alternative extraction media for recovering high-value phytochemicals from agro-industrial byproducts. In this study, a 5 × 22 factorial design was used to evaluate the influence of HBD type, molar ratio, and added water content for choline chloride-based NADES-UAE of neohesperidin from Citrus aurantium byproducts. Individual flavonoids were quantified by Ultrahigh-Performance Liquid Chromatograph (UPLC), while total polyphenolic content (TPC), total flavonoid content (TFC), vitamin C content (TAA), and antioxidant activity (AX) were determined by spectrophotometric assays. The highest extracted neohesperidin content, 995.98 ± 1.77 mg/100 g dry mass (DM), was achieved with lysine-based (HBD) NADES (1:2 mol/mol, 40% w/w). In addition, the flavonoid profile was dominated by hesperidin, quercetin + luteolin and diosmetin. Among the NADES formulations evaluated, the highest TPC and TFC with a fructose-based (HBD) concentration of 4436.03 ± 94.31 mg GAE/100 g DM (1:1 mol/mol, 40% w/w) and 1514.64 ± 53.11 mg QE/100 g DM (1:1 mol/mol, 60% w/w), respectively; for TAA, 2467.39 ± 52.18 mg AAE/100 g DM with citric acid (HBD); and finally for AX, 91.71 ± 0.86% was obtained by fructose (HBD) both with 1:2 mol/mol, 60% w/w. Changes in the NADES composition also altered the extraction of other flavonoids, generating distinct phytochemical profiles. Statistical analyses demonstrated that the solvent composition significantly affected the extraction selectivity and the resulting phytochemical profile. Overall, the fructose-based NADES provided one of the most effective systems for recovering bioactive compounds from Citrus aurantium byproducts, supporting the potential of NADES-UAE as an alternative extraction strategy for obtaining flavonoid-enriched extracts with potential relevance for future food, nutraceutical, and pharmaceutical applications.