Extraction optimization of Citrus medica L. whole fruit: phytochemical profile, pectin recovery and antioxidant activity in Caco-2 cells

• The extraction of Citrus medica fruit was optimized by Full Factorial Design • Using 1h, 85.30% EtOH 47.22°C an optimized antioxidant extract (OE) was obtained • Untargeted-Targeted analysis revealed the presence of antioxidant metabolites • Pectin was recovered from the exhausted material promoting the zero-waste theory • OE protected Caco-2 cells by increasing enzymes involved in antioxidant defenses This study aims to increase the antioxidant yield from fresh whole fruits of Citrus medica cv. Diamante Liscia applying the Full Factorial Design (FFD), which allowed to obtain an optimized extract (OE). The optimal extraction conditions obtained by FFD were 60min, 85.30% EtOH/H 2 O and 47.22°C, reporting the following data: 3.79±0.09 mgGAE/g, 4.45±0.03 mgTE/g and 10.62±0.26 mgTE/g for Total Polyphenol Content (TPC), 2,2-diphenyl-1-picryl hydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP), respectively. To provide a comprehensive metabolic profile of samples prepared as part of the experimental design, LC-HRMS-based untargeted analysis was performed using a combination of MS-DIAL, MS-CLEANR, and MS-FINDER software suites. Pearson correlation established the relationship between antioxidant activity evaluated by TPC, DPPH, and FRAP and peaks area of specific metabolites. Of these, rutin, hesperidin, and diosmin were confirmed and quantified as OE’s characteristic component. Furthermore, after recovering bioactive compounds of interest, pectin with a high yield of 14.78 ± 1.26 % was recovered from the exhausted material. The OE showed antioxidant properties on intestinal Caco-2 cells by stimulating antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, catalase, and NAD(P)H dehydrogenase. This study reports for the first time the antioxidant properties of C. medica whole fruit extract highlighting its potential as therapeutic agent in preventing oxidative stress-related diseases, such as inflammatory bowel disease.