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

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 60 min, 85.30 % EtOH/H2O 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, LCsingle bondHRMS-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.