Abstract

Calafate (Berberis microphylla), a native berry from the Patagonian region, is rich in phenolic compounds with potential health benefits. Most studies focus on free phenolics, whereas the insoluble-bound compound fraction, frequently discarded by the food industry, remains understudied. Evaluating the biological redox impact is crucial for conferring functionality to this byproduct, particularly improving the antioxidant defenses of mammalian cells against an oxidative challenge. This study started with a comprehensive characterization of phenolic composition in four fractions from calafate byproduct (free, esterified, etherified, and insoluble-bound) by HPLC-DAD and UPLC-ESI-MS/MS, confirming the presence of phenolic acids and flavonoids. Furthermore, the antioxidant capacity of these fractions was assessed using a set of chemical-based assays such as FRAP, DPPH, ABTS, and ORAC, which served to select the free and insoluble-bound fractions that elicited the highest total phenolic content and antioxidant capacity. To investigate whether the antioxidant capacity of these fractions has biological relevance, we evaluated their protective effect against hydrogen peroxide on Caco-2 cells. Additionally, we monitored the redox impact on Caco-2 cells expressing the HyPer biosensor, a molecular tool suitable for real-time measurements of thiol/disulfide balance. Our experimental strategy revealed that the insoluble-bound fraction [IC50 = 2.1 x 10-6 g/mL] provided greater oxidative protection than the free fraction [IC50 = 2.7 x 10-3 g/mL] and induced a rapid antioxidant shift at the cytoplasm of intact HyPer-expressing Caco-2 cells. These findings highlight the redox-modulating potential of calafate byproducts, particularly the insoluble-bound compound fraction, supporting their application in functional foods and contributing to the circular economy.