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Abstract

Coffee cascara, the dried skin of coffee cherries, is a by-product rich in polyphenols but remains underutilized. While this waste product contains potent bioactives, the actual antioxidant activity and importance to intestinal function are less studied. This study analyzed twelve cascara samples from seven countries for polyphenol content using high-performance liquid chromatography (HPLC). Antioxidant capacity of selected extracts was assessed with ABTS, ORAC assays, and cell-based assays (MTT and DCFHDA) in differentiated Caco-2 cells to measure cell viability and oxidative stress modulation. Fortification of these extracts with gallic acid (GA) and protocatechuic acid (PCA) was used to investigate their impact on the antioxidant response of the cascara matrix. Chemical attributes, such as total phenol content and H₂O₂ production, were correlated with biological antioxidant capacity and intracellular NRF2 expression, a key oxidative stress regulator measured using western blot, ELISA, and qRT-PCR. Intestinal barrier integrity was evaluated through TEER measurements, tight junction protein staining, qRT-PCR, and western blot. The results showed significant variation in polyphenol profiles among the samples, with higher antioxidant activities linked to elevated PCA and GA levels, in particular. Chemical antioxidant capacity did not always match biological results, but a significant correlation was found between H₂O₂ production derived from cascara samples and biological ROS inhibition (p