Intestinal oxidative stress mitigation and transepithelial anti-inflammatory bioactivity mediated by EtOH-modified supercritical-CO ₂ coffee pulp extract

This research examined the antioxidant and anti-inflammatory effects of ethanol-modified supercritical CO₂ (EtOH-modified sc-CO₂) coffee pulp extract after INFOGEST 2.0 <i>in vitro</i> simulated gastrointestinal digestion. EtOH-modified sc-CO₂ extract digesta (3.1 mg mL^-1) contained elevated concentrations of protocatechuic acid (61.2 µg mL^-1) and caffeine (287.6 µg mL^-1) compared to water and ethanol digesta, contributing to its strong ABTS radical scavenging capacity (4015 µg TE mL^-1) and superior cellular antioxidant activity (37.8%), along with cyclooxygenase 2 inhibition activity (37.2%). In a Caco-2/THP-1 co-culture model of intestinal inflammation induced by 4 h of LPS (1 μg mL^-1) stimulation, a 24 h treatment with the EtOH-modified sc-CO₂ digesta (diluted 1 : 14, v/v, in complete culture medium) demonstrated transepithelial anti-inflammatory effects, decreasing TNF-α, IL-6, CCL2, and IL-1α levels by 9.9%, 24.7%, 34.3%, and 17.0%, respectively, in comparison to the LPS-stimulated control group. Relative to the pure standard mixture digesta, the EtOH-modified sc-CO₂ digesta favored a balance between pro-inflammatory (IL-1α and IL-1β) and anti-inflammatory mediators (IL-10 and IL-1ra), highlighting the importance of matrix effects in inflammation regulation. Bioinformatic analysis revealed that protocatechuic acid, chlorogenic acid, and caffeine metabolites potentially inhibit NF-κB-driven signaling. These results demonstrate that coffee pulp extracts obtained through EtOH-modified sc-CO₂ extraction exhibit substantial bioavailability and bioactivity, reinforcing their potential application as functional food ingredients for the prevention of chronic diseases.