Metabolite remodeling of age-stratified immature citrus peel (Qingpi) by pectinase hydrolysis: antioxidant activity and protection against ethanol-induced HepG2 cell injury.

Abstract

Pectinase-assisted processing may enhance the functional value of immature citrus peel (Qingpi, QCP), but the links between matrix degradation, metabolite remodeling, and bioactivity remain unclear. QCP aged for 1, 3, 7, and 10 years and their corresponding pectinase hydrolysates (QCPEH) were evaluated using antioxidant assays, an ethanol-injured HepG2 model, metabolite profiling, chemical-space analysis, network pharmacology, molecular docking, single-compound validation, and RT-qPCR. Pectinase hydrolysis enhanced DPPH, ABTS, and FRAP responses at all aging periods, with 10-QCPEH showing the strongest overall activity. In HepG2 cells, 10-QCPEH reduced LDH release, ALT and AST activities, ROS and MDA levels, restored SOD activity, and partially recovered ADH and ALDH activities. Changes in IL-1β, IL-6, and TNF-α were not statistically significant. Pectinase treatment decreased pectin molecular weight and remodeled both VOC and NVOC profiles, supporting increased metabolite accessibility. Tangeretin, thymol, and nobiletin each reduced ROS accumulation and LDH release. Aromaticity was positively correlated with DPPH, ABTS, and FRAP activities (ρ = 0.90-0.95, q < 0.05). Network and docking analyses highlighted targets related to redox regulation, metabolism, inflammation, and cell survival. RT-qPCR further showed increased HMOX1 and PPARA and decreased PPARG, RELA, TNF, and PTGS2 expression, whereas AKT1 was not significantly altered. These findings support pectinase hydrolysis as a strategy to improve the functional potential of aged QCP through matrix depolymerization and metabolite remodeling.