Phytochemicals from Cocoa Shell Protect Mitochondrial Function and Alleviate Oxidative Stress in Hepatocytes via Regulation of ERK and PI3K-AKT Pathways

Abstract

This research aimed to assess the impact of an aqueous extract from the cocoa shell and its major phytochemicals on preventing oxidative stress and mitochondrial dysfunction in hepatocytes using an in vitro model of nonalcoholic fatty liver disease (NAFLD). The phytochemicals from cocoa shell were extracted using water and characterized by UPLC-MS/MS analysis. HepG2 cells were cotreated with either the aqueous extract from cocoa shell (CAE, 20–100 µg mL⁻¹) or 10–50 µmol L⁻¹ of pure theobromine, protocatechuic acid, procyanidin B2, epicatechin, and catechin in the presence or absence of palmitic acid (PA, 500 µmol L⁻¹) to mimic NAFLD conditions in vitro. Biomarkers of mitochondrial function and oxidative stress were evaluated 24 h after the cotreatment in cell supernatants and lysates using chemical, biochemical, and immunochemical techniques. CAE and the phytochemicals therein significantly (p < 0.05) protected mitochondrial content (15–100%) and preserved mitochondrial function, promoting O₂ consumption (1.2- to 1.8-fold) and ATP production (1.3- to 2.1-fold). Phytochemicals from cocoa shell significantly (p < 0.05) decreased PA-triggered oxidative stress. The mitochondrial membrane potential was maintained (62–100%), and the production of mitochondrial superoxide (26–100%) and total ROS (17–100%) was abrogated. CAE significantly (p < 0.05) modulated cell signaling pathways associated with ROS production and mitochondrial dysfunction, including an increase in the phosphorylation of ERK1/2 (2.8-fold), protein kinase B (AKT) (2.8-fold), GSK3 (2.3-fold), Raf-1 (1.9-fold), and mTOR (1.7-fold). In conclusion, results suggested that the cocoa shell’s phytochemicals could protect mitochondrial liver function and alleviate oxidative stress by modulating key pathways involved in their regulation.