Deep Eutectic Solvent Extraction of Lotus Peel Proanthocyanidins and Its Anti-Senescence Mechanism in t-BHP Induced HepG2 Cells

Oxidative stress-induced senescence is a key driver of aging-related diseases, particularly in the liver. Despite the importance of natural interventions, effective and safe options remain limited. To address this, researchers developed a green extraction method for lotus peel proanthocyanidins (LPPs) using deep eutectic solvents (NADESs). Among eight NADESs tested, glycerol-betaine (GL-BET, 1:1) proved optimal. The optimal extraction conditions were determined using response surface methodology, yielding a 10.97% LPP yield that was 5.23-fold higher than 70% ethanol. During simulated digestion, six monomers were identified, with proanthocyanidin B2 (PB2) showing the highest content at all stages. Proanthocyanidin B3 (PB3) peaked in gastric digestion, while proanthocyanidin B4 (PB4) exhibited the strongest stability during intestinal digestion. GL-BET, PB3, and PB4 were found to be non-cytotoxic to HepG2 cells at concentrations up to 150 μg/mL. Pretreatment with these compounds significantly restored viability in tert-butyl hydroperoxide (t-BHP)-damaged cells, with PB4 showing the highest recovery. PB4 reduced senescence-associated β-galactosidase-positive cells from 43.23 to 15.76%, decreased reactive oxygen species (ROS) and malondialdehyde (MDA), increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities. Western blotting confirmed that PB4 upregulated nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase 1 (NQO1), and downregulated Kelch-like ECH-associated protein 1 (Keap1), indicating modulation of the Nrf2/Keap1/antioxidant response element (ARE) pathway. The study suggests that ultrasound-assisted deep eutectic solvent extraction significantly improves LPPs yield and bioactivity, providing a basis for green extraction and application of LPPs.