Deep eutectic solvent extraction of lotus peel proanthocyanidins and its anti-senescence mechanism in t-BHP induced HepG2 cells.

Abstract

Oxidative stress-induced senescence is a key driver of aging-related diseases, particularly in the liver, yet effective and safe natural interventions remain limited. This study aimed to develop a green extraction method for lotus peel proanthocyanidins (LPPs) and evaluate their antioxidant activity, monomer release during digestion, and anti-aging mechanisms. Among eight natural deep eutectic solvents (NADESs), glycerol-betaine (GL-BET, 1:1) was optimal. Using response surface methodology, the optimal extraction conditions were: water content 35%, liquid-to-solid ratio 10 mL/g, sonication time 22 min, temperature 36 °C, yielding 10.97%, which was 5.23-fold higher than 70% ethanol. During simulated digestion, six monomers were identified; proanthocyanidin B2 (PB2) showed the highest content at all stages, proanthocyanidin B3 (PB3) peaked in gastric digestion, and proanthocyanidin B4 (PB4) exhibited the strongest stability (retaining 55.32% after 4 h intestinal digestion). GL-BET, PB3, and PB4 were non-cytotoxic to HepG2 cells at 0-150 μg/mL. Pretreatment with 150 μg/mL of each compound significantly restored viability in tert-butyl hydroperoxide (t-BHP)-damaged cells, with PB4 showing the highest recovery (88.98%). PB4 reduced senescence-associated β-galactosidase (SA-β-gal)-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. Among the three compounds tested in cellular assays (GL-BET, PB3, and PB4), PB4 exhibited the most potent anti-senescence effects. Thus, ultrasound-assisted deep eutectic solvent (DES) extraction significantly improves LPPs yield and bioactivity, providing a basis for green extraction and application of LPPs.