Purification and Identification of Peptides from Hydrilla verticillata (Linn. f.) Royle with Cytoprotective and Antioxidative Effect against H2O2-Treated HepG2 Cells.

Abstract

Antioxidant peptides were purified from Hydrilla verticillata (Linn. f.) Royle (HVR) protein hydrolysate by ultrafiltration, gel filtration chromatography, and semipreparative reversed-phase HPLC and identified by UPLC-ESI-MS/MS. Therein, TCLGPK and TCLGER were selected to be synthesized, and they displayed desirable radical-scavenging activity to ABTS (99.20 ± 0.56-99.20 ± 0.43%), DPPH (97.32 ± 0.59-97.56 ± 0.97%), hydroxyl radical (54.32 ± 1.27-70.42 ± 2.01%), and superoxide anion (42.93 ± 1.46-52.62 ± 1.11%) at a concentration of 0.96 μmol/mL. They possessed a cytoprotective effect against H2O2-induced oxidative stress in HepG2 cells in a dose-dependent manner. 1.6 μmol/mL of the two peptides could perfectly protect HepG2 cells from H2O2-induced injury. The TCLGPK exhibited higher antioxidant activity and cytoprotective effect than TCLGER. Western blot and molecular docking results indicated that the two peptides achieved antioxidant ability and cytoprotective effect by combining with Kelch-like ECH-associated protein 1 (Keap1) to activate the Keap1-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response elements signaling pathway, leading to the activity and expression of the related antioxidases in the pathway significantly up-regulating and the intracellular reactive oxygen species level, lipid peroxidation, and cell apoptosis rate significantly down-regulating.