| Summary: | In recent years, plant protein hydrolysates have gained increased attention due to their superior antioxidant activity and potential to prevent several chronic diseases associated with oxidative stress. This study aimed to investigate the antioxidant activity, functional properties, and cytoprotective effects of the tree peony seed protein hydrolysate (TPSPH) with different molecular weights (MWs). The antioxidant activities were evaluated by DPPH, hydroxyl radicals scavenging, Fe<sup>2+</sup> chelating, and inhibition of the β-carotene oxidation abilities. The protective effects and mechanism against oxidative stress were determined using H<sub>2</sub>O<sub>2</sub>-stressed HepG2 cells. MW > 30 kDa of TPSPH showed the highest radical scavenging (DPPH IC<sub>50</sub> = 0.04, hydroxyl IC<sub>50</sub> = 0.89 mg/mL) and inhibition of β-carotene oxidation (80.07% at 2.0 mg/mL) activity. Moreover, MW > 30 kDa possessed high hydrophobicity, emulsifying capacity, and abundant antioxidant amino acids (28.22% of hydrophobic amino acids and 8.3% of aromatic amino acids). MW 5–10 kDa exhibited more effective protection against H<sub>2</sub>O<sub>2</sub>-induced HepG2 cells, by reducing reactive oxygen species (ROS), malonaldehyde (MDA), lactate dehydrogenase (LDH), and activating antioxidant enzymes (superoxide dismutase and catalase). These results indicated the potential application of TPSPH as an antioxidant in food and functional foods.
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