Identification of Polyphenol Oxidase Inhibitors from the Root of Pueraria Lobata: Inhibitor Profiles, Kinetics Analysis and Molecular Interaction

Enzymatic browning induced by polyphenol oxidase (PPO) plays a significant role to quality reduction in fruit and vegetables. PPO inhibitors derived from natural products are gaining attention for their strong inhibitory abilities and low toxicity. This study aims to identify the component of phenolic-rich extract from Pueraria lobata root and evaluate their inhibitory capacity on PPO. The results showed that nine phenolics were identified by UPLC-QE-Orbitrap-MS method as PPO effective inhibitors based on their IC₅₀ values, in which kaempferol showed the strongest inhibitory effect. Among the inhibitors, kaempferol, daidzein, phloretin and luteolin with different inhibitory abilities were selected as representative flavonols, isoflavones, chalcones and flavonoids compounds to investigate the kinetics analysis and molecular interaction. Kaempferol and daidzein exhibited competitive inhibition on PPO, while phloretin and luteolin showed mixed-type and non-competitive inhibition, respectively. According to the fluorescence spectra results, these inhibitors were bound to PPO and quenching fluorescence, among which kaempferol showed the highest K_b value and accompanied by the blueshift. Molecular docking results indicated that the inhibitors interact with PPO primarily through hydrophobic interactions, van der Waals forces and hydrogen bonds, in which kaempferol showed the strongest binding ability with the lowest binding energy. This research provides a theoretical basis for the practical application of Pueraria lobata root extracts as natural PPO inhibitors in the food industry.