Mechanistic Insights into Polyphenol Oxidase Inhibition by Four Bioactive Constituents from Hydrangea macrophylla Serrata: Antioxidant Activity of Single Compounds Compared to the Whole-Leaf Extract

Abstract

Hydrangea macrophylla serrata (tea-hortensia) leaves are traditionally used to produce a sweet herbal tea (“Amacha”) and contain hydroxycinnamates and dihydroflavonols with potential bioactivities (e.g., anti-inflammatory, anti-allergy). In this study, from an extract of tea-hortensia leaves, we isolated and structurally characterized four major constituents: chlorogenic acid (CGA, 5-caffeoylquinic acid), hydrangenol (HG), hydrangeic acid (HGA), and phyllodulcin (PD). The four constituents and the extract were evaluated for antioxidant capacity via total phenolic content (TPC), ABTS radical scavenging (TEAC: Trolox equivalent antioxidant capacity), and oxygen radical absorbance capacity (ORAC) assays, and for anti-browning potential via polyphenol oxidase (PPO) inhibition assays. To elucidate the underlying mechanisms, we employed kinetic analysis to determine inhibition types, evaluated copper-chelating capacity, and performed molecular docking simulations to investigate ligand–PPO binding modes and key interacting residues. The tea extract exhibited significantly superior antioxidant capacity compared to all isolated compounds across all assays, with CGA being the most potent among the four compounds. For anti-browning activity, CGA and PD showed the strongest PPO inhibition (90%), while the tea extract (83% at 2 mg/mL) outperformed HG and HGA but was less potent than CGA and PD. Molecular docking further revealed that CGA formed stable interactions with key residues in the PPO active site. These findings provide a systematic comparative assessment of the anti-browning mechanisms of the major constituents of H. macrophylla serrata, highlighting the potential of this plant as a natural source of antioxidants and anti-browning agents for food applications. The integration of biochemical kinetics, copper-chelating analysis, and in silico docking offers a comprehensive mechanistic framework for understanding PPO inhibition by natural phenolic compounds.