A Comprehensive Experimental and Theoretical Investigation of the Antioxidant Properties of Hispidin and Isohispidin

Abstract

This study provides a comprehensive analysis of the antioxidant activity of hispidin (<b>His</b>) and its tautomer isohispidin (<b>IsoH</b>) using DFT calculations, corroborated by experimental data. Under physiological conditions, both tautomers demonstrated significant scavenging capacity for the HO^• radical, with <i>k</i>_overall ranging from 4.48 × 10⁹ to 2.06 × 10¹⁰ M^-1 s^-1 in lipid media and 3.24 × 10¹⁰ M^-1 s^-1 in water. Mechanistic analysis revealed that the radical adduct formation (RAF) mechanism is dominant in lipid environments, whereas both RAF and single electron transfer (SET) operate nonselectively in water. Hispidin also exhibited strong scavenging capacity for the HOO^• radical in water (<i>k</i>_overall = 1.40 × 10⁸ M^-1 s^-1), but its reactivity in lipid environments was comparatively lower, with <i>k</i>_overall of 1.40 × 10² M^-1 s^-1 for <b>His</b> and 1.94 × 10⁴ M^-1 s^-1 for <b>IsoH</b>. The <i>f</i>-HAT mechanism was identified as the predominant pathway in lipid media, while both <i>f</i>-HAT and SET contribute to HOO^• scavenging in water. Additionally, hispidin demonstrated a strong ability to chelate copper(II) ions, effectively inhibiting HO^• radical formation via the Fenton reaction. The theoretical results align well with the experimental data from the DPPH and ABTS assays, indicating that hispidin is a potent antioxidant under physiological conditions.