Glutathione peroxidases (GPxs) are essential antioxidant enzymes that catalyze the reduction of hydrogen peroxide and organic hydroperoxides, thereby maintaining cellular redox homeostasis. The search for small-molecule GPx mimetics has stimulated the development of biomimetic chemical assays capable of evaluating peroxide-reducing catalytic activity under controlled conditions. Among these, Iwaoka’s assay has emerged as a robust and mechanistically informative method. The assay monitors the catalyst-mediated oxidation of 1,4-dithiothreitol (DTTred) to its disulfide form (DTTox) in the presence of hydrogen peroxide, typically using 1H NMR, UV-Vis spectroscopy, or more recently hyphenated chromatographic techniques such as HPLC-DAD and GC-MS. Although originally designed for synthetic organoselenium compounds, Iwaoka’s assay has increasingly been applied to natural products, including phenylpropanoids and terpenophenolic cannabinoids. These studies reveal that certain natural scaffolds can promote peroxide reduction through redox cycling mechanisms. Importantly, the assay distinguishes true catalytic turnover from simple stoichiometric radical scavenging, offering mechanistic insight beyond conventional antioxidant tests. This review critically examines the principles, analytical evolution, and application of Iwaoka’s assay to natural products, highlighting methodological challenges, structure-activity relationships, and future directions aimed at improving standardization, biological relevance, and integration with cellular oxidative stress models.
Fiorito et al. (Fri,) studied this question.