Continuous fluorescence-based quantitative antioxidant assay using vegetable oil as an oxidizable substrate

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Several spectrophotometric assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC), are commonly used to assess antioxidant activity. However, these methods often lack real-world relevance as they do not inhibit autoxidation in actual food substrates. Although direct measurement of oxygen consumption or peroxide formation during inhibited autoxidation offers certain advantages, it is labor intensive and requires specialized equipment. In this study, we introduce a small-volume inhibited autoxidation approach that utilizes a standard microplate reader and a food-derived oxidizable substrate, specifically stripped sunflower oil (SSO), and styrene-conjugated BODIPY (STY-BODIPY) chromophores that oxidizes with the substrate, enabling straightforward monitoring of the reaction progress without interfering with it. The rate of initiation (Ri) was controlled by using azobis(isobutyronitrile) (AIBN) at 30 °C (Ri = 8.6 ± 0.5 × 10−10 M s−1) to accurately determine the rate constant of antioxidant reaction with peroxyl radicals (kinh). The method was standardized using the synthetic α-tocopherol analogue 2,2,5,7,8-pentamethyl-6-chromanol (PMC) as a reference antioxidant and was successfully applied to evaluate its synergistic interactions with γ-terpinene, quercetin, and caffeic acid. The rate constant for the reaction of peroxyl radicals with STY-BODIPY was determined, kST = 890 ± 52 M−1 s−1. Induction time (τ) of PMC increased in a concentration-dependent manner by the synergistic interactions of PMC/γ-terpinene, PMC/quercetin, and PMC/caffeic acid. The kinh value for PMC in SSO at 30 °C remained constant at 1.5 × 106 M−1 s−1. The validity of this approach was further confirmed using isothermal calorimetry, demonstrating its potential as a reliable and accessible tool for antioxidant testing in food systems.