Development of Flow-Injection Spin-Trapping ESR Methods for Kinetic Study of the O-2(-center dot) Radical Scavenging Reaction with Mixtures of Phenolic Antioxidants

The apparent second-order rate constants ((a)k(s)) of the reaction between the O-2(-center dot) radical and mixed solutions ((a)Aox) of caffeic acid (CA) and 4-hydroxy-coumaric acid (4CA) were estimated in this study; this was achieved by a flow-injection spintrapping ESR (FI-ESR) method that employed 5,5-dimethylpyrroline-N-oxide (DMPO) as the spin-trapping reagent. The (a)Aox solutions were prepared by varying the mixing molar ratio of CA (a = [CA]/([CA] + [4CA])). The FI-ESR signal intensities of the DMPO spin-adduct of the O-2(-center dot) radical (DMPO/O-2) were monitored in the absence (I-0) and presence (I) of aAox to estimate the inhibition ratio Y (%) (Y = 100 x I/I-0), and 50% inhibitory dose of (a)Aox ((ID50)-I-a). Further, the (a)k(s) values of aAox were evaluated using these (ID50)-I-a values through a competitive reaction kinetics treatment, which was conducted by using the second-order rate constant of the spin-trapping reaction (k(1)). The value obtained for the (a)k(s) of (a)Aox showed excellent agreement with the calculated values ((a)k(s)(cal)) that were estimated from the molar ratio (a) weighted mean of the k(s) values of CA ((CA)k(s)) and 4CA ((4CA)k(s)): (a)k(s)(cal) = a(CA)k(s) + (1 (1) a)(4CA)k(s). These results demonstrate that CA and 4CA contribute independently to the O-2(-center dot) radical scavenging reactions, and that the (a)k(s) values of (a)Aox completely satisfy the additive property depending on the mixing molar ratio (a). The O-2(-center dot) radical scavenging capacities of (a)Aox were also evaluated as equivalent concentrations of CA ((Y)[CA](eq).) using the observed inhibition ratio Y (%), and the 50% inhibitory dose of CA ((ID50)-I-CA): (Y)[CA](eq). = (ID50)-I-CA x (100 - Y)/Y. The estimated (Y)[CA](eq). values of (a)Aox agreed with the calculated equivalent concentrations of CA ((k)[CA](eq).) that were obtained on the basis of the additive property established for (a)Aox and (a)k(s): (k)[CA](eq). = [CA] + ((4CA)k(s)/(CA)k(s)) x [4CA].