The hydroxyl radical scavenging effects of REB and CH were analyzed by using a spin trapping technique. The spin trapping agent used in the experiments was 5, 5-dimethyl-1-pyrroline-
N-oxide (DMPO; Sigma-Aldrich, St. Louis, MO), which forms secondary radicals (spin adduct) with hydroxyl radicals. The Fenton system, containing diethylenetriaminepentaacetic acid (DETAPAC; Wako Pure Chemicals, Osaka, Japan), ferrous iron (Nakalai Tesque, Kyoto, Japan), and hydrogen peroxide (Nakalai Tesque), was used as a hydroxyl radical generating system. As previously described,
10 0.05 mM of ferrous iron, 0.125 mM of DETAPAC, the test sample, and 1.0 or 5.0 mM of DMPO were added to a 50 mM phosphate buffer solution (pH 7.8). The reaction was started by the addition of 1 mM hydrogen peroxide. An aliquot of the reaction mixture was then transferred into a flat cell, and ESR spectra were recorded by spectrometer (JES FR-30; JEOL, Tokyo, Japan). Measurements were performed under the following conditions: magnetic field, 341.0 ± 10.0 mT; power, 4.0 mW; frequency, 9420 MHz; modulation frequency, 100 kHz; modulation width, 0.25 mT; sweep time, 10 mT/min; time constant, 0.1 second; and received gain, ×100.