2.2. Measurement of Plasma Free Radical Species Scavenging Capacity

The plasma scavenging capacity for different free radical species was measured according to the method reported by Oowada et al. [12]. A spin trapping agent, 5-(2,2-dimethyl-1,3-propoxy cyclophosphory)-5-methyl-1-pyrroline N-oxide (CYPMPO, Radical Research Inc, Tokyo, Japan), was used to measure the scavenging capacity of each free radical with an X-band Microwave Unit ESR (RE SERIES, JEOL, Tokyo, Japan). WIN-RAD (Radical Research Inc, Version 1.30, Tokyo, Japan) was used for data analysis. Briefly, we prepared a solution containing 20 µL of 1:5 diluted plasma samples, 20 µL of 100 mM CYPMPO, 20 µL of 10 mM dimethylene triamine pentaacetic acid (DTPA, WAKO, Osaka, Japan), 20 µL of distilled water, and 20 µL of 100 mM H₂O₂ for hydroxy radical generation. This was exposed to UV light (SUPERCURE-203, SAN-EI ELECTRIC, Osaka, Japan) for 5 s (total reflection mirror, hot-cut filter). CYPMPO was used as a spin trapping reagent to capture any radicals produced in the solution. The height of the fifth signal of each sample was recorded. The fifth signal of control was set to 100% and compared with each sample to obtain a relative ratio. The standard calibration curve was determined using glutathione disulfide (GSSG, WAKO, Osaka, Japan). The relative ratios of the control and each concentration were set as I0 and I, respectively, when GSSG was added to each sample. In the graph created from these results, the y-axis and x-axis represented I0/I-1 and the concentration of GSSG (mM), respectively. For other free radicals, such as superoxide (O₂•⁻), alkoxy radical (RO•), methyl radical (CH₃•), alkylperoxyl radical (ROO•), and singlet oxygen (¹O₂), each suitable reaction mixture was used and measured for each condition. These standard curves are shown in Supplementary Figure S1. Typical spectrometer settings were as follows: field modulation width, 0.1 mT; microwave power, 6 mW; field scan width and rate, ±7.5 mT/2 min; and time constant, 0.1 s.