Sho-saiko-to is an herbal medicine that is known to have diverse pharmacological activities and has been used for the treatment of various infectious diseases. Here, we examined the effects of baicalin, a compound isolated from Sho-saiko-to, and the effects of the iron chelator quinolinic acid on the Fenton reaction. The control reaction mixture contained 0.1 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 0.2 mM H 2 O 2, 0.2 mM FeSO 4( NH 4)2 SO 4, and 40 mM sodium phosphate buffer (pH 7.4). Upon the addition of 0.6 mM baicalin or quinolinic acid to the control reaction mixture, the ESR peak heights of DMPO/OH radical adducts were measured as 32% ± 1% (baicalin) and 166% ± 27% (quinolinic acid) of that of the control mixture. In order to clarify why baicalin and quinolinic acid exerted opposite effects on the formation of hydroxyl radicals, we measured oxygen consumption in the presence of either compound. Upon the addition of 0.6 mM baicalin (or quinolinic acid) to the control reaction mixture without DMPO and H 2 O 2, the relative oxygen consumption rates were found to be 449% ± 40% (baicalin) and 18% ± 9% (quinolinic acid) of that of the control mixture without DMPO and H 2 O 2, indicating that baicalin facilitated the transfer of electrons from Fe (2+) to dissolved oxygen. Thus, the great majority of Fe (2+) turned into Fe (3+), and the formation of hydroxyl radicals was subsequently inhibited in this reaction.

Heme and nonheme high-valent Fe═O can mediate reactions of olefin epoxidation, alkane hydroxylation, aromatic hydroxylation, S-oxidation, P-oxidation, N-dealkylation, alkylaromatic oxidation, and alcohol oxidation. Bromocycloheptane forms as a product in the reaction of high-valent Fe═O with cycloheptane, suggesting that a cycloheptyl radical reacts with CClBr. However, the cycloheptyl radical has not yet been directly detected. To directly detect the radical intermediate in the reaction of the high-valent Fe═O, we analyzed reaction mixtures containing chloroiron tetraphenylporphyrin, iodosylbenzene, ethanol, and α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in 1,2-dichloroethane by an electron spin resonance (ESR) spin-trapping method. As a spin-trapping reagent, we used 4-POBN. Prominent ESR signals were observed in the reaction mixtures. To determine the structure of the radical, the reaction was performed using ethanol-1-C (or ethanol-2-C) instead of ethanol. ESR spectra with no additional hyperfine splitting were observed, indicating that the radical formed in complete reaction mixtures of the porphyrin π-cation-radical species (TPP)Fe═O (TPP = 5,10,15,20-tetraphenyl-21H,23H-porphine) with ethanol has an unpaired electron at neither the α-carbon nor the β-carbon. When the reaction mixture containing ethanol-d instead of ethanol was analyzed using high-performance liquid chromatography-ESR-mass spectrometry, the ions m/z 240 (4-POBN/OCHCH) shifted to m/z 245 (4-POBN/OCDCD). Thus, the radical formed in the complete reaction mixture of (TPP)Fe═O with ethanol has an unpaired electron at the oxygen atom in ethanol. We detected and identified the ethanol-derived oxygen-centered radicals in the reaction of (TPP)Fe═O with ethanol for the first time in this study.