Re-evaluation of superoxide scavenging capacity of xanthohumol

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3'-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.