Polyphenols and red wine as peroxynitrite scavengers: a chemiluminescent assay

A novel chemiluminescent assay for evaluating peroxynitrite (ONOO(-))-scavenging capacity was developed. The experimental protocol ensures sensitivity and reproducibility of measurements. The addition of 0-500 microM ONOO(-) to rat liver homogenate generated a luminous signal that was analyzed by chemiluminescence in a LKB Wallac liquid scintillation counter. The obtained optimal conditions were: 1-2 mg/mL of homogenate protein in 120 mM KCl, 30 mM phosphate buffer (pH 7.4), and 220 microM ONOO(-) at 30 degrees C. As polyphenols we used (+)-catechin, (-)-epicatechin, and myricetin. The most efficient of the compounds tested was myricetin with an IC(50) of 20 microM. The effectiveness of this method was verified by evaluating the antioxidant ability of three red wine samples to decrease peroxynitrite-initiated chemiluminescence. The ONOO(-)-scavenging activity of wines measured by this assay was related to the phenolic level of the samples. The quickness and reliability of this assay makes it particularly suitable for a large-scale screening of watery food extracts.