Hypochlorite scavenging activity of hydroxycinnamic acids evaluated by a rapid microplate method based on the measurement of chloramines

Scavengers of hypochlorite (XOCI) could have beneficial effects in diseases in which this oxidant plays a pathogenic role. It has been reported that ferulic acid and chlorogenic acid, the quinic ester of caffeic acid, are good hypochlorite scavengers, but a systematic evaluation of the naturally occurring hydroxycinnamic acids (HCAs), which these substances belong to, has not been performed yet. Thus, in this work we studied, by two different in-vitro methods, the antioxidant activity of five HCAs: p-coumaric acid, ferulic acid, sinapinic acid, caffeic acid and chlorogenic acid. The methods applied in this study were based on the oxidation of human serum albumin (HSA) by XOCI, a new microplate method based on the measurement of chloramines and a previously described carbonyl assay. Firstly, lysine-derived chloramines, in the presence or absence of the HCAs, were detected using 5-thio-2-nitrobenzoic acid (TNB), measuring the absorbance at 415 nm by a microplate reader. To remove excess XOCI, Trolox, a known XOCI scavenger, was added before TNB. Secondly, lysine-derived carbonyls, in the presence or absence of the HCAs, were detected by using 2,4-dinitro-phenylhydrazine. Hydroxycinnamic acids appeared active (caffeic≥sinapinic>chlorogenic≅ferulic> p-coumaric acid) by both methods, suggesting possible pharmacological applications for these compounds, which are present at high concentrations in the plant kingdom.