Differentiation between stoichiometric and anticatalytic antioxidant properties of benzoic acid analogues: A structure/redox potential relationship study Thierry Franck a,b,⇑ , Ange Mouithys-Mickalad a , Thierry Robert c , Gianangelo Ghitti c , Ginette Deby-Dupont a , Philippe Neven d , Didier Serteyn a,b a Center for Oxygen Research and Development, Institute of Chemistry B6a, University of Liège, 4000 Liège, Belgium b Department of Clinical Sciences, Equine Clinic, Veterinary Institute B41, University of Liège, 4000 Liège, Belgium c Analytical Chemistry and Electrochemistry B6a, University of Liège, 4000 Liège, Belgium d Department of Medicinal Chemistry B36, University of Liège, 4000 Liège, Belgium article info Article history: Received 6 March 2013 Received in revised form 17 June 2013 Accepted 11 September 2013 Available online 21 September 2013 Keywords: Benzoic acids Myeloperoxidase Voltammetry Antioxidant Stoichiometric Anticatalytic abstract We investigated the antioxidant activities of some phenolic acid derivatives on a cell free system and on cellular and enzymatic models involved in inflammation. The stoichiometric antioxidant activities of phe- nolic acid derivatives were studied by measuring their capacity to scavenge the radical cation 2,2 0 -azino- bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS Å+ ) and reactive oxygen species (ROS) produced by stimulated neutrophils. The anticatalytic antioxidant capacity of the molecules was evaluated on the activity of myeloperoxidase (MPO), an oxidant enzyme present in and released by the primary granules of neutrophils. The ROS produced by PMA-stimulated neutrophils were measured by lucigenin-enhanced chemiluminescence (CL) and the potential interaction of the molecules with MPO was investigated with- out interferences due to medium by Specific Immuno-Extraction Followed by Enzyme Detection (SIEFED). The antioxidant activities of the phenolic compounds were correlated to their redox potentials measured by differential pulse voltammetry (DPV), and discussed in relation to their molecular structure. The ability of the phenolic molecules to scavenge ABTS radicals and ROS derived from neutrophils was inversely correlated to their increased redox potential. The number of hydroxyl groups (three) and their position (catechol) were essential for their efficacy as stoichiometric antioxidants or scavengers. On MPO activity, the inhibitory capacity of the molecules was not really correlated with their redox potential. Likewise, for the inhibition of MPO activity the number of OH groups and mainly the elongation of the carboxylic group were essential, probably by facilitating the interaction with the active site or the struc- ture of the enzyme. The redox potential measurement, combined with ABTS and CL techniques, seems to be a good technique to select stoichiometric antioxidants but not anticatalytic ones, as seen for MPO, what rather involves a direct interaction with the enzyme. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Phenolic acids are aromatic secondary metabolites widely spread through the plant kingdom and involved in plant life pro- tection [22,42]. These compounds are ubiquitous in plant food (i.e. fruits, vegetables, leaves) and therefore a certain quantity of them is consumed in our daily diet [22,35]. The basic skeleton of phenolic acids remains the same, but the number of the hydroxyl groups on the aromatic ring and the modifications of the carboxylic acid function create the variety of the molecules (see the structures of the molecules in Fig. 1). The pharmacological functionality of phenolic acid derivatives has attracted much attention due to their antioxidant, anti-inflammatory, anti-fungal, anti-bacterial and antimutagenic properties that can be exploited in health [28,35,36,46]. One of the vast areas of interest of phenolic acids has been in food quality and preservation. In plants, flavonoids are the predominant phenolic class described which account for approximately the two-third of the dietary phenols. Phenolic acids account for almost all of the remaining third and there is an increasing interest in the antioxidant behaviour and potential health benefits associated with these simple phenolic acids [42] especially on the inflammatory response. Plasma concentration of an individual phenolic acid is dependent of the dietary intake and rarely exceeds 1 lM [34]. However, there is an evidence for microbial origin of some phenolic acids in blood and the total 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.09.009 ⇑ Corresponding author at: Center for Oxygen Research and Development, Institute of Chemistry B6a, University of Liège, 4000 Liège, Belgium. Tel.: +32 43663362; fax: +32 43662866. E-mail address: t.franck@ulg.ac.be (T. Franck). Chemico-Biological Interactions 206 (2013) 194–203 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint