Original Contribution TEAC ANTIOXIDANT ACTIVITY OF 4-HYDROXYBENZOATES BO˙ ZENA TYRAKOWSKA,* ANS E. M. F. SOFFERS, † HENRYK SZYMUSIAK,* SJEF BOEREN, † MARELLE G. BOERSMA, † KATARZYNA LEMA ´ NSKA,* JACQUES VERVOORT, † and IVONNE M. C. M. RIETJENS † *Faculty of Commodity Science, Poznan ´ University of Economics, Poznan ´, Poland and † Laboratory of Biochemistry and Division of Toxicology, Wageningen University, Wageningen, The Netherlands (Received 15 March 1999; Revised 9 August 1999; Accepted 30 August 1999) Abstract—The influence of pH, intrinsic electron donating capacity, and intrinsic hydrogen atom donating capacity on the antioxidant potential of series of hydroxy and fluorine substituted 4-hydroxybenzoates was investigated experimen- tally and also on the basis of computer calculations. The pH-dependent behavior of the compounds in the TEAC assay revealed different antioxidant behavior of the nondissociated monoanionic form and the deprotonated dianionic form of the 4-hydroxybenzoates. Upon deprotonation the radical scavenging ability of the 4-hydroxybenzoates increases significantly. For mechanistic comparison a series of fluorobenzoates was synthesized and included in the studies. The fluorine substituents were shown to affect the proton and electron donating abilities of 4-hydroxybenzoate in the same way as hydroxyl substituents. In contrast, the fluorine substituents influenced the TEAC value and the hydrogen atom donating capacity of 4-hydroxybenzoate in a way different from the hydroxyl moieties. Comparison of these experi- mental data to computer-calculated characteristics indicates that the antioxidant behavior of the monoanionic forms of the 4-hydroxybenzoates is not determined by the tendency of the molecule to donate an electron, but by its ability to donate a hydrogen atom. Altogether, the results explain qualitatively and quantitatively how the number and position of OH moieties affect the antioxidant behavior of 4-hydroxybenzoates. © 1999 Elsevier Science Inc. Keywords— 4-Hydroxybenzoates, Antioxidant activity, TEAC value, pH dependency, Mechanism, Computer calcu- lations, Quantitative structure activity relationship, Free radicals INTRODUCTION Phenolic antioxidants such as hydroxybenzoates and flavonoids are important classes of natural antioxi- dants [1–5]. The antioxidant efficiency of the phenolic acids and flavonoids have been related to the number of hydroxyl groups in the molecule and also to their hydrogen radical donating abilities [1,2,6,7]. How- ever, the detailed mechanism underlying the effect of additional hydroxyl moieties on the antioxidant poten- tial has not been studied in any detail. Furthermore, the possible influence of OH group deprotonation on the antioxidant potential of the phenolic antioxidants is generally not taken into account in studies on the antioxidant activity of phenolic antioxidants. Theoretically, upon deprotonation of a hydroxyl moi- ety, the additional negative charge generated in the mol- ecule can be expected to affect its radical scavenging properties. The additional negative charge may decrease the energy required for homolytic O-H bond dissocia- tion, and, thus, facilitate hydrogen atom donation reac- tions [8]. In contrast, the additional negative charge may increase the electron donating capacities of the molecule, reflected by a decreased ionization potential. In the latter case the antioxidant activity of phenolic antioxidants might become comparable to the mechanism for antiox- idant activity of carotenoids, being electron, rather than hydrogen donation [9 –13]. Furthermore, characteristics independent from the electron and hydrogen donating capacity of the molecules may determine their antioxi- dant action. Such characteristics may be the possibilities for donation of more than one electron, metal chelating properties, and/or possibilities for complex formation with the radical to be scavenged [14 –16]. Independent of the actual mechanism for antioxidant activity, a direct correlation between the structure of the Address correspondence to: B. Tyrakowska, Faculty of Commodity Science, Poznan ´ University of Economics, al. Niepodleglos ´ci 10, 60- 967 Poznan ´ Poland; Tel: +48 (61) 856-9040; Fax: +48 (61) 854-3993; E-Mail: btyrak@novci1.ae.poznan.pl. Free Radical Biology & Medicine, Vol. 27, Nos. 11/12, pp. 1427–1436, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter PII S0891-5849(99)00192-6 1427