Use of Reference Compounds in Antioxidant Activity Assessment NIKOLAOS NENADIS,OLGA LAZARIDOU, AND MARIA Z. TSIMIDOU* Department of Chemistry, Laboratory of Food Chemistry and Technology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece The choice of reference compounds is examined as a “critical control point” of antioxidant activity assessment. Gallic, caffeic, sinapic, uric, and ascorbic acids, isoeugenol, and Trolox were tested using different redox (FRAP, Folin-Ciocalteu) and radical scavenging (DPPH • , ABTS •+ , CBA, ORAC) assays. The ability to chelate transition metals was assessed to support some of the findings. Analytes were also tested in liposomes. On the basis of the findings, we do not recommend uric acid (due to solubility constrains) and ascorbic acid (due to fast degradation kinetics) as references. The behavior of the rest of the compounds could not always be attributed to typical structural characteristics. Selection of suitable reference compounds for in vitro antioxidant activity assays is not an easy task to achieve. The choice of reference compounds has to remain at the convenience of the researchers, with regard to the aim of the study. KEYWORDS: Antioxidants; antioxidant activity assessment; reference compound; radical scavenging assays; redox assays; liposome oxidation; gallic acid; caffeic acid; sinapic acid; uric acid; ascorbic acid; isoeugenol; Trolox INTRODUCTION Evaluation of antioxidant activity of matrixes such as plasma, beverages, vegetables, and fruits as well as of pure compounds (i.e., phenols, peptides) has become a rather disputable issue during the past decade. Methods and data are questioned for providing meaningful information to interested parties. Data are expressed either in absolute values or indirectly with regard to a control (e.g., antioxidant index) or a reference (e.g., Trolox equivalents) (1, 2). On the basis of analytical chemistry principles, apart from the antioxidant activity, a suitable reference should fulfill some other requirements. It should be stable, inexpensive, and structurally affiliated to the tested compounds to ensure reliability of results (3, 4). As a conse- quence, over the years, a series of antioxidants have been used as references depending on the aim of each study (1, 2, 5). Some of them were abandoned (e.g., BHT) with the advance of consumer preferences in natural products. Lately, Trolox (6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water- soluble analogue of vitamin E, gained an unequivocal position as a reference in antioxidant activity assays, mainly because of its effectiveness in both lipophilic and hydrophilic systems (6, 7). However, it is still under question as to whether its chemical structure substantiates this use, in particular, when the results of in vitro assays serve to extrapolate a potential in vivo activity (5). Our work aimed at examining the choice of reference compounds as a “critical control point” of the antioxidant activity assessment process and to add to the ongoing discussion on the issue. Gallic and caffeic acids were chosen as two representative natural phenolics (bearing a pyrogallol and a catechol moiety, respectively) often used as references in antioxidant activity studies (8, 9). Sinapic acid and isoeugenol were also included as prominent antioxidants (10, 11) and possible references in activity studies of methoxy-substituted phenolics. Uric (2,6,8-trioxypurine) and ascorbic (2-oxo-L-threo- hexono-1,4-lactone-2,3-enediol) acids were tested because of their known in vivo antioxidant potency (12). Trolox was included for the reasons previously stated. The assays chosen were redox (FRAP, F-C) as well as radical scavenging activity (DPPH • , ABTS •+ , ORAC, and CBA) ones. The behavior of all the compounds was also tested in liposomes, a model system that mimics cell membranes and shares common characteristics with the above assays. Other experiments were carried out where it was considered necessary. MATERIALS AND METHODS Materials. Ascorbic acid (99.7%) and caffeic acid (98%) were purchased from Riedel de Hae¨n (Seelze, Germany), and isoeugenol (98%) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (97%) were from Aldrich Chemical Co. (Steinheim, Germany). Gallic acid (99.5%) and sinapic acid (98%) were obtained from Sigma Chemical Co. (St. Louis, MO), and uric acid (98%) was from BDH Chemicals, Ltd. (Poole, England). 2,2′-Azobis (2-amino propane) dihydrochloride (AAPH) was purchased from Fluka Chemie (Buchs, * Corresponding author. Telephone: +30-23-10-99-77-96. Fax: +30- 23-10-99-77-79. E-mail: tsimidou@chem.auth.gr. 5452 J. Agric. Food Chem. 2007, 55, 5452-5460 10.1021/jf070473q CCC: $37.00 © 2007 American Chemical Society Published on Web 06/19/2007