pubs.acs.org/JAFC Published on Web 04/19/2010 © 2010 American Chemical Society J. Agric. Food Chem. 2010, 58, 5355–5362 5355 DOI:10.1021/jf100233y Inhibitory Effect of Gallic Acid and Its Esters on 2,2 0 -Azobis(2-amidinopropane)hydrochloride (AAPH)-Induced Hemolysis and Depletion of Intracellular Glutathione in Erythrocytes VALDECIR F. XIMENES,* ,†,‡ MARIANA G. LOPES, † MAICON SEGALLA PETRO ˆ NIO, § LUIS OCTAVIO REGASINI, § DULCE H. SIQUEIRA SILVA, § AND LUIZ M. DA FONSECA ‡ † Departamento de Quı´mica, Faculdade de Ci^ encias, Unesp - Univ Estadual Paulista, Bauru, S~ ao Paulo (SP), CEP 17033-360, Brazil, ‡ Departamento de Ana´lises Clı´nicas, Faculdade de Ci^ encias Farmac^ euticas, Unesp - Univ Estadual Paulista, Araraquara, S~ ao Paulo (SP), Brazil, and § Departamento de Quı´mica Org^ anica, Instituto de Quı´mica, Unesp - Univ Estadual Paulista, Araraquara, S~ ao Paulo (SP), Brazil The protective effect of gallic acid and its esters, methyl, propyl, and lauryl gallate, against 2,2 0 -azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellu- lar glutathione (GSH) in erythrocytes was studied. The inhibition of hemolysis was dose-dependent, and the esters were significantly more effective than gallic acid. Gallic acid and its esters were compared with regard to their reactivity to free radicals, using the DPPH and AAPH/pyranine free- cell assays, and no significant difference was obtained. Gallic acid and its esters not only failed to inhibit the depletion of intracellular GSH in erythrocytes induced by AAPH but exacerbated it. Similarly, the oxidation of GSH by AAPH or horseradish peroxidase/H 2 O 2 in cell-free systems was exacerbated by gallic acid or gallates. This property could be involved in the recent findings on pro- apoptotic and pro-oxidant activities of gallates in tumor cells. We provide evidence that lipophilicity and not only radical scavenger potency is an important factor regarding the efficiency of anti- hemolytic substances. KEYWORDS: Gallic acid; gallates; erythrocytes; hemolysis; glutathione; peroxyl radical; antioxidant; pro-oxidant activity INTRODUCTION Gallic acid and its derivatives are among the most abundant phenolic antioxidants in wines and green tea ( 1 -3 ). This phyto- chemical is well-known for its antioxidant, antibacterial, anti- inflammatory, antimutagenic, and chemopreventive properties ( 4 -7 ). The similar beneficial effects of the synthetic esters of gallic acid have been extensively studied, and in many cases, they are stronger than those of gallic acid itself. For instance, lauryl gallate (dodecyl gallate), a widely used food additive, is more efficient than gallic acid as an inhibitor of the enzyme xanthine oxidase, an endogenous source of the superoxide anion, and of mitochondrial lipid peroxidation induced by Fe III -NADPH ( 8 ). Methyl gallate is more effective than gallic acid at inhibiting oral bacterial growth and the formation of Streptococcus mutans biofilms ( 4 ). Methyl gallate inhibits the production of leukotriene C4 and prostaglandin D2 by bone-marrow-derived mast cells ( 9 ). Nonyl gallate is more effective than gallic acid against the growth of Salmonella choleraesuis ( 10 ). Lauryl gallate protects against the formation of dimethylbenzanthracene-induced skin tumors in mice and kills, selectively, tumor cells in established tumors ( 11 ). Such biological activities have been correlated with the amphipathic feature of these ester derivatives ( 12 ), because the in vitro antioxi- dant potential is nearly the same as that of gallic acid. Hence, it may be supposed that the accessibility of the gallates to the intracellular medium could be a determining factor for their effects. Another relevant property of gallates is their pro-oxidant characteristics. Indeed, the cytotoxic effect of octyl, dodecyl, and tetradecyl gallates on melanoma cells has recently been demonstrated ( 13 , 14 ). This biological effect has been associated with DNA fragmentation, activation of NF-κB, inhibition of cell adhesion, and alteration of the cell redox status by depletion of both glutathione (GSH) and ATP ( 13 ). Similarly, the inhibition of HeLa cell growth by propyl gallate was associated with depletion of intracellular GSH and increased production of the superoxide anion ( 14 ). The hemolysis of erythrocytes has been extensively used as an ex vivo model for studying reactive oxygen species (ROS)-induced disruption of cell membranes and the protective effect of antioxi- dants. One of the most frequently studied models uses the water- soluble azo compound 2,2 0 -azobis(2-amidinopropane)hydrochloride (AAPH), which decomposes at physiological temperature (37 °C) *To whom correspondence should be addressed: Departamento de Quı´mica, Faculdade de Ci^ encias, UNESP, Bauru, SP, CEP 17033-360, Brazil. Telephone: þ55-14-31036088. Fax: þ55-12-31036099. E-mail: vfximenes@fc.unesp.br.