pubs.acs.org/JAFC Published on Web 12/02/2009 © 2009 American Chemical Society J. Agric. Food Chem. 2010, 58, 135–140 135 DOI:10.1021/jf9031052 Powerful Protective Role of 3,4-Dihydroxyphenylethanol-Elenolic Acid Dialdehyde against Erythrocyte Oxidative-Induced Hemolysis FA ´ TIMA PAIVA-MARTINS,* ,†,‡ JOA ˜ O FERNANDES, ‡ VERA SANTOS, ‡ LISETE SILVA, †,‡ FERNANDA BORGES, †,‡ SUSANA ROCHA, #,§ LUIS BELO, #,§ AND ALICE SANTOS-SILVA #,§ † CIQ, ‡ Departamento de Quı´mica, Faculdade de Ci^ encias, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal, # Servic -o de Bioquı´mica, Faculdade de Farma´cia, Universidade do Porto (FFUP), Rua Anı´bal Cunha 164, 4050-047 Porto, Portugal, and § Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal The present work studied and compared the capacity of four important olive oil polyphenolic compounds, oleuropein, hydroxytyrosol, and the oleuropein aglycones 3,4-dihydroxyphenyletha- nol-elenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4- DHPEA-EDA), to protect red blood cells (RBCs) from oxidative hemolysis induced by the physiological initiator H 2 O 2 . The amount of hemolysis was evaluated spectrophotometrically. The compounds were also tested in the presence and absence of the naturally occurring antioxidant ascorbic acid. All compounds were revealed to significantly protect RBCs from oxidative hemolysis induced by H 2 O 2 at 40 and 80 μM, with the order of activity being 3,4-DHPEA-EDA > 3,4-DHPEA- EA > hydroxytyrosol = oleuropein. At 20, 10, and 5 μM, only 3,4-DHPEA-EDA showed a significant protection against the oxidative injury. In the presence of ascorbic acid at physiological concentra- tion, the addition of individual compounds at 40 μM increased the stability of erythrocytes. The addition of phenolic compounds at 20 and 10 μM did not produce further protection when compared with the protection given by ascorbic acid alone, except for 3,4-DHPEA-EDA. This compound was shown to produce further protection even at 5 μM. In summary, 3,4-DHPEA-EDA plays an important protective role against reactive oxygen species-induced oxidative injury in RBCs, and this effect is more potent than the one evidenced by hydroxytyrosol or oleuropein. KEYWORDS: Olea europaea; polyphenols; erythrocytes; olive oil; hydroxytyrosol; oleuropein; 3,4- DHPEA-EA; 3,4-DHPEA-EDA; ascorbic acid INTRODUCTION Antioxidants have received particular attention because of their potential to modulate oxidative stress associated with chronic disease. The lower incidence of coronary heart disease and some cancers in the Mediterranean area led to the hypothesis that a diet rich in fruits, vegetables, and grains has a beneficial effect on health. The major fat component of the so-called “Mediterranean diet” is virgin olive oil (VOO) ( 1 ). Several studies have suggested that phenolic compounds, although considered to be among the minor constituents of VOO, may contribute to the healthy nature of this diet ( 2 -5 ). In recent years, there has been much interest in antioxidants that retard oxidative modification of low-density lipoproteins (LDL), which is believed to be a key step in the development of atherosclerosis. The stability of LDL isolated from animals and humans fed VOO is increased, and this increased stability is attributable to the minor phenolic compounds in the oil ( 3 , 5 -7 ). The administration of high doses of hydroxytyrosol (10 mg/kg/ day) to apo E deficient mice, however, enhanced atherosclerotic lesion development ( 8 ). This fact points out the importance of the matrix, the combination of all antioxidants, and the knowledge of the activity of each polyphenolic compound present in natural foods such as virgin olive oil. Human red blood cells (RBC) are particularly useful in the evaluation of the antioxidant properties of several compounds, namely, olive oil polyphenols. RBCs are particularly susceptible to endogenous oxidative damage because of their specific role as oxygen carriers. In the normal metabolism of RBCs, around 0.3% of the oxygen molecule is shifted from its normal role with the production of superoxide anion. Moreover, during the course of inflammatory processes, superoxide radicals may be generated in large amounts. This occurrence may be due to the activation of mast cells, macrophages, eosinophils, and neutrophils ( 9 ). The superoxide radical is then rapidly converted into H 2 O 2 by super- oxide dismutase. H 2 O 2 is not an inherently reactive compound, but it can easily penetrate the membranes of surrounding cells and be converted into highly reactive and deleterious products ( 10 ). *Address correspondence to this author at the Departamento de Quı´mica, Faculdade de Ci^ encias, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal (telephone 351-22- 6082956/856; fax 351-22-6082959; e-mail mpmartin@fc.up.pt).