Acute Cardioprotective and Cardiotoxic Effects of Bilberry Anthocyanins in Ischemia–Reperfusion Injury: Beyond Concentration-Dependent Antioxidant Activity Lovro Ziberna • Mojca Lunder • Spela Moze • Andreja Vanzo • Federica Tramer • Sabina Passamonti • Gorazd Drevensek Published online: 27 October 2010 Ó Springer Science+Business Media, LLC 2010 Abstract Despite being reported to reduce the risk of cardiovascular diseases, little is known about acute direct effects of bilberry anthocyanins on whole mammalian heart under ischemia–reperfusion (I–R) conditions. Bilberry anthocyanins were prepared from the ripe bilberries and analyzed using HPLC–DAD. Their antioxidant activity was evaluated by measuring the intrinsic free radical–scavenging capacity and by cellular antioxidant assay (CAA) on endo- thelial cells, where we quantified the intracellular capacity to inhibit the formation of peroxyl radicals. Experiments on the isolated rat hearts under I–R were carried out according to the Langendorff method. Perfusion with low concentrations of bilberry anthocyanins (0.01–1 mg/L) significantly attenu- ated the extent of I–R injury as evidenced by decreasing the release rate of LDH, increasing the postischemic coronary flow, and by decreasing the incidence and duration of reperfusion arrhythmias. High concentrations (5–50 mg/L) diminished cardioprotection and show cardiotoxic activity despite having their radical scavenging and intracellular antioxidant capabilities increased in a concentration- dependent manner. This study reveals the biphasic concen- tration-dependent bioactivity of bilberry anthocyanins under I–R, which results in strong cardioprotective activity in low concentrations and cardiotoxic activity in high concentrations. Keywords Bilberry anthocyanins  Cellular antioxidant activity  Endothelium  Ischemia–reperfusion injury  Isolated rat heart Introduction Flavonoid-rich diets, such as among the Finnish [1], are associated with a decreased risk of cardiovascular dis- eases. Bilberries (Vaccinium myrtillus L.) are one of the richest dietary sources of anthocyanins, which are also considered the most pharmacologically active constituents [2, 3]. In cardiovascular system, bilberry anthocyanins exhibit a wide range of biologic effects, including antioxidant [4, 5], anti-inflammatory [5], and vasodilatory actions [6], among others. Interpretation of these results is, however, complicated since different extraction procedures, oxida- tion models, and corresponding experimental conditions were applied. Antioxidant activity of anthocyanins can be described directly, in terms of their per se activity as free radical scavengers, or indirectly as modulators of intracellular pro- and anti-oxidant enzymes [7]. However, in recent years, flavonoids were reported to have both antioxidant and pro- oxidant activity depending upon certain experimental conditions [8]. Often, these pro-oxidant effects involve interactions of polyphenols with transition metal ions, L. Ziberna (&)  M. Lunder  G. Drevensek Institute of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia e-mail: lovro.ziberna@mf.uni-lj.si S. Moze Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia A. Vanzo Central Laboratories, Agricultural Institute of Slovenia, Hacquetova 17, 1000 Ljubljana, Slovenia F. Tramer  S. Passamonti Department of Life Sciences, University of Trieste, via L. Giorgieri 1, 34127 Trieste, Italy Cardiovasc Toxicol (2010) 10:283–294 DOI 10.1007/s12012-010-9091-x