Lovro Ziberna

Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells

Berry anthocyanins have pronounced health effects, even though they have a low bioavailability. The common mechanism underlying health protection is believed to relate to antioxidant activity. Berry extracts, chemically characterised for their phenolic content, were prepared from bilberries (Vaccinium myrtillusL.) and blueberries (Vaccinium corymbosumL.); the bilberry extract was further purified to obtain the anthocyanin fraction. The antioxidant activity of each extract was examined at the cellular level. For this purpose a specific assay, known as cellular antioxidant activity assay (CAA), was implemented in different cell lines: human colon cancer (Caco-2), human hepatocarcinoma (HepG2), human endothelial (EA.hy926) and rat vascular smooth muscle (A7r5). Here we show for the first time that anthocyanins had intracellular antioxidant activity if applied at very low concentrations (<1 μg/l; nM range), thereby providing a long-sought rationale for their health protecting effects in spite of their unfavorable pharmacokinetic properties.

Transport and bioactivity of cyanidin 3-glucoside into the vascular endothelium.

Flavonoids are dietary components involved in decreasing oxidative stress in the vascular endothelium and thus the risk of endothelial dysfunction. However, their very low concentrations in plasma place this role in doubt. Thus, a relationship between the effective intracellular concentration of flavonoids and their bioactivity needs to be assessed. This study examined the uptake of physiological concentrations of cyanidin 3-glucoside, a widespread dietary flavonoid, into human vascular endothelial cells. Furthermore, the involvement of the membrane transporter bilitranslocase (TC No. 2.A.65.1.1) as the key underlying molecular mechanism for membrane transport was investigated by using purified anti-sequence antibodies binding at the extracellular domain of the protein. The experimental observations were carried out in isolated plasma membrane vesicles and intact endothelial cells from human endothelial cells (EA.hy926) and on an ischemia-reperfusion model in isolated rat hearts. Cyanidin 3-glucoside was transported via bilitranslocase into endothelial cells, where it acted as a powerful intracellular antioxidant and a cardioprotective agent in the reperfusion phase after ischemia. These findings suggest that dietary flavonoids, despite their limited oral bioavailability and very low postabsorption plasma concentrations, may provide protection against oxidative stress-based cardiovascular diseases. Bilitranslocase, by mediating the cellular uptake of some flavonoids, is thus a key factor in their protective activity on endothelial function.

The endothelial plasma membrane transporter bilitranslocase mediates rat aortic vasodilation induced by anthocyanins

BACKGROUND AND AIMS Anthocyanins, a sub-class of flavonoids, induce endothelium-dependent vasorelaxation, by activating endothelial nitric oxide synthase and consequently increasing production of the vasorelaxant agent nitric oxide. It is not yet clear if anthocyanin-induced vasorelaxation starts with their interaction with plasma membrane receptors in the extracellular compartment, or with their membrane transport toward intracellular molecular targets. We therefore investigated the possible role of bilitranslocase (TC 2.A.65.1.1), an endothelial plasma membrane carrier that transports flavonoids, in the vasodilation activity induced by anthocyanins. METHODS AND RESULTS Vascular reactivity was assessed in thoracic aortic rings obtained from male Wistar rats. Pre-treatment of aortic rings with anti-sequence bilitranslocase antibodies targeting the carrier, decreased vasodilation induced by cyanidin 3-glucoside and bilberry anthocyanins. CONCLUSION Here we show for the first time that bilitranslocase mediates a critical step in vasodilation induced by anthocyanins. This offers new insights into the molecular mechanism involved in endothelium-dependent vasorelaxation by flavonoids, and the importance of their specific membrane carriers.

Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells

Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with cardiovascular disease risk. Given the role of endothelial dysfunction in originating cardiovascular diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships. Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To confirm the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9–1.5 pmol mg−1 protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular bilirubin (3–5 pmol mg−1 protein) could be modulated by either extracellular bilirubin uptake, or by up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis. Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in the range of reported values of free serum bilirubin (8.5–13.1 nM). Biliverdin showed similar antioxidant properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates, and may thus be a dynamic factor of the endothelial function.

Role of endothelial cell membrane transport in red wine polyphenols-induced coronary vasorelaxation: involvement of bilitranslocase.

Red wine polyphenols (RWP) induce nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-mediated coronary vasodilatation involving the redox-sensitive PI3-kinase/Akt-dependent pathway in the endothelium. However, there is a gap of knowledge in explaining how bioactive polyphenols initialize their signalling pathway in endothelial cells. Here, we investigated the hypothesis that flavonoids act subsequently to their entry into the endothelium via the flavonoid membrane transporter bilitranslocase (TC 2.A.65.1.1). Thus, vascular reactivity studies were performed using isolated porcine coronary artery rings. We separately determined the NO- and EDH-mediated components of the relaxation in the presence of specific inhibitors. In either case, bilitranslocase antibodies significantly reduced the relaxations of coronary artery rings induced by RWP. Furthermore, bilitranslocase antibodies significantly reduced RWP-induced phosphorylation levels of Akt and eNOS, assessed in cultured endothelial cells from porcine coronary arteries by Western blot analysis. The present findings indicate that bilitranslocase-mediated membrane transport substantially contributes to the initial step of RWP-induced coronary vasodilatation.

Determination of cyanidin 3-glucoside in rat brain, liver and kidneys by UPLC/MS-MS and its application to a short-term pharmacokinetic study.

Anthocyanins exert neuroprotection in various in vitro and in vivo experimental models. However, no details regarding their brain-related pharmacokinetics are so far available to support claims about their direct neuronal bioactivity as well as to design proper formulations of anthocyanin-based products. To gather this missing piece of knowledge, we intravenously administered a bolus of 668 nmol cyanidin 3-glucoside (C3G) in anaesthetized Wistar rats and shortly after (15 s to 20 min) we collected blood, brain, liver, kidneys and urine samples. Extracts thereof were analysed for C3G and its expected metabolites using UPLC/MS-MS. The data enabled to calculate a set of pharmacokinetics parameters. The main finding was the distinctive, rapid distribution of C3G in the brain, with an apparently constant plasma/brain ratio in the physiologically relevant plasma concentration range (19–355 nM). This is the first report that accurately determines the distribution pattern of C3G in the brain, paving the way to the rational design of future tests of neuroprotection by C3G in animal models and humans.

Bioavailability of Flavonoids: The Role of Cell Membrane Transporters

Abstract Dietary flavonoids play an important role in the prevention of diseases related to oxidative stress in living systems. Although much attention has been focused on studying the protective functions of flavonoids, so far only limited biochemical research was done to tackle the fundamental issue of how flavonoids enter cells. Flavonoids have limited bioavailability, and consequently low plasma concentrations. Thus, in order to enter the intracellular compartment of cells in target tissues, flavonoids must exploit specific cell membrane transporter systems. This interaction between flavonoids and the proteins that mediate their transmembrane transport is of critical importance since it affects both the pharmacokinetic and pharmacodynamic properties of the flavonoids. Extensive knowledge of cell membrane transporters is therefore essential to fully understand the mechanisms of flavonoid-mediated protective activity in the cardiovascular, renal, hepatic, and central nervous systems, and also to elucidate flavonoid-drug interactions.Dietary flavonoids play an important role in the prevention of diseases related to oxidative stress in living systems. Although much attention has been focused on studying the protective functions of flavonoids, so far only limited biochemical research has been done to tackle the fundamental issues of how flavonoids enter cells. Flavonoids have limited bioavailability, and consequently low plasma concentrations. Thus, in order to enter the intracellular compartment of cells in target tissues, flavonoids must exploit specific cell membrane transporter systems. This interaction between flavonoids and the proteins that mediate their transmembrane transport is of critical importance since it affects both the pharmacokinetic and pharmacodynamic properties of flavonoids. Extensive knowledge on cell membrane transporters is, therefore, essential to fully understand the mechanisms of flavonoid-mediated protective activity in the cardiovascular, renal, hepatic and central nervous systems, and also to elucidate flavonoid-drug interactions.

Normothermic and hypothermic models for studying the deleterious effects of hypoxia-reoxygenation on EDHF-mediated relaxation in isolated porcine coronary arteries

INTRODUCTION The vasomotor response of the coronary artery is altered by hypoxia-reoxygenation (H-R) induced damage. The aim of our study was to compare and evaluate normothermic and hypothermic models which are suitable for future drug studies of vasoprotective action against H-R injury. METHODS Porcine coronary arterial rings were isolated and placed in Krebs-Henseleit (K-H) solution. Rings were exposed to normoxic conditions (control group) and two different H-R conditions: the first induced by a 95% N(2)-5% CO(2) gas mixture (40- and 60-min hypoxia) in a normothermic protocol, and the second induced by hypothermic (4 degrees C) hypoxia-reoxygenation in an air-tight beaker filled with K-H solution (24- and 48-hours hypoxia). Reoxygenation was applied by introducing K-H solution aerated with a 95% O(2)-5% CO(2) mixture under normothermic (37 degrees C) conditions. To test the EDHF-mediated relaxation by substance P, rings were first incubated in L-NNA, nitric oxide synthase inhibitor, and indomethacin, cyclooxygenase inhibitor, and then pre-contracted with thromboxane analogue U-46619. Analysis of the maximum relaxation of the arterial rings was performed by one-way ANOVA, followed by Bonferronis post-test. RESULTS Distal segments of the coronary artery responded faster to contraction induced by U-46619 and were relaxed by substance P to a greater extent than proximal segments. Maximal relaxations of arterial rings induced by a 10 nM solution of substance P were significantly reduced (p<0.001) from the values for normoxic rings (81.0+/-1.0%, n=30) after 40-min H-R (50.5+/-5.3%, n=30), 60-min H-R (32.1+/-3.5%, n=30), 24-hours hypothermic H-R (56.0+/-2.3%, n=30) and after 48-hours hypothermic H-R (38.5+/-5.1%, n=30). CONCLUSIONS The model employing 40-min normothermic H-R is as effective as 24-hours hypothermic H-R, and 60-min normothermic H-R as 48-hours hypothermic H-R for studying the deleterious effects of H-R on EDHF-mediated relaxation.

Phenolic composition of orange peels and modulation of redox status and matrix metalloproteinase activities in primary (Caco-2) and metastatic (LoVo and LoVo/ADR) colon cancer cells

Orange (Citrus sinensis) peels, consumed in the diet as wines, candies or as infusions, are rich sources of phenolic compounds. This study sought to investigate the protective ability of phenolic extracts from orange peels against oxidative stress in primary human colonic tumor (Caco-2) and metastatic cell lines (LoVo and LoVo/ADR). The effects of the extracts on glutathione reductase, glutathione peroxidase and total matrix metalloproteinase (MMP) activities in the cells were also investigated. The results revealed that the extracts displayed cellular antioxidant activities and increased the activities of glutathione reductase and glutathione peroxidase in the cells. Furthermore, the extracts inhibited total MMP activities in the cells in a dose-dependent manner. HPLC fingerprinting revealed the presence of flavonoids and hydroxycinnamic acid. This study showed that orange peels phenolic extracts exhibited cellular antioxidant activity and inhibited MMP activity in colon cancer cells.

Inhibition of metalloproteinase and proteasome activities in colon cancer cells by citrus peel extracts

Abstract Background: Citrus peels are consumed in the form of infusions, candy or wine, based on their well-documented nutritional and medicinal properties. This study sought to investigate the effect of some citrus peels’ [grapefruit (Citrus paradisii), orange (Citrus sinensis) and shaddock (Citrus maxima)] extracts on matrix metalloproteinase (MMP) and proteasome activities in primary human colonic tumor (Caco-2) and the metastatic cell lines (LoVo and LoVo/ADR) in a bid to explain the possible mechanism by which the peels could manage/prevent colon cancer. Methods: The inhibition of MMP and proteasome activities in the cells by the peel extracts, as well as the identification of phenolic compounds using high-performance liquid chromatography with diode-array detection (HPLC-DAD), was determined. Results: Orange peel extracts had the strongest inhibition of MMP in Caco-2 and LoVo cells, while shaddock had the least. Shaddock peel extracts also had the least MMP inhibition in LoVo/ADR lysates. Grapefruit had the least proteasome inhibition in Caco-2 and LoVo lysates, while there was no significant (p>0.05) difference in the proteasome inhibition of the peel extracts in LoVo/ADR lysates. The extracts inhibited proteasome activity in extract-treated cells, and HPLC fingerprinting of the extracts revealed the presence of some phenolic compounds such as quercetin, caffeic acid, kaempferol, catechin and naringin. Conclusions: The inhibition of MMP and proteasome activities in colon cancer cell lines suggests the potential use of citrus peels as functional food in the management and/or prevention of colon cancer.