Schlüsselwort: Antioxidantie

The role of reactive oxygen species during vasodilatation by tea polyphenols Green and black tea are attributed to numerous health promoting effects on the cardiovascular system. In particular, the protection against arteriosclerosis, hypertension and the positive effect on vascular function is discussed. In addition, antioxidant effects of tea polyphenols have been described. In vitro, however, prooxidative effects of tea polyphenols have also been observed. They can be oxidized themselves and thereby form reactive oxygen species (ROS) like hydrogen peroxide (H2O2) and superoxide (O2.-). Some studies assume autooxidation of tea polyphenols with the formation of dimers/quinones, which has been demonstrated in vitro only. In this context, it is still unclear how the described vasodilatory effects of polyphenols are mediated. It is unknown if receptor-mediated signaling pathways are involved or if autooxidation with the formation of ROS takes place. In endothelial cells, epigallocatechin-3-gallate (EGCG) activates the endothelial nitric oxide synthase (eNOS) by phosphorylation of protein kinases that results in increased nitric oxide (NO) production and vasodilatation. Phosphatidylinositol-3-OH-kinase (PI3-kinase), cAMP-dependent protein kinase (PKA) and protein kinase B (AKT) were identified as protein kinases involved in this process. It is discussed whether the NO-mediated relaxation of the vessels is a redox-sensitive reaction that requires the presence of H2O2 and O2.-. These could trigger the PI3-kinase/AKT-dependent eNOS phosphorylation as an early signal. In the present work, the possible autooxidation with the formation of ROS as a trigger of the PI3/AKT kinase signaling pathway was investigated. Previous results on the autooxidation of EGCG were extended to green and black tea, as well as to theaflavin-3,3´-digallate (TF3) as a component of black tea. The investigations were carried out in several steps in order to verify the results with different methods. All investigations were done with the rat aortic ring model, an ex vivo experimental model, which simulates the in vivo situation in the blood vessels. To investigate the involvement of the ROS H2O2 and O2.- in the vasodilation of tea polyphenols, a single dose of superoxide dismutase (SOD, 500 U/ml), catalase (200 U/ml) or mangan(III)tetrakis(1-methyl-)porphyrine (MnTMPyP, 10 μM) was added to organ baths at the beginning of each experiment. Control rings were treated with water only. To measure vasodilation, every 30 minutes EGCG (0.1 - 10 μM), TF3 (0.02 - 2 μM), green or black tea (5 -50 μl, which correspond to about 5 μM EGCG or 0.06 μM TF3 at 50 μl) were given to organ baths. Tea polyphenol-induced vasodilatation in the aortic ring model was completely prevented by catalase, but was significantly enhanced by SOD in black tee and not significantly enhanced in EGCG, TF3 and green tea. The results suggest that H2O2 is a major cause of vasodilatation. The stability measurements of EGCG and TF3 revealed a possible autooxidation of tea polyphenols. EGCG (1 and 10 μM) and TF3 (0.5 and 2 μM) were added as a single dose to organ bath and sampled after 1, 15 and 30 minutes. Measurements of EGCG and TF3 concentrations through ultra-high-pressure liquid chromatography revealed a strong reduction over time and thus support the hypothesis of autooxidation. This may also be suspected for the tea polyphenols in green and black tea. Catalase and SOD had a stabilizing effect on EGCG and TF3, so that both polyphenols were partly detectable in higher concentrations than without antioxidant. Furthermore, the Amplex Red Hydrogen Peroxide Assay was used to measure the amount of H2O2 induced by EGCG and TF3, as well as green and black tea. EGCG (1 and 10 μM), TF3 (0.5 and 2 μM), as well as green and black tea (50 μl) were added as a single dose to the organ bath and sampled after 1, 15 and 30 minutes. By using green and black tea, as well as EGCG and TF3, H2O2 was measured within a short time, that was diminished by pretreatment with catalase and SOD. However, the involvement of further ROS in the process of vasodilatation cannot be excluded. From the present results, an extracellular decay/autooxidation of tea polyphenols with concurrent formation of H2O2 is concluded. These extracellular generated H2O2 can diffuse into the cells and trigger the PI3/AKT signaling pathway followed by phosphorylation of eNOS and vasodilatation. The present work thus supports the assumption of prooxidative effects of tea polyphenols and contributes to the understanding of the mechanisms of vasodilatation by tea polyphenols. However, due to the contrary results regarding the prooxidative and antioxidant effects of tea polyphenols, further investigations are needed. In addition, the question must be investigated whether other ingredients or metabolites in green and black tea mediate vasodilation.