, 2008; Budzisz et al., 2009, 2010). All substances were reagent grade or better and were used without further purification. Trolox equivalent antioxidant capacity (TEAC)

assay with ABTS and K2 S2O8 The main mechanism of this test is the reduction of the ABTS (2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonate]) Decitabine order radical cation by antioxidants. The ABTS radical cation was obtained as a result of reaction of ABTS stock solution (7 mM in water) with 2.45 mM potassium persulfate. For measurements, the ABTS•+ solution was diluted with ethanol to an absorbance of 0.700 ± 0.020 at 754 nm. Stock solutions of the all compounds were diluted with DMSO. For the photometric assay 1,350 μL of the ABTS•+ solution and 150 μL of antioxidant solution were mixed for 45 s and absorbance was measured immediately after 1 min at 754 nm. The concentration of Cu(II) complexes was varied in the range 2–400 μM. The antioxidant activity of the tested compounds was calculated by determining the decrease in absorbance at different concentrations by using

the following equation (Schlesier et al., 2002): %antioxidant activity = ((E ABTS •+  − E Standard)/E ABTS •+ ) × 100. Blood sample preparation and enzymes activity measurement Examinated group comprise 50 individuals (aged 27–45 years). Blood was taken from AZD6244 supplier cubital vain on heparinized sample (5 mL). Blood was centrifuged 10 min at 3,000 rpm in room temperature. Obtained erythrocytes were three times washed 0.9 % sol NaCl at the same condition of centrifugation. After centrifugation and removal of the supernatant 920 μL of sample and 80 μL of Cu(II) complex solution were mixed. Next it was added to 1 mL glucose and incubated at 37 °C, after which the hemolysate were prepared and then frozen at −70 °C. Thus, prepared hemolysate was used for further experiments. The concentration of compounds

2a–c and 3a–c in experiment was 25 μg/mL of blood. Activity of CAT, GPx, SOD enzymes and TAS value were determined in blood samples (erythrocytes) treated by Cu(II) complexes and in control samples using spectrophotometric methods. All absorbance STK38 measurements were performed with a UV/Vis Spectrometer Lambda 14P (Perkin Elmer, USA). CAT activity in erythrocytes was determined according to spectrophotometric procedure by Beers and Sizer (1952) and expressed in Bergmeyer units (BU/g Hb). CAT activity was measured at 25 °C by recording H2O2 decomposition at 240 nm. One BU of CAT activity is defined as the amount of enzyme decomposing 1 g of H2O2/min. GPx activity in erythrocytes was measured according to Little and O’Brien (1968) methods and expressed in enzymatic units (U/g Hb). The difference in the rate of GPx reaction with glutathione and lumen in the sample is used for its activity determination by absorbance measurement at 412 nm.