Protection of erythrocytes against oxidative haemolysis in the presence of M1.

<p>Haemolysis of a 1% human erythrocytes suspension in the presence of the metabolite M1 (1 µM) was determined in an AAPH-assay. Erythrocytes were either co-incubated with M1 (left column) or pre-incubated with M1 for 60 min (right column), and delay of haemolysis was determined with reference to an incubation mixture without addition of M1. Columns represent the mean and standard deviation of three replicates.</p

Influence of the stop solution on the uptake of M1 into human erythrocytes.

<p>In an initial experiment the distribution of different concentrations of the metabolite M1 was analyzed in the absence and presence of glucose (100 mM) with and without addition of a stop solution containing phloretin (200 µM) and cytochalasin B (20 µM). Data points of the experiments with stop solution (solid lines) represent the mean and mean deviation of the mean of three replicates, the data points without stop solution (dashed lines) were single experiments.</p

Erythrocyte/plasma partitioning ratios of polyphenols.

<p>1.3 µM caffeic acid, 6 µM taxifolin, 80 µM ferulic acid and 6 µM of the Pycnogenol metabolite M1 were concomitantly incubated with a human blood mixture (hematocrit 0.43) at 37°C. Each data point represents the mean and standard deviation of five replicates.</p

Distribution of M1 into human erythocytes.

<p>Increasing concentrations of the metabolite M1 were incubated in the absence and presence of glucose (100 mM) with human erythrocytes (hematocrit 0.043) at 4°C. The reaction was stopped after one minute with phloretin (200 µM) and cytochalasin B (20 µM). For 0.3 to 1 µM M1 the uptake into erythrocytes was statistically significant higher in absence of glucose compared to the respective uptake (0.3 to 1 µM M1) in the presence of glucose (p<0.05) and also compared to the uptake of 10 µM M1 (p<0.001; one-way ANOVA with Bonferroni post-hoc test). Each data point represents the mean and mean deviation of the mean of six replicates.</p

MS/MS spectra of the M1-glutathione adduct.

<p><b>A</b>: MS/MS spectrum of the putative M1-glutathione adduct with [M+H]⁺<i>m/z</i> of 514 found in the erythrocyte lysate after incubation with the metabolite M1. <b>B</b>: MS/MS spectrum of the M1-glutathion adduct with [M+H]⁺<i>m/z</i> of 514 obtained after incubation of the metabolite M1 with glutathione and glutathione-S-transferase. Characteristic fragments for glutathione are pyrroglutamic acid [MH⁺-129], cysteine [MH⁺-103] and glycine [MH⁺-76] are present.</p