Cytotoxicity of flavones in monocytes.

<p>Effects of the four hydroxyflavones on the survival of THP-1 monocytes, as determined by the MTT assay. Cells were plated in 6-wells plates using 1×10⁶ cells/well and treated with the flavones (10 µM or 10 nM) for 30 min at 37° C, then cumene hydroperoxide was added at the final concentration of 200 µM for additional 30 min at 37°C. Then MTT solution (0.5 mg/ml final concentration) was added and incubation was carried out at 37°C for 3–4 h. Results represent cell viability and are given as percentage relative to untreated controls. Data are mean ± SD of 3 different experiments. The statistical significance was evaluated by a Student's <i>t</i> test; where not indicated the differences were not significant, except for the difference of Control <i>vs.</i> Cumene hydroperoxide (Cu) that was always significant. #not statistically different from Control; *<i>p</i><0.05 with respect to cumene hydroperoxide alone; **<i>p</i><0.01 with respect to cumene hydroperoxide alone, ***<i>p</i><0.001 with respect to cumene hydroperoxide alone.</p

Antioxidant molecules.

<p>Structures of the flavone ring system, the four flavones tested, and the flavonol quercetin.</p

Antioxidant activity measured by EPR spectroscopy.

<p><b>Upper panel:</b> EPR spectra showing the reaction between galvinoxyl and antioxidants after 5 min incubation. (<b>a</b>) 10 µM galvinoxyl in ethanol; (<b>b</b>) with the addition of 2 µM 5,6-dihydroxyflavone; (<b>c</b>) with the addition of 1.0 µM quercetin. For each spectrum 4 scans were accumulated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060796#pone.0060796-Pietta1" target="_blank">[18]</a>. The reduced product, galvinol, is not a radical, therefore it does not show an EPR spectrum. In theory it should be possible to see also the radical of the oxidized antioxidant, but almost all radicals of flavonoids and most other polyphenolic compounds are too reactive to be detected in this assay; their EPR spectra are not visible when the antioxidants are used in the micromolar range. The capacity of a compound to eliminate the galvinoxyl radical gives a relative measure of its general radical scavenging activity, although the intracellular antioxidant effect may depend on other factors and mechanisms. <b>Lower panel:</b> Kinetics of antioxidant activities of the four flavones measured by the EPR technique. The data show the concentration of galvinoxyl remaining in the samples at different times after addition of flavones, and are given as mean ± SD of 3 experiments. Control 10 µM galvinoxyl in ethanol (▪), and after addition of mosloflavone 10 µM (♦), negletein 1 µM (•), 5,6-dihydroxyflavone 1 µM (▴), baicalein 1 µM (▾), or baicalein 0.1 µM (▵). In samples containing 10 µM of either negletein, 5,6-dihydroxyflavone or baicalein the galvinoxyl signal disappeared completely within 30 s.</p

Long-term effects of flavones on proliferation.

<p>L-6 cells were grown for four days in the presence of 10 µM of either baicalein (▾), 5,6-dihydroxyflavone (▴), mosloflavone (♦) or negletein (•); an equivalent volume of DMSO was added to the control cells (▪).</p

Cytotoxicity of flavones in myoblasts.

<p>Effects of the four hydroxyflavones on the survival of L-6 cells, as determined by the MTT assay. The concentration of cumene hydroperoxide was 27.5 µM, since preliminary experiments showed that higher concentrations were too toxic for these cells, and incubation with MTT was carried out for 24 h. Data are given as optical density (O.D.) rather than viability, because the values measured also include a component due to proliferation of the myoblasts. The bar graph shows data from a representative experiment with measurements carried out in triplicate. Data are mean ± SD of 3 different experiments. #not statistically different from Control; **<i>p</i><0.01 with respect to cumene hydroperoxide alone.</p

Dose-dependence curves for elimination of ROS by 5,6-dihydroxyflavone.

<p>Wide concentration range dose-responses of the antioxidant activity of 5,6-dihydroxyflavone in THP-1 monocytes (<b>Upper panel</b>) and in L-6 myoblasts (<b>Lower panel</b>). The oxidative stress was induced and measured as in Fig. 2. In addition to monocytes (▪) the upper panel also includes results obtained for differentiated macrophages after PMA treatment for 24 h (▴) or 72 h (▾). Data are reported as mean ± SD of 5–10 different experiments. <i>p</i><0.05 at least, as from a Student's <i>t</i> test, starting from 10⁻¹⁰ M for both L-6 cells and THP-1 cells, at any stage of differentiation.</p