Biologically and chemically important hydrazino-containing imidazolines as antioxidant agents

<p>Biologically and chemically useful hydrazinoimidazolines were evaluated as antioxidant and antihaemolytic agents. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH^•), galvinoxyl radical (GOR), nitric oxide (NO) and hydrogen peroxide (H₂O₂) scavenging assays, ferric ions reducing power assay, and <i>ex vivo</i> model of rat erythrocytes exposed to 2,2′-azobis(2-methylpropionamidine)dihydrochloride (AAPH) or H₂O₂ were used. The most potent DPPH^• scavengers proved to be hydrazinoimidazolines <b>3</b>, <b>2</b>, and <b>4</b>, revealing excellent antiradical effects – superior or comparable to that of all antioxidant standards used. Moreover, these molecules showed strong NO neutralising potencies – better to that of ascorbic acid (AA) (<b>3</b>), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (<b>3</b> and <b>2)</b>, butylated hydroxytoluene (BHT) (<b>3</b> and <b>2</b>), and butylated hydroxyanisole (BHA) (<b>3</b>, <b>2</b>, and <b>4</b>). Compound <b>4</b> was also effective in GOR scavenging. The excellent scavenger of GOR, NO, and H₂O₂ proved to be structure <b>5</b>, with the potency superior or comparable to the majority of antioxidant standards used. In turn, compound <b>9</b> was effective in H₂O₂ and GOR neutralisation. All hydrazinoimidazolines revealed the reducing power that is higher than BHT. Moreover, the protective effects of most test compounds on oxidatively stressed erythrocytes were observed. Some structure–activity relationships were disclosed. A significance of the primary hydrazino group on antioxidant effects was confirmed. The most likely DPPH^• and GOR scavenging mechanisms for test compounds were propound. Among all the investigated molecules, hydrazinoimidazolines <b>5</b>, <b>3</b>, <b>2</b>, <b>4</b>, and <b>9</b>, due to their excellent or good antiradical activities, can represent promising antioxidant candidates with prospective utility for prevention of diseases related to reactive oxygen/nitrogen species.</p

Baseline clinical, anthropometric, and biochemical characteristics of the study populations.

<p>Group I–prediabetic, obese NAFLD with MS, group II—lean NAFLD without MS, group III—obese without MS, group IV—healthy individuals.</p><p>Data are expresses as means ± SD (normally distributed continuous variables).</p><p>*statistically significant at <i>p</i><0.005 in comparison to group IV (control)</p><p>- concentrations of circulating IL-6 levels in healthy subjects were below the detection threshold.</p><p>Baseline clinical, anthropometric, and biochemical characteristics of the study populations.</p

Expression of TLR4 (mean MFI) on blood monocytes of patients with NAFLD and control subjects treated ex vivo with LPS (100 ng/ml).

<p>Analysis of variance by Kruskall-Wallis method with Dunn’s test for statistical significance.</p><p>The influence of metformin (20 μM or 100 μM). A comparison of statistical significance between groups.</p

A comparison of statistical significance in the middle of groups.

<p>n.s not significant</p><p>Heparinized blood was diluted 1:5 with cell culture medium and incubated for 24 h at 37°C, 5% CO₂ with LPS (100 ng/ml) and two concentrations of AKG. Next cells were centrifuged (300xg, 5 min) and cytokine level was measured as described in Material and Methods.</p><p>A comparison of statistical significance in the middle of groups.</p

Production of proinflammatory cytokines ex vivo by blood leukocytes of patients with NAFLD, induced by LPS, and comparison to healthy control.

<p>Analysis of variance by Kruskall-Wallis method with Dunn’s test for statistical significance.</p><p>The influence of metformin (20 μM lub 100 μM). A comparison of statistical significance between groups.</p

Expression of TLR4 (mean MFI) on blood monocytes of patients with NAFLD and control subjects treated ex vivo with LPS (100 ng/ml).

<p>Analysis of variance by Kruskall-Wallis method with Dunn’s test for statistical significance.</p><p>The influence of PC (20 μg/ml or 100 μg/ml). A comparison of statistical significance between groups.</p

A comparison of statistical significance in the middle of the groups.

<p>n.s not significant</p><p>Expression of TLR4 was measured as mean MFI by flow cytometry on monocytes after incubation with 100 ng/ml of LPS and two doses of AKG.</p><p>A comparison of statistical significance in the middle of the groups.</p

Production of proinflammatory cytokines ex vivo by blood leukocytes of patients with NAFLD, induced by LPS, and comparison to healthy control.

<p>Analysis of variance by Kruskall-Wallis method with Dunn’s test for statistical significance.</p><p>The influence of alpha ketoglutarate (AKG 25 mM or 10mM). A comparison of statistical significance between groups.</p