Antioxidant activity evaluation by physiologically relevant assays based on haemoglobin peroxidase activity and cytochrome <i>c</i>-induced oxidation of liposomes

<p>Two new protocols for exploring antioxidant-related chemical composition and reactivity are described: one based on a chronometric variation of a haemoglobin ascorbate peroxidase assay and one based on cytochrome <i>c</i>-induced oxidation of lecithin liposomes. Detailed accounts are given on their design, application, critical correlations with established methods and mechanisms. These assays are proposed to be physiologically relevant and bring new information regarding a real sample, both qualitative and quantitative. The well-known assays used for evaluation of antioxidant (re)activity are revisited and compared with these new methods. Extracts of the <i>Hedera helix</i> L. are examined as test case, with focus on seasonal variation and on leaf, fruit and flower with respect to chromatographic, spectroscopic and reactivity properties. According to the set of assays performed, winter are the most antioxidant, followed by summer leaves, and then by flowers and fruits.</p

Phytoconstituent classification based on spectral similarities after chromatographic separation.

<p>Score plots of the first two principal components after applying PCA on the UV-vis mean spectra of the chromatographic peaks obtained by HPLC analysis of <i>G</i>. <i>verum</i> extract prior hydrolysis (<b>A</b>) and after hydrolysis (<b>B</b>). The UV-vis spectra of the two extracts, before separation and the standards are also indicated in both plots. The letter U stands for “unhydrolysed” and indicates compounds specific for this extract; the letter H stands for “hydrolysed” and indicates compounds found only in the hydrolysed extract, while the numbers lacking a letter indicate compounds that are found in both extracts. The number is attributed according to the retention order; the compounds are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200022#pone.0200022.t002" target="_blank">Table 2</a>.</p

Probing the polyphenolic components and alkali-generated radicals reactivity in the studied extract using EPR spectroscopy.

<p><b>A.</b> Time dependence of such generated radicals between 2 min (dead time due to spectrometer calibration prior measuring) and 20 min, at about 1.8 minutes interval. <b>B.</b> The dominant spectral fingerprint of the chlorogenic acid is visible in the extract with minor contributions of rutin and quercetin. Ferulic and coumaric acids gives no EPR spectrum while treated with sodium hydroxide as described in experimental section. The best fit for the EPR spectrum of the extract was obtained by a linear combination of the identified polyphenols in a 1/30/40 ratio (quercetin/rutin/chlorogenic acid) (model).</p

HPLC-UV-vis analysis of the unhydrolysed and hydrolysed <i>G verum</i> extract.

<p>(A) Heatmap of the chromatographic profile versus elution time. (<b>B</b>) Chromatograms of the two extracts and of the standards monitored at 320 nm. (<b>C</b>) UV-vis molecular absorption spectra of six of the employed standards.</p

Serum biochemistry of control and experimental animals.

<p>Values are expressed as mean ± SEM.</p

Evaluation of the pro-oxidant activity of the studied extract.

<p>The oxyHb (25 μM) is readily oxidized into met form in the presence of the extract (0.2 μg/mL final concentration) and laccase (100 nM) (<b>A</b>). Comparison between the prooxidant reactivity of the identified components of the extract all at the same molar concentration (5 μM) (<b>B</b>), their mixture in same ratios as in the extract prior hydrolysis (standard mixture 1) and after hydrolysis (standard mixture 2) and of the two analysed extracts (<b>C</b>). The comparison of the kinetic profile of the oxyHb oxidation in the presence of the two extracts and laccase depicted as first derivative of the measured curves (<b>D</b>).</p

Oxidative stress parameters of control and experimental animals.

<p>Values are expressed as mean ± SEM.</p

List of the compounds corresponding to each chromatographic peak (320 nm chromatogram) with their retention times (column 1) and their classification into two classes: Phenolic acids and flavonoids, based on spectral similarities^*.

<p>Assuming comparable molar absorptivities at 320 nm for both classes, their relative content was roughly estimated using areas of the peaks at this wavelength.</p