Elias Atala

The ORAC (oxygen radical absorbance capacity) index does not reflect the capacity of antioxidants to trap peroxyl radicals

In the present work we demonstrate from kinetic studies that under the experimental conditions proposed for the ORAC protocol, ORAC values do not correlate with the capacity of antioxidants to trap peroxyl radicals (ROO˙), suggesting a dominant role of alkoxyl radicals (RO˙) in the assay.

Quercetin and related flavonoids conserve their antioxidant properties despite undergoing chemical or enzymatic oxidation

Oxidation of a phenolic group in quercetin is assumed to compromise its antioxidant properties. To address this assumption, the ROS-scavenging, Folin-Ciocalteau- and Fe-reducing capacities of quercetin and thirteen structurally related flavonoids were assessed and compared with those of mixtures of metabolites resulting from their chemical and enzymatic oxidation. Regardless of the oxidation mode, the metabolites mixtures largely conserved the antioxidant properties of the parent molecules. For quercetin, 95% of its ROS-scavenging and over 77% of its Folin-Ciocalteau- and Fe-reducing capacities were retained. The susceptibility of flavonoids to oxidative disappearance (monitored by HPLC-DAD) and that of the mixtures to retain their antioxidant capacity was favourably influenced by the presence of a catechol (ring-B) and enol (ring C) function. This is the first study to report that mixtures resulting from the oxidation of quercetin and its analogues largely conserve their antioxidant properties.

Mechanism of Pyrogallol Red Oxidation Induced by Free Radicals and Reactive Oxidant Species. A Kinetic and Spectroelectrochemistry Study

Pyrogallol red (PGR) presents high reactivity toward reactive (radical and nonradical) species (RS). This property of PGR, together with its characteristic spectroscopic absorption in the visible region, has allowed developing methodologies aimed at evaluating the antioxidant capacity of foods, beverages, and human fluids. These methods are based on the evaluation of the consumption of PGR induced by RS and its inhibition by antioxidants. However, at present, there are no reports regarding the degradation mechanism of PGR, limiting the extrapolation to how antioxidants behave in different systems comprising different RS. In the present study, we evaluate the kinetics of PGR consumption promoted by different RS (peroxyl radicals, peroxynitrite, nitrogen dioxide, and hypochlorite) using spectroscopic techniques and detection of product by HPLC mass spectrometry. The same pattern of oxidation and spectroscopic properties of the products is observed, independently of the RS employed. Mass analysis indicates the formation of only one product identified as a quinone derivative, excluding the formation of peroxides or hydroperoxides and/or chlorinated compounds, in agreement with FOXs assays and oxygen consumption experiments. Cyclic voltammetry, carried out at different pHs, shows an irreversible oxidation of PGR, indicating the initial formation of a phenoxy radical and a second charge transfer reaction generating an ortho-quinone derivative. Spectroelectrochemical oxidation of PGR shows oxidation products with identical UV-visible absorption properties to those observed in RS-induced oxidation.

Use of Pyrogallol Red and Pyranine as Probes to Evaluate Antioxidant Capacities towards Hypochlorite

Hypochlorite is a strong oxidant able to induce deleterious effects in biological systems. The goal of this work was to investigate the use of PGR and PYR as probes in assays aimed at evaluating antioxidant activities towards hypochorite and apply it to plant extracts employed in Chilean folk medicine. The consumption of PGR and PYR was evaluated from the decrease in the visible absorbance and fluorescence intensity, respectively. Total phenolic content was determined by the Folin Ciocalteau assay. PGR and PYR react with hypochlorite with different kinetics, being considerably faster the consumption of PGR. Different stoichiometric values were also determined: 0.7 molecules of PGR and 0.33 molecules of PYR were bleached per each molecule of added hypochlorite. Both probes were protected by antioxidants, but the rate of PGR bleaching was too fast to perform a kinetic analysis. For PYR, the protection took place without changes in its initial consumption rate, suggesting a competition between the dye and the antioxidant for hypochlorite. Plant extracts protected PYR giving a PYR-HOCl index that follows the order: Fuchsia magellanica ≈ Marrubium vulgare ≈ Tagetes minuta > Chenopodium ambrosoides ≈ Satureja montana > Thymus praecox. Based on both the kinetic data and the protection afforded by pure antioxidants, we selected PYR as the best probe. The proposed methodology allows evaluating an antioxidant capacity index of plant extracts related to the reactivity of the samples towards hypochlorite.

International Regulations of Propolis Quality: Required Assays do not Necessarily Reflect their Polyphenolic-Related In Vitro Activities.

Propolis has been proposed as a polyphenolic-rich natural product potentially able to be used for human consumption or even for medicinal proposes. To guarantee a minimum phenolic and flavonoid content and as consequence of their related-biological activities, international requirements of propolis quality are commonly applied. In this work we assessed phenolic and flavonoid contents of propolis; the antioxidant capacity (toward peroxyl radicals and hypochlorous acid); the ability to generate nitric oxide (NO); and, finally the antimicrobial activity of 6 propolis samples from the VI region of Chile. Our results show that the total phenolic and flavonoid content of propolis samples are not always in agreement with their polyphenolic-associated in vitro activities. For example, P03 and P06 samples showed the lowest (25 ± 4 GAE/g propolis) and the highest (105 ± 3 GAE/g propolis) total phenolic content, respectively. This was in agreement with flavonoid content and their Oxygen Radical Absorbance Capacity (ORAC) activity. However, this dependence was not observed toward HOCl, NO release and antimicrobial activity. Based on our results, we consider that, in order to guarantee the antioxidant or antimicrobial in vitro effects, the international regulations of propolis quality should contemplate the convenience of incorporating other simple analytical test such as ORAC or antimicrobial tests.