Camilo López-Alarcón

Evaluating the antioxidant capacity of natural products: a review on chemical and cellular-based assays.

Oxidative stress is associated with several pathologies like cardiovascular, neurodegenerative, cancer and even aging. It has been suggested that a diet rich in antioxidants would be beneficial to human health and a lot of interest is focused on the determination of antioxidant capacity of natural products. Different chemical methods have been developed including the popular ORAC that evaluates the potential of a sample as inhibitor of a target molecule oxidation. Chemical-based methods are useful for screening, they are low cost, high-throughput and yield an index value (expressed as equivalents of Trolox) that allows comparing and ordering different products. More recently, nanoparticles-based assays have been developed to sense the antioxidant power of natural products. However, the antioxidant capacity indexes obtained by chemical assays cannot extrapolate the performance of the sample in vivo. Considering that antioxidant action is not limited to scavenging free radicals but includes upregulation of antioxidant and detoxifying enzymes, modulation of redox cell signaling and gene expression, it is necessary to move to cellular assays in order to evaluate the potential antioxidant activity of a compound or extract. Animal models and human studies are more appropriate but also more expensive and time-consuming, making the cell culture assays very attractive as intermediate testing methods. Cellular antioxidant activity (CAA) assays, activation of redox transcription factors, inhibition of oxidases or activation of antioxidant enzymes are reviewed and compared with the classical in vitro chemical-based assays for evaluation of antioxidant capacity of natural products.

Generation of superoxide radicals by copper–glutathione complexes: Redox-consequences associated with their interaction with reduced glutathione

The interaction between Cu(2+) ions and GSH molecules leads to the swift formation of the physiologically occurring Cu(I)-[GSH](2) complex. Recently, we reported that this complex is able to reduce molecular oxygen into superoxide in a reversible reaction. In the present study, by means of fluorescence, luminescence, EPR and NMR techniques, we investigated the superoxide-generating capacity of the Cu(I)-[GSH](2) complex, demonstrated the occurrence and characterized the chemical nature of the oxidized complex which is formed upon removing of superoxide radicals from the former reaction, and addressed some of the redox consequences associated with the interaction between the Cu(I)-[GSH](2) complex, its oxidized complex form, and an in-excess of GSH molecules. The interaction between Cu(I)-[GSH](2) and added GSH molecules led to an substantial exacerbation of the ability of the former to generate superoxide anions. Removal of superoxide from a solution containing the Cu(I)-[GSH](2) complex, by addition of Tempol, led to the formation and accumulation of Cu(II)-GSSG. Interaction between the latter complex and GSH molecules permitted the re-generation of the Cu(I)-[GSH](2) complex and led to a concomitant recovery of its superoxide-generating capacity. Some of the potential redox and biological implications arising from these interactions are discussed.

A novel and simple ORAC methodology based on the interaction of Pyrogallol Red with peroxyl radicals

Oxygen radicals absorbance capacities (ORAC) indexes are frequently employed to characterize the radical trapping capacity of pure compounds and their complex mixtures. A drawback of ORAC values obtained using phycoerythrin, fluorescein (FL) or c-phycocyanin as targets, makes it possible to conclude that for very reactive compounds they are much more related to stoichiometric factors than to the reactivity of the tested compound. In the present paper, we propose a simple methodology, based on the bleaching of Pyrogallol Red (PGR) absorbance that provides ORAC indexes that are almost exclusively determined by the reactivity of the tested compounds. This difference is due to the high reactivity of PGR and the high concentrations of this compound employed in the experiments.

Cu(I)-glutathione complex: a potential source of superoxide radicals generation.

Cu(2+) ions and GSH molecules interact swiftly to form the complex Cu(I)-glutathione. We investigated the potential capacity of such complex to reduce molecular oxygen. The addition of SOD to a solution containing Cu(I)-glutathione led to a sustained decline of the basal oxygen level. Such effect was partially reverted by the addition of catalase. The complex was able to induce the reduction of cytochrome c and the oxidation of dyhydroethidium into 2-hydroxyethidium. Both effects were totally blocked by SOD. The ability of the complex to generate superoxide radicals was confirmed by EPR spin-trapping. Cu(I)-glutathione induces no oxidation of fluorescein, a hydroxyl radical-sensitive probe. We conclude that in solutions containing the complex, oxygen is continually reduced into superoxide, and that-in absence of interceptors-the latter radicals are quantitatively re-oxidized into molecular oxygen. We suggest that, by functioning as a continuous source of superoxide, the complex could potentially affect a broad range of susceptible biological targets.

Interaction of pyrogallol red with peroxyl radicals. A basis for a simple methodology for the evaluation of antioxidant capabilities.

A competitive method to evaluate the reactivity of highly reactive antioxidants is reported. Pyrogallol red (PGR) and AAPH (2,2′-azo-bis-(2-amidinopropane) dihydrochloride) were employed as target-molecule and peroxyl radical source, respectively. In the zero-order kinetic limit in PGR, the dependence of the ratio R°/R (where R° is the rate of the process in the absence of additive and R is the rate of the process in the presence of additive) upon the additive concentration (Stern-Volmer like plots) was studied. Various polyphenols (n=10) and ascorbic acid (AA) were tested as additives. In PGR protection by AA, was observed a neat induction time, associated to the total protection of the target molecule. On the other hand, the experiments that were carried out in presence of phenolic compounds allowed a relative evaluation of their reactivity towards peroxyl radicals. This reactivity follows the order quercetin>gallic acid>Trolox > kaempferol. Data obtained employing quercetin and Trolox are compatible with a competitive protection by these antioxidants. Due to the high reactivity of PGR towards peroxyl radicals and its high extinction coefficient at long wavelengths, it is a very suitable molecule to be employed as target in the evaluation of the free radical scavenging capability of very reactive phenolic compounds.

First web-based database on total phenolics and oxygen radical absorbance capacity (ORAC) of fruits produced and consumed within the south Andes region of South America.

This paper reports the first database on antioxidants contained in fruits produced and consumed within the south Andes region of South America. The database ( www.portalantioxidantes.com ) contains over 500 total phenolics (TP) and ORAC values for more than 120 species/varieties of fruits. All analyses were conducted by a single ISO/IEC 17025-certified laboratory. The characterization comprised native berries such as maqui ( Aristotelia chilensis ), murtilla ( Ugni molinae ), and calafate ( Barberis microphylla ), which largely outscored all other studied fruits. Major differences in TP and ORAC were observed as a function of the fruit variety in berries, avocado, cherries, and apples. In fruits such as pears, apples, apricots, and peaches, a significant part of the TP and ORAC was accounted for by the antioxidants present in the peel. These data should be useful to estimate the fruit-based intake of TP and, through the ORAC data, their antioxidant-related contribution to the diet of south Andes populations.

Voltammetric oxidation of Hantzsch 1,4-dihydropyridines in protic and aprotic media: relevance of the substitution on N position

Abstract A detailed investigation on the electrochemical oxidation of some Hantzsch 1,4-dihydropyridine derivatives with the aim of study the influence of the hydrogen substituent on the N1 position of the heterocyclic ring have been carried out in protic and aprotic media. For this objective we have synthesized two series of compounds wherein the difference was the substituent (H or ethyl) on the N1-position of the heterocyclic ring. Voltammetry, UV–Vis spectroscopy, Controlled potential electrolysis, EPR, 1 H NMR and gas chromatography–mass spectrometry techniques in order to obtain evidences for postulate oxidation mechanisms in both protic and aprotic media have been used. Compounds having the ethyl substituent in the N1 position follow an oxidation mechanism obeying the sequence ECE with the second step as the r.d.e. in both, protic and aprotic media, thus producing the corresponding ethyl substituted pyridinium cation. On the other hand compounds having H in the N1 position follow the same ECE sequence only at acidic media. At basic media, the mechanism consisted of a DISP1 scheme in which rate determining step (r.d.s.) is the uptake of the proton in the N1 position by the OH − ion of the media. In aprotic media both type of compounds follow the same ECEC mechanism with the second step as the r.d.s. but only the H-substituted compounds generates an anionic species that is more easily oxidized than the parent compounds.

Antioxidant capacity of pure compounds and complex mixtures evaluated by the ORAC-pyrogallol red assay in the presence of Triton X-100 micelles.

The protective effect of different antioxidants and complex mixtures on the consumption of pyrogallol red (PGR) induced by peroxyl radicals was studied in the absence and presence of Triton X-100 micelles. The presence of micelles decreased significantly the protection of PGR afforded by lipophilic antioxidants (β-carotene, octyl gallate), while no effect of micelles was observed for hydrophilic antioxidants such as Trolox, caffeic acid, gallic acid, and ascorbic acid. In the presence of complex mixtures a clear effect of Triton X-100 micelles was also observed in the protection afforded by wines, tea infusions, and seed extracts of Eugenia jambolana and Myrciaria cauliflora. On the other hand, no effect of micelles was observed for orange juice and pulp fruit extracts. The ORAC (Oxygen Radical Absorbance Capacity) index was evaluated in the absence (ORAC-PGR) and presence of Triton X-100 micelles (ORAC-PGRMIC). Triton X-100 micelles affect ORAC-PGR values of antioxidants in a lipophilicity-dependent way. From the obtained results, we conclude that ORAC-PGR and ORAC-PGRMIC assays could be considered as an alternative to estimate the antioxidant ability (ORAC-PGR) and to infer the association to Triton X-100 micelles (ORAC-PGR/ORAC-PGRMIC) of pure antioxidants and their complex mixtures.

Antioxidant capacity of human blood plasma and human urine : Simultaneous evaluation of the ORAC index and ascorbic acid concentration employing pyrogallol red as probe

The oxygen radical absorbance capacity (ORAC) methodology has been employed to estimate the antioxidant capacity of human blood plasma and human urine using pyrogallol red (ORAC-PGR) as target molecule. Uric acid, reduced glutathione, human serum albumin, and ascorbic acid (ASC) inhibited the consumption of pyrogallol red, but only ASC generated an induction time. Human blood plasma and human urine protected efficiently pyrogallol red. In these assays, both biological fluids generated neat induction times that were removed by ascorbate oxidase. From these results, ORAC-PGR method could be proposed as a simple alternative to evaluate an ORAC index and, simultaneously, to estimate the concentration of ascorbic acid in human blood plasma or human urine.

ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM HYPERICUM TERNUM

In the present work we have studied the scavenging activity of several flavonoids isolated from Hypericum ternum A. St. Hil. The evaluation of the free radical scavenging capacity was based on the 2,2-diphenyl-l-picrylhydrazyl radical (DPPH) consumption elicited by their addition. Also, we have attempted to evaluate their capacity to delay pyrogallol red consumption promoted by peroxyl radicals. The compounds isolated and characterized were 13,118-biapigenin and five quercetin derivatives (quercetin 3-methyl ether, quercetin 3,7-dimethyl ether, hyperoside, isoquercitrin and guaijaverin). All compounds were able to scavenge DPPH radicals. The order in scavenging capacity (from highest to lowest) was: guaijaverin > hyperoside ≈ isoquercitrin > quercetin-3-methyl-ether. No protection of pyrogallol red was evidenced for all flavonoids derivatives at relatively high (100 µM) concentrations. This lack of protection contrasts with the efficient protection afforded by 10 µM quercetin, indicating that substitution at the 3-position in quercetin strongly reduces the capacity of the molecule to scavenge peroxyl radicals