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An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 μM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h(-1)), low operating costs and low generation of waste., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The current therapeutic strategies are based on the design of multifunctional drug candidates able to interact with various disease related targets. Drugs that have the ability to scavenge reactive oxygen species (ROS), beyond their main therapeutic action, may prevent the oxidative damage of biomolecules. Therefore, analytical approaches that monitor in a continuous mode the ability of drugs to counteract peroxidation of physiologically relevant biotargets are required. In the present work, a microplate spectrophotometric assay is proposed to evaluate the ability of selected cardiovascular drugs, including angiotensin-converting enzyme (ACE) inhibitors, β -blockers and statins to prevent protein peroxidation. Myoglobin, which is a heme protein, and peroxyl radicals generated from thermolysis of 2,2'-azo-bis(2-amidinopropane) dihydrochloride at 37 °C, pH 7.4 were selected as protein model and oxidative species, respectively. Myoglobin peroxidation was continuously monitored by the absorbance decrease at 409 nm and the ability of drugs to counteract protein oxidation was determined by the calculation of the area under the curve upon the myoglobin oxidation. Fluvastatin (AUC₅₀=12.5 ± 1.2 μM) and enalapril (AUC₅₀=15.2 ± 1.8 μM) showed high ability to prevent myoglobin peroxidation, providing even better efficiency than endogenous antioxidants such as reduced glutathione. Moreover, labetalol, enalapril and fluvastatin prevent the autoxidation of myoglobin, while glutathione showed a pro-oxidant effect., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Antioxidant capacity of food samples is usually assessed by different analytical methods, however the results attained even for the same method are strongly dependent on the selected reaction time and also on the standard compound used. To tackle this problem, we propose here a kinetic matching approach, associated to the conversion of results into equivalents of a common standard compound, as a universal way for expression of results. The methodology proposed was applied to methods based on different chemistries (Folin-Ciocalteu (F-C), CUPRAC, DPPH(•) and ABTS(•+) assays) and red wines (n=40) were chosen as a model of complex food sample. For implementation of the kinetic matching approach, the standard phenolic mixture (caffeic acid, (+)-catechin, hesperetin, morin and (-)-epigallocatechin gallate) was chosen for calibration in F-C, CUPRAC and DPPH(•) assays, while tannic acid was suitable for ABTS(•+) assay. Results showed that, for all methods, there was no statistical difference between results attained by the kinetic matching approach (after <10 min of reaction) and that at endpoint conditions (after 60 to 300 min). The repeatability and the reproducibility of the kinetic matching approach was <4.5%, for all antioxidant assays. The sample throughput increases from <18 (endpoint measurements) to >108 h(-1) using the proposed kinetic approach. Moreover, we have established here a way of converting results to equivalents of a common standard, providing values independent of its kinetic profile, by using the ratio between calibration sensitivities performed at endpoint conditions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

High-throughput cupric ion reducing antioxidant capacity (CUPRAC) methods were developed for assessment of total antioxidant capacity (TAC) in urine and serum, based on reduction of Cu(II)-neocuproine complex to highly colored Cu(I)-neocuproine complex, measured spectrophotometrically at 450 nm. The reaction time was significantly reduced from 30 to 4 min by application of a calibration compound (uric acid) with kinetic behavior similar to that shown by urine samples. The method was implemented in a microformat (96 well plates) and also in an automatic fashion (flow injection analysis, FIA). A determination throughput value of 288 h(-1) (microplate method) or of 15 h(-1) (automatic FIA) was attained. Application of both methods to human serum (SRM 909b, level I) and urines (n = 9) provided TAC values in agreement with those of the end-point batch method.

Lipophilic compounds structurally based on caffeic, hydrocaffeic, ferulic and hydroferulic acids were synthesized. Subsequently, their antioxidant activity was evaluated as well as their partition coefficients and redox potentials. The structure-property-activity relationship (SPAR) results revealed the existence of a clear correlation between the redox potentials and the antioxidant activity. In addition, some compounds showed a proper lipophilicity to cross the blood-brain barrier. Their predicted ADME properties are also in accordance with the general requirements for potential CNS drugs. Accordingly, one can propose these phenolic compounds as potential antioxidants for tackling the oxidative status linked to the neurodegenerative processes., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In the present work, an automatic method based on multisyringe flow injection analysis (MSFIA) was developed for determination of scavenging capacity against H(2)O(2) using a non-enzymatic fluorimetric assay for H(2)O(2) detection based on the formation of europium-tetracycline-H(2)O(2) complex. The MSFIA-fluorimetric methodology was developed to perform in-line the scavenging reaction of H(2)O(2) prior to reaction of the remaining H(2)O(2) with the fluorescent probe, using conditions close to those found in vivo regarding pH (6.9), temperature (37 degrees C) and H(2)O(2) concentration (25microM). This approach allowed the evaluation of scavenging capacity against H(2)O(2) in a non-competitive (antioxidant+H(2)O(2)) or a competitive (antioxidant+H(2)O(2)+biological target) scheme. Using the first strategy, IC(50) values determined for the antioxidant compounds glutathione reduced (1191+/-46microM) and pyruvate (446+/-49microM) were lower than those obtained for biological targets such as cysteine (2616+/-182microM), taurine (359+/-38mM) and adenine (2224+/-214microM), indicating that reactivity towards H(2)O(2) was higher for antioxidant compounds than for biological targets. However, when a competitive scheme was applied, the scavenging effectiveness against H(2)O(2) depended on the biological molecule present, showing that antioxidant assessment should also take into consideration the concomitant reactivity of biological molecules or structures that are prone to oxidative damage.

Cellular membranes, which contain abundant phospholipids, such as phosphatidylcholine, are major targets subjected to the damage caused by free radicals. Cellular damage due to lipid oxidation is strongly associated with ageing, carcinogenesis and other diseases. In addition, lipid oxidation is an important deteriorative reaction in the processing and storage of lipid-containing foods. Liposomes have been used extensively as biological models for in vitro lipid oxidation studies. The resemblance between the liposomal and membrane bilayer core makes liposomes a very useful tool to investigate the significance of the antioxidant-membrane interactions for antioxidant activity. The antioxidant activity of a compound is strongly influenced by numerous factors including the nature of the lipid substrate, the hydrophilic-lipophilic balance of the antioxidant, the physical and chemical environments of the lipids, and various other interfacial interactions. Thus, compounds that are effective antioxidants in one model system or food matrix may be unsuitable in other systems.This chapter describes fluorescent probes-based methods commonly used for testing antioxidant activity in liposomes and stresses the need to combine antioxidant assays and drug-membrane interaction studies to get a better description of the antioxidants' profile considering their location in lipid bilayer and their effect on membrane fluidity and consequently provide additional information to that obtained currently from assays performed in aqueous buffer media.

The role and importance of antioxidants in different fields, ranging from physiology to food technology, have become evident in the past years, requiring adequate analytical methodologies. Therefore, the determination of antioxidant capacity as a routine or screening analysis fosters its automation. In this context, several flow injection methods based on scavenging of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTS(+)) or 2,2-diphenyl-1-picrylhydrazyl radical (DDPH) or based on the determination of total reducing capacity have been proposed. The objective of the present review is to critically compare the different approaches, regarding their degree of automation, their performance vs. the respective batch procedure and its applicability to real samples.

This work stresses the need to combine antioxidant assays and drug-membrane interaction studies to describe more accurately the antioxidant profile of non-steroidal anti-inflammatory drugs (NSAIDs). Different experiments performed in liposomes and aqueous solution were compared and used to evaluate the protective effect of etodolac in lipid peroxidation. Lipid peroxidation was induced by the peroxyl radical (ROO*) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydroxyl radical (HO*) generated by the Fenton reaction and was assessed by the fluorescence intensity decay of three fluorescence probes with distinct lipophilic properties--fluorescein; hexadecanoyl aminofluorescein (HDAF) and diphenylhexatriene propionic acid (DPHPA). Membrane fluidity changes due to lipid peroxidation were also evaluated by steady-state anisotropy measurements. Interactions of etodolac with lipid bilayers were evaluated by membrane zeta-potential measurements. Results indicate a drug location near the membrane surface and show that etodolac can scavenge the radicals studied but to a variable extent, depending on the assayed media and reactive species. The use of different probes and liposomes as membrane mimetic systems allowed us to conclude that membrane lipoperoxidation is not only related to the scavenging characteristics of the antioxidants, but also to their ability to interact with lipid bilayers.

In this study the possibility of using liposomes as membrane mimetic systems was evaluated to estimate the antioxidant properties of oxicams and establish a relationship between the interactions of the drugs with the membrane and their consequent antioxidant activity. Different experiments were performed covering the study of the protective effect of oxicams in lipid peroxidation induced by the peroxyl radical (ROO) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and using two fluorescence probes with distinct lipophilic properties. Lipid peroxidation using the hydrophilic probe fluorescein was evaluated in lipid and aqueous media. Lipid systems labelled with the fluorescent probe diphenylhexatriene propionic acid (DPH-PA) were used to assess the effects of the drugs on membrane peroxidation simultaneously by fluorescence intensity decay and changes in membrane fluidity by steady-state anisotropy measurements. The use of different probes and liposomes as membrane mimetic systems allowed to conclude that membrane lipoperoxidation is related not only to the scavenging characteristics of the antioxidants but also to their ability to interact with the lipid bilayers.

Eight structurally similar dihydroxy and trihydroxyphenolic acids (protocatechuic acid, 3,4-dihydroxyphenylacetic acid, hydrocaffeic acid, caffeic acid, gallic acid, 3,4,5-trihydroxyphenylacetic acid, 3-(3,4,5-trihydroxyphenyl)propanoic acid and 3-(3,4,5-trihydroxyphenyl)propenoic acid) were examined for their total antioxidant capacity (TAC). Furthermore, their ability to scavenge peroxyl radicals, generated by AAPH in liposomes, was determined. The antioxidant/pro-oxidant activity of the compounds was screened using the 2'-deoxyguanosine assay. All compounds behave as radical scavengers, with 3,4,5-trihydroxyphenylacetic acid being the most potent. Nevertheless, in the lipid peroxidation assay an inverse ranking order was observed, 3,4-dihydroxyphenylacetic acid being the most effective compound. All the dihydroxylated compounds showed a pro-oxidant behaviour leading to an increase of 50% in 8-OH-dG induction. From the structure-antioxidant activity relationship studies performed it may be concluded that the number of phenolic groups and the type of the alkyl spacer between the carboxylic acid and the aromatic ring strongly influence the antioxidant activity.

The screening of natural flavonoids for their bioactivity as antioxidants is usually carried out by determinination of their profile as chain-breaking antioxidants, by the evaluation of their direct free radical-scavenging activity as hydrogen- or electron-donating compounds. Since this may not be the only mechanism underlying the antioxidant activity it is important to check the ability of these compounds to act as chelators of transition metal ions. Accordingly, in the present study the acidity constants of catechin and taxifolin, as well as the formation constants of the corresponding copper (II) complexes, were investigated by potentiometry and/or spectrophotometry. Moreover, a detailed quantitative examination of the coordination species formed is presented. In addition, the partition coefficients of both catechin and taxifolin in a biomimetic system (micelles) were determined, since these properties may also contribute to the antioxidant behavior of this type of compound. The log P values determined depend on the electrostatic interactions of the compounds with the differently charged micelles (the highest values were obtained for zwitterionic and cationic micelles). The prooxidant behavior of the compounds was assessed through the oxidation of 2'-deoxyguanosine, induced by a Fenton reaction, catalyzed by copper. The data obtained reveal that the flavonoids under study did not present prooxidant activity, in this particular system. The results obtained are evidence of a clear difference among the pKa, the complexation properties, and the lipophilicity of the flavonoids studied, which can partially explain their distinct antioxidant activity. The most stable geometries of the free compounds were determined by theoretical (ab initio) methods, in order to properly account for the electron correlation effects which occur in these systems, thus allowing a better interpretation of the experimental data.

Highlights: [•] Method developed for phenolic-rich food samples to measure antioxidant capacity. [•] A kinetic matching approach better assesses endpoint antioxidant capacity. [•] Method relies on standard selected with oxidation profile similar to food sample. [•] TEAC values were calculated from reactivity of standard and Trolox at endpoint. [•] Consumer-friendly index VCEAC (vitamin C equivalent antioxidant capacity) was given. [ABSTRACT FROM AUTHOR]

Abstract: The determination of hydrogen peroxide (H2O2) and the evaluation of scavenging capacity against this species were performed using five colorimetric reactions, which were adapted to flow injection analysis. The reactions chosen were based on the oxidation of iodide (I− method), on the formation of titanium-peroxide complex (TiP method), on the formation of titanium-xylenol orange-peroxide complex (TiXoP method), on the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB method) and on the co-oxidation of phenol-4-sulfonic acid and 4-aminoantipyrine (PSA/4-AAP method). The operational conditions were studied in order to improve the sensitivity of each method. Concerning to the method sensitivity, the ranking order was TMB method>I− method>TiXoP method ∼PSA/4-AAP method>TiP method. All methods showed an excellent repeatability (RSD<2%) and, except for I− method, relative deviations from the reference method were <1.9%. The FIA manifolds were adapted to perform the determination of scavenging capacity against H2O2 and glutathione (GSH) was applied as model compound. TiP and TiXoP methods were not suitable as no inhibition or an increase of analytical signal was attained. PSA/4-AAP method was chosen for further application to dietary phenolics and pharmaceutical compounds, providing IC50 values for those compounds that are fast reacting antioxidants. [Copyright &y& Elsevier]

Abstract: Several of the most commonly used methods for in vitro determination of antioxidant capacity are reviewed in the present paper. The chemical principles of methods based either on biological oxidants (peroxyl radical, superoxide radical anion, hydrogen peroxide, hydroxyl radical, hypochlorous acid, singlet oxygen, nitric oxide radical, and peroxynitrite) or on non-biological assays (scavenging of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation (TEAC assay), scavenging of 2,2-diphenyl-1-picrylhydrazyl radical (DPPHferric reducing antioxidant power (FRAP assay), Folin–Ciocalteu reducing capacity (FC assay), electrochemical total reducing capacity) are outlined and critically discussed. The scope of application, the advantages and shortcomings of each method are also highlighted. [Copyright &y& Elsevier]

Abstract: In the present work, an automatic flow procedure for the sequential spectrophotometric determination of Folin-Ciocalteu reducing capacity (FC assay) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS +) scavenging capacity expressed as the trolox equivalent (TEAC assay) is proposed for a comparative study of antioxidant properties in food products. Exploiting the flexibility of flow management associated to the computer control offered by multisyringe flow injection analysis, both methodologies were carried out in the same manifold using gallic acid and trolox as standard compounds. The proposed system configuration allowed the performance of each method separately or in tandem, providing 24 determinations per hour, which accounts for its application in routine analysis. The present methodology was applied to a large number of beverages (n =72), namely red and white wines, herbal and tea infusions, juices and beers. The results obtained showed that FC reducing capacity and TEAC values of red wines were significantly different from those obtained for the other beverages, while tea infusions provided significantly higher TEAC values when compared to white wines, herbal infusions, juices and beers. A good correlation was found between TEAC and FC reducing capacity (R >0.9) for red wines, herbal and tea infusions, and beers. For these beverages, similar slope values were found (106–140mgL−1 of gallic acid per mM of Trolox), except for beers that showed a higher response for FC assay. These results provided evidence that the correlation between these assays vary according to the type of sample, reinforcing the idea that more than one method should be used for evaluation of antioxidant capacity. [Copyright &y& Elsevier]

Abstract: An automatic system that performs two analytical procedures, allowing the evaluation of the relative antioxidant capacity of wine samples, was developed. Automation was carried out using a sequential injection analysis (SIA) system that allowed, thanks to its versatility, the development of two methodologies. One is based on the decolorization assay of the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical, using a spectrophotometric detector. A second methodology allowed the evaluation of the hydrogen peroxide scavenging activity by measuring the oxidation of homovanylic acid (HVA) to its fluorescent dimer, using a fluorescent detector. The developed automatic methodologies were evaluated using trolox as standard and subsequently using other antioxidant substances as gallic acid, caffeic acid, ascorbic acid, catechin and taxifolin which are abundant in wine and whose antioxidant activities were compared to that shown by trolox. The spectrophotometric and fluorimetric assays showed linearity intervals between 0.001 and 0.01mM, and 0.001 and 0.008mM of trolox, respectively. The evaluation of the antioxidant power of 20 white and red wine samples, from different Portuguese wine producing regions, was carried out sequentially, in the automatic system. The results were expressed in trolox equivalent antioxidant capacity (TEAC) and presented, for the ABTS and hydrogen peroxide scavenging activity methodologies, detection limits of 8.4×10-7 and 1.4×10-4mM and relative standard deviation (R.S.D. (%)) in the range 0.6–2.4 and 1–1.8, respectively. [Copyright &y& Elsevier]

A programmable flow system is proposed for assessment of total antioxidant capacity of biological fluids based on Cu(II) reduction. A high-sensitivity procedure was implemented by using a 1 m pathlength liquid core waveguide capillary cell (LWCC), fostering high oxidant to antioxidant ratio. This feature promoted a faster reaction development and the response of the developed method towards several model antioxidant compounds, including ascorbic acid, Trolox (soluble analogue of vitamin E), uric acid, cysteine, glutathione and serum albumin, was studied. Application to less studied biological samples available at low volumes (tear, saliva), and also to serum samples, was performed. Analytical features include the low limit of detection (200 nM) and application range up to 10 μM Trolox with intraday repeatability of < 3%. Trolox equivalent antioxidant capacity (TEAC) values between 26 ± 1 and 155 ± 7 μM were found for tears and saliva samples, while values between 421 ± 3 and 759 ± 14 μM were found for serum samples. The mean recovery was 101.9 ± 7.3% when uric acid was added to the different biological sample matrices, showing the suitability of the proposed method towards this application at low μM levels. [ABSTRACT FROM AUTHOR]

Abstract: Conventionally, measuring antioxidant capacity is time consuming, laborious and costly, especially in routine work. These limitations can be overcome using multi-syringe flow-injection analysis (MSFIA), which has succeeded in automating the most widely applied antioxidant-capacity assays that use chromogen radicals or reactive species found in vivo. We highlight the features of MSFIA and improved methodologies in using MSFIA to measure antioxidant capacity. [Copyright &y& Elsevier]

Abstract: Redox reactions are the heart of numerous biochemical pathways found in cellular chemistry, generating reactive oxygen species (ROS) and reactive nitrogen species (RNS), that includes superoxide anion radical (O2 −), hydrogen peroxide (H2O2), hydroxyl radical (HOt oxygen (1O2), hypochlorite anion (OCl−), peroxynitrite anion (ONOO−) and nitric oxide radical (NOasurement of scavenging capacity against these reactive species presents new challenges, which can be met by flow injection analysis (FIA). In the present review several methods based on FIA and also on its predecessors computer-controlled techniques (sequential injection analysis, multisyringe flow injection analysis, multicommutated and multipumping flow systems) are critically discussed. The selectivity and applicability of the methodology, the generation and detection of the target reactive species, the benefits and limitations of automation when compared to batch methods are some of the issues addressed. [Copyright &y& Elsevier]