Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols

Proteins are not considered as true antioxidants but are known to protect antioxidants from oxidation in various antioxidant activity assays. This study aims to investigate the contribution of proteins, especially thiol-containing proteins, to the observed overall antioxidant capacity measured by known methods. To determine the antioxidant properties of thiol-containing proteins, the CUPRAC method of antioxidant assay using the oxidizing reagent Cu(II)-neocuproine previously used for simultaneous analysis of cystine and cysteine was adopted. While the CUPRAC method is capable of determining all antioxidant compounds including thiols in complex sample matrices, the Ellman method of thiol quantitation basically does not respond to other antioxidants. The antioxidant quantities in the selected samples were assayed with the ABTS and FRAP methods as well as with the CUPRAC method. In all applied methods, the dilutions were made with a standard pH 8 buffer used in the Ellman method by substituting the Na₂EDTA component of the buffer with sodium citrate. On the other hand, the standard CUPRAC protocol was modified by substituting the pH 7 ammonium acetate buffer (at 1 M concentration) with 8 M urea buffer adjusted to pH 7 by neutralizing with 6 M HCl. Urea helps to partly solubilize and denaturate proteins so that their buried thiols be oxidized more easily. All methods used in the estimation of antioxidant properties of proteins (i.e., CUPRAC, Ellman, ABTS, and FRAP) were first standardized with a simple thiol compound, cysteine, by constructing the calibration curves. The molar absorptivities of these methods for cysteine were: ?_CUPRAC = 7.71 × 10³, ?_Ellman = 1.37 × 10⁴, ?_ABTS = 2.06 × 10⁴, and ?_FRAP = 2.98 × 10³ L mol⁻¹cm⁻¹. Then these methods were applied to various samples containing thiols, such as glutathione (reduced form:GSH), egg white, whey proteins, and gelatin. Additionally, known quantities of selected antioxidants were added to these samples to show the additivity of responses.     

Determination of total antioxidant capacity by a new spectrophotometric method based on Ce(IV) reducing capacity measurement

Dietary antioxidants widely found in fruits and vegetables may serve the task of reducing oxidative damage in humans induced by free radicals and reactive oxygen species under ‘oxidative stress’ conditions. The aim of this work is to develop a simple, low-cost, sensitive, and diversely applicable indirect spectrophotometric method for the determination of total antioxidant capacity of several plants. The method is based on the oxidation of antioxidants with cerium(IV) sulfate in dilute sulfuric acid at room temperature. The Ce(IV) reducing capacity of the sample is measured under carefully adjusted conditions of oxidant concentration and pH such that only antioxidants and not other organic compounds would be oxidized. The spectrophotometric determination of the remaining Ce(IV) was performed after completion of reaction with antioxidants. Quercetin and gallic acid were used as standards for flavonoids and phenolic acids, respectively, and results of antioxidant measurements were reported as trolox equivalents. The developed procedure was successfully applied to the assay of total antioxidant capacity due to simple compounds such as trolox, quercetin, gallic acid, ascorbic acid, catechin, naringin, naringenin, caffeic acid, chlorogenic acid, ferulic acid, and p-coumaric acid, and due to phenolic acids and flavonoids in the arieal parts of nettle (Urtica Dioica L.). Blank correction of significantly absorbing plant extracts at 320 nm could be made with the aid of spectrophotometric titration. Plant selection was made in respect to high antioxidant content, and extraction was made with water. The proposed method was reproducible, and the trolox equivalent antioxidant capacities (TEAC coefficients) of the tested antioxidant compounds were correlated to those found by reference methods such as ABTS and CUPRAC. Since the TEAC coefficients found with the proposed method of naringin-naringenin and rutin-catechin pairs were close to each other, this Ce(IV)-based assay probably caused the simultaneous hydrolysis of flavonoid glycosides to the corresponding aglycones and their subsequent oxidation such that the hydrolysis products exhibed antioxidant capacities roughly proportional the number of -OH groups contained in a molecule.     

Temperature-dependence of radical-trapping activity of phenoxazine,phenothiazine and their aza-analogues clarifies the way forward for new antioxidant design

The prediction and/or rationalization of diarylamine radical-trapping antioxidant (RTA) activity at the elevated temperatures where they are most useful presents a significant challenge, precluding the development of new technology. Whilst structure–activity relationships at ambient temperatures are well established, their predictive capacity at elevated temperatures is poor due to competing reactions. A striking example involves phenoxazine, which is a superior RTA relative to its sulfur analog phenothiazine at ambient temperature (e.g. k = 3.9 × 10⁷vs. 7.6 × 10⁶ M⁻¹ s⁻¹ at 37 °C, respectively), but is demonstrably inferior at elevated temperatures. Despite being inherently less oxidizable in electrochemical experiments and high-accuracy computations, phenoxazine is more rapidly consumed than phenothiazine in autoxidations at 160 °C – a result which we attribute to a lower reorganization energy barrier to oxidation. Given these observations, we surmised that incorporation of an electronegative N-atom into the phenoxazine ring system would decrease the driving force for oxidation and ‘rescue’ its activity. Indeed, this was found to be the case for nitrogen incorporation at any position, regardless of the impact on the inherent RTA activity. Analogous experiments were carried out on phenothiazines into which nitrogen atoms were incorporated, revealing little benefit at 160 °C. These results suggest that for highly reactive diarylamines (i.e. those with k > 10⁶ M⁻¹ s⁻¹), further enhancements in reactivity do not materially improve their ability to inhibit hydrocarbon autoxidation at elevated temperatures. Instead, their stability to one-electron oxidation determines their efficacy.

Studies of potent diarylamine antioxidants illustrate how their high-temperature activity can be limited by direct reaction with O₂, and how driving force and barrier to oxidation can be modulated for future antioxidant development.     

Automatic sequential determination of the hydrogen peroxide scavenging activity and evaluation of the antioxidant potential by the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation assay in wines by sequential injection analysis

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 0.01 mM, and 0.001 and 0.008 mM 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 × 10⁻⁴ mM and relative standard deviation (R.S.D. (%)) in the range 0.6-2.4 and 1-1.8, respectively.     

Thermal oxidation and molding feasibility of cycloolefin copolymers (COCs) with high glass transition temperature

Cycloolefin copolymers (COCs) with high glass transition temperature (T_g = 203 °C) have been synthesized and pelletized by extrusion molding. However, their colors change from transparent to yellow during extrusion molding because of thermal oxidation and generation of alkene groups. We have successfully blended several antioxidants (Irganox 1010, Irgafos 168, Irganox HP2225 and Irganox HP2921) into lab-made COCs to avoid the discoloration. The experimental results show that Irganox HP2921 is the best antioxidant among the antioxidants used and can effectively not only suppress thermal oxidation but also eliminate the color stain.     

Sequential injection analysis with electrochemical detection as a tool for economic and rapid evaluation of total antioxidant capacity

This work presents a new flow-based coupled electrochemical technique for evaluation of “total antioxidant capacity (TAC)”. A sequential injection (SI) with amperometric detection was applied to the TAC analysis of commercial instant ginger infusion beverages using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Besides having chromogenic properties, the ABTS reagent behaves as an electroactive species at the glassy carbon electrode in phosphate buffer pH 7.0, the decrease of the cathodic current signal of the ABTS⁺ radical after reaction with antioxidants can be monitored. The SI system, furnished with an in-house electrochemical detection cell (ECD), was optimized with respect to the applied potential, sample and reagent volume, and flow rate to the detector. Gallic acid was used as the standard antioxidant and the capacity was reported as gallic acid equivalent (GAE) unit. TAC measurements of ginger infusions at the optimum condition were performed using the proposed technique and also with the classical batch spectrophotometric ABTS assay. TAC values obtained from our method and the standard method are in good agreement (r² = 0.956). The SI-amperometric technique provided satisfactory precision (4.11% RSD) with rapid sample throughput (40 samples h⁻¹). Also using this method, the consumption of the expensive ABTS reagent was greatly reduced.     

Novel reactions for simple and sensitive spectrophotometric determination of nitrite

Nine spectrophotometric methods based on new reactions for the determination of tracer amounts of nitrite in environmental samples were developed. Replacement of toxic reagents was explored to attain the standards of clean chemistry. These methods utilize two classes of compounds namely; phenoxazines and sulphonamides, the well established drugs in the presence of limited amounts of hydrochloric acid. The methods were based on the oxidation of sulfanilamide (SAA), sulfadoxine (SDX) or sulfamethoxazole (SMX) by nitrite in hydrochloric acid medium and coupling with phenoxazine (PNZ), 2-chlorophenoxazine (CPN) or 2-trifluoromethylphenoxazine (TPN) which yielded red colored derivatives having an absorbance maximum in the range 530-540 nm and were stable for about 4 h. Beer's law was obeyed for nitrite in the concentration range 0.13-1.60 μg mL⁻¹. The reaction conditions and other important analytical parameters were optimized to enhance the sensitivity of the methods. Interference if any, by non-target ions was also investigated. The methods were applied determining nitrite in environmental samples. The performance of these methods were evaluated in terms of Student's t-test and variance ratio F-test to find out the significance of proposed methods over the reference spectrophotometric method.     

Solid-phase ultraviolet absorbance spectrophotometric multisensor for the simultaneous determination of butylated hydroxytoluene and co-existing antioxidants

Continuous flow injection and UV spectrophotometric detection have been proposed for simultaneous determination of the two binary mixtures, butylated hydroxytoluene (BHT)/n-propyl gallate (n-PG) and butylated hydroxytoluene (BHT)/butylated hydroxyanisole (BHA), in food and cosmetics samples. The method is based on the different residence times of each antioxidant when the flow cell is packed to a height of 25 mm with silica C₁₈ using methanol-water 50:50% (v/v) as a carrier with a flow rate of 1.25 and 1.10 ml min⁻¹, respectively. The determination of each antioxidant is based on the measurement of its absorbance at its maximum wavelengths using a DAD detector at 30 and 180 s for the mixture n-PG-BHT and 90 and 220 s for BHA-BHT. Calibration graphs were linear over the range 10.0-300.0 μg ml⁻¹ for each antioxidant in both mixtures. The relative standard deviations were 2.5% for BHT and 2.0% for the co-existing antioxidant. Resolution of the n-PG-BHT mixture in ratios between 1:8 and 8:1 and the BHA-BHT mixture in ratios between 1:10 and 10:1 is possible. The method was applied to the determination of both antioxidants in fat foods and cosmetics samples with recoveries ranging between 101 and 105%.     

Simultaneous determination of uric acid,xanthine, hypoxanthine and caffeine in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode

This paper describes the fabrication of graphene on glassy carbon electrode (GCE) by electrochemical reduction of graphene oxide (GO) attached through 1,6-hexadiamine on GCE and the simultaneous determination of structurally similar four purine derivatives using the resultant electrochemically reduced GO (ERGO) modified electrode. The electrocatalytic activity of ERGO was investigated toward the oxidation of four important purine derivatives, uric acid (UA), xanthine (XN), hypoxanthine (HXN) and caffeine (CAF) at physiological pH. The modified electrode not only enhanced the oxidation currents of the four purine derivatives but also shifted their oxidation potentials toward less positive potentials in contrast to bare GCE. Further, it successfully separates the voltammetric signals of the four purine derivatives in a mixture and hence used for the simultaneous determination of them. Selective determination of one purine derivative in the presence of low concentrations other three purine derivatives was also realized at the present modified electrode. Using differential pulse voltammetry, detection limits of 8.8 × 10⁻⁸ M, 1.1 × 10⁻⁷ M, 3.2 × 10⁻⁷ M and 4.3 × 10⁻⁷ M were obtained for UA, XN, HXN and CAF, respectively. The practical application of the modified electrode was demonstrated by simultaneously determining the concentrations of UA, XN, HXN and CAF in human blood plasma and urine samples.     

Laccase Based Synergistic Electrocatalytical System

Bioelectrodes employing the conversion of N‐substituted phenothiazine/hexacyanoferrate(II) and N‐substituted phenoxazine/hexacyanoferrate(II) that act as synergistic substrates were built using graphite electrode and recombinant laccase. The response of the bioelectrodes to low reactive substrate (hexacyanoferrate(II)) increases 2.9–97 times in presence of high reactive phenothiazine or phenoxazine mediator. The sensitivity of the bioelectrodes reaches 0.5–14.3 A/M?cm². To background a scheme of bioelectrodes action the kinetics of laccase‐catalyzed synergistic substrates oxidation was investigated in homogenous solution. A satisfactory agreement of experimentally determined rate constants and calculated using parameters of bioelectrocatalytical system confirms the scheme of bioelectrodes action.     

Study of antioxidant properties of a water-soluble Vitamin E derivative-tocopherol monoglucoside (TMG) by differential pulse voltammetry

Study of antioxidant properties of tocopherol monoglucoside (TMG), a water-soluble Vitamin E derivative, by differential pulse voltammetry has been carried out in this work. The pH influence on the antioxidant properties of TMG has been also investigated. It was observed that the antioxidant activity of TMG is greater at 6.90<pH<9.18. The reactions between the TMG and reactive oxygen species have been considered. Antioxidant activity of some standard antioxidants including Vitamin E was given for comparison. The results indicate that the TMG is an effective antioxidant in neutral solutions. The oxidation potential for TMG at pH 7.31 has been found (E=0.55±0.03 V versus Ag|AgCl|KCl_sat electrode). Finally the use of TMG for protection against oxidative stress has been recommended.     

Influence of nanodispersed boehmite on polypropylene photooxidation

Nanocomposites containing pure or organically modified nanoboehmites of different sizes were prepared by melt compounding with polypropylene. The samples were UV light irradiated in artificial accelerated conditions representative of solar irradiation (λ > 300 nm) at 60 °C in air. The chemical modifications resulting from photooxidation were followed by IR and UV-visible spectroscopies. The presence of pristine nanoboehmites was shown to change the rate of oxidation of polypropylene by reducing the oxidation induction period due to the presence of residual processing antioxidant. The differences of the oxidation induction periods between the nanocomposites and the pristine polymer disappear after solvent extraction of the antioxidant. The inefficiency of traditional antioxidant in retarding the photooxidation of polypropylene containing nanodispersed boehmite is proved. Antioxidant migration to the boehmite surface induced by the preferential interaction with the polar filler is proposed as an explanation. The oxidative behaviour of the organically modified boehmites was shown to depend on the type of organic substituent. p-Toluenesulfonate reduces the adsorption of antioxidants while the presence of a long-chain alkyl benzensulfonate increased the oxidation rate by generation of radical initiators.     

Biosensor based on laccase and an ionic liquid for determination of rosmarinic acid in plant extracts

Novel biosensors based on laccase from Aspergillus oryzae and the ionic liquids (ILs) 1-n-butyl-3-methylimidazolium hexafluorophosphate (BMIPF₆) and 1-n-butyl-3-methylimidazolium tetrafluoroborate (BMIBF₄) were constructed for determination of rosmarinic acid by square-wave voltammetry. The laccase catalyzes the oxidation of rosmarinic acid to the corresponding o-quinone, which is electrochemically reduced back to rosmarinic acid at +0.2 V vs. Ag/AgCl. The biosensor based on BMIPF₆ showed a better performance than that based on BMIBF₄. The best performance was obtained with 50:20:15:15% (w/w/w/w) of the graphite powder:laccase:Nujol:BMIPF₆ composition in 0.1 mol L⁻¹ acetate buffer solution (pH 5.0). The rosmarinic acid concentration was linear in the range of 9.99 × 10⁻⁷ to 6.54 × 10⁻⁵ mol L⁻¹ (r = 0.9996) with a detection limit of 1.88 × 10⁻⁷ mol L⁻¹. The recovery study for rosmarinic acid in plant extract samples gave values from 96.1 to 105.0% and the concentrations determined were in agreement with those obtained using capillary electrophoresis at the 95% confidence level. The BMIPF₆-biosensor demonstrated long-term stability (300 days; 920 determinations) and reproducibility, with a relative standard deviation of 0.56%.     

A novel method for flow injection analysis of total antioxidant capacity using enzymatically produced ABTS and biamperometric detector containing interdigitated electrode

Application of interdigitated array microelectrodes as electrochemical sensors for determination of antioxidant capacity is reported. Electrochemical measurements with interdigitated electrodes (IDE) were studied in both stationary solutions and the flow system. The method is based on biamperometric measurements using ABTS⁺|ABTS redox couple in phosphate buffer solution, pH 7.40. During analysis, the ABTS radical cation was enzymatically produced by peroxidase in a tubular flow-through reactor. The performance of bioreactor was tested at different concentrations of immobilized enzyme, ABTS and hydrogen peroxide. The influence of flow rate on proper operation of the bioreactor was also studied. The results of antioxidant activity were determined using Trolox as a standard. The applied IDE detector accomplished good sensitivity of 0.3 nA/μM of Trolox and offered linear range between 20 to 500 μM of Trolox.The comparison of results (R² = 0.9915) for antioxidant activity between spectroscopic and FIA biamperometric measurements by interdigitated electrodes confirmed the applicability of the proposed method for determination of antioxidant capacity.     

Comprehensive high-performance liquid chromatographic method for the measurements of lipophilic antioxidants in human plasma

Owing to the increasing interest in the health effects of antioxidant micronutrients on chronic diseases, a robust and rapid HPLC method for simultaneous measurement of coenzyme Q₁₀ (ubiquinone and ubiquinol), vitamin A (all-trans-retinol), vitamin E (tocopherols and tocotrienols) and carotenoids (lutein, zeaxanthin, β-cryptoxanthin, lycopene and β-carotene) was developed. Sample preparation and analytical conditions that would affect solubility and stability of these antioxidants were investigated and optimized. The mobile phase used was made up of acetonitrile, methanol, ethanol and tert-butanol without corrosive additives such as ammonium perchlorate and perchloric acid. Our results show that using two C₁₈ columns coupled with photodiode array, fluorescence and electrochemical detection, a comprehensive spectrum of 16 lipid-soluble antioxidants in 30 μL of plasma could be separated and quantified within 30 min. The chromatographic run time was about 3-fold faster and the sample size was about 5-fold smaller than when assays were performed separately using existing methods. The present method will be useful for dietary habit studies and for antioxidant status investigations.     

A new approach for the sequential injection spectrophotometric determination of the total antioxidant activity

A sequential injection system based on the ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic-acid) methodology was developed. The proposed method, incorporating a mixing chamber in the side port of the selection valve, was evaluated to measure the total antioxidant activity of several beverages and foods.The ABTS⁺ is generated by oxidation of ABTS with potassium persulfate and is reduced in presence of hydrogen-donating antioxidants converting into a colourless product. The applicability of the developed method was tested by measurement of the antioxidant activity of pure compounds as well as by analysing complex food and beverage samples. The antioxidant activity was presented as l(+) ascorbic acid equivalence. The values obtained by this methodology were not significantly different from the results obtained by the original spectrophotometric ABTS assay. For most of the studied antioxidants, antioxidant activity varied with pH and dilution. The proposed SIA system is suitable for screening direct or diluted total antioxidant activity of pure compounds or food samples.     

Analysis of the antioxidant activity of an indole library: cyclic voltammetry versus ROS scavenging activity

The electrochemical behavior of an indole library of compounds, including several tryptophan and tryptamine derivatives previously demonstrated to be active against several reactive oxygen species (ROS), was investigated. For this purpose, a voltammetry study was undertaken and the oxidation potential was correlated to the scavenging activity reported for the studied indoles. All the compounds showed an oxidation potential peak lower than that observed for indole (Epox = 1.035 V), but higher than that described for the antioxidant melatonin (Epox = 0.715 V). The electrochemical behavior showed a high correlation with the scavenging activity of peroxyl radical, for selected compounds.     

Ultrasound extracted flavonoids from four varieties of Portuguese red grape skins determined by reverse-phase high-performance liquid chromatography with electrochemical detection

Several flavonoids present in red grape skins from four varieties of Portuguese grapes were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) with electrochemical detection (ECD). Extraction of flavonoids from red grape skins was performed by ultrasonication, and hydrochloric acid in methanol was used as extraction solvent. The developed RP-HPLC method used combined isocratic and gradient elution with amperometric detection with a glassy carbon-working electrode. Good peak resolution was obtained following direct injection of a sample of red grape extract in a pH 2.20 mobile phase. Eleven different flavonoids: cyanidin-3-O-glucoside (kuromanin), delphinidin-3-O-glucoside (myrtillin), petunidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside (oenin), (+)-catechin, rutin, fisetin, myricetin, morin and quercetin, can be separated in a single run by direct injection of sample solution. The limit of detection obtained for these compounds by ECD was 20-90 pg/L, 1000 times lower when compared with photodiode array (PDA) limit of detection of 12-55 ng/L. RP-HPLC-ECD was characterized by an excellent sensitivity and selectivity, and appropriate for the simultaneous determination of these electroactive phenolic compounds present in red grape skins.     

Novel laccase redox mediators

The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4-methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3′-sulfophenyl)-3-methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3′-Sulfophenyl)-3-methylpyrazolone-5 was about 30–40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commerical interest for oxidoreductase-catalyzed biodegradation of organic compounds.