Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay

We report on the application of a simple and versatile antioxidant capacity assay for dietary polyphenols, vitamin C and vitamin E utilizing the copper(II)-neocuproine (Cu(II)-Nc) reagent as the chromogenic oxidant, which we term the CUPRAC (cupric reducing antioxidant capacity) method. It involves mixing the antioxidant solution (directly or after acid hydrolysis) with solutions of CuCl₂, neocuproine, and ammonium acetate at pH 7, and measuring the absorbance at 450 nm after 30 min. Slowly reacting antioxidants required an incubation at 50 °C for 20 min for color development. The flavonoid glycosides were hydrolyzed to their corresponding aglycones by refluxing in 1.2 M HCl-containing 50% MeOH for fully exhibiting their antioxidant potencies. Certain compounds also needed incubation after acid hydrolysis for color development. The CUPRAC absorbances of mixture constituents were additive, indicating lack of chemical deviations from Beer’s law. The CUPRAC antioxidant capacities of a wide range of polyphenolics are reported in this work and compared to those found by ABTS/persulfate and Folin assays. The trolox-equivalent capacities of the antioxidants were linearly correlated (r = 0.8) to those found by ABTS but not to those of Folin. The highest antioxidant capacities in the CUPRAC method were observed for epicatechin gallate, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, caffeic acid, epicatechin, gallic acid, rutin, and chlorogenic acid in this order, in accordance with theoretical expectations. The experiences of other CUPRAC users also are summarized. Correspondence: Reşat Apak, Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, TR-34320 Istanbul, Turkey     

Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection

A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 μM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples.     

Combined HPLC-CUPRAC (cupric ion reducing antioxidant capacity) assay of parsley, celery leaves, and nettle

This study aims to identify the essential antioxidant compounds present in parsley (Petroselinum sativum) and celery (Apium graveolens) leaves belonging to the Umbelliferae (Apiaceae) family, and in stinging nettle (Urtica dioica) belonging to Urticaceae family, to measure the total antioxidant capacity (TAC) of these compounds with CUPRAC (cupric ion reducing antioxidant capacity) and ABTS spectrophotometric methods, and to correlate the TAC with high performance liquid chromatography (HPLC) findings. The CUPRAC spectrophotometric method of TAC assay using copper(II)-neocuproine (2,9-dimethyl-1,10-phenanthroline) as the chromogenic oxidant was developed in our laboratories. The individual antioxidant constituents of plant extracts were identified and quantified by HPLC on a C18 column using a modified mobile phase of gradient elution comprised of MeOH-0.2% o-phosphoric acid and UV detection for polyphenols at 280 nm. The TAC values of HPLC-quantified antioxidant constituents were found, and compared for the first time with those found by CUPRAC. The TAC of HPLC-quantified compounds accounted for a relatively high percentage of the observed CUPRAC capacities of plant extracts, namely 81% of nettle, 60-77% of parsley (in different hydrolyzates of extract and solid sample), and 41-57% of celery leaves (in different hydrolyzates). The CUPRAC total capacities of the 70% MeOH extracts of studied plants (in the units of mmol trolox g⁻¹ plant) were in the order: celery leaves > nettle > parsley. The TAC calculated with the aid of HPLC-spectrophotometry did not compensate for 100% of the CUPRAC total capacities, because all flavonoid glycosides subjected to hydrolysis were either not detectable with HPLC, or not converted to the corresponding aglycons (i.e., easily detectable and quantifiable with HPLC) during the hydrolysis step.     

Measurement of xanthine oxidase inhibition activity of phenolics and flavonoids with a modified cupric reducing antioxidant capacity (CUPRAC) method

Various dietary polyphenolics have been found to show an inhibitory effect on xanthine oxidase (XO) which mediates oxidative stress-originated diseases because of its ability to generate reactive oxygen species (ROS), including superoxide anion radical (O₂⁻) and hydrogen peroxide. XO activity has usually been determined by following the rate of uric acid formation from xanthine-xanthine oxidase (X-XO) system using the classical XO activity assay (UV-method) at 295 nm. Since some polyphenolics have strong absorption from the UV to visible region, XO-inhibitory activity of polyphenolics was alternatively determined without interference by directly measuring the formation of uric acid and hydrogen peroxide using the modified CUPRAC (cupric reducing antioxidant capacity) spectrophotometric method at 450 nm. The CUPRAC absorbance of the incubation solution due to the reduction of Cu(II)-neocuproine reagent by the products of the X-XO system decreased in the presence of polyphenolics, the difference being proportional to the XO inhibition ability of the tested compound. The structure-activity relationship revealed that the flavones and flavonols with a 7-hydroxyl group such as apigenin, luteolin, kaempferol, quercetin, and myricetin inhibited XO-inhibitory activity at low concentrations (IC₅₀ values from 1.46 to 1.90 μM), while the flavan-3-ols and naringin were less inhibitory. The findings of the developed method for quercetin and catechin in the presence of catalase were statistically alike with those of HPLC. In addition to polyphenolics, five kinds of herbs were evaluated for their XO-inhibitory activity using the developed method. The proposed spectrophotometric method was practical, low-cost, rapid, and could reliably assay uric acid and hydrogen peroxide in the presence of polyphenols (flavonoids, simple phenolic acids and hydroxycinnamic acids), and less open to interferences by UV-absorbing substances.     

A microfluidic system for evaluation of antioxidant capacity based on a peroxyoxalate chemiluminescence assay

A microfluidic system incorporating chemiluminescence detection is reported as a new tool for measuring antioxidant capacity. The detection is based on a peroxyoxalate chemiluminescence (PO-CL) assay with 9,10-bis-(phenylethynyl)anthracene (BPEA) as the fluorescent probe and hydrogen peroxide as the oxidant. Antioxidant plugs injected into the hydrogen peroxide stream result in inhibition of the CL emission which can be quantified and correlated with antioxidant capacity. The PO-CL assay is performed in 800-μm-wide and 800-μm-deep microchannels on a poly(dimethylsiloxane) (PDMS) microchip. Controlled injection of the antioxidant plugs is performed through an injection valve. Of the plant-food based antioxidants tested, β-carotene was found to be the most efficient hydrogen peroxide scavenger (SA _HP of 3.27 × 10⁻³ μmol⁻¹ L), followed by α-tocopherol (SA _HP of 2.36 × 10⁻³ μmol⁻¹ L) and quercetin (SA _HP of 0.31 × 10⁻³ μmol⁻¹ L). Although the method is inherently simple and rapid, excellent analytical performance is afforded in terms of sensitivity, dynamic range, and precision, with RSD values typically below 1.5%. We expect our microfluidic devices to be used for in-the-field antioxidant capacity screening of plant-sourced food and pharmaceutical supplements. Figure Assembled PDMS microchip sandwiched between two glass plates with the top plate containing capillary reservoirs     

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.     

Evaluation of ORAC methodologies in determination of antioxidant capacity of binary combinations of quercetin and 3-(3,4,5-trihydroxybenzoyl) coumarin derivatives

Antioxidant capacity is used to refer to ability of compounds to react with free radicals, it is also described as ability to inhibit oxidation processes. There are different methods that evaluate the antioxidant capacity of compounds of natural origin or extracts. ORAC methodologies measure the ability to transfer hydrogen atoms to RO·/ROO· radicals generated by the AAPH thermolysis, in presence of a probe that accounts for oxidation of antioxidant. Despite extensive use of these methods to assess antioxidant capacity, they have been questioned by type of radical generated and information they can deliver, especially if it is a mixture of compounds. In this work, antioxidant capacity of binary combinations of quercetin with synthetic 3-phenylcoumarins was evaluated through ORAC-FL, PGR and quantification techniques after oxidation kinetics via free radicals, through HPLC. It was found through ORAC-FL that derived 3-(3,4,5-trihydroxybenzoyl) coumarin have greater antioxidant capacity than Trolox: 1 < 2 < 3 < quercetin. Determination of ORAC-FL indices of combination showed an antagonistic effect among the antioxidants. Also, ORAC-PGR method did not allow determination of antioxidant capacity of compounds, separately, indicated high reactivity of compounds. However, unusual behaviors were observed in combinations, unable to explain antagonistic effect observed in ORAC-FL. HPLC oxidation kinetics analysis showed that in the combination the consumption of the most reactive antioxidant dominated the antioxidant capacity and followed a similar trend as observed by ORAC-FL. Therefore, ORAC methodologies would not be useful in characterizing antioxidant capacity of mixture in relation to reactivity of metabolites present therein, but in relation to amount of hydroxyl groups available.     

Comparative evaluation of antioxidant capacities of thiol-based antioxidants measured by different in vitro methods

Thiol-type compounds are an important class of strong antioxidants and main determinants of total antioxidant capacity (TAC) of cellular homogenates. The TAC of thiol mixtures and the corresponding TEAC (trolox equivalent antioxidant capacity) values of individual thiols were determined by the CUPRAC (CUPric Reducing Antioxidant Capacity) method, and the results were compared with those found by reference assays for method validation. Synthetic mixtures of thiols were prepared, and the expected and found TAC values (in mM trolox (TR) equivalents) of these mixtures showed a good agreement. The technique of standard additions was performed for thiol mixtures and human serum, and the absorbance results confirmed that apparent chemical deviations from Beer's law were absent in the system. The CUPRAC results were compared with those of reference methods, namely 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/persulphate and Ferric Reducing Antioxidant Power (FRAP). As being a most important thiol (-SH) peptide at in vivo conditions, glutathione (GSH) showed a TEAC value of 0.57 in the CUPRAC method, as opposed to the corresponding value (1.51) in the ABTS/persulphate method. The ABTS/persulphate result was not in accordance with the reversible 1-e oxidation of GSH to the corresponding disulfide that is expected to occur under physiological conditions. FRAP did not give consistent results, and even at relatively high concentrations of GSH, the TEAC_FRAP value was only 0.07. The thiol-type antioxidant-bearing pharmaceuticals of Brunac eye drop, Trom and Mentopin effervescent tablets containing N-acetyl-l-cysteine (NAC) were assayed with HPLC for comparison, and the obtained results for NAC were in accordance with those found with CUPRAC.     

Determination of polyphenols in three Capsicum annuum L. (bell pepper) varieties using high-performance liquid chromatography-tandem mass spectrometry: their contribution to overall antioxidant and anticancer activity

A mixture of polyphenol components was isolated from the fruits of C. annuum L. cv. Cupra, C. annuum L. cv. Orange glory, and C. annuum L. cv. ST4712 (CLST), via 70% methanol extraction followed by column chromatography over silica gel. The polyphenol components of the mixture were analyzed via HPLC-MS/MS and compared with the reported data. Three cinnamic acid derivatives and five flavonoid components in the fruits of the three varieties were identified for the first time in this study. The antioxidant activity and anticancer effect of the polyphenol mixtures of the three fruits were determined. The antioxidant and anticancer activities of CLST were substantially higher than those of C. annuum L. cv. Cupra and C. annuum L. cv. Orange glory. The high activities of CLST were attributed to the much higher concentration of quercetin derivatives in CLST.     

Indirect electrochemical determination of antioxidant capacity with hexacyanoferrate(III) reduction using a gold nanoparticle-coated o-phenylenediamine-aniline copolymer electrode

In spite of the many unstandardized literature methods for the determination of the antioxidant activity/capacity (AOA/AOC) of food extracts, there are a very limited number of documented voltammetric nanosensors, despite the fact that commercial electrochemical devices for rapid AOA estimation are on the rise. The mechanism of the developed sensor is based on the chemical reduction of hexacyanoferrate(III) to hexacyanoferrate(II) by antioxidants, followed by the decrement of the cathodic current intensity of hexacyanoferrate(III) in proportion to antioxidant concentration. During voltammetric measurements, the surface of the glassy carbon electrode was coated with an o-phenylenediamine-aniline copolymer and gold nanoparticles were accumulated on this electrode surface to increase the conductivity. It was shown that the developed electrode gave a reversible voltammogram for the hexacyanoferrate(III)/(II) redox couple, and that the cathodic peaks due to strong antioxidants having a standard redox potential less than that of this couple (E^o < 0.36 V) continuously emerged at very close peak potentials. Single antioxidants as well as binary–ternary mixtures were analyzed with this electrode using square wave voltammetry. The trolox-equivalent antioxidant capacities of selected antioxidants were evaluated with this electrode. The modified voltammetric sensor allowed precise measurement of the total antioxidant capacity of plant tea samples such as green tea, lime, and coral moss, and was not interfered by the food preservative sulfite. The results of the developed voltammetric sensor were statistically compared with those of a reference differential pulse voltammetry-cupric reducing antioxidant capacity electrochemical method established in literature.     

Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium

Total protein assay was made using copper(II)–neocuproine (Nc) reagent in alkaline medium (with the help of a hydroxide-carbonate-tartarate solution) after 30 min incubation at 40 °C. The absorbance of the reduction product, Cu(I)–Nc complex, was recorded at 450 nm against a reagent blank. The absorptivity of the developed method for bovine serum albumin (BSA) was 0.023 l mg⁻¹ cm⁻¹, greater than that of Lowry assay (0.0098), and much greater than that of Cu(II)–bicinchoninic acid (BCA) assay (0.00077). The linear range of the developed method (8–100 mg l⁻¹ BSA) was as wide as that of Lowry, and much wider than that of BCA (200–1000 mg l⁻¹ BSA) assay. The sensitivity of the method was greater than those of Cu-based assays (biuret, Lowry, and BCA) with a LOD of 1 mg l⁻¹ BSA. The within-run and between-run precisions as RSD were 0.73 and 1.01%, respectively. The selectivity of the proposed method for protein was much higher than those of dye-binding and Lowry assays: Most common interferents to other protein assays such as tris, ethanolamine, deoxycholate, CsCl, citrate, and triton X-100 were tolerated at 100-fold concentrations in the analysis of 10 mg l⁻¹ BSA, while the tolerance limits for other interferents, e.g., (NH₄)₂SO₄ and acetylsalicylic acid (50-fold), SDS (25-fold), and glycerol (20-fold) were at acceptable levels. The redox reaction of Cu(II)–Nc as an outer-sphere electron transfer agent with the peptide bond and with four amino acid residues (cystine, cysteine, tryptophan, and tyrosine) was kinetically more favourable than that of Cu(II) alone in the biuret assay. Since the reduction product of Cu(II) with protein, i.e., Cu(I), was coordinatively saturated with Nc in the stable Cu(Nc)₂⁺ chelate, re-oxidation of the formed Cu(I) with Fenton-like reactions was not possible, thereby preventing a loss of chromophore. After conventional protein extraction, precipitation, and redissolution procedures, the protein contents of the minced meat (veal and turkey), sardine, various milk products, and egg white were analyzed with the proposed and Lowry methods, and the results correlated appreciably (r = 0.98). The method was validated by Kjeldahl analyses of the tested samples; the data sets of complex samples assayed by Cu(II)–Nc and Lowry correlated to the findings of Kjeldahl yielded correlation coefficients r = 0.96 and 0.97, respectively, with slopes being close to 1. Interferences of glucose and thiol compounds at relatively low concentrations could be compensated for by selecting a lower alkaline pH (i.e., pH 10) at a cost of slightly reduced sensitivity and adding an identical amount of interferent to the reagent blank, respectively, since the absorbances due to BSA and interferent were additive. Thus a novel spectrophotometric method for total protein assay using a stable reagent and chromophore, which was simple, rapid, sensitive, flexible, and relatively selective, was developed, and applied to a variety of food products.     

Spectrophotometric determination of ascorbic acid by the modified CUPRAC method with extractive separation of flavonoids-La(III) complexes

The proposed method for ascorbic acid: AA (Vitamin C) determination is based on the oxidation of AA to dehydroascorbic acid with the CUPRAC reagent of total antioxidant capacity assay, i.e., Cu(II)-neocuproine (Nc), in ammonium acetate-containing medium at pH 7, where the absorbance of the formed bis(Nc)-copper(I) chelate is measured at 450 nm. The flavonoids (essentially flavones and flavonols) normally interfering with the CUPRAC procedure were separated with preliminary extraction as their La(III) chelates into ethylacetate (EtAc). The Cu(I)-Nc chelate responsible for color development was formed immediately with AA oxidation. Beer's law was obeyed between 8.0 × 10⁻⁶ and 8.0 × 10⁻⁵ M concentration range, with the equation of the linear calibration curve: A_450 nm = 1.60 × 10⁴C (mol dm⁻³) − 0.0596. The relative standard deviation (R.S.D.) in the analysis of N = 45 synthetic mixtures containing 1.25 × 10⁻² mM AA with flavonoids was 5.3%. The Cu(II)-Nc reagent is a lower redox-potential and therefore more selective oxidant than the Fe(III)-1,10-phenanthroline reagent conventionally used for the same assay. This feature makes the proposed method superior for real samples such as fruit juices containing weak reductants such as citrate, oxalate and tartarate that may otherwise produce positive errors in the Fe(III)-phen method when equilibrium is achieved. The developed method was applied to some commercial fruit juices and pharmaceutical preparations containing Vitamin C + bioflavonoids. The findings of the developed method for fruit juices and pharmaceuticals were statistically alike with those of HPLC. The proposed spectrophotometric method was practical, low-cost, rapid, and could reliably assay AA in the presence of flavonoids without enzymatic procedures open to interferences by enzyme inhibitors.     

Comparative evaluation of Fe(III) reducing power-based antioxidant capacity assays in the presence of phenanthroline, batho-phenanthroline, tripyridyltriazine (FRAP), and ferricyanide reagents

The chemical diversity of antioxidants in complex matrices such as plant extracts makes it difficult to separate and quantify antioxidants from these solutions. Therefore it is desirable to establish methods that can measure the total antioxidant capacity (TAC) levels directly from plant extracts. Iron(III)-based TAC assays, especially the most widely used FRAP (ferric-reducing antioxidant power), play an important role in this regard. However, many problems have been reported in the application of the FRAP assay, the most serious one being the incomplete oxidation of a number of antioxidants during the time protocol of the assay. Thus, six different ferric ion-based total antioxidant capacity (TAC) assays have been comparatively tested, modified, and improved so as to obtain more sensitive and precise results for complex mixtures, namely: 1,10-phenanthroline (o-phen) method (with incubation), batho-phenanthroline method (with incubation), original FRAP method, modified FRAP method (with incubation), original ferricyanide method, and modified ferricyanide method (with incubation). Two new assays in this regard (i.e., o-phen and batho-phen) have been established, and the existing assays (FRAP and ferricyanide) have been modified so as to let the oxidation reactions of antioxidants reach completion. The molar absorptivity for a variety of antioxidants was highest for modified FRAP, batho-phen, and original FRAP methods. The absorption maximum wavelength shifted batochromically to a higher extent for modified ferricyanide, FRAP, and batho-phen procedures, decreasing the possibility of interferences due to organics absorbing in the near-UV range of the visible spectrum where most antioxidant assays are performed. The linear concentration ranges were shown to be further extended and linear correlation coefficients improved with respect to the most widely used ferric-based assay, FRAP. Of the six assays tested and developed, only the modified ferricyanide procedure gave high intercept values and low addivitity of TAC values of constituents in complex mixtures, requiring further attention of method optimization. Thus, it was shown that the most widely used FRAP could be effectively modified, and o-phen, batho-phen, and ferricyanide methods constitute cheaper alternatives to FRAP under certain conditions, with partly improved molar absorptivity (and thus sensitivity) for antioxidants, lower intercept values (and higher precision), broader linear range (and higher flexibility), and better additivity of TAC values of antioxidant constituents in mixtures.     

Stopped-flow method for assessment of pH and timing effect on the ABTS total antioxidant capacity assay

The pH and timing effect on the relative capacity of antioxidant compounds to scavenge the ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)] radical cation (ABTS⁺) expressed as the trolox equivalent antioxidant capacity (TEAC) is assessed for a wide range of antioxidants using a modification of the original ABTS assay at different pHs (4.6, 5.4 and 7.4).To study both fast and slow reacting antioxidants, a stopped-flow method based on a low cost laboratory-made system was used. For most of the tested antioxidants, total antioxidant capacity values were found to be a function of pH and time allowed for the reaction.Structurally similar compounds have the same time and pH-dependent behaviour even if they have significant differences in total antioxidant capacity values. The ABTS⁺ scavenging reaction rate was found to depend strongly on pH for p-coumaric acid, glutathione, BHT and albumin. Quercetin, gallic acid, (+)-catechin and (−)-epicatechin showed the higher activities at all pHs.The stopped-flow method can be utilized for screening of antioxidant compounds of unknown kinetics towards ABTS⁺ at different pHs and the results can be used to predict the total antioxidant capacity of structurally related compounds.     

Kinetic deoxyribose degradation assay and its application in assessing the antioxidant activities of phenolic compounds in a Fenton-type reaction system

Pro-oxidant properties of phenolic antioxidants, which are derived from their iron recycling reactivity, render the traditional deoxyribose degradation assay invalid to assess the hydroxyl radical-scavenging activity in Fenton-type reaction systems. In the present paper, we studied in detail the interactions between iron and phenolic compounds, and established a kinetic deoxyribose method by taking advantage of the distinct difference between the completion time of Fenton reaction and that of the iron-reducing process. With the newly established kinetic method, we investigated the effects of phenolics on hydroxyl radical formation in a Fenton-type system and determined successfully the second rate constants of hydroxyl radical-scavenging reactions. The site-specific and non-site-specific hydroxyl radical-scavenging ability suggested that both direct hydroxyl radical-scavenging potency and iron-chelating capacity accounted for their inhibitory effects on deoxyribose oxidation degradation. This method, more simple, time saving, and applicative than the traditional deoxyribose assay, produces as accurate results (RSD<0.05, with dynamic range from 7.5 to 575 μM) as typical methods, such as radiolysis technology, and may be of significance in evaluating and screen the hydroxyl radical-scavenging antioxidants.     

Green and highly extraction of phenolic compounds and antioxidant capacity from kinkeliba (Combretum micranthum G. Don) by natural deep eutectic solvents (NADESs) using maceration,ultrasound-assisted extraction and homogenate-assisted extraction

Kinkeliba (C. micranthum) is a tropical plant widely used for its tremendous phytochemicals and biological activities. In the present study, three green carboxylic acid-based natural deep eutectic solvents (NADESs) were used to assess the extraction of phenolic compounds in terms of total phenolic content (TPC), total flavonoid content (TFC), individual phenolic compounds and antioxidant capacity (DPPH and FRAP assays) from dried C. micranthum leaves. For the synthesis of NADESs choline chloride was used as hydrogen bond acceptors (HBA) in combination with lactic acid (ChLa), acetic acid (ChAa) and tartaric acid (ChTa) as hydrogen bond donors (HBDs). The conventional solvents including distilled water, pure methanol and pure ethanol were used for comparison. Three extraction methods including maceration extraction (ME), homogenate-assisted extraction (HAE) and ultrasound-assisted extraction (UAE) were tested to determine the best extraction conditions. The solvents combined with the extraction methods were successfully applied for the recovery of phenolic compounds from C. micranthum leaves. ChLa exhibited the highest performance giving the TPC (21.12 ± 0.13–23.62 ± 0.58 mg GAE/g, followed by ChAc (15.49 ± 0.13–18.85 ± 0.39 mg GAE/g), water (17.08 ± 0.32–18.13 ± 0.13 mg GAE/g), ChTa (14.49 ± 0.26–17.44 ± 0.19 mg GAE/g), methanol (7.46 ± 0.45–11.64 ± 0.32 mg GAE/g) and ethanol (2.88 ± 0.39–4.60 ± 0.39 mg GAE/g), respectively. For TFC, ChLa (4.38 ± 0.09–5.01 ± 0.09 mg ECE/g) was the most prominent solvent, followed by ChAc (2.84 ± 0.04–5.01 ± 0.36 mg ECE/g), methanol (1.93 ± 053–4.85 ± 0.04 mg ECE/g), ethanol (1.49 ± 0.36–4.16 ± 0.04 mg ECE/g), ChTa (1.09 ± 0.04–3.22 ± 0.13 mg ECE/g) and water (1.15 ± 0.04–1.37 ± 0.44 mg ECE/g), respectively. The acidic NADESs especially ChLa and ChAa exhibited the best efficiencies compared to the conventional solvents. Furthermore, UAE and HAE provided good extraction efficiency in a short extraction time (30 min) in terms of the TPC, TFC, individual phenolic compounds and the antioxidant capacity compared to ME which gave a similar yield with 12 h of extraction time. Principal component analysis (PCA) showed that C. micranthum extracts could clearly be discriminated in terms of phytochemical compounds and antioxidant capacity and UAE, HAE or ME combined with ChLa ChAc or ChTa were the best choices to higher extraction efficiency.     

Development and validation of a sequential-injection method with chemiluminescence detection for the high throughput assay of the total antioxidant capacity of wines

This work reports a sequential-injection analysis (SIA) method with chemiluminescence (CL) detection for the rapid assay of the total antioxidant capacity (TAC) in wines. The method exploited the Co(II)-catalysed CL reaction of luminol with hydrogen peroxide in alkaline medium. Zones of sample, hydrogen peroxide, catalyst (Co(II) solution) and alkaline luminol were sequentially aspirated into the holding coil of the SIA manifold. Then, the flow was reversed and the stacked zones were directed to the CL detector. As the zones overlapped, antioxidants in the samples scavenged a portion of hydrogen peroxide and the decrease in the CL intensity was monitored and related to the TAC. The chemical and geometric conditions were studied and the method was validated in terms of linearity, accuracy (trueness and precision), matrix effects, signal additivity and robustness. The reproducibility of the method (expressed as the between-days % relative standard deviation) was between 2.5 and 3.4% and the trueness (expressed as the % recovery in wines spiked with gallic acid) was in the range 96.7-97.3%. The sampling frequency was 60 samples h⁻¹. The proposed SIA-CL method was compared with the DPPH method and the Folin-Ciocalteau (FC) method for the analysis of 25 wine samples.     

Electrochemical Determination of the Antioxidant Capacity: The Ceric Reducing/Antioxidant Capacity (CRAC) Assay

The CRAC assay is a direct electron transfer test of antioxidant capacity for several organic compounds. The ability of eight different compounds in reducing Ce⁴⁺ was studied by chronoamperometric measurements of the remaining Ce³⁺ species. The following antioxidant classification was observed: tannic acid quercetin>rutin>gallic acid≈catechin>ascorbic acid>BHA>Trolox. These results agree with others already published and a good correlation (R²=0.937) was found with the classical spectrophotometric FRAP assay. The CRAC assay is simple, fast, free from sample pretreatment and applicable to nontransparent samples.     

Antioxidant capacity and sensory quality of soy-based powder drink mix enriched with functional hydrolysates of swiftlet (Aerodramus fuciphagus)

The enrichment with low amount of bioactive protein of spray-dried edible bird’s nest hydrolysates (EBNH) (3.0 %) in view of its cost and high solubility provided significant value added to the overall in vitro antioxidant capacity of soy-based powder drink mix (PDM). Its beverage (12.5 % concentration, consistency index 0.39 Pa.sⁿ) antioxidant capacity as measured by ABTS and FRAP was comparable (p > 0.05) but significantly higher than antioxidant assays of FCR and DPPH. The respective antioxidant capacity of the PDM beverage in terms of trolox equivalent (TE) and gallic acid equivalent (GAE) were 21.95 TE mg/g, 20.75 TE mg/g, 2.93 TE mg/g and 14.72 GAE mg/g for FRAP, ABTS, DPPH and FCR. Depending on antioxidant assay, EBNH in beverage of PDM contributed an increase in the range of 3.7–9.3 % (which was significant (p < 0.05) according to ABTS and FCR assays) or about 6.0 % to its overall antioxidant capacity. The interaction among the antioxidant activity of all the food product’s ingredients is antagonistic since the difference between the expected and observed total antioxidant potential is significantly higher (p < 0.05) for all antioxidants assays, except FCR. The beverage of PDM has excellent sensory quality. It is sugar free and high protein PDM that has excellent cocoa flavour and possesses sufficient sweetness with acceptable beany aroma and taste when served as hot beverage.     

Development of a cytochrome c-based screen-printed biosensor for the determination of the antioxidant capacity of orange juices

This paper describes the development of an amperometric cytochrome c (cyt c)-based biosensor and its later application to the quantification of the scavenging capacity of antioxidants. The enzymatic biosensor was constructed by covalently co-immobilizing both cyt c and XOD on a mercaptoundecanol/mercaptoundecanoic acid (MU/MUA) mixed self-assembled monolayer (SAM)-modified screen-printed gold electrode. The applicability of this method was shown by analyzing the antioxidant capacity of pure substances, such as ascorbic acid and Trolox, and natural sources of antioxidants, particularly 5 orange juices.