Characterization of plant phenolic compounds by cyclic voltammetry

Phenolic acids and flavonoids were characterized by cyclic voltammetry and total antioxidant activity in the reaction with the ABTS cation radical. Anode peak voltages (E_ap) and their pH dependences were determined for the studied phenolic acids and flavonoids. The E_ap and Trolox equivalent antioxidant capacity (TEAC) values were found to correlate for polyphenols, which react with the ABTS cation radical in two steps. Correlation between the half-wave potential (E_1/2) and TEAC was determined for electrochemically irreversible compounds. Mechanisms of the reaction of phenolics on the electrode involving one-and two-electron oxidation are proposed.     

Antioxidant activity applying an improved ABTS radical cation decolorization assay.

A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.     

Measurement of relative antioxidant activity of compounds: a methodological note

The relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS.+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS.+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS.+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS.+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.     

Measurement of relative antioxidant activityof compounds: a methodological note

Abstract

The relativeThe relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS^?+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS^?+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS^?+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS^?+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.     

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS*+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like L-tryptophan and derivatives (N-acetyltryptophan, L-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-beta-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS*+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic beta-carbolines (pyridoindoles), that did not scavenge ABTS*+. Radical scavenger activity of indoles against ABTS*+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

The antioxidant activities of seasonings used in Asian cooking. Powerful antioxidant activity of dark soy sauce revealed using the ABTS assay

Scavenging of the ABTS (2,2'-azinobis[3-ethylbenzothiazoline-6-sulphonate])-derived nitrogen-centred radical cation (ABTS^•+) was used to compare the total antioxidant activities of several seasonings used in Asian cooking. The results were expressed as Trolox equivalent antioxidant capacity (TEAC). The TEAC activities of dark soy sauces were found to be exceptionally high. In evaluating the TEAC of commercial products, attention must be paid to the addition of preservatives by manufacturers to the seasonings tested. Sodium benzoate (a preservative added to several seasonings) did not react significantly with ABTS^•+, but the sulphite content of certain white wines may have led to an over-estimation of their TEAC.     

The antioxidant activities of seasonings used in Asian cooking. Powerful antioxidant activity of dark soy sauce revealed using the ABTS assay

Scavenging of the ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulphonate])-derived nitrogen-centred radical cation (ABTS^?+) was used to compare the total antioxidant activities of several seasonings used in Asian cooking. The results were expressed as Trolox equivalent antioxidant capacity (TEAC). The TEAC activities of dark soy sauces were found to be exceptionally high. In evaluating the TEAC of commercial products, attention must be paid to the addition of preservatives by manufacturers to the seasonings tested. Sodium benzoate (a preservative added to several seasonings) did not react significantly with ABTS^?+, but the sulphite content of certain white wines may have led to an over-estimation of their TEAC.     

Flavonoid B-ring chemistry and antioxidant activity: fast reaction kinetics

Rapid scavenging of the model stable radical cation, ABTS(*+), has been applied to screen for the antioxidant activity of flavonoids. The reaction follows two distinct phases. For compounds with a monophenolic B-ring there is a rapid initial phase of reduction of ABTS(*+) within 0.1 s with no further change in the subsequent 2.9 s. In contrast, compounds with a catechol-containing B ring follow a fast initial scavenging phase with a slow secondary phase. Flavonoids with an unsubstituted B ring do not react within this time scale. The findings suggest that the structure of the B ring is the primary determinant of the antioxidant activity of flavonoids when studied through fast reaction kinetics.     

Endogenous and Dietary Indoles: A Class of Antioxidants and Radical Scavengers in the ABTS Assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS^?+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like l-tryptophan and derivatives (N-acetyltryptophan, l-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-β-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9?mM, usually higher than that for Trolox and ascorbic acid (1?mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS^?+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic β-carbolines (pyridoindoles), that did not scavenge ABTS^?+. Radical scavenger activity of indoles against ABTS^?+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer

Cancer prevention and treatment using traditional Chinese medicines have attracted increasing interest. This study characterizes antioxidant activity and phenolic compounds of traditional Chinese medicinal plants associated with anticancer, comprising 112 species from 50 plant families. The improved ABTS(*+) method was used to systematically assess the total antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC) of the medicinal extracts. The TEAC values and total phenolic content for methanolic extracts of herbs ranged from 46.7 to 17,323 micromol Trolox equivalent/100 g dry weight (DW), and from 0.22 to 50.3 g of gallic acid equivalent/100 g DW, respectively. A positive, significant linear relationship between antioxidant activity and total phenolic content (all R(2) values>/=0.95) showed that phenolic compounds were the dominant antioxidant components in the tested medicinal herbs. Major types of phenolic compounds from most of the tested herbs were preliminarily identified and analyzed, and mainly included phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids. These medicinal herbs exhibited far stronger antioxidant activity and contained significantly higher levels of phenolics than common vegetables and fruits. Traditional Chinese medicinal plants associated with anticancer might be potential sources of potent natural antioxidants and beneficial chemopreventive agents.     

Antioxidant properties of catechins and proanthocyanidins: Effect of polymerisation, galloylation and glycosylation

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.     

Antioxidant properties of catechins and proanthocyanidins: Effect of polymerisation,galloylation and glycosylation

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2′-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.     

Selective ABTS radical-scavenging activity of prenylated flavonoids from Cudrania tricuspidata

The antioxidative properties of five prenylated flavonoids, including new flavanone (2), from the root bark of Cudrania tricuspidata were examined against the ABTS, DPPH, and hydroxyl radicals. In most of the assays to determine their antioxidative properties, the ABTS activity was strongly correlated with DPPH because both methods are responsible for the same chemical property of hydrogen- or electron-donation to the antioxidant. On the other hand, the prenylated flavonoids (1-5) acted differently with both methods; namely, all the prenylated flavonoids strongly scavenged the ABTS radical (IC(50) < 10 microM), while they were inactive against the DPPH radical (IC(50) > 300 microM). Even though isolated 5,7,2',4',-tetrahydroxy-6,5'-diprenylflavanone (3) showed weak reducing power (746 mV) by cyclic voltammetry when compared to quercetin (394 mV), both had similar ABTS activity (IC(50) < 5 microM).     

Assessment of antioxidant activity by using different in vitro methods

In this study, six common tests for measuring antioxidant activity were evaluated by comparing four antioxidants and applying them to beverages (tea and juices): Trolox equivalent antioxidant capacity assay (TEAC I-III assay), Total radical-trapping antioxidant parameter assay (TRAP assay), 2,2-diphenyl- l -picrylhydrazyl assay (DPPH assay), N , N -dimethyl- p -phenylendiamine assay (DMPD assay), Photochemiluminescence assay (PCL assay) and Ferric reducing ability of plasma assay (FRAP assay). The antioxidants included gallic acid representing the group of polyphenols, uric acid as the main antioxidant in human plasma, ascorbic acid as a vitamin widely spread in fruits and Trolox ® as water soluble vitamin E analogue. The six methods presented can be divided into two groups depending on the oxidising reagent. Five methods use organic radical producers (TEAC I-III, TRAP, DPPH, DMPD, PCL) and one method works with metal ions for oxidation (FRAP). Another difference between these tests is the reaction procedure. Three assays use the delay in oxidation and determine the lag phase as parameter for the antioxidant activity (TEAC I, TRAP, PCL). They determine the delay of radical generation as well as the ability to scavenge the radical. In contrast, the assays TEAC II and III, DPPH, DMPD and FRAP analyse the ability to reduce the radical cation (TEAC II and III, DPPH, DMPD) or the ferric ion (FRAP). The three tests acting by radical reduction use preformed radicals and determine the decrease in absorbance while the FRAP assay measures the formed ferrous ions by increased absorbance. Gallic acid was the strongest antioxidant in all tests with exception of the DMPD assay. In contrast, uric acid and ascorbic acid showed low activity in some assays. Most of the assays determine the antioxidant activity in the micromolar range needing minutes to hours. Only one assay (PCL) is able to analyse the antioxidant activity in the nanomolar range. Black currant juice showed highest antioxidant activity in all tests compared to tea, apple juice and tomato juice. Despite these differences, results of these in vitro assays give an idea of the protective efficacy of secondary plant products. It is strongly recommended to use at least two methods due to the differences between the test systems investigated.     

湘西“蒿菜粑粑”原料植物鼠麴草总黄酮提取物体外抗氧化活性研究

为了解湘西特色食品“蒿菜粑粑”原料植物鼠麴草(Gnaphalium affine)总黄酮提取物体外抗氧化能力,采用DPPH、ABTS自由基清除实验,还原力实验和抑制β-胡萝卜素褪色实验等方法,测定鼠麴草总黄酮抗氧化活性。结果显示,鼠麴草总黄酮母液中总黄酮浓度为7.01 mg·mL–1mg/mL;总黄酮提取物对DPPH、ABTS自由基有较好的清除能力,其半数抑制浓度(IC50)分别为16.30 mg·L–1、30.16 mg·L–1,将胡萝卜素相对吸光度降为50%的时间延长至67.49 min,在还原能力、延缓胡萝卜素褪色和抑制脂质过氧化上也有较好效果。鼠麴草总黄酮提取物具有良好的体外抗氧化活性,可作为优质食用植物资源进一步开发与推广。     

The compositional characterisation and antioxidant activity of fresh juices from sicilian sweet orange (Citrus sinensis L. Osbeck) varieties

Epidemiological evidence has suggested that consumption of fruit and vegetables reduces the risk of both cancer and cardiovascular diseases, potentially through the biological actions of components such as vitamin C, vitamin E, flavonoids and carotenoids. Citrus species are extremely rich sources in vitamin C and flavanones, a class of compounds which belongs to the flavonoids family. A comparison of the phenolic compositions, the ascorbic acid contents and the antioxidant activities of fresh Sicilian orange juices from pigmented (Moro, Tarocco and Sanguinello) and non-pigmented (Ovale, Valencia and Navel) varieties of orange (Citrus sinensis L. Osbeck), was undertaken. The simultaneous characterisation and quantification of the major flavanone, anthocyanin and hydroxycinnamate components were attained by HPLC with diode array detection. Differences between varieties in terms of the flavanone glycoside content, particularly hesperidin, were observed, with the Tarocco juices reporting the highest content. Furthermore, cyanidin-3-glucoside and cyanidin-3-(6"-malonyl)-glucoside were predominant in all the pigmented varieties, but their concentration was higher in the juices of the Moro variety. Quantitatively, the major antioxidant component of all juices was ascorbic acid and its concentration was significantly correlated (r = 0.74, P < 0.001) with the total antioxidant activity of the juices, determined in vitro using the ABTS radical cation decolorization assay. Similarly, hydroxycinnamates (r = 0.73, P < 0.01) and anthocyanins (r = 0.98, P < 0.001) content showed a good correlation with the determined antioxidant capacity. Therefore orange juices, particularly those rich in anthocyanins, may represent a significant dietary source of flavonoids.     

Antioxidant properties of gallocatechin and prodelphinidins from pomegranate peel

Gallocatechins and a range of prodelphinidins were purified by high performance liquid chromatography from pomegranate peel. Gallocatechin, gallocatechin-(4-8)-catechin, gallocatechin-(4-8)-gallocatechin and catechin-(4-8)-gallocatechin were all identified, purified and quantified by LC-DAD-MS and MS-MS. The antioxidant properties of these compounds were assessed using two methods: (i) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; and (ii) scavenging of the radical cation of 2,2-azinobis (3-ethyl-benzothiazoline-6-sulphonate, ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). The prodelphinidin dimers were potent antioxidants in the aqueous phase, being much more effective than the gallocatechin monomer. However, in the lipid phase, only one of the dimers (gallocatechin-(4-8)-catechin) was significantly more effective than the monomer in the inhibition of lipid peroxidation of phosphatidylcholine vesicles. This study represents the first report on the antioxidant properties of prodelphinidins.     

A simple, post-additional antioxidant capacity assay using adenosine triphosphate-stabilized 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation in a G-quadruplex DNAzyme catalyzed ABTS-H2O2 system

The scavenging of 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation (ABTS(+)) by antioxidants has been widely used in antioxidant capacity assay. Because of ABTS(+) disproportionation, however, this radical cannot be prepared on a large scale and stored long-term, making it unsuitable for high-throughput detection and screening of antioxidants. We developed a modified "post-additional" antioxidant capacity assay. This method possessed two remarkable features: First, instead of natural peroxidases, an artificial enzyme, G-quadruplex DNAzyme, was used for the preparation of ABTS(+), thus greatly reducing the cost of the assay, and eliminating the strict demand for the storage of enzymes. Second, an ABTS(+) stabilizer, adenosine triphosphate (ATP), was used. In the presence of ATP, the disproportionation of ABTS(+) was effectively inhibited, and the lifetime of this radical cation was prolonged about 6-fold (12 days versus 2 days), making the large-scale preparation of ABTS(+) possible. Utilizing this method, the antioxidant capacities of individual antioxidants and real samples can be quantified and compared easily. In addition, this method can be developed as a high-throughput screening method for antioxidants. The screening results could even be judged by the naked eye, eliminating the need for expensive instruments.     

Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method

BACKGROUND: Tests measuring the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of the organism's capability to counteract reactive species known as prooxidants, resist oxidative damage and combat oxidative stress-related diseases. The selected chromogenic redox reagent for the assay of human serum should be easily accessible, stable, selective, respond to all types of biologically important antioxidants such as ascorbic acid, alpha-tocopherol, beta-carotene, reduced glutathione (GSH), uric acid and bilirubin, regardless of chemical type or hydrophilicity. Currently, there is no rapid method for total antioxidant assay of human serum meeting the above criteria.METHODS: Our recently developed cupric reducing antioxidant capacity (CUPRAC) spectrophotometric method for a number of polyphenols and flavonoids using the copper(II)-neocuproine reagent in ammonium acetate buffer was now applied to a complete series of plasma antioxidants for the assay of total antioxidant capacity (TAC) of serum, and the resulting absorbance at 450 nm was recorded either directly (e.g. for ascorbic acid, alpha-tocopherol and glutathione) or after incubation at 50 degrees C for 20 min (e.g. for uric acid, bilirubin and albumin), quantitation being made by means of a calibration curve. The lipophilic antioxidants, alpha-tocopherol and beta-carotene, were assayed in dichloromethane (DCM). Lipophilic antioxidants of serum were extracted with n-hexane from an ethanolic solution of serum subjected to centrifugation. Hydrophilic antioxidants of serum were assayed after perchloric acid precipitation of proteins in the centrifugate.Results: The molar absorptivities, linear ranges and trolox equivalent antioxidant capacity (TEAC) coefficients of the serum antioxidants were established with respect to the CUPRAC spectrophotometric method, and the results (TEAC, or TEAC coefficients) were evaluated in comparison to the findings of the ABTS/TEAC reference method using persulfate as oxidant. As for hydrophilic phase, a linear correlation existed between the CUPRAC and ABTS findings (r=0.58), contrary to current literature reporting that either serum ORAC or serum ferric reducing antioxidant potency (FRAP) does not correlate at all with serum TEAC. The analytical responses of serum antioxidants were shown to be additive, enabling a TAC assay. The intra- and inter-assay CVs were 0.7 and 1.5%, respectively, for serum.Conclusions: The CUPRAC assay proved to be efficient for glutathione and thiol-type antioxidants, for which the FRAP test was nonresponsive. The findings of CUPRAC completely agreed with those of ABTS-persulfate for lipophilic phase. The additivity of absorbances of all the tested antioxidants confirmed that antioxidants in the CUPRAC test did not chemically interact among each other so as to cause an intensification or quenching of the theoretically expected absorbance. As a distinct advantage over other electron-transfer based assays (e.g. Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH close to the physiological pH, favourable redox potential, accessibility and stability of reagents and applicability to lipophilic antioxidants as well as hydrophilic ones.