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Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Table 18.1

Antioxidant Composition of Different Types of Cereals


Antioxidant Compounds


Vanillic acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid,

p-coumaric acid, caffeic acid, sinapic acid, tocols (β-tocopherol, and

α-tocopherol), lysophosphatidylcholine

Choline, betain

p-coumaric acid, syringic acid, vanillic acid, protocatechuic acid, caffeic

acid, sinapic acid, α-tocopherol

Vitamin E, γ-oryzanol (Gamma oryzanol is a mixture of substances derived

from rice bran oil, including sterols and ferulic acid), tocols

(γ-tocotrienols, γ-tocopherol, and α-tocopherol), phosphatidylcholine,

sterols (β-sitosterol) similar cysteine and methionine

Cyanidin-3-glucoside, peonidin-3-glucoside

Phytic acid, avenanthramides (alkoloids containing phenolic groups),

tocols (α-tocotrienols, and α-tocopherol), phenolic acids (vanillic acid,

and p-hydroxybenzoic acid), phosphatidylcholine, similar cysteine,

methionine, phytic acid

Benzoic and cinnamic acid derivatives (ferulic acid), proanthocyanidins,

quinines, flavonols, chalcones, flavones, flavanones, amino phenolic

compounds, similar cysteine and methionine

Isoferulic acid, coumaric acid, syringic acid, p-hydroxybenzoic acid,

caffeic acid, sinapic acid, dimer 8-O-4-di ferulic acid,

phosphatidylinositol, tocols (β-tocopherol, and α-tocopherol), similar

cysteine and methionine

Tannins, anthocyanins (apigeninidin, luteolinidin), apigenin, luteolin,

vanillic acid, p-hydroxybenzoic acid, naringenin, carotenoids (lutein,

zeaxanthin, β-carotene), α-tocopherol, lysophospholipid

Flavones (C-glycosylvitexin, vitexin, and glycosylorientin), tocols

(α-tocotrienols, and α-tocopherol), lysophosphatidylcholine, and


Toasted wheat



Black rice






Major Component

Ferulic acid

Ferulic acid

trans-Ferulic acid

Ferulic acid and caffeic


Ferulic acid and

p-coumaric acid

Ferulic acid

p-coumaric acid and

ferulic acid

Ferulic acid , p-coumaric

acid, cinnamic acid and

gentisic acid

Source: White, P. J. and Xing, Y., Natural Antioxidants: Chemistry Health Effects, and Applications, 25–63,

Champaign, IL: AOCS Press, 1997.

(quercetin); (C6 –C3)2, lignans (matairesinol); (C6 –C3–C6)2, biflavonoids (agathisflavone); (C6 –C3)n,

lignins; (C6 –C3–C6)n, condensed tannins (procyanidin) (Harborne and Simmonds 1964).

Fruits and vegetables are usually mentioned as primary sources of phenolic compounds in food but

different cereals may be a good source of phenolic compounds as well. The cereals of primary economic

and nutritional importance in developed countries include wheat, rye, barley, oat and rice, whereas corn,

millet, and sorghum (that are more consumed in developing countries) are consumed much less (Stratil

et al. 2007). Whole grain cereals contain a much wider range of compounds with potential antioxidant

effects than do refined cereals (Table 18.1). These include vitamin E (mainly in the germ), folates, minerals (iron, zinc), trace elements (selenium, copper, and manganese), carotenoids, phytic acid, lignin and

other compounds such as betaine, choline, sulfur amino acids, alkylresorcinols, and lignans found mainly

in the bran fraction. Some, such as vitamin E, are considered to be direct free radical scavengers, while

others act as cofactors of antioxidant enzymes (selenium, manganese, and zinc), or indirect antioxidants

(folates, choline, and betaine). Whole-grain cereals are a major source of polyphenols, especially phenolic acids such as ferulic, vanillic, caffeic, syringic, sinapic, and p-coumaric acids (Fardet et al. 2008).

Definitions of Oxidative Stress, Antioxidant, and Prooxidant Terms

Halliwell’s perception of oxidative stress is somewhat vague, and defines it as “the biomolecular damage

that can be caused by direct attack of reactive species” (Halliwell and Whiteman 2004). Oxidative stress

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals


is caused by an imbalance between the production of reactive oxygen species (ROS; including hydroxyl

and superoxide anion radicals, hydrogen peroxide, and singlet oxygen) and a biological system’s ability

to readily detoxify the reactive intermediates or easily repair the resulting damage to all components of

the cell, including biological macromolecules like proteins, lipids, and DNA (Halliwell 2007). Oxidative

stress, as defined by Sies (1985, 1986), is a serious imbalance between oxidation and antioxidants, “a disturbance in the prooxidant–antioxidant balance in favor of the former, leading to potential damage.” An

antioxidant may be defined as “any substance that when present at low concentrations, compared with

those of the oxidizable substrate, significantly delays or inhibits oxidation of that substrate” (Gutteridge

1994). Thus antioxidants are health-beneficial compounds that may prevent chronic diseases resulting

from oxidative stress. For convenience, antioxidants have been traditionally divided into two classes; primary or chain-breaking antioxidants and secondary or preventative antioxidants (Madhavi et al. 1996).

On the other hand, prooxidants are chemicals that induce oxidative stress, either through creating ROS

or inhibiting antioxidant systems (Puglia and Powell 1984).

Primary (Chain Breaking) Antioxidants

Chain-breaking mechanisms are represented by:

L• + AH → LH + A•


LO• + AH → LOH + A•


LOO• + AH → LOOH + A•


Thus radical initiation (by reacting with a lipid radical: L•) or propagation (by reacting with alkoxyl:

LO• or peroxyl: LOO• radicals) steps are inhibited by the antioxidant: AH.

Secondary Antioxidants

Secondary (preventive) antioxidants retard the rate of oxidation. For example, metal chelators (e.g., ironsequesterants) may inhibit Fenton-type reactions (represented by Equation 18.4) that produce hydroxyl

radicals (Ames et al. 1993):


Fe2+  + H2O2 → Fe3+  + •OH + OH–


One important function of antioxidants toward free radicals such as OH, O 2 , and ROO is to suppress

free radical-mediated oxidation by inhibiting the formation of free radicals and/or by scavenging radicals. The formation of free radicals may be inhibited by reducing hydroperoxides and hydrogen peroxide

and by sequestering metal ions (Niki 2002) through complexation/chelation reactions. Radical scavenging action is dependent on both the reactivity and concentration of the antioxidant. In a multiphase

medium (such as an emulsion), the localization of the antioxidant at the interphases may be important.

The evaluation of antioxidant activity is complicated by the prooxidative effect of antioxidants in the

presence of unsequestered metal ions such as iron and copper. The lower oxidation states of these metals

[i.e., Fe(II) and Cu(I)] should not be present at significant levels in tests measuring antioxidant status so

as not to initiate Fenton-type reactions exemplified in Equation 18.4. The prooxidative effect of phenolic

antioxidants (ArOH), generally induced by transition metal ions like Cu(II) in the presence of dissolved

oxygen, gives rise to oxidative damage to lipids, and can be demonstrated by the following reactions

(Huang et al. 2005):

Cu(II) + ArOH → Cu(I) + ArO• + H + 


ArO• + LH → ArOH + L•


L• + O2 → LOO•


LOO• + LH → LOOH + L•


Cu(I) + LOOH → Cu(II) + LO• + OH–



Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

A reducing agent may even be a prooxidant if it reduces oxygen to free radicals or converts transition metal ions to lower oxidation states that may give rise to Fenton-type reactions (Halliwell and

Whiteman 2004). Currently, prooxidant activity assay methods are by no means adequate, as they

are primarily based on the measurement of reducibility of transition metal ion-complexes that give

rise to reactive species. The prooxidant activity of flavonoids is generally accepted to be concentration-dependent, and both the antioxidant and the copper-initiated prooxidant activities of a flavonoid

depend on the number and position of –OH substituents in its backbone structure (Cao et al. 1997).

Flavones and flavanones, which have no –OH substituents, showed neither antioxidant nor Cu-initiated

prooxidant activities in the automated ORAC assays set for the purpose (Cao et al. 1997). It was also

observed that Cu(II)-induced prooxidant activity of Ar–OH proceeds via intra- and intermolecular

electron transfer reactions accompanying ROS formation, and copper complexation followed by oxidation of resveratrol analogues (e.g., 3,4-dihyroxystilbene) ending up with quinone (Ar = O) products

(Zheng et al. 2006).

Total Antioxidant Capacity (TAC) Assays Applied

to Phenolics in Fruits and Cereals

The chemical diversity of phenolic antioxidants makes it difficult to separate and quantify individual

antioxidants (i.e., parent compounds, glycosides, and many isomers) from the plant-based food matrix.

Moreover, the total antioxidant power as an “integrated parameter of antioxidants present in a complex sample” (Ghiselli et al. 2000) is often more meaningful to evaluate health beneficial effects

because of the cooperative action of antioxidants. Therefore it is desirable to establish and standardize methods that can measure the total antioxidant capacity (TAC) level directly from plant-based

food extracts containing phenolics. By means of standardized tests for TAC, the antioxidant values

of foods, pharmaceuticals, and other commercial products can be meaningfully compared, and variations within or between products can be controlled. By considering the changes in TAC values of

human serum measured by standardized methods, one can detect diseases and monitor the course of

medical treatments. For the sake of simplicity, only spectrophotometric or fluorometric assays using

molecular probes (i.e., UV-Vis absorbing or fluorescent probes) will be discussed in this work. Due

to complexity and limitations of directly following reaction kinetics of the inhibited autoxidation of

lipids, molecular spectrometric assays that may or may not apply a suitable radical, but without a

chain-propagation step as in lipid autoxidation will be discussed. Antioxidant capacity assays may

be broadly classified as ET (electron transfer)-based assays and HAT (hydrogen atom transfer)-based

assays (Huang et al. 2005; Prior et al. 2005), though in some cases, these two mechanisms may not be

differentiated with distinct boundaries. In fact, most nonenzymatic antioxidant activity (e.g., scavenging of free radicals, inhibition of lipid peroxidation, etc.) is mediated by redox reactions (Pulido et al.

2000). In addition to these two basic classes considering mechanism, ROS scavenging assays will also

be taken into account.

HAT-Based Assays

The HAT-based assays measure the capability of an antioxidant to quench free radicals (generally peroxyl radicals) by H-atom donation (Table 18.2). The HAT mechanisms of antioxidant action in which

the hydrogen atom of a phenol (Ar–OH) is transferred to an ROO• radical can be summarized by the


ROO• + AH/ArOH → ROOH + A•/ArO•,


where the aryloxy radical (ArO•) formed from the reaction of antioxidant phenol with peroxyl radical is stabilized by resonance. The AH and ArOH species denote the protected biomolecules and phenolic antioxidants, respectively. Effective phenolic antioxidants need to react faster than biomolecules

with free radicals to protect the latter from oxidation. Since in HAT-based antioxidant assays, both the


Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals

Table 18.2

HAT-Based Antioxidant Capacity Methods and Basic Principles



ORAC (oxygen radical absorbance capacity) assay

TRAP (total radical trapping antioxidant parameter)

PCL (photochemiluminescence) assay

β-carotene/linoleate system

Crocin based assay

Calculating the net protection area under the time recorded

fluorescence decay curve of red-phycoerythrin or


Measuring the consumed oxygen

Measurement of chemiluminiscence of luminol radical

Measurement of bleaching of β-carotene

Measurement of bleaching of crocin

fluorescent probe and antioxidants react with ROO•, the antioxidant activity can be determined from

competition kinetics by measuring the fluorescence decay curve of the probe in the absence and presence

of antioxidants, and integrating the area under these curves (Huang et al. 2005; Prior et al. 2005).

HAT-based assays include oxygen radical absorbance capacity (ORAC) assay (Cao et al. 1995), total

peroxyl radical-trapping antioxidant parameter (TRAP) assay using R-phycoerythrin as the fluorescent

probe developed by Wayner et al. (1985) and further developed by Ghiselli et al. (1995, 2000), Crocin

bleaching assay using AAPH as the radical generator (Bors et al. 1984), and β-carotene bleaching assay

(Burda and Oleszek 2001), although the latter bleaches not only by peroxyl radical attack but by multiple

pathways (Prior et al. 2005).

In general, HAT reactions may be considered to be relatively independent from solvent- and pHeffects, and are completed in a short time (at the order of sec-min) as opposed to ET-based assays. On the

other hand, the ET mechanism of antioxidant action is based on the reaction:

ROO• + AH/ArOH → ROO – + AH•+ /ArOH•+


AH•+ / ArOH•+ + H2O ↔ A• / ArO• + H3O +


ROO  + H3O  ↔ ROOH + H2O



where the reactions are relatively slower than those of HAT-based assays, and are solvent- and pHdependent. The aryloxy radical (ArO•) is subsequently oxidized to the corresponding quinone (Ar = O).

The more stabilized the aryloxy radical is, the easier the oxidation will be from ArOH to Ar = O due to

the reduced redox potential.

Oxygen radical absorbance capacity (ORAC) assay (Cao et al. 1995) applies a competitive reaction

scheme in which antioxidant and substrate kinetically compete for thermally generated peroxyl radicals

through the decomposition of azo compounds such as ABAP (2,2′-azobis(2-aminopropane) dihydro­

chloride) (Huang et al. 2005; Prior et al. 2005). The net area under curve (AUC), found by subtracting the

AUC of blank from that of antioxidant-containing sample (the fluorescence decay of which is retarded),

is an indication of the total antioxidant concentration of the sample in the ORAC method. The fluorescent

probes used in the ORAC assay were initially β-phycoerythrin (Cao et al. 1993; Ghiselli et al. 1995; Glazer

1990), and later fluorescein (Ou et al. 2001), though TAC results obtained with the latter probe are much

higher than those reported with the former. The ORAC measures the inhibition of peroxyl radical induced

oxidations by antioxidants and thus reflects classical radical chain-breaking antioxidant activity by H-atom

transfer (Ou et al. 2001; Prior et al. 2005). The reaction was reported to go to completion so that both inhibition time and inhibition degree are considered in the quantification of antioxidants (Cao et al. 1995).

ET-Based Assays

In most ET-based assays, the antioxidant action is simulated with a suitable redox-potential probe; that

is, the antioxidants react with a fluorescent or colored probe (oxidizing agent) instead of peroxyl radicals.

Spectrophotometric ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced (Table 18.3). The degree of color change (either an increase or


Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Table 18.3

ET-Based Antioxidant Capacity Methods and Basic Principles


Basic Principle

DPPH (2,2- diphenyl-1-picrylhydrazyl) assay

TEAC (Trolox equivalent antioxidant

capacity)/ABTS [2,2′-azinobis-(3ethylbenzothiazoline-6-sulphonic acid)] assay

FRAP (Ferric reducing ability of plasma) assay

Folin method

CUPRAC (Cupric ion reducing antioxidant

capacity) method

Evaluation of scavenging activity of antioxidants by measurement of

change in absorbance at 515–517 nm

Measurement of inhibition of the absorbance of ABTS•+ radical cation

by antioxidants at 415 nm

Measurement of blue color of reduced [Fe2+-TPTZ tripyridyltriazine] at

593 nm at low pH

Measurement of reduction of Mo(VI) to Mo(V)

Measurement of orange-yellow color of reduced [Cu+-Neocuproine] at

450 nm at pH 7

decrease of absorbance at a given wavelength) is correlated to the concentration of antioxidants in the

sample. ABTS/TEAC (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid/trolox-equivalent antioxidant capacity) (Miller et al. 1993; Re et al. 1999) and DPPH (2,2-diphenyl-1-picrylhydrazyl) (Bondet et al.

1997; Brand-Williams et al. 1995; Sánchez-Moreno et al. 1998) are decolorization assays, whereas in Folin

total phenols assay (Folin and Ciocalteu 1927; Singleton et al. 1999), FRAP (ferric reducing antioxidant

power) (Benzie and Strain 1996; Benzie and Szeto 1999), and CUPRAC (cupric ion reducing antioxidant

capacity) (Apak et al. 2004, 2005) there is an increase in absorbance at a prespecified wavelength as the

antioxidant reacts with the chromogenic reagent (i.e., in the latter two methods, the lower valencies of iron

and copper, namely Fe(II) and Cu(I), form charge-transfer complexes with the ligands, respectively). The

basic chromophores used in Folin, ABTS/TEAC, FRAP, ferricyanide, ferric-phenanthroline, DPPH, and

CUPRAC assays are shown in Figure 18.1. There is no visible chromophore in the Ce4 + -reducing antioxidant capacity assay developed recently by Özyurt et al. (2007), as the remaining Ce(IV) in dilute sulfuric

acid solution after polyphenol oxidation under carefully controlled conditions was measured at 320 nm

(i.e., in the UV region of the electromagnetic spectrum). These assays generally set a fixed time for the concerned redox reaction, and measure thermodynamic conversion (oxidation) during that period. ET-based

assays, namely ABTS/TEAC, DPPH, Folin–Ciocalteu (FCR), FRAP, ferricyanide, and CUPRAC (though

ABTS/TEAC, DPPH are considered as mixed HAT–ET-based assays by some researchers) use different chromogenic redox reagents with different standard potentials. Although the reducing capacity of a

sample is not directly related to its radical scavenging capability, it is a very important parameter of antioxidants. The reaction equations of various ET-based assays can be summarized as follows:

Folin: Mo(VI) (yellow) + e – (from AH) → Mo(V) (blue)


(λmax = 765 nm) where the oxidizing reagent is a molybdophosphotungstic heteropolyacid comprised of

3 H2O – P2O5 – 13 WO3 – 5 MoO3 – 10 H2O, in which the hypothesized active center is Mo(VI).

FRAP: Fe(TPTZ)23+  + ArOH → Fe(TPTZ)22+  + ArO• + H+ 


(λmax = 595 nm) where TPTZ: 2,4,6-tripyridyl-s-triazine ligand.

Ferricyanide/Prussian Blue: Fe(CN)63– + ArOH → Fe(CN)64– + ArO• + H+


Fe(CN)64– + Fe 3+  + K+ → KFe[Fe(CN)6] (λmax = 700 nm)


ABTS/TEAC: ABTS + K2S2O8 → ABTS•+ (λmax = 734 nm)


ABTS•+ + ArOH → ABTS + ArO• + H+


where ABTS: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and TEAC is Trolox-equivalent

antioxidant capacity (also the name of the assay). Although other wavelengths such as 415 and 645 nm


Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals








Fe (II)





Tris(1,10-phenanthroline) iron(II)





FRAP: [Fe(II)(TPTZ)2]2+

(Ferrous tripyridyltriazine cation)









DPPH radical



CUPRAC: Bis(neocuproine)copper(I) chelate cation









Folin reagent





ABTS•+ radical cation

Figure 18.1  Basic chromophores used in TAC (total antioxidant capacity) assays.

have been used in the ABTS assay (Prior et al. 2005), the 734 nm peak wavelength has been predominantly preferred due to less interference from plant pigments.

DPPH: DPPH• + ArOH → DPPH + ArO• + H+ 


(λmax = 515 nm),

where DPPH•. is the 2,2-diphenyl-1-picrylhydrazyl stable radical.

CUPRAC: 2 n Cu(Nc)22+  + Ar(OH)n → 2 n Cu(Nc)2+  + Ar( = O)n + 2 n H + 


(λmax = 450 nm),

where the polyphenol with suitably situated Ar–OH groups is oxidized to the corresponding quinone,

and the reduction product [i.e., bis(neocuproine)copper(I) chelate] shows absorption maximum at 450

nm. It should be noted that not all phenolic –OH are reduced to the corresponding quinones, and the

efficiency of this reduction depends on the number and position of the phenolic –OH groups as well as

on the overall conjugation level of the polyphenolic molecule.

The ABTS–TEAC assay was first reported by Miller et al. (1993), which is based on the scavenging ability of antioxidants to the long-life radical anion ABTS•+. In this assay, ABTS is oxidized by

peroxyl radicals or other oxidants to its radical cation, ABTS•+, and the TAC is measured as the ability of test compounds to decrease the color reacting directly with the ABTS•+ radical. Originally, this

assay used metmyoglobin and H2O2 to generate ferrylmyoglobin, which then reacted with ABTS to form

ABTS•+ (Miller et al. 1993). ABTS•+ can be generated by either chemical reaction (e.g., potassium persulfate; Re et al. 1999) or enzyme reactions (e.g., horseradish peroxidase; Arnao et al. 1996). Generally,

the chemical generation requires a long time (e.g., up to 16 hours for potassium persulfate generation),

whereas enzymatic generation is faster and the reaction conditions are milder. In this assay, 415 and 734


Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

nm were adopted by most investigators to spectrophotometrically monitor the reaction between the antioxidants and ABTS•+. In terms of quantification methods, most recently revised methods measure the

absorbance decrease of ABTS•+ in the presence of a testing sample or Trolox at a fixed time point (1–6

min), and then antioxidant capacity was calculated as Trolox equivalents.

The FRAP assay was first developed by Benzie and Strain (1996). At a low pH, reduction of ferric

tripridyltriazine (Fe(III)-TPTZ) complex to ferrous form (which has an intense blue color) is monitored

by measuring the change in absorbance (increase in absorbance) at 593 nm.

The original Folin–Ciocalteu (F–C) method was first developed in 1927 and originated from chemical

reagents used for tyrosine analysis (Folin and Ciocalteu 1927) in which the oxidation of phenols by a

molybdotungstate reagent yields a colored product with λmax at 745–750 nm and designed to determine

the total content of phenolics (total phenols) (Singleton et al. 1999).

The DPPH method was first reported by Brand-Williams et al. (1995). The DPPH• radical bearing

a deep purple color is one of the few stable organic nitrogen radicals. This is a free radical scavenging

assay involving decoloration based on the measurement of the reducing ability of antioxidants toward

DPPH•. This assay spectrophotometrically measures the loss of DPPH color at 515 nm after a reaction

with antioxidant compounds.

Original CUPRAC (Cupric Ion Reducing Antioxidant Capacity) Method

The CUPRAC assay was developed in our laboratories and expanded with some modifications. The

chromogenic redox reagent used for the CUPRAC assay was bis(neocuproine)copper(II) chelate. This

reagent was useful at pH 7, and the absorbance of the Cu(I)-chelate formed as a result of redox reaction

with reducing polyphenols was measured at 450 nm. The color was due to the Cu(I)-Nc chelate formed

(see Figure 18.2). The reaction conditions such as the reagent concentration, pH, and oxidation time at

room and elevated temperatures were optimized (Apak et al. 2004, 2005).

The chromogenic oxidizing reagent of the developed CUPRAC method; that is, bis(neocuproine)

copper(II) chloride (Cu(II)-Nc), reacts with antioxidants (AOX) acting as reductants in the following


In this reaction, the reactive Ar–OH groups of polyphenolic antioxidants (AOX) are oxidized to the

corresponding quinones (Ar = O) and Cu(II)-Nc is reduced to the highly colored Cu(Nc)2+ chelate showing maximum absorption at 450 nm. Although the concentration of Cu2+ ions is in stoichiometric excess

of that of neocuproine in the CUPRAC reagent for driving the redox equilibrium reaction represented

by Figure 18.2 to the right, the actual oxidant is the Cu(Nc)22 + species and not the sole Cu2 + , because

the standard redox potential of the Cu(II/I)-neocuproine is 0.6 V, much higher than that of the Cu2 + /

Cu+ couple (0.17 V; Tütem et al. 1991). As a result, polyphenols are oxidized much more rapidly and

efficiently with Cu(II)-Nc than with Cu2 + , and the amount of colored product (i.e., Cu(I)-Nc chelate)














AOX Product


H 3C





Light blue CUPRAC reagent






Yellow–orange product, λmax = 450 nm

Figure 18.2  The CUPRAC reaction and chromophore: Bis(neocuproine)copper(I) chelate cation. (Protons liberated in

the reaction are neutralized by the NH4Ac buffer).

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals


emerging at the end of the redox reaction is equivalent to that of reacted Cu(II)-Nc. The liberated protons are buffered in an ammonium acetate medium. The CUPRAC reagent is capable of oxidizing

suitably situated phenolic –OH groups to the corresponding quinones as long as the corresponding conditional quinone–phenol potential is less than or close to that of cupric/cuprous-neocuproine in neutral


In the normal CUPRAC method (CUPRACN), the oxidation reactions were essentially complete

within 30 minutes. Flavonoid glycosides required acid hydrolysis to their corresponding aglycons for

fully exhibiting their antioxidant potency. Slow reacting antioxidants needed elevated temperature incubation so as to complete their oxidation with the CUPRAC reagent (Apak et al. 2004, 2005). Special

precautions to exclude oxygen from the freshly prepared and analyzed solutions of pure antioxidants

were not necessary since oxidation reactions with the CUPRAC reagent were much more rapid than with

dissolved O2 (i.e., the latter would not appreciably occur during the period of CUPRAC protocol since

there is a spin restriction for the ground state triplet of dioxygen molecule to participate in fast reactions).

However, plant extracts should be purged with N2 to drive off O2, and should be kept in a refrigerator if

not analyzed on the day of extraction, since complex catalyzed reactions with unpredictable kinetics may

take place in real systems. Additionally, the oxidation of ascorbic acid with dissolved oxygen may take

place more rapidly than with polyphenolics, especially in the presence of transition metal salts.

As a distinct advantage over other ET-based TAC assays (e.g., Folin, FRAP, ABTS, DPPH), CUPRAC

is superior in regard to its realistic pH close to the physiological pH, favorable redox potential, accessibility, and stability of reagents, flexibility, simplicity, low-cost, and applicability to lipophilic antioxidants

as well as hydrophilic ones. CUPRAC gives additive responses to antioxidants in regard to their contribution to TAC, and perfectly linear calibration curves (of absorbance vs. concentration) over a relatively

wide concentration range of antioxidants.

An example of the calculation of TAC for apricots with respect to the CUPRAC method is given below

(Gỹỗlỹ et al. 2006):

TAC (in àmol TR/g)=(Af/TR) (Vf/Vs) r (Vi/m)ì103,

where TR=1.67ì104 Lmol1cm1 (CUPRACN method); Vi = initial extract volume; m = grams of solid

apricot sample; r = extract dilution ratio; Vs = sample volume for analysis; Vf = final volume; Af = sample


Some Modifications of the CUPRAC Method

It should be remembered that the CUPRAC assay does not merely measure the TAC of an antioxidant

sample, but gives rise to many other modified assays of radical scavenging or activity measurement that

may be useful for antioxidant research (Demirci ầekiỗ et al. 2009; ệzyỹrek et al. 2007, 2008a, 2008b,

2009). In this regard, CUPRAC should be perceived as a train of antioxidant measurement methods in

varying media, one evolving from the other. This resembles the highly popular Russian stacking doll,

“Matrushka” (Figure 18.3).

Simultaneous Measurement of Lipophilic and Hydrophilic Antioxidants

Lipophilic and hydrophilic antioxidants can be assayed simultaneously by solubilizing lipophilic compounds such as β-carotene, vitamin E, and oil-soluble synthetic antioxidants and hydrophilic compounds

such as vitamin C and phenolic antioxidants as “host–guest” complexes with 2% methyl-β-cyclodextrin

(M-β-CD; w/v) in 90% aqueous acetone (Özyürek et al. 2008a). This method eliminates the wide variability in apparent antioxidant capabilities arising from different levels of accumulation of oil- and watersoluble antioxidants at emulsion interfaces, and assigns an objective TEAC value to each antioxidant that

depends only on its chemical character (i.e., electron donating ability).

Determination of Ascorbic Acid by the Modified CUPRAC Method

with Extractive Separation of Flavonoids-La(III) Complexes

The modified CUPRAC method (Özyürek et al. 2007) for ascorbic acid: AA (vitamin C) determination

is based on the oxidation of AA to dehydroascorbic acid with the CUPRAC reagent of TAC assay; that


Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Figure  18.3  CUPRAC assay resembles the famous Russian stacking doll, Matrushka, as the mother CUPRAC

method of TAC measurement has given rise to many other modified CUPRAC methods for activity/radical scavenging


is, 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 a 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.

Hydroxyl Radical Scavenging Assay of Phenolics and

Flavonoids with a Modified CUPRAC Method

A salicylate probe was used for detecting • OH generated by the reaction of iron(II)-EDTA complex with

H2O2. The produced hydroxyl radicals attack both the salicylate probe (see the formulas of dihydroxybenzoic acids: DHBAs produced from salicylate under hydroxyl radical attack, Figure 18.4) and the hydroxyl

radical scavengers that are incubated in a solution for 10 minutes. Added radical scavengers compete

with salicylate for the •OH produced, and diminish chromophore formation from Cu(II)-neocuproine. At

the end of the incubation period, the reaction was stopped by adding catalase, and the reaction products

were quantified with both CUPRAC and HPLC (see Figure 18.5 for the HPLC quantification of DHBAs).

With the aid of this reaction, a kinetic approach was adopted to assess the hydroxyl radical scavenging

properties of polyphenolics, flavonoids, and other compounds (e.g., ascorbic acid, glucose, and mannitol). A second-order rate constant for the reaction of the scavenger with • OH could be deduced from the

inhibition of color formation due to the salicylate probe (Özyürek et al. 2008b).

Measurement of Xanthine Oxidase Inhibition Activity of Phenolics

and Flavonoids with a Modified CUPRAC Method

Since some polyphenolics have a strong absorption from the UV to the 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 spectrophotometric

method at 450 nm (Özyürek et al. 2009). 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



Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals




2, 3-Dihydroxybenzoic acid





Salicylic acid


2, 4-Dihydroxybenzoic acid




2, 5-Dihydroxybenzoic acid

Figure 18.4  Major hydroxylation products formed from a salicylic acid probe upon the attack of • OH radicals.



Response (mAU)



















Time (min)







Figure 18.5  The HPLC chromatogram for salicylate and its hydroxylation products in the absence of hydroxyl ­radical

scavengers. The retention times were (a) 2,5-DHBA 9.38 min; (b) 2,4-DHBA 9.78 min; (c) 2,3-DHBA 11.20 min; and

(d) salicylate 17.65 min.

Modified Cupric Reducing Antioxidant Capacity (CUPRAC) Assay for Measuring the

Antioxidant Capacities of Thiol-Containing Proteins in Admixture with Polyphenols

In most assays measuring a TAC, proteins are not taken into account (e.g., in assays carried out in

the hydrophilic fraction of human serum) and remain in the precipitate (obtained by using perchloric acid, trichloroacetic acid, ammonium sulfate, etc.). Modified CUPRAC assay for proteins has


Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

verified that the contribution of proteins, especially thiol-containing proteins, to the observed TAC

is by no means negligible (Demirci ầekiỗ et al. 2009). Various protein fractions (egg white, whey

proteins, gelatin) and peptides (like glutathione: GSH) may either respond to TAC assays directly

via their free –SH groups, or indirectly after protein denaturation through their exposed (originally

buried) thiol groups. An 8 M urea buffer was used to expose these thiols to TAC assays. Urea—in

combination with SDS—maximized the reactivity of thiols and disulfides that may be buried within

the protein matrix. Urea partly denaturated proteins and significantly lowered the reduction potential

of disulfide/thiol couples in peptides facilitating thiol oxidizability. Among the tested TAC assays,

only CUPRAC and ABTS/H 2O2/HRP possessed the property of optical absorbance additivity (i.e.,

obeying Beer’s law). This study has reported, for the first time, the measurement of the TAC of thiolcontaining proteins in admixture with phenolic antioxidants after taking up the protein fractions

with a suitable buffer that neither causes the precipitation of proteins nor interferes with the selected

antioxidant assay (specifically CUPRAC assay), and is expected to be useful in estimating the TAC

values and hence food qualities of dairy products and other protein-containing food varieties in

further studies.

The comparison of methods for assessing antioxidant capacity summarizing the experiences of our

analytical chemistry laboratory at Istanbul University is presented in Table 18.4.

Other Antioxidant Activity Tests

As for molecular probes used in the colorimetric/fluorometric detection of ROS, nitro blue tetrazolium

(NBT) has been used for superoxide anion (O2•–), scopoletin for hydrogen peroxide (H2O2), deoxyribose/thiobarbituric acid (TBA) or modified CUPRAC reagent for hydroxyl radicals (• OH), and tetratert-butylphtalocyanine for singlet oxygen (1O2) (Huang et al. 2005). Ewing and Janero developed a

superoxide dismutase (SOD) microassay based on the spectrophotometric assessment of O2•– –mediated

NBT reduction by an aerobic mixture of NADH and phenazine methosulfate, which produces superoxide chemically at a nonacidic pH (Ewing and Janero 1995). Hydrogen peroxide has been assayed by its

ability to oxidize scopoletin, a naturally occurring fluorescent compound, in the presence of horseradish peroxidase as catalyst, to a nonfluorescent product, and the decrease in fluorescence is an indication of H2O2 at nanomolar levels (De la Harpe and Nathan 1985). Hydroxyl radicals generated from a

Fenton-reaction (Equation 18.4) were most frequently detected by means of their oxidative attack on a

deoxyribose probe producing malondialdehyde (MDA) as the end product; MDA was colorimetrically

detected by the formation of colored products with TBA, forming the basis of the TBARS (thiobarbituric

acid–reactive substances) method (Gutteridge 1981; Halliwell and Gutteridge 1981). Bektaşoğlu et al.

(2006) used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals

generated from an equivalent mixture of Fe(II) + EDTA with hydrogen peroxide. The produced hydroxyl

radicals attacked both the probe and the water-soluble antioxidants in 37°C-incubated solutions for

2 hours. The CUPRAC absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine

reagent by the hydroxylated probe decreased in the presence of •OH scavengers, the difference being

proportional to the scavenging ability of the tested compound (Bektaşoğlu et al. 2006).

Antioxidant Capacities of Regularly Consumed Fruits

Phenolic substances can be extracted from fruits using a sequence of solvents with divergent polarity. In

general, useful solvents with a decreasing order of polarity are: water, 80% methanol or 70% ethanol,

80% acetone, and ethyl acetate. Among antioxidant phenolics, certain classes of compounds such as

phenolic acids, hydroxycinnamic acids, flavonoids, and carotenoids require a decreasing order of solvent

polarity for extraction, respectively, although suitable solvent combinations may be tailored for specific

purposes. Moreover, the dielectric constant of the solvent, intra-/intermolecular hydrogen bonding associations and standard redox potential of phenolics and derived aryloxy radicals in a given solvent may

be important for electron transfer kinetics in antioxidant assays (Huang et al. 2005; Prior et al. 2005).

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