Review of Methods to Determine Antioxidant Capacities

Review of Methods to Determine Antioxidant Capacities

Ayse Karadag & Beraat Ozcelik & Samim Saner

Received: 2 September 2008 /Accepted: 25 November 2008 /Published online: 13 January 2009# Springer Science + Business Media, LLC 2009

Abstract Antioxidant capacity is related with compoundscapable of protecting a biological system against thepotentially harmful effect of processes or reactions involvingreactive oxygen and nitrogen species (ROS and RNS). Theseprotective effects of antioxidants have received increasingattention within biological, medical, nutritional, and agro-chemical fields and resulted in the requirement of simple,convenient, and reliable antioxidant capacity determinationmethods. Many methods which differ from each other interms of reaction mechanisms, oxidant and target/probespecies, reaction conditions, and expression of results havebeen developed and tested in the literature. In this review, themethods most widely used for the determination of antiox-idant capacity are evaluated, presenting the general princi-pals, recent applications, and their strengths and limitations.Analysis conditions, substrate, and antioxidant concentra-tion should simulate real food or biological systems asmuch as possible when selecting the antioxidant capacitymethod. The total antioxidant capacity value shouldinclude methods applicable to both lipophilic and hydro-philic antioxidants, with regards the similarity and differ-ences of both hydrogen atom transfer and electron transfermechanism. The methods including various ROS/RNSalso have to be designed to comprehensively evaluate theantioxidant capacity of a sample.

Keywords Antioxidant CapacityMethods . Review .

Reactive Oxygen Species

Introduction

After several studies on the importance of antioxidants inbiological systems by counteracting of oxidative stress thatcauses several human diseases such as atherosclerosis,diabetes mellitus, chronic inflammation, neurodegenerativedisorders, and certain types of cancer have been conducted,there is a great interest of quantification of antioxidants anddetermination of antioxidant capacities of a number ofspecific food compounds.

In food science, the antioxidant is defined as a substance infoods that when present at low concentrations compared tothose of an oxidizable substrate significantly decreases orprevents the adverse effects of reactive species, such asreactive oxygen and nitrogen species (ROS/RNS), on normalphysiological function in humans (Halliwell et al. 1995;Huang et al. 2005). According to this definition, not allreductants involved in a chemical reaction are antioxidants;only those compounds which are capable of protecting thebiological target from oxidation meet this criterion.

Mechanisms of antioxidant action include serving as (1)physical barriers to prevent ROS generation or ROS access toimportant biological sites, e.g., UV filters, cell membranes; (2)chemical traps/sinks that “absorb” energy and electrons,quenching ROS, e.g., carotenoids, anthocyanidins; (3) cata-lytic systems that neutralize or divert ROS, e.g., theantioxidant enzymes SOD (superoxide dismutase), catalase,and glutathione peroxidase (Chaudiere and Ferrari-Iliou1999); (4) binding/inactivation of metal ions to preventgeneration of ROS, e.g., ferritin, ceruloplasmin, catechins;and (5) chain-breaking antioxidants which scavenge and

Food Anal. Methods (2009) 2:41–60DOI 10.1007/s12161-008-9067-7

A. Karadag : B. OzcelikDepartment of Food Engineering,Faculty of Chemical and Metallurgical,Istanbul Technical University,34469 Maslak, Istanbul, Turkey

S. Saner (*)Kalite Sistem Laboratories Group,Ar Plaza B Blok Degirmen Sk. No:16 Kozyatagi,34742 Kadikoy, Istanbul, Turkeye-mail: [email protected]

destroy ROS, e.g., ascorbic acid (vitamin C), tocopherols(vitamin E), uric acid, glutathione, flavonoids (Benzie 2003).

Many methods have been developed and tested in theliterature, advantages and limitations of these methods havestill been discussed. It does not seem to have a consensusfor concluding the most convenient method as a standardmethod for claiming total antioxidant capacity, for example,the limitations for determination of hydrophilic antioxi-dants, the problems occurring in determination of reactionend point, the concern on light sensitivity of initiators orprobes, carrying out the analysis in the physiologicalirrelevance pH, possible interference from certain foodcomponents, the use of different standards for expressingresults that causes the difficulties in comparison. The scopeof this review is to summarize the principals of the mostcommonly used in vitro antioxidant capacity assays byevaluating their strengths and limitations.

Definitions

Different researchers used different terms to expressantioxidant capacity including total antioxidant efficiency,effectiveness, action, power, parameter, potential, potency,and activity. Antioxidant activity and antioxidant capacityare terms that are often used interchangeably; they havedifferent meanings though (MacDonald-Wicks et al.2006). The “activity” of a chemical would be pointlesswithout specific reaction conditions such as pressure andtemperature. The antioxidant capacity gives the informa-tion about the duration while the activity describes thestarting dynamics of antioxidant action (Roginsky andLissi 2005). The antioxidant capacity in complex hetero-geneous foods and biological systems is affected by manyfactors including the partitioning properties of the anti-oxidants between lipid and aqueous phases, the oxidationconditions and the physical state of the oxidizablesubstrate (Frankel and Meyer 2000). For example, antiox-idant protection significantly changes according to thesubstrate used to conduct evaluation. When brain homog-enate and linoleic acid emulsion are used as substrates, α-tocopherol showed a much better protection than Trolox(Castro et al. 2006).

A dietary antioxidant can sacrificially scavenge ROS/RNS to stop radical chain reactions, considered as primarychain-breaking antioxidants or free radical scavengers(FRS), or it can inhibit the reactive oxidants from beingformed in the first place, considered as secondary orpreventive antioxidants. Primary antioxidants, when presentin trace amounts, may either delay or inhibit the initiationstep by inactivating or scavenging free radicals, thusinhibiting initiation and propagation reactions by reactingwith peroxyl or alkoxyl radicals.

FRS or chain-breaking antioxidants are capable ofaccepting a radical from oxidizing lipids species such asperoxyl LOO�) and alkoxyl LO�) radicals by the followingreactions.

LOO� or LO� þ FRS ! LOOH or LOHþ FRS�Antioxidant efficiency is dependent on the ability of the

FRS to donate hydrogen to the free radical. As thehydrogen-bond energy of the FRS decreases, the transferof the hydrogen to the free radical is more energeticallypromising and rapid. The ability of an FRS to donatehydrogen to a free radical can be predicted from standardone-electron reduction potentials. The reduction of an FRSshould be lower than 600 mV, which is a reductionpotential of polyunsaturated fatty acid to work as antioxi-dant (Lee et al. 2003). Efficient FRS also produces radicals(FRS�) that do not react rapidly with oxygen to formperoxides. In foods, the efficiency of phenolic FRS alsodepends on additional factors such as volatility, pHsensitivity, and polarity (Akoh and Min 1998).

Preventive antioxidants, such as superoxide dismutase,catalase, and peroxidase, are described either as preventingintroduction of initiating radicals or as inhibiting the rate atwhich new chains are set up. There are many different“preventive” antioxidation pathways because of the distinctrange of available oxidation initiators. These pathwaysinclude chelation of transition metals, singlet-oxygendeactivation, enzymatic ROS detoxification, UV filtration,inhibition of prooxidant enzymes, antioxidant enzymecofactors, etc. (Laguerre et al. 2007). Metal chelators arepreventive antioxidants by complexing with transitionmetal ions, thereby delaying metal-catalyzed initiationreactions and decomposition of lipid hydroperoxides. Otherantioxidant mechanisms include singlet-oxygen quenching,oxygen scavenging, and blocking the prooxidant effects bybinding certain proteins containing catalytic metal sites(Frankel and Meyer 2000). Dietary antioxidants oftenbroadly include radical chain reaction inhibitors, metalchelators, oxidative enzyme inhibitors, and antioxidantenzyme cofactors (Huang et al. 2005).

Antioxidant Mechanisms

Due to the confusion in the literature for the reactionmechanisms, the need to provide a protocol involvingmeasurement of more than one property due to multipleactivities of polyphenols is outlined and the dominantactivity depends on the medium and type of antioxidants.The response of antioxidants to different radical or oxidantsources may be different. For example, carotenoids are notparticularly good quenchers of peroxyl radicals relative tophenolics but are exceptional singlet-oxygen scavengers.

42 Food Anal. Methods (2009) 2:41–60

Therefore, no single assay accurately reflects the mecha-nism of action of all radical sources or all antioxidants in acomplex system (Prior et al. 2005).

On the basis of the inactivation mechanism involved,major antioxidant capacity methods have been generallydivided into two categories though: (1) hydrogen atomtransfer (HAT) reaction and (2) electron transfer (ET)reaction-based methods. Bond dissociation energy andionization potential are two major factors that determinethe mechanism and the efficiency of antioxidants. ET andHAT mechanisms almost always occur together in allsamples and their difference can be made difficult. InHuang et al. (2005) and MacDonald-Wicks et al. (2006),total phenol assay by using the Folin-Ciocalteu Reagent(FCR), Trolox equivalent antioxidant capacity (TEAC), and2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavengingcapacity assays was considered under the methods utilizingET mechanism, whereas they were classified under themethods utilizing both ET and HAT mechanisms in Prior etal. (2005) due to difficulty in the interpretation of inhibitionmechanisms of these radicals.

HAT-based methods measure the classical ability of anantioxidant to scavenge free radicals by hydrogen donationto form stable compounds (Prior et al. 2005). HAT-basedmethods are more relevant to the radical chain-breakingantioxidant capacity.

AH þ X� ! XHþ A� ð1ÞRelative reactivity in HAT methods is determined by the

bond dissociation energy of the H-donating group in thepotential antioxidant and ionization potential. HAT reactionsare solvent and pH dependent and are generally quite rapid,typically completed in seconds to minutes. Most HAT-basedmethods monitor competitive reaction kinetics, and thequantitation is derived from the kinetic curves. HAT-basedmethods are generally composed of a synthetic free radicalgenerator (AAPH, 2,2′-azobis(2amidinopropane) dihydro-chloride (ABAP), 2,2′-azobis(2,4dimethylvaleronitrile(AMVN), 2,2′-azinobis(3-ethylbenzothiazolline-6-sulfonicacid (ABTS; Valkonen and Kuusi 1997; Wolfe and Liu2007)), an oxidizable molecular probe (dichlorofluorescein(DCFH; Adom and Lui 2005)), fluorescein (FL; Moore et al.2006), and an antioxidant.

The HAT-based assay using fluorescent probes has amechanistic similarity to lipid peroxidation, but, under theassay conditions, the substrate (probe) concentration isoften smaller than the antioxidant concentration. This is inconflict with real situations. In food systems, the antioxi-dant concentration is much smaller than substrate (e.g.,lipid) concentration. It is questionable whether the antiox-idant capacity measured using the HAT-based assay using amolecular probe can exhibit the situations in a real foodsystem (Huang et al. 2005).

ET-based methods detect the ability of a potentialantioxidant to transfer one electron to reduce any com-pound, including metals, carbonyls, and radicals.

M IIIð Þ þ AH ! AH� þM IIð Þ

In ET methods, relative reactivity is based ondeprotonation and ionization potential of the reactivefunctional group, so ET reactions are pH dependent. Ingeneral, ionization potential values decrease with in-creasing pH, reflecting increased electron-donating ca-pacity with deprotonation (Prior et al. 2005). The pHvalues have an important effect on the reducing capacityof antioxidants. At acidic conditions, the reducing capac-ity may be restrained due to protonation on antioxidantcompounds, whereas, in basic conditions, proton dissoci-ation of phenolic compounds would increase sample’sreducing capacity (Huang et al. 2005). ET reactions can berelatively slow and need long times to reach completion sotraditionally measure relative percent decrease in productrather than kinetics or total antioxidant capacity (Ozgen etal. 2006). Compared to HAT, the ET mechanism isstrongly solvent dependent due to solvent stabilization ofthe charged species (Ou et al. 2002a).

ET-based methods involve two components in thereaction mixture, antioxidants and oxidant (probe). Theprobe itself is an oxidant and abstracts an electron fromthe antioxidant, resulting in color changes of the probe.The degree of the color change is proportional to theantioxidant concentration. The reaction is reached to endpoint when color change stops. The change of absor-bance (ΔA) is plotted against the antioxidant concentra-tion to give a linear curve. The slope of the curveindicates the antioxidant’s reducing capacity, which isexpressed as Trolox equivalence or Gallic acid equiva-lent. To make the correlation, it is accepted thatantioxidant capacity is equal to reducing capacity.However, it is questionable how the assay results relateto total antioxidant capacity of a sample because there isno competitive reaction involved and there is no oxygenradical in the assays (Huang et al. 2005).

The question raised is which mechanism, ET or HAT,physiologically reflects the antioxidant preventive action.As cited in Ou et al. (2002a), Wright and co-workers used aprocedure based on density functional theory to calculategas-phase bond dissociation enthalpy and ionization poten-tial for molecules belonging to the class of phenolicantioxidants, including tocopherols, catechins, aminophe-nols, and stilbenes related to resveratrol. Their resultsdemonstrated that in most cases HAT mechanism isdominant. It is clear that HAT reaction concurs with ETreaction and plays a dominant role in biological redoxreactions.

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Antioxidant Capacity Methods

The features of any antioxidant capacity method are anoxidation initiator, a suitable substrate, and an appropriatemeasure of the end point. Initiators may include increasedtemperature (Laguerre et al. 2007) and partial pressure ofoxygen, addition of transition metal catalysts (Ou et al.2002a), exposure to light to promote photosensitizedoxidation by singlet oxygen (Min and Boff 2002; Lee et al.2004), and variable shaking to enhance reactant contact andfree radical sources (Pulido et al. 2000). However, it shouldbe realized that the analytical methods of measurement andthe conditions under which the analysis has occurred canlead to variable results for the same food types (Antolovichet al. 2002; Nilsson et al. 2005) For example, photosensitizedacceleration underestimates the effects of chain-breakingantioxidants (Frankel and Meyer 2000) and temperature raisecan change oxidation mechanism.

Several analytical strategies are possible for the end-point measurements. These include (1) measurement at afixed time point; (2) measurement of reaction rate; (3) lag-phase measurement where the length of the lag time to end-point change is measured, samples with higher antioxidantcapacity suppress the change longer; and (4) integrated ratemeasurement which involves integration of the end pointversus time curve and is used where the reaction kineticshave no simple order (Antolovich et al. 2002).

Several colorimetric and fluorometric antioxidant capac-ity assays based on HAT mechanism apply a radicalreaction without a chain propagation step which is anessential step in lipid autoxidation. Thus, the relevance ofthese approaches to radical chain-breaking antioxidantcapacity is disputable. In general, added antioxidantcompetes with probes (substrates) for the radicals andinhibits or retards the probe oxidation. Assays with thisfeature include total radical trapping antioxidant parameter(TRAP) assay, oxygen radical absorbance capacity (ORAC)assay, and crocin-bleaching assay (Huang et al. 2005)These assays have the following components: (a) thermo-labile azo-radical initiator, which gives radicals (R�) thatreact fast with O2 to give a steady flux of ROO� radicals(the most frequently applied radical generators are thewater-soluble AAPH and lipid-soluble AMVN); (b) oxidiz-able target, a molecular probe (UV or fluorescence) formonitoring reaction progress; (c) antioxidant, its presenceinhibits or retards the oxidation of probe. Therefore, in thebeginning of the assay, insignificant spectroscopic changesof the probe would be observed (induction period or lagphase). As the reaction proceeds, the antioxidants areconsumed by radicals and the oxidation of the probe wouldcontinue at a slower rate compared to control. Finally, whenthe antioxidants are depleted, the reaction rate of the probeoxidation will be similar to that obtained for the control (d)

reaction kinetic parameters (Huang et al. 2005; Magalhaeset al. 2008).

Antioxidant capacity can be measured by the effects of theantioxidant in controlling the extent of oxidation. Methodsshow extreme diversity. The antioxidant capacity assays canalso be based in the peroxyl radical scavenging such as ORAC(Ou et al. 2001) and TRAP (Schlesier et al. 2002), metalreducing power such as ferric reducing/antioxidant power(FRAP; Benzie et al. 1999) and cupric reducing/antioxidantpower (CUPRAC; Apak et al. 2004), hydroxyl radicalscavenging such as deoxyribose assay (Caillet et al. 2007),organic radical scavenging such as ABTS (Miller et al. 1993)and DPPH (Rivero-Perez et al. 2007), or quantification ofproducts formed during lipid peroxidation such as thiobarbi-turic acid reactive substances (Chumark et al. 2008; Perez-Jimenez and Sauro-Calixto 2008) and low-density lipoprotein(LDL) oxidation (Gheldof and Engeseth 2002). The majordifference among these assays is the quantitation approaches.For example, the ORAC assay applies the area under thekinetic curve approach; the TRAP assay depends on lag time,and the crocin-bleaching assay uses initial reaction rate(Huang et al. 2005).

Oxygen Radical Absorbance Capacity Assay

ORAC measures antioxidant inhibition of peroxyl-radical-induced oxidations and reflects classical radical chain-breaking antioxidant activity by H-atom transfer (Ou et al.2001). In the basic assay, the peroxyl radicals generated fromthermal decomposition of AAPH in aqueous buffer orhydroxyl radicals generated from Cu2+-H2O2 (Cao et al.1997) react with a fluorescent probe, an oxidizable pro-tein substrate, to form a nonfluorescent product, whichcan be quantitated easily by fluorescence. Probe reactionwith peroxyl radicals is followed by loss of the intensityof fluorescence by time. The first version of the ORACassay employed B-phycoerythrin (B-PE, a fluorescentprotein) as the probe. B-PE was chosen because of itsexcitation wavelengths, high fluorescent yield, sensitiv-ity to ROS, and water solubility (MacDonald-Wicks etal. 2006).

In addition to evaluation of antioxidant capacitysamples of fruits and vegetables, dietary supplements,wines, juices, and nutraceuticals, the ORAC assay havebeen used in plasma or serum samples. The proteinfraction of plasma or serum contributes significantly tothe antioxidant capacity, which may mask responses,particularly if the interest is in small-molecular-weightantioxidants. Therefore, protein removal is important(Prior et al. 2003).

In general, samples, controls, and standard (Trolox offour or five different concentrations for structuring thestandard curve) are mixed with fluorescein solution and


incubated at constant temperature (37 °C) before AAPHsolution is added to initiate the reaction (MacDonald-Wicks et al. 2006). Under this reaction conditions, 1 molof AAPH loses a dinitrogen to generate 2 mol of AAPHradical. In air-saturated solution, the generated AAPHradical reacts with O2 rapidly to give a more stableperoxyl radical ROO�. The loss of fluorescence of theprobe is an indication of the extent of damage from itsreaction with the peroxyl radical. The fluorescenceintensity [485 nm (ex)/525 nm (em)] is measured everyminute for 35 min at ambient conditions (pH 7.4, 37 °C).In the presence of antioxidant, the fluorescence decay isprevented (Ou et al. 2002a).

Advantages

Traditional antioxidant analyses followed extension ofthe lag phase only, but antioxidant effects often extendwell beyond early stages of oxidation (Niki 2002; Huanget al. 2005). For example, glutathione shows no clear lagphase and thus accurate calculations based solely on a lagphase are difficult (Cao et al. 1997). The protective effectof an antioxidant in ORAC assay is measured by the netintegrated area under the fluorescence decay curve of thesample compared to that of blank (AUCsample−AUCblank)and stands for lag time, initial rate, and total inhibition in asingle value (Prior et al. 2005). To avoid underestimationof antioxidant capacity and to account for potentialeffects of secondary antioxidant products, the ORACassay follows the reaction for extended periods, forexample, ≥30 min.

The advantage of AUC approach is that it operatesequally well for both antioxidants that exhibit distinctlag phases and those that have no lag phases. It isparticularly useful for samples which often containmultiple ingredients and have complex reaction kinetics.The FL probe is inexpensive and the use of a fullyautomated microplate fluorescence reader makes themethod readily accessible and the efficiency of theassay is enhanced with at least a tenfold increase insample throughput (Huang et al. 2002b).

Another advantage of ORAC assay is the capability ofemploying different free radical generators or oxidants. This isimportant because the measured antioxidant capacity of acompound depends on which free radical or oxidant is used inthe assay. For example, the ORAC assay using AAPH (anROO� generator) measures all traditional antioxidantsincluding ascorbic acid, α-tocopherol, β-carotene, glutathi-one, bilirubin, uric acid, and melatonin while the ORACassay using Cu2+-H2O2 (mainly an OH� generator) measurescompounds like mannitol, glucose, uric acid, and transitionmetal chelators but not ascorbic acid and α-tocopherol (Caoet al. 1997). ORAC assay measures only the antioxidant

capacity against peroxyl and hydroxyl radicals not thatagainst all reactive oxygen species (e.g., superoxides andsinglet oxygen; Apak et al. 2004).

Disadvantages

The use of B-PE in antioxidant assays has some limitationssuch having large interbatch differences, photobleaching ofB-PE after exposure to the excitation light, and interactionwith polyphenols by nonspecific protein binding. Thesefactors cause inconsistency in assay results and false lowvalues. To solve these disadvantages, Ou et al. (2001)replaced B-PE with FL. FL is a synthetic nonprotein probeand overcomes the shortcomings of B-PE. It is more stableand less reactive. However, FL is pH sensitive and thismust be carefully monitored (MacDonald-Wicks et al.2006). In addition, the reaction products of FL with peroxylradical have been characterized, and the product patternwas consistent with a classic HAT reaction mechanism(Prior et al. 2005).

As originally designed, the ORAC assay is limited tomeasurement of hydrophilic chain-breaking antioxidantcapacity against only peroxyl radicals. However, manyantioxidants are lipophilic and are particularly importantagainst lipid oxidation in all systems as well as otherradicals that are very active physiologically. It is alsoknown that the antioxidant capacity of a compound isdependent on reaction media; therefore, an organicsolvent-based ORAC assay has been adapted to measurelipophilic samples, too. To solve these disadvantages,Huang et al. (2002a) developed an assay for lipophiliccomponents using randomly methylated β-cyclodextrin asa solubility enhancer, which allows for the measurementof the antioxidant capacity of both hydrophilic andlipophilic components in a given sample separately usingthe same radical source. To obtain an accurate total ORACvalue of a given sample, both hydrophilic and lipophilicfractions need to be measured (Wu et al. 2004).

However, FL is not sufficiently lipid soluble, and itsfluorescence intensity in nonpolar organic solvent israther low. To overcome this problem, 4,4-difluoro-3,5bis(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene(BODIPY 665/676) is employed as a fluorescent probeand AMVN as a peroxyl radical generator (Naguib 2000).AMVN has the advantage of being highly soluble intoluene, methanol, ethanol, and hexane (Laguerre et al.2007). By applying this assay, the antioxidant capacity ofvarious carotenoids was quantified. However, this assay is100 times less sensitive than the classical ORAC assay,probably due to the low efficiency of the radical generator,AMVN. In addition, the fluorescent quenching mechanismof BODIPY by peroxy radical remains to be investigated(Huang et al. 2005).


Total TRAP Assay

Total TRAP assay monitors the ability of antioxidantcompounds to interfere with the reaction betweenperoxyl radicals generated by AAPH or ABAP and atarget probe (Schlesier et al. 2002). The TRAP assayuses R-phycoerythrin (R-PE) as a fluorescent probe andthe reaction progress of R-PE with AAPH was monitoredfluorometrically [(ex) 495 nm and (em) 575 nm)].

Valkonen and Kuusi (1997) applied dichlorofluores-cein-diacetate (DCFH-DA) as the fluorescent oxidizablesubstrate (probe). The oxidation of DCFH-DA by peroxylradicals yields the formation of highly fluorescentdichlorofluorescein (DCF) product (ex 480 nm, em526 nm) and also has absorbance at 504 nm. This enabledthe determination of serum TRAP by simple spectropho-tometry. The presence of antioxidant compounds compet-itively inhibits the increase of fluorescence signal. Adomand Liu (2005) also developed a rapid and sensitivehydrophilic peroxyl radical capacity (hydro-PSC) assayincorporating DCHF-DA as a fluorescent probe andmodified the hydro-PSC assay as a lipophilic peroxylradical scavenging capacity (lipo-PSC) assay, usingrandomly methylated β-cyclodextrin to increase thesolubility of lipophilic compounds.

Wolfe and Liu (2007) developed a method to measureantioxidant capacity in cell culture. Cellular antioxidantactivity (CAA) assay is a valuable new method and hasadvantages over the traditional chemistry antioxidant assaysbecause it is a more biologically relevant model. The assayutilizes HepG2 human hepatocarcinoma cells because theygive consistent results with lower coefficient of variation.Cells are pretreated with antioxidant compounds andDCFH-DA. The antioxidants bound to the cell membraneand/or passed through the membrane to enter the cell.DCFH-DA diffused into the cell where cellular esterasescleaved the diacetate moiety to form more polar DCFH,which was trapped within the cell. Cells were also treatedwith ABAP or AAPH which was able to diffuse into cells.They spontaneously decompose to form peroxyl radicals.These radicals attack the cell membrane to yield moreradicals and oxidized the intracellular DCFH to thefluorescent DCF and the level of fluorescence measuredupon excitation is proportional to the level of oxidation. Inaddition to peroxyl radicals and hydrogen peroxides,various other species (e.g., peroxynitrite, nitric oxide,superoxide radicals) have been found to oxidize DCFH toDCF in cell culture. In a study, where the CAA values of 25fruits were determined, it has been shown that the CAAvalues for fruits were significantly positively related to totalphenolic content and ORAC values (Wolfe et al. 2008).However, this is in contrast to a study of Eberhardt et al.(2005) involving broccoli extracts which showed that no

correlation was found between ORAC and cellular antiox-idant capacity methods.

Advantages

TRAP assay involves the initiation of lipid peroxidation bygenerating water-soluble peroxyl radicals and is sensitive toall known chain-breaking antioxidants; the concept hasbeen very useful for quantifying and comparing antioxidantcapacity.

Disadvantages

The method’s greatest problem is perhaps its greateststrength; too many different end points have been used,so comparisons between laboratories are difficult. Forexample, the method suffered from the drawback ofoxygen electrode end point in that the electrode wouldnot maintain its stability over the required time period(up to 2 h per sample; Apak et al. 2007). TRAP assay isrelatively complex and time-consuming to perform,requiring a high degree of expertise and experienceHowever, the TRAP assay has been criticized as employ-ing water-soluble peroxyl radicals (Frankel and Meyer2000), but the method can be adapted to use lipid-solubleinitiators (Prior et al. 2005).

Another shortcoming of TRAP assay is the use of thelag time that corresponds to the inhibition of accumu-lation of colored radical reagents in the presence ofantioxidants (e.g., the time period required for thecolored radical to emerge in the reaction medium; Apaket al. 2007) for quantifying antioxidant capacity since notevery antioxidant has an obvious lag phase (Magalhaes etal. 2008).

Moreover, the value obtained from the lag phase aloneoften underestimates antioxidant capacity considerablybecause the antioxidant value obtained after the lag phaseis totally ignored (Prior et al. 2005). Another importantlimitation of this assay and also of the ORAC assay is thatthe oxidative deterioration and antioxidant protection offluorescent probe does not mimic a critical biologicalsubstrate (Frankel and Meyer 2000).

In TRAP assay, the probe must be reactive withROO� at low concentrations and to maximize the sensi-tivity there must be a dramatic spectroscopic changebetween the native and oxidized state of probe, and noradical chain reaction beyond probe oxidation shouldoccur (Prior et al. 2005).

β-Carotene or Crocin-Bleaching Assay

Carotenoids bleach via autoxidation induced by light orheat or peroxyl radicals (e.g., AAPH or oxidizing lipids).


The addition of an antioxidant-containing sample, individ-ual antioxidants, or plant extracts causes the inhibition ofβ-carotene bleaching (Laguerre et al. 2007). The assaymeasures the decrease in the rate of β-carotene or crocindecay provided by antioxidants. Color loss followedoptically at 443 nm in phosphate buffer (pH 7.0), so nospecial instrumentation is required. The bleaching ratebecomes linear at ∼1 min after the addition of AAPH andis monitored for 10 min (Huang et al. 2005).

Advantages

The kinetic approach in the measurement allows thedetermination of total inhibiting effects and provides amore precise evaluation of the efficiency of antioxidantdefense, but two independent parameters, the reactivityand antioxidant capacity, cannot be determined sepa-rately. Later, a modified microplate-based version suit-able for routine determinations was suggested (Roginskyand Lissi 2005).

Disadvantages

Although β-carotene is often used as the target (probe), itsdiscoloration at 470 nm can occur by multiple pathways, sointerpretation of results can be difficult. To overcome thisproblem, carotenoid derivative crocin, a natural compoundwith extremely strong absorbance in visible range, hasbecome the reagent choice. Crocin has straightforwardreactions and undergoes bleaching only under the attack ofperoxyl radical (Prior et al. 2005).

It is not clear in the presence of some antioxidants,such as Trolox, ascorbic acid, or uric acid whether a lagphase, or a period of maximal protection of crocin fromoxidation by peroxyl radicals, can be seen. Theseantioxidants usually show a lag phase in the assaysystems that use lipids or proteins as oxidizablesubstrate for peroxyl radicals. The antioxidant capacityof ascorbic acid analyzed using this kinetic method wasreported to be 7.7 Trolox equivalents (Tubaro et al.1998), much higher than those obtained with any othermethods (Prior and Cao 1999).

This assay has found limited applications in foodsamples so far because crocin is not available commer-cially and must be extracted from saffron which mayresult to a low reproducibility between assays. More-over, crocin absorbs at a rather short wavelength(450 nm), and many food pigments, such as carote-noids, absorb at the same wavelength. To eliminatepossible interferences from the sample itself, a blank (amixture containing only AAPH and food sample) mustbe tested at the same time under the same wavelength(MacDonald-Wicks et al. 2006). Ordoudi and Tsimidou

(2006) have examined the crocin-bleaching assay perfor-mance and validation procedures.

Total Phenol Assay by Using the Folin-Ciocalteu Reagent

The exact chemical nature of the Folin-Ciocalteu reagentis not known, but it is accepted that it containsphosphomolybdic/phosphotungstic acid complexes. Thechemistry behind the FCR assay counts on the transfer ofelectrons in alkaline medium from phenolic compoundsand other reducing species to molybdenum, forming bluecomplexes that can be monitored spectrophotometricallyat 750–765 nm (Magalhaes et al. 2008). The phenoliccompounds react with FCR only under basic conditions(adjusted by a sodium carbonate solution to pH∼10;MacDonald-Wicks et al. 2006). Since Singleton and Rossiextended this assay to the analysis of total phenols inwine, it has found many applications (Huang et al. 2005).They also specified the conditions to minimize thevariability and eliminate inconsistent results: (1) propervolume ratio of alkali and FCR, (2) optimal reaction timeand temperature for color development, (3) monitoring ofoptical density at 765 nm that allows us to minimizeinterference from the sample matrix, which is oftencolored, (4) use of reference standards such as gallic acid(Prior et al. 2005).

Advantages

Despite the undefined chemical nature of FCR, the totalphenol assay by FCR is convenient, simple, and reproduc-ible (Huang et al. 2005). Several publications applied thetotal phenol assay by FCR and antioxidant capacity assay(e.g., DPPH, FRAP, TEAC, ORAC etc.) and often foundexcellent linear correlations between the “total phenolicprofiles” and “the antioxidant activity” (Gheldof andEngeseth 2002; De Beer et al. 2003; Madhujith et al.2006; Shahidi et al. 2006; Stratil et al. 2006).

Disadvantages

Instead of recommended gallic acid reference standardcatechin equivalents (Vinson et al. 2001; Katsube et al.2003), tannic acid equivalents (Nakamura et al. 2003),chlorogenic acid equivalents (Wang et al. 2003b), caffeicacid equivalents (Maranz et al. 2003), and ferulic acidequivalents (Velioglu et al. 1998; Stratil et al. 2006) havealso been used. Lack of standardization of methods can leadto several orders of magnitude difference. The finalabsorbance values are usually proportional to the numberof reacting phenolic hydroxyl groups and also depend onthe molecule structure. If the standard used for calibration ishighly reactive and gives a high absorbance, then the


measured values of samples will be low. For example, theabsorbance value for caffeic acid (two reacting OH) isapproximately twice and for catechin (three reacting OH)three times higher than that for phenol (one reacting OH;Stratil et al. 2006). The choice of standard as a “criticalcontrol point” of the antioxidant capacity assays isexamined in Nenadis et al. (2007) in detail.

The FCR-based assay has been known as the totalphenol assay. The FCR reagent is not specific for phenoliccompounds as it can also be reduced by many nonphenoliccompounds (MacDonald-Wicks et al. 2006). The totalphenol assay by FCR is carried out in water, an aqueousphase. For lipophilic antioxidants, this assay in its currentform is not applicable. The FCR actually measures asample’s reducing capacity, but this is not reflected in thename “total phenolic assay.” There is always the disagree-ment on what is being detected in total antioxidant capacityassays, whether only phenols or phenols plus reducingagents plus possibly metal chelators.

TEAC Assay

TEAC assay was first reported by Miller et al. (1993) andhas been improved and widely used in testing antioxidantcapacity in food samples. ABTS is a peroxidase substrate,which when oxidized by peroxyl radicals or other oxidantsin the presence of H2O2 generates a metastable radicalcation ABTS�þ) which is intensely colored and can bemonitored spectrophotometrically in the range of 600–750 nm. The antioxidant capacity is measured as the abilityof test compounds to decrease the color reacting directlywith ABTS�þ radical and expressed relative to Trolox(Roginsky and Lissi 2005).

The order of addition of reagents and sample wasmodified in the improved version. The sample to be testedwas added after generation of a certain amount of radicalcation and the remaining radical cation concentration afterreaction with antioxidant compound/sample was thenquantified to minimize the interference of compounds withoxidants during radical formation and prevent the possibleoverestimation (Magalhaes et al. 2008).

In further modified methods that utilize ABTS radical incommon, ABTS was generated by enzymatic (e.g., perox-idase, myoglobin) or chemical reactions (manganese diox-ide, potassium persulfate, ABAP; Schlesier et al. 2002).Generally, chemical reactions require a long time (e.g., upto 16 h for potassium persulfate generation) or hightemperatures (e.g., 60 °C for ABAP generation), whereasenzyme generation is faster and the reaction conditions aremilder (Prior et al. 2005).

The TEAC assay measures the antioxidant capacity ofthe parent compound plus that of reaction products. Thesereaction products may have considerable contribution to the

TEAC. For example, the relatively high TEAC of chrysincompared to its moderate antioxidant activity is due to theformation of reaction products that also scavenge ABTS�þ.The TEAC assay is often used to rank antioxidants and forthe construction of structure activity relationships (Arts etal. 2004). There is no relation between the TEAC value andthe number of electrons that an antioxidant can donate(MacDonald-Wicks et al. 2006).

Advantages

ABTS�þ can be solubilized in both aqueous and in organicmedia and is not affected by ionic strength, so theantioxidant capacity can be measured due to the hydrophilicand lipophilic nature of the compounds (Arnao 2000).TEAC method which is based on the oxidation of ABTS inthe presence of H2O2 and metmyoglobin only measureshydrophilic antioxidants. Preparation of ABTS radical byfiltering ABTS solution through manganese dioxide pow-der allows the assay to measure lipophilic antioxidants likecarotenoids and tocopherols. By changing the solvent fromwater to ethanol, TEAC method also enables the measure-ment of both kinds of antioxidants (Schlesier et al. 2002).

TEAC assay is operationally simple and has beenused in many research laboratories. Absorption maximawere shown to be at wavelengths of 414, 752, and842 nm in aqueous media and 414, 730, and 873 nm inethanolic media (Arnao 2000). Another advantage ofTEAC assay is that it permits study over a wide pH range.Although frequently used at pH 7.4, the stability of ABTSradical at this pH has been reported to be problematic. Forstandard antioxidants such as Trolox or ascorbic acid,ABTS radical at pH 7.4 provided reliable end-point valuesafter 10 min. However, with standard phenolics, theresults at 10 min are estimates only and do not representequilibrium end-point values based on oxidation. Also,with ABTS radical at pH 7.4, values for the antioxidantcapacity of the standard phenolics were 5–20% greaterthan the values determined at pH 4.5 (Ozgen et al. 2006).

Disadvantages

As for the limitations of this method, TEAC value actuallycharacterizes the capability of the tested sample to react withABTS�þ rather than to inhibit the oxidative process. TheTEAC reaction with many phenolics and samples of naturalproducts may take a long time to reach an end point. Thus,by taking this fixed end point of short duration (4 or 6 min),false low TEAC values can be read before the reaction isfinished (Huang et al. 2005). As studied by Perez-Jimenezand Saura-Calixto (2008), comparison between the antioxi-dant capacity obtained at a fixed end point and the kineticsbehavior of the samples, when both the concentration and the


time necessary to deplete the radical are considered, showssome differences. For example, BHA shows higher antiox-idant capacity than ferulic acid when results are obtained at afixed end point, but it has low value indicating slowerkinetics than ferulic acid. The fact that the ABTS radicalused in TEAC assays is not found in biological systems andis not similar to radicals found in those systems is alsoanother problem (MacDonald-Wicks et al. 2006).

DPPH Radical Scavenging Capacity Assay

The DPPH radical is a long-lived organic nitrogen radicaland has a deep purple color. It is commercially availableand does not have to be generated before assay. In thisassay, the purple chromogen radical is reduced by antiox-idant/reducing compounds to the corresponding pale yellowhydrazine. The reducing ability of antioxidants towardsDPPH can be evaluated by electron spin resonance or bymonitoring the absorbance decrease at 515–528 nm untilthe absorbance remains stable in organic media. Thiswidely used method was first reported by Brand-Williamset al. (1995). The percentage of the DPPH (%DPPHrem)remaining is calculated as:

%DPPHrem ¼ 100� DPPH½ �rem�DPPH½ �T¼0

%DPPHrem is proportional to the antioxidant concentra-tion, and the concentration that causes a decrease in theinitial DPPH concentration by 50% is defined as EC50. Thetime needed to reach the steady state with EC50 is definedas TEC50.

Based on TEC50 values, the kinetic behavior of theantioxidant is classified as follows: <5 min (rapid), 5–30 min (intermediate), and >30 min (slow; Sanchez-Moreno et al. 1998). Sanchez-Moreno et al. (1998) furtherintroduced another parameter to express antioxidant capac-ity, called “antiradical efficiency” [AE=(1/EC50) TEC50]which is more discriminative than TEC50 and more usefulbecause it takes into account the reaction time.

A similar and new parameter designed as “radicalscavenging efficiency (RSE), combining scavenging activ-ity in terms of the amount of radicals scavenged and initialscavenging rate, was suggested by De Beer et al. (2003).RSE is calculated as the ratio of the initial scavenging rate(obtained during the first minute) and the EC50 value. Themain limitation of determination is that the percentage ofscavenged radical is dependent on of the initial DPPHradical concentration.

Advantages

The DPPH assay is technically simple and rapid and needsonly a UV-Vis spectrophotometer that might explain its

widespread use in antioxidant screening. Analyses of alarge number of samples could be made by using micro-plates (Fukumoto and Mazza 2000).

Disadvantages

There are some drawbacks which limit its application. DPPHcan only be dissolved in organic media (especially in alcoholicmedia), not in aqueous media, which is an important limitationwhen interpreting the role of hydrophilic antioxidants (Arnao2000). Although widely used for measuring and comparingthe antioxidant status of phenolic compounds and foodstuffs,the evaluation of antioxidant capacity by the changes inDPPH absorbance should be carefully inferred since absor-bance of DPPH radical at 517 nm after reaction with anantioxidant is decreased by light (Ozcelik et al. 2003),oxygen, and type of solvent (Apak et al. 2007). It wasconcluded that above a certain limit of water content ofsolvent, the antioxidant capacity decreased, since a part ofthe DPPH coagulates and it is not easily accessible to thereaction with antioxidants (Magalhaes et al. 2008).

DPPH molecule has little similarity to the highlyreactive and transient peroxyl radicals. Also, manyantioxidants that may react quickly with peroxylradicals in vivo may react slowly or may even be inertto DPPH. The reaction kinetic between DPPH andantioxidants is not linear to DPPH concentration;therefore, to express antioxidant capacity using EC50 isproblematic. Moreover, interpretation of the results iscomplicated if the test compounds have spectra thatoverlap DPPH at 515 nm (e.g., in particular, carotenoids;Prior et al. 2005). It was also reported that the reaction ofDPPH with eugenol was reversible (Huang et al. 2005).This would result in falsely low readings for antioxidantcapacity of samples containing eugenol and other phenolsbearing a similar structure.

Originally, it was believed that the DPPH assay was ahydrogen transfer reaction but recent studies suggestedotherwise (MacDonald-Wicks et al. 2006). The rate-determining step for this reaction occurs very quickly asinitial electron transfer and subsequent hydrogen transferoccurs very slowly and depends on the neutral hydrogen-bond accepting solvent such as methanol and ethanol(Huang et al. 2005) In addition, basic and acidic impuritiesin the solvent may influence the ionization equilibrium ofphenols and therefore cause a reduction or enhancement ofthe measured rate constants (MacDonald-Wicks et al.2006).

FRAP Assay

FRAP assay is based on the ability of phenolics to reduceyellow ferric tripyridyltriazine complex (Fe(III)-TPTZ) to


blue ferrous complex (Fe(II)-TPTZ) by the action ofelectron-donating antioxidants (Benzie et al. 1999). Theresulting blue color measured spectrophotometrically at593 nm is taken as linearly related to the total reducingcapacity of electron-donating antioxidants. Ferric salt isused as an oxidant and its redox potential (<0.70 V) iscomparable to that of ABTS (0.68 V). Therefore, essential-ly, there is not much difference between TEAC assay andthe FRAP assay except TEAC is carried out at neutral pHwhereas FRAP assay needs acidic (nonphysiologically lowpH value=3.6) conditions to maintain the iron solubility.One FRAP unit is defined as the reduction of 1 mol of Fe(III) to Fe(II) (Huang et al. 2005).

Advantages

FRAP assay is simple, rapid, inexpensive, and robust anddoes not require specialized equipment. The FRAP assaycan be performed using automated, semiautomatic, ormanual methods (Prior et al. 2005).

Disadvantages

Fe(II) is a well-known “prooxidant.” It can react with H2O2

to produce hydroxyl radical (OH�), the most harmful freeradical found in vivo. The ability of a compound to produceFe(II) from Fe(III) defined as “antioxidant power” in theFRAP assay because it is probable that some antioxidants,such as ascorbic acid and uric acid, can reduce both reactivespecies and Fe(III) and their ability in reducing Fe(III) mayreflect their ability in reducing reactive species. But not allreductants that are able to reduce Fe(III) are antioxidants(Prior and Cao 1999). Any electron-donating substanceeven without antioxidant properties with redox potentiallower than that of the redox pair Fe(III)/Fe(II) cancontribute to the FRAP value and indicate falsely highvalues (Nilsson et al. 2005). In addition, an antioxidant thatcan effectively reduce prooxidants may not be able toefficiently reduce Fe(III). For example, the FRAP assaydoes not measure GSH (glutathione), an important antiox-idant in vivo (Prior and Cao 1999).

Potential problems take place as the mixture containsother Fe(III) species, which can bind to chelators in thefood extract and these complexes are capable of reactingwith the antioxidants. Results show that, similar toTEAC, there is no relation between the FRAP value andthe number of electrons that an antioxidant can donate(MacDonald-Wicks et al. 2006). The FRAP values forascorbic acid, α-tocopherol, and uric acid are identical(2.0). The FRAP value of bilirubin is onefold higher thanthat of ascorbic acid. These results suggest that 1 mol ofascorbic acid can reduce 2 mol of Fe(III) and 1 mol ofbilirubin can reduce 4 mol of Fe(III). This is in conflict

with the fact that both ascorbic acid and bilirubin are bothtwo-electron reductants (Huang et al. 2005).

FRAP cannot detect compounds that act by radicalquenching (H transfer), particularly thiols and proteins (Ouet al. 2002a). This causes a serious underestimation inserum. Both the FRAP and TEAC assays rely on thehypothesis that the redox reactions proceed so rapidly thatall reactions are complete within 4 and 6 min, respectively,but in fact this is not always true. Pulido et al. (2000)measured the FRAP values of several polyphenols in waterand methanol. However, for polyphenols with such behav-iors including caffeic acid, tannic acid, ferulic acid, ascorbicacid, and quercetin, the absorption (A593) does not stop at4 min; instead, it slowly increased even after several hoursof reaction time. Thus, determination of antioxidantcapacity by utilizing a fixed end point may not representa completed reaction.

Total Antioxidant Potential Assay Using Cu(II)as an Oxidant

The method is based on reduction of Cu(II) to Cu(I) byreductants (antioxidants) present in a sample. It hasbeen introduced as Bioxytech AOP-490 and CUPRACdeveloped by Apak et al. (2004). In the Bioxytech AOP-490 assay, bathocuproine (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) forms a 2:1 complex with Cu(I),producing a chromophore with maximum absorbance at490 nm. Reaction rate and concentration of products arefollowed by bathocuproine complexation of the Cu(I)produced (Prior et al. 2005).

The CUPRAC assay uses the copper(II)-neocuproinereagent as the chromogenic oxidant. It involves mixingthe antioxidant solution (directly or after acid hydroly-sis) with solutions of CuCl2, neocuproine, and ammoni-um acetate at pH 7, and measuring the absorbance at450 nm after 30 min. Flavonoid glycosides needed to behydrolyzed to their corresponding aglycons for fullyexhibiting their antioxidant capacity. Slow-reacting anti-oxidants needed increased temperature incubation tocomplete their oxidation with the CUPRAC reagent (Apaket al. 2008).

Advantages

CUPRAC values are comparable to TEAC values forpolyphenols, whereas FRAP values are usually consider-ably lower (Apak et al. 2004). Due to the lower redoxpotential of the CUPRAC reagent, reducing sugars andcitric acid—which are not true antioxidants but oxidizablesubstrates in other similar assays, e.g., FRAP—are notoxidized with the CUPRAC reagent (Apak et al. 2008). Atthe same time, the low redox potential enhances redox


cycling, so copper reduction may be and even moresensitive indicator of potential prooxidant activity ofantioxidants (Prior et al. 2005).

Other advantages of the CUPRAC method over othersimilar assays are: (a) the CUPRAC reagent is fast enough tooxidize thiol-type antioxidants, whereas the FRAP methoddoes not measure certain thiol-type antioxidants like glutathi-one; (b) the reagent is more stable and accessible than otherchromogenic reagents (e.g., ABTS, DPPH); (c) easily anddiversely applicable in rutin laboratories; (d) the absorbanceversus concentration curves are linear over a wide range,unlike those of other methods yielding polynomial curves; (d)the redox reaction yielding colored species is carried out atpH 7 buffer as opposed to the acidic conditions of FRAP(pH 3.6) or basic conditions of the Folin-Ciocalteu assay(pH 10); (e) the method can concurrently measure hydrophilicand lipophilic antioxidants (unlike FCR and DPPH; Apak etal. 2008). CUPRAC assay is complete in minutes forascorbic acid, uric acid, gallic acid, and quercetin butrequires 30–60 min for more complex molecules.

Disadvantages

The copper reduction assays still have similar problemswith a complex mixture of antioxidants in terms ofselecting an appropriate reaction time (Prior et al. 2005).

Methods that Measure the Inhibition of Induced LipidAutoxidation

This method artificially induces autoxidation of linoleic acidor LDL particles or serum proteins without prior isolation ofLDL particles (Gheldof and Engeseth 2002), by either atransition element such as Cu(II) or thermal decompositionof AAPH, a water-soluble diazo ROO� initiator, andperoxidation of the lipid components determined throughthe formation of conjugated dienes, followed at 234 nmspectrophotometrically (Hendelman et al. 1999). The use ofAAPH as peroxyl radical generator is preferred over Cu(II)since it bears a strong resemblance to oxidative reactions inbiological systems (Magalhaes et al. 2008). Basically, threemechanisms are involved in LDL antioxidant activity; freeradical scavenging activity, binding to critical sites on LDL,and metal chelation (Katsube et al. 2004). In general, in vitroLDL oxidation has been used effectively in severalresearches to characterize antioxidant capacity of a numberof different phytochemicals (Hu et al. 2000; Katsube et al.2004; Shahidi et al. 2006; Chumark et al. 2008).

Advantages

The reaction can be carried out in micelles or in organicsolvents. In micelles, reaction progress cannot be followed

directly by a UV spectrophotometer and sample workup isnecessary; this limits the efficiency of the method (Huanget al. 2005). On a limited group of samples, a goodrelationship was observed between LDL oxidation usingAAPH and the ORAC value (Hendelman et al. 1999);however, the relationship was not present when Cu(II) wasused as the oxidant.

Disadvantages

Problems arise because it is difficult to measure the small lagtimes that may occur, and many substances in foods alsoabsorb at 234 nm. Additionally, linoleic acid will formmicelles in the presence of water that can be overcome byusing methyl esters, although products of methyl esters canshow interbatch differences which are not desired in achemical quantitation method and further complicate theassay because the antioxidant distribution between two phasesis critical (MacDonald-Wicks et al. 2006).

Cu(II) alone does not induce the autoxidation of lipids.Instead, the reaction was initiated by antioxidants (e.g.,tocopherols) present in the LDL. Flavonoids might presentantioxidant or prooxidant activities depending on the radical-generating system and concentration (Hassimotto et al. 2005).

The method has a major drawback that LDL must beisolated on a regular basis, and obtaining blood samplesfrom different individuals is necessary. Thus, it is notpossible to get consistent preparations. According to Prioret al. (2005), this method is not much suitable for thedevelopment of a consistent reproducible high-throughputassimilable organic carbon (AOC) assay.

Chemiluminescence Assay

The general principle of these methods is based on theability of luminol and related compounds to lumines-cence under the attack of free radicals (chemilumines-cence (CL); Roginsky and Lissi 2005). This reactionproduces low-intensity light emission that may decayrapidly. The addition of para-iodophenol gives a moreintense, prolonged, and stable light emission. The lightemission is interfered by antioxidants but will be restoredwhen all the added antioxidants have been depleted (Priorand Cao 1999). Oxidant sources of peroxyl radicalsinclude the enzyme horseradish peroxidase (Whitehead etal. 1992) and H2O2-hemin (Bastos et al. 2003). Luminol isthe most widely used marker compound to trap oxidantsand convert weak emissions into intense, prolonged, andstable emissions (Whitehead et al. 1992), althoughlucigenin and bioluminescent proteins such as Pholasin®are becoming more popular. Pholasin emits light in thepresence of different systems capable of generating freeradicals such as superoxide and hydroxyl radical (but not


peroxyl radical) and certain oxidants (Reichl et al. 2001;Jaffar et al. 2006).

The addition of the antioxidant/sample brings on a CL-lag phase (time interval which CL emission was notmonitored). The magnitude of the lag phase was directlyrelated to the antioxidant concentration or it results to thedecrease of CL emission, expressed as inhibition percent-age (Magalhaes et al. 2008).

Advantages

By changing the initiator, the reaction can be altered todifferentiate quenching of specific oxidants, for example,O��2 , HO�, HOCl, ROO�, �OONO, and 1O

2. CL can bequite sensitive in detecting low-level reactions because itprovides a detectable response below the detection limit ofmost chemical assays (Prior et al. 2005). The promisingcharacteristic of CL methods is their productivity; com-monly, one run takes a few minutes only; in addition, theassay can be easily automated and run in microwell plates(Roginsky and Lissi 2005).

Disadvantages

CL detection needs special equipment that both set samplesclose to the detector and detects light at single-photon levelsand, in addition, provides temperature control. The mecha-nism for chemical processes resulting in CL is not known indetail which may create problems with interpreting dataobtained (Roginsky and Lissi 2005). The choice of emitter isalso a critical consideration. The use of luminol is acceptablewhen single oxidants are being measured. However, becausethe intensity of emissions changes considerably with theoxidant, use of luminol in systems with mixed oxidants isnot straightforward (Prior et al. 2005).

Photochemiluminescence Assay

This assay was described by Popov and Lewin, wascommercialized by Analytik Jena AG (Jena, Germany), andis commercialized as a complete system under the namePHOTOCHEM® (Prior et al. 2005). This assay involves thephotochemical generation of superoxide O��

2 free radicalscombined with CL detection. The optical excitation of aphotosensitizer results in the generation of the superoxideradical. The free radicals are monitored with a CL reagent.Luminol acts as a photosensitizer as well as an oxygenradical detection reagent.

Advantages

PCL-based methods principally differ from other antioxi-dant evaluation procedures because they do not require

oxidizing reagents for the generation of radical species(Vertuani et al. 2004). In contrast to other commonly usedAOC assays, the PHOTOCHEM method is not limited to aspecific pH value or temperature range (Prior et al. 2005).PCL assay has been used effectively in several investiga-tions to characterize antioxidant activity (Madhujith et al.2006; Sclesier et al. 2002). Other advantages of PCL are: itdoes not require high temperatures to generate radicals andit is more sensitive (nanomolar range) in measuring, in fewminutes (Vertuani et al. 2004).

Disadvantages

The weak correlation between ORAC and PCL and FRAPand PCL is not unexpected because different radicalsources are being evaluated (Sclesier et al. 2002). Thecomplete reaction mechanism is not known which makesthe interpretation of results difficult.

Total Oxidant Scavenging Capacity Assay

Total oxidant scavenging capacity (TOSC) assay is based onoxidation of α-keto-γ-methiolbutyric acid (KMBA) to ethyl-ene by peroxyl radicals produced from AAPH. The antioxi-dant capacity of a molecule is quantified from its ability toinhibit ethylene formation relative to a control. The timeduration of ethylene formation is followed by gas chromatog-raphy (GC) headspace (Regoli and Winston 1999).

The approach for quantification of antioxidant capacity isbased in the area under the curve that defines the inhibition ofethylene formation as a function of time, which can be up to300 min (Huang et al. 2005). The antioxidant or TOSC valueis from 0 to 100 (Prior and Cao 1999).

This method points out an important issue in terms of beingable to evaluate different antioxidants with different biolog-ically relevant radical sources, peroxyl radicals, hydroxylradicals, generated by the reaction of iron and ascorbate, andperoxynitrite generated from 3-morpholinosydnonimine N-ethylcarbamide (Regoli and Winston 1999; Prior et al. 2005).For example, reduced glutathione was an efficient scavengerof peroxyl radicals but a relatively poor scavenger ofperoxynitrite and hydroxyl radicals. Uric acid, Trolox, andascorbic acid were comparable scavengers of peroxynitriteand peroxyl radicals. TOSC assay is useful and robust indistinguishing the reactivity of various oxidants and therelative capacities of antioxidants to scavenge these oxidants(Regoli and Winston 1999).

Advantages

In addition to reproducibility, the main advantages of theTOSC assay are its effectiveness in detecting the scaveng-ing capacity of both hydrophilic and lipophilic antioxidants


and its ability to distinguish between fast- and slow-actingantioxidants (Regoli and Winston 1999).

Disadvantages

The long reaction time (>100 min), the instability of theassay solutions, and the necessity of multiple manualinjections from the same sample into a gas chromatographto monitor the ethylene formation make this assay notadaptable to routine analysis (Huang et al. 2005).

Measurement of Other Reactive Oxygen/NitrogenSpecies

Although HAT- and ET-based methods, designed to measurea sample’s capacity to react with peroxyl radical, are mostfrequently employed in the determination of antioxidantcapacity, the methods including other ROS have to bedesigned to comprehensively evaluate the antioxidant capac-ity of a sample. This fact is implied in several studies(Kumaran and Karunakaran 2006; Madhujith et al. 2006;Mathew and Abraham 2006; Rivero-Perez et al. 2007)

The main ROS and RNS causing an oxidative damage inbiological tissues are superoxide anion (O��2 ), hydroxylradical (HO�), hydrogen peroxide (H2O2), peroxyl radicals(ROO�), singlet oxygen (1O2), peroxynitrite (ONOO−),nitric oxide (NO�), and nitric dioxide (NO�2 ). Living cellshave a biological defense system consisting of enzymaticantioxidants that convert ROS to harmless species. Forexample, O��2 is converted to oxygen and hydrogenperoxide by superoxide dismutase (SOD) or reacts withnitric oxide (NO�) to form peroxynitrite. H2O2 can beconverted to water and oxygen by catalase enzyme.

Superoxide anion (O��2 ) is a reduced form of molecularoxygen by receiving one electron. Superoxide is a relativelyweak oxidant; therefore, the reason for the discovery ofcompounds that scavenge O��2 is that this radical candecompose to form more potent and reactive ROS such asthe hydroxyl radical (�OH) and peroxynitrite (ONOO−),singlet oxygen which initiate the lipid oxidation. Flavonoids,including epicatechin, myricetin, rutin, catechin, epigalloca-techin, quercetin, galangin, and morin can scavenge O��2(Hort et al. 2008), but the scavenging of O��2 is not the onlymechanism for inhibition of lipid oxidation in eitherbiological or lipid food systems (Frankel and Meyer 2000).

The analytical methods for determination of O��2scavenging capacity employ enzymatic or nonenzymaticO��2 generation systems. Enzymatic system uses xanthineoxidase (XOD)/hypoxanthine or xanthine to generate O��2radicals at pH 7.4 (Madhujith et al. 2006). In normal tissue,XOD is a dehydrogenase enzyme that transfers electrons tonicotinamide adenine dinucleotide. During times of stress,

this enzyme is converted to an oxidase enzyme andproduces superoxide and hydrogen peroxide. Nonenzymat-ic system utilizes a nonenzymatic reaction of phenazinemethosulfate in the presence of nicotinamide adeninedinucleotide (Rivero-Perez et al. 2007).

Detection methods may include spectrophotometricmeasurements using nitroblue tetrazolium (NBT), cyto-chrome c, hydroethidine (HE) as fluorescent probes inredox reactions; CL-based determinations; GC and electronspin resonance (ESR) spectroscopy.

In spectrophotometric measurements that uses NBT asprobe, O��2 may reduce NBT into formazan and bemeasured at 560 nm. Antioxidant compounds competewith NBT for O��2 and decrease the reaction rate.

Another spectrophotometric detection method competi-tion utilizes kinetics of O��2 reduction of cytochrome c(probe) and O��2 scavenger (sample). The kinetic analysis ofreduction of ferricytochrome c to ferrocytochrome c wasmonitored at 550 nm (Aruoma et al. 1993). Cytochrome ccan be reduced directly by antioxidants, which can alsoinhibit the xanthine oxidase. This is an important issue asfar as ferricytochrome c is concerned, as it is easily reducedby ascorbic acid. Furthermore, the reduced antioxidantformed by attack of O��2 could also reduce NBT orferricytochrome c. Therefore, this method is not suitablefor quantifying nonenzymatic antioxidant (Huang et al.2005; Magalhaes et al. 2008).

Another spectrophotometric detection method uses HEas a redox sensitive probe. Nonfluorescent HE is oxidizedby O��2 generated from XOD/xanthine system to form afluorescent marker product that has signal at 586 nm. Theoxidation mechanism still remains unclear (Benov et al.1998; Zhao et al. 2003). This approach can avoid theproblem of direct reduction of the probe by antioxidants,but possible inhibition of xanthine oxidase by antioxidants(sample) still exists (Magalhaes et al. 2008).

In CL-based determination of O��2 scavenging capacity,a pale yellow solution of O��2 in dimethyl sulfoxide(DMSO), obtained from potassium superoxide and 18-crown-6, stable at room temperature for at least 1 h, emitsa high CL. This reaction was considered a good methodfor examining antioxidative or prooxidative properties ofmany biologically important compounds (Aboul-Eneinet al. 2005).

In GC detection method, the reaction product of KMBAand O��2 , ethylene, is measured. O��2 is generated by usingXOD/xanthine-generating system (Lavelli et al. 1999).

The scavenging capacity of O��2 can also be detected byusing ESR spectroscopy. Here, the O��2 is trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and the resultantDMPO superoxide adduct (DMPO-OOH) is detected byESR using manganese oxide as the internal standard(Calliste et al. 2001). A major disadvantage of DMPO is


that the DMPO superoxide adduct (DMPO-OOH) sponta-neously decomposed to the DMPO hydroxyl adduct(DMPO-OH) that makes spectral interpretation moreconfusing. To overcome this potential drawback, Zhang etal. (2000) employed 5-ethoxycarbonyl-5-methyl-pyrroline-N-oxide (EMPO) to trap superoxide radical. The superoxideadduct of EMPO (EMPO-OOH) does not spontaneouslydecompose its corresponding hydroxyl adduct; thus, itselectron spectra is less complex and easier to interpret.

Rivero-Perez studied the antioxidant profile of 321wines by superoxide radical and hydroxyl radical scaveng-ing assays and compared the results to TEAC, DPPH,FRAP, and ORAC methods. While superoxide radicalscavenging assay showed positive correlations with ABTS,DPPH, and FRAP, hydroxyl radical scavenging assayshowed negative correlations with ORAC. These resultsconfirm the hypothesis that different mechanisms areinvolved in capturing both types of radicals or their relatedcompounds. The negative correlation implies that, ingeneral, the wines with the greatest ability to transferhydrogen atoms are the least effective at stabilizing thehydroxyl radical (Rivero-Perez et al. 2007).

Hydrogen peroxide (H2O2) can be generated through adismutation reaction from superoxide dismutase. Enzymessuch as amino acid oxidase and xanthine oxidase alsoproduce hydrogen peroxide from superoxide anion. Hydro-gen peroxide is regarded as poorly reactive because of itsweaker oxidizing and reducing capabilities; it is stableunder physiological pH and temperature in the absence ofmetal ions. Biologically, H2O2 is converted to oxygen andwater by catalase. It can generate the hydroxyl radical in thepresence of metal ions and superoxide anion. It also canproduce singlet oxygen through reaction with superoxideanion or with HOCl or chloramines in living systems.Hydrogen peroxide can degrade certain heme proteins, suchas hemoglobin, to release Fe ions (Lee et al. 2004).

In the most common methods for detection of H2O2,scavenging capacity depends on the change of the absor-bance value at 230 nm when H2O2 concentration isdecreased by scavenger compounds. Nevertheless, theremight be possible interferences from samples that alsoabsorb at the same wavelength; thus, performance of a“blank” measurement is measured (Magalhaes et al. 2008).Other common assay employs horseradish peroxidasewhich uses H2O2 to oxidize scopoletin into a nonfluores-cent product. In the presence of antioxidants, the oxidationis inhibited and this reaction can be fluorimetricallymonitored (Huang et al. 2005).

Another widely used fluorimetric method is based onhomovanillic acid (Magalhaes et al. 2008). The nature ofthe inhibition is ambiguous because there are severalpotential inhibition pathways. The antioxidants can inhibitthe reaction by (a) reacting directly with H2O2, (b) reacting

with intermediates formed from enzyme and H2O2, or (c)inhibiting the horseradish peroxidase from binding H2O2.Therefore, it is difficult to explain the actual chemicalmeaning of the data (Martinez-Tome et al. 2001).

Singlet oxygen (1O2) is a nonradical and excited state ofmolecular oxygen that has no unpaired electrons and it isknown to be a powerful oxidizing agent, reacting directlywith a wide range of biomolecules. Singlet oxygen isnormally generated in the presence of light and photo-sensitizers. It was reported that metastable phosphatidyl-choline hydroperoxides present in the living organism alsoproduced singlet oxygen during their breakdown in thepresence of Cu2+ in the darkness (Lee et al. 2004).

The singlet-oxygen detection during photosensitizedoxidation of foods is difficult due to the short lifetime.One of the most common detection methods is ESRspectroscopy, which is highly sensitive for the detectionof free radicals. A spin-trapping agent such as 2,2,6,6-tetramethyl-4-piperidone (TMPD) can react with singletoxygen to form a stable radical nitroxide radical adduct2,2,6,6-tetramethyl-4-piperiodine-N-oxyl (TAN) which ismeasured by ESR. Although other ROS such as superoxideand hydroxyl radicals can react with TMPD, they do notconvert TMPD to TAN. This method is highly specific tosinglet oxygen (Min and Boff 2002).

Singlet oxygen can be physically scavenged by transferringits excitation energy to another molecule. β-carotene is anexcellent physical scavenger of 1O2. The energy differencebetween singlet oxygen and ground-state oxygen can bereleased as a 1,270 nm photon, which is very specific tosinglet oxygen. The decay rate of the light intensity can beused to measure the singlet-oxygen scavenging ability ofcompounds (MacDonald-Wicks et al. 2006). Nevertheless,the intensity of luminescence based on the self-emission of1O2. is often insufficient to provide reproducible quantitativeinformation (Magalhaes et al. 2008).

A more sensitive method is reported where the quench-ing of singlet-oxygen-sensitized delayed fluorescence oftetra-t-butylphthalocyanine at 703 nm is monitored. Al-though this method may provide sensitive measurementwhere the 1,270-nm luminescence is difficult to detect, it isnot widely applied since it requires specialized equipment(Huang et al. 2005). The assay was applied to compoundswhich are well known as singlet-oxygen quenchers such asβ-carotene, α-tocopherol, and lauric acid (Magalhaes et al.2008). Cholesterol reacts with singlet oxygen to formspecific oxidative products via the “ene” reaction. Thismakes use of cholesterol as an effective indicator of singlet-oxygen oxidation where the use of other detection methodsis difficult (Min and Boff 2002).

Hydroxyl radical (HO�) is the most reactive free radicalknown and can react with everything in living organisms.These short-lived species can hydroxylate DNA, proteins,


and lipids. It can be formed from superoxide anion andhydrogen peroxide in the presence of metal ions such ascopper or iron. When a hydroxyl radical reacts witharomatic compounds, it can add on across a double bond,resulting in hydroxycyclohexadienyl radical. The resultingradical can undergo further reactions, such as reaction withoxygen, to give peroxyl radical or decompose to phenoxyl-type radicals by water elimination (Lee et al. 2004).

The direct scavenging of the hydroxyl radical by dietaryantioxidants in a biological system is not meaningfulbecause the cellular concentration of dietary antioxidantsis negligible and very high concentrations of scavenger arerequired to compete in vivo or in the food matrix for anyHO� generated. On the other hand, it is possible to preventthe formation of hydroxyl radicals by either deactivatingfree metal ions [e.g., Fe(II)] through chelation or convertingH2O2 to other harmless compounds (such as water andoxygen). For example, catalase converts H2O2 to O2 andH2O, and metal chelators bind metal ions so that theybecome inert toward H2O2. Thus, dietary nutrients performin this way would act as preventive antioxidants (Huang et al.2005; Magalhaes et al. 2008).

Moore et al. (2006) reviewed available HO�-generatingsystems and classified them into five categories including (1)the classic Fenton reaction including the pH 7.4 bufferedferric iron–EDTA/ascorbic acid/H2O2 system used in the“deoxyribose assay.” HO� attacks unsaturated membranelipids to form malonaldehyde, which may be detected by itsability to react with thiobarbituric acid (TBA) to form a pinkchromogen. The role of ascorbic acid is to reduce the Fe(III)to Fe(II) and thus to favor the Fenton reaction (Caillet et al.2007); (2) the superoxide-driven Fenton reaction using thehypoxanthine/xanthine oxidase system to generate HO�; (3)Fenton-like systems including the Co(II)/H2O2 (Aboul-Eneinet al. 2005) and the Cu(II)/H2O2 systems (Cao et al. 1997);(4) pulse radiolysis of water (Joshi et al. 2005); (5) ultravioletexcitation of compounds such as pyridine-N-oxides, oftenreferred to as “photoFenton” reagents. The generation ofhydroxyl radicals from such reagents has been identified bytrapping with coumarin-carboxylic acid and the consequentformation of the highly fluorescent 7-hydroxycoumarincarboxylate (Botchway et al. 2007). There are also someresearches in literature that have used γ-irradiation toproduce HO� radicals (Yoshioka et al. 2001; Saran andSummer 1999).

Biologically, the hydroxyl radical is widely believedto be generated when hydrogen peroxide reacts with Fe(II) ion (Fenton reaction). However, when the sample ismixed with Fe(II), it may alter the activity of Fe(II) bychelation. So, it is impossible to distinguish if theantioxidants are simply good metal chelators or HO�scavengers. To solve this problem, a chelating agent likediethylenetriaminepentaacetic acid (DETAPAC) was usu-

ally added to the solution. However, antioxidants likeEGCG have strong chelating activity, so the effect of theaddition of DETAPAC is doubtful. In addition, antiox-idants may consume H2O2. Actually, it is known thatEGCG scavenges H2O2. This action also disturbs theHO�-generating system (Yoshioka et al. 2001).

The detection methods of HO� radicals may include theseparation by high-performance liquid chromatography(HPLC) combined with electrochemical or UV detectors,pulse radiolysis, fluorometric methods, ESR, spectrophoto-metric detection, capillary electrophoresis (CE), and CL-based determination methods.

The hydroxylated products generated from the reac-tion of hydroxyl radical with aromatic compounds suchas benzoic acid or salicylic acid are separated by HPLCand detected with either electrochemical or UV detec-tion (Saran and Summer 1999). Though this method ishandy and sensitive, the multiple reaction products makeit complicated to quantitative detection of hydroxylradical. Hantzsch reaction method and TBA method aresimple and do not need expensive instruments; however,both of the reactions need high temperature (60–100 °C)and long reaction time (30–60 min; Zou et al. 2002).Moreover, the organic mobile phase of the HPLC assay, inwhich �HO is generally detected by determining thedihydroxylated benzoic acid products derived from sali-cylic acid, will possibly cause environmental pollution andharm to health (Cheng et al. 2003).

Zhu et al. (2000) reported a metal-independent organicFenton reaction that production of HO� by tetrachlorohy-droquinone (a major metabolite of the widely used biocidepentachlorophenol) in the presence of H2O2 was obtained.Salicylate was used as an HO� trap, which forms thehydroxylation products 2,3- and 2,5-dihydroxybenzoic acid(DHBA). The DHBA derivatives can be separated by HPLCand detected with an electrochemical detector. This methodis extremely sensitive and detects DHBAs in the picomolarrange. The process was inhibited by HO� scavenging agentswithout being affected by iron chelators. This new assay mayprovide a more direct way of measuring hydroxyl radicalscavenging capacity of antioxidants.

“Pulse radiolysis” is the most often used technique formeasuring the reaction between HO� and antioxidants,requires specialized equipment, and can be expensive(Huang et al. 2005). The radiolysis and photosensitizationsystems do not measure the HO� scavenging capacityunder physiologically relevant conditions. The hypoxan-thine/xanthine oxidase system generates superoxide inaddition to OH� and may have interference from enzymeinhibitors, which result in overestimation of HO� scav-enging capacity (Moore et al. 2006). The methods usingthe generation of hydroxyl radicals from photoFentonreagents have a potential problem. One-photon excitation


of photoFenton reagents such as 2-hydroxypyridine-N-oxide and 2-mercaptopyridine-N-oxide has their shortwavelength (UV) absorption maxima at ∼310 and∼330 nm, respectively, at physiological pH. Such wave-lengths are absorbed by several biochemical chromo-phores and have limited penetration in tissues. The useof multiphoton excitation in the near infrared (approxi-mately 700–900 nm) has the potential to avoid theseproblems (Botchway et al. 2007).

Ou et al. (2002a, b) developed a fluorometric methodtermed as hydroxyl (HO) radicals averting capacity(HORAC) for screening the metal chelating capacity ofdietary antioxidants. HO�was generated by a Co(II)-complex-mediated Fenton-like reaction and the HO� formation underthe experimental conditions was indirectly confirmed by thehydroxylation of p-hydroxybenzoic acid measured byHPLC–mass spectrometry. FL was used as the probe. Thefluorescence decay curve of FL is monitored in the absenceor presence of antioxidants; the quantitation method is thesame with ORAC assay except gallic acid is used as thestandard. This method has been validated for linearity,precision, accuracy, and ruggedness. HORAC and ORACassays measure two different but equally important aspects ofantioxidant properties; the HORAC primarily reflects metalchelating radical prevention ability, and the ORAC reflectsperoxyl radical absorption capacity. Therefore, no correlationfound between HORAC and ORAC values is expected.

Another simple and sensitive fluorometric hydroxylradical detection method is described. This methodemploys the reaction between HO� and DMSO toproduce formaldehyde, which then reacts with ammoniaand 1,3-cyclohexanedione at pH 4.5. The product yieldsthe characteristic fluorescence with its excitation andemission wavelength at 400.4 and 452.3 nm, respectively.The quantitative analysis of hydroxyl radical can be donethrough the determination of the fluorescence intensity.The temperature of 95 °C seems to be the major drawbackof this method and make it unsuitable for the analysis ofsome unstable biological materials (Tai et al. 2002).

The other novel hydroxyl radical scavenging capacitynamed HOSC was described and validated by Moore etal. (2006). The HOSC assay uses FL as the probe. HO� isgenerated under physiological pH using a Fenton-likereaction with a definite end point. The experimentalconditions were evaluated and confirmed using electronspin resonance with DMPO spin-trapping measurement.HOSC assay has acceptable sensitivity, ruggedness,precision, and accuracy. Compared to the commonly useddeoxyribose method, the HOSC method has bettercompatibility with acetone and more efficiently generateshydroxyl radicals under physiological pH.

ESR spectroscopy measures the electron paramagneticresonance spectrum of a spin adduct derivative after trapping.

This technique involves the inclusion in the experimentalsystem of a nitrone or nitroso compound (spin trap) that canreact with a short-lived free radical such as HO� to generate along-lived nitroxide free radical (spin adduct). The spinadduct is usually detected by ESR spectroscopy. The twonitrone spin traps that have found wide use are DMPO(Cheng et al. 2007) and phenyl-tert-butylnitrone (Ma et al.1999). In the spin-trapping method with DMPO, the ESRsignal of the DMPO-OH adduct formed by the Fentonreaction disappeared rapidly in the presence of Fe(II) ions,and this quenching effect was enhanced by phosphate ions(Li et al. 2004). Besides, the expensive instruments make itunsuitable for routine analysis.

The use of high-performance CE to determine HO�generated by Fenton reaction was reported. CE is animportant analytical separation technique due to its speed,efficiency, reproducibility, ultrasmall sample volume, andease of clearing up the contaminants. In addition, withelectrochemical detection (ED), CE-ED (Cao et al. 2003)and amperometric detection (Wang et al. 2003a) offerhigher sensitivity than CE-UV detection.

The CL-based determination of scavenging capacityagainst �OH using luminol has also been described. Themethods take advantage of the transient properties of theFenton reaction and chemiluminescent reaction between�OH and luminol. However, other ROS, including O��2 andH2O2, are generated at the same time, so it is difficult todetect �OH specifically with this detection process (Chenget al. 2003; Chen et al. 2006).

Peroxynitrite (ONOO−), the reaction product of nitricoxide (NO) and superoxide, is a potent and relatively long-lived oxidant with its high diffusibility across cell mem-branes. Its protonated form, peroxynitrous acid (ONOOH),is a very strong oxidant. The main route of damage is thenitration or hydroxylation of aromatic compounds, partic-ularly tyrosine. Under physiological conditions, peroxyni-trite also forms an adduct with carbon dioxide dissolved inbody fluid. The adduct is believed to be responsible for theoxidative damage of proteins.

Two fluorogenic probes, DCFH and dihydrorhodamine-123 (DHR-123), considered to be ideal, have been widelyemployed to monitor peroxynitrite in various systems.These methods are based on the oxidation of the reducednonfluorescent forms of fluorescent dyes such as fluores-cein and rhodamine by peroxynitrite to produce the parentdye molecule, resulting in a dramatic increase in fluores-cence response. Rhodamine derivatives have the advan-tages of having great photostability, pH insensitivity over abroad range (low to neutral pH), and giving high quantumin aqueous solution and being excitable at long wavelength(Yang et al. 2002). Although these assays are commonlyused, there are some debates in the literature concerning themechanism of peroxynitrite-mediated oxidation of fluores-


cent indicators. These fluorogenic indicators are also lesssuitable for peroxynitrite formation in vivo (Glebska andKoppenol 2003) and the synthesis of these probes is ratherdifficult and inconvenient. Additionally, the use of organicdyes is very likely to result in environmental contaminationand usually should be avoided (Liang et al. 2005).

A fluorometric method using folic acid as a fluorescentprobe has been proposed because folic acid can act as aperoxynitrite scavenger. Folic acid is a low-fluorescentsubstance, but the oxidation of folic acid by peroxynitritecan give high-fluorescent emission product. Compared withthe commonly used probe DHR-123, the fluorescent probereported here has some advantages including highersensitivity that is desirable for probing of peroxynitrite inlower concentrations, greater photostability, commercialavailability, and not being toxic to biological system(Huang et al. 2007).

Another novel spectrofluorimetric method for the deter-mination of peroxynitrite uses hemoglobin (Hb) as acatalyst and thiamine (Cao et al. 2005) or l-tyrosine as thesubstrate (Liang et al. 2005). Hb is used as the mimeticenzyme for substituting horseradish peroxidase for tworeasons: (1) for the purpose of the mildness of reactionconditions and the minimization of cost and (2) Hb is akind of nature protein in human body and it is much likelyto coexist with peroxynitrite in cell or organism (Liang etal. 2005). Other peroxynitrite determination methods aredirect voltammetric detection at a mercury film electrode atalkaline pH (Zakharova et al. 2007) and luminol-enhancedchemiluminescence methods (Magalhaes et al. 2008).

Conclusion

In this review, numerous antioxidant capacity methods arereported; they differ from each other in terms of reactionmechanisms, oxidant and target/probe species, reactionconditions, and expression of results. When selecting themost appropriate method, it should be considered thatanalysis conditions, substrate, and concentration of antiox-idants should simulate real food or biological systems asmuch as possible. Antioxidant activity occurs by differentmechanisms which means employing a method dependingon one mechanism may not reflect the true antioxidantcapacity. The total antioxidant capacity value shouldinclude assays applicable to both lipophilic and hydrophilicantioxidants and regards the similarity and differences ofboth HAT and ET. The assays including various ROS/RNSsuch as superoxide anion (O��2 ), hydroxyl radical (HO�),hydrogen peroxide (H2O2), singlet oxygen (1O2), peroxyni-trite (ONOO−), nitric oxide (NO�), nitric dioxide (NO�2 )have to be designed to comprehensively evaluate theantioxidant capacity of a sample.

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Review of Methods to Determine Antioxidant Capacities

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