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review radical scavenging.pdf

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InternationalFood Science and Technology
Food Science and Technology International 
C. Sánchez-Moreno
Review: Methods Used to Evaluate the Free Radical Scavenging Activity in Foods and Biological Systems
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at UNIV FEDERAL DO CEARA on October 25, 2012fst.sagepub.comDownloaded from 
 
Review: Methods Used to Evaluate the Free Radical ScavengingActivity in Foods and Biological Systems
C. S
A ´NCHEZ
-M
ORENO
*
Departmento de Metabolismo y Nutricio´ n, Instituto del Frı´ o, Consejo Superior deInvestigaciones Cientı´  ficas (CSIC). Ciudad Universitaria, 28040 Madrid, Spain
Free radical generation is directly related with oxidation in foods and biological systems. Therefore, thesearch for methods to determine free radical scavenging is important. In this work are describedthe methods used for this purpose in both substrates as well as in specific cases of their application. Themain methods comprise superoxide radicals scavenging (O
Á
À
2
); hydrogen peroxide scavenging (H
2
O
2
);hypochlorous acid scavenging (HOCl); hydroxyl radical scavenging (HO
Á
); peroxyl radical scavenging(ROO
Á
), among them are the methods that use azo-compounds to generate peroxyl radicals, such as the‘‘TRAP’’ method (Total Radical-Trapping Antioxidant Parameter) and the ‘‘ORAC’’ method (Oxygen-Radical Absorbance Capacity); the scavenging of radical cation 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) or the ABTS or the ‘‘TEAC’’ method (Trolox Equivalent Antioxidant Capacity); thescavenging of stable radical 2,2-diphenyl-1-picrylhydrazyl or DPPH
Á
method and the scavenging of radicalcation
N,N 
-dimethyl-
 p
-phenylenediamine or DMPD method. At present, in spite of the diversity of methods, there is a great need to standardize measurements of antioxidant activity. The search formore specific assays, giving us chemical information that could be related directly to oxidativedeterioration of foods and biological systems could be the objective of future research.
Key Words:
free radical scavenging, antioxidant activity, assays, vegetables, biological systemsLa generacio ´n de radicales esta ´directamente relacionada con la oxidacio ´n de lı ´pidos y sustratos biolo ´gicos, por tanto, es importante la bu´squeda de me ´todos para la determinacio ´n del secuestro de especies radicales.En este trabajo se describen los principales me ´todos utilizados con este fin en ambos sustratos, ası ´como susaplicaciones especı ´ficas: secuestro de radicales supero ´xido (O
Á
À
2
), secuestro de pero ´xido de hidro ´geno(H
2
O
2
); secuestro de a ´cido hipocloroso (HOCl); secuestro de radicales hidroxilo (HO
Á
); secuestrode radicales peroxilo (ROO
Á
), entre los que destacan los me ´todos que utilizan azo-compuestos parala generacio ´n de radicales peroxilo, tales como el me ´todo del ‘‘TRAP’’ (
Total Radical-Trapping AntioxidantParameter
) y el me ´todo del ‘‘ORAC’’ (
Oxygen-Radical Absorbance Capacity
); secuestro del catio ´n radical2,2-azinobis-(3-etilbenzotiazolin-6-sulfonato) o me ´todo del ABTS o del ‘‘TEAC’’ (
Trolox EquivalentAntioxidant Capacity
); secuestro del radical estable 2,2-difenil-1-picrilhidrazil o me ´todo del DPPH
Á
y secuestro del radical catio ´n
,
-dimetil-
 p
-fenilendiamina o me ´todo del DMPD. Actualmente, a pesar dela diversidad de me ´todos existentes, hay una gran necesidad de estandarizar estos me ´todos para ladeterminacio ´n de la actividad antioxidante. La bu´squeda de me ´todos ma ´s especı ´ficos, que proporcionenuna informacio´n quı´mica que se directamente relacionada con el deterioro oxidativo de alimentosy muestras biolo ´gicas, podrı ´a ser el objetivo de futuras investigaciones.
Palabras Clave:
secuestro de radicales libres, actividad antioxidante, me ´todos, vegetales, sistemasbiolo ´gicos
INTRODUCTION
The methods used to determine activity of foods andbiological antioxidants have evolved from chemicalassays with lipid substrates to more complex assays tomeasure total antioxidant capacity in fluids andbiological samples.In the case of foods it is necessary to determine theefficacy of natural antioxidants for food protectionagainst oxidative damage, to avoid deleterious changesand loss of commercial and nutritional value (Halliwellet al., 1995; De la Torre Boronat and Lo ´pez Tamames,1997; Halliwell, 1997). In addition, a rapid method fordetermining the potential antioxidant capacity invegetable foods is necessary. This method could bea useful tool to make a selection among differentspecies, varieties, maturation degree and culture condi-tions, in order to obtain high content of natural
*E-mail: csanchezm@if.csic.esReceived 10 October 2001; revised 23 March 2002.
Food Sci Tech Int
2002;8(3):121–137
ß
2002Sage PublicationsISSN: 1082-0132DOI: 10.1106/108201302026770
121
 at UNIV FEDERAL DO CEARA on October 25, 2012fst.sagepub.comDownloaded from 
 
antioxidants in foods (Fogliano et al., 1999; Leonardiet al., 2000). Thus total antioxidant capacity of an ediblevegetable product could be a parameter to evaluate thequality of vegetable foods (Arnao et al., 1998; Canoet al., 1998).In the case of biological systems, oxidative stress, animbalance between reactive oxygen species and defenceand repair antioxidant systems, has been shown tobe involved in the development of degenerativediseases. Thus, determination of the antioxidant statusin biological systems could contribute to prevention andevaluation of diseases related to aging. In addition toorganism defences, the intake of dietary antioxidantsand evaluation of the real contribution of foods toantioxidant status in biological systems must beevaluated (Halliwell and Gutteridge, 1989; Namiki,1990; Jacob, 1995; Gurr, 1996).Many different substrates, system compositionsand analytical methods are employed in screening teststo evaluate the effectiveness of antioxidants, evidencethat many different methods are necessary to evaluatedifferent antioxidant effects. The methodology forevaluating natural antioxidants must be carefullyinterpreted according to the system and to the analyticalmethod used to determine the extent and end-point of oxidation (Frankel, 1993; Arnao et al., 1999; Foglianoet al., 1999; Frankel and Meyer, 2000).Antioxidant effectiveness is measured by monitoringthe inhibition of oxidation of a suitable substrate. Afterthe substrate is oxidized under standard conditions, theextent of oxidation (an end-point) is measured bychemical, instrumental or sensory methods. Hence, theessential features of any test are a suitable substrate, anoxidation initiator and an appropriate measure of the end-point. The combination of substrate, initiatorand end-point which have been used are numerous, andeven with the same analytical techniques, severalanalytical strategies are possible (Arnao et al., 1999;Robards et al., 1999).We could classify antioxidant tests in foods andbiological systems into two groups: those assays usedto evaluate lipid peroxidation, in which a lipid orlipoprotein substrate under standard conditions is usedand the degree of oxidation inhibition is measured(Sa ´nchez-Moreno and Larrauri, 1998), and those assaysused to measure free radical scavenging ability.Description of the second group is the objective of thisreview.
METHODOLOGIES FOR TESTINGRADICAL SCAVENGING SPECIES
A free radical is any species that contains one or moreunpaired electrons and is capable of independentexistence (Halliwell et al., 1995). Free radicals such astrichloromethyl (CCl
Á
3
), superoxide (O
Á
À
2
), hydroxyl(HO
Á
), peroxyl (ROO
Á
), and nitric oxide (NO
Á
) areknown to be produced metabolically in living organ-isms. In addition, some non-radical derivatives of oxygen molecules (hydrogen peroxide (H
2
O
2
), hypo-chlorous acid (HOCl)), can be generated in foods andbiological systems. All of these reactive oxygen speciesparticipate in the chain reaction of free radicals, thustests of the ability of a substance to scavenge radicalspecies may be relevant in the evaluation of antioxidantactivity (Halliwell, 1990; Halliwell et al., 1995).
Scavenging of Superoxide Radical (
O
Á
À
2
)
Pathological processes are known to involve complexmechanisms. Xanthine oxidase is one of the mainenzymatic sources of reactive oxygen species (ROS)
invivo
. Although xanthine oxidase present in normal tissueis a dehydrogenase enzyme that transfers electrons tonicotinamideadeninedinucleotide (NAD), as it oxidizesxanthine or hypoxanthine to uric acid, under certainstress conditions the dehydrogenase is converted to anoxidase enzyme by oxidation of essential thiol groups orby limited proteolysis. Upon this conversion the enzymereacts with the same electron donors, but reducingoxygen instead of NAD, thus producing superoxide andhydrogen peroxide, and contributing to the initiationand progression of a number of pathological processes(Halliwell and Gutteridge, 1990; Halliwell et al., 1995;Hippeli and Elstner, 1999).The scavenging activity towards O
Á
À
2
of a wide rangeof antioxidants is measured in terms of inhibitionof generation of O
Á
À
2
with the hypoxanthine–xanthineoxidase superoxide generating system (HX–XO) (Robakand Gryglewski, 1988; Halliwell, 1990; Mitsuya et al.,1990; Constantino et al., 1992; Minamiyama et al., 1995;Kruedener et al., 1995; Yen and Chen, 1995;Ramanathan et al., 1996; Robak and Sato et al., 1996;Lavelli et al., 1999, 2000; Suh et al., 1999; Saint-Cricq deGaulejac et al., 1999a,b; Deguchi, 2000; Kubo et al.,2000; Lu and Foo, 2000; Unno et al., 2000; Wang andJiao, 2000; Yamaguchi et al., 2000a; Calliste et al., 2001;Kweon et al., 2001). To a minor extent, O
Á
À
2
is generatedusing a non-enzymatic reaction of phenazine methosul-phate in the presence of NADH and molecular oxygen(Nishikimi et al., 1972; Robak and Gryglewski, 1988;Yen and Chen, 1995; Yen and Hsieh, 1995; Yamaguchiet al., 2000b; Barthomeuf et al., 2001). In bothstrategies, O
Á
À
2
reduces nitro-blue tetrazolium (NBT)into formazan at pH 7.4 and room temperature, andformazan generation is followed by spectrophotometryat 560nm. Any added molecule capable of reacting withO
Á
À
2
, inhibits the production of formazan. Wang andJiao (2000) used hydroxylammonium chloride asoxidant agent instead of NBT. The consequent nitrileformation from hydroxylammonium is followed bymeasuring the absorbance due to nitrile at 530nm. In a
122
C. S
A ´NCHEZ
-M
ORENO
 at UNIV FEDERAL DO CEARA on October 25, 2012fst.sagepub.comDownloaded from 

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