Methods for testing antioxidant activity
Michael Antolovich, Paul D. Prenzler, Emilios Patsalides, Suzanne McDonald and Kevin Robards* School of Science and Technology, Charles Sturt University, Locked Bag 588, Wagga Wagga 2678, Australia. E-mail:; Fax: 02 6933 2737; Tel: 02 6933 2547 Received 15th November 2000, Accepted 19th October 2001 First published as an Advance Article on the web 23rd November 2001

Summary of Contents
1 2 3 4 4.1 5 5.1 5.2 5.3 5.4 5.5 5.6 5.6.1 5.6.2 5.6.3 5.7 5.7.1 5.7.2 5.7.3 6 Introduction Processes of lipid oxidation Antioxidants Measurement of antioxidant activity Expression of results Individual procedures Accelerated stability tests Peroxide value Diene conjugation Thiobarbituric acid reactive substances (TBARS) assay Measurement of hexanal and related end-products Measurement of free radicals Electron spin resonance spectrometry ABTS assay Diphenylpicrylhydrazyl radical Other measures of antioxidant activity FRAP assay Phycoerythyrin assay Total radical-trapping antioxidant parameter Summary

1 Introduction
The importance of oxidation in the body and in foodstuffs has been widely recognized. Oxidative metabolism is essential for the survival of cells. A side effect of this dependence is the production of free radicals and other reactive oxygen species that cause oxidative changes. There is increasing evidence for the involvement of such species in a variety of normal in vivo regulatory systems.1 When an excess of free radicals is formed, they can overwhelm protective enzymes such as superoxide dismutase, catalase and peroxidase and cause destructive and lethal cellular effects (e.g., apoptosis) by oxidizing membrane lipids, cellular proteins, DNA and enzymes, thus shutting down cellular respiration. Furthermore, reactive oxygen species seem to influence cell signalling pathways in ways that are only now being unravelled.2,3 Oxidation can also affect foods, where it is one of the major causes of chemical spoilage,4 resulting in rancidity and/or deterioration of the nutritional quality, colour, flavour, texture and safety of foods.5 It is estimated that half of the world’s fruit and vegetable crops are lost6 due to postharvest deteriorative reactions. Defence mechanisms against the effects of excessive oxidations are provided by the action of various antioxidants and the need to measure antioxidant activity is well documented. Methods of assessing antioxidant behaviour fall into two broad categories reflecting the focus on activity in foods or bioactivity in humans. In the case of food systems, the need is to assess the efficacy of an antioxidant(s) in providing protection for the food7 against oxidative spoilage. A subcategory involves measurement of activity in foods, particularly fruits, vegetables and beverages, but with a view to predicting dietary burden and in vivo activity.8,9 Oxidative stress in humans arises from an imbalance in the antioxidant status (reactive oxygen species versus defence and repair mechanisms). Among the endogenous defences are enzymes such as superoxide dismutase, catalase and glutathione peroxidase, plus vitamin E, uric acid and serum albumins. Besides these defences, consumption of dietary antioxidants is also important. An important distinction from food-based systems is the absence of a single, definable substrate in many instances in vivo. This review examines the various methods of measuring antioxidant activity particularly as they relate to lipid oxidation. This should be distinguished from the related process of measuring the concentration of an antioxidant(s) which is not considered here. However, it should be recognized that the two are related as antioxidants generally exhibit pro-oxidant effects at higher concentration. The term ‘activity’ as applied to antioxidants needs clarification as it can have a variety of meanings. Relevant aspects include: mechanistic intervention, e.g., free radical scavenger, catalytic decomposition, prooxidant suppression; rate of scavenging, e.g., near-diffusion or Analyst, 2002, 127, 183–198
This journal is © The Royal Society of Chemistry 2002

Kevin Robards is Associate Professor of Chemistry at Charles Sturt University Riverina. He obtained his PhD in analytical chemistry from the University of New South Wales in 1979. His research interests are focused on the application of analytical chemistry to food science and in particular the identification and role of naturally occurring phenolic compounds in fruits. Kevin Robards DOI: 10.1039/b009171p


mass change (mg) 184 Analyst. such ‘activities’ need to be supported by testing in the actual substrate and conditions of interest. It is also possible to use screening methods to identify the class of antioxidant (e. hexane formation. Following a brief introduction to oxidative processes and the mechanism of antioxidant action. For convenience. all acronyms used in this review are collected in Table 2.19 Lipid oxidation is important in food deterioration and oxidative modification of low-density lipoprotein (LDL) (Table 2). metabolic transformations. The extensive literature concerning antioxidants precludes exhaustive treatment and rather selected examples of different tests have been chosen to illustrate various points. In the case of natural antioxidants. The present review complements that by Frankel and Meyer. Target substances include oxygen. surface or lipid phase). the term seems to be loosely applied to identifying ‘activity’ as that measured by one or several common or standard tests such as listed in Table 1. mg kg21 (ppm w/w) as malondialdehyde 100 times the corrected absorbance in a l cm cell at 350 nm containing l g of oil or fat per 100 mL of isooctane–acetic acid solvent mg kg21 of product formed Inhibition time for appearance of radical cation under specified conditions or decay rate once formed Fluorescence intensity Intensity/rates of change in concentration of antioxidant or spin-trap derivative radicals Time required for development of autoxidation in a dynamic oxygen atmosphere at specified temperature Inhibition of fluorescence decay under specified conditions of autoxidation. an historical background to activity tests is provided.. (2) non-enzymatic. radical inhibitor. they may be multifunctional.13 the presence of competitive enzymes and antioxidants in addition to pro-oxidants may profoundly affect the in vivo activity of test antioxidants. entities tested and basic units used for antioxidant activity mcasurements Test Peroxide value Diene conjugation Thiobarbituric acid reactive substances (TBARS) Kreis test Anisidine value Hexanal formation. phenolic) or even its action12 by the use of spray reagents (e. The mechanism that is operative or dominant in a particular situation is dependent on conditions and yet this will affect the kinetics and hence the antioxidant activity.. For example. 2 Processes of lipid oxidation A number of chemical and physical phenomena can initiate oxidation which proceeds continuously in the presence of a suitable substrate(s) until a blocking defence mechanism occurs. The relationship of tests designed for food systems and their extension to physiological systems is presented. aqueous.g. complexing agent. ABTS4+ assay Total radical trapping antioxidant parameter (TRAP) Phycoerythrin assay Electron spin resonance (ESR) spin-trap test TG/DTA Measurement Peroxides and hydroperoxides 1. in vivo measurements are difficult owing to problems relating to cellular uptakes of the antioxidants and the transport processes.and slowacting group of antioxidants. etc. Lipid oxidation proceeds20 via three different pathways: (1) non-enzymatic free radical-mediated chain reaction. non-radical photo-oxidation and (3) enzymatic reaction. In vitro methods provide a useful indication of antioxidant activities but data obtained by these methods are difficult to apply to biological systems.14 An important distinction can be made between short. TLC screening may be used10. synergistic effect for other antioxidants. For instance.18 which emphasizes the need for multi-faceted testing of antioxidant activity. cholesterol and DNA. stable standard such as di-tert-butyl nitroxide) DT (°C). excretion. phospholipids. This is particularly important for in vivo testing where absorption. However.11 to identify components in extracts that exhibit such activity.4-Dienes produced by early stages in lipid autoxidation Thiobarbituric acid derivatives of malondialdehyde absorbing at 532–535 nm Phloroglucinol derivatives of malondialdehyde and other aldehydes absorbing at 546 nm Aldehydes (mainly alkenals) Specific oxidation end-product formed Absorbance of radical cation in aqueous medium at 734 nm or other suitable wavelength Units mequiv. polyunsaturated fatty acids. On the other hand.. This is related to the reaction kinetics15. In many cases neither a specific substrate nor a specific additive may be involved but extracts may be screened to identify those which exhibit antioxidant activity according to the test method(s) employed.and long-term antioxidant protection. concentration effectiveness (moles of free radicals scavenged per mole of antioxidant). Non-invasive techniques such as nuclear magnetic resonance (NMR) spectrometry may be useful but require relatively high antioxidant concentrations. These may involve in vitro or in vivo testing and in the latter case may involve either invasive or non-invasive techniques. medium or substrate selectivity (e. hydroperoxide decomposer). 127. kg21of active oxygen Absorbance/unit mass mg kg21 linoleic acid equivalents mg kg21 (ppm w/w) as malondialdehyde Red colour on Lovibond scale (empirical). These differences and particularly the variation in analytical procedures account for the inconsistent results that have been reported for a number of recognized antioxidants. 183–198 . An example of route (2) is the stoichiometric oxidation of oleic acid by singlet Table 1 Cornmon tests. In any case. 2002.g.View Online controlled. pentane formation. Can be expressed as trolox equivalents? mg L21 of radical species (cf. disappearance of the DPPH radical (Table 2) followed a doubleexponential equation in the presence of edible oils and oil fractions17 which suggested the presence of a fast.g.16 and the rate at which an antioxidant reacts with a specific radical versus the thermodynamics of the reaction and how completely the antioxidant reacts.

Therefore.5-Dibromo-4-nitrosobenzenesulfonic acid 5.View Online oxygen21. 183–198 185 The breakdown of lipid hydroperoxides often involves transition metal ion catalysis. aldehydes. destructive chain reactions. Branching: LOOH ? LO4 + HO4 2LOOH ? LOO4 + LO4 + H2O 3 Antioxidants An antioxidant may be defined35 as ‘any substance that when present at low concentrations. The oxidation of the lipid generates a highly reactive allyl radical (L4) that can rapidly react with oxygen to form a lipid peroxyl radical (LOO4) Propagation: L4 + O2 ? LOO4 LOO4 + LH ? L4 + LOOH The peroxyl radicals are the chain carriers of the reaction that can further oxidize the lipid. BHA. Chain-breaking antioxidants may occur naturally or they may be produced synthetically as in the case of BHT. for example. producing lipid hydroperoxides (LOOH). Nevertheless. which in turn break down to a wide range of compounds. significantly delays or inhibits oxidation of that substrate’. when present in trace amounts.2A-Azinobis(3-ethylbenzthiazoline)-6-sulfonic acid 2. with paraquat).4-dimethylvaleronitrile) Butylated hydroxyanisole Butylated hydroxytoluene tert-Butylnitrosobenzene Chemiluminescence 3.18 Primary antioxidants.27 The range of effects of free radicals is only a few ångströms. The synthetic antioxidants are widely used in the food industry20 and are included in the human diet. 2002. propagation. The singlet oxygen is produced by sensitizers such as myoglobin or chlorophyll.33 and hydrogen peroxide formation in vivo. hydroperoxides decompose readily and spontaneously at 160 °C and the peroxy radical concentration can become relatively high under such conditions. the reaction mechanism is different for emulsified and bulk lipids.37.32 chemically induced free radical formation (e. with R4 as the initiating oxidizing radical. may either delay or inhibit the initiation step by reacting with a lipid radical or inhibit the propagation step by reacting with peroxyl or alkoxyl radicals:36 L4 + AH ? LH + A4 LOO4 + AH ? LOOH + A4 LO4 + AH ? LOH + A4 The antioxidant free radical may further interfere with chainpropagation reactions by forming peroxy antioxidant compounds: A4 + LOO4 ? LOOA A4 + LO4 ? LOA The activation energy of the above reactions5 increases with increasing A–H and L–H bond dissociation energy. alkyl formates.36 Secondary or preventative antioxidants are compounds that retard the rate of oxidation. Pathway (3) involves the action of lipoxygenases on various substrates. This may be achieved in a number of ways including removal of substrate or singlet oxygen quenching.24 The process may be initiated by the action of external agents such as heat. The measurement of antioxidant activity of certain components in vivo requires the definition of the type of free radical formation. For instance. primary or chainbreaking antioxidants and secondary or preventative antioxidants.2-Diphenyl-1-picrylhydrazyl Ferric reducing antioxidant power Gas chromatography(–mass spectrometry) High-performance liquid chromatography Low-density lipoprotein Malondialdehyde Oxygen radical absorbance capacity a-(4-Pyridyl-1-oxide) N-tert-butylnitrone Peroxide value Reactive nitrogen species Reactive oxygen species Total antioxidant activity Thiobarbituric acid Thiobarbituric acid reactive substances tert-Butylhydroquinone Trolox equivalent antioxidant activity Thermogravimetry/differential thermal analysis Total radical trapping parameter . For convenience. branching and termination steps. The essential features of oxidation via a free radical-mediated chain reaction are initiation. whereas the action of the nonfree radical hydrogen peroxide is several nanometres and hydrogen peroxide can pass biological membranes freely.38 The use of naturally occurring antioxidants39 has been promoted Analyst. Termination: LO4 + LO4 ? LOO4 + LOO4 ? non-radical products LO4 + LOO4 ? There are obvious differences between the reactions occurring in vivo and in foods27–30 that may be exposed to elevated temperatures during storage and/or processing.26 including alcohols.31 mitochondrial lesions and pore reactions leading to apoptosis. there are essential features of the process that are similar in all cases. ketones and hydrocarbons and radicals including the alkoxyl radical (LO4). the efficiency of the antioxidant increases with decreasing A–H bond strength. yielding lipid peroxyl and lipid alkoxyl radicals: Table 2 List of acronynms used in this paper Acronymn AAPH ABTS AMVN BHA BHT BNB CL DBNBS DMPO DPPH FRAP GC(-MS) HPLC LDL MDA ORAC POBN PV RNS ROS TAA TBA TBARS TBHQ TEAC TG/DTA TRAP Name 2. At least four different types may be identified as: free iron and the Fenton reaction.22 to produce two allylic hydroperoxides via addition of oxygen at either end of the double bond.34 where LH represents the substrate molecule. thus leading to the formation of polymers. TBHQ and the gallates.g. in reactions analogous to that with hydrogen peroxide.25 Initiation: LH + R4 ? L4 + RH LOOH + Mn+ + H+ ? LO4 + M(n + 1)+ + H2O LOOH + M(n + 1)+ + OH2 ? LOO4 + Mn+ + H2O Termination reactions involve the combination of radicals to form non-radical products. Pathway 1 is the classical free radical route23 that leads to initiation of rapidly progressing.2A-Azobis(2-amidinopropane)hydrochloride 2.5-Dimethylpyrroline-N-oxide 2. compared with those of the oxidizable substrate. 127. light or ionizing radiation or by chemical initiation involving metal ions or metalloproteins.2A-Azobis(2. antioxidants have been traditionally divided into two classes. Similarly. a lipid. AH.

For instance. viscosity. In this context. not the least of which is duplicating actual conditions of use.8. Caution is necessary when extrapolating from in vitro tests on food components. a suitable substrate and an appropriate 186 Analyst. Lipid substrates have included various oils and fats. C18:2/ C16:0). for example. free fatty acid content.65 linoleic acid. the preferable approach involves in vivo measurement of LDL oxidation products such as hydroxyfatty acids or oxysterols or indirect indicators of lipid oxidation (e. potential measurements include PV.g.g. Initiators include increased temperature and partial pressure of oxygen. In system 3.63 Antioxidant activity in the lipophilic phase is a composite response to partitioning behaviour and rates of reaction with the relevant radical species. Photosensitized acceleration underestimates the effects of chain-breaking antioxidants. (3) lag phase measurement and (4) integrated rate measurement. the source of ROS and the target substrate must always be considered. whereas in 2. 183–198 measure of the end-point.59 The effect of substrate can be attributed to the strong influence of the unsaturation type and degree of the lipid system60 on the kinetics and mechanism of the antioxidative action. e. the presence of antioxidant in the reaction mixture reduces the change in end-point parameter.68 In the case of oils/ fats. the reagents are mixed and the end-point is measured after a predetermined time interval in 1. several analytical strategies are possible. plant biophenols) possessing antioxidant activity similar to or even higher than that of synthetic antioxidants. Nevertheless. The activity of an antioxidant on b-carotene will not be the same as on vegetable oil.44 thiobarbituric acid value.. polymer content. the rate of the reaction is monitored. (2) measurement of reaction rate.45.51 Most test procedures use accelerated oxidation involving an initiator to manipulate one or more variables in the test system. In other instances. Hence the essential features of any test are an oxidation initiator. there is a distinction between antioxidant activity and the antioxidant capacity (i. LDL represents an obvious substrate and many in vitro tests have been described68. Several studies have examined structure– activity relationships82–87 and Rice-Evans et al.43 an oxidant and an initiator. In systems 1 and 2. to the human in vivo situation as antioxidant activity is a complex interplay of several related factors. the morphology of the LDL particle is important and differences in antioxidant activity can often be rationalized in terms of partition coefficients and accessibility to the lipid peroxyl radicals. LDL is a very dubious substrate. for example.58 also influence the results. the latter based on measurement of TEAC. oxidation of linoleic acid followed by determination of diene conjugation. 2002. in monitoring antioxidant activity in a food.18.47–49 Alternatively. Methods show extreme diversity. In both cases. there is no clear distinction between the various steps in the procedure.46 However.52 exposure to light to promote photosensitized oxidation by singlet oxygen. respectively. oxidation mechanisms can change as temperatures are raised55 while substrate effects56 and analytical technique57. Despite extensive use.. serum) that this confers on the blood plasma and the effect on oxidative susceptibility. iodine value.78 For this reason. Aruoma et al. the immunological response to antigenic lipid oxidation products can be measured. In studying antioxidant activity. g-Tocopherol at a concentration of 11 mg g21 decreased70 hydroperoxide and secondary product formation to 46 and 39%. addition of transition metal catalysts. In rare instances. tests should be performed in both aqueous and lipophilic phase systems.64 These include (1) measurement at a fixed time point. the sum of all antioxidant activities of a mixture containing many antioxidants..88 have presented a detailed discussion of structure–activity effects in both lipophilic and aqueous phases...50 Thus. The features of an oxidation are a substrate. liposomes. instrumental or sensory methods. substrate and end-point that have been used are numerous and even with the same reagents.80.40. the more bland materials are usually employed69 and these preferably only after refining and deodorizing. These ions catalyse the initiation and decomposition of hydroperoxides61 resulting in high levels of volatile decomposition products.71 linoleic acid66 and methyl linoleate in silicone oil. Physiological activity can be assessed by in vitro measurements such as the susceptibility of isolated LDL to oxidation. absorption at 232 and 268 nm. since the vitamin E level in LDL may be an important factor for protection of peroxidation of the unsaturated fatty acid in LDL.66 fatty acid methyl esters67 and LDL.75 that exploit various end-points including measurement of conjugated dienes and hexanal. intermediates and final products and measurement of any one of these can be used to assess antioxidant activity. An antioxidant may protect lipids against oxidative damage whilst accelerating damage to other biological molecules. The use of a number of different measures of activity is becoming a feature of published studies. After the substrate is oxidized under standard conditions. F-2-isoprostanes).76 A considerable amount of evidence is accumulating to suggest that synergism between aqueous and lipophilic systems is the important factor77 (and this shift in attitude is reflected in a wholistic approach to the Mediterranean diet.54 However. This has important implications as the potential for synergism with residual materials in a refined oil always exists and has led to the use of model substrates. model substrates are not without problems. Various model substrates have been described including methyl linoleate.41 with natural alternatives (e.View Online because of concerns regarding the safety of synthetic antioxidants.71 Citronellal was recently used72. Some methods involve a distinct oxidation step followed by measurement of the outcome as. an initiator has been omitted and the scavenging of endogenous pre-formed hydroperoxides has been studied.g. of LDL. the length of the lag time to end-point change is measured. samples with higher antioxidant activity suppress the change far longer than those with less activity.g.e.63 The combinations of initiation. Moreover. The kinetics of the various reactions need to be considered as most radicals are highly reactive species and can diffuse only very short distances.74.42 4 Measurement of antioxidant activity Antioxidant activity cannot be measured directly but rather by the effects of the antioxidant in controlling the extent of oxidation. System 4 involves integration of the end-point versus time curve and is used where the reaction kinetics are not of a simple order.18 Metal ions such as copper and iron are the most common initiators in both food and biological systems. where the interest is in the relative bioactivity of an antioxidant.79 Data on the lipophilic phase derive from studies on fatty acids.81 which have been used extensively as in vitro cellular models for investigating antioxidant activity and especially LDL. colour.53 variable shaking to enhance reactant contact16 and free radical sources. either the extent or rate of oxidation (an end-point) is measured by chemical.73 as a substrate in an accelerated test based on measurement of its degradation product by gas chromatography. Antioxidant effectiveness in an in vitro LDL oxidation test62 varied greatly with the level of copper ions used as catalyst. 127. fatty acid composition and ratio of unsaturated to saturated fatty acids (e. or especially ill-defined extracts. . The use of a substrate is considered essential18 and tests such as the ABTS assay that generally do not include a substrate are artificial and do not adequately mimic the processes in food and biological systems.50 used several measures of antioxidant activity and posed a series of questions that serve as a guide in evaluating antioxidant efficacy.

Qualitative measures used in screening tests would be reported as ‘shows antioxidant activity’. concentration of antioxidant. 127. and a comparison of the quantitative effect or likely effect. ‘shows pro-oxidant activity’ or ‘shows no activity’ according to the test procedure. photoactivated riboflavin and lipoxygenase. e.. is demonstrated as an inhibitory effect on the extent or rate of consumption of reactants or the formation of products. In (a) and (b) antioxidant activity. by the oil substrates. substrate. Analyst. The third method of measuring antioxidant activity (c) assumes that oxidation is inhibited largely by the capture of initiating or propagating free radicals in autoxidation. assay. Similar expressions could be written involving rates of oxidation. partitioning behaviour) For a fixed set of conditions AA could be defined. temperature. or (c) formation or decay of probe free radicals. The activity of carnosine. d h. A variety of new parameters for expressing results therefore are used (see Table 4) which more or less serve the same purpose as those based on monitoring the extent of autoxidation. However.89 On the basis of MDA release in a liposome system. there appear to be no standard units for reporting such activity (efficiency. weak antioxidant activity during riboflavin 5A-phosphate sensitized oxidation and even a pro-oxidant effect during copper(II)-catalysed oxidation. action. the latter being more or less a direct function of antioxidant concentration. d mol kg21 hr21... TBARS assays or absorbance increase at 230–235 nm after a fixed time period. Furthermore. carnosine exhibited good antioxidant activity during methylene blue photosensitized oxidation. in turn. 2002. Consistent with this simple definition. Also. A high correlation should therefore exist between results for the two broad methods though this has still to be clearly demonstrated. etc.g. concentration of other substances. This can be expressed as RAA1 = AA1/AAREF where AA1 and AAREF are the activities of the test and reference antioxidants at the same molar concentration. at least at low concentrations.90 Ethanol has exhibited antioxidant activity in certain circumstances68 and this must be considered when measuring the antioxidant activity of alcoholic beverages or when lipophilic compounds have to be added as ethanolic solutions to a test substrate. effectiveness. A practical measure of activity must show at least two things: whether the test substance has a detectable antioxidant or pro-oxidant effect under the test conditions. The advantage of this definition is that common test methods such as those listed in Table 1 can be used to calculate activities in standard concentration terms based on the general methods described in Table 3.92 All.. (b) formation of oxidation products. ‘independently’ of the test method.91 Results are expressed in a variety of ways that make comparisons difficult.View Online There is a need to exercise caution in the interpretation of data and to measure a number of oxidation parameters18 to evaluate antioxidant activity better. a common method of comparing activities. of course. conductivity. which is a useful antioxidant in food systems. peroxides or other antioxidants/pro-oxidants. iron/ascorbate. capacity. g L21 d21 mol kg21. the methods used to follow oxidation and the concentrations of test compounds. involve direct or indirect measurement of the rate or extent of: (a) decay of substrate or probe substance or of oxygen consumption. tREF = time for untreated or 5 Individual procedures Various chemical and physico-chemical procedures are used to monitor oxidation processes. AA would not increase if it were directly proportional to [AH].g.g. however. Note that this definition of antioxidant activity encompasses the concept of efficiency rather than capacity. For quantitive measures most authors report activities as comparative results. a negative result would indicate a pro-oxidant action.) independent of the test procedure. oxygen. antioxidant effectiveness was significantly influenced by the type of system tested (bulk oils versus oil-in-water emulsions). A more meaningful measure in context might be relative antioxidant activity (RAA). e. In the case of rosemary extracts. etc. has been carefully examined with large differences in the results in model systems. They form the basis of the newer test methods such as the ABTS/TEAC. etc. whatever the mechanism. The antioxidant effect in liposomes decreased89 according to the catalyst in the following order: copper/ascorbate. as follows: AA = (t . a dipeptide. peroxide values. 183–198 187 .tREF)/[AH]tREF where t = time for treated substrate to reach a set level of oxidation according to test method. AA would be zero if tREF = t and would become larger if t increased. kg21 mg kg21 (ppm w/w) Absorbance. hydrogen peroxide activated haemoglobin. attempt to indicate the effectiveness of substances to hinder the oxidation of a substrate under specified conditions. reference substrate to reach the same level of oxidation and [AH] = concentration of antioxidant in suitable units. e. g L21 mequiv. This rearranges to AA1 = RAA1 3 AAREF which gives the activity equivalence of a test substance relative to the reference substance. 4. a function of many parameters: AA = f(time or rate. One approach is to examine directly free radical production and its inhibition by anti- Table 3 Methods of expressing results of antioxidant activity tests Method Induction time Time to reach a set level of oxidation (pre-induction period) Rate of oxidation (pre-induction period) Concentration to produce equivalent effect to reference antioxidant (preinduction period) Concentration of functional group after set time period Concentration of oxidation product after set time period Scale reading after set time period Results h. These.1 Expression of results Methods of expressing antioxidant activity appear to be as varied as the methods of measurement. induction times. respectively. Most methods for reporting activities are based on measurements using common test procedures such as those summarized in Table 1. DPPH radical and phycoerythrin assays. They therefore focus on monitoring the capacity of additives/extracts for radical capture or inhibition of radical formation rather than on monitoring the actual oxidation itself. of specified concentrations of different test materials on the substrate. Antioxidant activity (AA) is.

The chemistry of each of the more common procedures is described. There was also a good correlation (r = 0. For instance. no active aeration)103–105 or sunflower oil thin films in an accelerated oven test.g.g. 2002.101. These are based on measurement of the inhibition of the various intermediates or final reaction products of oxidation. mmol of Trolox equivalents Absorbance of Fe(II) complex at 593 nm produced by antioxidant reduction of corresponding tripyridyltriazine Fe(III) complex 188 Analyst. The activity of ethyl acetate extracts was comparable to that of caffeic acid and greater than that of BHT.55 Active Oxygen Method and Schaal oven test commonly involve accelerated deterioration tests.95 The desirable features of a test of antioxidant activity are the use of a substrate and conditions in the test that mimic the real situation and the ability to quantify the result by reference to a suitable standard.103. It was estimated110 from Rancimat data that o-diphenols contributed over 50% to the stability of virgin olive oil. Results from the two accelerated tests were similar.94 which can be quantified by defining the amount of a suitable standard needed to produce the same end-point as the compound or material being analysed. a-tocopherol plus phenolics) has been proposed114 as an alternative to Table 4 Various methods of expressing results for methods based on free radical capture or formation suppression Method Free stable radical quenching Free stable radical quenching Free stable radical quenching Total radical-trapping antioxidant parameter (TRAP) ABTS assay. A number of differences in activity were observed between the two systems and depending on whether lard or corn oil was used in the Rancimat. there may be synergism between antioxidants and examining one in isolation may not accurately reflect their combined action.time to decrease concentration of test free radical by 50% mmol peroxy radical deactivated L21 TEAC (mM Trolox equivalent to 1 mM test substance) ORAC. Heating an oil and periodically testing for weight gain was one of the oldest methods for evaluating oxidative stability. with longer induction indicating better antioxidant activity. 5. This raises the question as to whether results should be expressed on a mass or equimolar basis. This is frequently not the case and the relative activity of several synthetic and natural antioxidants differed when determined by Rancimat or a procedure entailing milder test conditions (lower temperature. oxygen radical absorbance capacity. edible oils100 or a model substrate such as methyl linoleate. Individual measurement of the antioxidant activity of all components in a sample is possible.97 sometimes as a result of the action of light or UV radiation. A flow injection procedure using amperometric detection of oxidizable substrate (e. food manufacture or domestic use. In the more usual approach.101 Following oxidation.109 on plant biophenols.View Online oxidants. Extracts and caffeic acid were much stronger scavengers of DPPH free radical than BHT on an equimolar basis.05) between the antioxidant activity of an Apsergillus extract101 measured by Rancimat and a linoleic acid oxidation system using the thiocyanate method.55 The usual substrates include lard. In addition. The results suggested that the Rancimat may be the least reliable method. The addition of an antioxidant results in the inhibition of oxidation.98 This can be used as a general method for antioxidant activity by selecting a pure substrate (e. The latter can be removed from the sample by preheating in an inert atmosphere. These accelerated tests are specific to the analysis of oxidation in foods with results usually expressed as an induction time. P < 0. Oxidation was followed by measuring PV. The oxidative stability of lard and tallow was examined113 with and without antioxidants by four accelated stability tests. the end-point is determined by measuring parameters such as conductivity.104 Similarly..96. the trends in antioxidant activity differed106 according to whether hydroperoxide formation (PV) or decomposition (hexanal and volatiles) was measured in accelerated stability tests on olive oil. Finally. peroxide value or diene conjugation. However. Hydroxycinnamic acids are an important group of antioxidants and their antioxidant and free radical scavenging activities were measured112 by Rancimat and the DPPH radical assay. tripalmitin or triolein) or other substrate and adding an antioxidant. Stability tests and their limitations have been reviewed by Frankel. There is intense interest in identifying natural antioxidants for use in foods and there has been considerable focus39.108. but much more commonly at elevated temperatures. but this can be time consuming and expensive. with a brief historical overview of the development of the method and its applications to food and/or biological systems as appropriate. it follows from the definition of an antioxidant that its test concentration must be significantly lower than that of the substrate.107 who summarized some of the published literature on the methods used in the evaluation of various natural antioxidants.97 particularly with extracts of low to intermediate antioxidant activity. The technique can be extended to more sophisticated continuous monitoring of mass and energy changes as in thermogravimetry/differential scanning calorimetry. phycoerythrin assay Phycoerythrin assay FRAP assay Results Percentage inhibition EC50. as in production processing. Results are quantified by measuring the induction time of a control and sample. it was recommended that more than one accelerated stability test should be used to determine antioxidant effectiveness.102 Antioxidant activity of grape extract in refined soybean oil was determined69 by the Rancimat and Schaal oven test in conjunction with PV determination.. These differences are not uncommon.1 Accelerated stability tests Stability tests on edible oils and fats such as the Rancimat. 127.99 This requires simple equipment and indicates directly oxygen consumption although the mass change may reflect other volatiles. concentration to decrease concentration of test free radical by 50% TEC50. various indirect measurements are used to assess the effectiveness of an antioxidant in preventing oxidative damage. 183–198 .93 It is therefore of interest to measure the TAA. Such tests are often highly relevant to the conditions to which oils and fats are subject.9702. Antioxidant activities of cell culture extracts were evaluated111 by the Schaal oven test in sunflower oil and using the DPPH radical. any problems associated with the procedure are highlighted.

Selectivity can be enhanced by separation of different diene conjugates using HPLC or by matrix subtraction using secondderivative spectroscopy. For these reasons other methods for determining peroxide oxygen56.View Online Rancimat and ABTS radical tests for the evaluation of antioxidant activity of olive oils. Diene conjugation resulting from lipid oxidation126 is now commonly used as an end-point for determining the antioxidant activity of a sample.11(trans)-dienoic acid. For example. antioxidant activities of sage. that monitoring diene conjugation emerged as a useful technique for the study of lipid oxidation. hesperidin and narirutin extracted from lemon fruit were examined122 using a liposome and an LDL oxidation system. As hydroperoxides are the primary products of lipid oxidation and play a central role in the further autoxidation of lipids. Linoleic acid and antioxidant122–124 were incubated at 40–50 °C for 7 d in the dark. but it will simply not be detected by this test procedure. the relative antioxidant activities of lime peel fibre and orange peel fibre123 were determined. antioxidants may still exert a significant inhibitory action on transient hydroperoxides. possible addition of iodine across unsaturated bonds leading to low results. it may be necessary to run control samples to establish that hydroperoxide build-up does indeed occur for the substrate and test conditions chosen. For the LDL system. kg21) and protection factors were measured and used to assign relative activities to the extracts. The use of HPLC to separate the UV-absorbing ‘diene conjugate’ material from human body fluids revealed131 that most or all of it consisted of an isomer of linoleic acid. Catechins and procyanidins from cocoa were also studied133 in two in vitro systems: liposomes and human LDL. octadeca-9(cis). Extraction of lipids into organic solvents before analysis is a common approach to this problem. The antioxidant activity of 44 different berry and fruit wines and liquors was compared126 by conjugated diene measurement with methyl linoleate as substrate. Gillam and co-workers demonstrated that natural fats develop an absorption peak near 230–235 nm on storage. oxidation of iodide by dissolved oxygen and variations in reactivity of different peroxides. AAPH or DDPH or the application of heat. Initially.2 Peroxide value This parameter represents the total hydroperoxide and peroxide oxygen content of lipids or lipid-containing materials. However. Moreover. It is commonly calculated from an iodometric titration developed over 60 years ago115 that is the basis of current standard methods116. however. The method should.118 have been investigated but the iodometric method119 still remains the standard procedure.132 such as haem proteins. The antioxidant activities of the flavonoids eriocitrin. providing little information about the structure of the compounds. Flavonoid glycosides generally exhibited weaker activity than the corresponding aglycones.125 both abnormal and normal. Removal of sugars from the samples was a necessary step to prevent interference during oxidation of the methyl linoleate. chlorophylls. Its metabolites by intestinal bacteria (the aglycone eriodictyol. In the liposome system. Diene conjugation measurements often cannot be performed directly on tissues and body fluids because many other interfering substances are present.127. Peroxide value. Limitations involving this procedure are well recognized118 and include poor sensitivity and selectivity. diene conjugation by UV spectroscopy is a generic measurement. Lag phase measurements and percentage inhibition have also been used129. A limitation in this approach is that hydroperoxides are unstable and extensive oxidation of a lipid can occur without an accompanying build-up in hydroperoxides.117 for determining PV. 3. is due to a single fatty acid. the inhibition of formation and/or action of these unstable species by antioxidants can be used44. with oxidation being initiated90. be of value in assessing antioxidant activity during the early stages of lipid oxidation under mild conditions. 5. 5.4-dihydroxyhydrocinnamic acid and phloroglucinol) exhibited weaker antioxidative activity but nevertheless exhibited greater activity than a-tocopherol in the LDL oxidation system and had approximately the same activity as (-)-epigallocatechin gallate. even in simple lipid systems. induction period (defined as the time when the PV reached 20 mequiv. It was not until the 1960s. Liposome oxidation (evaluated as TBARS formation) was initiated with AAPH.125 Two years later it was discovered that the peak arose from a diene conjugated bond. The usual substrate for the determination of conjugated dienes includes any substance containing polyunsaturated fatty acids.128 by the addition of copper ions.125 He suggested that serum diene conjugation might reflect oxidation in vivo. Therefore. 2002. 127. the lipid undergoes hydrogen abstraction from a CH2 group and the product is usually stabilized by a molecular rearrangement to form a conjugated diene. lipid oxidation was induced by AAPH and the extent of inhibition by added antioxidant was determined as TBARS at 532 nm. following which time the hydroperoxides from linoleic acid oxidation were determined56 by the iron thiocyanate method. DiLuzio showed that there is a considerable amount of diene conjugated material in human serum lipid extracts.130 to quantify results. iron ions. In this method hydroperoxides and peroxides oxidize aqueous iodide to iodine which is then titrated with standard thiosulfate solution and starch as end-point indicator.131 In either case. Advantages claimed for the proposed procedure are that it is based on the chemical structure of the antioxidant and does not involve accelerated test conditions. Eriocitrin exhibited the highest activity of all lemon constituents as measured by all three methods. The measurement of the formation of diene conjugation has the advantage that it measures an early stage in the oxidation process. diosmin. tissue fluids and tissues. 183–198 189 . Quantification of the conjugated dienes may be achieved90 by calculating the increase in absorbance per mass of sample at a fixed time. Antioxidant activity was expressed as a reduction in oxidation relative to a control (untreated) sample. however. the effect of antioxidant on lag time of the copper(II)-mediated oxidative modification of LDL was measured by monitoring conjugated diene formation at 234 nm. Using this approach.120 as a means of assessing antioxidant activity. The reactions and stoichiometries for this method are ROOH + 2H+ +2I2 ? I2 + ROH + H2O ROOR + 2H+ +2I2 ? I2 + 2ROH I2 + 2S2O322 ? S4O622 + 2I2 where ROOH is a lipid hydroperoxide and ROOR is a lipid peroxide.3 Diene conjugation In 1931. AMVN or iron/ascorbate and LDL oxidation (evaluated as formation of conjugated dienes) was initiated with Cu2+ or Analyst. However. purines and pyrimidines that absorb strongly in the UV region. 95% of diene conjugation in human serum. sweet grass and camomile were tested121 in rapeseed oil at 40 °C. As early as 1972. sensitivity may also be increased. The PV is then calculated as milliequivalents of peroxide oxygen per kilogram of sample.

.g. pentanal and hexanal with accompanying decrease in the content of linoleic and linolenic acids.140. dimer and trimer fractions were the most effective antioxidants. Fritsch and Gale157 showed that rancid odours occurred in ready-to-eat oat cereals when the hexanal concentration reached 5–10 mg g21. pentane. It is postulated that the formation of MDA from fatty acids with less than three double bonds (e. 5. Ethanol is added to aid in the mixing of the antioxidant with the linoleic acid. hexanal Propanal.. 2002.143 however. The chromophore(s) formed in the condensation of aldehydes with N-methyl2-phenylindole absorbs strongly close to 586 nm and the method can be used as an alternative to the TBARS method.5-dien-2-one are suggested to be the major contributors to off-flavours153–156 associated with the rancidity of many food products. linoleic acid) occurs via the secondary oxidation of primary carbonyl compounds (e. hydrocarbons and saturated and unsaturated aldehydes such as hexanal. 190 Analyst. anisidine value152 measures 2-alkenals and the oxidation of various oils was followed70. During rice storage at 40 °C. deca2.145) and then the extent of oxidation is determined by addition of TBA and spectrophotometric measurement of the product.141 including emulsions of linoleic acid with SDS or Tween. The TBARS procedure is widely used147.148 even though the reaction is not very specific and reaction conditions have a significant effect on colour development.View Online AAPH.4-dienal Hepta-2. 127. When liposome oxidation was initiated in the aqueous phase. the appearance of stale flavour158 corresponded to higher levels of propanal. This procedure measures the MDA formed as the split product of an endoperoxide of unsaturated fatty acids resulting from oxidation of a lipid substrate. ethane Photo-oxidation Dec-2-enal Hept-2-enal.g. Indeed.. 1) that is measured spectrophotometrically136 at its absorption maximum at 532–535 nm.. For instance. Results may also be described4 in terms of percentage inhibition of the oxidation. The higher molecular weight procyanidins were the most effective antioxidants when oxidation was initiated in the lipid domains. Results are typically quantified146 against a calibration curve for malondialdehyde bis(dimethylacetal) or malondialdehyde bis(diethylacetal).135 The MDA is reacted with thiobarbituric acid (TBA) to form a pink pigment (TBARS) (Fig. including tissue samples. deca-2. 183–198 .5 Measurement of hexanal and related end-products Decomposition of the primary products of lipid oxidation generates a complex mixture131 including epoxides. pentane.4-dienal and 4-hydroxyalkenals such as 4-hydroxynon-2-enal. but-2-enal Fig.152 by measurement of both anisidine value and PV. deca-2. 5. Studies by Belguendouz et al.4-dienal and octa-3. butanones.g. ketones (e. pentanones. This method apparently responds to both MDA and 4-hydroxyalkenals but is not specific to either group. gives rise to the secondary products which include hexanal. the intermediate hydroperoxides and the specific volatile secondary metabolites analysed for Table 5 Main secondary oxidation products for various fatty acid moieties159 Moiety Oleate Linoleate Linolenate Autoxidation Nonanal. 13-hydroperoxide of linoleate ester groups. It has yet to be applied to a wide variety of sample types. octanones).137.4-dienal. Numerous substrates137–139 have been used in the determination of TBARS. The procedure involves two distinct steps: the substrate is oxidized with the addition of a transition metal ion such as copper or iron or a free radical source such as AAPH (also referred to as ABAP144. The carbonyl compounds including pentanal.150 but this still does not identify the source of MDA in samples or eliminate the possibility of a compound with similar spectral properties coeluting. linoleic and other fatty acids and LDL. found that the presence or absence of ethanol did not influence the antioxidant activity of their samples.4 Thiobarbituric acid reactive substances (TBARS) assay The TBARS assay was proposed over 40 years ago and is now the most commonly used method134 to detect lipid oxidation. The addition of ethanol has recently come under discussion as there is growing evidence142 that ethanol is in itself an antioxidant.149. For instance. Other fatty acid moieties also give rise (via thermolysis of hydroperoxides) to a characteristic set of reaction products159 depending on the mode of oxidation (Table 5). non-2-enal). Oxidation is inhibited by the addition of an antioxidant and therefore a reduction in the absorbance is seen. 1 Chromophore formed by condensation of MDA with TBA. A number of model linoleic acid systems have been developed. Another method for detecting peroxidation in lipids of biological origin151 involves the so-called LPO-586 assay. Various measures of these more or less stable final products of oxidation are used. Frankel160 provided a detailed insight into the mechanisms and spectrum of products obtained by lipid autoxidation and such knowledge is useful in recognising the relationship between fatty acid moieties. the decomposition of the primary oxidation product. which acts as a source of MDA. octanal Hexanal. monomer. Selectivity of the TBARS procedure is improved by the use of HPLC to characterize the individual species.

127. The radical that is generated varies and systems have been described using horseradish peroxidase–H2O2. well-defined endproduct. the rate of oxidative destruction of b-carotene by degradation products of linoleic acid has been measured190–192 spectrophotometrically at 450–470 nm. For example. although intrinsically sensitive to stable free radicals such as di-tert-butyl nitroxide.199 End-point detection also varies and has been based on measurement of fluorescence inhibition. The degradation products of oxidation have also been measured indirectly. in which case measurement of the inhibitory effect of an antioxidant is based on detection of either the radical or the products of oxidation. However. although vitamin E and bcarotene supplementation186 decreased hydrocarbon excretion. however. unlike the synthetic antioxidants BHA and BHT. More selective tests based on derivatisation and HPLC. are frequently based on measuring such secondary oxidation products by headspace GC or GC-MS154. Rancidity studies of refined oils and snack foods. with transient concentrations below 1028 M). Where other antioxidant activity tests may be non-specific. short-lived free radicals involved in autoxidation (lifetimes vary from 1029 s for the hydroxyl radical to several seconds for the peroxyl radical. the effect of added antioxidant was seen196 as an increase in the lag phase. hydroxyl radical. The ability to scavenge specific radicals may be targeted as. The activity of phenolic components of wines75 has been assessed in this manner.206 continuous flow systems and spin trapping. 2002. the production of peroxyl free radicals by the thermal decomposition of AAPH can be coupled to the oxidation of 2. physico-chemical measurement of hexanal174 offers the advantage of analysing a single. for example. for example. a-phenyl-tert-butylnitrone (PBN). the generation of a radical is coupled to oxidation of a substrate. The quantification of aldehydes such as 4-hydroxynonenal is of great interest not only in that they may indicate levels of autoxidation and hence antioxidant activity but also in that they are extremely reactive and cytoxic. This extreme reactivity and metabolic conversion. Since pentane is a very stable end-product it may be more suitable than hexanal for monitoring antioxidant activities. These clinical markers of antioxidant and oxidative stress status were not correlated with normal concentrations of carotenoids in plasma and tissues. trichloromethyl peroxyl radical. For example. carotene and antioxidant were mixed and the results were used to measure antioxidant activity in wines193 and berries. There is ample evidence178–183 that ethane and pentane (endproducts of the oxidation of n . simple chemical tests such as the TBARS and LPO-586 tests are not specific for this substance. monitoring only one or two analytes may be cyclopian in approach.7-dichlorofluorescein.162–164 and correlating these with organoleptic data. Analysis of the full range of volatile secondary oxidation products (which can easily be done these days by GCMS) may be the preferred approach.4-dinitrophenylhydrazone derivatives. Jackson and Giacherio.50 DPPH128.6.View Online rancidity or antioxidant studies. a-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and 3.207–209 of which the last has been the most widely used.95 o-phenylenediamine–H2O2.3 and n . chemiluminescence. have shown that pentane is one of the main secondary oxidation products formed for soybean oil.197. An aqueous emulsion of the linoleic acid substrate.201 oxygen uptake and absorbance.7-dichlorofluorescin to the fluorescent 2. solid-phase microextraction has been applied168 to the determination.50 superoxide radical194 or nitric oxide radical.161 for example. Various techniques have been used to overcome this problem. Spin trapping involves addition to samples of a compound (the spin trap) which reacts with free radicals to form radicaladducts131.6 Measurement of free radicals Strategies have been developed for measuring the antioxidant activity as the ability to scavenge free radicals generated in aqueous and lipophilic phases. Antioxidant activity can be calculated as percentage inhibition of one or more of the secondary oxidation products relative to a control.176 for example. In this instance. EPR spectra have shown207 that the model esters methyl oleate. The naturally occurring phenolics showed pro-oxidant activity at low concentrations.188 such as stimulation of neutrophil chemotaxis and inhibition of many enzymes.192 Results from the b-carotene procedure were com- pared192 with MDA production as measured by HPLC and a free radical procedure using DPPH. Results from the various procedures were generally similar. The major difficulty is contamination from ambient-air ethane and pentane184 and the effective removal of ambient-air hydrocarbons from the subject’s lungs before collection becomes an important step in standardizing the collection procedure.6 polyunsaturated fatty acids. Applications which illustrate the potential of spin trapping methods in antioxidant action include the determination of the antioxidant potential of tea extracts in aqueous and organic Analyst. Hexanal is the most commonly measured end-product of lipid oxidation169–173 and both sensory and physico-chemical methods165 are used for its determination. Alternatively. 183–198 191 .210 that are considerably longer-lived than the original species and can be detected without difficulty by ESR. For instance. Several volatile carbonyl compounds were measured177 in human breath following trapping as their 2. Oxidative stress status was evaluated by breath pentane measurements185 whilst antioxidant status was evaluated by measurement of the total antioxidant capacity of the plasma. 5. 5.175 that show that hexanal formation is usually an order of magnitude higher than with most other secondary oxidation products. may make them unsuitable as test analytes for in vivo antioxidant activity studies except at high levels of oxidative stress. Selectivity has been improved165–167 by (isotope dilution) mass spectrometry. including pulse radiolysis and UV photolysis. GC or GC-MS189 are more suitable.5-dimethylpyrroline-N-oxide (DMPO).195 One approach involves95 the generation of a free radical species and direct measurement of its inhibition due to addition of antioxidant(s). linoleate and linolenate each formed three distinct radical adducts with PBN and confirmed that oxidation proceeded via different mechansims at high and low temperatures. An exception is pentane.198 and azo compounds such as the chromogenic redox indicator ABTS.1 Electron spin resonance spectrometry. In fact. Spin traps are usually nitroso compounds or nitrones and those commonly used in biological systems include tert-nitrosobutane (tNB).205 ESR is unfortunately insensitive to detecting the reactive.5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). Electron spin resonance (ESR) spectrometry is the only analytical technique that can specifically detect the free radicals202–204 involved in autoxidation and related processes. respectively) in expired air are useful markers of in vivo lipid peroxidation. which forms in concentrations comparable to those of hexanal (pentane formation is an alternative decomposition pathway for 13-hydroperoxide-linoleate). The significance of hexanal as an analyte for oxidation monitoring (or antioxidant efficiency studies) is indicated by data reported by Snyder et al. the cytotoxicity of 4-hydroxynonenal is exhibited in diverse processes187. Furthermore.64 5. copper(II)–cumene hydroperoxide. More recently.200. tert-butylnitrosobenzene (BNB).

Their popularity can be attributed to simplicity and speed of analysis.211 assessing the antioxidant contribution of quercetin and other flavanols to the antioxidant capacity of red wines.225 by monitoring the decrease in this absorbance. Miller and RiceEvans220 found that results of the myoglobin–ABTS assay and direct reduction of the ABTS radical cation were very similar establishing that the action of the antioxidants studied was via scavenging of the ABTS radical cation and not by inhibition of its formation through reduction of ferrylmyoglobin or reaction with hydrogen peroxide.95 reported no interferences at the optimal wavelength of 414 nm and this translated to better detection limits. in the case of orange juice. The DPPH radical absorbs at 517 nm and.213 Even with these limitations. TEAC assays have also been measured for flavonol and catechin metabolites as the antioxidant capacities of such metabolites may be significantly different to that of the original antioxidant223 for in vivo processes.212 specific assays for the hydroxyl or superoxide radicals in natural extracts or biological systems. 127. Another problem208. HPLC required preliminary filtration and the measured composition reflected the soluble222 flavonoid portion only. although formation of a dimer239 was a common occurrence.3 and 6. Its use. The course of subsequent reactions was dependent on the nature of the phenol. by measurement in the near-infrared region at 734 nm. Phenolic compounds generally exhibited significant scavenging effects against the DPPH free radical. Nevertheless. ascorbic acid and a-tocopherol for DPPH were studied231 by 13C NMR.214 the study of free radical transfer in fish lipid–protein systems215 and the measurement of antioxidant capacity from ascorbic acid in blood plasma. it has been shown that such traps can exhibit both oxidant131 and antioxidant217 action. For example. when oxidized in the presence of H2O2 generates a metastable radical cation95. the amount of antioxidant necessary to decrease by 50% the initial DPPH concentration.0 mM solution of the substance under investigation. namely antiradical efficiency.219 with a characteristic absorption spectrum and high molar absorptivity at 414 nm. was defined. Citrus oils were examined228 by HPLC using DPPH and results expressed in Trolox equivalents.224.64 is based on the formation of the ferrylmyoglobin radical (from reaction of metmyoglobin with hydrogen peroxide) which is then free to react (at a higher reaction rate) with ABTS to produce the ABTS radical cation.237 The antioxidant activity of pomegranate juices was evaluated235 by DPPH and ABTS and the results were compared with those of red wine and tea infusions.2 ABTS assay.View Online media. 183–198 5. was the major antioxidant.213.209 is that spin traps exhibit widely differing trapping efficiencies for different radicals. However. Unlike the latter method that used the metmyoglobin peroxidase activity.235 superoxide-anion scavenging and lipid oxidation. .210. as described by Rice-Evans and Miller. 2002.190. The TEAC64. antioxidant activity can be determined128. Results were reported as the EC50.86. In recognition of the effect of both parameters on antiradical capacity.95 is similar to that of Rice-Evans and Miller64 but differs in a number of important aspects. A relationship between potential and DPPH scavenging was observed for phenolic acids but not for flavonoids. with those in blackcurrant having the greatest vitamin C-sparing activity. which minimized interference from other absorbing components and from sample turbidity. Plant extracts were separated229 by HPLC and reacted post-column with DPPH and the bleaching was detected as a negative peak by an absorbance detector at 517 nm. a new parameter. The molecular mechanisms and radical scavenging activities of (+)-catechin. The TAA of orange and grapefruit juices95 were 4. The ABTS assay has been used93 to measure the total antioxidant activity in pure substances. that is.236. presumably to protect the phenolics against oxidation. 734 and 815 nm.221 is equal to the millimolar concentration of a trolox solution having the antioxidant capacity equivalent to a 1. The procedure based on inhibition of the production of the ABTS radical cation218 did not involve a substrate. (+)-Catechin reacted with DPPH to form an o-quinone structure in the B-ring.64 the TEAC reflects the relative ability of hydrogenor electron-donating antioxidants to scavenge the ABTS radical cation compared with that of Trolox. spin traps can perturb systems under investigation.6. can be measured spectrophotometrically. respectively. However.226 which combined both factors.198. The method of Arnao et al. However. Arnao et al. ABTS with an absorption maximum at 342 nm has high water solubility and chemical stability.3 Diphenylpicrylhydrazyl (DPPH) radical. Results were expressed by comparison with standard amounts of the synthetic antioxidant trolox (a water-soluble vitamin E analogue) to give rise to the TEAC. ROS and DPPH scavenging abilities of extracts of evening primrose227 and citrus essential oils228 have been studied.1 mM L21 ascorbic acid equivalents. The phenols form o-quinone intermediates upon H-atom abstraction from DPPH and subsequent radical disproportionation.6. Miller and Rice-Evans199 reported the TEAC of orange and apple juices and blackcurrant drink (Ribena) and also the contribution of individual phenolic antioxidants. It is a peroxidase substrate which.216 The specificity. while spin adducts can act as antioxidants. 5. The antioxidant activity of plant biophenols has been attributed238 to trapping of ROS and regeneration of endogenous membranebound a-tocopherol. but this is achieved at a potential price and the relevance of data generated with these procedures must be considered carefully. The authors concluded that the phenolic antioxidants protected vitamin C against oxidative decomposition. The accumulation of ABTS4+ can be inhibited by the presence of an antioxidant in the reaction medium. in a second substrate-free system. to an extent and on a time scale dependent on the antioxidant activity. there is no doubt that ESR will continue to provide valuable information on the complex roles and patterns of free radicals in biological oxidation processes. a commercial peroxidase was used by Arnao et al. ethyl gallate. As used by Rice-Evans and Miller. Furthermore. Other problems include the specialist nature and relatively large size and cost of the equipment and that such instrumentation has yet to be developed to the stage where short-lived radicals can be measured in vivo (as NMR imaging has for hydrogen nuclei). The time taken to reach the steady state to EC50 concentration (TEC50) was also calculated. in body fluids and in plant material. vitamin C 192 Analyst. whereas in both orange juice and Ribena. The relative ability of hydrogen-donating antioxidants to scavenge ABTS4+ generated in the aqueous phase. Hydrolysable tannins accounted for the high activity of juices. The bulk of the TAA of apple juice could be accounted for by chlorogenic acid and the phloretins. It is worth reiterating that the ABTS and DPPH methods are substrate-free. there are secondary absorption maxima in the wavelength regions of 645. applications are limited to date owing mainly to the sensitivity problem. the situation is complex and winemakers add ascorbic acid during fermentation as an anti-browning agent.232–234 DPPH reduction has been compared with other methods including the ABTS assay. Coulometric detection has also been used230 for phenolic plant extracts. ability to handle complex biological samples and the capacity to identify individual free radicals represent distinct advantages for ESR methods.

A number of factors 5. a condition that is seldom valid. Trolox. the flavonoid glycosides (including rutin. Final results can be calculated244–246 using the differences in areas under the phycoerythrin decay curves between the blank and a sample and are expressed in trolox equivalents. 1.241 is based on the reduction of a ferroin analog.247 as the automated oxygen radical absorbance capacity (ORAC) in micromoles of Trolox equivalents. ascorbate.1 FRAP assay. An oxygen electrode will not maintain its stability over the period of time required (up to 2 h per sample)218 and the TRAP assay was modified249 to use luminol-enhanced chemiluminescence (CL) as the end-point.7 Other measures of antioxidant activity 5. that the measured reducing capacity does not necessarily reflect antioxidant activity. Site specific damage to macromolecules results from the reaction Target–Cu2+ + HO4 ? damaged target + Cu2+ This assay is particularly useful in screening for compounds that protect against damage by chelating metal ions necessary for site-specific formation of the radical species. Indeed. in general.250 although the concept has been very useful for quantifying and comparing251 antioxidant capacity.242 to other biological fluids..18 however.9 mM and a half-peak reduction potential below 0. juices. 1. It was concluded that LDL TRAP assay may complement the other methods used to quantify the antioxidant capacity of LDL. Peroxyl radicals generated by the thermal decomposition of AAPH quench the fluorescence of the phycoerythrin while addition of an antioxidant that reacts rapidly with peroxyl radicals inhibits the loss of fluorescence intensity and this inhibition is proportional to the antioxidant activity. determine antioxidant activity including reactivity as a hydrogen.3 Total radical-trapping antioxidant parameter. Results can be standardized by addition of Trolox to the sample after consumption of natural antioxidants to produce a second induction period. Phenolic acids. 127. The measurement of serum TRAP196 was based on the determination of the length of time that a subject’s serum was able to resist artificially induced oxidation. the CL was extinguished.7. These involved determination of LDL TRAP in plasma AMVN-induced oxidation and measuring the extinction time of chemiluminescence. to the intensely blue coloured Fe2+ complex Fe(TPTZ)2+ by antioxidants in acidic medium. It provides instead a very useful ‘total’ antioxidant concentration. The contribution of vitamin C to the activity was < 15% except for kiwi fruit and honey dew melon. The highly fluorescent proteins b-phycoerythrin and R-phycoerythrin (PE). naringin and hesperidin) usually had low ORAC activities. Three different methods were also used253 for quantifying the antioxidant capacity of LDL ex vivo in dyslipidaemic patients with coronary heart disease. The ferric reducing antioxidant power (FRAP) method240. have been used243. There was significant variation in the TAA of several fruits with strawberry having the highest ORAC activity on the basis of both wet and dry weight of fruit. Wayner and co-workers248 followed oxidation by monitoring oxygen consumption in a thermostated oxygen electrode cell during oxidation of linoleate by free radicals.218 A major problem with this method lies in the oxygen electrode end-point. This value combined both inhibition time and the extent of inhibition into a single quantity244 whereas other methods use either the inhibition time at a fixed inhibition degree or the inhibition degree at a fixed time as the basis for quantifying results. This was rationalized on the basis that both electrochemical oxidation and hydrogen-donating free radical scavenging involve the rupture of the same phenolic bond. the duration of which was directly proportional to the radical trapping ability of the antioxidant sample. Thus.64. The antioxidant activity of four standard antioxidants (gallic acid. Antioxidant activities of several juices and fruits were reported244.2 Phycoerythyrin assay.0. The results were not comparable in that gallic acid was the strongest antioxidant in all three systems but the relative activity of the remaining compounds depended on the system. Hydroxyl radicals are generated from an ascorbate–Cu2+ system at copper-binding sites on macromolecules. The total radical-trapping antioxidant parameter (TRAP) assay of Wayner et al. without measurement and summation of the concentration of all antioxidants involved.244 as the target of free radical damage.248 has been widely used to determine TAA based on measuring oxygen consumption during a controlled lipid oxidation reaction induced by thermal decomposition of AAPH. The TRAP expresses results196 as the number of mmoles of peroxyl radicals trapped by 1 l of plasma. Tocopherol supplementation produced statistically significant changes in all antioxidant variables except those related to LDL ubiquinol. When ingested by healthy volunteers. urate.241 We are in agreement with Frankel and Meyer. etc.7. The method is time consuming and suffers a number of problems. derived from numerous species of red algae. flavonoids with efficient scavenging properties had a TEAC value exceeding 1. Trolox and ascorbic acid) was compared252 using TEAC and TRAP assays and LDL oxidation. The contribution of the fruit pulp fraction (extracted with acetone) to the total ORAC activity of a fruit was usually < 10%.7) and these must be taken into account when extrapolating results back to molar concentrations from TRAP values. plant extracts. Although phenols exert strong antioxidant activity. peroxyl radicals enhance the CL reaction. The authors claim the method to be simple and rapid and both manual and automated procedures have been described. with the exception of kaempferol. Phycoerythrin is also used to assess the effectiveness of antioxidants against hydroxyl radicals. However. In this system. 183–198 193 . 5. 2. Stoichiometric factors for pure antioxidants are different218 (e. in vivo evidence254 has produced contradictory results.2 mV. the Fe3+ complex of tripyridyltriazine Fe(TPTZ)3+. foods.View Online 5. 2002. This correlation may be fortuitous as the half-peak reduction potentials are thermodynamically meaningless unless the electrochemical processes are reversible. with the inhibition being proportional to the antioxidant activity. Results are obtained as absorbance increases at 593 nm and can be expressed as micromolar Fe2+ equivalents or relative to an antioxidant standard. uric acid.or electron-donating agent and this aspect relates to its reduction potential. The inhibition of oxidation by an antioxidant can be examined by the retardation of the loss of fluorescence. there is broad agreement92 between the half-peak reduction potential and the TAA as measured by TEAC. The method was originally applied to plasma but has been extended241. Most of the antioxidant capacity of these fruits was from the juice fractions.g. This led to enhanced precision and a greater ability for automation. When an antioxidant was added. had lower antioxidant activities against peroxyl radicals than flavonoids that contained multiple hydroxyl groups. ORAC values showed a significant positive linear correlation246 with electrochemical data obtained by HPLC with coulometric array detection.7. red wine and green tea were the most efficient in protecting LDL from oxidation driven by peroxyl Analyst. conjugated diene formation in copper-induced oxidation and consumption times of reduced a-tocopherol and ubiquinol in AMVN-induced oxidation.5.

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