RelevanceandStandardizationof InVitro AntioxidantAssays: ABTS… · 2019. 7. 30. · DPPH, ABTS,...

Research ArticleRelevance and Standardization of In Vitro Antioxidant AssaysABTS DPPH and FolinndashCiocalteu

Helena Abramovic Blaz Grobin Natasa Poklar Ulrih and Blaz Cigic

Department of Food Science and Technology Biotechnical Faculty University of Ljubljana Jamnikarjeva 1011000 Ljubljana Slovenia

Correspondence should be addressed to Blaz Cigic blazcigicbfuni-ljsi

Received 24 July 2018 Revised 18 September 2018 Accepted 26 September 2018 Published 29 October 2018

Academic Editor Serkan Selli

Copyright copy 2018HelenaAbramovic et alis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Trolox gallic acid chlorogenic acid caffeic acid catechin epigallocatechin gallate and ascorbic acid are antioxidants used asstandards for reaction with chromogenic radicals 22-diphenyl-1-picrylhydrazyl (DPPHmiddot) and 22prime-azino-bis-3-ethylbenzotiazolin-6-sulfonic acid (ABTSmiddot+) and FolinndashCiocalteu (FC) reagent e number of exchanged electrons has been analyzed as function ofmethod and solvent Amajority of compounds exchangemore electrons in FC assay than in ABTS andDPPH assays In reactionwithchromogenic radicals the largest number of electrons was exchanged in buffer (pH 74) and the lowest reactivity was in methanol(DPPH) and water (ABTS) At physiological pH the number of exchanged electrons of polyphenols exceeded the number of OHgroups pointing to the important contribution of partially oxidized antioxidants formed in the course of reaction to the antioxidantpotential For Trolox small impact on the number of exchanged electrons was observed confirming that it is more suitable asa standard compound than the other antioxidants

1 Introduction

Numerousmethods can be applied for determining the in vitroantioxidant potential (AOP) of single compounds or theirmixtures Due to their simplicity the spectrophotometricmethods based on reaction of the antioxidants with chro-mogenic radicals such as 22prime-azino-bis-3-ethylbenzotiazolin-6-sulfonic acid (ABTSmiddot+) and 22-diphenyl-1-picrylhydrazyl(DPPHmiddot) or with phospho-tungsto-molybdate in FolinndashCiocalteu (FC) reagent are widely applied Recent papersnevertheless point to the major drawbacks of such methodsas no relation to the in vivo antioxidant efficacy was ob-served [1ndash3] In AOP determinations the endpoint mea-surements in the in vitro assays correspond to the amountof hydrogenselectrons which certain compound or mixturecan exchange in the reaction with the oxidant probe ratherthan to the actual efficiency in the reaction with radicalswhich is crucial for in vivo function of this compound

All three methods are based on the transfer of an electronfrom the deprotonated antioxidant to the probe when AOPis determined in protic solvents [4] and the mechanism is

described as sequential proton loss electron transfer(SPLET) e fact that the overall reaction rate depends onthe proportion of ionized molecules of the antioxidant(typically the phenolate group) [5] implies that apart fromthe structure of the antioxidant molecule the type of solventand the pH of the assay solution in particular have a largeinfluence on the reactivity of antioxidants [6ndash10] Solventcomposition influences not only the rate of initial oxidationsteps but also the degree of secondary reactions of partiallyoxidized antioxidants that contribute significantly to thenumber of exchanged electrons [6 11 12]

A serious disadvantage and shortcoming of the abovemethods is that they are poorly standardized A survey of theliterature reveals that the reactions with a given probe areperformed in different solvents and for different periods oftime [13] Additionally the AOPs of samples are normalizedto different standard antioxidants e AOP determined bythe ABTS or the DPPHmethod is mostly expressed as molarequivalent of (plusmn)-6-hydroxy-2578-tetramethylchromane-2-carboxylic acid (Trolox) [14] but normalization toascorbic acid (AA) [15] catechin (CTH) [16] chlorogenic

HindawiJournal of ChemistryVolume 2018 Article ID 4608405 9 pageshttpsdoiorg10115520184608405

(CGA) [17] caffeic acid (CA) [18] gallic acid (GA) [19] andother antioxidants is not uncommon

AOP of food samples is usually evaluated by more thanone method and correlation analysis of AOPs obtained byDPPH ABTS and FC assays is often performed e cor-relations can be high and significant or weak [20ndash22]reflecting the lack of consistency of the results of suchanalyses e large influence of the experimental parameterson the reactivity of antioxidants in the samples undoubtedlycontributes to the observed discrepancies ese are po-tentiated when AOPs of samples with different compositionand reactivity of antioxidants in particular assays arecompared e fact that AOP of the samples determined byDPPH ABTS and FC assays is rarely normalized to thesame model antioxidant contributes additionally to theambiguity in this area of research It is almost impossible tocompare the AOP of samples determined by differentmethods on a quantitative basis To enable a relevantcomparison between the results of studies carried out withdifferent methods under various experimental conditionsthe uniform reactivity of the compound used as standard isof great importance

We report here how the method applied for AOP es-timation length of the assay and the composition of thesolvent affects the reactivity of selected antioxidants fre-quently used as standard compounds e aim of the studywas to find the standard compound with reactivity that is theleast affected by experimental conditions and could betherefore applied as a general standard for DPPH ABTSand FC assays

2 Materials and Methods

21 Materials Trolox caffeic acid gallic acid chlorogenicacid L-ascorbic acid dehydroascorbic acid (DHA) catechinhydrate iron(II) sulphate heptahydrate (FeSO4 times 7H2O)FolinndashCiocalteu reagent DPPHmiddot and ABTS were fromSigma-Aldrich (Steinheim Germany) Acetic acid sodiumhydroxide sodium carbonate (Na2CO3) sodium hydrogenphosphate dihydrate and methanol were from Merck(Darmstadt Germany) Manganese(IV) oxide (MnO2) wasfrom Kemika (Zagreb Croatia) Epigallocatechin gallate(EGCG) was from DSM Food Specialities BV (DelftNetherlands) Water was purified using a MilliQ system(resistivity gt18MΩmiddotcm Millipore)

Stock solutions (10mM) of Trolox CTH CA CGA GAand EGCG were prepared in MeOH AA and FeSO4 inMilliQ water and DHA in acetate buffer (50mM pH 50) Allfurther dilutions were made in the solvents used for par-ticular assay

22 e FolinndashCiocalteau Assay e FC assay was per-formed according to a modified method of Gutfinger [23]An appropriate volume of the antioxidant or FeSO4 solution(50 μL) was added into a 15mLmicrocentrifuge tube mixedwith MilliQ water (700 μL) and FC reagent (125 μL pre-viously diluted 1 2 (vv) with MilliQ water) After 5min at25degC a solution of Na2CO3 (125 μL 20 ww) was addedand the sample was mixed again and incubated for an

additional 55min e absorbance at 765 nm (A765) wasmeasured with a Varian Cary 100 BIO UV-VIS spectro-photometer in a 1 cm cell e concentration range of an-tioxidants and FeSO4 in the assay solution is given in Table 1e measurements were made in triplicate including thepreparation of sample solutions and reagents

23 e DPPH and ABTS Assays e DPPH assay wasperformed according to a modified method of Brand-Williams et al [24] and the ABTS assay according toa modified method of Re et al [25] e DPPHmiddot solution wasprepared in MeOH and diluted to the concentration thatwould give an absorbance of 24 at 520 nm in a cuvette with1 cm path length ABTSmiddot+ was produced by reaction of ABTSin aqueous solution with MnO2 followed by centrifugationand filtration the solution was then diluted with MilliQwater to the concentration that would give an absorbance of24 in the cuvette with 1 cm path length at 734 nm All thesolutions buffers and solvents were incubated at 25degC priorto analysis e assay solutions were prepared in a 15mLmicrocentrifuge tube by mixing DPPHmiddot or ABTSmiddot+ solution(500 μL) with 450 μL of the solvent (MilliQ water MeOHand acetate buffer (250mM pH 50) or phosphate buffer(50mM pH 74) for the DPPH assay and MilliQ water andacetate buffer (250mM pH 50) or phosphate buffer(50mM pH 74) for the ABTS assay) e reactions werestarted by adding 50 μL of the antioxidant solution into theassay medium with thorough mixing After 60min in-cubation at 25degC the absorbance at 520 nm for DPPHmiddot (A520)and at 734 nm for ABTSmiddot+ (A734) was measured e mea-sured value was subtracted from the corresponding value forthe control (the selected solvent added into the assay me-dium instead of the antioxidant solution) and the dataexpressed as ΔA520 or ΔA734 e concentration range ofantioxidants in assay solution is given in Table 1 Allmeasurements were carried out in triplicate including thepreparation of sample solutions and reagents

e reaction kinetics of model antioxidants CGA (10 μM)CA (10 μM) Trolox (10 μM) and GA (70 μM) was analyzedin 1 cm quartz cuvettes with the stopper to prevent evapo-ration e solvent compositions were the same as those forendpoint measurements explained above e A520 and A734were continuously measured at 15 s intervals over 180mine first time point was measured 15 s after mixing the an-tioxidant with radical probe e measured absorbances weresubtracted from the corresponding absorbances of the con-trols at appropriate time points and obtainedΔA520 andΔA734values were normalized to the number of exchanged electronsper molecule as explained in Section 3124 Statistical Analysis e slope of the calibration curveobtained by linear regression analysis with program Origin(Microsoft) prepared with each model antioxidant ina particular type of assay and used to calculate the number ofexchanged electrons was the average value obtained fromthree independent experiments including the preparation ofsample solutions and reagents Relative standard deviationof the average slope was not larger than 5

Pearson correlation coefficients (r) were calculated withthe program Excel (Microsoft)

2 Journal of Chemistry

Mean absolute errors (MAE) and mean absolute percenterrors (MAPE) in the number of exchanged electrons (n) forseven model antioxidants (a) were calculated (Equations (1)and (2)) for each pair of antioxidant assays (X Y) Data forDHA were not included in the calculation since the low re-activity of DHA in all conditions except in the FC assay woulddistort MAPE values e antioxidant assay which gave thelowest n value for the particular antioxidant is designated as Y

MAPE 100

j1113944

j

a1

nXa minus nYa

nYa

11138681113868111386811138681113868111386811138681113868

11138681113868111386811138681113868111386811138681113868 (1)

MAE 1j

1113944

j

a1nXa minus nYa

11138681113868111386811138681113868111386811138681113868 (2)

3 Results and Discussion

31 Quantification of Exchanged Electrons in the Reaction ofAntioxidants with the Oxidant Probe e AOP of testedcompounds quantified as the number of exchanged elec-trons (n) for the reaction between antioxidants and DPPHmiddot

or ABTSmiddot+ radical was estimated as the molar ratio of thequenched radical to the tested antioxidant after 60minincubation It was calculated considering molar absorptioncoefficients (ε) of the radical in the solvent used and the slopeof the calibration curve (kantioxidant) prepared with modelantioxidants (dependence of ΔA520 and ΔA734 on antioxi-dant concentration in assay solution (cantioxidant)) as shownin Equations (3)ndash(5) For all antioxidants analyzed thedependence of ΔA520 and ΔA734 was linear in the concen-tration range as shown in Table 1

ΔAλ ελ times Δcradical times l (3)

ΔAλ k times Δcantioxidant (4)

n ΔcradicalΔcantioxidant

k

ελ times l (5)

where cradical represents the concentration of the radical inthe assay solution and l represents the cuvette path length Incalculations of n for the reaction between DPPHmiddot and an-tioxidants in MeOH water or buffer (pH 50) we used the ε

value of 12000 Lmiddotmolminus1middotcmminus1 [26]e corresponding n valuefor reaction with the ABTSmiddot+ radical in the tested solventswas calculated taking into account the ε value of (15000 plusmn549) Lmiddotmolminus1middotcmminus1 [25]

e contribution of the reaction product DPPH2 to theA520 in MeOH or in the mixture of MeOH and acetate buffercan be neglected and therefore a change in absorbance can beattributed solely to the change in concentration of DPPHmiddot Ata neutral pH the absorbance of formed DPPH2 shouldhowever be considered in calculations as observed by otherauthors [27] Indeed when we recorded the absorptionspectrum for the DPPHmiddot solution with large molar excess ofTrolox in the test in phosphate buffer (pH 74) we found thatthe DPPH2 absorbs at 520nm and significantly contributes tothemeasured valuee absorbance of DPPH2 contributes 41of the absorbance of a DPPHmiddot radical at 520nm It is importantto emphasize that the actual pH in the mixture of buffer andMeOH can be higher than in the pure aqueous buffer [28]enumber of exchanged electrons was calculated by consideringthe contribution of DPPH2 to the measured value of A520 andof ε for DPPHmiddot at neutral and basic pH of 10700Lmiddotmolminus1middotcmminus1[27] and applying Equation (6) instead of Equation (3)

ΔAλ ελ times Δcradical times l times 059 (6)

For the FC assay the AOP ie the value of n in the oxi-dation of antioxidants with phospho-tungsto-molybdate cannotbe calculated from the corresponding molar absorption co-efficient as ε of the products is not known In order to evaluatethe reactivity of the investigated compounds a calibration curvewith FeSO4was prepared inwhich Fe2+ ions in the reactionwithFC are oxidized to Fe3+ (one electron exchange) It was pre-viously shown that Fe2+ reacts in the FC assay [29]

ΔA765 kFe2+ times ΔcFe2+ (7)

ΔA765 kantioxidant times Δcantioxidant (8)

n ΔcFe2+

Δcantioxidant

kantioxidant

kFe2+

(9)

e n value in reaction of investigated antioxidants withFC reagent was estimated with Equations (7)ndash(9) using theslopes of the calibration curves for antioxidants (kantioxidant)and for FeSO4 (kFe2+ 00034 plusmn 3 times 10minus5 Lmiddotμmolminus1)

Table 1 e range of concentration of antioxidants in assay solution (cantioxidant)

Samplecantioxidant range (μmolL)

DPPH ABTS+FC

MeOH H2O pH 5 pH 74 H2O pH 5 pH 74CA 8ndash40 8ndash40 8ndash40 3ndash15 8ndash40 8ndash40 8ndash40 3ndash15CGA 6ndash30 6ndash30 6ndash30 2ndash10 6ndash30 6ndash30 4ndash20 6ndash30GA 26ndash13 26ndash13 26ndash13 26ndash13 16ndash80 16ndash80 16ndash80 6ndash30CTH 16ndash64 3ndash15 3ndash15 16ndash8 16ndash64 16ndash64 16ndash64 3ndash15EGCG 14ndash7 14ndash7 14ndash7 08ndash4 14ndash7 08ndash4 08ndash4 3ndash15AA 4ndash40 4ndash40 4ndash40 4ndash40 4ndash40 4ndash40 4ndash40 4ndash40DHA 100ndash500 100ndash500 100ndash500 20ndash200 100ndash500 100ndash500 20ndash200 10ndash100Trolox 5ndash35 5ndash35 5ndash35 5ndash35 5ndash35 5ndash35 5ndash35 5ndash35FeSO4 mdash mdash mdash mdash mdash mdash mdash 20ndash120

Journal of Chemistry 3

32 Number of Exchanged Electrons Greatly Varies withSolvent and Type of AOP Assay e AOPs for each of the 8model antioxidants in 4 variants of DPPH assay 3 variants ofABTS assay and 1 variant of the FC assay are shown inFigure 1 Large variations are observed in dierent types ofassay and also within subvariants of ABTS and DPPH assaysIn reaction with chromogenic radicals the largest number ofelectrons are exchanged in buer (74) while the lowest onewas observed in MeOH (DPPHmiddot) and in water (ABTSmiddot+) It isevident that for the majority of antioxidants higher n valueswere observed in the FC assay than in ABTS and DPPHassays e exceptions are phenolic compounds with py-rogallol group GA and EGCG

Large variations in the number of exchanged electronsfor dierent antioxidants are to be expected since antioxi-dants dier in the number of OH groups bound to aromaticrings or to an unsaturated carbon atom Typical oxidation ofpolyphenols is best depicted by the oxidation of a diphenoliccompound (catechol) into the corresponding quinone(Equation (10)) and of enediol (ascorbic acid) into vicinaldiketone (Equation (11))

OHOH O

O

+ 2H+ + 2endash (10)

OHO

OH

O

HO OH

OHO

OH

O

O O

+ 2H+ + 2endash

(11)

Oxidation of one OH group on an aromatic ringenoletherefore involves transfer of one electron on the radical(DPPHmiddot and ABTSmiddot+) or other oxidants (phospho-tungsto-molybdate) e results shown in Table 2 nevertheless revealthat n per OH can be substantially higher than 1 For GA thevalue of n per OH group in the FC test and with chro-mogenic radicals ranged from 17 to 35 being highest forABTSmiddot+ at pH 74 For the compound bearing two pyrogallolgroups in its structure EGCG n per OH group ranged from11 to 20 being the highest for DPPH and ABTS at pH 74CA CGA and CTH the phenolic compounds with a cate-chol group also exchanged more than 1 electron per OHgroup under the majority of assay conditions being thehighest for DPPH at pH 74 and with the FC assay where forCGA n per OH group amounted to almost 4 DHA reactsonly in FC assay where AA also shows higher AOP mostlikely as a result of formation of hydroxy furanones [30]

which are redox active substances [31] In the FC assay that isperformed at basic pH a sucient amount of secondaryantioxidants which contribute to AOP is formed

All the polyphenolic antioxidants analyzed exchangedmore than 1 electron per OH group which conrmed thatsecondary reactions under certain conditions contributeeven more to AOP than primary oxidations of the phenolicsA relevant question is whether such reactions can be relatedto the eciency of the antioxidants in food matrices andpotentially in vivo where antioxidants are enzymatically andnonenzymatically modied In the light of the fact that thehigh rate is crucial for the eciency of the antioxidantreacting with the radical [2] one could argue that analysis ofAOP which is based on oxidation in the second phase isirrelevant in this respect ere are serious and justiedconcerns about the current practice that assays with radicalsare allowed to proceed for long reaction periods [9]However a slow reaction rate in the second phase does notnecessarily mean that compounds that are formed frompartially oxidized phenolic compounds react at slow ratesper se if we assume that the rate-limiting step is the for-mation of these compounds It was previously shown thatthe product formed from partially oxidized chrysin in re-action with ABTSmiddot+ reacts faster with the radical than theparent molecule [12] e lack of relevant information re-lated to this topic means that these secondary reactions haveto be considered relevant Additionally we have shown thatat neutral pH which is encountered in large proportion ofthe gastrointestinal tract body uids and cellular com-partments reactivity of antioxidants in the second phase isincreased (Section 33)

Of all the standard antioxidants analyzed the number ofexchanged electrons for Trolox was the least dependent onthe type of assay solvent composition and pH e n valuedetermined in all conditions regardless of the method wasin the range of 175 to 225 (Table 2) For the reaction be-tween DPPHmiddot and Trolox the highest n (192) was observedin buer at pH 74 followed by buer solution at pH 5 andwater with the lowest one inMeOHe oxidation of Troloxby ABTSmiddot+ in water resulted in n 179 Comparable valueswere also obtained for the reaction at pH 5 and at pH 74 Inthe FC assay Trolox exchanged 225 electrons us theaverage value of n in all tests was 188 plusmn 017 and taking intoconsideration Trolox purity (97) it amounted to 194 whichis comparable with literature values of between 19 and 2[5 8 24] Analysis of oxidation in aqueous and alcoholicsolvents revealed that the chromane ring is broken upon ox-idation and Trolox quinone with two CO groups is formed(Equation (12)) resulting in two electron oxidation [32]

O

HO

COOH

O

O

OH

COOH+ H2O + 2H+ + 2endash

(12)


Trolox is formally a compound with one OH grouphowever when hydrolysis of the ether bond in the chromanering resulting in the formation of 14-hydroquinone is takeninto the account it is oxidized as a typical diphenoliccompound (Equation (10)) with one electron exchanged perOH group e extent of additional oxidation reactions istherefore relatively small as only in the FC assay there areslightly more than two electrons exchanged

Large difference in AOP determined at 60min can beexemplified by mean absolute errors and mean absolutepercent errors in the n for model antioxidants for each pairof the antioxidant assays (Supplementary file)

Since there are considerable differences in n values al-ready between variants of a particular assay (Table 2) controlof experimental parameters such as pH and solvent com-position is therefore particularly important Often thoseparameters are not controlled and even not reported in the

papers which complicates the comparison of obtained AOPfor similar samples in different studies Especially DPPHassay is often performed in the absence of buffer and there islarge probability that only with the added sample theconditions regarding water content and pH can be drasti-cally changed resulting in changed reactivity [6] To increasethe robustness of the assays with chromogenic radicals theyshould be preferentially performed in buffered solvents

33 e Influence of Solvent on the Kinetics of Reaction ofAntioxidants with ABTSmiddot+ and DPPHmiddot Radicals We haveanalyzed the kinetics of reaction of CGA CA GA andTrolox with chromogenic radicals in solvents giving thelowest (MeOH for DPPHmiddot and water for ABTSmiddot+) and thehighest (buffer (pH 74) for both) AOP after 60min eresults shown in Figure 2 reveal that the solvent and type ofthe assay have large influence on the amplitude and thekinetics of the reaction Additionally it is clearly shown thatsecondary reactions of partially oxidized antioxidants con-tribute significantly to the number of exchanged electronsalready at the minute time scale Accordingly it is notpossible to discriminate the contribution of primary oxi-dation of polyphenols to quinones from the contribution ofsecondary antioxidants to the AOP without stopped-flowmachine

For GA secondary reactions are quantitatively relevantunder all conditions analyzed already at the subminuterange Caffeic acid and its ester (CGA) have similar yetkinetically different profiles On the contrary to GA con-tribution of secondary reactions in DPPH assay at pH 74 is

0

2

4

6

8

10

12

14

16

18

CA CGA GA CTH EGCG AA DHA Trolox

Elec

tron

sm

olec

ule

DPPH MeOHDPPH H2ODPPH pH 5

DPPH pH 74ABTS H2OABTS pH 5

ABTS pH 74FC

Figure 1 e number of exchanged electrons per molecule of caffeic acid (CA) chlorogenic acid (CGA) gallic acid (GA) catechin (CTH)epigallocatechin gallate (EGCG) ascorbic acid (AA) dehydroascorbic acid (DHA) and Trolox in DPPH ABTS and FC assays Numbers ofexchanged electrons were determined after 60min incubation of antioxidant with the probe at 25degC in the appropriate solvent

Table 2 Number of exchanged electrons (n) per OH group indifferent antioxidant assays

SamplenOH

DPPH ABTSFC

MeOH H2O pH 5 pH 74 H2O pH 5 pH 74CA 11 11 11 31 12 11 12 29CGA 10 11 12 31 10 11 15 38GA 18 21 21 17 21 27 35 19CTH 08 10 14 21 14 17 20 23EGCG 12 13 14 20 11 14 19 15AA 09 09 09 10 09 09 09 21Trolox 18 19 19 19 18 18 17 22


themost pronounced For ABTS assay of CA at physiologicalpH the rate of reaction at prolonged incubation time isincreased which cannot be explained only by the reaction ofan antioxidant with the ABTSmiddot+ It is possible that H2O2 thatis formed in the course of CA oxidation [33] reoxidizesABTS into ABTSmiddot+ and therefore results in overall smallerdΔAdt at shorter incubation times until it is used upTrolox quinone (Equation (12)) or its degradation productsdo not react further with ABTSmiddot+ or DPPHmiddot as approximatelytwo electrons are exchanged per molecule of Trolox over180min

We have observed the sample specic kinetic prolesalso for dierent food matrices [22] e dΔAdt on the timescale of few tens of minutes when samples as tea coeecranberry and apple juice were analyzed was even largerthan observed for antioxidant compounds in this study Asroutine AOP measurements are often performed withoutstrict temperature and time control more reproducibleresults can be obtained if longer incubation times (60min)are applied when dΔAdt is smaller Still we have to be awarethat AOP values are not the measure of the antioxidantproperties of the molecules (in the kinetic term) but ratherreect the capacity of molecules to exchange certain amountof electrons in the reaction with oxidants under chosenconditions

34 Correlation Analysis of AOP of Model AntioxidantsDetermined by DPPH ABTS and FC Assays e results ofcorrelation analysis between the n values for the eightmodel compounds obtained by dierent assays are shown inTable 3 All correlation coecients are signicant at α 005e highest correlations are observed between dierentvariants within DPPH or ABTS assays e notable ex-ception is DPPH assay at pH 74 with weaker correlationsWhen variants of DPPH and ABTS assays are compared toeach other the Pearson correlation coecients are lowerthan within each type of assay but are still signicant at α 0001 When the results of FC assay are correlated to AOPobtained by DPPH and ABTS assays weaker correlationsmost of them signicant at α 005 are observed eexception is again DPPH assay at pH 74 which shows thehighest correlation with FC assay (α 0001)

In general the correlation between determined AOP ofinvestigated antioxidants (Table 4) which are structurallydierent compounds such as hydroxycinnamic acids tri-hydroxybenzoic acid avanols and their derivativesvitamers of vitamin C and synthetic chromanol is muchpoorer than correlation between the types of assays For GAeven negative correlation coecients are observed withpractically all other antioxidants A weak positive correlationbetween GA and its derivative EGCG could be attributed to

0

2

4

6

8

10

12

0 30 60 90 120 150 180

Elec

tron

sm

olec

ule

Time (min)

(a)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(b)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(c)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(d)

Figure 2 e number of exchanged electrons per molecule as a function of incubation time at 25degC for caeic acid (CA) chlorogenic acid(CGA) gallic acid (GA) and Trolox in DPPH assay inmethanol (thin purple) or DPPH assay in the mixture of methanol and buer (pH 74)(thick purple) and ABTS assays in MilliQ water (thin green) or buer (pH 74) (thick green) Black lines correspond to the number of OHgroups in the molecule (a) Caeic acid (CA) (b) chlorogenic acid (CGA) (c) gallic acid (GA) (d) trolox


the similar chemical structure In the same way statisticallysignificant correlation of EGCG is found with CTH that isanother structural element of EGCG e important role ofcompoundrsquos structure on correlation strength is particularlyconfirmed in the case of AA and its oxidized form DHASimilar considerations can be applied to the correlationbetween the CA and its ester CGA both significant at α

0001 Lower r values obtained for correlation among AOPsof investigated compounds (Table 4) than for correlationamong AOPs determined in different tests and differentsolvents (Table 3) indicate that despite similar influence ofsolvents the AOP still greatly depends on the structure of thecompounds

4 Conclusions

Regardless of persistent critiques of the in vitro antioxidantassays and lack of correlations that would exist with in vivoantioxidant properties this research area is lively as ever Inthe year 2017 017 of all manuscripts published in the SCIjournal (based on Web of Science database) containedDPPH ABTS or Folin in the abstracts However in the largeproportion of those manuscripts the methodology is poorlydescribed and comparison with work of others is practicallyimpossible Due to inconsistency of published results theadequacy of in vitro antioxidant assays is becoming ques-tionable [34]

e measured AOP should in principle give the esti-mation of the amount of the compounds that can be oxi-dized under conditions of the assays e number ofexchanged electrons in the reactions with chromogenicradicals is dependent on solvent composition pH of reaction

media length of assay and chemical structure of the anti-oxidant Secondary reactions of partially oxidized antioxi-dants contribute significantly to the number of exchangedelectrons We have found that the only exception is Troloxcompound with uniform number of electrons exchangedin applied assays which is therefore the most suitablecompound as standard for AOP determination ofsingle compounds or their mixtures in ABTS DPPH andFolinndashCiocalteu assays For practically all antioxidants withthe exception of Trolox the number of exchanged electronsunder the most favorable conditions typically the FC assayis more than two-fold higher than under the least favorableconditions in the majority of cases the DPPH assay inMeOH

Data Availability

e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

is work was supported by the Slovenian Research Agency(contract no P4-0121) e authors would like to thankRoger H Pain for his valuable suggestions and discussion ofthe paper

Supplementary Materials

Mean absolute errors and mean absolute percentage errorsin the number of exchanged electrons of model antioxidantsin different antioxidant assays (Supplementary Materials)

References

[1] R G Berger S Lunkenbein A Strohle and A HahnldquoAntioxidants in food mere myth or magic medicinerdquoCritical Reviews in Food Science and Nutrition vol 52 no 2pp 162ndash171 2012

[2] M C Foti ldquoUse and abuse of the DPPH radicalrdquo Journal ofAgricultural and Food Chemistry vol 63 no 40 pp 8765ndash8776 2015

[3] R Apak M Mustafa Ozyurek K Kubilay Guccedillu and E EsraCcedilapanoglu ldquoAntioxidant activitycapacity measurement 1

Table 4 e values of Pearson correlation coefficient for corre-lation among AOPs of investigated antioxidants determined in allvariants of ABTS DPPH and FC assays

Sample CA CGA GA CTH EGCG AA DHACGA 096lowastlowastlowastGA minus043 minus032CTH 072lowast 077lowast 026EGCG 056 055 035 074lowastAA 067 081lowast minus031 055 007DHA 079lowast 090lowastlowast minus037 061 018 098lowastlowastlowastTrolox 076lowast 083lowast minus051 052 015 090lowastlowast 091lowastlowastlowastlowastlowastValues are significant at the α 0001 level lowastlowastvalues are significant at theα 001 lowastvalues are significant at the α 005 level

Table 3 Values of the Pearson correlation coefficient for correlation between AOPs determined in different antioxidant assays

DPPHmiddot ABTS_+

MeOH H2O pH 5 pH 74 H2O pH 5 pH 74

DPPH_

H2O 0997lowastlowastlowastpH 5 0982lowastlowastlowast 0993lowastlowastlowastpH 74 0877lowastlowast 0887lowastlowast 0909lowastlowastlowast

ABTS_+H2O 0950lowastlowastlowast 0968lowastlowastlowast 0985lowastlowastlowast 0872lowastlowastpH 5 0954lowastlowastlowast 0972lowastlowastlowast 0985lowastlowastlowast 0853lowastlowast 0997lowastlowastlowastpH 74 0964lowastlowastlowast 0979lowastlowastlowast 0987lowastlowastlowast 0858lowast 0990lowastlowastlowast 0997lowastlowastlowast

FC 0796lowast 0816lowast 0856lowastlowast 0958lowastlowastlowast 0847lowastlowast 0821lowast 0820lowastlowastlowastlowastValues are significant at the α 0001 level lowastlowastvalues are significant at the α 001 lowastvalues are significant at the α 005 level


Classification physicochemical principles mechanisms andelectron transfer (ET)-based assaysrdquo Journal of Agriculturaland Food Chemistry vol 64 no 5 pp 997ndash1027 2016

[4] D Huang B Ou and R L Prior ldquoe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005

[5] M Musialik and G Litwinienko ldquoScavenging of DPPHmiddot

radicals by vitamin E is accelerated by its partial ionizationthe role of sequential proton loss electron transferrdquo OrganicLetters vol 7 no 22 pp 4951ndash4954 2005

[6] L Bertalanic T Kosmerl N Poklar Ulrih and B CigicldquoInfluence of solvent composition on antioxidant potential ofmodel polyphenols and red wines determined with 22-diphenyl-1-picrylhydrazylrdquo Journal of Agricultural and FoodChemistry vol 60 no 50 pp 12282ndash12288 2012

[7] A L Dawidowicz D Wianowska and M Olszowy ldquoOnpractical problems in estimation of antioxidant activity ofcompounds by DPPHbull method (Problems in estimation ofantioxidant activity)rdquo Food Chemistry vol 131 no 3pp 1037ndash1043 2012

[8] T Prevc N Segatin N Poklar Ulrih and B Cigic ldquoDPPHassay of vegetable oils and model antioxidants in protic andaprotic solventsrdquo Talanta vol 109 pp 13ndash19 2013

[9] J Xie and K M Schaich ldquoRe-evaluation of the 22-diphenyl-1-picrylhydrazyl free radical (DPPH) assay for antioxidantactivityrdquo Journal of Agricultural and Food Chemistry vol 62no 19 pp 4251ndash4260 2014

[10] H Abramovic T Kosmerl N Poklar Ulrih and B CigicldquoContribution of SO2 to antioxidant potential of white winerdquoFood Chemistry vol 174 pp 147ndash153 2015

[11] P Goupy C Dufour M Loonis and O Dangles ldquoQuan-titative kinetic analysis of hydrogen transfer reactions fromdietary polyphenols to the DPPH radicalrdquo Journal ofAgricultural and Food Chemistry vol 51 no 3 pp 615ndash6222003

[12] M J Arts G R Haenen H P Voss and A Bast ldquoAntioxidantcapacity of reaction products limits the applicability of thetrolox equivalent antioxidant capacity (TEAC) assayrdquo Foodand Chemistry Toxicology vol 42 no 1 pp 45ndash49 2004

[13] L M Magalhaes M A Segundo S Reis and J L F C LimaldquoMethodological aspects about in vitro evaluation of anti-oxidant propertiesrdquo Analytica Chimica Acta vol 613 no 1pp 1ndash19 2008

[14] Kaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysisvol 19 no 6-7 pp 669ndash675 2006

[15] A K C Saw W S Yam K C Wong and C S Lai ldquoAcomparative study of the volatile constituents of southeastasian Coffea arabica Coffea liberica and Coffea robusta greenbeans and their antioxidant activitiesrdquo Journal of Essential OilBearing Plants vol 18 no 1 pp 64ndash73 2015

[16] J A Vinson M Mandarano M Hirst J R Trevithick andP Bose ldquoPhenol antioxidant quantity and quality in foodsbeers and the effect of two types of beer on an animal model ofatherosclerosisrdquo Journal of Agricultural and Food Chemistryvol 51 no 18 pp 5528ndash5533 2003

[17] C Delgado-Andrade J A Rufian-Henares and F J MoralesldquoAssessing the antioxidant activity of melanoidins from coffeebrews by different antioxidant methodsrdquo Journal of Agri-cultural and Food Chemistry vol 53 no 20 pp 7832ndash78362005

[18] G Spigno L Tramelli and D M De Faveri ldquoEffects of ex-traction time temperature and solvent on concentration andantioxidant activity of grape marc phenolicsrdquo Journal of FoodEngineering vol 81 no 1 pp 200ndash208 2007

[19] F Shahdadi H O Mirzaei and A Daraei GarmakhanyldquoStudy of phenolic compound and antioxidant activity of datefruit as a function of ripening stages and drying processrdquoJournal of Food Science and Technology vol 52 no 3pp 1814ndash1819 2015

[20] L Fu B T Xu X R Xu et al ldquoAntioxidant capacities andtotal phenolic contents of 62 fruitsrdquo Food Chemistry vol 129no 2 pp 345ndash350 2011

[21] A Floegel D O Kim S J Chung S I Koo and O K ChunldquoComparison of ABTSDPPH assays to measure antioxidantcapacity in popular antioxidant-rich US foodsrdquo Journal ofFood Composition and Analysis vol 24 no 7 pp 1043ndash10482011

[22] H Abramovic B Grobin N Poklar Ulrih and B Cigic ldquoemethodology applied in DPPH ABTS and Folin-Ciocalteauassays has a large influence on the determined antioxidantpotentialrdquo Acta Chimica Slovenica vol 64 no 2 pp 491ndash4992017

[23] T Gutfinger ldquoPolyphenols in olive oilsrdquo Journal of theAmerican Oil Chemists Society vol 58 no 11 pp 966ndash9681981

[24] W Brand-Williams M E Cuvelier and C Berset ldquoUse of freeradical method to evaluate antioxidant activityrdquo LWT-FoodScience and Technology vol 28 no 1 pp 25ndash30 1995

[25] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9ndash10 pp 1231ndash1237 1999

[26] P Molyneux ldquoe use of the stable free radical diphenylpi-crylhydrazyl (DPPH) for estimating antioxidant activityrdquoSongklanakarin Journal of Science and Technology vol 26no 2 pp 211ndash219 2004

[27] O Friaa and D Brault ldquoKinetics of the reaction between theantioxidant Troloxreg and the free radical DPPHmiddot in semi-aqueous solutionrdquo Organic and Biomolecular Chemistryvol 4 no 12 pp 2417ndash2423 2006

[28] P R Mussini T Mussini and S Rondinini ldquoReference valuestandards and primary standards for pH measurements inD2O and aqueous organic solvent mixtures new accessionsand assessments (Technical Report)rdquo Pure and AppliedChemistry vol 69 no 5 pp 1007ndash1014 1997

[29] J D Everette Q M Bryant A M Green Y A AbbeyG W Wangila and R B Walker ldquoorough study of re-activity of various compound classes toward theFolinminusCiocalteu reagentrdquo Journal of Agricultural and FoodChemistry vol 58 no 14 pp 8139ndash8144 2010

[30] J P Yuan and F Chen ldquoDegradation of ascorbic acid inaqueous solutionrdquo Journal of Agricultural and Food Chem-istry vol 46 no 12 pp 5078ndash5082 1998

[31] I Zabetakis J W Gramshaw and D S Robinson ldquo25-Dimethyl-4-hydroxy-2H-furan-3-one and its derivativesanalysis synthesis and biosynthesis ndash a reviewrdquo FoodChemistry vol 65 no 2 pp 139ndash151 1999

[32] E N Delicado A S Ferrer and F G Carmona ldquoA kineticstudy of the one-electron oxidation of Trolox C by thehydroperoxidase activity of lipoxygenaserdquo Biochimica etBiophysica Acta vol 1335 no 1-2 pp 127ndash134 1997

[33] A F Hanham B P Dunn and H F Stich ldquoClastogenicactivity of caffeic acid and its relationship to hydrogen


peroxide generated during autooxidationrdquo Mutation Re-search vol 116 no 3-4 pp 333ndash339 1983

[34] D Granato F Shahidi R Wrolstad et al ldquoAntioxidant ac-tivity total phenolics and flavonoids contents should we banin vitro screening methodsrdquo Food Chemistry vol 264pp 471ndash475 2018


TribologyAdvances in

Hindawiwwwhindawicom Volume 2018


International Journal ofInternational Journal ofPhotoenergy


Journal of

Chemistry


Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of


NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of



Biochemistry Research International


Enzyme Research


Journal of

SpectroscopyAnalytical ChemistryInternational Journal of


MaterialsJournal of



BioMed Research International Electrochemistry

International Journal of


Na

nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom

(CGA) [17] caffeic acid (CA) [18] gallic acid (GA) [19] andother antioxidants is not uncommon

AOP of food samples is usually evaluated by more thanone method and correlation analysis of AOPs obtained byDPPH ABTS and FC assays is often performed e cor-relations can be high and significant or weak [20ndash22]reflecting the lack of consistency of the results of suchanalyses e large influence of the experimental parameterson the reactivity of antioxidants in the samples undoubtedlycontributes to the observed discrepancies ese are po-tentiated when AOPs of samples with different compositionand reactivity of antioxidants in particular assays arecompared e fact that AOP of the samples determined byDPPH ABTS and FC assays is rarely normalized to thesame model antioxidant contributes additionally to theambiguity in this area of research It is almost impossible tocompare the AOP of samples determined by differentmethods on a quantitative basis To enable a relevantcomparison between the results of studies carried out withdifferent methods under various experimental conditionsthe uniform reactivity of the compound used as standard isof great importance

We report here how the method applied for AOP es-timation length of the assay and the composition of thesolvent affects the reactivity of selected antioxidants fre-quently used as standard compounds e aim of the studywas to find the standard compound with reactivity that is theleast affected by experimental conditions and could betherefore applied as a general standard for DPPH ABTSand FC assays

2 Materials and Methods

21 Materials Trolox caffeic acid gallic acid chlorogenicacid L-ascorbic acid dehydroascorbic acid (DHA) catechinhydrate iron(II) sulphate heptahydrate (FeSO4 times 7H2O)FolinndashCiocalteu reagent DPPHmiddot and ABTS were fromSigma-Aldrich (Steinheim Germany) Acetic acid sodiumhydroxide sodium carbonate (Na2CO3) sodium hydrogenphosphate dihydrate and methanol were from Merck(Darmstadt Germany) Manganese(IV) oxide (MnO2) wasfrom Kemika (Zagreb Croatia) Epigallocatechin gallate(EGCG) was from DSM Food Specialities BV (DelftNetherlands) Water was purified using a MilliQ system(resistivity gt18MΩmiddotcm Millipore)

Stock solutions (10mM) of Trolox CTH CA CGA GAand EGCG were prepared in MeOH AA and FeSO4 inMilliQ water and DHA in acetate buffer (50mM pH 50) Allfurther dilutions were made in the solvents used for par-ticular assay

22 e FolinndashCiocalteau Assay e FC assay was per-formed according to a modified method of Gutfinger [23]An appropriate volume of the antioxidant or FeSO4 solution(50 μL) was added into a 15mLmicrocentrifuge tube mixedwith MilliQ water (700 μL) and FC reagent (125 μL pre-viously diluted 1 2 (vv) with MilliQ water) After 5min at25degC a solution of Na2CO3 (125 μL 20 ww) was addedand the sample was mixed again and incubated for an

additional 55min e absorbance at 765 nm (A765) wasmeasured with a Varian Cary 100 BIO UV-VIS spectro-photometer in a 1 cm cell e concentration range of an-tioxidants and FeSO4 in the assay solution is given in Table 1e measurements were made in triplicate including thepreparation of sample solutions and reagents

23 e DPPH and ABTS Assays e DPPH assay wasperformed according to a modified method of Brand-Williams et al [24] and the ABTS assay according toa modified method of Re et al [25] e DPPHmiddot solution wasprepared in MeOH and diluted to the concentration thatwould give an absorbance of 24 at 520 nm in a cuvette with1 cm path length ABTSmiddot+ was produced by reaction of ABTSin aqueous solution with MnO2 followed by centrifugationand filtration the solution was then diluted with MilliQwater to the concentration that would give an absorbance of24 in the cuvette with 1 cm path length at 734 nm All thesolutions buffers and solvents were incubated at 25degC priorto analysis e assay solutions were prepared in a 15mLmicrocentrifuge tube by mixing DPPHmiddot or ABTSmiddot+ solution(500 μL) with 450 μL of the solvent (MilliQ water MeOHand acetate buffer (250mM pH 50) or phosphate buffer(50mM pH 74) for the DPPH assay and MilliQ water andacetate buffer (250mM pH 50) or phosphate buffer(50mM pH 74) for the ABTS assay) e reactions werestarted by adding 50 μL of the antioxidant solution into theassay medium with thorough mixing After 60min in-cubation at 25degC the absorbance at 520 nm for DPPHmiddot (A520)and at 734 nm for ABTSmiddot+ (A734) was measured e mea-sured value was subtracted from the corresponding value forthe control (the selected solvent added into the assay me-dium instead of the antioxidant solution) and the dataexpressed as ΔA520 or ΔA734 e concentration range ofantioxidants in assay solution is given in Table 1 Allmeasurements were carried out in triplicate including thepreparation of sample solutions and reagents

e reaction kinetics of model antioxidants CGA (10 μM)CA (10 μM) Trolox (10 μM) and GA (70 μM) was analyzedin 1 cm quartz cuvettes with the stopper to prevent evapo-ration e solvent compositions were the same as those forendpoint measurements explained above e A520 and A734were continuously measured at 15 s intervals over 180mine first time point was measured 15 s after mixing the an-tioxidant with radical probe e measured absorbances weresubtracted from the corresponding absorbances of the con-trols at appropriate time points and obtainedΔA520 andΔA734values were normalized to the number of exchanged electronsper molecule as explained in Section 3124 Statistical Analysis e slope of the calibration curveobtained by linear regression analysis with program Origin(Microsoft) prepared with each model antioxidant ina particular type of assay and used to calculate the number ofexchanged electrons was the average value obtained fromthree independent experiments including the preparation ofsample solutions and reagents Relative standard deviationof the average slope was not larger than 5

Pearson correlation coefficients (r) were calculated withthe program Excel (Microsoft)



MAPE 100

j1113944

j

a1

nXa minus nYa

nYa

11138681113868111386811138681113868111386811138681113868

11138681113868111386811138681113868111386811138681113868 (1)

MAE 1j

1113944

j

a1nXa minus nYa

11138681113868111386811138681113868111386811138681113868 (2)







k

ελ times l (5)








n ΔcFe2+

Δcantioxidant

kantioxidant

kFe2+

(9)




DPPH ABTS+FC





OHOH O

O

+ 2H+ + 2endash (10)

OHO

OH

O

HO OH

OHO

OH

O

O O

+ 2H+ + 2endash

(11)





O

HO

COOH

O

O

OH


(12)








0

2

4

6

8

10

12

14

16

18


Elec

tron

sm

olec

ule



ABTS pH 74FC



SamplenOH

DPPH ABTSFC







0

2

4

6

8

10

12

0 30 60 90 120 150 180

Elec

tron

sm

olec

ule

Time (min)

(a)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(b)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(c)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(d)





4 Conclusions




Data Availability




Acknowledgments




References







DPPHmiddot ABTS_+


DPPH_














































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





MAPE 100

j1113944

j

a1

nXa minus nYa

nYa

11138681113868111386811138681113868111386811138681113868

11138681113868111386811138681113868111386811138681113868 (1)

MAE 1j

1113944

j

a1nXa minus nYa

11138681113868111386811138681113868111386811138681113868 (2)







k

ελ times l (5)








n ΔcFe2+

Δcantioxidant

kantioxidant

kFe2+

(9)




DPPH ABTS+FC





OHOH O

O

+ 2H+ + 2endash (10)

OHO

OH

O

HO OH

OHO

OH

O

O O

+ 2H+ + 2endash

(11)





O

HO

COOH

O

O

OH


(12)








0

2

4

6

8

10

12

14

16

18


Elec

tron

sm

olec

ule



ABTS pH 74FC



SamplenOH

DPPH ABTSFC







0

2

4

6

8

10

12

0 30 60 90 120 150 180

Elec

tron

sm

olec

ule

Time (min)

(a)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(b)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(c)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(d)





4 Conclusions




Data Availability




Acknowledgments




References







DPPHmiddot ABTS_+


DPPH_














































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






OHOH O

O

+ 2H+ + 2endash (10)

OHO

OH

O

HO OH

OHO

OH

O

O O

+ 2H+ + 2endash

(11)





O

HO

COOH

O

O

OH


(12)








0

2

4

6

8

10

12

14

16

18


Elec

tron

sm

olec

ule



ABTS pH 74FC



SamplenOH

DPPH ABTSFC







0

2

4

6

8

10

12

0 30 60 90 120 150 180

Elec

tron

sm

olec

ule

Time (min)

(a)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(b)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(c)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(d)





4 Conclusions




Data Availability




Acknowledgments




References







DPPHmiddot ABTS_+


DPPH_














































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls










0

2

4

6

8

10

12

14

16

18


Elec

tron

sm

olec

ule



ABTS pH 74FC



SamplenOH

DPPH ABTSFC







0

2

4

6

8

10

12

0 30 60 90 120 150 180

Elec

tron

sm

olec

ule

Time (min)

(a)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(b)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(c)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(d)





4 Conclusions




Data Availability




Acknowledgments




References







DPPHmiddot ABTS_+


DPPH_














































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








0

2

4

6

8

10

12

0 30 60 90 120 150 180

Elec

tron

sm

olec

ule

Time (min)

(a)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(b)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(c)

Elec

tron

sm

olec

ule

0

2

4

6

8

10

12

0 30 60 90 120 150 180Time (min)

(d)





4 Conclusions




Data Availability




Acknowledgments




References







DPPHmiddot ABTS_+


DPPH_














































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






4 Conclusions




Data Availability




Acknowledgments




References







DPPHmiddot ABTS_+


DPPH_














































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls













































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




RelevanceandStandardizationof InVitro AntioxidantAssays: ABTS… · 2019. 7. 30. · DPPH, ABTS,...

Documents

Transcript of RelevanceandStandardizationof InVitro AntioxidantAssays: ABTS… · 2019. 7. 30. · DPPH, ABTS,...