MethodologicalMethodologicalMethodologicalMethodological aspectsaspectsaspectsaspects aboutaboutaboutaboutin in in in vitrovitrovitrovitro evaluationevaluationevaluationevaluation of of of of antioxidant capacityantioxidant capacityantioxidant capacityantioxidant capacity

Training SchoolTraining SchoolTraining SchoolTraining School““““Building Skill on the Determination of the Overall Antioxidant CBuilding Skill on the Determination of the Overall Antioxidant CBuilding Skill on the Determination of the Overall Antioxidant CBuilding Skill on the Determination of the Overall Antioxidant Capacity of Thermally apacity of Thermally apacity of Thermally apacity of Thermally

Treated FoodsTreated FoodsTreated FoodsTreated Foods””””Olsztyn, 26Olsztyn, 26Olsztyn, 26Olsztyn, 26----30 January 200930 January 200930 January 200930 January 2009

The most The most The most The most commonlycommonlycommonlycommonly used methods for in used methods for in used methods for in used methods for in vitrovitrovitrovitrodetermination of antioxidant capacitydetermination of antioxidant capacitydetermination of antioxidant capacitydetermination of antioxidant capacity

•PeroxylPeroxylPeroxylPeroxyl radical scavenging capacity assaysradical scavenging capacity assaysradical scavenging capacity assaysradical scavenging capacity assays

•SuperoxideSuperoxideSuperoxideSuperoxide radical anion scavenging capacity assaysradical anion scavenging capacity assaysradical anion scavenging capacity assaysradical anion scavenging capacity assays

•HydrogenHydrogenHydrogenHydrogen peroxideperoxideperoxideperoxide scavenging capacity assaysscavenging capacity assaysscavenging capacity assaysscavenging capacity assays

•HydroxylHydroxylHydroxylHydroxyl radical scavenging capacity assayradical scavenging capacity assayradical scavenging capacity assayradical scavenging capacity assay

•HypochlorousHypochlorousHypochlorousHypochlorous acid scavenging capacity assaysacid scavenging capacity assaysacid scavenging capacity assaysacid scavenging capacity assays

•SingletSingletSingletSinglet oxygenoxygenoxygenoxygen scavenging capacity assaysscavenging capacity assaysscavenging capacity assaysscavenging capacity assays

•NitricNitricNitricNitric oxideoxideoxideoxide radical scavenging capacity assaysradical scavenging capacity assaysradical scavenging capacity assaysradical scavenging capacity assays

•PeroxynitritePeroxynitritePeroxynitritePeroxynitrite scavenging scavenging scavenging scavenging cpacitycpacitycpacitycpacity assaysassaysassaysassays

•Scavenging of Scavenging of Scavenging of Scavenging of 2,22,22,22,2’’’’----azinobisazinobisazinobisazinobis----(3(3(3(3----ethylbenzothiazolineethylbenzothiazolineethylbenzothiazolineethylbenzothiazoline----6666----sulfonate) radical cation sulfonate) radical cation sulfonate) radical cation sulfonate) radical cation (ABTS(ABTS(ABTS(ABTS••••+) +) +) +) or Trolox equivalent antioxidant capacity (TEAC) assayor Trolox equivalent antioxidant capacity (TEAC) assayor Trolox equivalent antioxidant capacity (TEAC) assayor Trolox equivalent antioxidant capacity (TEAC) assay

•Scavenging of Scavenging of Scavenging of Scavenging of 2,22,22,22,2----diphenyldiphenyldiphenyldiphenyl----1111----picrylhydrazyl radical (DPPHpicrylhydrazyl radical (DPPHpicrylhydrazyl radical (DPPHpicrylhydrazyl radical (DPPH•••• assay)assay)assay)assay)

•FolinFolinFolinFolin----CiocalteuCiocalteuCiocalteuCiocalteu reducingreducingreducingreducing capacity (FC assay)capacity (FC assay)capacity (FC assay)capacity (FC assay)

•Total Total Total Total reducingreducingreducingreducing capacity estimated by electrochemical methodscapacity estimated by electrochemical methodscapacity estimated by electrochemical methodscapacity estimated by electrochemical methods

Scavenging capacity assays Scavenging capacity assays Scavenging capacity assays Scavenging capacity assays againstagainstagainstagainst specific ROS/RNSspecific ROS/RNSspecific ROS/RNSspecific ROS/RNS

Scavenging capacity assays Scavenging capacity assays Scavenging capacity assays Scavenging capacity assays againstagainstagainstagainst stable, stable, stable, stable, nonnonnonnon----biologicalbiologicalbiologicalbiological radicalsradicalsradicalsradicals

Total Total Total Total reductionreductionreductionreduction capacity assayscapacity assayscapacity assayscapacity assays

The proposed The proposed The proposed The proposed methoddmethoddmethoddmethodd includeincludeincludeinclude the the the the followingfollowingfollowingfollowing requirementsrequirementsrequirementsrequirements/criteria:/criteria:/criteria:/criteria:

(I)(I)(I)(I) measuremetmeasuremetmeasuremetmeasuremet of the of the of the of the chemistrychemistrychemistrychemistry actuallyactuallyactuallyactually occuringoccuringoccuringoccuring in potential in potential in potential in potential applicationsapplicationsapplicationsapplications;;;;(II)(II)(II)(II) utilizationutilizationutilizationutilization of of of of biologicalbiologicalbiologicalbiological revelantrevelantrevelantrevelant moleculesmoleculesmoleculesmolecules;;;;(III)(III)(III)(III) technicallytechnicallytechnicallytechnically simple;simple;simple;simple;(IV)(IV)(IV)(IV) with a with a with a with a defineddefineddefineddefined endpoint and chemical endpoint and chemical endpoint and chemical endpoint and chemical mechanismmechanismmechanismmechanism;;;;(V)(V)(V)(V) readilyreadilyreadilyreadily availableavailableavailableavailable instrumentationinstrumentationinstrumentationinstrumentation;;;;(VI)(VI)(VI)(VI) goodgoodgoodgood repeatabilityrepeatabilityrepeatabilityrepeatability and and and and reproducibilityreproducibilityreproducibilityreproducibility;;;;(VII)(VII)(VII)(VII) adaptableadaptableadaptableadaptable for assay of both hydrophilic and lipophilic antioxidants;for assay of both hydrophilic and lipophilic antioxidants;for assay of both hydrophilic and lipophilic antioxidants;for assay of both hydrophilic and lipophilic antioxidants;(VIII)(VIII)(VIII)(VIII) adaptableadaptableadaptableadaptable to to to to highhighhighhigh----throughputthroughputthroughputthroughput analysisanalysisanalysisanalysis

These criteria are in accordance to the guidelines proposed by These criteria are in accordance to the guidelines proposed by These criteria are in accordance to the guidelines proposed by These criteria are in accordance to the guidelines proposed by Prior et al. (2005) for Prior et al. (2005) for Prior et al. (2005) for Prior et al. (2005) for standardization of the determination of antioxidant capacity:standardization of the determination of antioxidant capacity:standardization of the determination of antioxidant capacity:standardization of the determination of antioxidant capacity:Prior, R.L.; Wu, X.; Prior, R.L.; Wu, X.; Prior, R.L.; Wu, X.; Prior, R.L.; Wu, X.; SchaichSchaichSchaichSchaich, K. Standardized methods for the determination of , K. Standardized methods for the determination of , K. Standardized methods for the determination of , K. Standardized methods for the determination of antioxidant capacity and antioxidant capacity and antioxidant capacity and antioxidant capacity and phenolicsphenolicsphenolicsphenolics in foods, and dietary supplements. in foods, and dietary supplements. in foods, and dietary supplements. in foods, and dietary supplements. J. Agric. Food J. Agric. Food J. Agric. Food J. Agric. Food Chem.Chem.Chem.Chem. 2005, 53, 42902005, 53, 42902005, 53, 42902005, 53, 4290----4302.4302.4302.4302.

„From From From From evaluationevaluationevaluationevaluation of data of data of data of data presentedpresentedpresentedpresented at the at the at the at the FirstFirstFirstFirst InternationalInternationalInternationalInternational CongressCongressCongressCongress on on on on Antioxidant Methods in 2004 and in the literature, as Antioxidant Methods in 2004 and in the literature, as Antioxidant Methods in 2004 and in the literature, as Antioxidant Methods in 2004 and in the literature, as wellwellwellwell as as as as considerationconsiderationconsiderationconsideration end end end end usesusesusesuses of antioxidants, it is proposed that of antioxidants, it is proposed that of antioxidants, it is proposed that of antioxidants, it is proposed that proceduresproceduresproceduresprocedures and and and and applicationsapplicationsapplicationsapplications for three for three for three for three assays be assays be assays be assays be consideredconsideredconsideredconsidered for standardization: the for standardization: the for standardization: the for standardization: the oxygenoxygenoxygenoxygen radical radical radical radical absorbanceabsorbanceabsorbanceabsorbance capacity capacity capacity capacity (ORAC) assay, the (ORAC) assay, the (ORAC) assay, the (ORAC) assay, the FolinFolinFolinFolin----CiocalteuCiocalteuCiocalteuCiocalteu method, and method, and method, and method, and possiblypossiblypossiblypossibly the Trolox equivalent the Trolox equivalent the Trolox equivalent the Trolox equivalent antioxidant capacity (TEAC) assayantioxidant capacity (TEAC) assayantioxidant capacity (TEAC) assayantioxidant capacity (TEAC) assay””””....

The practical guidelines for the deterThe practical guidelines for the deterThe practical guidelines for the deterThe practical guidelines for the determination of antioxidant capaciy ination of antioxidant capaciy ination of antioxidant capaciy ination of antioxidant capaciy

Determination of antioxidant capacity of Determination of antioxidant capacity of Determination of antioxidant capacity of Determination of antioxidant capacity of foodsfoodsfoodsfoods includesincludesincludesincludes three steps: sample preparation and three steps: sample preparation and three steps: sample preparation and three steps: sample preparation and extraction of antioxidants, measurement of antioxidant capacity,extraction of antioxidants, measurement of antioxidant capacity,extraction of antioxidants, measurement of antioxidant capacity,extraction of antioxidants, measurement of antioxidant capacity, and expression results.and expression results.and expression results.and expression results.

DuringDuringDuringDuring sample preparation, the loss of antioxidants in the drying and sample preparation, the loss of antioxidants in the drying and sample preparation, the loss of antioxidants in the drying and sample preparation, the loss of antioxidants in the drying and milling steps must be milling steps must be milling steps must be milling steps must be kept to a minimum.kept to a minimum.kept to a minimum.kept to a minimum.

In the extraction of antioxidants, at least two extraction cycleIn the extraction of antioxidants, at least two extraction cycleIn the extraction of antioxidants, at least two extraction cycleIn the extraction of antioxidants, at least two extraction cycles with mixtures of different s with mixtures of different s with mixtures of different s with mixtures of different polarity of water and organic solvents must be combined.polarity of water and organic solvents must be combined.polarity of water and organic solvents must be combined.polarity of water and organic solvents must be combined.

Determination of total antioxidant capacity must be perfoDetermination of total antioxidant capacity must be perfoDetermination of total antioxidant capacity must be perfoDetermination of total antioxidant capacity must be performed both in aqueousmed both in aqueousmed both in aqueousmed both in aqueous----organic organic organic organic extracts and in their corresponing residues, which may exhibit hextracts and in their corresponing residues, which may exhibit hextracts and in their corresponing residues, which may exhibit hextracts and in their corresponing residues, which may exhibit higher antioxidant capacity igher antioxidant capacity igher antioxidant capacity igher antioxidant capacity than the aquoeousthan the aquoeousthan the aquoeousthan the aquoeous----organic extracts, a fact usually ignored in te literature.organic extracts, a fact usually ignored in te literature.organic extracts, a fact usually ignored in te literature.organic extracts, a fact usually ignored in te literature.

Antioxidant capacity values should only be compared where the meAntioxidant capacity values should only be compared where the meAntioxidant capacity values should only be compared where the meAntioxidant capacity values should only be compared where the method, the solvent and the thod, the solvent and the thod, the solvent and the thod, the solvent and the analytical conditions are the same.analytical conditions are the same.analytical conditions are the same.analytical conditions are the same.

Possible interference from certain food constituents must be taPossible interference from certain food constituents must be taPossible interference from certain food constituents must be taPossible interference from certain food constituents must be taken into account when ken into account when ken into account when ken into account when determining antioxidant capacity.determining antioxidant capacity.determining antioxidant capacity.determining antioxidant capacity.

At least two assays should be performed tAt least two assays should be performed tAt least two assays should be performed tAt least two assays should be performed to determine antioxidant capacity.determine antioxidant capacity.determine antioxidant capacity.determine antioxidant capacity.

J. J. J. J. PerezPerezPerezPerez----JimenezJimenezJimenezJimenez et al.. Food et al.. Food et al.. Food et al.. Food ResearchResearchResearchResearch InternationalInternationalInternationalInternational 41 (2008) 27441 (2008) 27441 (2008) 27441 (2008) 274----285285285285

Scheme of the extraction of antioxidants from a plant foodScheme of the extraction of antioxidants from a plant foodScheme of the extraction of antioxidants from a plant foodScheme of the extraction of antioxidants from a plant food

J. J. J. J. SerranoSerranoSerranoSerrano et al.. Food et al.. Food et al.. Food et al.. Food ResearchResearchResearchResearch InternationalInternationalInternationalInternational 40 (2007) 1540 (2007) 1540 (2007) 1540 (2007) 15----21212121

Scheme of the extraction of antioxidants from a plant foodScheme of the extraction of antioxidants from a plant foodScheme of the extraction of antioxidants from a plant foodScheme of the extraction of antioxidants from a plant food

J. J. J. J. PerezPerezPerezPerez----JimenezJimenezJimenezJimenez et al.. Food et al.. Food et al.. Food et al.. Food ResearchResearchResearchResearch InternationalInternationalInternationalInternational 41 (2008) 27441 (2008) 27441 (2008) 27441 (2008) 274----285285285285

Schematic of the methodology used to estimate the bioacceeibility of antioxidant capacity. N = number of samples for each food group

Food antioxidant capacity determined by chemical methods may underestimate the physiological antioxidant capacity

J. J. J. J. SerranoSerranoSerranoSerrano et al.. Food et al.. Food et al.. Food et al.. Food ResearchResearchResearchResearch InternationalInternationalInternationalInternational 40 (2007) 1540 (2007) 1540 (2007) 1540 (2007) 15----21212121

Methods for the measurement of antioxidant activity/capacityMethods for the measurement of antioxidant activity/capacityMethods for the measurement of antioxidant activity/capacityMethods for the measurement of antioxidant activity/capacity

Signal

Inductiontime

K

[A] 1

[A] 2

„t” –fixed time point

Inductiontime

Inhibition of reaction after time „t”

(%)

Concentration of antioxidant

Concentration of antioxidant

n

n

n

n

n

n

n

n

n

n

n

n

TargetTargetTargetTargetcompoundcompoundcompoundcompound

OxygenOxygenOxygenOxygen consumptionconsumptionconsumptionconsumption

MethodMethodMethodMethod –––– end point end point end point end point observedobservedobservedobserved

OxygenOxygenOxygenOxygen electrode (ORAC)electrode (ORAC)electrode (ORAC)electrode (ORAC)

ΒΒΒΒ----phycoerythrinphycoerythrinphycoerythrinphycoerythrin SpectrofluorymetrySpectrofluorymetrySpectrofluorymetrySpectrofluorymetry (ORAC, TRAP)(ORAC, TRAP)(ORAC, TRAP)(ORAC, TRAP)

(ABTS+ABTS+ABTS+ABTS+•••• ) () () () (2,22,22,22,2’’’’----azinobisazinobisazinobisazinobis----(3(3(3(3----ethylbenzothiazolineethylbenzothiazolineethylbenzothiazolineethylbenzothiazoline----6666----sulfonate)sulfonate)sulfonate)sulfonate)

SpectrophotometrySpectrophotometrySpectrophotometrySpectrophotometry (TEAC)(TEAC)(TEAC)(TEAC)

LuminolLuminolLuminolLuminol ChemiluminescenceChemiluminescenceChemiluminescenceChemiluminescence (CL)(CL)(CL)(CL)

TargetTargetTargetTargetcompoundcompoundcompoundcompound

2,22,22,22,2----diphenyldiphenyldiphenyldiphenyl----1111----picrylhydrazylpicrylhydrazylpicrylhydrazylpicrylhydrazyl (DPPH)(DPPH)(DPPH)(DPPH)

MethodMethodMethodMethod –––– end point end point end point end point observedobservedobservedobserved

SpectrophotometrySpectrophotometrySpectrophotometrySpectrophotometry

DPPHDPPHDPPHDPPH EPR EPR EPR EPR spectrometryspectrometryspectrometryspectrometry

(ABTS+(ABTS+(ABTS+(ABTS+••••)))) SpectrophotometrySpectrophotometrySpectrophotometrySpectrophotometry –––– decolorization assaydecolorization assaydecolorization assaydecolorization assay

N,NN,NN,NN,N---- dimethyldimethyldimethyldimethyl----pppp----phenyldiaminephenyldiaminephenyldiaminephenyldiamine

cation radicalcation radicalcation radicalcation radicalSpectrophotometrySpectrophotometrySpectrophotometrySpectrophotometry –––– decolorization decolorization decolorization decolorization assayassayassayassay

Expression of the resultsExpression of the resultsExpression of the resultsExpression of the results

The expression of results of antioxidant capacity assays can be The expression of results of antioxidant capacity assays can be The expression of results of antioxidant capacity assays can be The expression of results of antioxidant capacity assays can be summarized in three categories: results based on measurements atsummarized in three categories: results based on measurements atsummarized in three categories: results based on measurements atsummarized in three categories: results based on measurements at a a a a

fixed endfixed endfixed endfixed end----point compared to an standard, results expressed considering point compared to an standard, results expressed considering point compared to an standard, results expressed considering point compared to an standard, results expressed considering laglaglaglag----phase, and results based on kinetic parameters.phase, and results based on kinetic parameters.phase, and results based on kinetic parameters.phase, and results based on kinetic parameters.

O

OH

CH3

R1

R2

CH3

CH3

CH3CH3

CH3

O

OH

Wayner i inni, 1985Wayner i inni, 1985Wayner i inni, 1985Wayner i inni, 1985

Trolox Trolox Trolox Trolox –––– a hydrosoluble analoque of witamin Ea hydrosoluble analoque of witamin Ea hydrosoluble analoque of witamin Ea hydrosoluble analoque of witamin E

((((±±±±))))----6666----HydroxyHydroxyHydroxyHydroxy----2,5,7,82,5,7,82,5,7,82,5,7,8----tetramethylchromanetetramethylchromanetetramethylchromanetetramethylchromane----2222----carboxylic acid carboxylic acid carboxylic acid carboxylic acid

Trolox equivalentTrolox equivalentTrolox equivalentTrolox equivalent

Trolox equivalent antioxidant capacity is the milimolar concentrTrolox equivalent antioxidant capacity is the milimolar concentrTrolox equivalent antioxidant capacity is the milimolar concentrTrolox equivalent antioxidant capacity is the milimolar concentration of a Trolox ation of a Trolox ation of a Trolox ation of a Trolox solution having the antioxidant capacity equivalent to a 1.0 mM solution having the antioxidant capacity equivalent to a 1.0 mM solution having the antioxidant capacity equivalent to a 1.0 mM solution having the antioxidant capacity equivalent to a 1.0 mM solution of the solution of the solution of the solution of the substance under investigation.substance under investigation.substance under investigation.substance under investigation.

Trolox equivalent Trolox equivalent Trolox equivalent Trolox equivalent –––– µµµµmol of Trolox necessary to provide the same antioxidant mol of Trolox necessary to provide the same antioxidant mol of Trolox necessary to provide the same antioxidant mol of Trolox necessary to provide the same antioxidant capacity as a gram of the sample. capacity as a gram of the sample. capacity as a gram of the sample. capacity as a gram of the sample.

RiceRiceRiceRice----Evans and Miller, Methods in Enzymology 234 (1994) 279Evans and Miller, Methods in Enzymology 234 (1994) 279Evans and Miller, Methods in Enzymology 234 (1994) 279Evans and Miller, Methods in Enzymology 234 (1994) 279----293.293.293.293.

CaoCaoCaoCao et al.., 1998; Re et al.., 1999; et al.., 1998; Re et al.., 1999; et al.., 1998; Re et al.., 1999; et al.., 1998; Re et al.., 1999; OuOuOuOu et al.., 2001et al.., 2001et al.., 2001et al.., 2001

Useful informationUseful informationUseful informationUseful information

Trolox (2.5 mM; F.W. 250.28) is prepared by dissolving 0.15641 gTrolox (2.5 mM; F.W. 250.28) is prepared by dissolving 0.15641 gTrolox (2.5 mM; F.W. 250.28) is prepared by dissolving 0.15641 gTrolox (2.5 mM; F.W. 250.28) is prepared by dissolving 0.15641 g of Trolox of Trolox of Trolox of Trolox in 250 mL of solvent. When PBS is used as a solvent, at the pH sin 250 mL of solvent. When PBS is used as a solvent, at the pH sin 250 mL of solvent. When PBS is used as a solvent, at the pH sin 250 mL of solvent. When PBS is used as a solvent, at the pH solution is is olution is is olution is is olution is is near the upper limit of Trolox solubility, and gentle ultrasonicnear the upper limit of Trolox solubility, and gentle ultrasonicnear the upper limit of Trolox solubility, and gentle ultrasonicnear the upper limit of Trolox solubility, and gentle ultrasonication is required ation is required ation is required ation is required to dissolve the crystals.to dissolve the crystals.to dissolve the crystals.to dissolve the crystals.

Experiments have shown that frozen Trolox at this concentration Experiments have shown that frozen Trolox at this concentration Experiments have shown that frozen Trolox at this concentration Experiments have shown that frozen Trolox at this concentration is stable for is stable for is stable for is stable for more than 8 months.more than 8 months.more than 8 months.more than 8 months.

Fresh working standards (0.5, 1.0, 1.5, 2.0 mMFresh working standards (0.5, 1.0, 1.5, 2.0 mMFresh working standards (0.5, 1.0, 1.5, 2.0 mMFresh working standards (0.5, 1.0, 1.5, 2.0 mM) are prepared daily by mixing are prepared daily by mixing are prepared daily by mixing are prepared daily by mixing 2.5 mM Trolox with solvent.2.5 mM Trolox with solvent.2.5 mM Trolox with solvent.2.5 mM Trolox with solvent.

Scavenging of Scavenging of Scavenging of Scavenging of 2,22,22,22,2’’’’----azinobisazinobisazinobisazinobis----(3(3(3(3----ethylbenzothiazolineethylbenzothiazolineethylbenzothiazolineethylbenzothiazoline----6666----sulfonate) radical cation (ABTSsulfonate) radical cation (ABTSsulfonate) radical cation (ABTSsulfonate) radical cation (ABTS••••+) +) +) +) or Trolox equivalent or Trolox equivalent or Trolox equivalent or Trolox equivalent

antioxidant capacity (TEAC) assayantioxidant capacity (TEAC) assayantioxidant capacity (TEAC) assayantioxidant capacity (TEAC) assay

The first method was developed by Rice-Evans and Miller and published as follows:

Rice-Evans C.A., Miller N.M. Total antioxidant status in plasma and body fluids. Methods in Enzymology 234 (1994) 279-293.

Rice-Evans C.A., Miller N.M., Paganda G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20 (7)(1996) 933-956.

Miller N.J., Rice-Evans C.A. Spectrophotometric determination of antioxidant activity. Redox Report 2 (3), (1996) 161-171.

ABTS (2,2ABTS (2,2ABTS (2,2ABTS (2,2’’’’----azinoazinoazinoazino----bis(3bis(3bis(3bis(3----ethylbenzethylbenzethylbenzethylbenz----thiazolinethiazolinethiazolinethiazoline----6666----sulfonic acid)sulfonic acid)sulfonic acid)sulfonic acid)

ABTS λλλλmax342 nm

ABTS+••••λλλλmax417 nm

λλλλmax645 nm

λλλλmax734 nm

λλλλmax815 nm

ABTS+2 λλλλmax513 nm

Absorption spectra of Absorption spectra of Absorption spectra of Absorption spectra of ABTSABTSABTSABTS++++••••

Benefits of ABenefits of ABenefits of ABenefits of ABTSBTSBTSBTS++++••••

- the radical cation is stable,

- experiment is easy to repeat,

- results are comparable,

- the sensitivity of the assay depends on the ABTS concentration.

λλλλmax417 nm

λλλλmax645 nm

λλλλmax734 nm

λλλλmax815 nm

Useful informationUseful informationUseful informationUseful information

ABTS (F.W. 548.68) can be prepared in water, PBS, methanol and eABTS (F.W. 548.68) can be prepared in water, PBS, methanol and eABTS (F.W. 548.68) can be prepared in water, PBS, methanol and eABTS (F.W. 548.68) can be prepared in water, PBS, methanol and ethanol.thanol.thanol.thanol.

The extinction coefficient of ABTS+•••• at 734 nm has been calculated in water as 1.5 x 104 mol-1 L cm-1 ± 549 (mean = SD)

The extinction coefficient of ABTS+•••• at 734 nm has been calculated in ethanol as 1.6 x 104 mol-1 L cm-1 ± 606 (mean = SD)

The ABTS+•••• radical cation is stable for more than two days when stored in the dark at room temperature.

Re et al.., Free Radical Biology & Medicine 26(1999) 1231Re et al.., Free Radical Biology & Medicine 26(1999) 1231Re et al.., Free Radical Biology & Medicine 26(1999) 1231Re et al.., Free Radical Biology & Medicine 26(1999) 1231----1237123712371237

ABTSABTSABTSABTS••••+ + + + radical cation can be generated by:radical cation can be generated by:radical cation can be generated by:radical cation can be generated by:

1) chemical reaction using:

• manganase dioxide

• AAPH

• potasium persulfate (Re at. 1999)

2) enzymatic reaction using:

• metmyoglobin

• horseradish peroxidase

3) electrochemical generation

Reaction times ranging from 1 to 30 min have been adopted througReaction times ranging from 1 to 30 min have been adopted througReaction times ranging from 1 to 30 min have been adopted througReaction times ranging from 1 to 30 min have been adopted throughout the protocols. Concerning the hout the protocols. Concerning the hout the protocols. Concerning the hout the protocols. Concerning the wavelength of detectcion, the determination at 734 nm is preferrwavelength of detectcion, the determination at 734 nm is preferrwavelength of detectcion, the determination at 734 nm is preferrwavelength of detectcion, the determination at 734 nm is preferred bacause the interference from ed bacause the interference from ed bacause the interference from ed bacause the interference from other absorbibg components and from sample turbidity is minimizeother absorbibg components and from sample turbidity is minimizeother absorbibg components and from sample turbidity is minimizeother absorbibg components and from sample turbidity is minimized. The d. The d. The d. The ABTSABTSABTSABTS••••+ + + + scavenging can bescavenging can bescavenging can bescavenging can beevaluated over a wide pH range. evaluated over a wide pH range. evaluated over a wide pH range. evaluated over a wide pH range.

Total Antioxidant StatusTotal Antioxidant StatusTotal Antioxidant StatusTotal Antioxidant Status or TEAC Ior TEAC Ior TEAC Ior TEAC IMiller and Rice Evans (1996)Miller and Rice Evans (1996)Miller and Rice Evans (1996)Miller and Rice Evans (1996)

Assay PrincipleAssay PrincipleAssay PrincipleAssay Principle

Reaction inhibited,Reaction inhibited,Reaction inhibited,Reaction inhibited,so lessso lessso lessso less ABTS ABTS ABTS ABTS ++++

producedproducedproducedproduced

ABTSABTSABTSABTS + + + + ++++ HX HX HX HX ---- Fe Fe Fe Fe 3+ 3+ 3+ 3+

(Blue(Blue(Blue(Blue----green radical cation green radical cation green radical cation green radical cation measured at 6measured at 6measured at 6measured at 633330 nm)0 nm)0 nm)0 nm)

MbFe(IIIMbFe(IIIMbFe(IIIMbFe(III))))(metmyoglobin)(metmyoglobin)(metmyoglobin)(metmyoglobin)

HHHH2222OOOO2222(hydrogen peroxide)(hydrogen peroxide)(hydrogen peroxide)(hydrogen peroxide)+

MbFeMbFeMbFeMbFe (IV)(IV)(IV)(IV)= O = O = O = O FerrylmyoglobinFerrylmyoglobinFerrylmyoglobinFerrylmyoglobin radicalradicalradicalradical

No antioxidants presentNo antioxidants presentNo antioxidants presentNo antioxidants presentAntioxidantsAntioxidantsAntioxidantsAntioxidants presentpresentpresentpresent

+ ABTSABTSABTSABTS(2,2(2,2(2,2(2,2’’’’----azobis(3azobis(3azobis(3azobis(3----thylbenzolinethylbenzolinethylbenzolinethylbenzoline----6666----sulfonic acid)sulfonic acid)sulfonic acid)sulfonic acid)

Peroxidase Cycle of Myoglobin; HA is a SubstratePeroxidase Cycle of Myoglobin; HA is a SubstratePeroxidase Cycle of Myoglobin; HA is a SubstratePeroxidase Cycle of Myoglobin; HA is a Substrate

This method has been This method has been This method has been This method has been commercialized by Randox commercialized by Randox commercialized by Randox commercialized by Randox Laboratories (San Laboratories (San Laboratories (San Laboratories (San Francisco, USA) as the Francisco, USA) as the Francisco, USA) as the Francisco, USA) as the worldworldworldworld’’’’s first kit for the total s first kit for the total s first kit for the total s first kit for the total antioxidant status antioxidant status antioxidant status antioxidant status measurement in an measurement in an measurement in an measurement in an individualindividualindividualindividual’’’’s serum or s serum or s serum or s serum or plasma.plasma.plasma.plasma.

This commercial kit for the This commercial kit for the This commercial kit for the This commercial kit for the TEAC assay is expensive; TEAC assay is expensive; TEAC assay is expensive; TEAC assay is expensive; t h e r e a g e n t c os t pe r t h e r e a g e n t c os t pe r t h e r e a g e n t c os t pe r t h e r e a g e n t c os t pe r sample estimated in the sample estimated in the sample estimated in the sample estimated in the RandoxRandoxRandoxRandox----TEAC assay is TEAC assay is TEAC assay is TEAC assay is approximately nine times approximately nine times approximately nine times approximately nine times higher that in the ORAC that in the ORAC that in the ORAC that in the ORAC a s s a y .a s s a y .a s s a y .a s s a y .

Different analytical strategies were apparentDifferent analytical strategies were apparentDifferent analytical strategies were apparentDifferent analytical strategies were apparent

• decolorization assaydecolorization assaydecolorization assaydecolorization assay

• inhibition assay (fixed time point)inhibition assay (fixed time point)inhibition assay (fixed time point)inhibition assay (fixed time point)

• inhibition assay (reaction rate) inhibition assay (reaction rate) inhibition assay (reaction rate) inhibition assay (reaction rate)

• lag phase measurement lag phase measurement lag phase measurement lag phase measurement

RiceRiceRiceRice----Evans C.A.Evans C.A.Evans C.A.Evans C.A., , , , Miller N.M.Miller N.M.Miller N.M.Miller N.M. Total antioxidant status in plasma and body Total antioxidant status in plasma and body Total antioxidant status in plasma and body Total antioxidant status in plasma and body fluids. fluids. fluids. fluids. Methods in EnzymologyMethods in EnzymologyMethods in EnzymologyMethods in Enzymology 234 (1994) 279234 (1994) 279234 (1994) 279234 (1994) 279----293.293.293.293.

RiceRiceRiceRice----Evans C.A.Evans C.A.Evans C.A.Evans C.A.,,,, Miller N.M.Miller N.M.Miller N.M.Miller N.M., , , , Paganda G. StructurePaganda G. StructurePaganda G. StructurePaganda G. Structure----antioxidant activity antioxidant activity antioxidant activity antioxidant activity relationships of flavonoids and phenolic acids. relationships of flavonoids and phenolic acids. relationships of flavonoids and phenolic acids. relationships of flavonoids and phenolic acids. Free Radic. Biol. Med.Free Radic. Biol. Med.Free Radic. Biol. Med.Free Radic. Biol. Med. 20 20 20 20 (7)(7)(7)(7) (1996) (1996) (1996) (1996) 933933933933----956.956.956.956.

Antioxidant TEAC I [mM]

Bilirubin Urate

Ascorbate αααα-Tocopherol

Albumin Glutathione

N-Acetylcysteine

1.50 1.02 0.99 0.97 0.63 0.90 1.43

O

OOH

OH

OH

O

OOH

OH

OHOH

O

OOH

OH

OH O

OOH

OH

OH

OH

O

OOH

OH

OH

OH

OOH

OH

OH

O

OH

OOH

OH

OH

OH

O

OOH

OH

OH

Apigenin TEAC = 1.45 Luteolin TEAC = 2.1

Kaempferol TEAC = 1.34

Quercetin TEAC = 4.72

Galangin TEAC = 1.38

Naringenin TEAC = 1.5Floretin TEAC = ?

Genistein TEAC = ?

Rice-Evans i inni, 1996

OH

OH

OH

OOH

3' 4'

345

27

HO

QuercetinTEAC = 4.72

O-dihydroxy structure

Rice-Evans i inni, 1996

ElementElementElementElements of flavonoids structure important for their s of flavonoids structure important for their s of flavonoids structure important for their s of flavonoids structure important for their antioxidant activity antioxidant activity antioxidant activity antioxidant activity

O23

56

7

2'3'

4'5'

6'

O

OOOOHHHHOOOOHHHH

O23

56

7

2'3'

4'

5'6'

OOOO

OOOO

6'5'

4'3'

2'

7

65

3

2O

OOOOOOOO

HHHH HHHH

Bors, 1998

quercetin-4’-O-ββββ-glucoside(Q4’G)

O

O

HO

OH

OOH

OH

OOH

CH2OH

OHOH

O

O

HO

OH

OHOH

O

OOH

CH2OH

OHOH

quercetin-3-O-ββββ-glucoside(Q3G)

TEAC = 3.5 TEAC = 0.74

Antioxidant activity of Antioxidant activity of Antioxidant activity of Antioxidant activity of quercetin quercetin quercetin quercetin glucosidesglucosidesglucosidesglucosides

Rice-Evans i inni, 1996

TEAC [mM] = 2.5 ±±±± 0.02TEAC [mM] = 3.8 ±±±± 0.06

TEAC [mM] = 4.9 ±±±± 0.02 TEAC [mM] = 4.8 ±±±± 0.06

Rice-Evans i inni, 1996

Antioxidant activity of green tea catechinsAntioxidant activity of green tea catechinsAntioxidant activity of green tea catechinsAntioxidant activity of green tea catechins

TEAC [mM] = 1.90 ±±±± 0.01

TEAC [mM] = 2.90 ±±±± 0.1

Re i inni, 1999

1O2 + ββββ-karoten →→→→ 3O2 + 3ββββ-karoten* 3ββββ-karoten* →→→→ ββββ-karoten + energia termiczna

TEAC II

ABTS MnO2+

Antioxidants ABTS+••••+Reduction

ABTS

(radical cation)

TEAC III

ABTS K2S2O8+

Antioxidants

ABTS+••••

+Reduction

ABTSABTS+••••/EtOH

ABTS+••••/PBSAntioxidants +Reduction

ABTS

Disadvantages of TEAC assaysDisadvantages of TEAC assaysDisadvantages of TEAC assaysDisadvantages of TEAC assays

This same antioxidant can exhibit different TEAC value, dependinThis same antioxidant can exhibit different TEAC value, dependinThis same antioxidant can exhibit different TEAC value, dependinThis same antioxidant can exhibit different TEAC value, depending of the way g of the way g of the way g of the way of of of of ABTSABTSABTSABTS++++•••• radical cation generation and end point measurement.radical cation generation and end point measurement.radical cation generation and end point measurement.radical cation generation and end point measurement.

Antioxidant →→→→

TEAC assay ↓↓↓↓

Trolox Gallic acid Ascorbic acid Uric acid

TEAC I

TEAC II

TEAC/EtOH

TEAC/ PBS

1.00

1.00

1.00

1.00

2.24 ±±±± 0.27

4.35 ±±±± 0.22

3.83 ±±±± 0.10

3.35 ±±±± 0.32

1.06 ±±±± 0.09

0.99 ±±±± 0.09

0.31 ±±±± 0.03

1.14 ±±±± 0.06

0.86 ±±±± 0.13

0.86 ±±±± 0.11

0.83 ±±±± 0.06

0.99 ±±±± 0.17

Schlesier i inni, 2002Schlesier i inni, 2002Schlesier i inni, 2002Schlesier i inni, 2002

Disadvantages of TEAC assaysDisadvantages of TEAC assaysDisadvantages of TEAC assaysDisadvantages of TEAC assays

TTTThe ABTS radical used in TEAC assays is not found in mammalian biology and thus represents a „nonphysiological” rad ical source.

Schlesier i inni, 2002Schlesier i inni, 2002Schlesier i inni, 2002Schlesier i inni, 2002

TTTThermodynamically, a compound can reduce ABTS•+ if it has a redox potential lower than that of ABTS (0.68 V). Many ph enolic compounds have low redox potentials and thus react with ABTS• + .

TTTThe TEAC reaction may not be the same for slow react ions, and it may take a long time to reach endpoint. Thus, by us ing an endpoint of short duration (4 or 6 min), one may be reading before the reaction is finished and result in lowered TEAC values.

Comparison between the antioxidant activity as Comparison between the antioxidant activity as Comparison between the antioxidant activity as Comparison between the antioxidant activity as TEAC (mM) at specific timeTEAC (mM) at specific timeTEAC (mM) at specific timeTEAC (mM) at specific time----pointspointspointspoints

Advantages of TEAC assaysAdvantages of TEAC assaysAdvantages of TEAC assaysAdvantages of TEAC assays

TEAC assay is operationally simple, it has been used in many resTEAC assay is operationally simple, it has been used in many resTEAC assay is operationally simple, it has been used in many resTEAC assay is operationally simple, it has been used in many research earch earch earch laboratories for studying antioxidant capacity.laboratories for studying antioxidant capacity.laboratories for studying antioxidant capacity.laboratories for studying antioxidant capacity.

Prior et al.., 2005

TEAC values of many compounds and food samples have been reporteTEAC values of many compounds and food samples have been reporteTEAC values of many compounds and food samples have been reporteTEAC values of many compounds and food samples have been reportedddd.

TEAC reaction can be automated and adapted to microplates, to flTEAC reaction can be automated and adapted to microplates, to flTEAC reaction can be automated and adapted to microplates, to flTEAC reaction can be automated and adapted to microplates, to flow ow ow ow injection, and to stopped flow.injection, and to stopped flow.injection, and to stopped flow.injection, and to stopped flow.

ABTSABTSABTSABTS••••++++ reacts rapidly with antioxidants, typically within 30 min.reacts rapidly with antioxidants, typically within 30 min.reacts rapidly with antioxidants, typically within 30 min.reacts rapidly with antioxidants, typically within 30 min.

ABTSABTSABTSABTS••••++++ is soluble in both aqueous and organic solvents and is not affeis soluble in both aqueous and organic solvents and is not affeis soluble in both aqueous and organic solvents and is not affeis soluble in both aqueous and organic solvents and is not affected cted cted cted by ionic strength, by ionic strength, by ionic strength, by ionic strength, sosososo can be used in multiple media to determine both can be used in multiple media to determine both can be used in multiple media to determine both can be used in multiple media to determine both hydrophilic and lipophilic antioxidant capacities of extracts anhydrophilic and lipophilic antioxidant capacities of extracts anhydrophilic and lipophilic antioxidant capacities of extracts anhydrophilic and lipophilic antioxidant capacities of extracts and body fluids.d body fluids.d body fluids.d body fluids.

Pellegrini N., Del Rio D., Colombi B., Bianchi M., Brighenti F. Pellegrini N., Del Rio D., Colombi B., Bianchi M., Brighenti F. Pellegrini N., Del Rio D., Colombi B., Bianchi M., Brighenti F. Pellegrini N., Del Rio D., Colombi B., Bianchi M., Brighenti F. Application of the 2,2Application of the 2,2Application of the 2,2Application of the 2,2’’’’----Azinobis(3Azinobis(3Azinobis(3Azinobis(3----ethylbenzothiazolineethylbenzothiazolineethylbenzothiazolineethylbenzothiazoline----6666----sulfonic acid) Radical Cation Assay to a Flow sulfonic acid) Radical Cation Assay to a Flow sulfonic acid) Radical Cation Assay to a Flow sulfonic acid) Radical Cation Assay to a Flow Injection System for the Evaluation of Antioxidant Activity of SInjection System for the Evaluation of Antioxidant Activity of SInjection System for the Evaluation of Antioxidant Activity of SInjection System for the Evaluation of Antioxidant Activity of Some Pure Compounds ome Pure Compounds ome Pure Compounds ome Pure Compounds and Beverages. Journal of Agricultural and Food Chemistry 2003, and Beverages. Journal of Agricultural and Food Chemistry 2003, and Beverages. Journal of Agricultural and Food Chemistry 2003, and Beverages. Journal of Agricultural and Food Chemistry 2003, 51, 26051, 26051, 26051, 260----264. 264. 264. 264.

Milardovic S., Kerekovic I., Derrico R., Rumenjak V. A novel metMilardovic S., Kerekovic I., Derrico R., Rumenjak V. A novel metMilardovic S., Kerekovic I., Derrico R., Rumenjak V. A novel metMilardovic S., Kerekovic I., Derrico R., Rumenjak V. A novel method for flow hod for flow hod for flow hod for flow injection analysis of total antioxidant capacity using enzymaticinjection analysis of total antioxidant capacity using enzymaticinjection analysis of total antioxidant capacity using enzymaticinjection analysis of total antioxidant capacity using enzymatically produced ally produced ally produced ally produced ABTSABTSABTSABTS••••++++ and bioamperometric detector containing interdigitated electrodeand bioamperometric detector containing interdigitated electrodeand bioamperometric detector containing interdigitated electrodeand bioamperometric detector containing interdigitated electrode. . . . Talanta 2007, 71, 213Talanta 2007, 71, 213Talanta 2007, 71, 213Talanta 2007, 71, 213----220.220.220.220.

Antioxidant capacity of fruits

020406080

100120140160180

Strawbe

rryPlum

sOra

nge

Grape

fruit

Grape

fruit

Tomat

oGra

pes

Grape

s

umol

TE

/g d

.m.

Wang et al.., 1996

Antioxidant capacity of different juices

0

2468

101214

16

Juice

umol

TE

/mL Grapes

GrapefrutTomatoOrangeApple

(Wang i inni, 1996)

Antioxidant capacity of vegetables

Vegetable

(µµµµmol TE/ g fresk mass]

Curry kale

Garlic Spinach

Brussels sprouts Broccoli Red beet Paprica

Corn Onion

Cabbage Cauliflower

Potato Lettuce Carrot Celery

Cucumber

24.1 23.2 17.0 15.8 12.9 8.1 8.1 7.2 5.6 4.8 4.6 4.6 4.4 2.8 1.1 1.1

Horubała, 1999

0

5

10

15

20

25

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st

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st

Cru

mb

Flo

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100% 96% 85% 70%

[m

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Tro

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d.m

.]

Antioxidant capacity of rye breads

Antioxidant capacity of different types of beer

0

12

3

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6

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EB Pils

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Antioxidant capacity of different types of wine

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Antioxidant capacity of different types of vodka

0

0.5

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Bartosz, 2004

Buckwheat material and roastingBuckwheat material and roastingBuckwheat material and roastingBuckwheat material and roasting

Raw whole buckwheat seeds (Fagopyrum esculentum Moench var. KoraRaw whole buckwheat seeds (Fagopyrum esculentum Moench var. KoraRaw whole buckwheat seeds (Fagopyrum esculentum Moench var. KoraRaw whole buckwheat seeds (Fagopyrum esculentum Moench var. Kora), ), ), ), roasted whole seeds and roasted groats were provided by the locaroasted whole seeds and roasted groats were provided by the locaroasted whole seeds and roasted groats were provided by the locaroasted whole seeds and roasted groats were provided by the local industry l industry l industry l industry from Northfrom Northfrom Northfrom North----East Poland. The roasting included raising the moisture content East Poland. The roasting included raising the moisture content East Poland. The roasting included raising the moisture content East Poland. The roasting included raising the moisture content of of of of the whole raw seeds to 22% of dry matter followed by simultaneouthe whole raw seeds to 22% of dry matter followed by simultaneouthe whole raw seeds to 22% of dry matter followed by simultaneouthe whole raw seeds to 22% of dry matter followed by simultaneously steaming sly steaming sly steaming sly steaming (water vapour at 588 kPa) and heating (at 160(water vapour at 588 kPa) and heating (at 160(water vapour at 588 kPa) and heating (at 160(water vapour at 588 kPa) and heating (at 160ºººº C for 30 min) and then the C for 30 min) and then the C for 30 min) and then the C for 30 min) and then the roasted whole seeds were dehulled resulted in roasted groats as roasted whole seeds were dehulled resulted in roasted groats as roasted whole seeds were dehulled resulted in roasted groats as roasted whole seeds were dehulled resulted in roasted groats as previously previously previously previously reported. Raw groats was obtained in the laboratory by manually reported. Raw groats was obtained in the laboratory by manually reported. Raw groats was obtained in the laboratory by manually reported. Raw groats was obtained in the laboratory by manually dehulling of dehulling of dehulling of dehulling of raw whole buckwheat seeds before roasting. The raw whole buckwheat seeds before roasting. The raw whole buckwheat seeds before roasting. The raw whole buckwheat seeds before roasting. The simplified flow diagram of simplified flow diagram of simplified flow diagram of simplified flow diagram of roasting of whole buckwheat seeds is shown on roasting of whole buckwheat seeds is shown on roasting of whole buckwheat seeds is shown on roasting of whole buckwheat seeds is shown on f igureigureigureigure. . . . The buckwheat material The buckwheat material The buckwheat material The buckwheat material was f reezewas f reezewas f reezewas f reeze ---- d r i ed , g round and s to red a t d r i ed , g round and s to red a t d r i ed , g round and s to red a t d r i ed , g round and s to red a t ---- 20202020ºººº C un t i l ana lys i s .C un t i l ana lys i s .C un t i l ana lys i s .C un t i l ana lys i s .

The whole buckwheat grains consist of a true seed (groat) surrounded by a thick hull.

raw buckwheat whole seeds

roasted whole seeds

roasting

160 °C, 30 min, 588 kPa

raw groats

manually dehulled

(non-thermal process)

roasted groats (30 min)

industrialy dehulled

(non-thermal process)

40 min50 min

roasted groats (40 min)roasted groats (50 min)

COST 927 Training School

Sample code(1) Raw buckwheat groat

(2) Roasted buckwheat groat (160 °C/30 min)(3) Roasted buckwheat groat (160 °C/40 min)(4) Roasted buckwheat groat (160 °C/50 min

Buckwheat seeds

Roasted groats

Whole buckwheat seeds

Thermally treated whole buckwheat

Sample extractionTEAC, FCR, DPPHAbout 250 mg of dried and pulverized buckwheat samples was extracted with 1 mL of 80% methanol by 60 s

sonication. Next, the mixture was vortexed for 60 s, again sonicated and centrifuged for 5 min ( 5 000 x g, 4º C). That step was repeated on the residue with next volume of 2.5 mL of the solvent. Supernatants were collected in 5 mL flask. Finally, all extracts were kept at -80º C prior to further analysis. (50 mg/mL)

ORACAbout 100 mg of dried and pulverized buckwheat samples was extracted with 1 mL of 75 mmol/L phosphate buffer (pH

7.4) by 30 s sonication. Next, the mixture was vortexed for 30 s, again sonicated and vortexed, and centrifuged for 5 min (5 000 x g at 4ºC). That step was repeated 5 times, supernatant was collected in 5 mL flask and it was used for direct ORAC assay. (20 mg/mL)

PCL ACWAbout 100 mg of dried and pulverized buckwheat samples was extracted with 1 mL of 80% methanol by 30 s

sonication. Next, the mixture was vortexed for 30 s, again sonicated and vortexed, and centrifuged for 5 min ( 5 000 x g at 4ºC). That step was repeated 5 times and supernatants were collected in 5 mL flask. (20 mg/mL)

PCL ACLAbout 100 mg of dried and pulverized buckwheat samples was extracted with with mixture of 200 µL n-hexane and 800

µL methanol by 30 s sonication. Next, the mixture was vortexed for 30 s, again sonicated and vortexed, and centrifuged for 5 min ( 5 000 x g at 4ºC). That step was repeated 5 times and supernatants were collected in 5 mL flask. (20 mg/mL)

CVAbout 500 mg of dried and pulverized buckwheat samples was extracted with 2.5 mL of 80% methanol by 60 s

sonication. Next, the mixture was vortexed for 60 s, again sonicated and centrifuged for 5 min ( 5 000 x g, 4º C). That step was repeated on the residue with next volume of 2.5 mL of the solvent. Supernatants were collected in 5 mL flask. Finally, all extracts were kept at -80º C prior to further analysis. (100 mg/mL)

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