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181 Czech J. Food Sci. Vol. 29, 2011, No. 2: 181–189 Antioxidant Capacity and Antioxidants of Strawberry, Blackberry, and Raspberry Leaves Lucie BUřIčOVá 1 , Mirjana ANDJELKOVIC 2 , Anna čERMáKOVá 1 , Zuzana RéBLOVá 1 , Ondřej JURčEK 3,4,5 , Erkki KOLEHMAINEN 4 , Roland VERHé 2 and František KVASNIčKA 6 1 Department of Food Chemistry and Analysis, 3 Department of Chemistry of Natural Compounds and 6 Department of Food Preservation and Meat Technology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology in Prague, Prague, Czech Republic; 2 Department of Organic Chemistry, Ghent University, Ghent, Belgium; 4 Department of Chemistry, Laboratory of Organic Chemistry, University of Jyväskylä, Jyväskylä, Finland; 5 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Isotope Laboratory, Prague, Czech Republic Abstrakt Buřičová L., Andjelkovic M., Čermáková A., Réblová Z., Jurček O., Kolehmainen E., Verhé R., Kvasnička F. (2011): Antioxidant capacity and antioxidants of strawberry, blackberry, and raspberry leaves. Czech J. Food Sci., 29: 181–189. The total phenolic content (Folin-Ciocalteu method), free radical scavenging ability expressed as DPPH value, ferric re- ducing antioxidant capacity (FRAP), and oxygen radical absorbance capacity (ORAC) were determined in water extracts of leaves from Rosaceae family plants (Fragaria vesca L., Rubus fructicosus L., and Rubus idaeus L.). The antioxidant capacities of the extracts (in the order of the above mentioned methods) were 73.6–88.9%, 60.1–71.4%, 49.7–78.0% re- spectively, and 45.3–66.5% of that of green tea water extract. Further, the presence of 15 compounds (gallic acid, rutin, ellagic acid, caffeic acid, p-coumaric acid, quercetin, kaempferol, myricetin, quercetin-3-d-glucoside, ascorbic acid, (+)-catechin, (–)-epicatechin, epicatechingallate, epigallocatechin, procyanidin B1) was studied by HPLC-ECD and their antioxidant capacities were compared to the antioxidant capacity of the extracts. Out of the compounds studied, mostly (+)-catechin, ellagic acid, and (–)-epicatechin participated in the antioxidant capacities of the studied plant leaves water extracts. The antioxidant capacity of leaves infusions (determined by DPPH method) was lower than those of red wines and tea infusions, but comparable to the antioxidant capacities of white wines and fruit beverages. Keywords: DPPH; Folin-Ciocalteau method; Fragaria vesca; Rubus fructicosus; Rubus ideaus; FRAP; HPLC; ORAC Supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project No. MSM 6046137305, and by specific university research of Ministry of Education, Youth and Sports of the Czech Republic, Project No. 21/2010. In the screening study comparing the antioxidant capacities (AC) (measured by DPPH method) of seventeen Czech medicinal plants, the leaves of strawberry ( Fragaria vesca L.), blackberry (Rubus fructicosus L.) and raspberry ( Rubus idaeus L.) belonged to the most efficient plants tested (Buři- čová & Réblová 2008). The AC of their extracts were comparable to and in some cases even higher than AC of the studied plants from the family Lamiaceae (oregano, sweet balm, thyme, dead-

description

recipe for extract

Transcript of Blackberry Extract

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Antioxidant Capacity and Antioxidants of Strawberry, Blackberry, and Raspberry Leaves

Lucie BuřičoVá1, Mirjana ANdJeLkoViC 2, Anna čerMákoVá1, Zuzana réBLoVá1, ondřej Jurček 3,4,5, erkki koLehMAiNeN 4, roland Verhé 2

and František kVASNičkA6

1department of Food Chemistry and Analysis, 3department of Chemistry of Natural Compounds and 6department of Food Preservation and Meat Technology, Faculty of Food and Biochemical Technology, institute of Chemical Technology in Prague, Prague, Czech republic;

2department of organic Chemistry, Ghent university, Ghent, Belgium; 4department of Chemistry, Laboratory of organic Chemistry, university of Jyväskylä, Jyväskylä, Finland;

5institute of experimental Botany, Academy of Sciences of the Czech republic, isotope Laboratory, Prague, Czech republic

Abstrakt

BuřičováL.,AndjelkovicM.,ČermákováA.,RéblováZ.,JurčekO.,KolehmainenE.,VerhéR.,KvasničkaF.(2011):Antioxidant capacity and antioxidants of strawberry, blackberry, and raspberry leaves. CzechJ.FoodSci., 29:181–189.

Thetotalphenoliccontent(Folin-Ciocalteumethod),freeradicalscavengingabilityexpressedasDPPHvalue,ferricre-ducingantioxidantcapacity(FRAP),andoxygenradicalabsorbancecapacity(ORAC)weredeterminedinwaterextractsof leavesfromrosaceae familyplants(Fragaria vescaL.,rubus fructicosusL.,and rubus idaeusL.).Theantioxidantcapacitiesoftheextracts(intheorderoftheabovementionedmethods)were73.6–88.9%,60.1–71.4%,49.7–78.0%re-spectively,and45.3–66.5%ofthatofgreenteawaterextract.Further,thepresenceof15compounds(gallicacid,rutin,ellagic acid, caffeic acid, p-coumaric acid, quercetin, kaempferol, myricetin, quercetin-3-d-glucoside, ascorbic acid,(+)-catechin,(–)-epicatechin,epicatechingallate,epigallocatechin,procyanidinB1)wasstudiedbyHPLC-ECDandtheirantioxidantcapacitieswerecomparedtotheantioxidantcapacityoftheextracts.Outofthecompoundsstudied,mostly(+)-catechin,ellagicacid,and(–)-epicatechinparticipatedintheantioxidantcapacitiesofthestudiedplantleaveswaterextracts.Theantioxidantcapacityofleavesinfusions(determinedbyDPPHmethod)waslowerthanthoseofredwinesandteainfusions,butcomparabletotheantioxidantcapacitiesofwhitewinesandfruitbeverages.

Keywords:DPPH;Folin-Ciocalteaumethod;Fragaria vesca; rubus fructicosus; rubus ideaus; FRAP;HPLC;ORAC

SupportedbytheMinistryofEducation,YouthandSportsoftheCzechRepublic,ProjectNo.MSM6046137305,andbyspecificuniversityresearchofMinistryofEducation,YouthandSportsoftheCzechRepublic,ProjectNo.21/2010.

Inthescreeningstudycomparingtheantioxidantcapacities(AC)(measuredbyDPPHmethod)ofseventeenCzechmedicinalplants,theleavesofstrawberry(Fragaria vescaL.),blackberry(rubus fructicosusL.)andraspberry(rubus idaeusL.)

belongedtothemostefficientplantstested(Buři-čová&Réblová2008).TheACoftheirextractswerecomparabletoandinsomecasesevenhigherthanACofthestudiedplantsfromthefamilyLamiaceae(oregano,sweetbalm,thyme,dead-

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nettle,andmint),whichareknownasrichsourcesofantioxidants(Dormanet al.2004;Capeckaet al.2005).GoodACofthelistedplantsfromtherosaceaefamilywasalsoconfirmedbyotherarticles.Katalinicet al.(2006)determinedtheAC(ferricreducingantioxidantcapacity(FRAP))andphenoliccontentof70medicinalplants.Theleavesofraspberry,blackberry,andstrawberrywereamongelevenofthemosteffectiveplants.Inanotherstudy(Wang&Lin2000), theAC(oxygenradicalabsorbancecapacity(ORAC))ofleavesandberriesofstrawberry,blackberry,andraspberrywerecompared.Theleaveswerearichersourceofantioxidantsandhadagreatercontentofphenolicsubstancesthanberries.

However,fortheestimationofthepossibleanti-oxidanteffectin vivoisitimportanttoknownotonlytheACofherbextracts,butalsothecontentsofparticularantioxidantsandtheparticipationofthesecompoundsintheACofextracts.AlotofcompoundspossessingACwereidentifiedintheleavesofstrawberry,raspberry,andblackberryasprocyanidinB1,epigallocatechin, (+)-cate-chin,procyanidinB2,(–)-epicatechin,astringin,epicatechin-3-gallate,piceid,quercetin-4-glu-coside,trans-resveratrol(Mudnicet al.2009),ellagicacid(Gudej&Rychlínska1996;Gudej&Tomczyk2004;Skupien&Oszmianski2004;Hukkanenet al.2007),p-coumaricacid(Gudej2003;Skupien&Oszmianski2004;Hukkanenet al.2007),quercetin,kaempferol(Gudej&Rych-línska1996;Gudej2003;Gudej&Tomczyk2004;Skupien&Oszmianski2004),myricetin(Skupien&Oszmianski2004),gallicacid,agri-moniin,casuarictin,lambertianinC,potentillin,sanguiinH-10,nobotaninA(Hukkanenet al.2007),ascorbicacid(Wang1999),quercetin-3-o-β-d-glucopyranoside,quercetin-3-o-β-d-galacto-pyranoside,quercetin-3-O-α-l-arabinopyranose,kaempferol-3-o-β-d-galactopyranoside,kaempfe-rol-3-o-α-l-arabinopyranose(Gudej&Rychlín-ska1996;Gudej2003),quercetin-3-o-glucoside,rutin,quercetinglucuronide(Venskutoniset al.2007),andsomeotherphenylethanolderivativesofphenylpropanoidglucosides(Hanhinevaet al.2009).However,thecontentsofthesecompoundsinwaterextractsandinfusionshavenotbeenstudiedsufficiently,aswellastheirparticipationintheantioxidantcapacitiesoftherespectiveplants.

Withrespecttotheprevioustext,theaimofthisresearchwastovalidatethehighACofstrawberryleaves,blackberryleaves,andraspberryleaveswater

extractsusingfourdifferentmethodsappliedtothesamesamples.Fortheevaluationofthepossibleeffectsoftheextractsin vivo,quantificationoftheselectedphenoliccompounds(gallicacid,rutin,ellagicacid,caffeicacid,p-coumaricacid,quercetin,kaempferol,myricetin,quercetin-3-d-glucoside,ascorbicacid,(+)-catechin,(–)-epicatechin,epica-techingallate,epigallocatechin,procyanidinB1)inthestudiedextractswasdoneandtheparticipati-onofthesecompoundsinACoftheextractswascalculated.Further,theAC(measuredbyDPPHmethod)ofleaveswaterinfusionswerecomparedwithACofothersourcesofantioxidantsinhumandietsuchasgreenandblackteas,whiteandredwines,andfruit(vegetable)beverages.

MAteRiAL And MethodS

Materials.Threeleafsamplesofeachofthemedicinalplantsstudied,i.e.strawberryleaves(Fragaria vesca L.),blackberryleaves(rubus fru-ticosus L.),andraspberryleaves(rubus idaeus L.),werepurchasedfromaCzechproducerandmedicinalherbsdistributor(Natura,Děčín,CzechRepublic), frompharmacies(respectively),theplantshavingbeengrownintheareaoftheCzechRepublic.Greenandblackteas,redandwhitewi-nes,fruitandvegetablebeverageswerepurchasedinordinaryCzechshopsorspecialisedteashops.Allthesampleswereusedwithintherecommendedconsumptionperiod.

AllstandardsusedforHPLC,Folin-Ciocalteureagent,2,4,6-tripyridyl-S-triazine,ferroussul-phate,Trolox,2,2-diphenyl-1-picrylhydrazyl,andformicacid,andallchemicalsforelectrophoresiswerepurchasedfromSigma-AldrichChemie(Stein-heim,Germany).Sodiumcarbonate,AAPH,andfluoresceinwerepurchasedfromAcros(Geel,Belgium),sodiumchloridewasfromLachemaNeratovice,CzechRepublic.Organicsolventswereofanalyticalgrade,acetonitrilewasofHPLCgrade.AllsolventswerepurchasedfromMerck(Darmstadt,Germany).

Samples preparation.Forthewaterextractspreparation,theleavesofthemedicinalplants(orgreentea)weregroundand1gofthegroundleaveswasleft in50mlofdeionisedwaterforextractionduring20minutes.Thetemperatureofthewaterwas98°C.

Waterinfusionsofmedicinalplantsandgreenandblackteaswerepreparedasrecommendedby

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theproducers.Theamountofthesampleusedforinfusionanditsdurationwereadjustedaccordingtotheplanttypeinthefollowingmanner:1.5–2gofthemedicinalplantsper250mlwaterwithinfusionperiodof15min;2gofteaper150mlor250mlwaterwithinfusionperiodof2–4min(greentea)or3–5min(blacktea).

Afterthepreparation,theextractsandinfusionsweredilutedwiththerespecttothedetectionmethodusedandwereanalysedshortlyafterthepreparation.Redandwhitewinesandfruitandvegetablebeveragesweredilutedwithrespectthedetectionmetodused.

Total phenolics assay (Folin-Ciocalteu method, TPC).TotalphenolicscontentwasmeasuredusingtheFolin-CiocalteucolorimetricmethoddescribedbySingletonet al.(1999).Theabsorbancewasmeasured at 760 nm with Varian UV-VisibleSpectrophotometer(Cary50BIO,Mulgrave,Aus-tralia).Theresultswereexpressedasmgofgallicacidequivalentsper1gofdrysample.

Ferric reducing antioxidant capacity (FRAP).TheferricreducingabilitywasdeterminedusingtheassaydescribedbyBenzieandStrain(1996).Theabsorbancewasmeasuredevery10sduring30minreading(spectrophotometerCary100BioVarian,PaloAlto,USA).TheresultswereexpressedasmmolofFeSO4perlitre.

Oxygen radical absorbance capacity (ORAC).ThedeterminationofoxygenradicalabsorbancecapacitywasbasedonthemethodusedbyHuanget al.(2002).ThemeasurementswerecarriedoutonamicroplatefluorimeterSPECTRAmaxGeminixS(MolecularDeviceCorporation,Sunnyvale,USA)usingthe490nmexcitationand515nmemissi-onfilters.ThefluorescencewasrecordedeveryminuteaftertheadditionofAAPHuntilitsvaluewas<5%oftheinitialreading.TheresultswereexpressedasmicromoleTroloxequivalentsper1gofdrysample.

Free radical scavenging ability by the use of a stable DPPH radical.TotalantioxidantcapacitywasmeasuredusingDPPHmethod,whichpresentsradicalscavengingabilityofDPPH(2,2-diphenyl-1-picrylhydrazyl) radicals.ThismethodwasdescribedpreviouslybyGadowetal.1997,andwasmodifiedindetailbyBuřičováandRéblová(2008).Theabsorbanceat522nmwasmeasuredbyaVarianUV-VisibleSpectrophotometer(Cary50BIO,Mul-grave,Australia).Theresultswereexpressedasmgofascorbicacidper1gofdryplantmaterialorasmgofascorbicacidper100mlofthebeverage.

Determination of selected antioxidants.TheextractswereanalysedbyHPLC-RP-ECD,speci-ficallywithanamperometricdetectorHP1049A(HewlettPackard,Avondale,USA)equippedwithaglassy-carbonelectrode(operatingatapotentialof+0.8V),areferenceAg/AgClelectrodeandaplatinumcounterelectrode,andanisocraticnon-steelpumpLCP4020.31(ECOM,Prague,CzechRepublic).ThedatawererecordedusingaClari-ty2.6.4.402chromatographysystem(DataApex,Prague,CzechRepublic).ThecompoundswereseparatedonaRPC18column(HypersilODS:4.0mm×250mm,5μm;Agilent,USA)maintainedattheroomtemperature.Isocraticelutionwasperformedattheflowrateof1ml/min,usingamixtureofacetonitrileand0.13%(m/m)formicacidcontainingsodiumchloride(0.005mol/l)asamobilephase.Theproportionnofacetonitrileinthemobilephasedependedonthecompoundofinterest(5%acetonitrile(ACN):gallicacid;10%ACN:caffeicacid,p-coumaricacid,(+)-catechin,epigallocatechin,(–)-epicatechin,procyanidinB1;20%ACN:rutin,ellagicacid,quercetin-3-d-galactoside,quercetin-3-d-glucoside,(–)-epica-techingallate;30%ACN:quercetin,kaemferol,myricetin;0%ACNandchangeddetectionpotentialto+0.3:ascorbicacid).Theinjectionvolumewas20μl.Thesamples,standards,andspikedsampleswereanalysed.Thepeaksidentificationwasper-formedbythecomparisonoftheretentiontimeswiththoseofreferencestandards.

Isolation and confirmation of (+)-catechin.TheextractofstrawberryleaveswaspurifiedusingSPEC18(Supelco,USA),wherewaterwasusedforpurificationandmethanolfortheelutionofthesamplefromthecolumn.Thepurifiedextractwasconcentratedwithavacuumevaporator.ThesubsequentisolationofcatechinwasdonebyHPLC-RP-DADtechniqueusingUV/VISphotodiodearraydetectorSPD-M20A(Shimadzu,Kyoto,Japan),anautomaticsamplerinjectorSIL-10AP(Shimadzu,Kyoto,Japan),apreparativeLCpumpunitLC-8A(Shimadzu,Kyoto,Japan),afractioncollectormoduleFRC-10A(Shimadzu,Kyoto,Japan),andLCworkstationLabSolutions/LCsolutionversion1.2.ThecompoundswereseparatedonaRPC18column(HypersilODS:4.0mm×250mm,5μm;Agilent,SantaClara,USA)and10%acetonitrilecontained0.13%(m/m)formicacidwasusedasamobilephase.Theabsorbancewasreadat280nm.

Fortheconfirmationofcatechin,MSandNMRanalyseswereused.Themassspectrawererecorded

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onaBrukermicromassLCT.Thesamplesweredis-solvedinmethanoltoanappropriateconcentration,ionisedinapositiveandnegativeESImodes,andanalysedwithTOFdetector.NMRspectraweremeasuredinthesamplesdilutedindeuteratedwaterat303,thesolventsignalwassuppressed.1HNMR,protondecoupled(waltz-16)13CNMR,PFG1H,13CHMQC,andHMBCmeasurements(detailedacquisitionandprocessingparametersareavailableonrequest)werecarriedoutonaBrukerAvanceDRx500spectrometer(BrukerBioSpinAG,Fällanden,Switzerland)equippedwithaninversedetection5mmprobeheadandz-gradientaccessory.

EnantioseparationofcatechinwascarriedoutonaHewlett-Packard3DCEcapillaryelectrophore-ticsystem(HPSTLtd.,Prague,CzechRepublic)equippedwithadiodearraydetector(Konfiket al.2007).Theseparationwasperformedinafusedsilicacapillaryofthetotallengthof750mm(665mmeffectivelength,50μm i.d.),theconstantvoltageappliedtothecapillarywas+30kV.Thesampleswereinjectedbypressure(25mbarfor10s)andtheseparatedcompoundsweredetectedat280nm.Theelectrolytecontained63.4mMH3BO3+9.3mMNa2B4O7·10H2O(pH8.5)andchi-ralselectormaltosyl-β-cyclodextrin(0.5mM).

ReSuLtS And diSCuSSion

Antioxidant capacity of the plants water extracts determined using different methods

TheACofthewaterextractsoftheplantsstudiedasobtainedusingfourselectedmethods(TPC,DPPH,FRAP,ORAC)areshowninTable1.TheACgenerallydependsonthemethodusedandeventheresultsobtainedbythesamemethod

maynotbereproducibleindifferentlaboratories(forexampledependingonthepHofthereactionmedium,solventsused,calibrationstandardsorwayoftheresultsexpression)(Huanget al.2005;Roginsky&Lissi2005).Duetothis,itisdifficulttointerpretthedataofAC.Therefore,theACofthestudiedleaveswaterextractswerecomparedwiththeACofgreentea,whichisknownasarichsourceofantioxidant(Linet al.1996;Kris-Ether-tonet al.2002).TheACofwaterplantextractswere73.6–88.9% (TPC),60.1–71.4% (DPPH),49.7–78.0%(FRAP),and45.3–66.5%(ORAC)oftheACofgreenteaextract.Itisobviousthatwaterextractsofstrawberry,blackberry,andraspberryleavesshowverygoodACindependentlyofthemetodused.

Compounds participated on the AC of the plant extracts studied

Thecontentsoftheselectedantioxidantsinthestudiedextracts(togetherwiththeparticipationofthesecompoundsintheACoftheextracts)isshowedinTable2.Notallofthecompoundsstudied(i.e.gallicacid,rutin,ellagicacid,caf-feicacid,p-coumaricacid,quercetin,kaempferol,myricetin,quercetin-3-d-glucoside,ascorbicacid,(+)-catechin,(–)-epicatechin,epicatechingallate,epigallocatechin,procyanidinB1)wereidentifiedintheplantwaterextracts.Themainreasonforthisdisproportioncanbethewayofextraction.Thestudiedcompoundswereidentifiedinthepreviousstudiesintheextractspreparedusuallybyusingorganicsolventsasbenzene,chloroform(Gudej&Rychlínska1996;Gudej2003),ace-tone(Hukkanenet al.2007),methanol(Gudej&Tomczyk2004)orethanol(Venskutoniset al. 2007).However,fromthenutritionalpointof

Table1.ACofmedicinalplantswaterextractsdeterminedusingfourmethodsandtheircomparisonwiththeanti-oxidantcapacityofgreenteawaterextract

Medicinalplant DPPH FRAP ORAC TPC

Strawberry 110.1±16.6 23.3±1.4 1062.0±143.9a 62.4±1.0

Blackberry 125.2±2.9a 36.7±1.1 1304.3±232.4a 75.4±1.2

Raspberry 105.2±12.9 26.6±1.0 888.0±106.8a 68.9±5.6

Greentea 175.2±20.9b 47.0±2.4 1628.6±62.8 84.8±1.0

Dataareexpressedasmean±SD(n=3,an=4,bn=6);DPPHinmgascorbicacid/gofdrysample;FRAPinmmolFeSO4/l;ORACinμmolTrolox/gofdrysample;TPCinmggallicacid/gofdrysample

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view,wewereinterestedinthisstudyinwaterextractsinordertomimicthecommonprepara-tionofherbsinfusion.

Onthecontrary,forthefirsttime,inthisstudy(–)-epicatechin,(+)-catechin,andprocyanidinB1weredetectedinbothraspberryandblackberryleaveswhileepicatechingallatewaspresentonlyinblackberryleaves.Thesecompoundswereprevi-ouslydetectedonlyinstrawberryleaves(Mudnicet al.2009).

Thecontentsofthecompoundsvariedinthestudiedextractsandweredependentonthetestedplantsandevenontheindividualtestedsamples

ofthesameplant.Evenso,theAC(DPPH)oftheindividualextractspreparedfromdifferentsamplesofthesameplantdidnotlargelydeviate(Table2).Ellagicacidoccurredinsignificantamountsintheextractsofallsamples(ofalltestedplants).Itscon-tentrangedfrom10to35mg/l,whichcorrespondsto0.5–1.8mg/gofdryleaves.Intheextractsofstrawberryleaves,butnotinthoseofotherleavesstudied,(+)-catechinwasalsopresentinsignificantamountsrangingfrom29mg/lto99mg/l,whichcorrespondsto1.5–4.9mg/gofdryleaves.Thisismorethaninapreviousstudy(Mudnicet al.2009),where(+)-catechinwasdeterminedintheamount

Table2.Concentrationofantioxidantsinleaveswaterextractsandtheirparticipation(%AC)intotalantioxidantcapacitiesoftheextracts

Medicinalplant/compound c(mg/l) %AC c(mg/l) %AC c(mg/l) %AC

Strawberry sampleI(2202±332)*

sampleII(3295±357)*

sampleIII(2241±225)*

Haklicacid 1.2 <1 5.9 <1 2.0 <1

Ellagicacid 21.9±0.1 9.9 34.5±3.6 10.5 21.2±1.3 9.7

(+)-Catechin 45.6±2.4 10.1 29.8±12.2 4.4 98.8±9.9 21.6

Epigallocatechin 3.1 <1 4.1 <1 8.0±1.3 1.6

ProcyanidinB1 3.0 <1 11.8±0.2 1.8 5.4±0.8 1.4

Total >20.0 >16.7 >34.3

Blackberry sampleI(2504±58)*

sampleII(3112±110)*

sampleIII(3091±255)*

Gallicacid 1.4 <1 1.1 <1 1.6 <1

Ellagicacid 22.7±2.1 8.6 15.7±1.6 5.0 14.0±1.5 4.7

Quercetin-3-d-glucoside 6.2 <1 23.7 <1 21.8 <1

(+)-Catechine - - 2.6 <1 1.2 <1

(–)-Epicatechine 6.8±1.9 1.4 70.9±4.5 11.6 4.6 <1

Epicatechingallate 0.8 <1 3.6 <1 1.8 <1

ProcyanidinB1 8.5±0.0 1.8 2.8 <1 3.6 <1

Total >11.8 >15.6 >4.7

Raspberry sampleI(2104±258)*

sampleII(2181±463)*

sampleIII(1344±176)*

Gallicacid 1.2 <1 1.9 <1 1.5 <1

Ellagicacid 16.4±0.9 7.8 19.7±4.6 11.0 10.8±0.1 7.7

(+)-Catechine 0.6 <1 0.3 <1 0.5 <1

(–)-Epicatechine 3.6 <1 1.9 <1 1.5 <1

ProcyanidinB1 3.8 <1 3.7 <1 - -

Total >7.8 >11.0 >7.7

Thedataareexpressedasmean±SD(n=3forcompoundswith%AC>1);*Antioxidantcapacities(DPPH)ofleaveswaterextractsareexpressedasmeans±SD(n=3),mgascorbicacid/l

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of0.5mg/gofadrysampleofstrawberryleaves.Thecontentsofotherantioxidantsweregenerallylowerthan10mg/l(0.5mg/gofdryleaves)withtheexceptionsof(–)-epicatechin(71mg/l)inoneextractofblackberryleavesandprocyanidinB1(12mg/l)inoneextractofstrawberryleaves.

(+)-Catechinwasdetectedasthedominantcom-poundpresentintheextractsofstrawberryleaves(andinverysmallamounts inotherextracts).Therefore, itmaybeusedasamarker for thedistinctionofstrawberryleavespreparationsfromotherrelatedplantspreparations(ofblackberryandraspberryleaves).Consequently,(+)-cate-chin(Figure1)wasconfirmedintheseextractsbyMSandNMRaftertheisolation.ESI-TOFMSm/z(%):289.08(100),578.98(56),868.95(10),145.02(12),245.11(11).1HNMR(D2O,δppm):2.36(1H,dd,H-4a),2.75(1H,dd,H-4b),3.96(1H,m,H-3),4.47(1H,d,H-2),5.85(1H,s,H-8),5.95(1H,s,H-6),6.73(1H,d,H-5'),6.82(1H,d,H-6'),6.84(1H,s,H-2');13CNMR(D2O,δppm):26.89(C-4),66.75(C-3),80.96(C-2),95.08(C-8),95.94(C-6),100.56(C-10),115.09(C-2'),116.26(C-5'),120.11(C-6'),130.35(C-1'),144.07(C-4'),144.34(C-3'),154.83(C-9),155.07(C-5),155.14(C-7).TheseNMRresultswereconfirmedbyNMRdatapredictor(softwareACD/ChemSketchC+HNMRPredictorsandDB(Productversion10.04))andbycomparisonwiththepublishedresults(Mendoza-Wilson&Glossman-Mitnik2006).Enantiomerof (+)-catechinwasconfirmedbychiralelectrophoresis.

The contents of the compounds weres alsoexpressedas%oftheirparticipationinDPPHantioxidantcapacityintherespectiveextracts(Table2).DPPHmethodwasusedforthispurposebecauseitappearedtosuffertheleastsynergism

interferencesincomparisontoothermethods(Murakamiet al. 2003).Thestudiedcompoundsintheleavesofrosaceaeplantsmoderatelycon-tributedtotheantioxidantcapacityoftheirwaterextracts.Futureresearchshouldenlightentheremainingfractionsoftheantioxidantcapacitiestoofferacomprehensiveoverviewofthehealthandnutritionalvaluesoftheseplants.

Ofthecompoundsstudied,ellagicacid(mainly),(+)-catechin,and(–)-epicatechinparticipatedintheACofthewaterextractsofstrawberry,raspberry,andblackberryleaves.Ellagicacidisassumedtopossessantioxidant,anti-mutagenic,anti-inflam-matory,andcardioprotectiveactivities(Priya-darsiniet al. 2002).Catechinandepicatechinareantioxidantswhichaffectplasmaantioxidantbiomarkersandenergymetabolism(Williamson&Manach2005).Providedthatthesecompoundshavebeendetectedinthisstudy,theabovementi-onedmayimplyapossibleeffectofwaterrosacaeplantextractsorinfusionsin vivo.

Comparison of the antioxidant capacities of herbal infusions with other sources

of antioxidants

Antioxidantsarerecommendedtobeprimari-lyconsumedaspartofadailydiet(Halliwell2006).Therefore,theACofherbalinfusionswerecomparedtothecapacitiesofothersourcesofantioxidantsintheformsinwhichtheyaretakeninacommondiet(Figure2).Theselectedbevera-gesliketea,wine,vegetable,andfruitbeveragesarepreferredamongconsumersandsignificantlycontributetothetotalintakeofantioxidants(Svi-laaset al.2004;Pellegriniet al.2007).ThemethodwiththestableDPPHradicalwasusedforthedeterminationoftheAC.InFigure2,averyhighvariabilityoftheACinsidethegroupofwine,tea,andfruitbeveragesamplescanbeobserved.Thisvariabilityofbeveragesreflectsthedifferencesinthetypeoftheplantmaterial,typeofwine,localitiesandgrowingconditions,industryproceduresofbeveragespreparation,portionofrawmaterialinfruitandvegetablebeveragesetc.Moreover,someantioxidantsareaddedtosomefruitbeveragesduringtheproductionwhichmaycontributetothefinalresult.

TheAC(radicalscavengingcapacities)oftheplantsinfusionsstudiedaretypicallythreetimeslowerthanthecapacityofgreentea infusionsFigure1.Structureof(+)-catechin

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andtwotimeslowerthanthecapacityofblackteainfusions.Asmentionedabove,greenteaisprobablythemostefficientsourceofantioxidantsinourdiet(Linet al.1996;Kris-Ethertonet al.2002;Farhooshet al.2007).However,greenteaaswellasblackteacontainscaffeine(Farhooshet al.2007)andanexcessiveintakeofcaffeinecanberelatedtohypertension,dehydration,anxiety,orinsomnia(Gardneret al.2007),whereasnoadverseeffecthasbeenobservedondrinkingbeve-ragesmadefromleavesofstrawberry,blackberry,andraspberry.Therefore,thestudiedplantsmaybeseenasanattractivealternativesourceofanti-oxidantsforthegroupofconsumersconsciouslyselectingtheintakeofhigherlevelsofantioxidantswithoutcaffeine.

TheresultsofACinwineshowedthatredwinehadhigherACthanwhitewine.ThisagreeswiththereportofKatalinicet al.(2004).Moreover,besidesgreentea,redwineisseenasoneofthemostsignificantsourcesofantioxidants(Kata-linicet al.2004;deLangeet al.2007;Loganet al.2008).TheACofthestudiedplantinfusionswerecomparabletotheACofwhitewineandwere,onaverage,fourtimeslowerthanthatofredwine.However, inthecaseof“monitoringdribling”(whendailynomorethan100–200mlofwineisrecommended),twocupsofthetestedmedicinalplantsinfusiongiveusthesameamountofantioxidantslikeredwine.

TheACofthestudiedplantinfusionswerehigherorcomparabletothecapacitiesofdifferentfruit(vegetable)beverages.However,accordingtosomeauthors,fruitandvegetablebeveragescontributetothetotal intakeofantioxidantsthroughthediettoasmallerextentthanwine,tea,orcoffee(Svilaaset al.2004;Pellegriniet al.2007).

ConCLuSion

TheAC(TPC,DPPH,ORAC,FRAP)ofthestu-diedwaterextractsofstrawberry,blackberry,andraspberryleavesweredeterminedtobeintherangefrom49.7%to89.9%oftheantioxidantcapacityofgreenteawaterextract,andgoodanti-oxidantcapacitiesofthestudiedherbsinfusions(determinedbyDPPHmethod)wereobservedincomparisonwiththoseofteainfusions,wines,andotherbeverages.

Forthefirsttime(–)-epicatechin,(+)-catechin,andprocyanidinB1weredetectedinraspberryleaves,andallthesecompoundstogetherwithepicatechingallateinblackberryleaves,whereasbiologicallyactive(+)-catechin,ellagicacid,and(–)-epicatechinsignificantlyparticipatedintheACoftheleaveswaterextractsofallplantsstudied.Therefore,theanalysedmedicinalplantscanbeconsideredtobeagoodsourceofantioxidants.Theycanbeusedfordirectconsumptionasvariouskindsofbeveragesorasextractsofantioxidantstoincreasethenutritionalvalueofdifferentfoodsanddiets.

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Corresponding author:

Ing.LucieBuřičová,Vysokáškolachemicko-technologickávPraze,Fakultapotravinářskéabiochemickétechnologie,Ústavchemieaanalýzypotravin,Technická5,16628Praha6,ČeskáRepublikatel.:+420220445120,e-mail:[email protected]

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