Anthocyanincontent and antioxidant activity of various red lruit juices

LidijaJakobek#, Mariian Seruga, Martina Medvidovi6-Kosanovic and lvanaNovak DepartmentofApplied Chemistry and Ecology, Faculty of Food Technology,J.J. Strossmayer University ofOsijek, Kuhadeva I 8, HR-31 000 0sijek/Croatia

Summary im Bereichvon 4 pmolTroloxaquivalent/ml im SLlBkirschensaft biszu Anthocyaninc0ntent and antioxidant activity of variousred fruit juices 72umol TroloxdquivalenVml imAroniasaft. Eswurde eine starke Korrela- (blackcurrant, red raspberry,blackberry, sour cherry, sweet cherry, tionzwischen der antioxidativen Aktivitat und der Anthocyan-Konzentra- strawberry,chokeberry, and elderberry juice) has been evaluated inthis tionin den gepriiften Sdften aus Beerenfr0hten undKirschen lestgestellt. studyby usingHPLC, pH-differential, andDPPH method. AlleErgebnisse weisen darauf hin, dass Sdfte aus Beerenfrilchten und werethe predominant phenolic comp0nents (66% in elderberryjuice, Kirscheneine geeignete 0uelle an bioaktivenPhytochemikalien frirdie 56%in blackcurrant juice) or representedconsiderable portion in to- menschlicheErndhrung darstellen. Aronia-, Holunderbeer- undschwar- talpolyphenol content of somejuices (40% in blackberry juice, 33% in zerJohannisbeersaft enthalten die groBte Anthocyane-Konzentration und chokeberryjuice). Amount of anthocyanins determined byHPLC method zeigendie starkste antioxidative Aktivitiit Diese Safte sind daher zur Her- rangedfrom 202to 6287 mg I 1in strawberryand elderberry juice, re- stellungfunktioneller Lebensmittel geeignet. spectively.Anthocyanins present ininvestigated redfruit juices were de- rivativesofcyanidin, , andpeonidin. Chokeberry, Keywords:Anthocyanins, redfruit juices, berry juices, antioxidant activ- elderberry,blackberry and sour cherry juice were characterized bycyani- ity/ Anthocyane,Beerenf rtrchtesdfte, antioxidative Aktivitait dinderivatives, black currant juice by delphinidin and derivatives andstrawberry juice by pelargonidin derivatives. The major anthocyanins in redraspberry and sweet cherry juice were derivatives of cyanidin al- juice) though (in sweetcherry andpelargonidin derivatives Introduction (in redraspberry juice) were found in lowamount. Antioxidant activity variedfrom 4 Io 72 pnolTE/ml in sweetcherry and chokeberry juice, Considerableinterest has beendeveloped over the yearsin respectively,High correlation was observed between antioxidant activity fruits and vegetablesdue to their potentialbiological and andtotal c0ntent of investigatedredfruit juices. Overall re- health-promotingeffects. Numerous epidemiological sultsshowed that red fruit juices can serve as a goodsource of bioactive stud- phytochemicalsin human diet. Chokeberry, elderberry and black currant ies indicatethat an increasein the consumptionof fruits juicewere the richest in anthocyanin content and showed the strongest and i'egetablesis associatedwith a decreasein the incidence antioxidantactivity, as well. Therefore, these three juices can be regarded of variousdiseases like cardiovasculardisease. stroke. and asgood candldates forraw materials inproduction offunctional foods. cancerl5'. The protectiveeffect of fruits and vegetableshas beenattributed to their bioactiveanrioxidant constiruenrs, including vitamins, carotenoids,polyphenols'.o). Among Zusammenfassung variousantioxidants present in fruits and vegetables,poly- In dieserStudie wurden Anthocyane-Konzentration sowieantioxida- phenols (includinganthocyanins) have receivedmuch at- tiveAktivitiit von Fruchtsdfte aus verschiedenen BeerenfrUchte-sorten tention sincebeing reported to have a positive influenceon undKirschen (schwarze Johannisbeere, Himbeere, Brombeere, Sauer- humanhealthLr. undSilBkirschen, Erdbeere, Aronia und Holunderbeere) mittels HPLC, Anthocyaninsbelong to the classof phenoliccompounds. pH-DifferenzialundDPPH-Methode bestimmt. In einigenSiiften bilden They are water-solubleglycosides and acylglycosidesof Anthocyaneden groBten Anteil der Gesamtpolyphenole (2.B.66% im .The most commonnaturally occurring an- Holunderbeersaft,56% im schwarzenJohannisbeersaft), oderstellen thocyaninsare the 3-O-glycosides einenbedeutenden Anteil am Gesamtpolyphengehaltmancher Sdfte or 3,5-di-O-glycosides (40%im Brombeersaft,33% im Aroniasaft)dar. Die Anthocyane-Kon- of cyanidin,delphinidin, peonidin, , pelargonidin zentration,mittels HPLC bestimmt, variierte uon 202 mg/l im Erdbeer- and malvidinT).Anthocyanins are importanr polyphenolic saftbis 6287 mg/l im Holunderbeersaft. Beiden aus Beerenfrilchten und componentsof fruits,especiaily berries8-l3). They are potent Kirschenhergestellten Fruchsaften vorhandenen Anthocyanen handelt antioxidantsin uitrota)and may be protectiveagainst many essich um Cyanidin-, Delphinidin-, Pelargonidin-, undPeonidinderivate. degenerativediseasesT). Numerous studies have shown that Sowurden im Aronia-, Holunderbeere-, Brombeere- und Sauerkirschen- anthocyaninsare absorbedin their original glycosylated gefunden. saftCyanidinderivate lm schwarzenJohannisbeersaft wurden forms in humansl-s-18).They appearin urine after supple- Delphinidin-und Cyanidin-, im ErdbeersaftPelargonidinderivate gefun- mentation with berries or berry extracts but in very low den.Die im Himbeer-und SriBkirschensaft enthaltenen Anthocyane sind groBtenteilsCyanidinderivate, obwohl auch darin, aber nur in kleinen Mengen,Peonidinderivate (im SLiBkirschensaft) undPelargonidinderi- #Corresponding author, E-mail: [email protected] vate(im Himbeersaft) vorhanden sind. Die antioxidative Aktivitiit variierte Tel.:+385-31 -224-300, Fax: +385-31 -207-1 15

58r 0riginalarbeiten DeutscheLebensmitlel-Rundschau r 103.Jahrgang, H1II2, 2007 concentrationsri,r6,rs).Anthocyanins have also been found (Darmstadt/Germany)and ortho-phosphoricacid (85%, in human plasma in very low concentrationsls-l8r.Recent HPLC grade) was purchased from Fluka (Bnchs/Switzer- researchemphasized the importance of anthocyaninsin land). Hydrochloricacid (36.5o%),potassium chloride, deveiopmentof cancer therapy, becauseit was found that sodium acetatetrihydrate, Folin-Ciocalteaureagent were the anthocyaninscould decreasethe in uitro invasivenessof obtainedfrom Kemika (ZagreblCroatia).Trolox ((=)-6-hy- aancercellsle). droxy-2,5,7,8-tetramethylchromane-2-carboxylic acid ) and Besidesfruits and vegetables,a relevantpart of intake of 2,2-diphenyl-1-picrylhydrazylradical (DPPH ) were pur- polyphenolicphytochemicals (including anthocyanins)is chasedlrom Sigma-Aldrich(St. Louis, MOfuSA). suppliedby fruit juices.Juices are suitablefood products in terms of ingestionof health protectivephytochemicals. Samples Broactivecomponents may even be better absorbedfrom Fruits Iblackcurrant (Ribesnigrum), red raspberry(Rubus luicesthan from plant tissues,as it was demonstratedfor idaeus\,blackberry (Rubus fruticosus), sour cherry(Prunus ..rscorbicacid2O). Some studies have shown that the intakeof cerasus),slveet cherry (Prunus auium), strawberry (Fragaria ilntioxidantberry juice (containingwhite grape,black cur- anannassa),chokeberry (Aronia melanocarpa),and elder- rant, elderberry,sour cherry,blackberry and aronia juice) berry (Sambucusnigrdl) rvereharvested in Slavoniaregion can increaseplasma antioxidant capacity,which suggests (Croatia)at the commercialmaturity stage.Immediately af- :rn improvementin antioxidant statusand indicatesthat ter harvesting,fruits rverefrozen and storedat -20" C until antioxidant constituentsof the juice might decreaselipid analysis. oridation within plasmacompartment20). Consumption of polvphenolrich juice (containingapple, orange and mango Sampleprep(uation extr:lcts)enhanced antioxidant status,reduced oxidative For total anthocyanin,total polyphenolsand antioxidantac- DNA damageand stimulatedimmune cell functions2rr. tivity determination,fruits (-500 g) were thawed at room Thereis still not enoughknowledge about healtheffects of temperaturefor 30 min and then processedin juiceextrac- fruits,vegetables, juices or antioxidantconcentrates. Much tor (Juicer,Philips) in order to obtain natural fruit juice. more researchis thereforeneeded on the compositionof Three replicatesof juice (20 ml each)were centrifugedat antioxidantsin naturalantioxidant mixtures. iuices or fruits 4000 rpm for a t h. All luiceswere analysedthe sameday and on antioxidanteffects of theseproducts2rl. Many reports when they wereprepared. have beenwritten about the antioxidant activity and antho- cyaninprofiles of variousfruits or fruit extractss-t+,rr-r7),but D eterminationof tcttalpolyp h enols relativeiyfew havebeen based on red fruit juiceszs'ust.A need Total polyphenolswere determined by Folin-Ciocalteaumi- for suchdata still existsbecause of increasingpopularity of cro method'rOr.An aliquot (20 pl) of appropriatelydiluted redfruit juiceconsumption during the lasttime, and because fruit juice (red raspberry,blackberry, sour cherry, sweet of increasingconsumer awareness concerning the nutritional cherry, strawberry,1:1 (v/v); black currant 1:2 (v/v); el- valueof all foods(including these juices). derberry,chokeberry I:6 (vlv) with HrO) was mixed with Therefore,the objectiveof this work wasto evaluatevarious 1580 pl of distilledwater and 100 prlof Folin-Ciocalteau naturalred fruit juices(black currant, red raspberry,black- reagent.300 pl of sodiumcarbonate solution (200 g/l) was berrS sour cherry sweet cherry, strawberry chokeberry, addedto the mixture which was then shaken.After incuba- and elderberryjuice) regarding the amountof anthocyanins tion at 40'C for 30 min in water bath, absorbanceof the and antioxidantactivity and to determineindividual antho- mixture was read againstthe preparedblank at 765 nm. cyaniccompounds present in thesejuices. Furthermore, a Total polyphenolicswere expressedas mg of gallic acid concentrationof total polyphenolicswas measuredin order equivalents(GAE) per 1 of fruit juice. Data presentedare to determinethe portion of anthocyaninsin total polyphe- meana standarddeviation (SD). nol contentof redfruit iuices. D eterminationof total antho cy anins Total anthocyaninswere estimatedby a pH-differential Materials and methods methodrlr.Two dilutions of natural fruit juiceswere pre- pared,one with potassiumchloride buffer (pH 1.0) (1.86g Chemicals KCI in 1 I of distilledwater, pH valueadjusted to 1.0 with For this work, anthocyaninstandards cyanidin-3-O-gluco- concentratedHCI), and the other with sodiumacetate buf- sidechloride (kuromanin chloride), cyanidin-3-O-rutinoside fer (pH 4.5) (54.43g CH,CO.Na.3H.O in 1 I of distilied chloride (keracyaninchloride), delphinidin-3-O-glucoside water, pH value adjustedto 4.5 with concentratedHCI), chloride ( chloride), pelargonidin-3-O-glucosidediluting eachby the previouslydetermined dilution factor chloride (callistephinchloride) and peonidine-3-O-gluco- (sweetcherry 1:5 (v/v);strawberry 1:10 (v/v);red raspberry sidechloride) were purchasedfrom Extrasynthise(Genayl 1:20 (v/v),sour cherryand blackberry1:30 (v/v);black cur- France).Methanol (HPLC grade)was obtainedfrom Merck rant 1:25 (v/v);elderberry and chokeberry1:200 (v/v)).Ab-

DeutscheLebensmiltel-Rundschau r 103. Jahrgang, Hett 2,2007 0riginalarbeiten| 59 sorbance\\ras measured simultaneously :it 510 and 700 nm inner diameter,5 pm, Varian,USA) protectedwith guard after l-i min of incubationat room temperature.The con- column (ChromSep1 cm x 3 mm, Yarian,USA). The data tent of total anthocyaninswas expressedin mg of c,vani- were collectedand analysedon IBM computing sysrem din-3-O-glucosideequivalents (CGE) per I of fruit juice equippedwith Star Chromatographl.\Torkstation software usinga molar extinctioncoefficient (e) of cyanidin-3-O- (version-5.52). I I glucosideof 26 900 1 mol cm and molar weight (MW) Elr-rtionrvas emploved with a mobile phaseA consistingof r). (449.2g mol Data presentedare mean r standardde, 0..5% phosphoricacid in warerand mobilephase B consist- (SD). viation ing of 100% HPI-C grademethanol as follou,s:0-38 min ol, from 3 B to 65 "h B; from 38-45 min, 65 % B; with flow Determination of antioxida.ntactittitl, rate = 1 ml min-r. Operatingconditions were as follows: Antioxidantactiviry rvas determined using the 2,2-diphenvl- column temperature20"C; injectionvolumes, 10 pl of the 1-picrylhydrazyl(DPPH) mchod. In the DPPH merhode,. standardsand samples.A 10-minutere-equilibration period five dilutions of each fruit juice with two replicareswere was used betweenindividual runs. UV-Vis spectrawere re- analyzed.Reilction solution was preparedby mixing 50 prl corded in rvavelengthrange from 190-600 nn.r(detection of dilutedfruit juicervith 300 pl of methanolicDPPH solu- wavelengtl.rr,vas 520 nm). tion (1rnX'I)and brought to 3 ml with methanol.The solu- Identificationand peak assignmenrof anthocyaninsin all tion was kept in dark at room temperaturefor 15 minutes. fruit juicesn'as based on comparisonof their retentiontimes The absorban..(A,,,,..) r,vas read against the preparedblank and spectraldata (190-600nm) with thoseof aurhentic (-50pl dilutedfruit juice,29-50pl methanol)at 517nm. A standards.Additional identificationof anthocyanrnspres- DPPH blank solutionwas prepared(300 pl of 1mM DPPH ent in fruit juices'uvascarried out by spikingthe juiceswith solution,2.7 mI of methanol)and measureddaily. Percenr anthocyaninstandards. Anthocyanin profiles of fruit juices inhibitionof DPPH radicalwas calcularedfor eachdilution were comparedwith those found in literatures'l012't1'2r 2e1 of juiceaccording to fornrula: which gaveadditional information on anthocvaninidenti- fication.Calibration curves of the standardswere made by % inhibition = [(Ao.n,,-Ai,,..)/A,),,,,n)lx 100 diluting stock standardsin 0.1o1'HCI solutionprepared in rvhereA,,,,,,,,, is the absorbancevalue of the DPPH blarrkso- methanolto yield 1-100 mg I I (cyanidin-3-glucoside);0.5- lution, A is absorbancevalue of the samplesolution. 200 mg i r (cyanidin-3-rutinoside);5-100 mg I I (peonidin- ".. Calibrationcurve of Trolox was constructedby linear re- 3-glucoside);1-100 mg l1 (delphinidin-3-glucoside)and gressionof the absorbancevalue versusconcentration (0- 1-100 mg l-1 (pelargonidin-3-glucoside).Identified antho- 2500 pmol l-r).For eachdilution of fruit juice,antioxidant cyaninswere quantified using calibration curve of cyanidin- activitv'uvascalculated on the basisof the Trolox calibration 3-glucoside.Data presentedare mean * standarddeviation curveand expressedin pLmolof Trolox equivalent(TE) per (SD). ml of fruit juice.Percent of inhibition of DPPH radicalof eachdilution of fruit juice rvasplotted againstanrioxidant Analytical quality control activity(pnol Tt/rnl). Usingrhe curve obtaincd, antioxidant All anthocyaniccompounds showed a linearresponse within activitywas calculatedfor 50'% of DPPH inhibition.Final rangestudied 1r'= 0.9958-0.9997).Precision of method resultsare expressed as pmol of TE per ml of juiceneeded to was evaluatedby determiningwithin-day variation of the reduceDPPH radicalbv 50 %. HPI-C analysis(within-day precision-repeatability). To gain the data for studyingrepeatability, each juice samplewas HPLC artall'sisctf anthocyanins analysedthree times within one day. Coefficientsof varia- For HPLC analysis,fruits (--500g) (randomselection) were tion (CV) of peakareas varied between 0.04 and 5.95%. thawedat room temperaturefor 30 min and then processed Recoverieswere rreasured by adding known amounts in juiceextractor (Juicer, Philips) in order to obtain natural of standards(10-50 mg l') to fruit juicesprior to HPLC juice. fruit Three replicatesof juice (20 ml each)were cen- analysis.The recoveriesranp5ed from 80 to 92"t, for cyani- trifugedat 4000 rpm for a t hour and diluted1:3 (v/v)with din-3-glucoside,from 74 to 84 % for cyanidin-3-rutinoside, 0.1% solutionof HCI preparedin methanol.The samples from 90 to 95 "/ofor peonidin-3-glucoside,from 89 t<.t93"/,, were filteredthrough a 0.45 pm syringefilter (VariSepPTFE, for delphinidin-3-glucosideand from 95 to 101o/" lor pel- 0.45 prm, 25 mm-Varian) before they were injected into argonidin-3-glucoside.In the calculationof final results,no HPLC apparatus.All juiceswere analysedon HPLC system correctionfor recoverywas appliedto data.The following immediatell',the sirmeday when they were prepared. limits of detectionwere estimaredusing a signal-to-noise The analyticalHPI-C systemempioyed consisted of a Var- ratio of 3:0.64mg I I by cyanidin-3-glucoside,1.18 mg 1-1 ian LC system(USA) equipped r,vith a ProStar230 solvent by cyanidin-3-rutinoside,0.41 mg l-1 by peonidin-3-gluco- deliverymodule, ProStar 310 UV-VisDetecror, and ProStar side,0.35 mg l-' by delphinidin-3-glucosideand 0.92 mg l-r 330 PDA Detector.Anthocyanin compounds separauon by pelargondin-3-glucoside. 'nvasdone in an OmniSpherClu column (250 x 4.6 mm

60r 0rigrnalarbeiten DeutscheLebensmitlel-Rundschau I 103.Jahrgang, HeII 2, 2007 \t,ttictirnl nnnlv

DeutscheLebensmillel-Rundschau r 103.Jahrgang, HefI 2, 2007 Originalarbeitenr 61 Tab.2 Concentratlonsofanthocyanins innatural red fruit juices (mg/l),de- compoundspresent in red fruit juices,HPI,C merhodwas terminedbyHPLC method and percentage distribution 0fanthocvanins applied.The HPLC chromatogramsof red fruit juicesre- Juice ConcenlrationasCy-3- Totalanlhocyanins cordedat 520 nm are presentedin Figure 1. The amounts gluequivalents Img/l] I%l of anthocyaninsin red fruit juicesare shownin Table2. Chokeberry Chokeberry,elderberry, blackberry and sour cherry juices '1816.6 cy-3-gal t 4.9 68.9 containedonly cyanidinbased pigments. Chokeberry juice cy-3-glu 74.3x0.1 2.8 containeda mixture of four differentcyanidin-glycosides: cy-3-ara 647.1t 1.0 24.5 3-galactoside,3-arabinoside, 3-glucoside and 3-xyloside cy-3-xyl 99.8t 0.1 3.8 of cyanidin. Cyanidin-3-galactosideand cyanidin-3-ara- Tolal 2637.8 t 6.1 100.0 binosidewere found in high concenrrations(1817 mg I ,, r, Elderberry 647 mg 1 respectively,>93 "/" of total anthocyanlncon- cy-3-sam-5-glu 949.1r 9.9 15.1 tent) whereas the concentrarionsof cyanidin-3-xyloside cy-3-sam+ cy-3-glu 5226.6 t 18.9 831 (100 mg 1 r) and cyanidin-3-glucoside(74 mg I 1)were rela- cy-3-rut 111.7 t 5.9 1.8 tivelylow. The data presentedby otheraurhors are showing Total 6287.4 x 34.7 100.0 that the main anthocyaninsin chokeberryare cyanidin-3- Blackberry galactosideand cyanidin-3-arabinoside;the following are cy-3-glu 743.3t2.7 90.4 cyanidin-3-rylosideand cyanidin-3-glucosides't:,zs),which is cy-3-rut 10.8t 0.0 LJ consistentwith our results. cy-3-xyl 68.1t 1.2 8.3 The major anrhocyaninsin elderberryjuice were cyanidin Total 822.2x3.9 100.0 based Icyanidin-3-sambubiosideand cyanidin-3-glucoside SoutCherry '11.7 elutedtogether 15227 mg l'), cyanidin-3-sambubioside-5- cy-3-sopho t 0,8 39 glucoside(949 mg I r) and cyanidin-3-rutinoside(112 mg cy-3-glu-rut 1833 r 0.7 o t.4 l-1)1,with cyanidin-3-sambubiosideand cyanidin-3-glucoside cy-3-glu 106t0.3 3.5 cy-3-rut 93.0t 1.6 31.2 predominating(83 % of total anthocyaninamount). Data TotaI 298.6t 3 4 1000 presentedby other authors also confirmed that the major BlackCurrant anthocyaninsin elderberryar:e cyanidin-based, with cyani- din-3-sambubiosideand cyanidin-3-glucosidepredominat- dp-3-glu 222.3l-10.9 13.8 dp-3-rut 706.4t 389 43.9 ings't:.r8.rvrlike in our study. cy-3-glu 99.9t L7 6.2 Cyanidin-3-glucosidewas the major anthocyaninin black- cy-3-rul 579.5t 12.3 36.0 berryjuice (743 mgl-t, 90"/o of total anthocyanincontenr) Total 1608.1r 63.8 99.9 whereasthe concentrationsof cyanidin-3-xyloside(68 mg 1) Strawberry I and cyanidin-3-rutinoside(11 mg I r) wereconsiderably cy-3-glu 7.3x0.7 3.6 lower.Previous study confirmed that the main anthocyanin pg-3-glu 159.2t 2.0 78.7 in blackberryis cyanidin-3-glucoside(from 44 ro 95"/, of pg-3-rut 359t0.0 17.7 total peakarea in variousblackberry samples)23r. Cyanidin- Total 202.4x2.7 100.0 3-rutinosideranged from tr:aceamount to 53"/o,whereas RedRaspberry cyanidin-3-xyloside,cyanidin-3-malonylglucoside and cy- cy-3-sopho 239.4t2.8 79.8 anidin-3-dioxalylglucosidewere deteced but at much lower pg-3-sopho 18.0t 0.5 6.0 concentrationsl3r. cy-3-glu 42.5x0.7 t1.l Sour cherry juice contained-3-glucosylrutinoside (183 mg Tolal 299.9t 4.0 100.0 I t,61 .47n), -3-rutinoside (93 mg l-t, 31.2y"), -3-giucoside SweetCherry (11 mg i-1,3.5 "/") and-3-sophoroside (12 mg l-r,3.9./"1of cy-3-glu / r.ot t.c 20.2 cyanidin.These data are in accordancewith thosefound in cy-3-rut 252.9x 8.0 71.2 litera t ure2a'2'i'2 e). pn-3-glu 16.0t11 AE Black currant juice was characrerizedby the presenceof pn-3-rul 14.7t 0.2 4.1 rutinosidesand glucosidesof delphinidin and cyanidin Tolal 355.2t 10.8 100.0 with the rutinosidesbeing the most abundant(delphinidin- 3-rutinoside706 mg l-t, cyanidin,3-rutinoside580 mg l-t; 79.9% of total anthocyanincontent). The data reported in the literatureare showingthat the main anthocyaninsin black currant are rutinosidesand glucosidesof delphinidin and cyanidin8'12,r4.28'2e)which agrees with our results. (18" ), sourcherry (18 %), sweercherry (16%) and sfraw- Pelargonidinderivatives predominated in strawberryjuice berry juice(16"1) was considerablylower (Tab. 1). (pelargonidin-3-glucoside,159 mg l-r, pelargonidin-3-rutin- '). In order to separareand determineindividual anthocyanic oside,36 mg I Theseanthocyanins represented rogerher

62r0riginalarbeiten DeutscheLebensmittel-Rundschau r 103.Jahrgang, Heft 2, 2007 > 96"/" of total anthocyaninamount. The concentration of cyanidin-3-glucosidewas low (7 mg 1,, 4oA).Previous 80 studiesconfirmed that strawberry is characterizedby the 70 =0.9745*** samepelargonidin and cyanidinglycosides as in our study and that pelargonidinglycosides predominare in srraw- 360 berryt t'zot. 850 F The main anthocyaninfound in red raspberryjuice, which E+o represented79.8 % of total anthocyanincontent, was cyani- din-3-sophoroside(239 mg l-'). Cyanidin-3-glucosideand 3go +r pelargonidin-3-sophorosidewere found in relativelylower amount (43 mg l-r, 18 mg l-1,respecively). Previous study tzo confirmed that anthocyaninspresenr in red raspberry(early 10 cultivar Heritage) are cyanidin-3-sophoroside,cyanidin-3- 0 glucosideand pelargonidin-3-sophorosidewhich is in ac- 2000 4000 6000 8000 10000 cordancewith our results2T).However, anthocyaninprofiles Total polyphenols(mg/l) of various raspberrycultivars are very heterogenous;the main red raspberryanfhocyanins are cyanidin and pelargo- 11,27'2er rridin derivatives but in some late cultivars 80 and delphinidinderivatives were found aswell27). The dominant anthocyaninin sweetcherry juice was cyanl- 70 r0.94J4*** din-3-rutinoside(253 mg l-t), with cyanidin-3-glucosidebe- ^60 ing second(72 mg l-r).These two anthocyaninsrepresented rogether o=so 91,.4% of total anrhocyaninamount, while the F concentrationsof peonidin-3-rutinosideand peonidin- E40 3-glucosidewere low (15 mg l-1, 16 mg l-1,respectively). e30 The data reported in the literature are showing that sweet F cherry containscyanidin, peonidin and pelargonidinderiva- t20 tivesl0'11).The major anthocyaninsare cyanidin-3-rutinoside 10 and cyanidin-3-glucosidelike in our study, while 3-rutino- sideand 3-glucosideof pelargonidinand peonidinare pres- 0 ent but at much lower concentrationsr0'11). 1000 2000 3000 4000 5000 In order to evaluateantioxidant activiry of chosenred fruit Total anthocyanins(mg/l) juices,DPPH assaywas applied and the resultsare pre- Fig.2 Correlationplots of DPPH values versus total polyphenol andtotal sentedin Table 1. All investigatedjuices exhibited potent anthocyanincontents ofred fruit juices. The correlation coefficients (r) radical scavengingactivity. The strongestradical scavenging aremarked ineach plot; significant correlations aremarked with asterisks activity showed chokeberry,elderberry, and black currant (... =significance atP < 0.001) juice (72 pmol TE/ml, 62 pmol TE/ml pmol, 30 pmol TE/ml respectively)while activities of other juices were consider- ably lower (sour cherry 13 pmol TE/ml; blackberry 9 pmol (r = 0.95ooo)but the correlationcoefficient was lower for TE/ml; red raspberry8 pmol TE/ml; strawberry4 pmolTE/ total anthocyaninsversus antioxidant activity than for the ml; sweetcherry 4 pmol TE/ml). There are already a num- total polyphenoisversus antioxidant activity. According to ber of reports on the antioxidant activity of berry exrracrs the data presentedby others, the best linear relationship by severalmethods such as oxygenradical absorbancecapa- was observedbetween antioxidanr capacity and total poly- citytztot DPPH radical scavengingcapacitya'r) indicating phenolsof variousred fruits (r2= 0.95) than betweenantr- that chokeberry,elderberry and black currant possesssrrong oxidant capacityand total anthocyanins(r2 = 0.95;tzt*hi.6 antiradical activities. Antioxidant activity of chokeberry agreeswith our results. juice concentrateagainst DPPH radical was stronger than Red fruit juices evaluatedin this srudy are showing signifi- antioxidant activity of black curranr, elderberry,red cur- cant variations in anthocyanin content and profile. Some rant, strawberrS red raspberryand cherry concentrate2s). juices have low amounr of anthocyanins,like strawberry The correlation betweenantioxidant activity measuredby and red raspberryjuice, whereasthe amount of thesephy- DPPH methods and total polyphenolsand total anthocya- tochemicalsis very high in chokeberry,elderberry and black nins are presentedin Figure 2. The concentrationof total currant juice. Anthocyanins are the predominant phenolic polyphenolswere found to correlate with the antioxidant componentsin elderberryand black currant juice or re- activitiesof juices(r = 0.97*"o). The concentrationof ro- present significant part in total polyphenol concentratron tal anthocyaninscorrelate with antioxidant acrivity as well of chokeberryand blackberryjuice. Every juice evaluated

DeulscheLebensmittel-Rundschau r 103.Jahrgang, Hett2, 2007 0riginalarbeitenr63 possessunique anthocyanin pattern. ChokeberrS elder- of anthocyaninsbyHPLC-ESl-MS/MS in common ioods in theUnited berr.v,blackberry and sour cherryjuice contain only cyani- States:fruits and berries. J.Agr. Food Chem. 53, 2589-2599 (2005). 12) Wu,X., L. R.L. Prior,and McKay. of din basedpigments, black currant juiceis characterizedby Gu, S. Characterizationanthocyanins andproanthocyanidins insome cultivars oI Ribes,Aronia, and Sambucus delphinidinand cyanidin,and strawberryjuice by pelargo- andtheir antioxidant capacity. J.Agr. Food Chem. 52, 7846-7856 (2004). nidin derivatives.The major anthocyaninsin red raspberry 13) Kim,D. 0., H. J. Heo,Y. J. Kim,H. S. Yang,and C. Y Lee:Sweet and sour and sweetcherry juice are derivativesof cyanidinalthough cherryphenolics and their protective effects on neuronal cells. J. Agr. peonidin (in sweetcherry juice) and pelargonidinderiva- FoodChem. 53, 9921-9927 (2005) tives (in red raspberryjuice) are presentin low amount. 14) Klihkdnen,M. P, J. Heinamaki,V.0llilainen, and M. Hetnlnen:Berry an- thocyanins:isolation, identification andantioxidant activities. J.Sci. Food Although all juicescan serveas a good sourceof bioactive Agr.83, 1403-141 1 (2003). phytochemicalsin the human diet, chokeberry,elderberry 15\Netzel, M,, G. Strass, M. Herbst, H. Dietrich, R. Bitsch,and T.Frankfhe and black currant juicestand out in high anthocyanincon- excreti0nand biological antioxidant activity of elderberryantioxidants in tent and high antioxidant activity.From the view of the healthyhumans. Food Res, Int.38,905-910 (2005). anthocyanincontent and antioxidant activity thesejuices 16) Mulleder,U., M. Murkovic,and W. Pfannhausen Urinary excretion of cya- glycosides. (2002) can be regardedas good candidatesfor raw materialsin nidin J.Biochem. Biophys. Methods 53, 61-66 17) Miyazawa,T..K. Nakagawa, M.Kudo, K. Muraishi,and K.Someya:Direcl productionof healthbeneficial functional foods. Moreover, intestinalabsorption ofred fruit anthocyanins, cyanidin-3-glucoside and delphinidin-3-glucoside,delphinidin-3-rutinoside and cy- cyanidin-3,5-diglucoside,intorats and humans. J.Agr. Food Chem. 47, anidin-3-glucosidewere reported to havestrong antiradical 1093-1091(1999). activity among various anthocyaninsr2r.Therefore, black 18\ Milbury,P. E.. G. Cao, B. L. Prior, and J. Blumberg:Bioavailability of el- currant (which is abundantin delphinidinderivatives) and derberryanthocyanins. Mech. Ageing Dev, 123, 997-1006, (2002) 19\ Chen,P.N., S. C.Chu, H. L. Chiou,W. H. Kuo,C. L. Chiang,and Y S. blackberry (abundant in cyanidin-3-glucoside)juice can Hsleh:Mulberry anthocyanins. cyanidin 3-rutinoside andcyanidin 3-glu- good serveas sourceof theseindividual anthocvanins. coside.exhibited aninhibitory effect 0n the migration and invasion ofa humanlung cancer cell line. Cancer Lett.235, 248-259 (2006). Acknowledgements 20) Netzel,M., G. Strass,C. Kaul.l. Bitsch,H. Dietrich,and R. Bitsch:ln This work was supportedby the CroatianMinistry of Sci- vivoantioxidative capacity 0fa compositeberry juice, Food Res. Int. 35, (2002). ence,Education, and Sports(Project number 0113006). 213-216 21\ Bub,4.,B. Watzl,M. Blockhaus,K. Briviba,U. Liegibel, H. Mitller, B. L. Pool-Zobel,and G. Rechkemner Fruil iuice consumption modulates an- References tioxidantstatus, immune status and DNA damage. J.Nutr. Biochem. 14, 1) Kris-Etherton,P M., K. D. Hecker, A. Bonanome,S.M. Coval, A. E. Binko- 90-98(2003). ski,K E Hilpert,A. E. Griel, and T. D. Etherton: Bioactive compounds in 22)Heinonen, L M., and A, S. Meyer. Antioxidants infruits, berries and ve- foods:their role in the prevention ofcardiovascular disease and cancer. getables,lnJongen, lzl (ed.): Fruit and vegetable processing, pp.23-51, Am,J Med.113,71-88(2002) WoodheadPublishing Limited, Cambridge (2002). 2) Joshipura,K. J., F.B. Hu, J. E.Manson, M. J. Stampfer,E. B. Rimm,F. 23)Fan-Chiang, H.J., and R. E. Wrolstad. Anthocyanin pigment composition E.Speizer, G. Colditz, A. Ascheiro, B. Rosner, D. Spiegelman, and W. C. ofblackberries. J.Food Sci.70, C19B-C202 (2005). Willefffhe effect of fruit and vegetable intake on risk for coronary heart 24) Blando,F., C. Gerardi, and l. Nicoletti.Sour cherry (Prunus cerasus L.) disease.Ann. Intern. Med. 134, 1106-1114 (2001). anthocyaninsasingredients forlunctional foods. J. Biomed. Biotechnol. 3) Knekt,P, J. Kunpulainen,B. Jlirvinen.,H. Rrssanen,M. Heli1vaara,A. 5,253-258 (2004) Reunanen,and T. 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Introducing natural antioxidants. In Po- Forsch.A208, 33-38 (1 999). korny,J., N. Yanishlieva, andM. Gordon (eds.): Antioxidants infood, pp. 28\ Bermildez-9ltl,M., and F. A. Tomds-Barberdn'.Evalualion of commercial 147-158,Woodhead Publishing Limited, Cambridge (2001). redfruit juice concentrates asingredients forantioxidant functional jui- 7) Clifford,M tV.:Anthocyanins-nature occurence and dietary burden. J. ces,Eur. Food Res. Technol. 219, 133-141 (2004). SciFood Agr. 80, 1063-1072 (2000). 29) Goiffon,J., P.P Mouly,and E. M. Gaydou.Anlhocyanic pigment deter- 8) Nakajima,J., L Tanaka,S. Seo,M. Yamazaki,and K. Saito: LC/PDA/ESl- minationin red fruit juices, concentrated juices and syrups using liquid MSprofiling and radical scavenging activity ol anthocyaninsinvarious chromatography.Anal.Chim. Acta 382, 39-50 (1 999). berries.J.Biomed. Biotech nol. 5, 241-247(2004\. 30) Waterhouse,4.;(May, 1Oth 2006). http:/iwaterh0use.ucdavis,edu/phenol/ 9) Benvenuti,5.,E Pellati,M. Melegar| and D. Bertelli: Polyphenols, antho- folinmicro,htm cyanins,ascorbic acid, and radical scavenging activity oI Rubus,Ribes, 31\ Giusti,M. M.,and R. E. Wrolstad:Anthocyanins. Characterization and andAronia. J,Food Sci,69,164-169 (2004). measurementwithUV-visible spectroscopy. ln Wrolstad, B.E (ed.):Cur- 10)Mozetii, 8., andP Trebie:ldenlitication ol sweet cherry anthocyanins rentprotocols infood analytical chemistry, pp Fl.2.1 -F1 .2.13. Wiley, andhydroxycinnamic acids using HPLC coupled with DAD and MS NewYork (2001 ). detector.Acta Chim. Slov. 51, 151-158 (2004) 32)Kdhkonen, M.P., and M. Heinlnen. Antioxidant activity of anthocyanins 11) Wu,X., and R. L. Prior.Systematic identilication and characterization andtheir aglycons. J.Agr. Food Chem. 51, 628-633 (2003)

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