Corvallis, OR97331-6602.Directinquiries toauthor Technology, State Univ., 100 Wiegand Hall, Wrolstad arewiththeDept.ofFood Scienceand Accepted 9/26/03. MS 20030394Submitted7/11/03,Revised 9/26/03, I A. C of FreshandProcessedCherries Anthocyanin andPolyphenolicComposition Wrolstad (E-mail: 3-sophoroside havebeenidentified insour noside, cyanidin-3-glucoside,and cyanidin- nidin-3-glucosylrutinoside, cyanidin-3-ruti- Mazza 1995;Mozeticandothers2002).Cya- (Lynn ry andLuh 1964;Gao and been identifiedinBingandothersweetcher- side, andpelargonidin-3-rutinosidehave peonidin-3-rutinoside, peonidin-3-gluco- anidin-3-rutinoside, cyanidin-3-glucoside, and sourcherrieshavebeenidentified.Cy- Seeram andothers2002). and others1999c; Wang andothers2000; byinvestigated Michigan workers (Wang erties ofsourcherrieshavebeenextensively prop- antioxidant The 2003). and Wrolstad their antioxidantproperties(Chaovanalikit phenolic contentsofcherriesalongwith gated theanthocyaninpigmentandtotal 2002). Ourlaboratoryhasrecentlyinvesti- ram andFuhrman2002;BorsMichel and others1995;Porter andothers2001;Avi- ers 1993;Halliwellandothers1995;Hortog and ellagic-acidderivatives(Amesoth- anidins, flavonolglycosides,phenolicacids, anthocyanin pigments,flavan-3-ols,procy- tenoids andpolyphenolics,whichinclude cribed tophytochemicalssuchascaro- and dementia.Thesehealthbenefitsareas- disease, heart cancer,ing coronary stroke, of severaloxidativestressdiseases,includ- fruits andvegetableswillreducetherisk t iswidelyacceptedthatadietrichin The majoranthocyaninpigmentsinsweet ABSTRACT: ( The distribution ofanthocyaninpigmentsandpolyphenolicsin1sourcherry polyphenolics weretransferredtothesyrupwithcanning,andnearlyallbrineduringbrining. affected byprocessingandstorage,whereasflavonolglycosideswerequitestable.Halfoftheanthocyanins sweet ,whereasflavanolsareinthemajorityMontmorencycherries.Hydroxycinnamatesweregreatly qualitatively withrespecttotheminoranthocyanins.Hydroxycinnamatesaremajorclassofpolyphenolicsin detection. Changesduringfrozenstorage,canning,andbriningwerealsomonitored.Sweetcherrycultivarsdiffered sweet ( HAOVANALIKIT Keywords: anthocyanins,polyphenolics,canning,brining,frozenstorage Introduction [email protected]).

Authors Chaovanalikitand avium

AND R.E. W ) cultivarswasdeterminedbyhigh-performanceliquidchromatographywithdiodearray ROLSTAD sour cherries. sweet andsourcherries andprocyanidinsin (1998) foundtheflavanolepicatechin inboth side ofquercetinincherries.Friedrich andLee quercetin andkaempferolthe rhamno- cherries andidentifiedthegalactoside of sides ofkaempferolandquercetin fromsweet Herrmann (1980)isolatedthe3,4 glucoside (tentative).Henningand galactoside (tentative),andkaempferol-4 4 (1973b) reportedthepresenceofquercetin- 1980). Inaddition,ShrikhandeandFrancis Francis 1973b;HenningandHerrmann (Schaller and Von 1970; Shrikhande and Elbe by severalworkersinsweetandsourcherries of quercetinandkaempferolbeingreported polyphenolics, therutinosidesandglucosides sides areanotherimportantclassofcherry and Lee1998).Flavonolsflavonolglyco- cherries hasalsobeenreported(Friedrich ries and4 caffeoyltartatric acidinsweetandsourcher- Mozetic andothers2002).Thepresenceof (Gao andMazza1995;FriedrichLee1998; nic acidhavebeenidentifiedinsweetcherries Neochlorogenic acidand3 maric acidinMontmorencycherries. coumaroylquinic acid,caffeicand mers ofcaffeoylquinicacid,4isomers Schaller and Von Elbe(1970)identified6iso- polyphenolics thatarepresentincherries. Chandra andothers2001). zos 1970;ShrikhandeandFrancis1973a; present (Schallerand Von 1968;Deka- Elbe tinoside havealsobeenreportedtobe anidin-3-gentiobioside, andpeonidin-3-ru- 1992); cyanidin-3-arabinosylrutinoside,cy- cherries (Dekazos1970;Chandraandothers Ј glucoside, kaempferol-3-rhamnoside-4 Hydroxycinnamates areamajorclassof Ј - p -coumaroylquinic acidinsour Ј - p -coumaroylqui- Ј -bigluco- p -cou- p Ј Ј - - - frozen storage,canning,andbrining. tatively andquantitativelyaffected with would liketomonitorhowthey werequali- tion inskins,flesh,andpits.Inaddition,we sour cherriesandtomeasuretheirdistribu- thocyanins andpolyphenolicsinsweet antioxidant properties. tionship betweenpolyphenolicsandtheir cessing mayhelptounderstandtherela- polyphenolic classesareaffectedbypro- ing howindividualcompoundsand total phenolicsshowedlittlechange.Know- idant propertiesactuallyincreased,while all 3parameters.Insomeinstances,antiox- 2004). Processingclearlyhadanimpacton peratures (Chaovanalikit and Wrolstad ning andbriningstoredatfreezingtem- when sweetcherriesareprocessedbycan- properties ofsweetcherriesarealtered anins, totalphenolics,andantioxidant ceutical orantioxidantuse. tential sourceofpolyphenolicsfornutra- cherry-processing wasteandcouldbeapo- reported. Pitsinparticularareasubstantial classes inskins,flesh,andpitshasnotbeen individual phenolicsandpolyphenolic Ann andMontmorency. of The distribution Bing, andothersdonot,forexample,Royal pigmentation intheflesh,forexample, kit and Wrolstad 2003).Somevarieties have amounts varytremendously(Chaovanali- anthocyanin pigmentationintheskins, Standards Our objectivesweretoidentifythean- We recently reported how totalanthocy- Although allcherrycultivarshavesome Phenolic standards (protocatechuicacid, Materials andMethods Prunus cerasusPrunus ) and3

Food Chemistry and Toxicology HCl were add- Millipore filter N M Chemstation soft- HCl was added to ␮ 2D N The saponification was performed by the A high-performance liquid chromatogra- Acid hydrolysis was performed by the pro- The ethyl acetate fraction containing the The ethyl acetate fraction paper (Type HA, Millipore Corp., Bedford, Corp., HA, Millipore paper (Type Mass., U.S.A.) before high-performance liq- uid chromatography (HPLC) injection. ware (Waldbron, Germany) was used with si- ware multaneous detection at 520 nm for antho- cyanins, saponified anthocyanins, and anthocyanidins and at 260, 280, 320, 370, and 520 nm for saponified phenolics and phenolic characterization and quantifica- ed to purified anthocyanins (about 1 mL) in a screw-cap test tube, flushed with nitrogen, The purified compound was and capped. °C and then hydrolyzed for 30 min at 100 The hy- immediately cooled in an ice bath. drolysate was purified by solid-phase extrac- cartridge (Waters tion using a C-18 Sep-Pak Assoc.) as previously described. Alkaline hydrolysis of anthocyanins and polyphenolics Wrol- and Durst as described by procedure or polyphe- The anthocyanin stad (2001). nolic isolate (about 1 mL) was saponified in a screw-cap test tube with 5 mL of 10% at room tempera- for 8 min in the dark KOH Then, 5 mL of 2 ture. was The hydrolysate the solution. neutralize purified by solid-phase extraction using a C- cartridge (Waters Assoc.) as de- 18 Sep-Pak scribed earlier. HPLC analytical system phy Perkin Elmer Series 400 (Norwalk, Conn., U.S.A.), equipped with a Hewlett- Packard 1040A photodiode array detector and Gateway 2000 P5-90 computer with Hewlett-Packard HPLC Acid hydrolysis of anthocyanins Wrolstad and Durst as described by cedure (2001). Five milliliters of 2 adsorbed onto a C-18 Sep-Pak mini-column C-18 Sep-Pak onto a adsorbed U.S.A.), Mass., Assoc., Milford, (Waters water-sol- acids, and other whereas sugars, by washing were removed uble compounds 0.01% aque- with 10 mL of the mini-column was Then, the mini-column ous HCl (v/v). were nitrogen gas. Polyphenolics dried with with 5 mL of ethyl ac- subsequently eluted were eluted with 5 mL etate. Anthocyanins of acidified methanol. methanol fraction polyphenolics and the were concen- containing the anthocyanins a Büchi rotavapor-R trated to dryness using N.Y., Westbury, Instruments, (Brinkmann and an- The polyphenolics U.S.A.) at 40 °C. in acidified thocyanins were redissolved °C. Samples were –70 water and stored at filtered through a 0.45- for 20 min by g

ϫ Royal Ann and Bing Royal Ann The purification of anthocyanins and Anthocyanins and polyphenolics were Samples were liquid nitrogen powdered Brined cherries. Brined cherries. Anthocyanin and polyphenolic purification polyphenolics was conducted as described (2001). Wrolstad and Rodriguez-Saona by The aqueous extract (about 2 mL) was passed through a C-18 Sep-Pack cartridge U.S.A.), Mass., Assoc., Milford, (Waters which had been previously activated with methanol followed by 0.01% aqueous HCl (v/v). Extraction of anthocyanins and polyphenolics Pow- Blender. Waring using a stainless-steel dered samples (about 10 g) were blended with 20 mL of acetone, sonicated (Ultrason- Cleaning Equipment Branson ic cleanser, Corp., Shelton, Conn., U.S.A.) for 10 min, and then filtered on a Büchner funnel using Whatman nr 1 paper (Whatman Clif- Inc., ton, N.J.). The filter cake was re-extracted with 10 mL of 70% acetone (30% water and 70% acetone, v/v) twice. Filtrates were com- bined, mixed with 80 mL of chloroform, and then centrifuged at 170 IEC Intl. Centrifuge (Model UV, Intl. equip- Intl. UV, (Model Centrifuge IEC Intl. ment Co., Boston, Mass., U.S.A.). The aque- ous phase was collected and evaporated in vacuo at 40 the residual acetone res- °C until idue was removed (about 10 min). The frac- tion was made up to 25 mL with acidified water and stored at –70 °C until further an- alyzed. Sample extractions were replicated twice. immersed in water and heated at 100 at 100 and heated in water immersed °C for cooled by canned fruits were 12 min. The in 25 placing them and then stored °C water canning experiment °C. The °C and 22 at 2 twice. was replicated were each stems (about 1 kg) cherries with The bisulfite brine was placed in 4-L glass jars. jars until the added to the fruit-containing brine solution fruits were covered. Bisulfite 220 g of sodium was prepared by dissolving calcium chloride, and metabisulfite, 202 g of a This gave L of water. 10 g citric acid in 10 2.2% sodium met- solution, which was chloride, and 0.1% abisulfite, 2% calcium 3.0. Plastic The pH was about citric acid. jars to allow for air wrap covered the glass fruitThe brined was stored for 12 diffusion. cherries were °C. After 12 mo, mo at 22 5 washed with running cold water for about d, until sulfur dioxide content of cherries Washed was less than 200 parts per million. brined cherries were frozen with liquid nitro- The spent brine °C. at –70 gen and stored solution was also stored at –70 °C. - p Stemmed Bing cher- Stemmed Bing cherries Frozen cherries. Canned cherries. Cherry samples (250 g) were separated by Rainier sweet cherries were provided by Rainier sweet cherries ries (2.5 kg) were washed, destemmed, and Pitted pitted using a hand cherry pitter. cherries were filled into cans nr 303 with dark fruit enamel. Sucrose syrup (C&H Su- perfine sugar diluted to 19 °Brix with water, C&H Sugar Co., Crockett, Calif., U.S.A.) was added, and the filled cans were heated to a temperature of 80 °C. Cans were exhausted on a steam bath for 2 min 30 s and sealed with nr 303 lids by Canco 006 Steam-flow Can seamer (Pegasus Food Machinery were U.S.A.). Cans Nev., City, Carson Corp., Cherry processing (2 kg) were washed, destemmed, and pitted Pitted a household hand cherry pitter. by cherries were then frozen in liquid nitrogen. Samples (120 g) were packaged into 12 plas- tic Nalgene containers (Nalge Nunc Intl., U.S.A.), closed, and stored N.Y., Rochester, °C. °C and –70 at –23 hand into skins, flesh, and pits. The skins, flesh, and pits. hand into skins, flesh, and pits were frozen separately in liq- °C uid nitrogen, weighed, and kept at –70 pre- The samples were analysis. until further pared in 2 replications. Fresh cherries Fresh the Mid-Columbia Experiment Station, the Mid-Columbia cher- varieties of sweet Two Ore. River, Hood ries, Bing and Royal Ann, and 1 variety of harvested were sour cherries, Montmorency, Or- Farm, Horticultural at the Lewis Brown Cor- Dept. of Horticulture, Univ. egon State vallis, Ore. Oregon Cherry Growers, Inc., Salem, Ore., supplied Bing and Royal Ann cherries for the brining experiment and also provided Bing cherries for canning and fro- zen storage experiments. Cherries were de- Science and to the Dept. of Food livered Corvallis, Univ., State Oregon Technology, °C before Ore., and then were stored at 2 sample preparation or processing. Sources of cherry samples Sources of cherry coumaric acid, and quercetin-3-rutinoside) coumaric acid, Chemical Co. Sigma from purchased were Quercetin-3-gluco- U.S.A.). Mo., Louis, (St. kaempfereol- side, quercetin-3-rhamnoside, and kaempferol-3-rutinoside 3-glucoside, (Genay, Extrasynthese from purchased were juice concentrate France). Concord grape U.S.A.) Mass., Concord, Inc., Food (Welch local supermarket, was purchased at a rasp- red black currant, cranberry, whereas and strawberry juice concentrates berry, Concentrates Inc. were provided by Kerr (Salem, Ore., U.S.A.). chlorogenic acid, epicatechin, caffeic acid, caffeic acid, epicatechin, acid, chlorogenic Cherry anthocyanin and polyphenolic composition . . . . composition polyphenolic and anthocyanin Cherry

Food Chemistry and Toxicology strawberry juice concentrates. obtained fromacid hydrolysisofgrapeand and retentiontimeswith6anthocyanidins matching UV-visiblewas madefrom spectra from 10%to30%Afor20min.Identification used. Theprogramwasalinear gradient described previouslyforanthocyanins, were guard column(Alltech).SolventsAandB,as sphere 10mm inner dia(Phenomenex),fittedwithanAll- ODS-3 column(5 standards. with knownanthocyaninsfromfruitjuice ing UV-visible spectraandretention times 5 min.Identificationwasmadefrommatch- and alineargradientfrom20%to40%Afor ear gradientfrom0%to20%Afor15min, gram wasisocraticat0%Afor5min,alin- inwater. (v:v:v) and 5%acetonitrile The pro- phosphoric acid,10%aceticacid(glacial), grade acetonitrileandsolventBwas1% Ill., U.S.A.).SolventAwas100%HPLC- dia ODS-2guardcolumn(Alltech,Deerfield, with anAllsphere10mm (Phenomenex, Torrance, Calif., U.S.A.)fitted column (5 nins wereseparatedusingaProdigyODS-3 HPLC separationofanthocyanidins and saponifiedanthocyanins HPLC separationofanthocyanins The flowratewas1mL/min. as thatforpolyphenolicanalysiswas20 for anthocyaninanalysiswas50 Wilmington, Del.,U.S.A.).Injectionvolume autosampler (Agilent Series Technologies tion. SampleswereinjectedbyAgilent1100 b a ACN CultivarPortion morency Table 1—Relativeanthocyanincompositionsofskins,flesh,andpits4cherrycultivars:Bing,RoyalAnn,Rainier, andMont- Cherry anthocyaninandpolyphenoliccomposition... Bing Skins 60.6 nd nd 19.0 19.0 nd nd 60.6 Skins Bing ane kn . 1.17 2.1 3.42 Skins 2.2 Rainier Skins AnnFlesh0.1ndndndRoyal nd ndndndnd mrnyFeh00n dn dn dn nd nd nd nd nd nd nd nd 0.0 Skins Flesh morency Mont- Each valueintableismean Anthocyanin contentwerereportedasmgcyanidin-3-glucoside/100gfreshweight(fw)(ChaovanalikitandWrolstad2003). Anthocyanidin separationusedaProdigy Anthocyanins andsaponifiedanthocya- Flesh 26.0 nd nd 36.7 36.7 nd nd 26.0 Flesh Flesh0.0ndndnd nd ndndndnd ␮ is02n d12.3 nd 51.4 nd nd 0.2 nd Pits 10.4 Pits Pits 0.1 nd nd 8.13 8.13 nd 13.0 nd 0.8 Pits 0.1 Pits m) 250mm ϫ 4.6-mm innerdiaODS-2 ␮ m) 250mm 36.5 0.97 0.97 36.5 ϫ a Ϯ 4.6-mm innerdia ohrsd ti sophoroside ϫ standard deviation( yndn3 gl Cyanidin-3- 4.6-mm inner 4.6-mm ␮ ϫ Ϯ Ϯ Ϯ Ϯ L, where- 4.6-mm 00 d2.51 nd 3.34 0.09 nd 0.14 10 61.7 1.06 63.0 0.03 ␮ yndn3 Pel Cyanidin-3- L. ucosylru- oiegl noside n = 2); nd = not detected. nd =not =2); 0.07 HPLC-grade acetonitrilewhileSolventBwas guard column(Alltech).SolventAwas100% sphere 10mm inner dia(Phenomenex),fittedwithanAll- with authenticstandards(whenavailable). the UV-visible spectra andretention time min. Identificationwasmadebymatching 3 min,andfinallyisocraticat50%Afor5 then alineargradientfrom25%to50%Afor linear gradientfrom5%to25%Afor30min, program wasisocraticat5%Afor5min,a was 1%aceticacidindeionized water. The HPLC-grade acetonitrile,whereassolventB guard column(Alltech).SolventAwas100% as quercetin-3-rutinoside(rutin) at260nm. catechin at280nm,andflavonol glycosides nm, epicatechinandprocyanidins asepi- standard methodaschlorogenicacidat320 cinnamates werequantifiedbytheexternal ent from35%to55%Afor5min.Hydroxy- to 35%Afor27min,andthenalineargradi- at 5%Afor3min,alineargradientfrom etry (ESMS).The instrumentwasaPerkin obtained usingelectrospray massspectrom- gy ODS-3column(5 Synergi Hydro-RP(4 sphere 10mm inner dia(Phenomenex)fittedwithanAll- polyphenolics HPLC quantificationof and saponifiedpolyphenolics HPLC separationofpolyphenolics Electrospray massspectrometry centrated H Ϯ Ϯ 08 10.5 0.81 1.65 0.83 Low-resolution massspectrometry was Phenolics wereseparatedusingaProdi- Polyphenolics wereseparatedusinga M KH yndn3 yndn3 di Cyanidin-3- Cyanidin-3- 2 PO 3 csd rutinoside ucoside PO 4 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ adjustedtopH2.4withcon- ϫ ϫ 4 07 78.8 0.76 02 93.6 0.21 59.1 0.73 87.5 0.06 30.8 55.9 2.43 1.97 07 11.5 0.73 28.7 0.10 56.2 0.54 . Theprogramwasisocratic 4.6-mm innerdiaODS-2 4.6-mm innerdiaODS-2 ␮ ␮ % totalpeakareaat520mn m) 250mm m) 250mm Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 09 0.33 0.96 11 dn d28.6 nd nd 1.76 8.52 nd nd 5.70 nd 0.21 nd 1.18 nd 0.04 0.34 3.07 2.43 18 dn d35.7 nd nd 1.55 0.76 nd 0.89 0.53 1.85 ϫ ϫ 4.6-mm 4.6-mm glucoside roi eagn-Poii-Peonidin- Peonidin- Pelargoni- argoni- --dn3 -3- 3- din-3- n-3- Ϯ Ϯ Ϯ Ϯ 01 dn 1.80 nd nd 0.12 00 0.54 0.03 00 0.30 0.03 00 dn 4.88 nd nd 0.02 100- tions wereintroducedintotheESMSbya thocyanin andpurifiedpolyphenolicfrac- operated inthepositivemode.Purifiedan- voltage = 60). Themassspectrometer was an ionsprayinterface(ISV = 4000, orifice Ont., Can.) equippedwith lyzer (Thornhill, Elmer SCIEXAPIIIIbimolecularmassana- od ( models wascarriedoutusing Tukey’s meth- cant treatment levelmeansinallANOVA The pairwisecomparisonbetweensignifi- the factorsasfollows:timeandtemperature. tent, a2-factorANOVA modelwasusedwith canned storageonindividualphenoliccon- temperatures. To evaluate theeffectof and cannedcherriesat2levelsofstorage levels ofthefactorsasfollows:freshcherries a 1-factorANOVA modelwasusedwiththe of canningonindividualphenoliccontent, ature andstorage time. To evaluate theeffect ries andthecombinationoffrozentemper- model wasused,thefactorbeingfreshcher- vidual phenoliccontent,a1-factorANOVA storage onanthocyaninpeakareaandindi- Wash., U.S.A.). To assesstheeffectoffrozen variance usingS-Plus4.5(MathSoft,Seattle, tion wereseparatelyanalyzedbyanalysisof anin peakareaandpolyphenoliccomposi- sition inskins,flesh, andpitsofcherries. antioxidant activitiesandphenolic compo- used todeterminethecorrelation among lyze separately. The Pearson correlation was variety,each cherry a brined cherriesorspent solutionfor thocyanin peakareainfreshcherries and Statistical analyses fusion pumpattherateof12.55 Effects ofprocessingoncherryanthocy- P ␮ b

uioieguoierutinoside glucoside rutinoside Յ L glasssyringeconnectedwiththein- 0.05). To compare themeanofan- Ϯ Ϯ Ϯ Ϯ 00 0.23 0.03 00 d0.92 nd nd 0.08 nd 0.24 1.34 0.03 t testwasusedtoana- Ϯ Ϯ Ϯ 00 1.11 0.02 07 9.29 0.75 5.42 0.42 ␮ L/min. Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 0.20 0.20 0.05 0.91 0.67 0.20 0.21 1.75

Food Chemistry and Toxicology The polyphenolics were separated from Polyphenolic composition of Polyphenolic composition cherries the anthocyanins by solid-phase extraction on C-18 resin using ethyl acetate. Polyphe- nolics are eluted with ethyl acetate, whereas the anthocyanins remain absorbed until being removed by acidified methanol. A large number of peaks were separated by HPLC as illustrated by Figure 2 and 3, showing HPLC anthocyanin in Bing pits was cyanidin-3-glu- pits was in Bing anthocyanin much 51%). Pits contained coside (about of peonidin-3-glucoside higher proportions Anthocyanins and peonidin-3-rutinoside. in the flesh of Montmoren- are not present Skins cherries. Ann, and Rainier Royal cy, concentra- anthocyanin have the highest also found in the pits. tion, but pigments are skins and pits is The distribution between containing less cyani- different with the pits more cyanidin-3-glu- din-3-rutinoside and the distribution is differ- Moreover, coside. being ent with cyanidin-3-sophoroside proportions in present in much higher skins, whereas cya- Montmorency pits than is not present in Rain- nidin-3-sophoroside ier and Royal Ann pits. Gao and Mazza (1995) and Mozetic and (1995) and Mozetic Gao and Mazza Bing is the only containing an- Figure 1—HPLC anthocyanin profile of Bing skins (a), flesh (b), and pits (c) at 520 Figure 1—HPLC anthocyanin profile of Bing skins (a), flesh (b), and pits (c) at 3 = pelargonidin-3-glucoside; 2 = cyanidin-3-rutinoside; 1 = cyanidin-3-glucoside; nm. 6 = peonidin-3-rutinoside 5 = peonidin-3-glucoside; 4 = pelargonidin-3-rutinoside; knowledge, this is the 1st report on the an- report on the is the 1st this knowledge, and Roy- composition of Rainier thocyanin al Ann cherries. of identified the anthocyanins others (2002) cy- as cyanidin-3-rutinoside, sweet cherries pelargonidin-3-rutino- anidin-3-glucoside, and peonidin- side, peonidin-3-rutinoside, was not 3-glucoside. Peonidin-3-glucoside sweet cherries detected in the light-colored Sour cherries con- (Gao and Mazza 1995). and cyani- tained cyanidin-3-sophoroside along with most din-3-glucosylrutinoside in sweet cherries ex- anthocyanins present (Dekazos cept for pelargonidin-3-rutinoside 1990a; Chandra Wrolstad and 1970; Hong and others 1992). Cyanidin-3-arabinosylru- in Balaton cherries tinoside has been found 2001). (Chandra and others thocyanins in skins, flesh, and pits, with the skins being richest in anthocyanin concen- the HPLC 1 shows 1). Figure (Table tration anthocyanin profiles for Bing skins, flesh, and pits. The distribution pattern differs the major anthocyanin of with considerably, skins and flesh being cyanidin-3-rutinoside (about 79% and 56%), whereas the major Results and Discussion and Results Eight different anthocyanins were iden- anthocyanins were Eight different Cyanidin-3-rutinoside is the major an- Anthocyanin composition of composition Anthocyanin cherries 1). and sour cherries (Table tified in sweet matching are based on Peak assignments times with spectra and retention UV-visible fruit juices that have anthocyanins from cyanidin-3-gluco- been well characterized: and peonidin- side, cyanidin-3-rutinoside, (Slimestad 3-rutinoside from blackcurrants and Solheim 2002); cyanidin-3-sophoroside from red and cyanidin-3-glucosylrutinoside 1987); Wrolstad and (Spanos raspberries and pelargonidin- pelargonidin-3-glucoside (Bridle and 3-rutinoside from strawberries and peonidin-3-glu- García-Viguera 1997); Wrolstad and cranberries (Hong coside from 1990b). HPLC analysis of the acid hydroly- sis products of the anthocyanin isolates showed cyanidin to be the major anthocya- nidin with trace amounts of peonidin and pelargonidin. Saponification showed no change in the anthocyanin pigment profile, indicating that none of the anthocyanins was acylated with cinnamic or aliphatic or- ganic acids. In addition, electrospray mass spectra confirmed the identities of cyani- din-3-glucoside (m/z 449), cyanidin-3-ruti- noside (m/z 595), cyanidin-3-sophoroside (m/z 611), cyanidin-3-glucosylrutinoside (m/z 757), pelargonidin-3-rutinoside (m/z 579), and peonidin-3-rutinoside (m/z 609). The % relative intensity of minor pigments was insufficient to get definitive mass spec- tra. These assignments confirmed identifi- Wagen- (Li and workers previous cations by 1964; Dekazos and Luh knecht 1958; Lynn 1970; Shrikhande and Francis 1973a; Chan- dra and others 1992; Gao and Mazza 1995; and others and others 1997b; Mozetic Wang 2002) with the exception that this is the 1st report for the presence of pelargonidin-3- glucoside. thocyanin in sweet cherries, with cyanidin- 3-glucoside being second. Sour cherries have a very different profile with cyanidin- 3-glucosylrutinoside being the major pig- ment and cyanidin-3-rutinoside the 2nd largest pigment. This is consistent with pre- 1990a; Wrolstad and (Hong vious reports Gao and Mazza 1995; Chandra and others 2001; Mozetic and others 2002). The 3 sweet cherry cultivars vary with respect to trace anthocyanins. Cyanidin-3-sophoroside was found in Royal Ann and Rainier cherries but not in Bing cherries. Pelargonidin-3-gluco- side and peonidin-3-glucoside were found in Bing cherries but not in Royal Ann and Rainier cherries. Peonidin-3-rutinoside was our To Ann cherries. not detected in Royal Cherry anthocyanin and polyphenolic composition . . . . composition polyphenolic and anthocyanin Cherry

Food Chemistry and Toxicology phenolics werereportedasmggallicacid/100gfreshweight(fw). capacity (ORAC) reducingantioxidant andferric inaprevious power (FRAP)values, reported whichwere publication (Chaovanalik 7 =kaempferol-3-rutinoside; HC =unidentified hydroxycinnamates. 6 =quercetin-3-glucoside; 5 =quercetin-3-rutinoside; 4 =epicatechin; nm. 1 =neochlorogenic acid; 2 =3 Figure 2—HPLCpolyphenolic profileofBingskins(a),flesh(b),andpits(c)at280 acterized astocompoundclassaccording polyphenolics, respectively. Peaks were char- chromatograms ofBingandMontmorency e d b a — 185.0 Edible Bing Cultivar and FRAPinskins,flesh,pitsofsweetcherriessour Table 2—Total phenoliccontent,%area quantified,hydroxycinnamates, epicatechin,procyanidins, flavonolglycosides,ORAC, Cherry anthocyaninandpolyphenoliccomposition. oa n dbe29— 229 Edible Royal Ann ane dbe7 033 . .548292 498 1.15 7.2 3.35 30 — 75 Edible Rainier otoec dbe47— 407 Edible Montmorency Oxygen radical absorbance capacity(ORAC) andferric as reducingantioxidant power reported (FRAP)were % Areadenotesthepercentageoftotalpeakareathatwasmeasured,forexample,unidentifiedpeaksaccounts6.9%th Amount intheedibleportion(skinsplusflesh)wascalculatedfromproportionateweightofskinsandfleshexceptfortota Each valueintablerepresentsmean b oto TP Portion kn 3 99.6 333 Skins kn 5 98.4 351 Skins kn 4 93.8 142 Skins kn 5 98.4 558 Skins ls 0 97.1 301 Flesh 93.1 134 Flesh ls 7 97.0 176 Flesh ls 590.3 65 Flesh is1770.6 157 Pits 65.2 92 Pits is1463.2 104 Pits is5 68.1 54 Pits c Ϯ standard deviation( % Area Ϯ Ј Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ - p- 00 144 0.03 1355.7 1.3 48.2 0.9 0712.7 0.7 8.53 3.5 0.3 196. 196. 0.3 0383.0 0.3 8514.5 8.5 3172.1 3.1 0828.4 0.8 085.58 0.8 01139 0.1 omryqii cd = chlorogenic acid; = coumaroylquinic acid;3 d yrxcnaae Epi Hydroxycinnamates by matchingretentiontimewithauthentic stances specificcompoundswereidentified their UV-visible spectra, andinsomein- m hooei (mg (mg chlorogenic cd10g 10g /100 g) /100 acid/100 g) n 6882 d4.06 nd 8.29 56.8 701. 023.72 20.2 11.9 87.0 821. 9511.2 49.5 19.6 58.2 = 2); nd = not detected. nd =not =2); Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 11.79 19.6 19.6 11.79 72 18.5 7.21 22 50.5 2.24 04 17.7 0.45 7.71 6.03 02 8.15 0.23 2.51 2.02 10 13.5 1.02 12 2.70 1.28 26 4.70 2.63 03 3.77 0.37 03 1.22 0.38 pctci (mgepicatechin epicatechin aehnPoyndn Fl Procyanidins catechin Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 10 d17.2 nd 1.07 12 50.3 108 1.25 2.61 1.59 nd 0.70 03 22.5 0.38 4.70 0.49 24 24.6 2.49 05 25.3 0.52 02 7.34 0.25 07 0.97 0.75 04 9.90 0.44 01 4.83 0.17 a ␮ moles Trolox equivalent/100 gfw. Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ der notbeingidentified orquantitated. were 65.2%ofthetotalpeakarea, theremain- nonanthocyanin polyphenolics inBingpits 99.6% ofthetotalpeakarea,whereas the cyanin polyphenolicsinBingskins represent ple, thepeakareasmeasuredfor nonantho- the measuredpeaksisalsolisted.Forexam- percentage oftotalpeakarearepresenting because ofitsnutritionalrelevance.The edible portion(skinplusflesh)isalsoshown cultivars. Thepolyphenoliccontentforthe for skins,flesh,andpitsofthe4different pares amountsofthedifferentpolyphenolics asrutin). sides (determined Table 2com- mined asepicatechin),andflavonolglyco- acid), epicatechinandprocyanidins(deter- ycinnamates (determinedaschlorogenic for bothsweetandsourcherrieswerehydrox- Mozetic andothers2002). and others1999a; Wang andothers1999b; Mazza 1995;Friedrich andLee1998; Wang ature (Schallerand Von Elbe 1970;Gao and previous identificationsreportedintheliter- These peakassignmentsareconsistentwith confirmed byElecrospraymassspectra. (465) inBingandMontmorencyskinswere well asthepresenceofquercetin-3-glucoside side (m/z611)insweetandsourcherriesas tinoside (m/z595),andquercetin-3-rutino- roylquinic acid(m/z339),kaempferol-3-ru- chlorogenic acid(m/z355),3 side. Thepresenceofepicatechin(m/z291), tin-3-glucoside, andkaempferol-3-rutino- genic acid,quercetin-3-rutinoside,querce- standards, forexample,epicatechin,chloro- 40 63.2 4.00 51 d10 1381 1500 nd 5.13 18 d97848 997 nd 1.86 504 594 nd 0.81 27 20.64 2.75 11 d11 903 1310 nd 1.13 08 d58498 568 nd 0.88 02 8.49 0.27 04 d42227 462 nd 0.44 05 d38200 338 nd 0.56 )(grtn10g ORAC (mgrutin/100g) g) All sweetcherry skins contained9hydrox- The majorclassesofphenoliccompounds l phenolics,oxygenradicalabsorbance glycosides e totalpeakareaforBingflesh. it and Wrolstad 2003).| avonol Ϯ Ϯ Ϯ Ϯ Ϯ 15 862105 2826 1.51 41 124796 5102 4.17 02 0 728 907 0.21 09 00592 1050 0.96 31 741708 2744 3.19 441590 1494 491553 1449 573756 2557 c Ј Total - p- e couma- FRAP e

Food Chemistry and Toxicology coumaroylquinic acid; p- - Ј Three flavonol glycoside peaks in sweet glycoside peaks Three flavonol skins. Cherry pits contained high proportion contained Cherry pits skins. Procyanidin (29% to 52.5%). of procyanidins cherries identified in sour B2 has been and Lee 1998). (Friedrich as querce- were identified and sour cherries quercetin-3-glucoside, tin-3-rutinoside, which agrees and kaempferol-3-rutinoside, Francis (1973b), with Shrikhande and and Wang Elbe (1970), and Von Schaller and and others (1999b). Quercetin-3-rutinoside were found in all kampferol-3-rutinoside cultivars with querce- sweet and sour cherry found only in Bing tin-3-glucoside being Henning and Her- and Montmorency skins. that the gluco- rmann (1980) also reported of both quercetin and sides and rutinosides in sweet cherries. kaempferol were present and quercetin-3- Kaempferol-3-glucoside rhamnoside have been reported for Mont- Elbe Von cherries (Schaller and morency 1999b), but we did and others Wang 1970; not detect these compounds in our study by comparing retention time with authentic standards. There were additional unidenti- fied peaks with characteristic flavonol spec- -coumaroylquinic acid (24.8% and acid (24.8% -coumaroylquinic p - Ј Epicatechin was identified from match- was identified from Epicatechin by match- Procyanidins were identified 3 = chlorogenic acid; = epicatechin; 4 5 = quercetin-3-rutinoside; 6 = querce- tin-3-glucoside; 7 = kaempferol-3-rutinoside; HC = unidentified hydroxy- cinnamates; P = unidentified flavanols; F = unidentified flavonols. Figure 3—HPLC polyphenolic profile of Montmorency skins (a), flesh (b), and Figure 3—HPLC polyphenolic profile of Montmorency skins (a), flesh (b), 3 = neochlorogenic acid; 2 = pits (c) at 280 nm. 1 18.5%), chlorogenic acid (8.95% and 4.72%), acid (8.95% 18.5%), chlorogenic and 3.49%). acid (8.15% and neochlorogenic time and retention spectra ing UV-visible was present standard. It with an authentic epicatechin The presence of in all cultivars. others (Friedrich and was in agreement with 2000). Epicatechin Lee 1998; Arts and others in cherries, is another major polyphenolic and in skins, being found in all cultivars the hydroxycinnama- flesh, and pits. As with concentrations tes, skins had the highest contained slightly and the edible portion more than the flesh alone. with epicatechin spectra ing UV-visible 1996). There were (Bartolomé and others with respect to pro- large cultivar differences cyanidins. No procyanidins were detected in Bing skins and flesh, whereas Royal Ann contained substantial quantities in both skins and flesh. Rainier contained traces of procyanidins in skins and moderate amounts in the flesh. Montmorency was particularly high in procyanidins in the were 3 were - p - Ј -couma- p -coumaric - Ј p -caffeoylquinic acid) Ј coumaric acid. These p- -coumaroylquinic acid. p - Ј -coumaroylquinic acid (6% to 10%, 13% p Montmorency skins contained 7 hydrox- Skins contained the highest concentra- - Ј to 18%, and 7% to 19%), and chlorogenic acid (4% to 5%, 1% to 12%, and 1.1% to 1.4%). ycinnamate peaks, 6 flavanol peaks, and 4 flavonol glycoside peaks (Figure 3), the ma- jor peaks being identified as 3 roylquinic acid (peak 2), chlorogenic acid (peak 3), and neochlorogenic acid (peak 1). The identification is in agreement with liter- Elbe Von identifications (Schaller and ature 1970; Friedrich and Lee 1998). The major hydroxycinnamates in sour cherry skins, flesh, and pits were different. Of the quan- tified non-anthocyanin polyphenolics, the major hydroxycinnamates in sour cherry skins were chlorogenic acid (14.1%), 3 and peak 2 as 3 previous reports (Gao This is consistent with and Lee 1998; and Mazza 1995; Friedrich Gao and Mazza Mozetic and others 2002). (1995) and Mozetic and others (2002) report- ed that the major phenolic of sweet cherries was neochlorogenic acid, whereas Friedrich and Lee (1998) reported that the major phe- nolic in sweet and sour cherries was caffeoyl- tartaric acid, which was not evident from ESMS mass spectra data in our investigation. The remaining 6 peaks with spectra charac- teristic of hydroxycinnamates were mea- sured as hydroxycinnamates. tions of hydroxycinnamates with substan- that Note 2). tially less in the flesh (Table the amounts in the edible portion were slightly greater than in flesh by itself be- cause the skins are a relatively small propor- in low were pits Whereas tion of the fruits. hydroxycinnamates compared with skins and flesh, they still accounted for a sub- stantial proportion of the polyphenolics in sweet cherry pits. Of the quantified non- anthocyanin polyphenolics, the major hy- droxycinnamates in sweet cherry skins, flesh, and pits were neochlorogenic acid (54% to 57%, 42% to 56%, and 25% to 31%) 3 Cherry anthocyanin and polyphenolic composition . . . . composition polyphenolic and anthocyanin Cherry 2 fla- peak, and 1 flavanol peaks, ycinnamate an peaks. Bing contained vonol glycoside whereas flavonol glycoside peak, additional an addi- and Rainier contained Royal Ann (data not peak tional hydroxycinnamate chlorogenic 3 was identified as shown). Peak to its matching UV-visible acid according time with authentic spectra and retention a matching UV spec- standards. Peak 1 had UV 2’s acid while Peak trum to chlorogenic spectrum matched coumaroylquinic acid (11.6%), and neochlo- rogenic acid (6.9%); in flesh and pits they peaks completely disappeared with saponi- peaks completely disappeared acid and fication, with caffeic 1 was assigned to be acid being formed. Peak neochlorogenic acid (3

Food Chemistry and Toxicology UP =unidentified polyphenolics; F =unidentified flavonols P =procyanidins; hydroxycinnamates; HC =unidentified 3-rutinoside; 3 =chlorogenic acid; 4 =epicatechin; 5 =quercetin-3-rutinoside; 6 =kaempferol- 3 = neochlorogenicacid; 2 = brine solution(b)at280nm.1 Figure 4—HPLCpolyphenolics profileofRoyalAnncherries(a)andspent hydroxycinnamates (70.7%to82.1%),epi- major compoundsofsweetcherrieswere ble 2).Intheedibleportion(skinplusflesh), ofpolyphenolicsareportions different (Ta- sweet andsourcherriesare similar, thepro- inBingcentration fleshwasrelatively low. or pits(Table 2). The flavonol glycoside con- only intheskinsandnotdetectedflesh tion ofBings,flavonolglycosideswerefound tra. Astrikingfindingisthatwiththeexcep- Cherry anthocyaninandpolyphenoliccomposition. deviation ( reig–06 3 6 –70 — a 3 –70 Freezing –23 — Freezing –23 Freezing Freezing Fresh °C) °Cand–70 tures (–23 Table 3—Relativeanthocyanincompositionoffresh andfrozenBingcherriesstored at3moand62different tempera- Anthocyanin contentswerereportedasmgcyanidin-3-glucoside/100gfw(ChaovanalikitandWrolstad2003).Eachvalueintablei Whereas thequalitativecompositionof n = 2). Thevalueswithdifferentlettersinacolumnindicatesignificantdifferenceat epTm C m yndn3 yndn3 Pel Cyanidin-3- Cyanidin-3- ACN(mg Time Temp °)(o 10g gl g) /100 (mo) (°C) a 56.4 9.36 9.36 9.40 7.56 56.4 8.90 57.4 8.05 8.02 21.5 63.7 csd rutinoside ucoside cherries, Bingcherriescontainedthehigh- cinnamates (87mg/100g). Within sweet contained thehighestcontentofhydroxy- sides (11.2mg/100g).RoyalAnncherries nidins (49.5mg/100g),andflavonolglyco- tent ofepicatechin(19.6mg/100g),procya- Montmorency cherrieshadthehighestc