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Research ArticlePhenolic Compounds and Antioxidant Activity of Juices fromTen Iranian Pomegranate Cultivars Depend on Extraction
Hamidreza Akhavan1 Mohsen Barzegar2 Helmut Weidlich3 and Benno F Zimmermann34
1Department of Food Science and Technology Faculty of Agriculture Shahid Bahonar University of KermanPO Box 76169-133 Kerman Iran2Department of Food Science and Technology Faculty of Agriculture Tarbiat Modares UniversityPO Box 14115-336 Tehran Iran3Institut Prof Dr Georg Kurz GmbH Stockheimer Weg 1 50829 Koln Germany4Department of Nutritional and Food Sciences University of Bonn Romerstraszlige 164 53117 Bonn Germany
Correspondence should be addressed to Benno F Zimmermann bennozimmermannuni-bonnde
Received 17 September 2015 Revised 5 November 2015 Accepted 8 November 2015
Academic Editor Patricia Valentao
Copyright copy 2015 Hamidreza Akhavan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Phenolic compounds and antioxidant activities of ten juices from arils and whole pomegranate cultivars grown in Iran werestudied Phenolic contents and antioxidant activities of juices from whole pomegranate fruit were significantly higher than juicesfrom pomegranate arils but the variety has a greater influence than the processing method The main phenolics in the studiedjuices were punicalagin A (540ndash285mgL) punicalagin B (259ndash884mgL) and ellagic acid (174ndash928mgL) The major andminor anthocyanins of cyanidin 35-diglucoside (07ndash947mgL) followed by cyanidin 3-glucoside (05ndash525mgL) pelargonidin35-diglucoside + delphinidin 3-glucoside (0ndash103mgL) delphinidin 35-diglucoside (0ndash768mgL) pelargonidin 3-glucoside (0ndash940mgL) and cyanidin-pentoside (0ndash113mgL) were identified the latter anthocyanin as well as cyanidin-pentoside-hexosideand delphinidin-pentoside were detected for the first time in Iranian pomegranatesThe total phenolic contents were in the range of220ndash2931mg100mLThe results indicate that the pomegranate phenolics are not only influenced by extraction method but alsomdashand even moremdashaffected by the cultivar Moreover a good correlation was observed between total phenolic content and ABTS andFRAP methods in all pomegranate juices (gt090) The results of current research can help to select the pomegranate cultivars forcommercial juice production
1 Introduction
Pomegranate fruit (Punica granatum L) and its productsare rich sources of bioactive compounds such as flavonoidsellagitannins mainly punicalagins ellagic acid andpunicalins that also affect visual appearance and flavourof pomegranate juices [1 2] These compounds are discussedas health promoting but excessive amounts of polyphenolscan form undesirable astringent taste This fruit is one ofthe most important commercial fruits in Iran and its totalproduction in 2013 was sim910000 tons [3] The pomegranateis consumed worldwide as fresh fruit juice jam and jellyand nutritional supplements
In recent years the physicochemical properties ofpomegranate fruit such as total phenolic total anthocyaninand total tannin contents and antioxidant activity assaywith different methods [1 2 4ndash11] and individual phenoliccompounds such as flavonoids (including anthocyanins)ellagitannins mainly punicalagins ellagic acid and puni-calins [1 2 4 5 11ndash14] have been published in literature
Thus the major phenolic compounds of pomegranatejuices have been documented but no information is availableabout identification and quantification of phenolic com-pounds and antioxidant activities of juices obtained fromarils only and fromwhole Iranian pomegranate cultivarsThisstudy can help to select pomegranate cultivars for commercial
Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 907101 7 pageshttpdxdoiorg1011552015907101
2 Journal of Chemistry
use Therefore the aims of this study were (1) to quan-tify individual phenolic compounds (2) to determine totalphenolic content as well as antioxidant activity and (3) tostudy correlation of these parameters in pomegranate juicesobtained from arils and whole pomegranates of differentcultivars
2 Materials and Methods
21 Plant Material The following ten pomegranate culti-vars were harvested in the last week of September 2011from mature trees (16 years old) growing in the Agricul-tural Research Center of Yazd province Iran (31∘5410158405410158401015840N54∘1610158403710158401015840E) Vahshe Kane Tehran (VKT) Gorche ShahvarYazdi (GSY) Malase Yazdi (MY) Mesri Torshe Kazeron(MTK) Jangali Pust Germeze Rodbare Torsh (JPGRT) Tor-she Mamoli Lasjer (TML) Ardestani Torshe Semnan (ATS)Sefeede Robi Aval Brojen (SRAB) Pust Syahe Yazd (PSY) andMalase Porbarij Stahban (MPS)
The trees were grown under the same environmentalconditions basin irrigation (sim2-3 times per month duringgrowing season from March to September 2011) and routinecultural practices suitable for commercial fruit productionThe trees were planted with an average of 6m within rowsand 3m between them Yazd province is located in a desertenvironment with an annual precipitation of lt100mm Theaverage temperature the amount of rainfall and relativehumidity in growing season of 2011were 250∘C 140mm and236 respectively The soil texture is sandy-loam with EC= 412 dSmminus1 and soil pH = 721 Ten fruits (approximately25 kg) from four trees (2-3 fruits from each tree) wereharvested when fully matured according to commercialpractice It should be noted that the pomegranate fruits inYazd provincewere fullymatured and harvested between 15thand 30th September [12]
22 Chemicals Punicalagin and cyanidin 3-O-glucosidestandards were purchased from Phytolab (Vestenbergs-greuth Germany) and ellagic acid was purchased fromDr Jacob (Taunusstein Germany) The Folin-Ciocalteursquosphenol reagent (Merck Germany) ABTS [220-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] DPPH radical (11-diphenyl-2-picrylhydrazyl) and TPTZ (246-tripyridyl-s-triazine) were purchased from Sigma-Aldrich USA All sol-vents used as UHPLC eluents were LC-MS grade optigradewater was obtained from Promochem (Wesel Germany)acetonitrile fromMallinckrodt Baker (DeventerThe Nether-lands) and formic acid from FlukaSigma Aldrich
23 Preparation of Pomegranate Juice Each pomegranatecultivar was washed in cold tap water and drained Thefruits were pressed using a self-made press consisting ofa movable car lifter and a stainless steel pot creating apressure of 674 kpcm2 The supernatant juice was decantedand frozen at minus20∘C until analysis Juices were filteredthrough disposable membrane filters (regenerated cellulose02 120583m pore size Macherey + Nagel Duren Germany) andanalysed without any further sample pretreatment Suffixes
A and W (such as MYA and MYW) at each abbreviationof pomegranate cultivars indicate the juices obtained fromarils (ie without the exocarp and the endocarp) and wholepomegranate juices respectively Approximately 25 kg ofpomegranate fruits was cut in two halves in order to obtainaril juice and whole pomegranate juice from the same fruitsFor the aril juices the arils were separated manually and theinner white parts of the fruit were removed
24 UHPLC Analysis Identification and Quantification ofPhenolic Compounds For analysis of phenolic compoundsan Acquity UPLC-UV-MS system (Waters Milford MAUSA) was used as described by Feuereisen et al [15]
Anthocyanins were separated using an Acquity HSS-T3RP18 column (100mm times 21mm 18 120583m particle size) fromWaters with a guard column (5mm times 21mm) Eluent A waswater1 formic acid eluent B was acetonitrile01 formicacid The gradient program at a flow rate of 045mLminwas as follows 0ndash02min 0 B 06min 5 B 20min12 B 21ndash24min 95 B and 25ndash27min 0 B The massspectrometer was tuned using a solution of cyanidin 3-O-glucoside The following parameters were obtained capillaryvoltage 16 kV cone voltage 32V extractor voltage 30VRF voltage 13 V source temperature 150∘C desolvationtemperature 450∘C cone gas (nitrogen) flow 50 Lh anddesolvation gas (nitrogen) flow 800 Lh The collision gas(argon) flow used in tandemmass spectrometry experimentswas 03mLmin MS detection was used for confirmation ofpeak identity in selected reaction monitoring (SRM) modeFor quantification an external calibration of cyanidin 3-O-glucoside was used (detection wavelength of 500 nm) Eachsample was analyzed once All compounds were identified byMS (SRM) and quantified by UV-Vis
For the separation of punicalagin and ellagic acid anAcquity BEH Phenyl column (50mm times 21mm particle size17 120583m) from Waters with a guard column (5mm times 21mm)was used Eluent A was water01 formic acid eluent B wasacetonitrile01 formic acid The gradient program at a flowrate of 05mLminwas as follows 0ndash3min 0B 13min 35B 132min 95 B 132ndash14min 95 B and 142ndash15min 0B Each sample was analysed in duplicate
The mass spectrometer was tuned using a solution ofpunicalagin resulting in the following parameters capillaryvoltage minus20 kV cone voltage 34V extractor voltage 20VRF voltage 03 V source temperature 150∘C desolvationtemperature 450∘C cone gas (nitrogen) flow 50 Lh anddesolvation gas (nitrogen) flow 900 Lh The collision gas(argon) flow used in tandemmass spectrometry experimentswas 03mLmin For quantification an external calibration ofpunicalagin and ellagic acid was used (detection wavelengthof 360 nm) All compounds were identified byMS (SRM) andquantified by UV
25 Determination of Total Phenolic Content (TPC) andAntioxidant Activity of Juices Total phenolic content wasmeasured using Folin-Ciocalteumethod described by Tezcanet al [8] using gallic acid as reference The antioxidantcapacity of pomegranate juices was evaluated based on free
Journal of Chemistry 3
Table 1 Contents of individual phenolic compounds (mgL) in pomegranate juices (PJ)
PJ from arils Punicalagin A Punicalagin B Ellagic acid PJ from whole fruits Punicalagin A Punicalagin B Ellagic acidVKTA 159 plusmn 01eflowast 367 plusmn 26gh 748 plusmn 06g VKTW 665 plusmn 06e 2196 plusmn 06d 2459 plusmn 49d
MPSA 508 plusmn 09c 1492 plusmn 30e 1233 plusmn 16c MPSW 1809 plusmn 39b 6501 plusmn 13b 2618 plusmn 26dlowastDifferent letters in the same column present significant difference at 119901 lt 005
radical scavenging capacity by DPPH radical (expressed asinhibition percentage) and ABTS radical (expressed as mg L-ascorbic acid100mL juice) and with iron-reducing capacityby FRAP method (expressed in mmol Fe2+100mL juice)[7 8]
26 Statistical Analysis The data were subjected to analysesof variance (ANOVA) using SPSS 170 For the comparison ofsignificance between cultivars Duncanrsquos multiple range test(119875 lt 005) was used as post hoc test Results are the means plusmnSD of two determinations for each juice
3 Results and Discussion
31 Punicalagins and Ellagic Acids In this study the majorindividual phenolic compounds of ten pomegranate cultivarswere identified by HPLCPDAMS2 namely punicalagin Apunicalagin B and ellagic acid (Table 1) besides anthocyanins(see the next paragraph)These results are in accordance withpreviously reports on other pomegranate cultivars [1 2 4 1116]
Gallic acid gallagic acid and galloyl glucose were foundin minor amounts only and therefore not considered in thefollowing evaluation A typical chromatogram of the detectedphenolic compounds of pomegranate juice (MYWcultivar) isshown in Figure 1
The individual and total phenolic contents of all stud-ied cultivars were significantly different (119901 lt 001) Theconcentrations of the main phenolics showed wide rangespunicalagin B 259ndash884mgL ellagic acid 174ndash928mgLand punicalagin A 540ndash285mgL which is in agreementwith previous findings of juices from other pomegranatecultivars [1 4 13 14] Pearsonrsquos correlation coefficients werehigh (gt098) between punicalagin A and punicalagin B atsame juices (arils or whole pomegranate) but ellagic acidvalue was not significantly correlated with the other phenoliccompounds In each cultivar the total individual phenoliccontent of whole pomegranate juices was increased by 25ndash1136 compared to pomegranate aril juices In this regard theMY and JPGRT had increased 25 and 35 respectively butother samples showed higher increases (gt100) The highestincrease (1136) was awarded to GSY cultivar
()
0
5955413
4915413
5646013
7383011
6633011
7733011
200 300 400 500 600 700 800100
Time (min)Pu
nica
lagi
n A
Puni
cala
gin
B
Ella
gic a
cid
TIC452e6
Figure 1 Typical chromatogramof the nonanthocyanidin phenolicsin pomegranate juice (MYW) as detected by MS in selected ionmonitoring (SIM) mode Punicalagins appear as doubly chargedions Ions with mz = 301 other than ellagic acid are in-sourcefragments of other ellagitannins
The highest punicalagin contents among aril and wholefruit juices were found in JPGRTA (607mgL) and GSYW(1169mgL) respectively (Table 1) Punicalagin is the mostcharacteristic ellagitannin because it is found almost exclu-sively in pomegranate However pomegranate arils containlow or no punicalagins but pomegranate rind parts are anabundant source The higher punicalagin content of com-mercial pomegranate juices was due to the entrance of thesecompounds from rind parts of the fruit during processing[1 11] The phenolic content of four types of ldquoWonderfulrdquopomegranate juices including juices from fresh arils frozenarils and whole pomegranates and concentrate were evalu-ated [1] Punicalagin A in four types of pomegranate juiceswas 127 144 421 and 435mgL punicalagin B was 101 111839 and 918mgL and total ellagic derivatives were 332 265121 and 264mgL respectively The four major hydrolyzabletannins of punicalagin ellagic acid punicalin and gallagicacid were detected in 29 different pomegranate accessionsby Tzulker et al [11] The contents of these compounds
4 Journal of Chemistry
significantly differed in the whole fruit homogenates Thecontent of punicalagin was about 10-fold higher than thepunicalin and gallagic acid contents and 100-fold higher thanthe content of ellagic acid Although punicalagin punicalinand gallagic acid were also found in aril juices (ellagic acidwas below the detection level) these contents were very lowin comparison to the whole fruit homogenates [11]
In literature punicalagins content was reported to belower in pomegranate arils than in whole pomegranate fruitor even absent in the former [1 2 11] But in the current studythe cultivar JPGRT had such a high punicalagin content thatthe juice made from the arils of JPGRT had a higher content(607mgL) than the juice made from whole pomegranate ofcertain cultivars (MYW TMLW SRABW and PSYW)There-fore the content (or lack) of punicalagins in pomegranatejuice is not only affected by extractionmethod but alsomdashandeven moremdashby the pomegranate cultivar
Fischer et al [2] identified 48 compounds in 3 variantsof pomegranate juices among which 9 were anthocyanins2 gallotannins 22 ellagitannins 2 gallagyl esters 4 hydrox-ybenzoic acids and 7 hydroxycinnamic acids and 1 wasdihydroflavonol The phenolic compounds of pomegranatejuice prepared from whole pomegranate at 10 bars juiceprepared from whole pomegranate at 150 bars and juiceprepared from the isolated arils at 250 bars were as followstotal gallotannins (12ndash91mgL) total ellagitannins (932ndash2074mgL) total hydroxybenzoic acids (11ndash106mgL) andtotal hydroxycinnamic acids (214ndash283mgL) In anotherstudy punicalagin levels of juices from the pomegranate cul-tivar ldquoMollarrdquo ranged between 504 and 763mgL and puni-calinswere found in a lower range between 240 and 365mgLThe free ellagic acid level was between 269 and 390mgL [16]
Also Mena et al [14] have identified 75 compoundsby UHPLC-MS119899 of which 21 compounds were tentativelyidentified for the first time in pomegranate juice of Mollarde Elche cultivar The separated compounds included 37ellagitannins 6 gallotannins 14 noncoloured flavonoids 5phenolic acid derivatives 6 anthocyanins and a flavanol-anthocyanin adduct 6 lignans and 3 organic acids
32 Individual Anthocyanin Content The individual antho-cyanin contents of ten pomegranate cultivars were deter-mined using UHPLC A typical chromatogram of indi-vidual anthocyanins of MYA cultivar is shown in Fig-ure 2 The applied chromatographic conditions success-fully separated both the major and minor anthocyaninsof pomegranate juice The detected anthocyanins werecyanidin 3-glucoside (Cy 3G) cyanidin 35-diglucoside (Cy35dG) delphinidin 3-glucoside (Dp 3G) delphinidin 35-diglucoside (Dp 35dG) pelargonidin 3-glucoside (Pg 3G)pelargonidin 35-diglucoside (Pg 35dG) cyanidin-pentoside(Cy-pent) cyanidin-pentoside-hexoside (Cy-pent-hex) anddelphinidin-pentoside (Dp-pent)
Table 2 shows the individual anthocyanin contentsof the studied cultivars The JPGRTA cultivar had thehighest (175mgL) anthocyanin content whereas those ofMPSA ATSA PSYA and PSYW were lower than 5mgLIn some cultivars juice extraction of whole pomegranate
()
0400 600 800 1000 1200 1400 1600 1800 2000200
Time (min)
Dp3
5dG Cy
35
dGPg
35
dGD
p3
G
Cy-p
ent-h
exCy
3G
Pg3
G
Dp-
pent
Cy-p
ent
Figure 2 Typical chromatogram of the anthocyanidins inpomegranate juice (MYA) as detected by MS in SRMmode
juices increased the anthocyanin content ATSW (134)GSYW (477) MPSW (347) MTKW (223) and PSYW(196) In contrast the anthocyanin contents of SRABW(214) TMLW (104) MYW (440) and JPGRTW(390) were decreased Based on these results it is evidentthat the anthocyanin content of pomegranate juices is affectedby extraction methods
According to Figure 2 nine major and minoranthocyanins were identified namely cyanidin 3-glucosidecyanidin 35-diglucoside delphinidin 3-glucoside delphini-din 35-diglucoside pelargonidin 3-glucoside pelargonidin35-diglucoside cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside In terms of quantitythe main anthocyanin in most of the cultivars was Cy 35dG(07ndash947mgL) followed by Cy 3G (05ndash525mgL) Pg35dG + Dp 3G (0ndash103mgL) Dp 35dG (0ndash940mgL) Pg3G (0ndash400mgL) and Cy-pent (0ndash113mgL)
Except for Cy-pent Dp-pent and Cy-pent-hex theanthocyanin profiles were consistent with previouslyreported researches about other pomegranate cultivars[1 12 17ndash19] In the same pomegranate cultivars Alighourchiet al [12] reported the content of 6 anthocyanins inaril juices as follows Dp 3G (219ndash163mgL) Dp 35dG(236ndash631mgL) Pg 3G (026ndash136mgL) Pg 35dG(001ndash811mgL) Cy 3G (578ndash304mgL) and Cy 35dG(439ndash166mgL) Thus achieving different anthocyaninscontent in the same pomegranate cultivars indicated theeffect of agricultural and environmental conditions on thephysicochemical characteristic of pomegranate fruit
To our knowledge Dp-pent has not been reportedin pomegranate fruit so far It is tentatively identified bythe mass transition 435gt303 Fischer et al [2] identified9 anthocyanins of Dp 35dG (04ndash205mgL) Cy 35dG(954ndash271mgL) Pg 35dG (137ndash267mgL) Dp 3G (10ndash168mgL) Cy 3G (91ndash279mgL) Pg 3G (24ndash49mgL)a cyanidin-pentoside-hexoside (11ndash30mgL) cyanidin 3-rutinoside (00ndash12mgL) and a cyanidin-pentoside (02ndash06mgL) in the three pomegranate juice variants Gil et al[1] reported the main pigments in the juice of theWonderful
Journal of Chemistry 5
Table 2 Contents of individual anthocyanins (mgL) in pomegranate juices
cultivar as follows Cy 3G (595ndash128mgL) Dp 3G (236ndash952mgL) Cy 35dG (314ndash714mgL) Dp 35dG (211ndash611mgL) and Pg 3G (39ndash85mgL)Mena et al [4] reportedCy 3G (33) as the major anthocyanin followed by Dp 35dG(24) Dp 3G (21) and Cy 35dG (19) and Pg 3G andPg 35dG were only 3 of the total anthocyanin content Inaddition four major anthocyanins of Dp 3G Dp 35dG Cy3G and Cy 35dG andminor amounts of Pg 3G and Pg 35dGwere determined in pomegranate juices [20]
The main anthocyanin in all the cultivars was Cy 35dGexcept for SRABA in which Cy 3G was dominant It isknown that the main anthocyanin of pomegranate dependson the cultivar The main anthocyanins were Cy 3G [1]in the American Wonderful cultivar Cy 3G (40) and Cy35dG (38) in the Spanish Mollar cultivar [19] Dp 3G inthe Portuguese Assaria cultivar [17] Cy 35dG in 15 Iraniancultivars [12] Dp 35dG in 8 other Iranian cultivars [5] andCy 35dG in a Peruvian pomegranate fruit [2]
Among different species or even cultivars of the samespecies the anthocyanin content varies considerably affectedby genetic make-up light temperature and agronomic fac-tors [21] Intrinsic properties of the product and the processsuch as pH temperature light oxygen enzymes ascorbicacids sugars metal ions and copigmentation (intermolecu-lar and intramolecular complex formations self-associationand metal complexation) as well as chemical structure andconcentration of anthocyanins can affect anthocyanin stabil-ity [22]
The variation of anthocyanin content in juices from arilsand whole pomegranate can be affected by the entrance ofanthocyanins from the peel into the juices Therefore the
amount of anthocyanins of the same cultivar varied withextraction methods
33 Total Phenolic Content and Antioxidant CapacityAccording to the results presented in Table 3 total phenoliccontents of juices from whole pomegranate fruit (942ndash2931mgL) were significantly higher than those in arilsjuice (220ndash1267mgL) The GSYW cultivar had the highest(2931mgL) total phenolic content In other studies the totalphenolic contents of various pomegranate cultivars showedsignificant differences and were 296ndash985mg100 g [9] 208ndash344mg100 g [7] 144ndash1009mg100 g [8] 237ndash930mg100 g[5] 2015ndash5186mgL [2] and 1136ndash3581mgL [16] Differentamounts of total phenolic content in various studies can beattributed to analytical methods cultivar maturity stageand environmental conditions and also overestimation ofthe total individual phenolic compounds by Folin-Ciocalteureagent due to interference with other reducing factors [2]
The antioxidant properties were measured by ABTSFRAP and DPPH assay (Table 3) The results showed con-siderable antioxidant activity of juice samples Antioxidantactivities of juices from whole pomegranate fruit were sig-nificantly higher than juices from pomegranate arils thatindicated the importance of the juice extraction methodsThese results are in agreement with previously reportedfindings [1 11]The antioxidant capacity of pomegranate juice(and other fruit juices) depends on cultivar growing regionfruit maturation and agricultural factors The technologyused for juice processing may affect antioxidant capacityof pomegranate juice [7] In this regard the antioxidantactivity of commercial pomegranate juice was 50 higher
6 Journal of Chemistry
Table 3 Evaluation of total polyphenol content (mg gallic acid equivalentsL) and antioxidant activities based on ABTS (mg ascorbicacid100mL) FRAP (mmol Fe2+100mL) and DPPH (AA) in pomegranate juices
MPSW 19541 plusmn 271cd 12492 plusmn 265c 306 plusmn 18c 550 plusmn 18clowastDifferent letters in the same column present significant difference at 119901 lt 005
than juices from pomegranate arils due to the entranceof phenolic compounds from the peel into juices [1] AlsoTzulker et al [11] reported that antioxidant activity of wholefruit homogenates is about 20-fold higher than that foundin the arils These results indicated that hydrolysable tanninsincluding punicalagin were themain compounds influencingthe antioxidant capacity of pomegranate juices [1 11] Thiscompound shows a high antioxidant activity due to the 16phenolic hydroxyl groups in the molecule [1]
Moreover a good correlation was observed betweentotal phenolic content and ABTS and FRAP methods inall pomegranate juices (gt090) Correlations between totalphenolic and DPPH in juices of arils and whole pomegranatewere 092 and 086 respectively However Pearson corre-lation showed that the correlation of three measurementantioxidant methods is affected by pomegranate cultivarsas mentioned in previous research [7 11] In this regardTzulker et al [11] reported that the antioxidant activity (FRAPand DPPH methods) of juices from whole pomegranatehomogenate and arils alone significantly correlated withthe polyphenol level but it did not correlate with antho-cyanins level The highest correlation value was obtainedbetween antioxidant activity and punicalagin in the wholepomegranate homogenate The FRAP method comparedto DPPH method gave the highest correlation with totalpolyphenol content
4 Conclusion
The individual and total phenolic contents of all stud-ied cultivars were significantly different In this study
among nine major and minor anthocyanins the cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside were identified for the first time in Iranianpomegranate cultivars The phenolic contents and antioxi-dant activity of whole pomegranate juice were significantlyhigher than those of juices from pomegranate arils from thesame cultivar due to the entrance of phenolic compoundsfrom the rind parts of pomegranate to the juices Thevariation between the cultivars is very wide therefore thecontents of ellagitannins and anthocyanins in the juice fromwhole pomegranates of the low-polyphenol cultivars (asMY)are lower than those in the juices made from arils only ofthe high-polyphenol cultivars (as JPGRT) In otherwords thecultivar has a higher influence on the phenolic content of thejuice than the extraction method
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to acknowledge Dr Vazifeshenas forhis assistance in afforded pomegranate cultivars
References
[1] M I Gil F A Tomas-Barberan B Hess-Pierce D M Holcroftand A A Kader ldquoAntioxidant activity of pomegranate juice
Journal of Chemistry 7
and its relationship with phenolic composition and processingrdquoJournal of Agricultural and Food Chemistry vol 48 no 10 pp4581ndash4589 2000
[2] U A Fischer R Carle and D R Kammerer ldquoIdentificationand quantification of phenolic compounds from pomegranate(Punica granatum L) peel mesocarp aril and differently pro-duced juices by HPLC-DAD-ESIMS119899rdquo Food Chemistry vol127 no 2 pp 807ndash821 2011
[3] Anonymous Iran Statistical Year Book 2013 2013 httpeamarsciorgirindex easpx
[4] P Mena C Garcıa-Viguera J Navarro-Rico et al ldquoPhytochem-ical characterisation for industrial use of pomegranate (Punicagranatum L) cultivars grown in Spainrdquo Journal of the Science ofFood and Agriculture vol 91 no 10 pp 1893ndash1906 2011
[5] G Mousavinejad Z Emam-Djomeh K Rezaei and M HH Khodaparast ldquoIdentification and quantification of pheno-lic compounds and their effects on antioxidant activity inpomegranate juices of eight Iranian cultivarsrdquo Food Chemistryvol 115 no 4 pp 1274ndash1278 2009
[6] O A Fawole U L Opara and K I Theron ldquoChemicaland phytochemical properties and antioxidant activities ofthree pomegranate cultivars grown in South Africardquo Food andBioprocess Technology vol 5 no 7 pp 2934ndash2940 2012
[7] M Cam Y Hısıl and G Durmaz ldquoClassification of eightpomegranate juices based on antioxidant capacity measured byfourmethodsrdquo FoodChemistry vol 112 no 3 pp 721ndash726 2009
[8] F Tezcan M Gultekin-Ozguven T Diken B Ozcelik and FB Erim ldquoAntioxidant activity and total phenolic organic acidand sugar content in commercial pomegranate juicesrdquo FoodChemistry vol 115 no 3 pp 873ndash877 2009
[9] A Tehranifar M Zarei Z Nemati B Esfandiyari and MR Vazifeshenas ldquoInvestigation of physico-chemical propertiesand antioxidant activity of twenty Iranian pomegranate (PunicagranatumL) cultivarsrdquo ScientiaHorticulturae vol 126 no 2 pp180ndash185 2010
[10] M Ozgen C Durgac S Serce and C Kaya ldquoChemical andantioxidant properties of pomegranate cultivars grown in theMediterranean region of Turkeyrdquo Food Chemistry vol 111 no3 pp 703ndash706 2008
[11] R Tzulker I Glazer I Bar-Ilan D Holland M Aviramand R Amir ldquoAntioxidant activity polyphenol content andrelated compounds in different fruit juices and homogenatesprepared from 29 different pomegranate accessionsrdquo Journal ofAgricultural and Food Chemistry vol 55 no 23 pp 9559ndash95702007
[12] H Alighourchi M Barzegar and S Abbasi ldquoAnthocyaninscharacterization of 15 Iranian pomegranate (Punica granatumL) varieties and their variation after cold storage and pasteur-izationrdquo European Food Research and Technology vol 227 no 3pp 881ndash887 2008
[13] S Chrubasik-Hausmann C Vlachojannis and B Zimmer-mann ldquoPomegranate juice and prostate cancer importanceof the characterisation of the active principlerdquo PhytotherapyResearch vol 28 no 11 pp 1676ndash1678 2014
[14] P Mena L Calani C DallrsquoAsta et al ldquoRapid and comprehen-sive evaluation of (Poly)phenolic compounds in pomegranate(Punica granatum L) Juice by UHPLC-MSnrdquoMolecules vol 17no 12 pp 14821ndash14840 2012
[15] M M Feuereisen J Hoppe B F Zimmermann F Weber NSchulze-Kaysers andA Schieber ldquoCharacterization of phenoliccompounds in Brazilian pepper (Schinus terebinthifolius Raddi)
exocarprdquo Journal of Agricultural and FoodChemistry vol 62 no26 pp 6219ndash6226 2014
[16] SVegaraNMartı J Lorente et al ldquoChemical guide parametersfor Punica granatum cv lsquoMollarrsquo fruit juices processed atindustrial scalerdquo Food Chemistry vol 147 pp 203ndash208 2014
[17] G Miguel S Dandlen D Antunes A Neves and D MartinsldquoThe effect of two methods of pomegranate (Punica granatumL) juice extraction on quality during storage at 4∘Crdquo Journalof Biomedicine and Biotechnology vol 2004 no 5 pp 332ndash3372004
[18] A Perez-Vicente P Serrano P Abellan and C Garcıa-VigueraldquoInfluence of packaging material on pomegranate juice colourand bioactive compounds during storagerdquo Journal of the Scienceof Food and Agriculture vol 84 no 7 pp 639ndash644 2004
[19] NMartı A Perez-Vicente andCGarcıa-Viguera ldquoInfluence ofstorage temperature and ascorbic acid addition on pomegranatejuicerdquo Journal of the Science of Food and Agriculture vol 82 no2 pp 217ndash221 2002
[20] C U Pala and A K Toklucu ldquoEffect of UV-C light on antho-cyanin content and other quality parameters of pomegranatejuicerdquo Journal of Food Composition and Analysis vol 24 no 6pp 790ndash795 2011
[21] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsCRC Press Boca Raton Fla USA 2004
[22] A Patras N P Brunton S Da Pieve and F Butler ldquoImpact ofhigh pressure processing on total antioxidant activity phenolicascorbic acid anthocyanin content and colour of strawberryand blackberry pureesrdquo Innovative Food Science amp EmergingTechnologies vol 10 no 3 pp 308ndash313 2009
use Therefore the aims of this study were (1) to quan-tify individual phenolic compounds (2) to determine totalphenolic content as well as antioxidant activity and (3) tostudy correlation of these parameters in pomegranate juicesobtained from arils and whole pomegranates of differentcultivars
2 Materials and Methods
21 Plant Material The following ten pomegranate culti-vars were harvested in the last week of September 2011from mature trees (16 years old) growing in the Agricul-tural Research Center of Yazd province Iran (31∘5410158405410158401015840N54∘1610158403710158401015840E) Vahshe Kane Tehran (VKT) Gorche ShahvarYazdi (GSY) Malase Yazdi (MY) Mesri Torshe Kazeron(MTK) Jangali Pust Germeze Rodbare Torsh (JPGRT) Tor-she Mamoli Lasjer (TML) Ardestani Torshe Semnan (ATS)Sefeede Robi Aval Brojen (SRAB) Pust Syahe Yazd (PSY) andMalase Porbarij Stahban (MPS)
The trees were grown under the same environmentalconditions basin irrigation (sim2-3 times per month duringgrowing season from March to September 2011) and routinecultural practices suitable for commercial fruit productionThe trees were planted with an average of 6m within rowsand 3m between them Yazd province is located in a desertenvironment with an annual precipitation of lt100mm Theaverage temperature the amount of rainfall and relativehumidity in growing season of 2011were 250∘C 140mm and236 respectively The soil texture is sandy-loam with EC= 412 dSmminus1 and soil pH = 721 Ten fruits (approximately25 kg) from four trees (2-3 fruits from each tree) wereharvested when fully matured according to commercialpractice It should be noted that the pomegranate fruits inYazd provincewere fullymatured and harvested between 15thand 30th September [12]
22 Chemicals Punicalagin and cyanidin 3-O-glucosidestandards were purchased from Phytolab (Vestenbergs-greuth Germany) and ellagic acid was purchased fromDr Jacob (Taunusstein Germany) The Folin-Ciocalteursquosphenol reagent (Merck Germany) ABTS [220-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] DPPH radical (11-diphenyl-2-picrylhydrazyl) and TPTZ (246-tripyridyl-s-triazine) were purchased from Sigma-Aldrich USA All sol-vents used as UHPLC eluents were LC-MS grade optigradewater was obtained from Promochem (Wesel Germany)acetonitrile fromMallinckrodt Baker (DeventerThe Nether-lands) and formic acid from FlukaSigma Aldrich
23 Preparation of Pomegranate Juice Each pomegranatecultivar was washed in cold tap water and drained Thefruits were pressed using a self-made press consisting ofa movable car lifter and a stainless steel pot creating apressure of 674 kpcm2 The supernatant juice was decantedand frozen at minus20∘C until analysis Juices were filteredthrough disposable membrane filters (regenerated cellulose02 120583m pore size Macherey + Nagel Duren Germany) andanalysed without any further sample pretreatment Suffixes
A and W (such as MYA and MYW) at each abbreviationof pomegranate cultivars indicate the juices obtained fromarils (ie without the exocarp and the endocarp) and wholepomegranate juices respectively Approximately 25 kg ofpomegranate fruits was cut in two halves in order to obtainaril juice and whole pomegranate juice from the same fruitsFor the aril juices the arils were separated manually and theinner white parts of the fruit were removed
24 UHPLC Analysis Identification and Quantification ofPhenolic Compounds For analysis of phenolic compoundsan Acquity UPLC-UV-MS system (Waters Milford MAUSA) was used as described by Feuereisen et al [15]
Anthocyanins were separated using an Acquity HSS-T3RP18 column (100mm times 21mm 18 120583m particle size) fromWaters with a guard column (5mm times 21mm) Eluent A waswater1 formic acid eluent B was acetonitrile01 formicacid The gradient program at a flow rate of 045mLminwas as follows 0ndash02min 0 B 06min 5 B 20min12 B 21ndash24min 95 B and 25ndash27min 0 B The massspectrometer was tuned using a solution of cyanidin 3-O-glucoside The following parameters were obtained capillaryvoltage 16 kV cone voltage 32V extractor voltage 30VRF voltage 13 V source temperature 150∘C desolvationtemperature 450∘C cone gas (nitrogen) flow 50 Lh anddesolvation gas (nitrogen) flow 800 Lh The collision gas(argon) flow used in tandemmass spectrometry experimentswas 03mLmin MS detection was used for confirmation ofpeak identity in selected reaction monitoring (SRM) modeFor quantification an external calibration of cyanidin 3-O-glucoside was used (detection wavelength of 500 nm) Eachsample was analyzed once All compounds were identified byMS (SRM) and quantified by UV-Vis
For the separation of punicalagin and ellagic acid anAcquity BEH Phenyl column (50mm times 21mm particle size17 120583m) from Waters with a guard column (5mm times 21mm)was used Eluent A was water01 formic acid eluent B wasacetonitrile01 formic acid The gradient program at a flowrate of 05mLminwas as follows 0ndash3min 0B 13min 35B 132min 95 B 132ndash14min 95 B and 142ndash15min 0B Each sample was analysed in duplicate
The mass spectrometer was tuned using a solution ofpunicalagin resulting in the following parameters capillaryvoltage minus20 kV cone voltage 34V extractor voltage 20VRF voltage 03 V source temperature 150∘C desolvationtemperature 450∘C cone gas (nitrogen) flow 50 Lh anddesolvation gas (nitrogen) flow 900 Lh The collision gas(argon) flow used in tandemmass spectrometry experimentswas 03mLmin For quantification an external calibration ofpunicalagin and ellagic acid was used (detection wavelengthof 360 nm) All compounds were identified byMS (SRM) andquantified by UV
25 Determination of Total Phenolic Content (TPC) andAntioxidant Activity of Juices Total phenolic content wasmeasured using Folin-Ciocalteumethod described by Tezcanet al [8] using gallic acid as reference The antioxidantcapacity of pomegranate juices was evaluated based on free
Journal of Chemistry 3
Table 1 Contents of individual phenolic compounds (mgL) in pomegranate juices (PJ)
PJ from arils Punicalagin A Punicalagin B Ellagic acid PJ from whole fruits Punicalagin A Punicalagin B Ellagic acidVKTA 159 plusmn 01eflowast 367 plusmn 26gh 748 plusmn 06g VKTW 665 plusmn 06e 2196 plusmn 06d 2459 plusmn 49d
MPSA 508 plusmn 09c 1492 plusmn 30e 1233 plusmn 16c MPSW 1809 plusmn 39b 6501 plusmn 13b 2618 plusmn 26dlowastDifferent letters in the same column present significant difference at 119901 lt 005
radical scavenging capacity by DPPH radical (expressed asinhibition percentage) and ABTS radical (expressed as mg L-ascorbic acid100mL juice) and with iron-reducing capacityby FRAP method (expressed in mmol Fe2+100mL juice)[7 8]
26 Statistical Analysis The data were subjected to analysesof variance (ANOVA) using SPSS 170 For the comparison ofsignificance between cultivars Duncanrsquos multiple range test(119875 lt 005) was used as post hoc test Results are the means plusmnSD of two determinations for each juice
3 Results and Discussion
31 Punicalagins and Ellagic Acids In this study the majorindividual phenolic compounds of ten pomegranate cultivarswere identified by HPLCPDAMS2 namely punicalagin Apunicalagin B and ellagic acid (Table 1) besides anthocyanins(see the next paragraph)These results are in accordance withpreviously reports on other pomegranate cultivars [1 2 4 1116]
Gallic acid gallagic acid and galloyl glucose were foundin minor amounts only and therefore not considered in thefollowing evaluation A typical chromatogram of the detectedphenolic compounds of pomegranate juice (MYWcultivar) isshown in Figure 1
The individual and total phenolic contents of all stud-ied cultivars were significantly different (119901 lt 001) Theconcentrations of the main phenolics showed wide rangespunicalagin B 259ndash884mgL ellagic acid 174ndash928mgLand punicalagin A 540ndash285mgL which is in agreementwith previous findings of juices from other pomegranatecultivars [1 4 13 14] Pearsonrsquos correlation coefficients werehigh (gt098) between punicalagin A and punicalagin B atsame juices (arils or whole pomegranate) but ellagic acidvalue was not significantly correlated with the other phenoliccompounds In each cultivar the total individual phenoliccontent of whole pomegranate juices was increased by 25ndash1136 compared to pomegranate aril juices In this regard theMY and JPGRT had increased 25 and 35 respectively butother samples showed higher increases (gt100) The highestincrease (1136) was awarded to GSY cultivar
()
0
5955413
4915413
5646013
7383011
6633011
7733011
200 300 400 500 600 700 800100
Time (min)Pu
nica
lagi
n A
Puni
cala
gin
B
Ella
gic a
cid
TIC452e6
Figure 1 Typical chromatogramof the nonanthocyanidin phenolicsin pomegranate juice (MYW) as detected by MS in selected ionmonitoring (SIM) mode Punicalagins appear as doubly chargedions Ions with mz = 301 other than ellagic acid are in-sourcefragments of other ellagitannins
The highest punicalagin contents among aril and wholefruit juices were found in JPGRTA (607mgL) and GSYW(1169mgL) respectively (Table 1) Punicalagin is the mostcharacteristic ellagitannin because it is found almost exclu-sively in pomegranate However pomegranate arils containlow or no punicalagins but pomegranate rind parts are anabundant source The higher punicalagin content of com-mercial pomegranate juices was due to the entrance of thesecompounds from rind parts of the fruit during processing[1 11] The phenolic content of four types of ldquoWonderfulrdquopomegranate juices including juices from fresh arils frozenarils and whole pomegranates and concentrate were evalu-ated [1] Punicalagin A in four types of pomegranate juiceswas 127 144 421 and 435mgL punicalagin B was 101 111839 and 918mgL and total ellagic derivatives were 332 265121 and 264mgL respectively The four major hydrolyzabletannins of punicalagin ellagic acid punicalin and gallagicacid were detected in 29 different pomegranate accessionsby Tzulker et al [11] The contents of these compounds
4 Journal of Chemistry
significantly differed in the whole fruit homogenates Thecontent of punicalagin was about 10-fold higher than thepunicalin and gallagic acid contents and 100-fold higher thanthe content of ellagic acid Although punicalagin punicalinand gallagic acid were also found in aril juices (ellagic acidwas below the detection level) these contents were very lowin comparison to the whole fruit homogenates [11]
In literature punicalagins content was reported to belower in pomegranate arils than in whole pomegranate fruitor even absent in the former [1 2 11] But in the current studythe cultivar JPGRT had such a high punicalagin content thatthe juice made from the arils of JPGRT had a higher content(607mgL) than the juice made from whole pomegranate ofcertain cultivars (MYW TMLW SRABW and PSYW)There-fore the content (or lack) of punicalagins in pomegranatejuice is not only affected by extractionmethod but alsomdashandeven moremdashby the pomegranate cultivar
Fischer et al [2] identified 48 compounds in 3 variantsof pomegranate juices among which 9 were anthocyanins2 gallotannins 22 ellagitannins 2 gallagyl esters 4 hydrox-ybenzoic acids and 7 hydroxycinnamic acids and 1 wasdihydroflavonol The phenolic compounds of pomegranatejuice prepared from whole pomegranate at 10 bars juiceprepared from whole pomegranate at 150 bars and juiceprepared from the isolated arils at 250 bars were as followstotal gallotannins (12ndash91mgL) total ellagitannins (932ndash2074mgL) total hydroxybenzoic acids (11ndash106mgL) andtotal hydroxycinnamic acids (214ndash283mgL) In anotherstudy punicalagin levels of juices from the pomegranate cul-tivar ldquoMollarrdquo ranged between 504 and 763mgL and puni-calinswere found in a lower range between 240 and 365mgLThe free ellagic acid level was between 269 and 390mgL [16]
Also Mena et al [14] have identified 75 compoundsby UHPLC-MS119899 of which 21 compounds were tentativelyidentified for the first time in pomegranate juice of Mollarde Elche cultivar The separated compounds included 37ellagitannins 6 gallotannins 14 noncoloured flavonoids 5phenolic acid derivatives 6 anthocyanins and a flavanol-anthocyanin adduct 6 lignans and 3 organic acids
32 Individual Anthocyanin Content The individual antho-cyanin contents of ten pomegranate cultivars were deter-mined using UHPLC A typical chromatogram of indi-vidual anthocyanins of MYA cultivar is shown in Fig-ure 2 The applied chromatographic conditions success-fully separated both the major and minor anthocyaninsof pomegranate juice The detected anthocyanins werecyanidin 3-glucoside (Cy 3G) cyanidin 35-diglucoside (Cy35dG) delphinidin 3-glucoside (Dp 3G) delphinidin 35-diglucoside (Dp 35dG) pelargonidin 3-glucoside (Pg 3G)pelargonidin 35-diglucoside (Pg 35dG) cyanidin-pentoside(Cy-pent) cyanidin-pentoside-hexoside (Cy-pent-hex) anddelphinidin-pentoside (Dp-pent)
Table 2 shows the individual anthocyanin contentsof the studied cultivars The JPGRTA cultivar had thehighest (175mgL) anthocyanin content whereas those ofMPSA ATSA PSYA and PSYW were lower than 5mgLIn some cultivars juice extraction of whole pomegranate
()
0400 600 800 1000 1200 1400 1600 1800 2000200
Time (min)
Dp3
5dG Cy
35
dGPg
35
dGD
p3
G
Cy-p
ent-h
exCy
3G
Pg3
G
Dp-
pent
Cy-p
ent
Figure 2 Typical chromatogram of the anthocyanidins inpomegranate juice (MYA) as detected by MS in SRMmode
juices increased the anthocyanin content ATSW (134)GSYW (477) MPSW (347) MTKW (223) and PSYW(196) In contrast the anthocyanin contents of SRABW(214) TMLW (104) MYW (440) and JPGRTW(390) were decreased Based on these results it is evidentthat the anthocyanin content of pomegranate juices is affectedby extraction methods
According to Figure 2 nine major and minoranthocyanins were identified namely cyanidin 3-glucosidecyanidin 35-diglucoside delphinidin 3-glucoside delphini-din 35-diglucoside pelargonidin 3-glucoside pelargonidin35-diglucoside cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside In terms of quantitythe main anthocyanin in most of the cultivars was Cy 35dG(07ndash947mgL) followed by Cy 3G (05ndash525mgL) Pg35dG + Dp 3G (0ndash103mgL) Dp 35dG (0ndash940mgL) Pg3G (0ndash400mgL) and Cy-pent (0ndash113mgL)
Except for Cy-pent Dp-pent and Cy-pent-hex theanthocyanin profiles were consistent with previouslyreported researches about other pomegranate cultivars[1 12 17ndash19] In the same pomegranate cultivars Alighourchiet al [12] reported the content of 6 anthocyanins inaril juices as follows Dp 3G (219ndash163mgL) Dp 35dG(236ndash631mgL) Pg 3G (026ndash136mgL) Pg 35dG(001ndash811mgL) Cy 3G (578ndash304mgL) and Cy 35dG(439ndash166mgL) Thus achieving different anthocyaninscontent in the same pomegranate cultivars indicated theeffect of agricultural and environmental conditions on thephysicochemical characteristic of pomegranate fruit
To our knowledge Dp-pent has not been reportedin pomegranate fruit so far It is tentatively identified bythe mass transition 435gt303 Fischer et al [2] identified9 anthocyanins of Dp 35dG (04ndash205mgL) Cy 35dG(954ndash271mgL) Pg 35dG (137ndash267mgL) Dp 3G (10ndash168mgL) Cy 3G (91ndash279mgL) Pg 3G (24ndash49mgL)a cyanidin-pentoside-hexoside (11ndash30mgL) cyanidin 3-rutinoside (00ndash12mgL) and a cyanidin-pentoside (02ndash06mgL) in the three pomegranate juice variants Gil et al[1] reported the main pigments in the juice of theWonderful
Journal of Chemistry 5
Table 2 Contents of individual anthocyanins (mgL) in pomegranate juices
cultivar as follows Cy 3G (595ndash128mgL) Dp 3G (236ndash952mgL) Cy 35dG (314ndash714mgL) Dp 35dG (211ndash611mgL) and Pg 3G (39ndash85mgL)Mena et al [4] reportedCy 3G (33) as the major anthocyanin followed by Dp 35dG(24) Dp 3G (21) and Cy 35dG (19) and Pg 3G andPg 35dG were only 3 of the total anthocyanin content Inaddition four major anthocyanins of Dp 3G Dp 35dG Cy3G and Cy 35dG andminor amounts of Pg 3G and Pg 35dGwere determined in pomegranate juices [20]
The main anthocyanin in all the cultivars was Cy 35dGexcept for SRABA in which Cy 3G was dominant It isknown that the main anthocyanin of pomegranate dependson the cultivar The main anthocyanins were Cy 3G [1]in the American Wonderful cultivar Cy 3G (40) and Cy35dG (38) in the Spanish Mollar cultivar [19] Dp 3G inthe Portuguese Assaria cultivar [17] Cy 35dG in 15 Iraniancultivars [12] Dp 35dG in 8 other Iranian cultivars [5] andCy 35dG in a Peruvian pomegranate fruit [2]
Among different species or even cultivars of the samespecies the anthocyanin content varies considerably affectedby genetic make-up light temperature and agronomic fac-tors [21] Intrinsic properties of the product and the processsuch as pH temperature light oxygen enzymes ascorbicacids sugars metal ions and copigmentation (intermolecu-lar and intramolecular complex formations self-associationand metal complexation) as well as chemical structure andconcentration of anthocyanins can affect anthocyanin stabil-ity [22]
The variation of anthocyanin content in juices from arilsand whole pomegranate can be affected by the entrance ofanthocyanins from the peel into the juices Therefore the
amount of anthocyanins of the same cultivar varied withextraction methods
33 Total Phenolic Content and Antioxidant CapacityAccording to the results presented in Table 3 total phenoliccontents of juices from whole pomegranate fruit (942ndash2931mgL) were significantly higher than those in arilsjuice (220ndash1267mgL) The GSYW cultivar had the highest(2931mgL) total phenolic content In other studies the totalphenolic contents of various pomegranate cultivars showedsignificant differences and were 296ndash985mg100 g [9] 208ndash344mg100 g [7] 144ndash1009mg100 g [8] 237ndash930mg100 g[5] 2015ndash5186mgL [2] and 1136ndash3581mgL [16] Differentamounts of total phenolic content in various studies can beattributed to analytical methods cultivar maturity stageand environmental conditions and also overestimation ofthe total individual phenolic compounds by Folin-Ciocalteureagent due to interference with other reducing factors [2]
The antioxidant properties were measured by ABTSFRAP and DPPH assay (Table 3) The results showed con-siderable antioxidant activity of juice samples Antioxidantactivities of juices from whole pomegranate fruit were sig-nificantly higher than juices from pomegranate arils thatindicated the importance of the juice extraction methodsThese results are in agreement with previously reportedfindings [1 11]The antioxidant capacity of pomegranate juice(and other fruit juices) depends on cultivar growing regionfruit maturation and agricultural factors The technologyused for juice processing may affect antioxidant capacityof pomegranate juice [7] In this regard the antioxidantactivity of commercial pomegranate juice was 50 higher
6 Journal of Chemistry
Table 3 Evaluation of total polyphenol content (mg gallic acid equivalentsL) and antioxidant activities based on ABTS (mg ascorbicacid100mL) FRAP (mmol Fe2+100mL) and DPPH (AA) in pomegranate juices
MPSW 19541 plusmn 271cd 12492 plusmn 265c 306 plusmn 18c 550 plusmn 18clowastDifferent letters in the same column present significant difference at 119901 lt 005
than juices from pomegranate arils due to the entranceof phenolic compounds from the peel into juices [1] AlsoTzulker et al [11] reported that antioxidant activity of wholefruit homogenates is about 20-fold higher than that foundin the arils These results indicated that hydrolysable tanninsincluding punicalagin were themain compounds influencingthe antioxidant capacity of pomegranate juices [1 11] Thiscompound shows a high antioxidant activity due to the 16phenolic hydroxyl groups in the molecule [1]
Moreover a good correlation was observed betweentotal phenolic content and ABTS and FRAP methods inall pomegranate juices (gt090) Correlations between totalphenolic and DPPH in juices of arils and whole pomegranatewere 092 and 086 respectively However Pearson corre-lation showed that the correlation of three measurementantioxidant methods is affected by pomegranate cultivarsas mentioned in previous research [7 11] In this regardTzulker et al [11] reported that the antioxidant activity (FRAPand DPPH methods) of juices from whole pomegranatehomogenate and arils alone significantly correlated withthe polyphenol level but it did not correlate with antho-cyanins level The highest correlation value was obtainedbetween antioxidant activity and punicalagin in the wholepomegranate homogenate The FRAP method comparedto DPPH method gave the highest correlation with totalpolyphenol content
4 Conclusion
The individual and total phenolic contents of all stud-ied cultivars were significantly different In this study
among nine major and minor anthocyanins the cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside were identified for the first time in Iranianpomegranate cultivars The phenolic contents and antioxi-dant activity of whole pomegranate juice were significantlyhigher than those of juices from pomegranate arils from thesame cultivar due to the entrance of phenolic compoundsfrom the rind parts of pomegranate to the juices Thevariation between the cultivars is very wide therefore thecontents of ellagitannins and anthocyanins in the juice fromwhole pomegranates of the low-polyphenol cultivars (asMY)are lower than those in the juices made from arils only ofthe high-polyphenol cultivars (as JPGRT) In otherwords thecultivar has a higher influence on the phenolic content of thejuice than the extraction method
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to acknowledge Dr Vazifeshenas forhis assistance in afforded pomegranate cultivars
References
[1] M I Gil F A Tomas-Barberan B Hess-Pierce D M Holcroftand A A Kader ldquoAntioxidant activity of pomegranate juice
Journal of Chemistry 7
and its relationship with phenolic composition and processingrdquoJournal of Agricultural and Food Chemistry vol 48 no 10 pp4581ndash4589 2000
[2] U A Fischer R Carle and D R Kammerer ldquoIdentificationand quantification of phenolic compounds from pomegranate(Punica granatum L) peel mesocarp aril and differently pro-duced juices by HPLC-DAD-ESIMS119899rdquo Food Chemistry vol127 no 2 pp 807ndash821 2011
[3] Anonymous Iran Statistical Year Book 2013 2013 httpeamarsciorgirindex easpx
[4] P Mena C Garcıa-Viguera J Navarro-Rico et al ldquoPhytochem-ical characterisation for industrial use of pomegranate (Punicagranatum L) cultivars grown in Spainrdquo Journal of the Science ofFood and Agriculture vol 91 no 10 pp 1893ndash1906 2011
[5] G Mousavinejad Z Emam-Djomeh K Rezaei and M HH Khodaparast ldquoIdentification and quantification of pheno-lic compounds and their effects on antioxidant activity inpomegranate juices of eight Iranian cultivarsrdquo Food Chemistryvol 115 no 4 pp 1274ndash1278 2009
[6] O A Fawole U L Opara and K I Theron ldquoChemicaland phytochemical properties and antioxidant activities ofthree pomegranate cultivars grown in South Africardquo Food andBioprocess Technology vol 5 no 7 pp 2934ndash2940 2012
[7] M Cam Y Hısıl and G Durmaz ldquoClassification of eightpomegranate juices based on antioxidant capacity measured byfourmethodsrdquo FoodChemistry vol 112 no 3 pp 721ndash726 2009
[8] F Tezcan M Gultekin-Ozguven T Diken B Ozcelik and FB Erim ldquoAntioxidant activity and total phenolic organic acidand sugar content in commercial pomegranate juicesrdquo FoodChemistry vol 115 no 3 pp 873ndash877 2009
[9] A Tehranifar M Zarei Z Nemati B Esfandiyari and MR Vazifeshenas ldquoInvestigation of physico-chemical propertiesand antioxidant activity of twenty Iranian pomegranate (PunicagranatumL) cultivarsrdquo ScientiaHorticulturae vol 126 no 2 pp180ndash185 2010
[10] M Ozgen C Durgac S Serce and C Kaya ldquoChemical andantioxidant properties of pomegranate cultivars grown in theMediterranean region of Turkeyrdquo Food Chemistry vol 111 no3 pp 703ndash706 2008
[11] R Tzulker I Glazer I Bar-Ilan D Holland M Aviramand R Amir ldquoAntioxidant activity polyphenol content andrelated compounds in different fruit juices and homogenatesprepared from 29 different pomegranate accessionsrdquo Journal ofAgricultural and Food Chemistry vol 55 no 23 pp 9559ndash95702007
[12] H Alighourchi M Barzegar and S Abbasi ldquoAnthocyaninscharacterization of 15 Iranian pomegranate (Punica granatumL) varieties and their variation after cold storage and pasteur-izationrdquo European Food Research and Technology vol 227 no 3pp 881ndash887 2008
[13] S Chrubasik-Hausmann C Vlachojannis and B Zimmer-mann ldquoPomegranate juice and prostate cancer importanceof the characterisation of the active principlerdquo PhytotherapyResearch vol 28 no 11 pp 1676ndash1678 2014
[14] P Mena L Calani C DallrsquoAsta et al ldquoRapid and comprehen-sive evaluation of (Poly)phenolic compounds in pomegranate(Punica granatum L) Juice by UHPLC-MSnrdquoMolecules vol 17no 12 pp 14821ndash14840 2012
[15] M M Feuereisen J Hoppe B F Zimmermann F Weber NSchulze-Kaysers andA Schieber ldquoCharacterization of phenoliccompounds in Brazilian pepper (Schinus terebinthifolius Raddi)
exocarprdquo Journal of Agricultural and FoodChemistry vol 62 no26 pp 6219ndash6226 2014
[16] SVegaraNMartı J Lorente et al ldquoChemical guide parametersfor Punica granatum cv lsquoMollarrsquo fruit juices processed atindustrial scalerdquo Food Chemistry vol 147 pp 203ndash208 2014
[17] G Miguel S Dandlen D Antunes A Neves and D MartinsldquoThe effect of two methods of pomegranate (Punica granatumL) juice extraction on quality during storage at 4∘Crdquo Journalof Biomedicine and Biotechnology vol 2004 no 5 pp 332ndash3372004
[18] A Perez-Vicente P Serrano P Abellan and C Garcıa-VigueraldquoInfluence of packaging material on pomegranate juice colourand bioactive compounds during storagerdquo Journal of the Scienceof Food and Agriculture vol 84 no 7 pp 639ndash644 2004
[19] NMartı A Perez-Vicente andCGarcıa-Viguera ldquoInfluence ofstorage temperature and ascorbic acid addition on pomegranatejuicerdquo Journal of the Science of Food and Agriculture vol 82 no2 pp 217ndash221 2002
[20] C U Pala and A K Toklucu ldquoEffect of UV-C light on antho-cyanin content and other quality parameters of pomegranatejuicerdquo Journal of Food Composition and Analysis vol 24 no 6pp 790ndash795 2011
[21] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsCRC Press Boca Raton Fla USA 2004
[22] A Patras N P Brunton S Da Pieve and F Butler ldquoImpact ofhigh pressure processing on total antioxidant activity phenolicascorbic acid anthocyanin content and colour of strawberryand blackberry pureesrdquo Innovative Food Science amp EmergingTechnologies vol 10 no 3 pp 308ndash313 2009
MPSA 508 plusmn 09c 1492 plusmn 30e 1233 plusmn 16c MPSW 1809 plusmn 39b 6501 plusmn 13b 2618 plusmn 26dlowastDifferent letters in the same column present significant difference at 119901 lt 005
radical scavenging capacity by DPPH radical (expressed asinhibition percentage) and ABTS radical (expressed as mg L-ascorbic acid100mL juice) and with iron-reducing capacityby FRAP method (expressed in mmol Fe2+100mL juice)[7 8]
26 Statistical Analysis The data were subjected to analysesof variance (ANOVA) using SPSS 170 For the comparison ofsignificance between cultivars Duncanrsquos multiple range test(119875 lt 005) was used as post hoc test Results are the means plusmnSD of two determinations for each juice
3 Results and Discussion
31 Punicalagins and Ellagic Acids In this study the majorindividual phenolic compounds of ten pomegranate cultivarswere identified by HPLCPDAMS2 namely punicalagin Apunicalagin B and ellagic acid (Table 1) besides anthocyanins(see the next paragraph)These results are in accordance withpreviously reports on other pomegranate cultivars [1 2 4 1116]
Gallic acid gallagic acid and galloyl glucose were foundin minor amounts only and therefore not considered in thefollowing evaluation A typical chromatogram of the detectedphenolic compounds of pomegranate juice (MYWcultivar) isshown in Figure 1
The individual and total phenolic contents of all stud-ied cultivars were significantly different (119901 lt 001) Theconcentrations of the main phenolics showed wide rangespunicalagin B 259ndash884mgL ellagic acid 174ndash928mgLand punicalagin A 540ndash285mgL which is in agreementwith previous findings of juices from other pomegranatecultivars [1 4 13 14] Pearsonrsquos correlation coefficients werehigh (gt098) between punicalagin A and punicalagin B atsame juices (arils or whole pomegranate) but ellagic acidvalue was not significantly correlated with the other phenoliccompounds In each cultivar the total individual phenoliccontent of whole pomegranate juices was increased by 25ndash1136 compared to pomegranate aril juices In this regard theMY and JPGRT had increased 25 and 35 respectively butother samples showed higher increases (gt100) The highestincrease (1136) was awarded to GSY cultivar
()
0
5955413
4915413
5646013
7383011
6633011
7733011
200 300 400 500 600 700 800100
Time (min)Pu
nica
lagi
n A
Puni
cala
gin
B
Ella
gic a
cid
TIC452e6
Figure 1 Typical chromatogramof the nonanthocyanidin phenolicsin pomegranate juice (MYW) as detected by MS in selected ionmonitoring (SIM) mode Punicalagins appear as doubly chargedions Ions with mz = 301 other than ellagic acid are in-sourcefragments of other ellagitannins
The highest punicalagin contents among aril and wholefruit juices were found in JPGRTA (607mgL) and GSYW(1169mgL) respectively (Table 1) Punicalagin is the mostcharacteristic ellagitannin because it is found almost exclu-sively in pomegranate However pomegranate arils containlow or no punicalagins but pomegranate rind parts are anabundant source The higher punicalagin content of com-mercial pomegranate juices was due to the entrance of thesecompounds from rind parts of the fruit during processing[1 11] The phenolic content of four types of ldquoWonderfulrdquopomegranate juices including juices from fresh arils frozenarils and whole pomegranates and concentrate were evalu-ated [1] Punicalagin A in four types of pomegranate juiceswas 127 144 421 and 435mgL punicalagin B was 101 111839 and 918mgL and total ellagic derivatives were 332 265121 and 264mgL respectively The four major hydrolyzabletannins of punicalagin ellagic acid punicalin and gallagicacid were detected in 29 different pomegranate accessionsby Tzulker et al [11] The contents of these compounds
4 Journal of Chemistry
significantly differed in the whole fruit homogenates Thecontent of punicalagin was about 10-fold higher than thepunicalin and gallagic acid contents and 100-fold higher thanthe content of ellagic acid Although punicalagin punicalinand gallagic acid were also found in aril juices (ellagic acidwas below the detection level) these contents were very lowin comparison to the whole fruit homogenates [11]
In literature punicalagins content was reported to belower in pomegranate arils than in whole pomegranate fruitor even absent in the former [1 2 11] But in the current studythe cultivar JPGRT had such a high punicalagin content thatthe juice made from the arils of JPGRT had a higher content(607mgL) than the juice made from whole pomegranate ofcertain cultivars (MYW TMLW SRABW and PSYW)There-fore the content (or lack) of punicalagins in pomegranatejuice is not only affected by extractionmethod but alsomdashandeven moremdashby the pomegranate cultivar
Fischer et al [2] identified 48 compounds in 3 variantsof pomegranate juices among which 9 were anthocyanins2 gallotannins 22 ellagitannins 2 gallagyl esters 4 hydrox-ybenzoic acids and 7 hydroxycinnamic acids and 1 wasdihydroflavonol The phenolic compounds of pomegranatejuice prepared from whole pomegranate at 10 bars juiceprepared from whole pomegranate at 150 bars and juiceprepared from the isolated arils at 250 bars were as followstotal gallotannins (12ndash91mgL) total ellagitannins (932ndash2074mgL) total hydroxybenzoic acids (11ndash106mgL) andtotal hydroxycinnamic acids (214ndash283mgL) In anotherstudy punicalagin levels of juices from the pomegranate cul-tivar ldquoMollarrdquo ranged between 504 and 763mgL and puni-calinswere found in a lower range between 240 and 365mgLThe free ellagic acid level was between 269 and 390mgL [16]
Also Mena et al [14] have identified 75 compoundsby UHPLC-MS119899 of which 21 compounds were tentativelyidentified for the first time in pomegranate juice of Mollarde Elche cultivar The separated compounds included 37ellagitannins 6 gallotannins 14 noncoloured flavonoids 5phenolic acid derivatives 6 anthocyanins and a flavanol-anthocyanin adduct 6 lignans and 3 organic acids
32 Individual Anthocyanin Content The individual antho-cyanin contents of ten pomegranate cultivars were deter-mined using UHPLC A typical chromatogram of indi-vidual anthocyanins of MYA cultivar is shown in Fig-ure 2 The applied chromatographic conditions success-fully separated both the major and minor anthocyaninsof pomegranate juice The detected anthocyanins werecyanidin 3-glucoside (Cy 3G) cyanidin 35-diglucoside (Cy35dG) delphinidin 3-glucoside (Dp 3G) delphinidin 35-diglucoside (Dp 35dG) pelargonidin 3-glucoside (Pg 3G)pelargonidin 35-diglucoside (Pg 35dG) cyanidin-pentoside(Cy-pent) cyanidin-pentoside-hexoside (Cy-pent-hex) anddelphinidin-pentoside (Dp-pent)
Table 2 shows the individual anthocyanin contentsof the studied cultivars The JPGRTA cultivar had thehighest (175mgL) anthocyanin content whereas those ofMPSA ATSA PSYA and PSYW were lower than 5mgLIn some cultivars juice extraction of whole pomegranate
()
0400 600 800 1000 1200 1400 1600 1800 2000200
Time (min)
Dp3
5dG Cy
35
dGPg
35
dGD
p3
G
Cy-p
ent-h
exCy
3G
Pg3
G
Dp-
pent
Cy-p
ent
Figure 2 Typical chromatogram of the anthocyanidins inpomegranate juice (MYA) as detected by MS in SRMmode
juices increased the anthocyanin content ATSW (134)GSYW (477) MPSW (347) MTKW (223) and PSYW(196) In contrast the anthocyanin contents of SRABW(214) TMLW (104) MYW (440) and JPGRTW(390) were decreased Based on these results it is evidentthat the anthocyanin content of pomegranate juices is affectedby extraction methods
According to Figure 2 nine major and minoranthocyanins were identified namely cyanidin 3-glucosidecyanidin 35-diglucoside delphinidin 3-glucoside delphini-din 35-diglucoside pelargonidin 3-glucoside pelargonidin35-diglucoside cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside In terms of quantitythe main anthocyanin in most of the cultivars was Cy 35dG(07ndash947mgL) followed by Cy 3G (05ndash525mgL) Pg35dG + Dp 3G (0ndash103mgL) Dp 35dG (0ndash940mgL) Pg3G (0ndash400mgL) and Cy-pent (0ndash113mgL)
Except for Cy-pent Dp-pent and Cy-pent-hex theanthocyanin profiles were consistent with previouslyreported researches about other pomegranate cultivars[1 12 17ndash19] In the same pomegranate cultivars Alighourchiet al [12] reported the content of 6 anthocyanins inaril juices as follows Dp 3G (219ndash163mgL) Dp 35dG(236ndash631mgL) Pg 3G (026ndash136mgL) Pg 35dG(001ndash811mgL) Cy 3G (578ndash304mgL) and Cy 35dG(439ndash166mgL) Thus achieving different anthocyaninscontent in the same pomegranate cultivars indicated theeffect of agricultural and environmental conditions on thephysicochemical characteristic of pomegranate fruit
To our knowledge Dp-pent has not been reportedin pomegranate fruit so far It is tentatively identified bythe mass transition 435gt303 Fischer et al [2] identified9 anthocyanins of Dp 35dG (04ndash205mgL) Cy 35dG(954ndash271mgL) Pg 35dG (137ndash267mgL) Dp 3G (10ndash168mgL) Cy 3G (91ndash279mgL) Pg 3G (24ndash49mgL)a cyanidin-pentoside-hexoside (11ndash30mgL) cyanidin 3-rutinoside (00ndash12mgL) and a cyanidin-pentoside (02ndash06mgL) in the three pomegranate juice variants Gil et al[1] reported the main pigments in the juice of theWonderful
Journal of Chemistry 5
Table 2 Contents of individual anthocyanins (mgL) in pomegranate juices
cultivar as follows Cy 3G (595ndash128mgL) Dp 3G (236ndash952mgL) Cy 35dG (314ndash714mgL) Dp 35dG (211ndash611mgL) and Pg 3G (39ndash85mgL)Mena et al [4] reportedCy 3G (33) as the major anthocyanin followed by Dp 35dG(24) Dp 3G (21) and Cy 35dG (19) and Pg 3G andPg 35dG were only 3 of the total anthocyanin content Inaddition four major anthocyanins of Dp 3G Dp 35dG Cy3G and Cy 35dG andminor amounts of Pg 3G and Pg 35dGwere determined in pomegranate juices [20]
The main anthocyanin in all the cultivars was Cy 35dGexcept for SRABA in which Cy 3G was dominant It isknown that the main anthocyanin of pomegranate dependson the cultivar The main anthocyanins were Cy 3G [1]in the American Wonderful cultivar Cy 3G (40) and Cy35dG (38) in the Spanish Mollar cultivar [19] Dp 3G inthe Portuguese Assaria cultivar [17] Cy 35dG in 15 Iraniancultivars [12] Dp 35dG in 8 other Iranian cultivars [5] andCy 35dG in a Peruvian pomegranate fruit [2]
Among different species or even cultivars of the samespecies the anthocyanin content varies considerably affectedby genetic make-up light temperature and agronomic fac-tors [21] Intrinsic properties of the product and the processsuch as pH temperature light oxygen enzymes ascorbicacids sugars metal ions and copigmentation (intermolecu-lar and intramolecular complex formations self-associationand metal complexation) as well as chemical structure andconcentration of anthocyanins can affect anthocyanin stabil-ity [22]
The variation of anthocyanin content in juices from arilsand whole pomegranate can be affected by the entrance ofanthocyanins from the peel into the juices Therefore the
amount of anthocyanins of the same cultivar varied withextraction methods
33 Total Phenolic Content and Antioxidant CapacityAccording to the results presented in Table 3 total phenoliccontents of juices from whole pomegranate fruit (942ndash2931mgL) were significantly higher than those in arilsjuice (220ndash1267mgL) The GSYW cultivar had the highest(2931mgL) total phenolic content In other studies the totalphenolic contents of various pomegranate cultivars showedsignificant differences and were 296ndash985mg100 g [9] 208ndash344mg100 g [7] 144ndash1009mg100 g [8] 237ndash930mg100 g[5] 2015ndash5186mgL [2] and 1136ndash3581mgL [16] Differentamounts of total phenolic content in various studies can beattributed to analytical methods cultivar maturity stageand environmental conditions and also overestimation ofthe total individual phenolic compounds by Folin-Ciocalteureagent due to interference with other reducing factors [2]
The antioxidant properties were measured by ABTSFRAP and DPPH assay (Table 3) The results showed con-siderable antioxidant activity of juice samples Antioxidantactivities of juices from whole pomegranate fruit were sig-nificantly higher than juices from pomegranate arils thatindicated the importance of the juice extraction methodsThese results are in agreement with previously reportedfindings [1 11]The antioxidant capacity of pomegranate juice(and other fruit juices) depends on cultivar growing regionfruit maturation and agricultural factors The technologyused for juice processing may affect antioxidant capacityof pomegranate juice [7] In this regard the antioxidantactivity of commercial pomegranate juice was 50 higher
6 Journal of Chemistry
Table 3 Evaluation of total polyphenol content (mg gallic acid equivalentsL) and antioxidant activities based on ABTS (mg ascorbicacid100mL) FRAP (mmol Fe2+100mL) and DPPH (AA) in pomegranate juices
MPSW 19541 plusmn 271cd 12492 plusmn 265c 306 plusmn 18c 550 plusmn 18clowastDifferent letters in the same column present significant difference at 119901 lt 005
than juices from pomegranate arils due to the entranceof phenolic compounds from the peel into juices [1] AlsoTzulker et al [11] reported that antioxidant activity of wholefruit homogenates is about 20-fold higher than that foundin the arils These results indicated that hydrolysable tanninsincluding punicalagin were themain compounds influencingthe antioxidant capacity of pomegranate juices [1 11] Thiscompound shows a high antioxidant activity due to the 16phenolic hydroxyl groups in the molecule [1]
Moreover a good correlation was observed betweentotal phenolic content and ABTS and FRAP methods inall pomegranate juices (gt090) Correlations between totalphenolic and DPPH in juices of arils and whole pomegranatewere 092 and 086 respectively However Pearson corre-lation showed that the correlation of three measurementantioxidant methods is affected by pomegranate cultivarsas mentioned in previous research [7 11] In this regardTzulker et al [11] reported that the antioxidant activity (FRAPand DPPH methods) of juices from whole pomegranatehomogenate and arils alone significantly correlated withthe polyphenol level but it did not correlate with antho-cyanins level The highest correlation value was obtainedbetween antioxidant activity and punicalagin in the wholepomegranate homogenate The FRAP method comparedto DPPH method gave the highest correlation with totalpolyphenol content
4 Conclusion
The individual and total phenolic contents of all stud-ied cultivars were significantly different In this study
among nine major and minor anthocyanins the cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside were identified for the first time in Iranianpomegranate cultivars The phenolic contents and antioxi-dant activity of whole pomegranate juice were significantlyhigher than those of juices from pomegranate arils from thesame cultivar due to the entrance of phenolic compoundsfrom the rind parts of pomegranate to the juices Thevariation between the cultivars is very wide therefore thecontents of ellagitannins and anthocyanins in the juice fromwhole pomegranates of the low-polyphenol cultivars (asMY)are lower than those in the juices made from arils only ofthe high-polyphenol cultivars (as JPGRT) In otherwords thecultivar has a higher influence on the phenolic content of thejuice than the extraction method
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to acknowledge Dr Vazifeshenas forhis assistance in afforded pomegranate cultivars
References
[1] M I Gil F A Tomas-Barberan B Hess-Pierce D M Holcroftand A A Kader ldquoAntioxidant activity of pomegranate juice
Journal of Chemistry 7
and its relationship with phenolic composition and processingrdquoJournal of Agricultural and Food Chemistry vol 48 no 10 pp4581ndash4589 2000
[2] U A Fischer R Carle and D R Kammerer ldquoIdentificationand quantification of phenolic compounds from pomegranate(Punica granatum L) peel mesocarp aril and differently pro-duced juices by HPLC-DAD-ESIMS119899rdquo Food Chemistry vol127 no 2 pp 807ndash821 2011
[3] Anonymous Iran Statistical Year Book 2013 2013 httpeamarsciorgirindex easpx
[4] P Mena C Garcıa-Viguera J Navarro-Rico et al ldquoPhytochem-ical characterisation for industrial use of pomegranate (Punicagranatum L) cultivars grown in Spainrdquo Journal of the Science ofFood and Agriculture vol 91 no 10 pp 1893ndash1906 2011
[5] G Mousavinejad Z Emam-Djomeh K Rezaei and M HH Khodaparast ldquoIdentification and quantification of pheno-lic compounds and their effects on antioxidant activity inpomegranate juices of eight Iranian cultivarsrdquo Food Chemistryvol 115 no 4 pp 1274ndash1278 2009
[6] O A Fawole U L Opara and K I Theron ldquoChemicaland phytochemical properties and antioxidant activities ofthree pomegranate cultivars grown in South Africardquo Food andBioprocess Technology vol 5 no 7 pp 2934ndash2940 2012
[7] M Cam Y Hısıl and G Durmaz ldquoClassification of eightpomegranate juices based on antioxidant capacity measured byfourmethodsrdquo FoodChemistry vol 112 no 3 pp 721ndash726 2009
[8] F Tezcan M Gultekin-Ozguven T Diken B Ozcelik and FB Erim ldquoAntioxidant activity and total phenolic organic acidand sugar content in commercial pomegranate juicesrdquo FoodChemistry vol 115 no 3 pp 873ndash877 2009
[9] A Tehranifar M Zarei Z Nemati B Esfandiyari and MR Vazifeshenas ldquoInvestigation of physico-chemical propertiesand antioxidant activity of twenty Iranian pomegranate (PunicagranatumL) cultivarsrdquo ScientiaHorticulturae vol 126 no 2 pp180ndash185 2010
[10] M Ozgen C Durgac S Serce and C Kaya ldquoChemical andantioxidant properties of pomegranate cultivars grown in theMediterranean region of Turkeyrdquo Food Chemistry vol 111 no3 pp 703ndash706 2008
[11] R Tzulker I Glazer I Bar-Ilan D Holland M Aviramand R Amir ldquoAntioxidant activity polyphenol content andrelated compounds in different fruit juices and homogenatesprepared from 29 different pomegranate accessionsrdquo Journal ofAgricultural and Food Chemistry vol 55 no 23 pp 9559ndash95702007
[12] H Alighourchi M Barzegar and S Abbasi ldquoAnthocyaninscharacterization of 15 Iranian pomegranate (Punica granatumL) varieties and their variation after cold storage and pasteur-izationrdquo European Food Research and Technology vol 227 no 3pp 881ndash887 2008
[13] S Chrubasik-Hausmann C Vlachojannis and B Zimmer-mann ldquoPomegranate juice and prostate cancer importanceof the characterisation of the active principlerdquo PhytotherapyResearch vol 28 no 11 pp 1676ndash1678 2014
[14] P Mena L Calani C DallrsquoAsta et al ldquoRapid and comprehen-sive evaluation of (Poly)phenolic compounds in pomegranate(Punica granatum L) Juice by UHPLC-MSnrdquoMolecules vol 17no 12 pp 14821ndash14840 2012
[15] M M Feuereisen J Hoppe B F Zimmermann F Weber NSchulze-Kaysers andA Schieber ldquoCharacterization of phenoliccompounds in Brazilian pepper (Schinus terebinthifolius Raddi)
exocarprdquo Journal of Agricultural and FoodChemistry vol 62 no26 pp 6219ndash6226 2014
[16] SVegaraNMartı J Lorente et al ldquoChemical guide parametersfor Punica granatum cv lsquoMollarrsquo fruit juices processed atindustrial scalerdquo Food Chemistry vol 147 pp 203ndash208 2014
[17] G Miguel S Dandlen D Antunes A Neves and D MartinsldquoThe effect of two methods of pomegranate (Punica granatumL) juice extraction on quality during storage at 4∘Crdquo Journalof Biomedicine and Biotechnology vol 2004 no 5 pp 332ndash3372004
[18] A Perez-Vicente P Serrano P Abellan and C Garcıa-VigueraldquoInfluence of packaging material on pomegranate juice colourand bioactive compounds during storagerdquo Journal of the Scienceof Food and Agriculture vol 84 no 7 pp 639ndash644 2004
[19] NMartı A Perez-Vicente andCGarcıa-Viguera ldquoInfluence ofstorage temperature and ascorbic acid addition on pomegranatejuicerdquo Journal of the Science of Food and Agriculture vol 82 no2 pp 217ndash221 2002
[20] C U Pala and A K Toklucu ldquoEffect of UV-C light on antho-cyanin content and other quality parameters of pomegranatejuicerdquo Journal of Food Composition and Analysis vol 24 no 6pp 790ndash795 2011
[21] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsCRC Press Boca Raton Fla USA 2004
[22] A Patras N P Brunton S Da Pieve and F Butler ldquoImpact ofhigh pressure processing on total antioxidant activity phenolicascorbic acid anthocyanin content and colour of strawberryand blackberry pureesrdquo Innovative Food Science amp EmergingTechnologies vol 10 no 3 pp 308ndash313 2009
significantly differed in the whole fruit homogenates Thecontent of punicalagin was about 10-fold higher than thepunicalin and gallagic acid contents and 100-fold higher thanthe content of ellagic acid Although punicalagin punicalinand gallagic acid were also found in aril juices (ellagic acidwas below the detection level) these contents were very lowin comparison to the whole fruit homogenates [11]
In literature punicalagins content was reported to belower in pomegranate arils than in whole pomegranate fruitor even absent in the former [1 2 11] But in the current studythe cultivar JPGRT had such a high punicalagin content thatthe juice made from the arils of JPGRT had a higher content(607mgL) than the juice made from whole pomegranate ofcertain cultivars (MYW TMLW SRABW and PSYW)There-fore the content (or lack) of punicalagins in pomegranatejuice is not only affected by extractionmethod but alsomdashandeven moremdashby the pomegranate cultivar
Fischer et al [2] identified 48 compounds in 3 variantsof pomegranate juices among which 9 were anthocyanins2 gallotannins 22 ellagitannins 2 gallagyl esters 4 hydrox-ybenzoic acids and 7 hydroxycinnamic acids and 1 wasdihydroflavonol The phenolic compounds of pomegranatejuice prepared from whole pomegranate at 10 bars juiceprepared from whole pomegranate at 150 bars and juiceprepared from the isolated arils at 250 bars were as followstotal gallotannins (12ndash91mgL) total ellagitannins (932ndash2074mgL) total hydroxybenzoic acids (11ndash106mgL) andtotal hydroxycinnamic acids (214ndash283mgL) In anotherstudy punicalagin levels of juices from the pomegranate cul-tivar ldquoMollarrdquo ranged between 504 and 763mgL and puni-calinswere found in a lower range between 240 and 365mgLThe free ellagic acid level was between 269 and 390mgL [16]
Also Mena et al [14] have identified 75 compoundsby UHPLC-MS119899 of which 21 compounds were tentativelyidentified for the first time in pomegranate juice of Mollarde Elche cultivar The separated compounds included 37ellagitannins 6 gallotannins 14 noncoloured flavonoids 5phenolic acid derivatives 6 anthocyanins and a flavanol-anthocyanin adduct 6 lignans and 3 organic acids
32 Individual Anthocyanin Content The individual antho-cyanin contents of ten pomegranate cultivars were deter-mined using UHPLC A typical chromatogram of indi-vidual anthocyanins of MYA cultivar is shown in Fig-ure 2 The applied chromatographic conditions success-fully separated both the major and minor anthocyaninsof pomegranate juice The detected anthocyanins werecyanidin 3-glucoside (Cy 3G) cyanidin 35-diglucoside (Cy35dG) delphinidin 3-glucoside (Dp 3G) delphinidin 35-diglucoside (Dp 35dG) pelargonidin 3-glucoside (Pg 3G)pelargonidin 35-diglucoside (Pg 35dG) cyanidin-pentoside(Cy-pent) cyanidin-pentoside-hexoside (Cy-pent-hex) anddelphinidin-pentoside (Dp-pent)
Table 2 shows the individual anthocyanin contentsof the studied cultivars The JPGRTA cultivar had thehighest (175mgL) anthocyanin content whereas those ofMPSA ATSA PSYA and PSYW were lower than 5mgLIn some cultivars juice extraction of whole pomegranate
()
0400 600 800 1000 1200 1400 1600 1800 2000200
Time (min)
Dp3
5dG Cy
35
dGPg
35
dGD
p3
G
Cy-p
ent-h
exCy
3G
Pg3
G
Dp-
pent
Cy-p
ent
Figure 2 Typical chromatogram of the anthocyanidins inpomegranate juice (MYA) as detected by MS in SRMmode
juices increased the anthocyanin content ATSW (134)GSYW (477) MPSW (347) MTKW (223) and PSYW(196) In contrast the anthocyanin contents of SRABW(214) TMLW (104) MYW (440) and JPGRTW(390) were decreased Based on these results it is evidentthat the anthocyanin content of pomegranate juices is affectedby extraction methods
According to Figure 2 nine major and minoranthocyanins were identified namely cyanidin 3-glucosidecyanidin 35-diglucoside delphinidin 3-glucoside delphini-din 35-diglucoside pelargonidin 3-glucoside pelargonidin35-diglucoside cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside In terms of quantitythe main anthocyanin in most of the cultivars was Cy 35dG(07ndash947mgL) followed by Cy 3G (05ndash525mgL) Pg35dG + Dp 3G (0ndash103mgL) Dp 35dG (0ndash940mgL) Pg3G (0ndash400mgL) and Cy-pent (0ndash113mgL)
Except for Cy-pent Dp-pent and Cy-pent-hex theanthocyanin profiles were consistent with previouslyreported researches about other pomegranate cultivars[1 12 17ndash19] In the same pomegranate cultivars Alighourchiet al [12] reported the content of 6 anthocyanins inaril juices as follows Dp 3G (219ndash163mgL) Dp 35dG(236ndash631mgL) Pg 3G (026ndash136mgL) Pg 35dG(001ndash811mgL) Cy 3G (578ndash304mgL) and Cy 35dG(439ndash166mgL) Thus achieving different anthocyaninscontent in the same pomegranate cultivars indicated theeffect of agricultural and environmental conditions on thephysicochemical characteristic of pomegranate fruit
To our knowledge Dp-pent has not been reportedin pomegranate fruit so far It is tentatively identified bythe mass transition 435gt303 Fischer et al [2] identified9 anthocyanins of Dp 35dG (04ndash205mgL) Cy 35dG(954ndash271mgL) Pg 35dG (137ndash267mgL) Dp 3G (10ndash168mgL) Cy 3G (91ndash279mgL) Pg 3G (24ndash49mgL)a cyanidin-pentoside-hexoside (11ndash30mgL) cyanidin 3-rutinoside (00ndash12mgL) and a cyanidin-pentoside (02ndash06mgL) in the three pomegranate juice variants Gil et al[1] reported the main pigments in the juice of theWonderful
Journal of Chemistry 5
Table 2 Contents of individual anthocyanins (mgL) in pomegranate juices
cultivar as follows Cy 3G (595ndash128mgL) Dp 3G (236ndash952mgL) Cy 35dG (314ndash714mgL) Dp 35dG (211ndash611mgL) and Pg 3G (39ndash85mgL)Mena et al [4] reportedCy 3G (33) as the major anthocyanin followed by Dp 35dG(24) Dp 3G (21) and Cy 35dG (19) and Pg 3G andPg 35dG were only 3 of the total anthocyanin content Inaddition four major anthocyanins of Dp 3G Dp 35dG Cy3G and Cy 35dG andminor amounts of Pg 3G and Pg 35dGwere determined in pomegranate juices [20]
The main anthocyanin in all the cultivars was Cy 35dGexcept for SRABA in which Cy 3G was dominant It isknown that the main anthocyanin of pomegranate dependson the cultivar The main anthocyanins were Cy 3G [1]in the American Wonderful cultivar Cy 3G (40) and Cy35dG (38) in the Spanish Mollar cultivar [19] Dp 3G inthe Portuguese Assaria cultivar [17] Cy 35dG in 15 Iraniancultivars [12] Dp 35dG in 8 other Iranian cultivars [5] andCy 35dG in a Peruvian pomegranate fruit [2]
Among different species or even cultivars of the samespecies the anthocyanin content varies considerably affectedby genetic make-up light temperature and agronomic fac-tors [21] Intrinsic properties of the product and the processsuch as pH temperature light oxygen enzymes ascorbicacids sugars metal ions and copigmentation (intermolecu-lar and intramolecular complex formations self-associationand metal complexation) as well as chemical structure andconcentration of anthocyanins can affect anthocyanin stabil-ity [22]
The variation of anthocyanin content in juices from arilsand whole pomegranate can be affected by the entrance ofanthocyanins from the peel into the juices Therefore the
amount of anthocyanins of the same cultivar varied withextraction methods
33 Total Phenolic Content and Antioxidant CapacityAccording to the results presented in Table 3 total phenoliccontents of juices from whole pomegranate fruit (942ndash2931mgL) were significantly higher than those in arilsjuice (220ndash1267mgL) The GSYW cultivar had the highest(2931mgL) total phenolic content In other studies the totalphenolic contents of various pomegranate cultivars showedsignificant differences and were 296ndash985mg100 g [9] 208ndash344mg100 g [7] 144ndash1009mg100 g [8] 237ndash930mg100 g[5] 2015ndash5186mgL [2] and 1136ndash3581mgL [16] Differentamounts of total phenolic content in various studies can beattributed to analytical methods cultivar maturity stageand environmental conditions and also overestimation ofthe total individual phenolic compounds by Folin-Ciocalteureagent due to interference with other reducing factors [2]
The antioxidant properties were measured by ABTSFRAP and DPPH assay (Table 3) The results showed con-siderable antioxidant activity of juice samples Antioxidantactivities of juices from whole pomegranate fruit were sig-nificantly higher than juices from pomegranate arils thatindicated the importance of the juice extraction methodsThese results are in agreement with previously reportedfindings [1 11]The antioxidant capacity of pomegranate juice(and other fruit juices) depends on cultivar growing regionfruit maturation and agricultural factors The technologyused for juice processing may affect antioxidant capacityof pomegranate juice [7] In this regard the antioxidantactivity of commercial pomegranate juice was 50 higher
6 Journal of Chemistry
Table 3 Evaluation of total polyphenol content (mg gallic acid equivalentsL) and antioxidant activities based on ABTS (mg ascorbicacid100mL) FRAP (mmol Fe2+100mL) and DPPH (AA) in pomegranate juices
MPSW 19541 plusmn 271cd 12492 plusmn 265c 306 plusmn 18c 550 plusmn 18clowastDifferent letters in the same column present significant difference at 119901 lt 005
than juices from pomegranate arils due to the entranceof phenolic compounds from the peel into juices [1] AlsoTzulker et al [11] reported that antioxidant activity of wholefruit homogenates is about 20-fold higher than that foundin the arils These results indicated that hydrolysable tanninsincluding punicalagin were themain compounds influencingthe antioxidant capacity of pomegranate juices [1 11] Thiscompound shows a high antioxidant activity due to the 16phenolic hydroxyl groups in the molecule [1]
Moreover a good correlation was observed betweentotal phenolic content and ABTS and FRAP methods inall pomegranate juices (gt090) Correlations between totalphenolic and DPPH in juices of arils and whole pomegranatewere 092 and 086 respectively However Pearson corre-lation showed that the correlation of three measurementantioxidant methods is affected by pomegranate cultivarsas mentioned in previous research [7 11] In this regardTzulker et al [11] reported that the antioxidant activity (FRAPand DPPH methods) of juices from whole pomegranatehomogenate and arils alone significantly correlated withthe polyphenol level but it did not correlate with antho-cyanins level The highest correlation value was obtainedbetween antioxidant activity and punicalagin in the wholepomegranate homogenate The FRAP method comparedto DPPH method gave the highest correlation with totalpolyphenol content
4 Conclusion
The individual and total phenolic contents of all stud-ied cultivars were significantly different In this study
among nine major and minor anthocyanins the cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside were identified for the first time in Iranianpomegranate cultivars The phenolic contents and antioxi-dant activity of whole pomegranate juice were significantlyhigher than those of juices from pomegranate arils from thesame cultivar due to the entrance of phenolic compoundsfrom the rind parts of pomegranate to the juices Thevariation between the cultivars is very wide therefore thecontents of ellagitannins and anthocyanins in the juice fromwhole pomegranates of the low-polyphenol cultivars (asMY)are lower than those in the juices made from arils only ofthe high-polyphenol cultivars (as JPGRT) In otherwords thecultivar has a higher influence on the phenolic content of thejuice than the extraction method
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to acknowledge Dr Vazifeshenas forhis assistance in afforded pomegranate cultivars
References
[1] M I Gil F A Tomas-Barberan B Hess-Pierce D M Holcroftand A A Kader ldquoAntioxidant activity of pomegranate juice
Journal of Chemistry 7
and its relationship with phenolic composition and processingrdquoJournal of Agricultural and Food Chemistry vol 48 no 10 pp4581ndash4589 2000
[2] U A Fischer R Carle and D R Kammerer ldquoIdentificationand quantification of phenolic compounds from pomegranate(Punica granatum L) peel mesocarp aril and differently pro-duced juices by HPLC-DAD-ESIMS119899rdquo Food Chemistry vol127 no 2 pp 807ndash821 2011
[3] Anonymous Iran Statistical Year Book 2013 2013 httpeamarsciorgirindex easpx
[4] P Mena C Garcıa-Viguera J Navarro-Rico et al ldquoPhytochem-ical characterisation for industrial use of pomegranate (Punicagranatum L) cultivars grown in Spainrdquo Journal of the Science ofFood and Agriculture vol 91 no 10 pp 1893ndash1906 2011
[5] G Mousavinejad Z Emam-Djomeh K Rezaei and M HH Khodaparast ldquoIdentification and quantification of pheno-lic compounds and their effects on antioxidant activity inpomegranate juices of eight Iranian cultivarsrdquo Food Chemistryvol 115 no 4 pp 1274ndash1278 2009
[6] O A Fawole U L Opara and K I Theron ldquoChemicaland phytochemical properties and antioxidant activities ofthree pomegranate cultivars grown in South Africardquo Food andBioprocess Technology vol 5 no 7 pp 2934ndash2940 2012
[7] M Cam Y Hısıl and G Durmaz ldquoClassification of eightpomegranate juices based on antioxidant capacity measured byfourmethodsrdquo FoodChemistry vol 112 no 3 pp 721ndash726 2009
[8] F Tezcan M Gultekin-Ozguven T Diken B Ozcelik and FB Erim ldquoAntioxidant activity and total phenolic organic acidand sugar content in commercial pomegranate juicesrdquo FoodChemistry vol 115 no 3 pp 873ndash877 2009
[9] A Tehranifar M Zarei Z Nemati B Esfandiyari and MR Vazifeshenas ldquoInvestigation of physico-chemical propertiesand antioxidant activity of twenty Iranian pomegranate (PunicagranatumL) cultivarsrdquo ScientiaHorticulturae vol 126 no 2 pp180ndash185 2010
[10] M Ozgen C Durgac S Serce and C Kaya ldquoChemical andantioxidant properties of pomegranate cultivars grown in theMediterranean region of Turkeyrdquo Food Chemistry vol 111 no3 pp 703ndash706 2008
[11] R Tzulker I Glazer I Bar-Ilan D Holland M Aviramand R Amir ldquoAntioxidant activity polyphenol content andrelated compounds in different fruit juices and homogenatesprepared from 29 different pomegranate accessionsrdquo Journal ofAgricultural and Food Chemistry vol 55 no 23 pp 9559ndash95702007
[12] H Alighourchi M Barzegar and S Abbasi ldquoAnthocyaninscharacterization of 15 Iranian pomegranate (Punica granatumL) varieties and their variation after cold storage and pasteur-izationrdquo European Food Research and Technology vol 227 no 3pp 881ndash887 2008
[13] S Chrubasik-Hausmann C Vlachojannis and B Zimmer-mann ldquoPomegranate juice and prostate cancer importanceof the characterisation of the active principlerdquo PhytotherapyResearch vol 28 no 11 pp 1676ndash1678 2014
[14] P Mena L Calani C DallrsquoAsta et al ldquoRapid and comprehen-sive evaluation of (Poly)phenolic compounds in pomegranate(Punica granatum L) Juice by UHPLC-MSnrdquoMolecules vol 17no 12 pp 14821ndash14840 2012
[15] M M Feuereisen J Hoppe B F Zimmermann F Weber NSchulze-Kaysers andA Schieber ldquoCharacterization of phenoliccompounds in Brazilian pepper (Schinus terebinthifolius Raddi)
exocarprdquo Journal of Agricultural and FoodChemistry vol 62 no26 pp 6219ndash6226 2014
[16] SVegaraNMartı J Lorente et al ldquoChemical guide parametersfor Punica granatum cv lsquoMollarrsquo fruit juices processed atindustrial scalerdquo Food Chemistry vol 147 pp 203ndash208 2014
[17] G Miguel S Dandlen D Antunes A Neves and D MartinsldquoThe effect of two methods of pomegranate (Punica granatumL) juice extraction on quality during storage at 4∘Crdquo Journalof Biomedicine and Biotechnology vol 2004 no 5 pp 332ndash3372004
[18] A Perez-Vicente P Serrano P Abellan and C Garcıa-VigueraldquoInfluence of packaging material on pomegranate juice colourand bioactive compounds during storagerdquo Journal of the Scienceof Food and Agriculture vol 84 no 7 pp 639ndash644 2004
[19] NMartı A Perez-Vicente andCGarcıa-Viguera ldquoInfluence ofstorage temperature and ascorbic acid addition on pomegranatejuicerdquo Journal of the Science of Food and Agriculture vol 82 no2 pp 217ndash221 2002
[20] C U Pala and A K Toklucu ldquoEffect of UV-C light on antho-cyanin content and other quality parameters of pomegranatejuicerdquo Journal of Food Composition and Analysis vol 24 no 6pp 790ndash795 2011
[21] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsCRC Press Boca Raton Fla USA 2004
[22] A Patras N P Brunton S Da Pieve and F Butler ldquoImpact ofhigh pressure processing on total antioxidant activity phenolicascorbic acid anthocyanin content and colour of strawberryand blackberry pureesrdquo Innovative Food Science amp EmergingTechnologies vol 10 no 3 pp 308ndash313 2009
cultivar as follows Cy 3G (595ndash128mgL) Dp 3G (236ndash952mgL) Cy 35dG (314ndash714mgL) Dp 35dG (211ndash611mgL) and Pg 3G (39ndash85mgL)Mena et al [4] reportedCy 3G (33) as the major anthocyanin followed by Dp 35dG(24) Dp 3G (21) and Cy 35dG (19) and Pg 3G andPg 35dG were only 3 of the total anthocyanin content Inaddition four major anthocyanins of Dp 3G Dp 35dG Cy3G and Cy 35dG andminor amounts of Pg 3G and Pg 35dGwere determined in pomegranate juices [20]
The main anthocyanin in all the cultivars was Cy 35dGexcept for SRABA in which Cy 3G was dominant It isknown that the main anthocyanin of pomegranate dependson the cultivar The main anthocyanins were Cy 3G [1]in the American Wonderful cultivar Cy 3G (40) and Cy35dG (38) in the Spanish Mollar cultivar [19] Dp 3G inthe Portuguese Assaria cultivar [17] Cy 35dG in 15 Iraniancultivars [12] Dp 35dG in 8 other Iranian cultivars [5] andCy 35dG in a Peruvian pomegranate fruit [2]
Among different species or even cultivars of the samespecies the anthocyanin content varies considerably affectedby genetic make-up light temperature and agronomic fac-tors [21] Intrinsic properties of the product and the processsuch as pH temperature light oxygen enzymes ascorbicacids sugars metal ions and copigmentation (intermolecu-lar and intramolecular complex formations self-associationand metal complexation) as well as chemical structure andconcentration of anthocyanins can affect anthocyanin stabil-ity [22]
The variation of anthocyanin content in juices from arilsand whole pomegranate can be affected by the entrance ofanthocyanins from the peel into the juices Therefore the
amount of anthocyanins of the same cultivar varied withextraction methods
33 Total Phenolic Content and Antioxidant CapacityAccording to the results presented in Table 3 total phenoliccontents of juices from whole pomegranate fruit (942ndash2931mgL) were significantly higher than those in arilsjuice (220ndash1267mgL) The GSYW cultivar had the highest(2931mgL) total phenolic content In other studies the totalphenolic contents of various pomegranate cultivars showedsignificant differences and were 296ndash985mg100 g [9] 208ndash344mg100 g [7] 144ndash1009mg100 g [8] 237ndash930mg100 g[5] 2015ndash5186mgL [2] and 1136ndash3581mgL [16] Differentamounts of total phenolic content in various studies can beattributed to analytical methods cultivar maturity stageand environmental conditions and also overestimation ofthe total individual phenolic compounds by Folin-Ciocalteureagent due to interference with other reducing factors [2]
The antioxidant properties were measured by ABTSFRAP and DPPH assay (Table 3) The results showed con-siderable antioxidant activity of juice samples Antioxidantactivities of juices from whole pomegranate fruit were sig-nificantly higher than juices from pomegranate arils thatindicated the importance of the juice extraction methodsThese results are in agreement with previously reportedfindings [1 11]The antioxidant capacity of pomegranate juice(and other fruit juices) depends on cultivar growing regionfruit maturation and agricultural factors The technologyused for juice processing may affect antioxidant capacityof pomegranate juice [7] In this regard the antioxidantactivity of commercial pomegranate juice was 50 higher
6 Journal of Chemistry
Table 3 Evaluation of total polyphenol content (mg gallic acid equivalentsL) and antioxidant activities based on ABTS (mg ascorbicacid100mL) FRAP (mmol Fe2+100mL) and DPPH (AA) in pomegranate juices
MPSW 19541 plusmn 271cd 12492 plusmn 265c 306 plusmn 18c 550 plusmn 18clowastDifferent letters in the same column present significant difference at 119901 lt 005
than juices from pomegranate arils due to the entranceof phenolic compounds from the peel into juices [1] AlsoTzulker et al [11] reported that antioxidant activity of wholefruit homogenates is about 20-fold higher than that foundin the arils These results indicated that hydrolysable tanninsincluding punicalagin were themain compounds influencingthe antioxidant capacity of pomegranate juices [1 11] Thiscompound shows a high antioxidant activity due to the 16phenolic hydroxyl groups in the molecule [1]
Moreover a good correlation was observed betweentotal phenolic content and ABTS and FRAP methods inall pomegranate juices (gt090) Correlations between totalphenolic and DPPH in juices of arils and whole pomegranatewere 092 and 086 respectively However Pearson corre-lation showed that the correlation of three measurementantioxidant methods is affected by pomegranate cultivarsas mentioned in previous research [7 11] In this regardTzulker et al [11] reported that the antioxidant activity (FRAPand DPPH methods) of juices from whole pomegranatehomogenate and arils alone significantly correlated withthe polyphenol level but it did not correlate with antho-cyanins level The highest correlation value was obtainedbetween antioxidant activity and punicalagin in the wholepomegranate homogenate The FRAP method comparedto DPPH method gave the highest correlation with totalpolyphenol content
4 Conclusion
The individual and total phenolic contents of all stud-ied cultivars were significantly different In this study
among nine major and minor anthocyanins the cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside were identified for the first time in Iranianpomegranate cultivars The phenolic contents and antioxi-dant activity of whole pomegranate juice were significantlyhigher than those of juices from pomegranate arils from thesame cultivar due to the entrance of phenolic compoundsfrom the rind parts of pomegranate to the juices Thevariation between the cultivars is very wide therefore thecontents of ellagitannins and anthocyanins in the juice fromwhole pomegranates of the low-polyphenol cultivars (asMY)are lower than those in the juices made from arils only ofthe high-polyphenol cultivars (as JPGRT) In otherwords thecultivar has a higher influence on the phenolic content of thejuice than the extraction method
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to acknowledge Dr Vazifeshenas forhis assistance in afforded pomegranate cultivars
References
[1] M I Gil F A Tomas-Barberan B Hess-Pierce D M Holcroftand A A Kader ldquoAntioxidant activity of pomegranate juice
Journal of Chemistry 7
and its relationship with phenolic composition and processingrdquoJournal of Agricultural and Food Chemistry vol 48 no 10 pp4581ndash4589 2000
[2] U A Fischer R Carle and D R Kammerer ldquoIdentificationand quantification of phenolic compounds from pomegranate(Punica granatum L) peel mesocarp aril and differently pro-duced juices by HPLC-DAD-ESIMS119899rdquo Food Chemistry vol127 no 2 pp 807ndash821 2011
[3] Anonymous Iran Statistical Year Book 2013 2013 httpeamarsciorgirindex easpx
[4] P Mena C Garcıa-Viguera J Navarro-Rico et al ldquoPhytochem-ical characterisation for industrial use of pomegranate (Punicagranatum L) cultivars grown in Spainrdquo Journal of the Science ofFood and Agriculture vol 91 no 10 pp 1893ndash1906 2011
[5] G Mousavinejad Z Emam-Djomeh K Rezaei and M HH Khodaparast ldquoIdentification and quantification of pheno-lic compounds and their effects on antioxidant activity inpomegranate juices of eight Iranian cultivarsrdquo Food Chemistryvol 115 no 4 pp 1274ndash1278 2009
[6] O A Fawole U L Opara and K I Theron ldquoChemicaland phytochemical properties and antioxidant activities ofthree pomegranate cultivars grown in South Africardquo Food andBioprocess Technology vol 5 no 7 pp 2934ndash2940 2012
[7] M Cam Y Hısıl and G Durmaz ldquoClassification of eightpomegranate juices based on antioxidant capacity measured byfourmethodsrdquo FoodChemistry vol 112 no 3 pp 721ndash726 2009
[8] F Tezcan M Gultekin-Ozguven T Diken B Ozcelik and FB Erim ldquoAntioxidant activity and total phenolic organic acidand sugar content in commercial pomegranate juicesrdquo FoodChemistry vol 115 no 3 pp 873ndash877 2009
[9] A Tehranifar M Zarei Z Nemati B Esfandiyari and MR Vazifeshenas ldquoInvestigation of physico-chemical propertiesand antioxidant activity of twenty Iranian pomegranate (PunicagranatumL) cultivarsrdquo ScientiaHorticulturae vol 126 no 2 pp180ndash185 2010
[10] M Ozgen C Durgac S Serce and C Kaya ldquoChemical andantioxidant properties of pomegranate cultivars grown in theMediterranean region of Turkeyrdquo Food Chemistry vol 111 no3 pp 703ndash706 2008
[11] R Tzulker I Glazer I Bar-Ilan D Holland M Aviramand R Amir ldquoAntioxidant activity polyphenol content andrelated compounds in different fruit juices and homogenatesprepared from 29 different pomegranate accessionsrdquo Journal ofAgricultural and Food Chemistry vol 55 no 23 pp 9559ndash95702007
[12] H Alighourchi M Barzegar and S Abbasi ldquoAnthocyaninscharacterization of 15 Iranian pomegranate (Punica granatumL) varieties and their variation after cold storage and pasteur-izationrdquo European Food Research and Technology vol 227 no 3pp 881ndash887 2008
[13] S Chrubasik-Hausmann C Vlachojannis and B Zimmer-mann ldquoPomegranate juice and prostate cancer importanceof the characterisation of the active principlerdquo PhytotherapyResearch vol 28 no 11 pp 1676ndash1678 2014
[14] P Mena L Calani C DallrsquoAsta et al ldquoRapid and comprehen-sive evaluation of (Poly)phenolic compounds in pomegranate(Punica granatum L) Juice by UHPLC-MSnrdquoMolecules vol 17no 12 pp 14821ndash14840 2012
[15] M M Feuereisen J Hoppe B F Zimmermann F Weber NSchulze-Kaysers andA Schieber ldquoCharacterization of phenoliccompounds in Brazilian pepper (Schinus terebinthifolius Raddi)
exocarprdquo Journal of Agricultural and FoodChemistry vol 62 no26 pp 6219ndash6226 2014
[16] SVegaraNMartı J Lorente et al ldquoChemical guide parametersfor Punica granatum cv lsquoMollarrsquo fruit juices processed atindustrial scalerdquo Food Chemistry vol 147 pp 203ndash208 2014
[17] G Miguel S Dandlen D Antunes A Neves and D MartinsldquoThe effect of two methods of pomegranate (Punica granatumL) juice extraction on quality during storage at 4∘Crdquo Journalof Biomedicine and Biotechnology vol 2004 no 5 pp 332ndash3372004
[18] A Perez-Vicente P Serrano P Abellan and C Garcıa-VigueraldquoInfluence of packaging material on pomegranate juice colourand bioactive compounds during storagerdquo Journal of the Scienceof Food and Agriculture vol 84 no 7 pp 639ndash644 2004
[19] NMartı A Perez-Vicente andCGarcıa-Viguera ldquoInfluence ofstorage temperature and ascorbic acid addition on pomegranatejuicerdquo Journal of the Science of Food and Agriculture vol 82 no2 pp 217ndash221 2002
[20] C U Pala and A K Toklucu ldquoEffect of UV-C light on antho-cyanin content and other quality parameters of pomegranatejuicerdquo Journal of Food Composition and Analysis vol 24 no 6pp 790ndash795 2011
[21] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsCRC Press Boca Raton Fla USA 2004
[22] A Patras N P Brunton S Da Pieve and F Butler ldquoImpact ofhigh pressure processing on total antioxidant activity phenolicascorbic acid anthocyanin content and colour of strawberryand blackberry pureesrdquo Innovative Food Science amp EmergingTechnologies vol 10 no 3 pp 308ndash313 2009
Table 3 Evaluation of total polyphenol content (mg gallic acid equivalentsL) and antioxidant activities based on ABTS (mg ascorbicacid100mL) FRAP (mmol Fe2+100mL) and DPPH (AA) in pomegranate juices
MPSW 19541 plusmn 271cd 12492 plusmn 265c 306 plusmn 18c 550 plusmn 18clowastDifferent letters in the same column present significant difference at 119901 lt 005
than juices from pomegranate arils due to the entranceof phenolic compounds from the peel into juices [1] AlsoTzulker et al [11] reported that antioxidant activity of wholefruit homogenates is about 20-fold higher than that foundin the arils These results indicated that hydrolysable tanninsincluding punicalagin were themain compounds influencingthe antioxidant capacity of pomegranate juices [1 11] Thiscompound shows a high antioxidant activity due to the 16phenolic hydroxyl groups in the molecule [1]
Moreover a good correlation was observed betweentotal phenolic content and ABTS and FRAP methods inall pomegranate juices (gt090) Correlations between totalphenolic and DPPH in juices of arils and whole pomegranatewere 092 and 086 respectively However Pearson corre-lation showed that the correlation of three measurementantioxidant methods is affected by pomegranate cultivarsas mentioned in previous research [7 11] In this regardTzulker et al [11] reported that the antioxidant activity (FRAPand DPPH methods) of juices from whole pomegranatehomogenate and arils alone significantly correlated withthe polyphenol level but it did not correlate with antho-cyanins level The highest correlation value was obtainedbetween antioxidant activity and punicalagin in the wholepomegranate homogenate The FRAP method comparedto DPPH method gave the highest correlation with totalpolyphenol content
4 Conclusion
The individual and total phenolic contents of all stud-ied cultivars were significantly different In this study
among nine major and minor anthocyanins the cyanidin-pentoside cyanidin-pentoside-hexoside and delphinidin-pentoside were identified for the first time in Iranianpomegranate cultivars The phenolic contents and antioxi-dant activity of whole pomegranate juice were significantlyhigher than those of juices from pomegranate arils from thesame cultivar due to the entrance of phenolic compoundsfrom the rind parts of pomegranate to the juices Thevariation between the cultivars is very wide therefore thecontents of ellagitannins and anthocyanins in the juice fromwhole pomegranates of the low-polyphenol cultivars (asMY)are lower than those in the juices made from arils only ofthe high-polyphenol cultivars (as JPGRT) In otherwords thecultivar has a higher influence on the phenolic content of thejuice than the extraction method
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to acknowledge Dr Vazifeshenas forhis assistance in afforded pomegranate cultivars
References
[1] M I Gil F A Tomas-Barberan B Hess-Pierce D M Holcroftand A A Kader ldquoAntioxidant activity of pomegranate juice
Journal of Chemistry 7
and its relationship with phenolic composition and processingrdquoJournal of Agricultural and Food Chemistry vol 48 no 10 pp4581ndash4589 2000
[2] U A Fischer R Carle and D R Kammerer ldquoIdentificationand quantification of phenolic compounds from pomegranate(Punica granatum L) peel mesocarp aril and differently pro-duced juices by HPLC-DAD-ESIMS119899rdquo Food Chemistry vol127 no 2 pp 807ndash821 2011
[3] Anonymous Iran Statistical Year Book 2013 2013 httpeamarsciorgirindex easpx
[4] P Mena C Garcıa-Viguera J Navarro-Rico et al ldquoPhytochem-ical characterisation for industrial use of pomegranate (Punicagranatum L) cultivars grown in Spainrdquo Journal of the Science ofFood and Agriculture vol 91 no 10 pp 1893ndash1906 2011
[5] G Mousavinejad Z Emam-Djomeh K Rezaei and M HH Khodaparast ldquoIdentification and quantification of pheno-lic compounds and their effects on antioxidant activity inpomegranate juices of eight Iranian cultivarsrdquo Food Chemistryvol 115 no 4 pp 1274ndash1278 2009
[6] O A Fawole U L Opara and K I Theron ldquoChemicaland phytochemical properties and antioxidant activities ofthree pomegranate cultivars grown in South Africardquo Food andBioprocess Technology vol 5 no 7 pp 2934ndash2940 2012
[7] M Cam Y Hısıl and G Durmaz ldquoClassification of eightpomegranate juices based on antioxidant capacity measured byfourmethodsrdquo FoodChemistry vol 112 no 3 pp 721ndash726 2009
[8] F Tezcan M Gultekin-Ozguven T Diken B Ozcelik and FB Erim ldquoAntioxidant activity and total phenolic organic acidand sugar content in commercial pomegranate juicesrdquo FoodChemistry vol 115 no 3 pp 873ndash877 2009
[9] A Tehranifar M Zarei Z Nemati B Esfandiyari and MR Vazifeshenas ldquoInvestigation of physico-chemical propertiesand antioxidant activity of twenty Iranian pomegranate (PunicagranatumL) cultivarsrdquo ScientiaHorticulturae vol 126 no 2 pp180ndash185 2010
[10] M Ozgen C Durgac S Serce and C Kaya ldquoChemical andantioxidant properties of pomegranate cultivars grown in theMediterranean region of Turkeyrdquo Food Chemistry vol 111 no3 pp 703ndash706 2008
[11] R Tzulker I Glazer I Bar-Ilan D Holland M Aviramand R Amir ldquoAntioxidant activity polyphenol content andrelated compounds in different fruit juices and homogenatesprepared from 29 different pomegranate accessionsrdquo Journal ofAgricultural and Food Chemistry vol 55 no 23 pp 9559ndash95702007
[12] H Alighourchi M Barzegar and S Abbasi ldquoAnthocyaninscharacterization of 15 Iranian pomegranate (Punica granatumL) varieties and their variation after cold storage and pasteur-izationrdquo European Food Research and Technology vol 227 no 3pp 881ndash887 2008
[13] S Chrubasik-Hausmann C Vlachojannis and B Zimmer-mann ldquoPomegranate juice and prostate cancer importanceof the characterisation of the active principlerdquo PhytotherapyResearch vol 28 no 11 pp 1676ndash1678 2014
[14] P Mena L Calani C DallrsquoAsta et al ldquoRapid and comprehen-sive evaluation of (Poly)phenolic compounds in pomegranate(Punica granatum L) Juice by UHPLC-MSnrdquoMolecules vol 17no 12 pp 14821ndash14840 2012
[15] M M Feuereisen J Hoppe B F Zimmermann F Weber NSchulze-Kaysers andA Schieber ldquoCharacterization of phenoliccompounds in Brazilian pepper (Schinus terebinthifolius Raddi)
exocarprdquo Journal of Agricultural and FoodChemistry vol 62 no26 pp 6219ndash6226 2014
[16] SVegaraNMartı J Lorente et al ldquoChemical guide parametersfor Punica granatum cv lsquoMollarrsquo fruit juices processed atindustrial scalerdquo Food Chemistry vol 147 pp 203ndash208 2014
[17] G Miguel S Dandlen D Antunes A Neves and D MartinsldquoThe effect of two methods of pomegranate (Punica granatumL) juice extraction on quality during storage at 4∘Crdquo Journalof Biomedicine and Biotechnology vol 2004 no 5 pp 332ndash3372004
[18] A Perez-Vicente P Serrano P Abellan and C Garcıa-VigueraldquoInfluence of packaging material on pomegranate juice colourand bioactive compounds during storagerdquo Journal of the Scienceof Food and Agriculture vol 84 no 7 pp 639ndash644 2004
[19] NMartı A Perez-Vicente andCGarcıa-Viguera ldquoInfluence ofstorage temperature and ascorbic acid addition on pomegranatejuicerdquo Journal of the Science of Food and Agriculture vol 82 no2 pp 217ndash221 2002
[20] C U Pala and A K Toklucu ldquoEffect of UV-C light on antho-cyanin content and other quality parameters of pomegranatejuicerdquo Journal of Food Composition and Analysis vol 24 no 6pp 790ndash795 2011
[21] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsCRC Press Boca Raton Fla USA 2004
[22] A Patras N P Brunton S Da Pieve and F Butler ldquoImpact ofhigh pressure processing on total antioxidant activity phenolicascorbic acid anthocyanin content and colour of strawberryand blackberry pureesrdquo Innovative Food Science amp EmergingTechnologies vol 10 no 3 pp 308ndash313 2009
and its relationship with phenolic composition and processingrdquoJournal of Agricultural and Food Chemistry vol 48 no 10 pp4581ndash4589 2000
[2] U A Fischer R Carle and D R Kammerer ldquoIdentificationand quantification of phenolic compounds from pomegranate(Punica granatum L) peel mesocarp aril and differently pro-duced juices by HPLC-DAD-ESIMS119899rdquo Food Chemistry vol127 no 2 pp 807ndash821 2011
[3] Anonymous Iran Statistical Year Book 2013 2013 httpeamarsciorgirindex easpx
[4] P Mena C Garcıa-Viguera J Navarro-Rico et al ldquoPhytochem-ical characterisation for industrial use of pomegranate (Punicagranatum L) cultivars grown in Spainrdquo Journal of the Science ofFood and Agriculture vol 91 no 10 pp 1893ndash1906 2011
[5] G Mousavinejad Z Emam-Djomeh K Rezaei and M HH Khodaparast ldquoIdentification and quantification of pheno-lic compounds and their effects on antioxidant activity inpomegranate juices of eight Iranian cultivarsrdquo Food Chemistryvol 115 no 4 pp 1274ndash1278 2009
[6] O A Fawole U L Opara and K I Theron ldquoChemicaland phytochemical properties and antioxidant activities ofthree pomegranate cultivars grown in South Africardquo Food andBioprocess Technology vol 5 no 7 pp 2934ndash2940 2012
[7] M Cam Y Hısıl and G Durmaz ldquoClassification of eightpomegranate juices based on antioxidant capacity measured byfourmethodsrdquo FoodChemistry vol 112 no 3 pp 721ndash726 2009
[8] F Tezcan M Gultekin-Ozguven T Diken B Ozcelik and FB Erim ldquoAntioxidant activity and total phenolic organic acidand sugar content in commercial pomegranate juicesrdquo FoodChemistry vol 115 no 3 pp 873ndash877 2009
[9] A Tehranifar M Zarei Z Nemati B Esfandiyari and MR Vazifeshenas ldquoInvestigation of physico-chemical propertiesand antioxidant activity of twenty Iranian pomegranate (PunicagranatumL) cultivarsrdquo ScientiaHorticulturae vol 126 no 2 pp180ndash185 2010
[10] M Ozgen C Durgac S Serce and C Kaya ldquoChemical andantioxidant properties of pomegranate cultivars grown in theMediterranean region of Turkeyrdquo Food Chemistry vol 111 no3 pp 703ndash706 2008
[11] R Tzulker I Glazer I Bar-Ilan D Holland M Aviramand R Amir ldquoAntioxidant activity polyphenol content andrelated compounds in different fruit juices and homogenatesprepared from 29 different pomegranate accessionsrdquo Journal ofAgricultural and Food Chemistry vol 55 no 23 pp 9559ndash95702007
[12] H Alighourchi M Barzegar and S Abbasi ldquoAnthocyaninscharacterization of 15 Iranian pomegranate (Punica granatumL) varieties and their variation after cold storage and pasteur-izationrdquo European Food Research and Technology vol 227 no 3pp 881ndash887 2008
[13] S Chrubasik-Hausmann C Vlachojannis and B Zimmer-mann ldquoPomegranate juice and prostate cancer importanceof the characterisation of the active principlerdquo PhytotherapyResearch vol 28 no 11 pp 1676ndash1678 2014
[14] P Mena L Calani C DallrsquoAsta et al ldquoRapid and comprehen-sive evaluation of (Poly)phenolic compounds in pomegranate(Punica granatum L) Juice by UHPLC-MSnrdquoMolecules vol 17no 12 pp 14821ndash14840 2012
[15] M M Feuereisen J Hoppe B F Zimmermann F Weber NSchulze-Kaysers andA Schieber ldquoCharacterization of phenoliccompounds in Brazilian pepper (Schinus terebinthifolius Raddi)
exocarprdquo Journal of Agricultural and FoodChemistry vol 62 no26 pp 6219ndash6226 2014
[16] SVegaraNMartı J Lorente et al ldquoChemical guide parametersfor Punica granatum cv lsquoMollarrsquo fruit juices processed atindustrial scalerdquo Food Chemistry vol 147 pp 203ndash208 2014
[17] G Miguel S Dandlen D Antunes A Neves and D MartinsldquoThe effect of two methods of pomegranate (Punica granatumL) juice extraction on quality during storage at 4∘Crdquo Journalof Biomedicine and Biotechnology vol 2004 no 5 pp 332ndash3372004
[18] A Perez-Vicente P Serrano P Abellan and C Garcıa-VigueraldquoInfluence of packaging material on pomegranate juice colourand bioactive compounds during storagerdquo Journal of the Scienceof Food and Agriculture vol 84 no 7 pp 639ndash644 2004
[19] NMartı A Perez-Vicente andCGarcıa-Viguera ldquoInfluence ofstorage temperature and ascorbic acid addition on pomegranatejuicerdquo Journal of the Science of Food and Agriculture vol 82 no2 pp 217ndash221 2002
[20] C U Pala and A K Toklucu ldquoEffect of UV-C light on antho-cyanin content and other quality parameters of pomegranatejuicerdquo Journal of Food Composition and Analysis vol 24 no 6pp 790ndash795 2011
[21] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsCRC Press Boca Raton Fla USA 2004
[22] A Patras N P Brunton S Da Pieve and F Butler ldquoImpact ofhigh pressure processing on total antioxidant activity phenolicascorbic acid anthocyanin content and colour of strawberryand blackberry pureesrdquo Innovative Food Science amp EmergingTechnologies vol 10 no 3 pp 308ndash313 2009