Research Article Phenolic Compounds and Antioxidant ...downloads.hindawi.com/journals/jchem/2015/907101.pdf · Research Article Phenolic Compounds and Antioxidant Activity of Juices

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

GSYA 132 plusmn 01f 485 plusmn 03g 1081 plusmn 33f GSYW 2850 plusmn 28a 8843 plusmn 529a 9283 plusmn 202a

MYA 731 plusmn 14b 2589 plusmn 46b 1155 plusmn 07de MYW 746 plusmn 05e 2631 plusmn 51d 1212 plusmn 01f

MTKA 698 plusmn 18b 2407 plusmn 26c 671 plusmn 21h MTKW 1346 plusmn 19c 4838 plusmn 61c 1385 plusmn 21f

JPGRTA 1290 plusmn 24a 4784 plusmn 63a 1326 plusmn 14b JPGRTW 1751 plusmn 60b 6519 plusmn 199b 1776 plusmn 40e

TMLA 194 plusmn 07e 484 plusmn 09g 1201 plusmn 16cd TMLW 1192 plusmn 30d 4496 plusmn 57c 3399 plusmn 01c

ATSA 365 plusmn 16d 1013 plusmn 38f 1559 plusmn 26a ATSW 1825 plusmn 24b 6085 plusmn 95b 4605 plusmn 41b

SRABA 543 plusmn 05c 1903 plusmn 29d 1107 plusmn 04ef SRABW 1153 plusmn 31d 4341 plusmn 01c 3154 plusmn 26c

PSYA 54 plusmn 00g 259 plusmn 11h 174 plusmn 19i PSYW 233 plusmn 10f 1328 plusmn 36e 2673 plusmn 10d

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


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


Table 2 Contents of individual anthocyanins (mgL) in pomegranate juices

Pomegranate juice Dp 35dG Cy 35dG Pg 35dG + Dp 3G Cy 3G Pg 3G Cy-pent Total anthocyaninsVKTA 00 117 08 66 05 00 196GSYA 00 91 10 55 08 00 164MYA 15 633 103 448 94 08 1302MTKA 00 159 20 110 15 03 307JPGRTA 50 932 87 626 47 11 1754TMLA 00 198 24 42 08 00 271ATSA 00 24 08 05 00 00 37SRABA 00 43 10 50 03 00 105PSYA 00 07 04 06 01 00 18MPSA 00 23 00 19 00 00 42VKTW 00 110 09 76 06 00 201GSYW 00 119 17 96 10 00 243MYW 17 598 94 438 91 07 1245MTKW 00 197 25 133 16 04 375JPGRTW 77 947 87 525 40 10 1685TMLW 00 176 19 41 07 00 243ATSW 00 60 12 16 00 00 88SRABW 00 33 10 37 03 00 83PSYW 00 08 05 07 02 00 21MPSW 00 31 00 26 00 00 57

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


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

Pomegranate juice Total phenolic (mgL) ABTS (mg100mL) FRAP (mmol100mL) DPPH (AA)VKTA 3605 plusmn 145elowast 2223 plusmn 68f 158 plusmn 04de 256 plusmn 17de

GSYA 5903 plusmn 251cd 4070 plusmn 170d 202 plusmn 13bc 355 plusmn 13bc

MYA 9060 plusmn 127b 5517 plusmn 166b 236 plusmn 18ab 373 plusmn 21bc

MTKA 8270 plusmn 297b 4974 plusmn 136c 229 plusmn 03ab 398 plusmn 24ab

JPGRTA 12668 plusmn 442a 6200 plusmn 198a 259 plusmn 13a 468 plusmn 26a

TMLA 5620 plusmn 92d 3124 plusmn 65e 171 plusmn 09cd 312 plusmn 01cd

ATSA 5903 plusmn 209cd 3113 plusmn 75e 207 plusmn 03bc 351 plusmn 18bc

SRABA 6713 plusmn 216c 3982 plusmn 96d 196 plusmn 17cd 347 plusmn 18bc

PSYA 2200 plusmn 14f 1171 plusmn 33g 136 plusmn 01e 188 plusmn 14e

MPSA 5363 plusmn 173d 3642 plusmn 47d 166 plusmn 13cd 281 plusmn 17de

VKTW 12708 plusmn 295f 6147 plusmn 236e 233 plusmn 07d 451 plusmn 28d

GSYW 29315 plusmn 825a 16401 plusmn 663a 599 plusmn 43a 713 plusmn 49a

MYW 18547 plusmn 323de 12470 plusmn 240c 407 plusmn 21b 623 plusmn 47bc

MTKW 16988 plusmn 620e 12212 plusmn 149c 370 plusmn 16b 536 plusmn 32c

JPGRTW 25680 plusmn 529b 14317 plusmn 442b 548 plusmn 30a 656 plusmn 50bc

TMLW 19551 plusmn 694cd 12225 plusmn 190c 357 plusmn 21bc 605 plusmn 50c

ATSW 21361 plusmn 480c 11398 plusmn 131cd 338 plusmn 29c 635 plusmn 20bc

SRABW 18343 plusmn 528de 9970 plusmn 184d 316 plusmn 18c 580 plusmn 33c

PSYW 9429 plusmn 434g 4962 plusmn 130f 226 plusmn 06d 366 plusmn 23e

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


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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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SpectroscopyInternational Journal of


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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



















()

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










()

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








































4 Conclusion





Acknowledgment


References



































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



















()

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










()

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








































4 Conclusion





Acknowledgment


References



































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of








()

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








































4 Conclusion





Acknowledgment


References



































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



































4 Conclusion





Acknowledgment


References



































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

























4 Conclusion





Acknowledgment


References



































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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Research Article Phenolic Compounds and Antioxidant ...downloads.hindawi.com/journals/jchem/2015/907101.pdf · Research Article Phenolic Compounds and Antioxidant Activity of Juices