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AfricanJournalofBiotechnologyVol.
9(3),pp.
326-330,11January2010Availableonlineathttp://www.
academicjournals.
org/AJBDOI:10.
5897/AJB09.
1229ISSN1684–53152010AcademicJournalsFullLengthResearchPaperFlavonoid,hesperidine,totalphenoliccontentsandantioxidantactivitiesfromCitrusspeciesMohammedFadlinizalAbdGhafar,K.
NagendraPrasad,KongKinWengandAminIsmail*DepartmentofNutritionandDietetics,FacultyofMedicineandHealthSciences,UniversitiPutraMalaysia,43400Serdang,Selangor,Malaysia.
Accepted7October,2009Citrushaslongbeenregardedasafoodandalsoasamedicinalplant.
FruitsoffourspeciesofcitruswhicharecommonlyavailableinMalaysia,namelyC.
hystrix(wildlime),C.
aurantifolia(commonlime),C.
microcarpa(musklime)andC.
sinensis(orange),werechosentoinvestigatetheirtotalphenolic,flavonoidandhesperidinecontents.
Additionally,theantioxidantactivitieswerealsodeterminedbyferricreducingantioxidantpower(FRAP)and1,1-diphenyl-2-picrylhydrazyl(DPPH)radicalscavengingactivity.
C.
hystrixhadthehighestflavonoidandtotalphenoliccontentswhileC.
aurantifoliahadthehighesthesperidinecontent.
TheantioxidantactivityofC.
hystrixwashighestdeterminedbyFRAPandDPPHassayscomparedtoothercitrusspecies.
AstrongpositivecorrelationofR2=0.
9090betweentotalphenoliccontentandFRAPvalueswasobservedinthisinvestigation.
ThisstudyindicatedthatC.
hystrixexhibitedthehighestantioxidant,flavonoidandphenoliccontentandcanbeusedpotentiallyasareadilyaccessiblesourceofnaturalantioxidant.
Keywords:Antioxidantactivity,citrusjuice,flavonoids,hesperidine,phenolics.
INTRODUCTIONFreeradicalshavebeenclaimedtoplayanimportantroleinaffectinghumanhealthbycausingseveraldiseasesin-cludingcancer,hypertension,heartattackanddiabetes.
Thesefreeradicalsaregeneratedduringbodymeta-bolism.
Exogenousintakeofantioxidantscanhelpthebodyscavengefreeradicalseffectively.
Furthermore,manystudieshaveshownthatincreaseddietaryintakeofnaturalphenolicscorrelateswithreducedcoronaryheartdisease,cancermortalitywithlongerlifeexpectancy(Halliwell,2007).
Moreover,thesepolyphenoliccom-poundshavebeenfoundeffectiveinmanyhealth-relatedproperties,suchasantioxidant,anticancer,antiviralandanti-inflammatoryactivities(Aminetal.
,2006).
Onotherhand,concernaboutthesafetyofthecommonlyusedsyntheticantioxidantssuchasbutylatedhydroxyanisole(BHA)andtertiarybutylhydroquinone(TBHQ)haveledtoincreasedinterestonnaturalantioxidantswhichoccurinplantsassecondarymetabolites.
ThegenuscitrusbelongingtothefamilyRutaceaecomprisesabout40specieswhicharedistributedinIndia,*Correspondingauthor.
E-mail:amin@medic.
upm.
edu.
my.
Tel:+603-89472435.
Fax:+603-89426769.
China,Malaysia,SrilankaandAustralia.
Citrusisoneofthemostimportantworldfruitcropsandisconsumedmostlyasfreshorasjuicebecauseofitsnutritionalvalueandspecialflavor.
Consumptionofcitrusjuiceisfoundtobebeneficialinpreventingcoronarydiseasesandchronicasthma(DugoandGiacomo,2002).
Citrusfruitextractsarealsofoundtohaveantioxidant,anti-inflammatory,anti-tumor,anti-fungalandbloodclotinhibitionactivities(Gargetal.
,2001;KaurandKapoor,2001;Abeysingheetal.
,2007).
Thehealthbenefitsofcitrusfruithavemainlybeenattributedtothepresenceofbioactivecom-pounds,suchasferulicacid,hydrocinnamicacid,cyani-dinglucoside,hisperidine,vitaminC,carotenoidandnaringincontent(Abeysingheetal.
,2007;Kelebeketal.
,2008;Xuetal.
,2008).
TheconsumptionofcitrusjuicesinMalaysiaisincrea-singrapidly.
Previousstudiesonbiochemicalactivitiesfromcitrusweremainlyfocusedonitsessentialoilswhichincludeantimicrobialproperties(Chanthaphonetal.
,2008),anti-aflatoxigenicactivity(Razzaghi-Abyanehetal.
,2009).
However,acomparisonofantioxidantacti-vity,phenolic,flavonoidandhesperidinecontentbetweenseveralspeciesofcitruswhicharecommonlyavailableinMalaysiaisstillunknownandhence,weinvestigatedthisstudyforthefirsttime.
ThisstudycanhelpthefoodGhafaretal327Table1.
Physicalcharacteristicsofdifferentspeciesofcitrus.
CitrusspeciesColorSizeShapeTasteTextureC.
hystrixYellowgreen5-6cmPearSourSmoothC.
aurantifoliaGreenishyellow5-10cmRoundSourFibrousC.
microcarpaGreenishyellow4-5cmRoundSweetFibrousC.
sinensisOrange5-10cmRoundSweetSmoothindustrytouseitasanaturalcompoundforantioxidantactivity,whichmightbeusedasanalternativetosyn-theticantioxidantssinceitisenvironmentallyfriendlyandsafeforconsumption.
MATERIALSANDMETHODSPreparationofjuicesampleFreshfruitsofCitrushystrix,C.
aurantifolia,C.
microcarpaandC.
sinensisatthecommercialmaturestagewereharvestedfromacommercialorchardinthemonthofNovember-December,fromPasarBorongandSeriKembangan,Selangor,Malaysia.
Healthyfruitswereselectedrandomlyforuniformityofshapeandcolor.
PhysicalcharacteristicsofeachspeciesofcitrusfruitaredescribedandgiveninTable1.
Thefruitswerewashedthoroughlyinpotablewaterandthenair-dried.
Thecitrusfruitjuicewasextractedbycuttingthefruitinhalfandcarefulhand-squeezingtoobtainthejuice.
Thejuicewaspassedthroughastrainertoremovepulpandseeds.
Thefreshlysqueezedjuicewascentrifugedat1610gfor20minandthesupernatantwasthencollectedandstoredat–20Cuntilfurtheranalysis.
Chemicalsandreagents1,1-Diphenyl-2-picrylhydrazyl(DPPH),2,4,6-tripyridyl-s-triazine(TPTZ),hesperidine,gallicacidandaluminiumtrichloridewerefromSigmaChemicalCo.
(StLouis,MO,USA).
Folin-Ciocalteaureagent(FCreagent),sodiumcarbonateandferroussulphate,werefromMerck(Darmstadt,Germany).
Allotherchemicalsandsolventsusedinthisstudywereofanalyticalgrade.
DeterminationoftotalphenoliccontentThetotalphenoliccontentsofthedifferentspeciesofcitrusweredeterminedwithsomemodificationasdescribedbyVeliogluetal.
(1998).
Variousspeciesofcitrusjuicesamples(100L)wasmixedwith0.
75mLofFolinCiocalteureagent(10-folddilutionwithdistilledwater)andallowedtostandat22Cfor5min.
Then,0.
75mLofsodiumcarbonate(60g/L)solutionwasaddedtothemixture.
Following90minincubationat22C,absorbancewasmeasuredat725nmusingaUV-visiblespectrophotometer(Prim,SECOMAM,France).
Thetotalphenoliccontentwasdeterminedusingastan-dardcurveofgallicacidat0.
02-0.
1mg/mLconcentrations.
Totalphenoliccontentwascalculatedforeachsampleandexpressedasmilligramsofgallicacidequivalentper100mLofjuice.
DeterminationofflavonoidcontentThetotalflavonoidcontentwasdeterminedusingthemethodofQuettieretal.
(2000)withminormodifications.
Inbrief,1mLof2%aluminiumtrichloridewasmixedwiththesamevolumeofcitrusjuice.
Absorbancereadingsat430nmweretakenafter10minagainstablanksampleconsistingof1mLofsamplesolutionand1mLofdistilledwaterwithoutaluminiumtrichloride.
Thetotalflavor-noidcontentwasdeterminedusingastandardcurveofhesperidineat0–50mg/mL.
Theaverageofthreereadingswasusedandthenexpressedasmilligramsofhesperidineequivalentsper100mLofjuice.
Identificationofhisperidinebyhighperformanceliquidchromatography(HPLC)HighperformanceliquidchromatographycoupledtodiodearraydetectorwasusedtoanalyzehisperidinecontentofcitrussamplesaccordingtomethodofOrtunaetal.
(1997)usingseparationmodule(Agilent,HP1100,Delware,USA)equippedwithaC18column(-Bondapak,Waters,USA,250*4mm,5mparticlesize).
Thesampleswereelutedusingisocraticsystemofwater:methanol:acetonitrile:aceticacid(75:10:10:5,v/v/v/v)asthemobilephaseataflowrateof1.
0mL/min.
Thetemperatureofthecolumnwasmaintainedat35°Candtheinjectionvolumewas20L.
Thepeakofstandardhisperidinewasmonitoredat280nmforquantification.
Identificationandquantificationofhisperidinewasaccomplishedbycomparingtheretentiontimesofpeaksinsamplestothoseofstandard.
Calculationofhisperidineconcentration(expressedasmg/100mLofjuice)wascarriedoutbyanexternalstandardmethodusingcalibrationcurvesofstandardhisperidine.
DPPHradicalscavengingactivityThescavengingactivityofcitrusspecieswasestimatedaccordingtotheproceduremodifiedbyShimadaetal.
(1992).
Analiquotof0.
5mlofjuiceandascorbicacidatdifferentconcentrations(25,50,75,100and150mg/mL)wasmixedwith2.
9mLof100MDPPH(dissolvedin80%ethanol).
Themixturewasvigorouslyshakenandlefttostandatroomtemperaturefor30mininadarkroom.
ThecontrolcontainedonlyDPPHsolutioninsteadofsamplewhile80%ethanolwasusedastheblank.
Absorbancewasreadat515nmbyusingUV-visspectrophotometer.
Thescavengingeffectwascalcu-latedusingthefollowingequation:Scavengingeffect(%)=1–[(Absorbancesample/Absorbancecontrol)x100]EC50valuewasdeterminedfromtheplottedgraphofscavengingactivityagainsttheconcentrationsofthecitrussamples,whichisdefinedastotalantioxidantnecessarytodecreasetheinitialDPPHradicalby50%.
TriplicatemeasurementswerecarriedoutandEC50wascalculatedbasedonthepercentageofDPPHradicalssca-venged.
Ferricreducingpower(FRAP)assayFirst,ferricreducingantioxidantpower(FRAP)reagentwasprepared328Afr.
J.
Biotechnol.
Table2.
Totalphenolic,flavonoidandhisperidinecontentsofdifferentspeciesofcitrus.
CitrusspeciesCommonnameMalayannameTPCFCHCC.
hystrixWildlimeLimaupurut490.
74±1.
75a22.
25±0.
20a7.
01±0.
83bC.
aurantifoliaCommonlimeLimaunipis211.
70±0.
0b10.
67±0.
27b16.
67±2.
57aC.
microcarpaMusklimeLimaukesturi105.
0±3.
0d8.
70±0.
13cNDC.
sinensisOrangeLimaumanis135.
3±0c2.
99±0.
09d5.
58±0.
66cTPC,Totalphenoliccontentexpressedasmilligramsofgallicacidequivalent/100mLofjuice;FC,Flavonoidcontentexpressedasmilligramsofhesperidineequivalent/100mLofjuice;HC,Hesperidinecontentexpressedasmg/100mLofjuice;ND,Notdetected.
Foreachtreatment,themeanswithinthecolumnfollowedbydifferentlettersweresignificantlydifferentatPC.
aurantifolia>C.
sinensis>C.
microcarpa.
Inthepresentstudy,C.
hystrix(wildvariety)demonstratedthegreatestreducingpowersignificantlyhigher(p<0.
05)thanothercitrusspecies.
ThisresultisinagreementwiththosereportedbyScalzoetal.
(2005)whoreportedhighantioxidantactivityofwildstrawberrythanthoseofcultivatedvarieties.
Jayaprakashaetal.
(2008)demonstratedthatthereducingpowerofcitrusfractionswereduetopresenceofphenoliccom-pounds.
Theantioxidantactivitiesofcitrusspeciesareinaccor-dancewiththeiramountofphenolics.
C.
hystrixcontainedhighphenoliccontentcomparedtoothercitrusspecies,whichwasresponsibleforitshighantioxidantactivity.
Severalreportsshowedacloserelationshipbet-weentotalphenoliccontentandhighantioxidantactivity(Prasadetal.
,2005;Aminetal.
,2006;Lietal.
,2009).
Thephenoliccompoundsexhibitextensivefreeradicalscavengingactivitiesthroughtheirreactivityashydrogenorelectron-donatingagentsandmetalionchelatingproperties(Rice-EvansandBourdon,1993).
AhighlinearcorrelationsofR2=0.
9090and0.
9470betweenferricreducingpower,DPPHradicalscavengingactivityandtotalphenoliccontentwereobservedinthisinvestigation.
Lietal.
(2009and2008)havereportedtheexistenceofsimilarlinearrelationshipsbetweenreducingpowerandtotalphenolcontent.
However,anegativecor-relationcoefficientsbetweentotalflavonoidsandDPPHradicalscavengingactivityweredeterminedtobeR2=-330Afr.
J.
Biotechnol.
0.
9580,muchlowerthanthoseoftotalphenolics.
SimilarresultsofnegativecorrelationwerereportedbyMedaetal.
(2005)betweenflavonoidcontentandantioxidantacti-vity.
Thenegativecorrelationbetweencitrusspeciesandflavonoidcontentareprobablybecauseofthepresenceofotherphenolicsinthecitrussampleswhichmighthavecontributedtoantioxidantactivity.
ConclusionsInthepresentstudy,theantioxidantactivityfromfourspeciesofcitruswasevaluated.
C.
hystrixpossessedhighphenolicandflavonoidcontentandexhibitedgoodantioxidantactivitybyDPPHandFRAPmethods.
TheuseofC.
hystrixasanaturalantioxidantsourceappearstobeanalternativetosyntheticantioxidants.
ThisstudywasthefirstreportabouttheantioxidantactivitiesofdifferentspeciesofcitruswhicharecommonlyavailableinMalaysia.
Furtherinvestigationtodetermineanti-oxidantactivitybyin-vivomethodsofcitrusspeciescouldbeconsidered.
ACKNOWLEDGEMENTTheauthorsaregratefultotheDepartmentofNutritionandDietetics,FacultyofMedicineandHealthSciences,UniversitiPutraMalaysiaforlaboratoryfacilitiestocarryoutthisstudy.
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