JNatSciBiolMed.2010JulDec1(1):2934.

PMCID:PMC3217280
doi:10.4103/09769668.71670

AntioxidantandfreeradicalscavengingeffectsoffruitsofDregeavolubilis
MoulishaBiswas,PallabKantiHaldar, 1andAshokeKumarGhosh2

BengalInstituteofPharmaceuticalSciences,Kalyani,Nadia,India
1
DepartmentofPharmaceuticalTechnology,JadavpurUniversity,Kolkata,India
2
BengalSchoolofTechnology,DelhiRoad,Sugandha,Hooghly,India
Addressforcorrespondence:Dr.MoulishaBiswas,BengalInstituteofPharmaceuticalSciences,Kalyani,Nadia741235,India.Email:
moulisha_biswas@yahoo.co.in

CopyrightJournalofNaturalScience,BiologyandMedicine

ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNoncommercialShareAlike3.0Unported,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.

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Abstract Goto:

Thisstudyevaluatedtheinvitroantioxidantpotentialofpetroleumether(6080C),chloroform,andmethanol
extractofthefruitsofDregeavolubilisBenth(Asclepiadaceae).Thedifferentantioxidantassays,includingtotal
antioxidantactivity,reducingpower,freeradical,superoxideanionradical,nitricoxidescavenging,lipid
peroxidation,andtotalphenoliccontentwerestudied.Theextractsexhibitedpotenttotalantioxidantactivitythat
increasedwithincreasingamountofextractconcentration,whichwascomparedwithstandarddrugvitaminCat
differentconcentrationsasextracts.ThedifferentconcentrationsofalltheextractsandvitaminCshowedinhibition
onlipidperoxidation.Inaddition,alltheextractshadeffectivereducingpower,freeradicalscavenging,superoxide
anionscavenging,nitricoxidescavenging,lipidperoxidation,andtotalphenoliccontentdependingon
concentration.ThesevariousantioxidantactivitieswerecomparedwithstandardantioxidantsuchasvitaminCat
differentconcentrationasdifferentextracts.

Keywords:Dregeavolubilis,freeradicalscavenging,invitroantioxidant

INTRODUCTION Goto:

Antioxidantsareprovidedtolivingorganismstoprotectthemfromdamagecausedbyuncontrolledproductionof
reactiveoxygenspecies(ROS)andtheconcomitantlipidperoxidation,proteindamage,andDNAstrandbreaking.
Currentinterestisfocusedonthepotentialroleofantioxidantsandantioxidantenzymesinthetreatmentand
preventionofatherosclerosis,heartfailure,neurodegenerativedisorders,aging,cancer,diabetesmellitus,and
severalothersdiseases.[1]

Antioxidantsareaddedtoavarietyoffoodstopreventordeterfreeradicalinducedlipidperoxidation,whichis
responsibleforthedevelopmentofoffflavorsandtheundesirablechemicalcompoundsinfood.[2]TheseROS
causedestructiveandirreversibledamagetothecomponentsofacell,suchaslipids,proteins,andDNA.Although
normalcellspossessantioxidantdefensesystems,ROSproducedinthecellsinducesdiseasessuchascancerand
aging.[3]

ROSareformedanddegradedbyallaerobicorganisms.ROScanreadilyreactwithmostbiomoleculesincluding
proteins,lipids,lipoproteins,andDNA.Exogenouschemicalandendogenousmetabolicprocessesinthehuman
bodyorinthefoodsystemmightproduceveryeffectiveROS,whicharecapableofoxidizingbiomolecules,
resultingintissuedamageandcelldeath.Whenthemechanismofantioxidantprotectionbecomesunbalancedby
exogenousandendogenousfactors,itresultsininflammation,diabetes,genotoxicity,cancer,andaccelerating
aging.[4]

Antioxidantsupplementsorfoodcontainingantioxidantsmaybeusedtohelpthehumanbodyreduceoxidative
damage.ThemostcommonlyusedantioxidantsareBHAButylatedhydroxylanisole,BHTButylatedhydroxyl
toluene,propylgallate,andtertbutylhydroquinone.[5]However,theyhavebeensuspectedofbeingresponsible
forliverdamageandcarcinogenesisinlaboratoryanimals.Therefore,thedevelopmentanduseofmoreeffective
antioxidantsaredesired.

Traditionalmedicineworldwideisbeingreevaluatedbyextensiveresearchondifferentplantspeciesandtheir
therapeuticprinciples.Plantsproduceantioxidantstocontroltheoxidativestresscausedbysunbeamsandoxygen
theycanrepresentasourceofnewcompoundswithantioxidantactivity.

DregeavolubilisBenth(Asclepiadaceae)iscommonlyknownasJuktiinBengal.Itisatallwoodyclimberof11
mofheightand95cmingirthwithdenselylenticulatebranches,occurringthroughoutthehotterpartsofIndiaand
CarNicoberislandsascendingtoanaltitudeof1500m.Inthetraditionalsystemofmedicines,thejuiceoftheplant
isusedasasternutatoryandleavesareemployedintheapplicationforboilsandabscesses.[6]Therootsandtender
stalksareusedasemeticandexpectorant.[7]Itisreportedthatanalcohol(50%)extractoftheplantshowedactivity
onthecentralnervoussystemaswellasanticanceractivityagainstSarcoma180inmice.Themaximumtolerated
dosewasfoundtobe500mg/kgbodyweightsofalbinomice.[7]Twopentacyclictriterpenoidstaraxeroneand
taraxerolisolatedfromthisplant,showedantitumoractivitiesagainstK562leukemiccellline.[8]

Thisstudyevaluatedthetotalantioxidantactivity,reductiveability,freeradicalscavenging,superoxideanion
radicalscavenging,hydroxylradicalscavenging,andlipidperoxidationofpetroleumether,chloroform,and
methanolextractofD.volubilis.Animportantobjectiveofthisresearchwastocompareinvitroantioxidative
potentialofpetroleumether,chloroform,andmethanolextractofD.volubilis.

MATERIALSANDMETHODS Goto:

Plantmaterial
ThefruitsofD.volubiliswerecollectedduringAugust2008fromSouth24Paraganas,WestBengal,India.The
plantmaterialwastaxonomicallyidentifiedbyDr.LakhmiNarashimhan,Scientist,BotanicalSurveyofIndia,
CentralNationalHerbarium,Howrah,WestBengal,India.Thevoucherspecimen[CNH/I
I/(267)/2008/Tech.II/267]wasmaintainedinourlaboratoryforfuturereference.

Preparationofplantextract
Theplantmaterialwasshadedriedwithoccasionalshiftingandthenpowderedwithmechanicalgrinder,passing
throughsieveno40,andstoredinatightcontainer.

Thedriedpowdermaterial(450gm)oftheplantD.volubiliswasextractedwithpetroleumether(6080C)for72
hintheconeshapedpercolatorat33C.Thesolventwasdistilledinreducedpressureandresultingsemisolidmass
wasvacuumdriedusingrotaryflashevaporatortoyieldasolidresidue(5.33%,w/w).Thesameplantpartresidue
wasthenextractedwithchloroformandmethanolaccordinglyandwasdistilledinreducedpressure,andthe
resultingsemisolidmasswasvacuumdriedbyusingrotaryflashevaporatortoyieldasolidresidue.Theextractive
valueofchloroformandmethanolweresubsequently(10.59%,w/w)and(20.29%,w/w).Thepreliminary
phytochemicalanalysiswasperformedtoidentifythephytoconstituentspresentintheextract.[9]

Chemical
AmmoniumthiocyanatewaspurchasedfromEMerck(Mumbai,India)andSigmaChemicalCo.Ltd.(Mumbai,
India).Ferrouschloride,ferricchloride,1,1diphenyl2picrylhydrazyl(DPPH),VitaminC,nitrobluetetrazolium
(NBT),thiobarbituricacid(TBA),tricholoaceticacid(TCA),phenazinemethosulfate(PMS),andpotassiumferric
cyanidewerepurchasedfromSigmaChemicalCo.Ltd.(St.Louis,MO,USA).Allotherchemicalsandreagent
wereofanalyticalgrade.

Freeradicalscavengingactivitymeasuredby1,1diphenyl2picrylhydrazil
Thefreeradicalscavengingactivityofalloftheextractsweremeasuredby1,1diphenyl2picrylhydrazil(DPPH).
[10]Briefly,an0.1mMsolutionofDPPHinmethanolwasprepared,and1mlofthissolutionwasaddedto3ml
ofthealloftheextractssolutionrespectivelyinpetroleumetherandwateratdifferentconcentrations(10,20,40,
80,160and320g/ml).Themixturewereshakenvigorouslyandallowedtostandatroomtemperaturefor30min.
Thentheabsorbancewasmeasuredat517nmbyusingaUVvisiblespectrophotometer(Genesys10UV:The
locationofThermoElectronCorporationisUSA).Lowerabsorbancevaluesofreactionmixtureindicatehigher
freeradicalscavengingactivity.ThecapabilitytoscavengetheDPPHradicalwascalculatedusingthefollowing
equation:

DPPHscavengingeffect(%)=[(A0A1)/A0)100],

whereA0istheabsorbanceofthecontrolreaction,andA1istheabsorbanceofpresenceofalloftheextract
samplesandstandard.

TotalreductioncapabilitybyFe3+Fe2+transformation

Thetotalreducingpowersofalltheextractsweredetermined[11]withdifferentconcentrationsofalltheextracts
(10,20,40,80,160,320g/ml)in1mlofdistilledwatermixedwithphosphatebuffer(2.5ml,0.2M,pH6.6)and
potassiumferricyanide[K3Fe(CN)6](2.5ml,1%).Themixturewasincubatedat50Cfor20min.Trichloroacetic
acid(2.5ml,10%)wasaddedtothemixture,whichwascentrifugedfor10minat1000g(RemiT8A,Mumbai,
India).Theupperlayerofsolution(2.5ml)wasmixedwithdistilledwater(2.5ml)andFeCl3(0.5ml.0.1%),and
theabsorbancewasmeasuredat700nmusingaUVVisspectrophotometer(Genesys10UV:ThermoElectron
Corporation).Higherabsorbanceofthereactionmixtureindicatedgreaterreducingpower.

SuperoxideanionradicalscavengingactivityinPMSNADHsystem
Measurementsofsuperoxideanionscavengingactivityofalltheextractswerebasedonthemethod[11]taking1
mlofNBTsolution(156MNBTin100mMphosphatebuffer,pH7.4),andvariousconcentrations(10,20,40,
80,160,320g/ml)ofsamplesolutionsofalltheextracts,respectively,inpetroleumether,chloroform,and
methanolweremixed.Thereactionstartedbyadding100lPMSsolution(60MPMSin100mMphosphate
buffer,pH7.4)tothemixture.Themixtureswerethenincubatedat25Cfor5min,andtheabsorbanceat560nm
wasmeasuredagainstblanksamples.Adecreaseintheabsorbanceofreactionsampleindicatedincreasedsuper
oxideanionscavengingactivityandvitaminCwasusedasastandarddrugtakingatdifferentconcentrationsasthe
differentextracts.

Nitricoxideradicalscavengingassay
InvitronitricoxidegeneratedfromsodiumnitroprussideinaqueoussolutionatphysiologicalpHinteractswith
oxygentoproducenitriteions,whichwasmeasured[11]withreactionmixture(3ml)containingsodium
nitroprusside(10mM)inphosphatebufferedsaline(PBS)andvariousconcentrations(20,40,80,160,320g/ml)
oftheplantextractwereincubatedat25Cfor150min.Attheendofincubation,0.5mlofthereactionmixture
wasremovedand0.5mlofGriessreagent(1%sulfanilamide,2%H3PO4,and0.1%naphthylethylenediamine
dihydrochloride)wasadded.Theabsorbanceofchromophoreformedwasmeasuredat546nm.Thepercentage
inhibitionofnitriteoxidegeneratedwasmeasuredbycomparingtheabsorbancevaluesofcontrolandtest
compounds.
Determinationofinhibitionoflipidperoxidation
Ratliverhomogenatewasusedasasourceofpolyunsaturatedfattyacidsfordeterminingtheextentoflipid
peroxidation.[12]Ratwassacrificedbycervicaldislocation.Theliverwascollectedandwashomogenizedwith40
mMTrisHClbuffer(pH7.0)andcentrifugedat3000gfor10min.Clearsupernatantwastakenandtoit100l
ofeachof0.15MKCl,15mMFeSO4,and6mMascorbicacidwereadded,andthesupernatantwasincubatedat
37Cfor1h.OnemilliliterofTCA(10%)wasaddedtothemixtureandthesamplewascentrifugedat3000g
for20minat4Ctoremovetheinsolubleprotein.Twomillilitersofthesupernatantwasremovedand1.0mlTBA
(0.8%)wasaddedtothisfractionfollowedbyheatingat90Cfor20mininawaterbath.Aftercooling,thecolored
TBAMDAcomplexwasextractedwithorganicsolvent(2.0mlbutanol)andabsorbancewasmeasuredat532
nm.

Determinationoftotalphenoliccompounds
ThetotalphenoliccompoundsinthefruitsofD.volubilisatdifferentconcentrationsweredeterminedwithFolin
Ciocalteureagentbyusingpyrocatecholasastandardphenoliccompound.Briefly,1mgofalltheextractssolution
(1000gofextract)wasplacedina100mlErlenmeyerflaskdilutedwithdistilledwater(46ml).FolinCiocalteu
reagent(1ml)wasaddedandthecontentsoftheflaskweremixedthoroughly.After3min,3mlofNa2CO3(2%)
wasadded,thenthemixturewasallowedtostandfor2hwithintermittentshaking.Theabsorbancewasmeasured
at760nmbyaspectrophotometer(Genesys10UVThermoElectronCorporation).[12]Theamountoftotal
phenoliccompoundsinthefruitsofDregeavolubiliswasdeterminedinmicrogramsofpyrocatecholequivalent,
usingtheequationobtainedfromthestandardpyrocatecholgraph:

Absorbance=0.001pyrocatechol(g)+0.0033

Statisticalanalysis
ExperimentalresultsweremeanS.E.M.ofthreeparallelmeasurements.Statisticalanalysiswasestimatedby
usingStudent'sttestfollowedbyANOVAmethod.ThevaluesforP<0.05wereconsideredassignificantand
valuesforP<0.001asverysignificant.

RESULTSANDDISCUSSIONS Goto:

ThequalitativechemicalanalysisofthefruitsofD.volubilisshowedpositiveresultsforthepresenceofsteroids,
triterpenoids,flavonoids,tannins,andglycosides.Antioxidantmethodsandmodificationshavebeenproposedto
evaluateantioxidantcharacteristicsandtoexplainhowantioxidantsfunction.Ofthese,antioxidantactivity,
reducingpower,freeradicalscavenging,superoxideanionradicalscavenging,andnitricoxideradicalinhibition
activitiesandestimationofphenolcontentsaremostcommonlyusedfortheevaluationofthetotalantioxidant
behavioroftheextracts.

Effectsontotalantioxidantactivity
Theantioxidantactivityofratliverperoxidationduetothepresenceofphytoconstituentssuchasflavonoidsand
biflavoneshasbeenreported.Therefore,thisstudysuggeststhattheantioxidantactivityofalltheextractsmaybe
attributedtothereductionoffreeradicals,chelationofmetalions,oracombinationthereofpresenceof
phytoconstituents.

EffectonscavengingDPPHradical
ThestableDPPHradicalmodelisawidelyused,relativelyquickmethodfortheevaluationoffreeradical
scavengingactivity.TheeffectsofantioxidantsonDPPHradicalscavengingisthoughttobeduetotheirhydrogen
donatingability.DPPHisastablefreeradicalthatacceptsanelectronorhydrogenradicaltobecomeastable
diamagneticmolecule.TheabsorptionmaximumofastableDPPHradicalinmethanolwas517nm.Thedecrease
inabsorbanceofDPPHradicalcausedbyantioxidants,becauseofthereactionbetweenantioxidantmoleculesand
radicalprogressed,resultsinthescavengingoftheradicalsbyhydrogendonation.Itisvisuallynoticeablethatasa
changeincolorfrompurpletoyellow.Hence,DPPHisusuallyusedasasubstratetoevaluatetheantioxidant
activityofantioxidants.[12]Ithasbeenreportedthatoxidativestress,whichoccurswhenfreeradicalformation
exceedsthebody'sabilitytoprotectitself,formsthebiologicalbasisofchronicconditionssuchasarteriosclerosis.
[13]

Basedonthedataobtainedfromthisstudy,alltheextractswereeffectivefreeradicalinhibitororscavenger,aswell
asaprimaryantioxidantthatreactswithfreeradicals,whichmaylimitfreeradicaldamageoccurringinthehuman
body.Table1showsasignificant(P<0.001)decreaseintheconcentrationofDPPHradicalsduetothe
scavengingabilityoftheextractsandstandard.Freeradicalscavengingactivityalsoincreasedwithincreasing
concentrationintherangeof10320g/ml.

Table1
DPPHscavengingpowerofthedifferentextractsofD.volubilisandof
vitaminCasstandard

Effectonreductiveability
Forthemeasurementofthereductive,weinvestigatedFe3+Fe2+transformationinthepresenceofextracts.The
reducingcapacityofacompoundmayserveasasignificantindicatorofitspotentialantioxidantactivity.[14]The
antioxidantactivityofputativeantioxidantshasbeenattributedtovariousmechanisms,amongwhichthe
preventionofchaininitiation,thebindingoftransitionmetalioncatalysts,decompositionofperoxides,the
preventionofcontinuedhydrogenabstraction,thereductivecapacity,andradicalscavengingaresomeexamples.
[15]Liketheantioxidantactivity,thereducingpowerofalltheextractsincreasedwithincreasingconcentrationof
samples.Table2showsthereductivecapabilitiesofalltheextractscomparedwithvitaminC.Alltheextracts
concentrationstestedshowedhigheractivitiesthanthecontrol,andthesedifferenceswerestatisticallysignificant(P
<0.001).

Table2
ReducingpowerofthedifferentextractsofD.volubilisandofvitaminC

Effectsonsuperoxideanionradicalscavengingactivity
Superoxideanionradical(O2 )areformedbyactivatedphagocytes,suchasmonocytes,macrophages,eosinophils,
andneutrophills,andtheproductionofO2 isanimportantfactorinthekillingofbacteriabyphagocytes.Inthe
PMSNADHNBTsystem,superoxideanion,derivedfromdissolvedoxygenfromthecouplingreactionofPMS
NADH,reducesNBT.[16]Thedecreaseinabsorbanceat560nmwithantioxidantsindicatestheconsumptionof
superoxideanioninthereactionmixtures,Table3showsthepercentinhibitionofsuperoxideradicalgenerationby
20,40,60,80,100g/mlofalltheextractcomparedwithvitaminC.Theconcentrationsofalltheextractshavea
significantlevel(P<0.001)ofsuperoxideradicalscavengingactivitycomparedwithcontrol.

Table3
SuperoxidescavengingpowerofthedifferentextractsofD.volubilisandof
vitaminC

Effectsonnitricacid(NO)scavengingactivity
Nitricoxideandsuperoxideanioncauseischemicrenalinjuryseparately,andtheseradicalsworktogethertobring
furtherdamage.ThetoxicityanddamagecausedbyNOandO2ismultipliesastheyreacttoproducereactive
peroxynitrite,whichleadstoserioustoxicreactionswithbiomolecules,suchasprotein,lipids,andnucleicacids.
Highconcentrationofnitricoxide(NO)hasdeleteriouseffects,andtherefore,itisnecessarythattheproductionof
NObetightlyregulated.[17]WhenNOisproducedbymacrophages,thenitricoxideradicalcanbeconvertedinto
peroxynitrites,whichwillcausediversechemicalreactionsinabiologicalsystemincludingnitrationoftyrosine
residueofprotein,triggeringlipidperoxidation,inactivationofaconites,inhibitionofmitochondrialelectron
transportandoxidationofbiologicalthiolcompound.[18]

SuppressionofNOreleasedmaybepartiallyattributedtodirectNOscavenging,asconcentrationsoftheextracts
decreasestheamountofnitritegeneratedfromthedecompositionofsodiumnitroprussideatphysiologicpHwas
foundtobeinhibitedbyalltheextracts.Table4showsthepercentageinhibitionofnitricoxideproduction.The
extractsshowedsignificantlevel(P<0.001)ofinhibitionofnitricoxideproductionbyactivatedperitoneal
macrophageswithinvitroconditions.

Table4
NitricoxidescavengingpowerofthedifferentextractsofD.volubilisandof
vitaminC

Effectsonlipidperoxidation
Alltheextractsretardtheperoxidationoflinoleicacid(LH).TheperoxidationofLHiswellknowntobeachain
reactioninwhichthechainsarecarriedbylinoleylperoxylradicals,LOOandtheproductsarelinoleyl
hydroperoxides.[19]TheretardationofLHperoxidationbytheextractshadbeenfoundtobeduetorapidchain
terminationviaaveryfastcrossreactionbetweenHOOandLOOradicals.Thisantioxidantmechanismis
completelydifferentfromthemechanismofantioxidantactionofvitaminE.SincevitaminEbecomesaprooxidant
athighconcentrations,theadditionoftheextractstoediblelipidsmayprovideanalternativeorsupplementary
strategyforobtaininglargeincreasesintheiroxidativestabilityandshelflife,somethingthatcannotbeachievedby
simplyaddingmoreandmorevitaminC.Table5showsthepercentageinhibitionoflipidperoxidation.

Table5
InhibitionoflipidperoxidationofthedifferentextractsofD.volubilisandof
VitaminC

Effectsoftotalphenoliccompounds
Phenolicconstituentsareveryimportantinplantsbecauseoftheirscavengingabilityduetotheirhydroxylgroups.
[20]
Anumberofstudieshavefocusedonthebiologicalactivitiesofphenoliccompounds,whicharepotential
antioxidantsandfreescavengers.Inaddition,ithasbeenreportedthatphenoliccompoundsareassociatedwith
antioxidantactivityandplayanimportantroleinstabilizinglipidperoxidation.Onemilligramofeachconcentration
ofalltheextractscontained95.03gofpyrocatecholequivalentsofphenols.Thesephenoliccompoundsmay
contributedirectlytotheantioxidativeaction.Ithasbeensuggestedthatupto1gofpolyphenoliccompounds(from
adietrichinfruitsandvegetables)ingesteddailyhasinhibitoryeffectsonmutagenesisandcarcinogenesisin
humans.[20]

CONCLUSIONS Goto:

Basedontheresultsofthisstudy,itisclearthatalloftheextractshavepowerfulinvitroantioxidantscapacity
againstvariousantioxidantsystems.Fromtheresults,itcanbeconcludedthattheantioxidantactivityofallthe
extractswereconcentrationdependentwithinhibitionoflipidperoxidation.Fromtheaboveanalyses,thepossible
mechanismofantioxidantactivityofalltheextractsincludereductiveability,hydrogendonatingability,and
scavengingofsuperoxide,nitricoxideandfreeradicals,whichmaybeduetothepresenceofphytoconstituents
suchasflavonoidsandpolyphenolspresentinthepetroleumether,chloroform,andmethanolextractofD.
volubilis.

Footnotes Goto:

SourceofSupport:Nil

ConflictofInterest:Nonedeclared.

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