International Journal of PharmTech Research

CODEN (USA): IJPRIF

Vol.6, No.2, pp 575-581,

Study Of The Antibacterial Activities Of

Soursop (Annona muricata L.)
Leaves
Ginda Haro*, Niky Puji Utami, and Erly Sitompul
Faculty of Pharmacy, University of Sumatera Utara,
Jl. Tri Dharma No. 5, Pintu 4, Kampus USU, Medan, Indonesia, 20155

*Corres. Author : gindaharo@yahoo.com

Phone number : +62 812 6477 1909

Abstract : Background: The use of natural product medicines has emerged from traditional to modern

therapyinordertoincreasethequalityofhealthworldwide.Naturederivedmedicinesareconsideredsafer.The
leavesofsoursop(Annonamuricata L.)haslongbeenusedbycertainlocalcommunitiesinIndonesiaasan
alternativetreatmentofbacterialdiseases.
Objective:Objectiveofthisstudywastheinvestigationofantibacterialactivitiesofmethanolextractand
chloroformfractionoftheleavesofsoursop(AnnonamuricataL.)ofthefamilyofAnnonaceaeagainst
EscherichiacoliandStaphylococcusaureus
Methods:Themethanolextractandchloroformfractionwereobtainedbymeansofmacerationmethodusing
methanolassolventandthenfractionated,withchloroform.Theantibacterialactivitiesweremeasuredinvitro
bymeansofagardiffusionmethodusingpaperdisc.
Results: Methanol extract of soursop leaves at concentration of 150 mg/ml could inhibit the growth of
Staphylococcusaureus withinhibitionzonediameterof14.1mmandof13.1for Escherichiacoli.Butthe
methanolextractofsoursopleavesatconcentrationof250mg/mlcouldinhibitthegrowthofEscherichiacoli
withinhibitionzonediameterof14.5mm.ItfulfilledtherequirementofFarmakopeIndonesia.Chloroform
fractionofsoursopleavesatconcentrationof150mg/ml couldonlyinhibit the growthof Staphylococcus
aureuswithinhibitionzonediameterof9.9mmandof9.2mmforEscherichiacoli.
Conclusion:Themethanolextractindicatedahigherinhibitionzonediameterthanchloroformfractionagainst
StaphylococcusaureusandEscherichiacoli.
Keywords:soursopleaves,antibacterialactivities,EscherichiacoliandStaphylococcusaureus.

Introduction
Wepreviouslyhavereportedourinvestigationaboutthecharacterizationandthephytochemicalscreeningof

the leaves of soursop (Annona muricata L.). The alkaloids, flavonoids, tannins, saponins, glycosides and
steroid/triterpenoidswerepresentontheleaves.TheantibacterialactivitiesagainstStaphylococcusaureus,
Stapylococcusepidermidis,PropionibacteriumacneandPseudomonasaeruginosawerealsopositivelyfound1.
Soursop(AnnonamuricataL.)isatropicalplantandfamiliartotheIndonesiancertainlocalcommunities.This
planthasgreatbenefitsforhumanlifewhichisfullwithnutrition.Inthefoodindustrysoursopcanbe
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Ginda Haro et al /Int.J.PharmTech Res.2014,6(2),pp 575-581.

processedintojam,fruitjuice,syrup 2.Soursopleavescontainflavonoid,tannin,alcaloid,saponin,calcium,
phosphor,carbohydrate,vitamineA,BandC,phytosterol,calciumoxalate 3.
Theseleavesaretraditionallyusedtopreventandtreatarthritis,asthma,bronchitisbiliarydisorder,diabetic,
heartdiseases,hypertension,wormdisease,liverdisorder,malaria,rheumatism,sedative,tumor,andcancer 4.
Theleavesarealsousedforthetreatmentofseveraltypesofdiseasescausedbybacteriasuchaspneumonia,
diarrhea,urinarytractinfection,andotherkindsofskindiseases 5.
Staphylococcus aureus is a gram positive bacteria that is commonly found on human skin and mucous
membranes. This bacteria can cause skin infection 6. Escherichia coli is a gram negative bacteria that is
commonlyfoundinthehumancolon.Thisbacteriaisoneofthemostcommonpathogenicbacteriainfood
causingtheprimaryinfectionoftheintestinesuchasdiarrhea 7.
Basedonthosefacts,inordertocontinueourinvestigationwereporthereourstudyabouttheantibacterial
activitiesofsoursop(AnnonamuricataL.)leavesagainstEscherichiacoliandStaphylococcusaureus.

ExperimentalMethods
Materials
Materialsusedinthisstudyweresoursop(daunsirsak,AnnonamuricataL.)leaves,agarnutrientmedia,broth
nutrient media, Staphylococcus aureus (ATCC No. 6538) and Escherichia coli (ATCC No. 25922), and
aquadest.
Apparatus
Apparatus used in this study were glasses, autoclave (Fisons), blender (Philips), freeze dryer (Modulio),
incubator(FiberScientific),filterpaper,LaminarAirFlowCabinet(AstecHLF1200L),refrigerator(Toshiba),
oven(Memmert),heater,micropipet(Eppendorf), rotaryevaporator (HaakeD),visiblespectrophotometer
(Dynamic),digitalbalance(MettlerToledo).
TheCollectionandTheTreatmentofSoursopLeaves
The leaves of soursop (Annona muricata L.) were purposively collected from Kelurahan Tanjung Mulia,
KecamatanMedanDeli,Medancity,IndonesiainJanuary2013.Thefourthandthefifthleaveswerepickedout
ofthepointofayoungleaf.
TheIdentificationofSoursopLeaves
ItwasconductedbytheHerbariumMedanense,UniversityofSumateraUtara.
ThePreparationofSymplex
Theleavesofsoursopwasweighedafterwashinganddryinginopenair.Theywereblendedtoobtainapowder
massandkeptinaclosedpocketofplastics.
ThePreparationofExtractofSoursop(AnnonamuricataL.)Leaves
500gofsymplexpowderofsoursopleavesweremaceratedfor5daysoutoflight,occasionallystirred,by
macerationmethodusingmethanolassolvent.After5days,themixturewasfilteredandtheresiduewashedby

usingmethanolandtreatedfor2daysofthesametreatmentasbefore.Themacerateswerethenmixedtogether
and concentrated by means of rotary evaporator with the temperature not exceeding 40C untill obtained
spissumextractbymeansoffreezedryer 8.
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Ginda Haro et al /Int.J.PharmTech Res.2014,6(2),pp 575-581.

ThePreparationofMedia
Agarnutrientmedia
Composition:Lablemcopowder

1g

Yeastextract

2g

Peptone

5g

Sodiumchloride5g
Agar

15g

Thepreparation9: 28 g of agar nutrient media was suspendedin 1000 ml of distilledwater andheatedto

completelydissolved.Themediawasthenputintoflaskandsterillizedfor15minutesat121C.
Brothnutrientmedia
Composition:Lablemcopowder

1g

Yeastextract

2g

Peptone

5g

Sodiumchloride

5g

Thepreparation9 :13gofbrothnutrientmediawassuspendedin1000mlofdistilledwaterandheatedto
completelydissolved.Themediawasthenputintoflaskandsterillizedfor15minutesat121C.
TheSterilizationofApparatus
Theglassesusedinthisantibacterialactivitiestestwassterillizedinovenfor1houratthetemperatureof
170C.Themediawassterillizedinautoclavefor15minutesat121C.Alatalatyangdigunakandalamuji
aktivitasantibakteriini,disterilkanterlebihdahulusebelumdipakai.Theosesyringeandpincetweresterillized
bymeansofBunsenlamp10.
ThePreparationofChloroformFractionofSoursop(AnnonamuricataL.)Leaves
10mlofmethanolsolventwasaddedinto10gofmethanolextract,thenhomogenouslystirredandremoved
intoseparatingfunneland20mlofdistilledwaterand40mlofnhexanewereadded,stirredandletitseparated
andfractionatedcompletelyby nhexane,theresidueofmethanolextractwasthenfractionatedby40mlof
chloroform.Theresultofchloroformfractionationwasthenevaporatedonawaterbathtoobtainadryextractof
chloroformfraction.
ThePreparationofBacteriaCultureStock11
Thecolonyofbacteriawastakenbysterillizedosesyringe,thenplantedintoslopingagarnutrientmediaby
scratchingit.Itwasthenincubatedinaincubatorfor1824hoursatthetemperatureof3637C.
ThePreparationofBacteriaInoculumn
Thecolonyofbacteriawastakenfromculturestockbysterillizedosesyringe,thensuspendedintotesttubeof
brothnutrientmediaof10ml.Theturbidityofthesolutionwasthenmeasuredatthewavelengthof580nm
untillobtainedthetransmittanceof25%equivalentwith10 6CFU(ColonyFormingUnits)11.
ThePreparationofSolutionTestsofMethanolExtractandChloroformFractionwithVarious

Concentrations
3gofmethanolextractwasdissolvedintoDMSOuntill10mlofvolumeinordertoobtaintheconcentrationof
theextractof300mg/ml.Thedilutionwasthenmadeinordertoobtainextractswiththeconcentrationsof250
mg/ml;200mg/ml;150mg/ml;100mg/ml;50mg/ml;25mg/ml;10mg/ml;5mg/ml.Thesameprocedurewas
donetochloroformfraction.
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Ginda Haro et al /Int.J.PharmTech Res.2014,6(2),pp 575-581.

TheInVitroAntibacterialActivitiesTests
0.1mlofinokulumwasputintopetridiscwasthenadded20mlofsterillizedliquidagarnutrientmediaheated
gentlyto45C,homogenizedandletitstandinordertoobtainasolidmedia.Thepaperdiscwithdiameterof6
mmwassoakedintosolutiontestsofvariousconcentrations,driedandletthemonthesurfaceofagarnutrient
media.Itwasthenincubatedfor1824hoursatthetemperatureof3637C.Thesameprocedurewasdoneto
chloroformfraction.Theinhibitionzonediameteraroundthepaperdiscwasthenmeasuredandrecordedby
whatsocalledjangkasorong.Thetestswererespectivelydone3times 11.Thetestsofantibacterialactivitiesof
methanol extract and chloroform fraction of soursop (Annona muricata L.) leaves against Staphylococcus
aureusandEscherichiacoliaredepictedinthenextFigure1,2,3and4.
muricata L.) leaves
against Escherichia
coli

Fig 2. The test of

Fig.1.Thetestofantibacterialactivitiesofmethanolextractofsoursop antibacterialactivities
ofmethanolextractof
(AnnonamuricataL.)leavesagainstStaphylococcusaureus
soursop (Annona
Staphylococ
cusaureus
Fig.3. Thetestof
antibacterial
activities of
chloroform
fraction of
soursop
(Annona
muricataL.)
leaves
against

their
relatio
ResultsAndDiscussion nship.
The
The antibacterial activitiesresults
testwasconductedinvariousof
concentrations to investigateantiba

cterial metha
activit nol
ies
extract
exami and
nation chlorof
showe orm
d thatfractio

Fig.3. Thetestof
antibacterial
activities of
chloroform
fraction of
soursop
(Annona
muricata L.)
leaves
against
Escherichia
coli

n gavecoli and Staphylococcus

antibac aureus. They were denoted
terial bytheexistenceofinhibition
effect zone around the paperdisc.
againts Theresultsoftheaverageof
Escher inhibition zone diameter of
ichia methanol extract and

chloroform fractions ofStaph

soursop leaves forylococ

cus s and
aureu Escher

ichia can completely be seen on

coli Tabel1andTabel2below.

Ginda Haro et al /Int.J.PharmTech Res.2014,6(2),pp 575-581.

579

Tabel1.TheresultsoftheaverageofinhibitionzonediameterofthegrowthofStaphylococcus
aureusandEscherichiacoliofmethanolextractofsoursopleaves
Methanolextract
concentration
(mg/ml)
300
250
200
150
100
50
25
10
5
Blank

InhibitionZoneDiameter
(mm)*
Staphylococcusaureus
16.4
15.4
14.7
14.1
13.1
12.2
11.2
9.8
8.6

Escherichiacoli
15.3
14.5
13.8
13.1
12.3
11.5
10.7
8.7
8.0

Informations:(*)=Theaverageresultsoftriplemeasurements,()=noinhibition

OntheTabel1aboveitisobviousthattheincreaseofextractconcentrationswillenhancetheirantibacterial
activitiescausedbytheincreaseofbioactivecontentsoftheextract.
Theresultsofantibacterialactivitiesexaminationshowedthatmethanolextractatconcentrationof150mg/ml
could inhibit the growth of Staphylococcus aureus with inhibition zone diameter of 14.1 mm and at
concentrationof250mg/mlcouldinhibitthegrowthofEscherichiacoliwithinhibitionzonediameterof14.5
mm.TheMinimumInhibitionConcentration(MIC)ofmethanolextractatconcentrationof5mg/mlcould
inhibitStaphylococcusaureuswithinhibitionzonediameterof8.6mmandof8.0mmforEscherichiacoli.
Theinhibitionzonediameterofbacterialgrowthofmethanolextractofsoursopwaslargerthanofethanol
extract1causedbythestrengthofmethanolcomparedwithethanolasasolventofbioactivecompound 12.
Tabel2.TheresultsoftheaverageofinhibitionzonediameterofthegrowthofStaphylococcus
aureusandEscherichiacoliofchloroformfractionsofsoursopleaves
Chloroformfractions InhibitionZoneDiameter
concentration
(mm)*
Staphylococcusaureus
Escherichiacoli
(mg/ml)
300
12.1
11.5
250
11.5
10.7
200
10.5
9.8
150
9.9
9.2
100
9.3
8.8
50
8.8
8.3
25

10

Blank

Informations:(*)=Theaverageresultsoftriplemeasurements,()=noinhibition
Thechloroformfractionsatconcentrationof300mg/mlshowedtheinhibitionzonediameterof12.1mmfor
Staphylococcusaureusandof11.5mmforEscherichiacoli.TheMinimumInhibitionConcentration(MIC)at
concentrationof50mg/mlshowedtheinhibitionzonediameterof8.8mmforStaphylococcusaureusandof

8.3mmforEscherichiacoli.
Acompoundwithinhibitionzonediameterabout14through16mmissaidtohaveasatisfiedinhibitionzone
diameter11.Theinhibitionzonediameterofmethanolextractfulfilledtherequirement.Itcouldbeunderstood,
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Ginda Haro et al /Int.J.PharmTech Res.2014,6(2),pp 575-581.

maybebecauseofmethanolextractcontainingtanninandflavonoids,whereaschloroformfractioncontaining
onlyflavonoidsinsmallamount.Themechanismoftanninwithantibacterialactivitiesonlowconcentration
wasbydestroyingthecytoplasmmembranecausingtheleakofcell,onhighconcentrationthetanninwould
coagulatewithcellularprotein13.Theflavonoidscoulddamagethepermeabilityofcellwallofbacteria 14.
Theresultofthisstudyindicatedthat Staphylococcusaureus aspositivegrambacteriaismoresensitiveto
chemicalsthan Escherichiacoli asnegativegrambacteria.Thisthingcouldbecausedbythedifferencein
compositionandstructureofcellwallofthosebacteria.Thestructureofcellwallofpositivegrambacteria
consistsoflipidonly(14%),whereasofnegativegrambacteriaconsistsofmultilayersoflipoprotein,
liposaccharideandpeptidoglicanwithahighlipidcontent(1112%).

Conclusion
Thisstudyindicatedthatmethanolextractofsoursopleavesatconcentrationof300mg/mlcouldinhibitthe
growthof Staphylococcusaureus withinhibitionzonediameterof16.4mmandof15.3mmfor Escherichia
coli.Chloroformfractionofsoursopleavesatconcentrationof300mg/mlcouldonlyinhibitthegrowthof
Staphylococcusaureuswithinhibitionzonediameterof12.1mmandof11.5mmforEscherichiacoli.
Methanolextractofsoursopleavesatconcentrationof150mg/mlcouldinhibitthegrowthofStaphylococcus
aureuswithinhibitionzonediameterof14.1mmandof13.1forEscherichiacoli.Butthemethanolextractof
soursopleavesatconcentrationof250mg/mlcouldinhibitthegrowthofEscherichiacoliwithinhibitionzone
diameterof14.5mm.ItfulfilledtherequirementofFarmakopeIndonesia,theyareof14through16mm.The
minimuminhibitoryconcentration(MIC)ofmethanolextractatconcentrationof5mg/mlcouldinhibitthe
growthofStaphylococcusaureuswithinhibitionzonediameterof8.6mmandof8.0mmforEscherichiacoli
respectively. The MIC of chloroform fraction at concentration of 5 mg/ml did not inhibit growth of
StaphylococcusaureusandEscherichiacoliatall.WhereastheMICofchloroformfractionatconcentrationof
50mg/mlcouldonlyinhibitStaphylococcusaureuswithinhibitionzonediameterof8.8mmandof8.3mmfor
Escherichiacoli.
The methanol extract indicated a more effective bacterial inhibition than chloroform fraction of the same
concentration.

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