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6/20/2017 AntibacterialandantifungalactivitiesfromleafextractsofCassiafistulal.

:Anethnomedicinalplant

JAdvPharmTechnolRes.2011AprJun2(2):104109. PMCID:PMC3217694
doi:10.4103/22314040.82956

AntibacterialandantifungalactivitiesfromleafextractsofCassiafistula
l.:Anethnomedicinalplant
NayanR.BhalodiaandV.J.Shukla
DepartmentofPharmaceutical,I.P.G.TandR.A,GujaratAyurvedUniversity,Jamnagar,Gujarat,India
Addressforcorrespondence:Dr.NayanRameshbhaiBhalodia,PanchavatiGaushala,95B,SarusectionRoad,Opp.GitaBunglow,
Jamnagar361008,Gujarat,India.Email:nayanbhalodia@gmail.com

Copyright:JournalofAdvancedPharmaceuticalTechnology&Research

ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNoncommercialShareAlike3.0Unported,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.

Abstract
Thisstudywascarriedoutwithanobjectivetoinvestigatetheantibacterialandantifungalpotentialsofleaves
ofCassiafistulaLinn.Theaimofthestudyistoassesstheantimicrobialactivityandtodeterminethezone
ofinhibitionofextractsonsomebacterialandfungalstrains.Inthepresentstudy,themicrobialactivityof
hydroalcoholextractsofleavesofCassiafistulaLinn.(anethnomedicinalplant)wasevaluatedforpotential
antimicrobialactivityagainstmedicallyimportantbacterialandfungalstrains.Theantimicrobialactivitywas
determinedintheextractsusingagardiscdiffusionmethod.Theantibacterialandantifungalactivitiesof
extracts(5,25,50,100,250g/ml)ofCassiafistulaweretestedagainsttwoGrampositive
Staphylococcusaureus,StreptococcuspyogenestwoGramnegativeEscherichiacoli,Pseudomonas
aeruginosahumanpathogenicbacteriaandthreefungalstrainsAspergillusniger,Aspergillusclavatus,
Candidaalbicans.Zoneofinhibitionofextractswerecomparedwiththatofdifferentstandardslike
ampicillin,ciprofloxacin,norfloxacin,andchloramphenicolforantibacterialactivityandnystatinand
griseofulvinforantifungalactivity.Theresultsshowedthattheremarkableinhibitionofthebacterialgrowth
wasshownagainstthetestedorganisms.Thephytochemicalanalysesoftheplantswerecarriedout.The
microbialactivityoftheCassiafistulawasduetothepresenceofvarioussecondarymetabolites.Hence,
theseplantscanbeusedtodiscoverbioactivenaturalproductsthatmayserveasleadsinthedevelopmentof
newpharmaceuticalsresearchactivities.

Keywords:Cassiafistula,invitroantibacterialactivity,antifungalactivity,secondarymetabolites

INTRODUCTION
Antibioticsareoneofourmostimportantweaponsinfightingbacterialinfectionsandhavegreatlybenefited
thehealthrelatedqualityofhumanlifesincetheirintroduction.However,overthepastfewdecades,these
healthbenefitsareunderthreatasmanycommonlyusedantibioticshavebecomelessandlesseffective
againstcertainillnessesnot,onlybecausemanyofthemproducetoxicreactions,butalsoduetoemergence
ofdrugresistantbacteria.Itisessentialtoinvestigatenewerdrugswithlesserresistance.Drugsderivedfrom
naturalsourcesplayasignificantroleinthepreventionandtreatmentofhumandiseases.Inmanydeveloping
countries,traditionalmedicineisoneoftheprimaryhealthcaresystems.[1,2]Herbsarewidelyexploitedin
thetraditionalmedicineandtheircurativepotentialsarewelldocumented.[3]About61%ofnewdrugs
developedbetween1981and2002werebasedonnaturalproductsandtheyhavebeenverysuccessful,
especiallyintheareasofinfectiousdiseaseandcancer.[4]Recenttrends,however,showthatthediscovery
rateofactivenovelchemicalentitiesisdeclining.[5]Naturalproductsofhigherplantsmaygiveanewsource
ofantimicrobialagentswithpossiblynovelmechanismsofaction.[6,7]Theeffectsofplantextractson
bacteriahavebeenstudiedbyaverylargenumberofresearchersindifferentpartsoftheworld.[8]Much
workhasbeendoneonethnomedicinalplantsinIndia.[9]

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Plantsarerichinawidevarietyofsecondarymetabolitessuchastannins,terpenoids,alkaloids,flavonoids,
glycosides,etc.,whichhavebeenfoundinvitrotohaveantimicrobialproperties.[10,11]

Herbalmedicineshavebeenknowntomanforcenturies.Therapeuticefficacyofmanyindigenousplantsfor
severaldisordershasbeendescribedbypractitionersoftraditionalmedicine.[12]Antimicrobialpropertiesof
medicinalplantsarebeingincreasinglyreportedfromdifferentpartsoftheworld.TheWorldHealth
Organizationestimatesthatplantextractsortheiractiveconstituentsareusedasfolkmedicineintraditional
therapiesof80%oftheworld'spopulation.[13]Theharmfulmicroorganismscanbecontrolledwithdrugs
andtheseresultsintheemergenceofmultipledrugresistantbacteriaandithascreatedalarmingclinical
situationsinthetreatmentofinfections.Thepharmacologicalindustrieshaveproducedanumberofnew
antibioticsresistancetothesedrugsbymicroorganismshasincreased.Ingeneral,bacteriahavethegenetic
abilitytotransmitandacquireresistancetosyntheticdrugswhichareutilizedastherapeuticagents.[14]

Inanefforttoexpandthespectrumofantibacterialagentsfromnaturalresources,Cassiafistulabelongingto
Leguminosaefamilyhasbeenselected.IntheIndianliterature,thisplanthasbeendescribedtobeuseful
againstskindiseases,livertroubles,tuberculosesglandsanditsuseintothetreatmentofhematemesis,
pruritus,leucoderma,anddiabeteshasbeensuggested.[15,16]Ithasbeenconcludedthatplantpartscouldbe
usedasatherapeuticagentinthetreatmentofhypercholesterolemiapartiallyduetotheirfiberandmucilage
content.[17]Besidesitspharmacologicaluses,theplantextractisalsorecommendedasapestanddisease
controlagentsinIndia.[1820]Thisplantiswidelyusedbytribalpeopletotreatvariousailmentsincluding
ringwormandotherfungalskininfections.[21]Theleavesarelaxative,antiperiodic,depurative,anti
inflammatory,andareusefulinskindiseases,boils,carbuncles,ulcers,intermittentfever,goutyarthritis,and
rheumatalgia.Cassiafistulaplantorgansareknowntobeanimportantsourceofsecondarymetabolites,
IndianpeopleareusingtheleavestotreatinflammationCassiafistulaplantorgansareknowntobean
importantsourceofsecondarymetabolites,notablyphenoliccompounds.[22]

Cassiafistulaexhibitedsignificantantimicrobialactivityandshowedpropertiesthatsupportfolkloricusein
thetreatmentofsomediseasesasbroadspectrumantimicrobialagents.[23]Thus,Cassiafistulaiswell
anchoredinitstraditionaluseshasnowfoundwidespreadacceptanceacrosstheworld.

Inthecurrentinvestigationcarriedout,ascreeningofhydroalcoholicextractsofCassiafistulaleavesagainst
pathogenicbacteriaandfungiisdoneinordertodetectnewsourcesofantimicrobialagents.

MATERIALSANDMETHODS

CollectionofPlantMaterials
ThefreshandhealthyleavesoftheplantCassiafistulawerecollectedbetweenJuneandAugust,2009from
variousareasofJamnagardistrict,Gujarat,India.Theplantspecimenswereidentifiedindepartmentof
PharmacognosyI.P.G.TandR.A.(Instituteofpostgraduateteachingandresearchinayurveda),Jamnagar.
PlantpartswerecollectedonthebasisoftheinformationprovidedintheethnobotanicalsurveyofIndia.
Eachspecimen/plantmaterialwaslabeled,numbered,anotedwiththedateofcollection,locality,andtheir
medicinaluseswererecorded.

PreparationofPlantExtract

Extraction
TheextractionoftheCassiafistulaleaveswascarriedoutusingknownstandardprocedures.[24]Theplant
materialsweredriedinshadeandpowderedinamechanicalgrinder.Thepowder(25.0g)oftheplant
materialswereinitiallydefattedwithpetroleumether(6080C),followedby900mlofhydroalcoholby
usingaSoxhletextractorfor72hoursatatemperaturenotexceedingtheboilingpointofthesolvent.The
extractswerefilteredusingWhatmanfilterpaper(No.1)whilehot,concentratedinvacuumunderreduced
pressureusingrotaryflaskevaporator,anddriedinadesiccator.Thehydroalcoholicextractyieldsadark
greenishsolidresidueweighing5.750g(23.0%w/w).Moreyieldsofextractswerecollectedbythismethod
ofextractions.Theextractswerethenkeptinsterilebottles,underrefrigeratedconditions,untilfurtheruse.
Thedryweightoftheplantextractswasobtainedbythesolventevaporationandusedtodetermine

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concentrationinmg/ml.Theextractwaspreservedat2to4C.Thiscrudeextractsofhydroalcoholwas
usedforfurtherinvestigationforpotentialofantimicrobialproperties.

PreliminaryPhytochemicalScreening
Theextractsweresubjectedtopreliminaryphytochemicaltestingtodetectforthepresenceofdifferent
chemicalgroupsofcompounds.Airdriedandpowderedplantmaterialswerescreenedforthepresenceof
saponins,tannins,alkaloids,flavonoids,triterpenoids,steroids,glycosides,anthraquinones,coumarin,
saponins,gum,mucilage,carbohydrates,reducingsugars,starch,protein,andaminoacids,asdescribedin
literatures.[2527]

TestMicroorganismsandGrowthMedia

Thefollowingmicroorganisms
Staphylococcusaureus(MTCC96),Streptococcuspyogenes(MTCC442),Escherichiacoli(MTCC443),
Pseudomonasaeruginosa(MTCC424)andfungalstrainsAspergillusniger(MTCC282),Aspergillus
clavatus(MTCC1323),Candidaalbicans(MTCC227)werechosenbasedontheirclinicaland
pharmacologicalimportance.[28]ThebacterialstrainsobtainedfromInstituteofMicrobialTechnology,
Chandigarh,wereusedforevaluatingantimicrobialactivity.Thebacterialandfungalstockcultureswere
incubatedfor24hoursat37Connutrientagarandpotatodextroseagar(PDA)medium(Microcare
laboratory,Surat,India),respectively,followingrefrigerationstorageat4C.Thebacterialstrainswere
growninMuellerHintonagar(MHA)platesat37C(thebacteriaweregrowninthenutrientbrothat37C
andmaintainedonnutrientagarslantsat4C),whereastheyeastsandmoldsweregrowninSabouraud
dextroseagarandPDAmedia,respectively,at28C.Thestockculturesweremaintainedat4C.

AntimicrobialActivity

Determinationofzoneofinhibitionmethod
Invitroantibacterialandantifungalactivitieswereexaminedforhydroalcoholextracts.Antibacterialand
antifungalactivitiesofplantpartextractsagainstfourpathogenicbacteria(twoGrampositiveandnegative)
andthreepathogenicfungiwereinvestigatedbytheagardiskdiffusionmethod.[2931]Antimicrobial
activitytestingwascarriedoutbyusingagarcupmethod.Eachpurifiedextractsweredissolvedindimethyl
sulfoxide,sterilizedbyfiltrationusingsinteredglassfilter,andstoredat4C.Forthedeterminationofzoneof
inhibition,pureGrampositive,Gramnegative,andfungalstrainsweretakenasastandardantibioticfor
comparisonoftheresults.Alltheextractswerescreenedfortheirantibacterialandantifungalactivities
againsttheEscherichiacoli,Pseudomonasaeruginosa,Staphylococcusaureus,Streptococcuspyogenesand
thefungiCandidaalbicans,Aspergillusniger,andAspergillusclavatus.Thesetsoffivedilutions(5,25,50,
100,and250g/ml)ofCassiafistulaextractandstandarddrugswerepreparedindoubledistilledwater
usingnutrientagartubes.MuellerHintonsterileagarplateswereseededwithindicatorbacterialstrains(108
cfu)andallowedtostayat37Cfor3hours.Controlexperimentswerecarriedoutundersimilarcondition
byusingampicillin,chloramphenicol,ciprofloxacin,andnorfloxacinforantibacterialactivityandnystatin
andgriseofulvinforantifungalactivityasstandarddrugs.Thezonesofgrowthinhibitionaroundthedisks
weremeasuredafter18to24hoursofinincubationat37Cforbacteriaand48to96hoursforfungiat
28C.Thesensitivitiesofthemicroorganismspeciestotheplantextractsweredeterminedbymeasuringthe
sizesofinhibitoryzones(includingthediameterofdisk)ontheagarsurfacearoundthedisks,andvalues<8
mmwereconsideredasnotactiveagainstmicroorganisms.

RESULTSANDDISCUSSION

Results

Preliminaryphytochemicalscreening
ItwasfoundthathydroalcoholicextractsofCassiafistulaleavescontainedtannins,flavonoids,saponins,
triterpenoids,steroids,glycosides,anthraquinones,reducingsugars,carbohydrates,proteins,andamino
acids.
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Microbialactivity
TheantimicrobialactivityoftheextractsofCassiafistulawerestudiedindifferentconcentrations(5,25,50,
100,and250g/ml)againstfourpathogenicbacterialstrains,twoGrampositive(Staphylococcusaureus
MTCC96,StreptococcuspyogenesMTCC442)andtwoGramnegative(EscherichiacoliMTCC443,
PseudomonasaeruginosaMTCC424),andthreefungalstrains(AspergillusnigerMTCC282,Aspergillus
clavatusMTCC1323,CandidaalbicansMTCC227).Thesestrainshavebeenselectedforthebasisofits
applicationpurposeoffurtherformulationstudy.

Antibacterialandantifungalpotentialofextractswereassessedintermsofzoneofinhibitionofbacterial
growth.TheresultsoftheantibacterialandantifungalactivitiesarepresentedinTables14.

Theantibacterialandantifungalactivitiesoftheextractsincreasedlinearlywithincreaseinconcentrationof
extracts(g/ml).Ascomparedwithstandarddrugs,theresultsrevealedthatintheextractsforbacterial
activity,S.pyogenesandS.aureusweremoresensitiveascomparedwithE.coliandP.aeruginosa,andfor
fungalactivity,C.albicansshowsgoodresultascomparewithA.nigerandA.clavatus.Thegrowth
inhibitionzonemeasuredrangedfrom11to20mmforallthesensitivebacteria,andrangedfrom14to20
mmforfungalstrains[Figures17].

TheresultsshowthattheextractsofCassiafistulawerefoundtobemoreeffectiveagainstallthemicrobes
tested.

Discussion
Antimicrobialpropertiesofmedicinalplantsarebeingincreasinglyreportedfromdifferentpartsoftheworld.
TheWorldHealthOrganizationestimatesthatplantextractortheiractiveconstituentsareusedasfolk
medicineintraditionaltherapiesof80%oftheworld'spopulation.Inthepresentwork,theextractsobtained
fromCassiafistulashowstrongactivityagainstmostofthetestedbacterialandfungalstrains.Theresults
werecomparedwithstandardantibioticdrugs.Inthisscreeningwork,extractsofCassiafistulawerefound
tobenotinactiveagainstanyorganism,suchasGrampositive,Gramnegative,andfungalstrainswere
resistanttoalltheextractsofCassiafistula.

TheaboveresultsshowthattheactivityofhydroalcoholextractsofCassiafistulashowssignificant
antibacterialandantifungalactivities.Thisstudyalsoshowsthepresenceofdifferentphytochemicalswith
biologicalactivitythatcanbeofvaluabletherapeuticindex.Theresultofphytochemicalsinthepresent
investigationshowedthattheplantcontainsmoreorlesssamecomponentslikesaponin,triterpenoids,
steroids,glycosides,anthraquinone,flavonoids,proteins,andaminoacids.Resultsshowthatplantrichin
tanninandphenoliccompoundshavebeenshowntopossesantimicrobialactivitiesagainstanumberof
microorganisms.

CONCLUSION
Inthecurrentinvestigation,thehydroalcoholextractintheratioof8:2hasbeenselectedafterstudyofsuch
aselectedplantwithwaterextractsandmethanolextracts,hydroalcoholextractgavehigheryieldof
chemicalconstituentsexpectedforthisresearchworktheoriginalityofthisworkisthatgoodresultshave
beenfoundwithhydroalcoholratio,anditwillbehelpfultocarryoutotherdatawithMICandother
formulationstudy,becauseincomparisonofmethanolorwaterextracts,hydroalcoholismoresuitablefor
clinicalstudy.ThehydroalcoholicextractsofCassiafistulawerefoundtobeactiveonmostoftheclinically
isolatedmicroorganismandfungi,ascomparewithstandarddrugs.Thepresentstudyjustifiedtheclaimed
usesofleavesinthetraditionalsystemofmedicinetotreatvariousinfectiousdiseasecausedbythemicrobes.
However,furtherstudiesareneededtobetterevaluatethepotentialeffectivenessofthecrudeextractsasthe
antimicrobialagents.Thepresentresultswillformthebasisforselectionofplantspeciesforfurther
investigationinthepotentialdiscoveryofnewnaturalbioactivecompounds.Furtherstudieswhichaimedat
theisolationandstructureelucidationofantibacterialactiveconstituentsfromtheplanthavebeeninitiated.

ACKNOWLEDGMENT
TheauthorsofthispaperarethankfultotheDirector,I.P.G.T.andR.A.,GujaratAyurvedUniversity,
Jamnagar,Gujarat,India,fortheirinvaluablesupportandforprovidingalltheresearchfacilities.Theauthors

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arealsothankfultotheMycrocarelaboratory,Surat,Gujarat,India,forhelpingandprovidingnecessary
facilitiesforthisresearchwork.

Footnotes
SourceofSupport:Nil

ConflictofInterest:Nil.

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FiguresandTables

Table1

AntibacterialactivitiesofhydroalcoholicextractsofleavesofCassiafistulaagainstbacterialtestorganism

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Table2

Antibacterialactivityofstandarddrugsagainstbacterialtestorganism

Table3

AntibacterialactivitiesofhydroalcoholicextractsofleavesofCassiafistulaagainstfungaltestorganism

Table4

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Antifungalactivityofstandarddrugsagainstfungaltestorganism

Figure1

AntibacterialactivityagainstS.pyogenes(MTCC442)

Figure2

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AntibacterialactivityagainstS.aureus(MTCC96)

Figure3

AntibacterialactivityagainstE.coli(MTCC443)

Figure4

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AntibacterialactivityagainstP.aeruginosa(MTCC424)

Figure5

AntifungalactivityagainstA.niger(MTCC282)

Figure6

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AntifungalactivityagainstA.clavatus(MTCC1323)

Figure7

AntifungalactivityagainstC.albicans(MTCC227)CMLHydroalcoholicextractofleaves

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