: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
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217694/?report=printable 4/11
6/20/2017 AntibacterialandantifungalactivitiesfromleafextractsofCassiafistulal.:Anethnomedicinalplant
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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