Biomolecules2020,10,982;doi:10.3390/biom10070982

www.
mdpi.
com/journal/biomoleculesArticleTumoricidalandBactericidalPropertiesofZnONPsSynthesizedUsingCassiaauriculataLeafExtractKollurShivaPrasad1,*,ShashankaKPrasad2,MohammadAzamAnsari3,*,MohammadAAlzohairy4,MohammadNAlomary5,SamiAlYahya6,ChandrashekarSrinivasa7,MahadevamurthyMurali8,VeenaMalligereAnkegowda9andChandanShivamallu2,*1DepartmentofSciences,AmritaSchoolofArtsandSciences,AmritaVishwaVidyapeetham,MysuruCampus,Mysuru,Karnataka–570026,India2DepartmentofBiotechnologyandBioinformatics,SchoolofLifeSciences,JSSAcademyofHigherEducationandResearch,Mysuru,Karnataka–570015,India;shashankaprasad@jssuni.
edu.
in3DepartmentofEpidemicDiseaseResearch,InstitutesforResearchandMedicalConsultations(IRMC),ImamAbdulrahmanBinFaisalUniversity,Dammam31441,SaudiArabia4DepartmentofMedicalLaboratories,CollegeofAppliedMedicalSciences,QassimUniversity,Qassim51431,SaudiArabia;dr.
alzohairy@gmail.
com5NationalCenterforBiotechnology,LifeScienceandEnvironmentalResearchInstitute,KingAbdulazizCityforScienceandTechnology,P.
O.
Box6086,Riyadh,SaudiArabia;malomary@kacst.
edu.
sa6NationalCenterforBiotechnology,KingAbdulazizCityforScienceandTechnology,Riyadh,P.
O.
Box6086,SaudiArabia;salyahya@kacst.
edu.
sa7DepartmentofBiotechnology,DavangereUniversity,Shivagangotri,Karnataka–577007,India;chandru.
s@davangereuniversity.
ac.
in8DepartmentofStudiesinBotany,UniversityofMysore,Manasagangotri,Mysuru570006,Karnataka,India;botany.
murali@gmail.
com9DepartmentofChemistry,BangaloreInstituteofTechnology,K.
R.
Road,VVPuram,Karnataka,Bangalore560004,India;veenamdy12@gmail.
com*Correspondence:shivachemist@gmail.
com(K.
S.
P.
);maansari@iau.
edu.
sa(M.
A.
A.
);chandans@jssuni.
edu.
in(C.
S.
)Received:2June2020;Accepted:25June2020;Published:30June2020Abstract:Inthiswork,weaimedtosynthesizezincoxidenanoparticles(ZnONPs)usinganaqueousextractofCassiaauriculataleaves(CAE)atroomtemperaturewithouttheprovisionofadditionalsurfactantsorcappingagents.
Theformationofas‐obtainedZnONPswasanalyzedbyUV–visible(ultraviolet)absorptionandemissionspectroscopy,X‐rayphotoemissionspectroscopy(XPS),X‐raydiffractionanalysis(XRD),energydispersiveX‐raydiffraction(EDX),thermogravimetricanalysis/differentialthermalanalysis(TGA‐DTA),scanningelectronmicroscopy(SEM),transmissionelectronmicroscopy(TEM),high‐resolutiontransmissionelectronmicroscopy(HRTEM),andselectedareaelectrondiffraction(SAED).
TheXRDresultsreflectthewurtzitestructureofas‐preparedZnONPs,whichproduceddiffractionpatternsshowinghexagonalphases.
TheSEMimagesindicatethatthemorphologyofas‐preparedZnONPsiscomposedofhexagonalnanostructureswithanaveragediameterof20nm.
TheHR‐TEMresultshowsthattheinter‐planardistancebetweentwolatticefringesis0.
260nm,whichcoincideswiththedistancebetweentheadjacent(d‐spacing)ofthe(002)latticeplaneofZnO.
ThefluorescenceemissionspectrumofZnONPsdispersedinethanolshowsanemissionmaximumat569nm,revealingthesemiconductornatureofZnO.
As‐obtainedZnONPsenhancedthetumoricidalpropertyofCAEinMCF‐7breastcancercellswithoutsignificantinhibitionofnormalhumanbreastcells,MCF‐12A.
Furthermore,wehavestudiedtheantibacterialeffectsofZnONPs,whichshoweddirectcellsurfacecontact,resultinginthedisturbanceofbacterialcellintegrity.
Biomolecules2020,10,9822of14Keywords:zincoxidenanoparticles(ZnONPs);Cassiaauriculata;anticancerproperty;X‐rayphotoemissionspectroscopy(XRD)pattern1.
IntroductionInrecentyears,nanoscienceandnanotechnologyresearchhasgainedampleattentionfromresearchers,asitoffersinnovativesolutionsinthefieldsofmaterialsscience,electronics,andmedicine[1–3].
Thesignificanceofnanomaterialsisduetotheirsuperiorphysicochemicalandbiologicalpropertiesovertheirbulkphase.
Moreover,thesizeofthesematerials(20KeV)allowsthedetectionofallelements(withtheexceptionofHandHe)atalllocationssampledbythebeam,whichprovidesasignificantadvantagewithcomplexmicrostructures.
ThepeaksrelatedtothepresenceofZnandOcanbeclearlyseenintheEDXspectrum,andthepercentagechemicalcompositionoftheas‐obtainedmaterialisdepictedinTable1.
ThiswasalsoevidentfromFT‐IRstudies(SupplementaryFigure1).
ThechemicalstoichiometryofZnONPsreportedhereisaffirmedtobeZn:O≈1:1.
Biomolecules2020,10,9826of14Figure2.
XRDdiffractionpatternofas‐grownZnONPsobtainedusingaqueousextractofC.
auriculataleaves.
Figure3.
Energy‐dispersiveX‐raydiffractionspectrumofZnONPsunderstudy.
Table1.
TheEDXanalysisdepictingweight%andatomic%ofzincandoxygenelementspresentintheas‐obtainedZnONPs.
ElementWeight%Atomic%Zinc29.
3215.
40Oxygen19.
6633.
633.
3.
ScanningElectronMicroscopy(SEM)AnalysisTheSEMmicrographofas‐obtainedZnONPsisdisplayedinFigure4.
ThesizeandshapeofZnONPsweredeterminedfromtheSEMimage.
TheSEMresultsindicatethattheas‐preparedZnONPsarecomposedofhexagonalnanostructureswithanaveragediameterof20nm.
Biomolecules2020,10,9827of14Figure4.
TheSEMimageofas‐obtainedZnONPsusingCassiaauriculataleafextract.
3.
4.
X‐RayPhotoemissionStroscopyAnalysisTheXPSspectrumofas‐obtainedZnONPswasanalyzedtoinvestigatethechemicalstatesofZn.
AsdisplayedinFigure5a,weobservedtheexperimentalZn2p3/2andZn2p1/2photoelectronpeaksofZnONPspreparedatroomtemperature.
Thepeaksobservedat1029.
6eVand1054.
3eVcorrespondtoZn2p3/2andZn2p1/2species,respectively,whichcoincideswiththereportedvalues[28].
Furthermore,spectraldeconvolutionresultedinanasymmetricpeakobservedat528.
9and230.
2eVintheO1sspectrumofas‐obtainedZnONPs(Figure5b),whichisusuallyrelatedtotheO2‐bondingwithmetals.
Thus,inthiscase,itisaZn‐Ocrystallinelattice(OL)[29,30].
Figure5.
TheX‐rayphotoemissionspectrashow(a)Zn2pand(b)O1sofas‐obtainedZnONPs.
3.
5.
ThermogravimetricAnalysisThethermogravimetricanalysisanddifferentialthermalanalysisofZnONPssynthesizedusingtheaqueousextractofC.
auriculataleaveswereperformedinthetemperaturerangebetween25and800°C.
ThecurvesofTGA(greencurve)andDTA(bluecurve)areshowninFigure6.
FromtheTGAcurve,itcanbeclearlyseenthattheweightlossstartsat~150°C,indicatingtheevaporationofwater.
Asignificantlossisobservedbetween285and460°C,whichisduetothedecompositionoforganicgroupspresentinthesampleduringgreensynthesis.
Furtherincreaseinthetemperatureleadstonoadditionaldecompositioninthesample,whichindicatesthecompleteremovaloforganicsubstances,leavingbehindZnO.
ThemajorexothermicpeakobservedintheDTAcurvebetween285and460°Crevealsthemaximumat395°C,whichrepresentstheburn‐outoforganicsubstancepresentinthesample.
Additionally,thereisnosignificantexothermicorendothermicpeakintheDTApattern.
Biomolecules2020,10,9828of14Figure6.
TGA/DTAgraphshowingthedecompositionofas‐obtainedZnONPs.
3.
6.
TransmissionElectronMicroscopyInvestigationsInordertoconfirmthesizeandmorphologyofas‐obtainedZnONPs,TEManalysiswasperformed.
AsshowninFigure7a,theas‐obtainedZnONPshaveahexagonalshapewithparticlesizesbetween18and20nm.
Furthermore,thecrystallinityobservedintheXRDspectrumwascomplementedbyhigh‐resolutionTEM(HRTEM)studies.
AsshowninFigure7b,theobserveddiffractionlatticefringesinZnONPsshowd‐spacingwithaninter‐planardistanceof0.
260nmbetweentwofringes,whichcorrespondstod‐spacingofthe(002)crystalplaneofZnO[20].
Moreover,ithasbeenreportedthatadecreaseintheparticlesizeincreasesthefunctionalityofantimicrobialandanticanceragentsduetothelargersurface‐to‐volumeratio[13].
Figure7.
(a)TEMand(b)HR‐TEMimageswithSAED(inset)ofas‐obtainedZnONPs.
3.
7.
BactericidalActivityThediskdiffusionassaywasperformedtoanalyzethebactericidalactivityoftheas‐obtainedZnONPsagainstthetestbacterialspeciesunderstudy.
TheresultsfromthisstudyrevealthattheantibacterialactivityexhibitedbytheZnONPspreventedthegrowthofthesebacteriaatdifferentconcentrations,whichcanbevisualizedintheformofaclearzoneofinhibition.
ThebactericidalactivityoftheZnONPswasthehighestagainstKlebsiellapneumoniaincomparisonwiththestandarddrug,ampicillin.
AmoderatezoneofinhibitionwasdisplayedforE.
coli.
Significantactivitywasobservedbycomparingthestandardagainsttheplantpathogenselectedforthestudyviz.
,RalstoniaBiomolecules2020,10,9829of14solanacearumandXanthomonasvesicatoria(Table2andFigure8).
Furthermore,wecomparedourresultswithaliteraturereportonchemicallysynthesizedZnONPstoaccountforthesignificanceofas‐obtainedZnONPsusinganaqueousextractofC.
auriculataleaves.
Theresultsdemonstratethattheas‐obtainedZnONPsshowedagreaterantibacterialpotencyascomparedtothecommerciallyavailableZnONPs[31].
Table2.
ThewelldiffusionassayatdifferentZnONPconcentrationsafter24hincubationat37°C.
Thepositiveandnegativecontrolswereampicillin(0.
02mg/mL)andMilli‐Qwater,respectively.
TestOrganismZnONPs(mg/mL)Positivecontrol0.
51.
02.
0(0.
02mg/mL)Escherichiacoli12.
03±0.
1012.
06±0.
0518.
00±0.
3021.
60±0.
37Klebsiellapneumonia17.
00±0.
4016.
23±0.
8727.
10±0.
4728.
00±0.
45Ralstoniasolanacearum12.
06±0.
1513.
00±0.
9815.
06±0.
0518.
40±0.
15Xanthomonasvesicatoria10.
00±0.
2011.
06±0.
1012.
03±0.
1518.
00±0.
30Figure8.
ZoneofinhibitiongraphbyZnONPsagainstselectedbacterialstrains.
3.
8.
StudyofGrowthKineticsagainstZnONPsThegrowthofalltestorganismswasanalyzedinthepresenceandabsenceofZnONPs,andampicillinwasusedasastandarddrugagainstallofthepathogens.
AsignificantdeclineovertimewasobservedinthegrowthofallbacterialculturestreatedwithZnONPscomparedtothatoftheuntreatedone.
ThisstudysuggeststhattheZnONPshaveactivityagainstthegrowthofthetestorganisms.
Thegrowthcurve(Figure9)ofKlebsiellapneumoniadisplayssimilarinhibitiontothatresultingfromampicillin.
Thebacterialgrowthoftheothertestorganismswasmodestlyaffected(SupplementaryFigureS2).
Biomolecules2020,10,98210of14Figure9.
AcomparativedisplayofgrowthcurvesoftestbacteriaintheabsenceandpresenceofsynthesizedZnONPs.
Experimentswereperformedintriplicate.
3.
9.
ZnONPsSensitizedtheCassiaAuriculataLeavesCytotoxicityinMCF‐7CellsWhileallofthetreatmentgroups,involving10,20,40,80,160,and320μg/mLconcentrationsofCAEandZnONPs,showeddose‐dependentanti‐breastcanceractivity,thecytotoxicityofCAEwasinsignificantatlowerconcentrations,andnearly50%viablecellsremainedevenattheveryhighconcentrationof320μg/mL.
However,upondelivery,theZnONPscausedadrasticshiftinthetumoricidalpotencyoftheplantextract.
TheIC50oftheZnONPswasfoundtobeatleast8‐foldlowercomparedtotheindependenttreatmentwithCAE.
AsreportedbyPrasannaandcolleagues,theC.
auriculataplantextractisindeedcytotoxictoMCF‐7cellsonlyathigherdoses[18,19](Figure10).
However,thiscanbecircumventedwiththeaidofZnONPstoenhancetheanti‐tumorigenicpotentialofCAEatlowdoses.
Nanoparticle‐aideddeliveryofphyto‐compoundshasbeenfrequentlyreportedtoreducetheirIC50valueininvitroaswellasinvivomodels[25],therebysuggestingthattheZnOnanoparticle‐guideddeliveryofCAEimprovesitsanti‐tumorigenicactivity.
0501001502002503003504004505000.
250.
500.
751.
001.
251.
501.
75Time(inmin)Absorbanceat605nmComparativeGrowthCurveK.
pneumonia(ZnONP)K.
pneumonia(Amp)K.
pneumonia(Control)R.
solanacearum(ZnONP)R.
solanacearum(Amp)R.
solanacearum(Control)X.
vesicatoria(ZnONP)X.
vesicatoria(Amp)X.
vesicatoria(Control)E.
coli(ZnONP)E.
coli(Amp)E.
coli(Control)Biomolecules2020,10,98211of14Figure10.
CytotoxicityofCAEandZnONPsonbreastcancerMCF‐7cells.
Resultsarereportedasmean±SEMforn=3,andap‐valueof<0.
05wasconsideredtobesignificant;*p=<0.
033,**p=<0.
002,***p=<0.
001,ns=notsignificant.
3.
10.
NeitherCassiaAuriculataLeavesnorZnONPsShowedSignificantToxicityonMCF‐12ACellsAlthoughas‐obtainedZnONPsenhancedanti‐breastcanceractivity,nosignificantgrowthinhibitionwasobservedinasimilartreatmentinnormalbreastcells,MCF‐12A.
Inthisfirst‐of‐its‐kindstudyonthecytotoxicityofC.
auriculatainnormalhumancells,weidentifiedthattheplantextracthadnonoteworthyeffectonthegrowthofMCF‐12Acells(Figure11).
Surprisingly,theCAEextractsignificantlyattenuatedthenormalcellcytotoxicityofZnONPs,confirmingthechemoprotectivepotentialofCAE[26].
Biomolecules2020,10,98212of14Figure11.
CytotoxicityofCAEandZnONPsonnormalhumanbreastMCF‐12Acells.
Resultsarereportedasmean±SEMforn=3,andap‐valueof<0.
05wasconsideredtobesignificant;*p=<0.
033,**p=<0.
002,***p=<0.
001,ns=notsignificant.
4.
ConclusionsInthepresentstudy,weobtainedzincoxidenanoparticlesbyaconvenientgreenapproachusingtheaqueousextractofC.
auriculataleavesasreducingandcappingagentsbycontinuousstirringfor4hatroomtemperature.
TheZnONPssynthesizedusingCAEimproveditstumoricidalpotentialinbreastcancerMCF‐7cellswhilenotsignificantlyaffectingnormalhumanbreastMCF‐12Acellgrowth.
Therefore,thisimpliesthattheas‐obtainedZnONPsarepotentialtherapeuticcandidatesforbreastcancer.
However,evaluationofinvivotumorreductionpotentialandmechanisticelucidationoftheobservedanti‐tumorigeniceffectisdeemednecessaryfortheZnONPs.
Furthermore,theZnONPsweretestedagainstbothplantandanimalpathogens,andthepotentialeffectivenessofthenanoparticlesresultedininhibitionofalltheselectedtestorganisms,ascomparedwiththestandarddrug.
Thus,thepresentlyreportedsyntheticrouteandmaterialcouldfindsignificantimportanceinpharmaceuticalapplications.
SupplementaryMaterials:Thefollowingareavailableonlineatwww.
mdpi.
com/2218‐273X/10/7/982/s1,FigureS1:FT‐IRspectrumofas‐obtainedZnONPsusingCEAandFigureS2:ComparativedisplayofgrowthcurvesoftestbacteriainabsenceandpresenceofsynthesizedZnONPs.
Experimentswereperformedintriplicate.
AuthorContributions:Conceptualization,MethodologyandInvestigation:K.
S.
P.
,S.
K.
P.
,C.
S.
;SoftwareandValidation:M.
A.
A.
M.
M.
;FormalanalysisandDatacuration:C.
S.
,V.
M.
A.
;Resources:C.
S.
;Supervision:K.
S.
P.
;Writing‐originaldraftpreparation:KSPandSKP;Writing‐reviewandediting:C.
S.
,C.
S.
,V.
M.
A.
;Fundingacquisition:M.
A.
A.
,M.
A.
A.
,M.
N.
A.
,S.
A.
Y.
,M.
M.
Allauthorshavereadandagreedtothepublishedversionofthemanuscript.
Biomolecules2020,10,98213of14Acknowledgments:TheauthorsthanktheDirector,IOE,UniversityofMysore,Mysuruforprovidinganalyticalfacilities.
KSPgratefullythanktheDirector,AmritaVishwaVidyapeetham,Mysurucampus,Mysuruforinfrastructuralfacilities.
CSandSKPacknowledgethesupportandinfrastructureprovidedbytheJSSAcademyofHigherEducationandResearch(JSSAHER),Mysuru,India.
TheauthorMMwouldliketoacknowledgetheUniversityGrantsCommission(UGC),NewDelhi,India,forprovidingthefinancialsupportunderUGCPost‐DoctoralFellowshipforSC/STCandidates(No.
F/PDFSS‐2015‐17‐KAR‐11846).
WearealsogratefultotheInstituteforResearchandMedicalConsultations(IRMC),ImamAbdulrahmanBinFaisalUniversity,Damamm,SaudiArabiaforprovidinginstrumentationfacilities.
ConflictsofInterest:Nopotentialconflictofinterestbetweenauthorsinpublishingthiswork.
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