rangehttp

ORIGINALARTICLEOpenAccessSize-controlledgreensynthesisofsilvernanoparticlesmediatedbygumghatti(Anogeissuslatifolia)anditsbiologicalactivityArunaJyothiKora1,SashidharRaoBeedu2*andArunachalamJayaraman1AbstractBackground:Gumghattiisaproteinaceousedible,exudatetreegumofIndiaandisalsousedintraditionalmedicine.
Afacileandecofriendlygreenmethodhasbeendevelopedforthesynthesisofsilvernanoparticlesfromsilvernitrateusinggumghatti(Anogeissuslatifolia)asareducingandstabilizingagent.
Theinfluenceofconcentrationofgumandreactiontimeonthesynthesisofnanoparticleswasstudied.
UV–visiblespectroscopy,transmissionelectronmicroscopyandX-raydiffractionanalyticaltechniqueswereusedtocharacterizethesynthesizednanoparticles.
Results:Byoptimizingthereactionconditions,wecouldachievenearlymonodispersedandsizecontrolledsphericalnanoparticlesofaround5.
7±0.
2nm.
ApossiblemechanisminvolvedinthereductionandstabilizationofnanoparticleshasbeeninvestigatedusingFouriertransforminfraredspectroscopyandRamanspectroscopy.
Conclusions:ThesynthesizedsilvernanoparticleshadsignificantantibacterialactiononboththeGramclassesofbacteria.
Asthesilvernanoparticlesareencapsulatedwithfunctionalgrouprichgum,theycanbeeasilyintegratedforvariousbiologicalapplications.
Keywords:Antibacterial,Autoclaving,Gumghatti,Silvernanoparticles,Surface-EnhancedRamanScattering(SERS)BackgroundAsurveyofearlierliteraturesuggeststhatvariousnat-uralpolymerssuchasstarch[1],chitosan[2],andtannicacid[3]havebeenreportedasreducingagentsforthesynthesisofsilverandgoldnanoparticles.
Ithasbeendemonstratedthattheplant-basedexudategumssuchasgumAcacia[4]andgumkondagogu[5]canbeutilizedasreducingandstabilizingagentsforthesilvernanopar-ticlebiosynthesis.
Gumgellan,amicrobialheteropolysac-charide,wasemployedforsimilarpurposeinthecaseofgoldnanoparticles[6].
Gumghattiisanaturallyoccur-ringwatersoluble,complexpolysaccharidederivedasanexudatefromthebarkofAnogeissuslatifolia(Combreta-ceaefamily),anativetreeoftheIndiansub-continent.
Thenamegumghattihasoriginatedfromitstransporta-tionthroughmountainpassesorghats.
ThisnativeIn-diangumiscollectedfromtheforestsbythetribalsandmarketedthroughgovernmentorganizationssuchasGirijanCo-operativeCorporationLtd.
,Visakhapatnam,India.
Theworldproductionofgumghattiisabout1,000–1,500MT/year[7,8].
Thisbiopolymerisanarabi-nogalactantypeofnaturalgumanditsmorphological,structural,physico-chemical,compositional,solution,thermal,rheological,andemulsifyingpropertieshavebeenwelldocumentedandstudied[9-17].
Thisbiopoly-merisahigh-arabinose,proteinrich,acidicheteropoly-saccharide,occurringinnatureasmixedcalcium,magnesium,potassium,andsodiumsalt[12-14,16].
Theprimarystructureofthisgumiscomposedofsugarssuchas,L-arabinose,D-galactose,D-mannose,D-xylose,andD-glucuronicacidinamolarratioof48:29:10:5:10andhttp://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17http://www.
orgmedchemlett.
com/content/2/1/17sidechain.
Ithasamolecularweightof8.
94*107g/mol[12,13,15,16].
ThegumghattiwithaCASnumber9000-28-6isrecog-nizedas"generallyrecognizedassafe"(GRAS)andapprovedasafoodingredient(Code184.
1333)bytheFoodandDrugAdministration,USA,underthefunctionofemulsifierandemulsifiersalt.
ItsuseinfoodisalsoapprovedinJapan,China,SouthKorea,Singapore,Russia,Australia,SouthAfrica,Iran,SaudiArabia,LatinAmerica,andothercountries.
But,itisnotapprovedasafoodaddi-tiveinEuropeanUnionandnotbeenaccordedaEuropeanfoodsafetyEnumber.
ItisconsideredasafoodgradeadditivesoffoodbytheBureauofIndianStandards,IndiaunderIndianStandardIS7239:1974[13,15,16].
InIndia,theapplicationofthishydrocolloidintraditionalmedicineandfoodpreparationsiswellknownforcenturies.
Thegumisfedtothelactatingmothersintheformofladdutoenhancethenutrientsinmilkaswellastopreventthepost-deliverybackache[18].
Thegumladduisalsoeatenasaheatingagentduringwinterseason[18,19].
Thegumghattiiscomprisedofaround80%solubledietaryfiberandactsaprebioticbysupplyingthematrixrequiredtosustainthebacterialfloraofthehumancolon.
Thishydro-colloidisresistanttogastrointestinalenzymesandknowntobedegradedenzymaticallyonlybythespecificmicro-floraofthecolonsuchasBifidobacteriumlongum,therebyaidinginbifidusfermentation[20-22].
Thisgumisalsogivenforthetreatmentofdiarrheaanddiabetes[23].
Earl-ierstudiesongumghattifedwhiteleghorncockerelsandalbinoratshaveestablishedthehypolipidemicactivityofgumghatti[24,25].
Recentstudieshaveestablishedthatgumghattihasapotentialapplicationasareleasemodifierforcontrolleddrugdelivery[26].
Gumghattihaslongbeenusedinnon-foodapplications,suchas,calicoprinting,explosives,varnishes,carpolishes,ceramics,cosmetics;andinpharmaceutical,textile,paper,petroleum,andmin-ingindustries.
Also,thisbiopolymeraidsinvariousphoto-electricdeterminations[7,8,13,16,23].
Theattractivefeaturesofgumghattipromptedustousethisbiopolymerforthesynthesisandstabilizationofsilvernanoparticlesduetoits(i)ediblenatureandGRAS[13];(ii)naturalavailabilityandlowcost[23];(iii)inter-mediateviscositybetweengumarabicandgumkaraya[14,15];(iv)greaterstabilitytopHacidification,electro-lyteaddition,andhigh-pressuretreatment[15,17];(v)higheremulsificationabilityandsuperioremulsionstor-agestabilityatlowerconcentrations[15],and(vi)excep-tionalinterfacialcharacteristicswithfasterkinetics[17].
Thegreensynthesisofinherentlysafersilvernanoparti-clesdependsontheadoptionofthebasicrequirementsofgreenchemistry;thesolventmedium,thebenignre-ducingagent,andthenon-hazardousstabilizingagent[1,27].
Inthiscontext,wehaveexploredanddevelopedafacileandgreensyntheticroutefortheproductionofsilvernanoparticlesusingaproteinaceous,edible,renew-ablenaturalplantpolymer,gumghattiasboththeredu-cingandstabilizingagents.
Beinganaturalpolymer,gumghattiisamenableforbiodegradation.
Thesynthesiswascarriedoutinaqueousmediumbyautoclaving,withouttheadditionofanyexternalchemicalreducingagent.
Inthisstudy,autoclavingwasadoptedasasyntheticroutetoproducesterilesilvernanoparticlesthatarecompletelyfreefrombacteria,viruses,andspores,whichwouldsuitbiologicalapplications.
Thefocusofthisstudywason(i)thesynthesis,(ii)characterization,and(iii)cappingandstabilizationofsilvernanoparticles.
Inaddition,wehavealsodemonstratedtheantibacterialactivityofthepreparednanoparticlesonGram-positiveandGram-negativebacteriaforfindingoutthepotentialofthegen-eratednanoparticlesforvariousenvironmentalandbio-medicalapplications.
MethodsCharacterizationofsynthesizedsilvernanoparticlesInordertostudytheformationofsilvernanoparticles,theUV–Visibleabsorptionspectraofthepreparedcol-loidalsolutionswererecordedusinganElicoSL196spectrophotometer(Hyderabad,India),from250to800nm,againstautoclavedgumblank.
Theabsorptionspectraofgumbeforeandafterautoclavingwerealsorecordedagainstultrapurewaterblank.
ThesizeandshapeofthenanoparticleswereobtainedwithHitachiH7500(Tokyo,Japan)andJEOL3010(Tokyo,Japan)transmissionelectronmicroscopes(TEM),operatingat80and200kV,respectively.
Sampleswerepreparedbydepositingadropofcolloidalsolutiononacarbon-coatedcoppergridanddryingatroomtemperature.
TheX-raydiffraction(XRD)analysiswasconductedwithaRigaku,UltimaIVdiffractometer(Tokyo,Japan)usingmonochromaticCuKαradiation(λ=1.
5406)runningat40kVand30mA.
Theintensitydataforthenanopar-ticlesolutiondepositedonaglassslidewerecollectedovera2θrangeof35–85°withascanrateof1°/min.
ThenanoparticleswererecoveredfromthesynthesizedsolutionsbycentrifugationandmadeintopowdersusingaFTSSystems,Dura-DryTMMPfreezedryer(NewYork,USA).
TheIRspectraofthelyophilizedsampleswererecordedusingaBrukerOptics,TENSOR27FT-IRspectrometer(Ettlingen,Germany);overaspectralrangeof400–4000cm–1.
TheRamanspectrumofthesynthe-sizednanoparticleswasrecordedatroomtemperatureusingthe532-nmlinefromaSUWTECH,G-SLMdiodelaser(Shanghai,China).
ThescatteredlightwascollectedanddetectedusingaCCD-basedmonochromator,cover-ingaspectralrangeof150–1700cm–1.
Thesamplesolu-tionwastakeninastandard1cm*1cmcuvetteandplacedinthepathofthelaserbeam.
Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page2of10http://www.
orgmedchemlett.
com/content/2/1/17ResultsanddiscussionSynthesisofsilvernanoparticlesThepresentexperimentalinvestigationreportsthegreensynthesisofsilvernanoparticlesusinggumghattibyautoclaving.
Thismethodutilizesaproteinaceous,edible,renewable,andwatersolublebiopolymer;gumghattiwhichfunctionsasbothreducingandstabilizingagentsduringsynthesis.
Byvirtueofbeinganaturalpolymer,thisgumisalsoamenableforbiodegradation.
Theprocessofautoclavingmakesthesilvernanoparticlesin-trinsicallysafeandsterile,inenvironmentallybenignsolventwater.
Moreover,generationofgum–silvernano-particlesbyautoclavingisaprerequisiteforbiologicalapplications.
Thus,theadoptedmethodismeetingtherequirementsofgreenchemistryprinciples.
ProposedmechanismofreductionDuringautoclavingat121°Cundertheinfluenceoftemperatureandpressure(103kPa),thisbiopolymerexpandsandbecomesmoreaccessibleforthesilverionstointeractwiththeavailablefunctionalgroupsonthegumasobservedearlierforstarch[1].
Thegumhasbeencategorizedunderarabinogalactanduetotheabundanceofarabinoseandgalactose.
Thisacidicheteropolysac-charideisknowntoberichinuronicacidcontentandshowsapHof4.
5–5.
5[8,14-17].
Thepresenceofhy-droxylandcarboxylicgroupsonthisbiopolymer[28]facilitatesthecomplexationofsilverions.
Subsequently,thesesilverionsoxidizethehydroxylgroupstocarbonylgroups,duringwhichthesilverionsarereducedtoelem-entalsilver.
Inadditiontothisinherentoxidation,thedissolvedairmayalsocausesoxidationoftheexistinghydroxylgroupstocarbonylgroupssuchasaldehydesandcarboxylates.
Inturn,thesepowerfulreducingalde-hydegroupsalongwiththeotherexistingcarbonylgroupsreducemoreandmoreofsilverionstoelementalsilver.
Further,thesenanoparticlesareprobablycappedandstabilizedbythepolysaccharidesalongwiththepro-teinspresentinthegum.
Asthesecarbohydratepoly-mersareverycomplex,itismostlikelythatmorethanonemechanismisinvolvedinthecomplexationandsub-sequentreductionofsilverionsbygumghattiduringautoclaving.
Silverioncomplexationbyhydroxylgroupsanditssubsequentreductionbyaldehydegroupsarereportedforstarch,inwhichsilvernanoparticleswereproducedbyautoclaving[1].
Silvernanoparticlespro-ducedusinggumAcacia,carboxylategroupsinvolvingcomplexationofsilverionsanditssubsequentreductionbyhydroxylgroupswerereported[4].
Thereductionofsilverionsbythisgumevenatroomtemperaturewasobserved.
But,theformednanoparticleswerenotstableandaggregatedduetolackofstabilizationofthesynthesizednanoparticles.
Itwasnoticedthattheautoclavingat121°Cand103kPaofpressure,increasedtheextentofsynthesisandstabilizationofthenanoparticles.
Itisknownthatele-vatedtemperatureandpressureacceleratethesynthesisofnanoparticles[1].
Besides,thisprocesscomplexlyeliminatesthemicrobialcontaminationpossiblyacquiredduringgumsecretion,collection,handling,andtransportation.
CharacterizationofsynthesizedsilvernanoparticlesUV–VisiblespectroscopyTheUV–Visabsorptionspectroscopyisoneofthemostwidelyusedsimpleandsensitivetechniquesfortheob-servationofnanoparticlesynthesis.
Inordertomonitortheformationofsilvernanoparticles,theabsorptionspectraofsynthesizedsilvernanoparticleswererecordedagainstrespectiveautoclavedgumblanks.
Figure1isin-dicating(a)gumtearsofgrade1quality,(b)gumpowdersievedto38μmparticlesize,and(c)centrifugedgumso-lutionof0.
5%.
Tooptimizethenanoparticlesynthesis,theinfluenceofparameterssuchasconcentrationofgumandreactiontimewasstudied.
Theroleofgumconcentrationonthesynthesiswasstudiedbyautoclav-ingthesegumsolutions(0.
1–0.
5%)containing1mMofsilvernitratefor30min.
Figure2ashowstheUV–Visspectraoftheproducedsilvernanoparticleswithdiffer-entconcentrationsofgum(0.
1–0.
5%)at1mMAgNO3and30minofautoclaving.
Afterautoclavingthesilvernitratecontaininggumsolutions,theappearanceofyel-lowcolorinthereactionmixtureswasobserved.
Thisisaclearindicationfortheformationofsilvernanoparti-clesbythegum.
Itrevealsthattheefficiencyofnanopar-ticlesynthesisincreaseswithincreasingconcentrationofgum.
Thesynthesiswasalsoevaluatedbyvaryingthere-actiontime(10–60min)andreductionwasstudiedwith0.
5%gumat1mMAgNO3(Figure2b).
Itwasnoticedthatthereductioncapacityofthegumincreasedwithre-actiontime.
Astheautoclavingtimeincreases,possiblymoreandmoreofhydroxylgroupsarebeingconvertedtocarbonylgroupsbyairoxidation,whichinturnreducethesilverions.
IntheUV-Visspectraasinglestrongpeakwithamaximumaround412nmwasobserved,whichcorrespondstothetypicalsurfaceplasmonreson-ance(SPR)ofconductingelectronsfromthesurfaceofsilvernanoparticles.
TheSPRabsorptionofmetalnano-particleslikegoldandsilverisverysensitivetothechangesofthesizeandshapeofthenanoparticlesformed[29].
TransmissionelectronmicroscopyFigure3showstheTEMimagesofthesilvernanoparticlessynthesizedwith0.
5%gumand1mMAgNO3autoclavedfor30min.
Thesenanoparticlesarespherical,polydisperse,aggregated,andtheaverageparticlesizeobtainedfromthesemicrographswasabout31.
6±21.
7nm(Figure3c).
TheKoraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page3of10http://www.
orgmedchemlett.
com/content/2/1/17influenceofgumconcentrationonthemorphologyofthenanoparticleswasinvestigatedwith0.
1%gumand1mMAgNO3,autoclavedfor30min(Figure4).
Thesenanoparti-clesweresphericalinshapeandnearlyisotropicinnature.
Theaverageparticlesizeobtainedfromthecorrespondingdiameterdistributionwasabout5.
7±0.
2nm(Figure4e).
Theeffectofautoclavingtimeontheshapeandsizeofthenanoparticleswasconfirmedwith0.
1%gumsolution,auto-clavedfor60minat1mMAgNO3(Figure5).
TheTEMobservationsofthissampleindicatetheshapeanisotropyandthenanoparticlesdisplayarichvarietyofshapesinvaryingsizes.
Inadditiontonanospheres,hexagonal,andpolygonalnanoprisms,ellipsoidalandunevenshapednano-particleswereobserved.
Thesenanoparticlesarepolydis-perse,aggregated,andtheaverageparticlesizeobtainedfromthesemicrographswasabout27.
2±11.
5nm,for60minofreactiontime(Figure5e).
Theselected-areaelec-trondiffraction(SAED)patternsdepictedinFigures4dand5dexhibitconcentricringswithintermittentbrightdots,in-dicatingthatthesenanoparticlesarehighlycrystallineinna-ture.
Theseringscanbeattributedtothediffractionfromthe(111),(200),(220),and(311)planesofface-centeredFigure2TheUV–Visabsorptionspectraofsilvernanoparticlessynthesized:(a)byautoclavingdifferentconcentrationsofgumghattisolutionsat1mMAgNO3concentrationfor30min;insetplotofAmaxversusgumconcentrationand(b)with0.
5%(w/v)gumghattisolutionsat1mMAgNO3concentrationfordifferentdurationsofautoclaving;insetplotofAmaxversusautoclavingtime.
Figure1Adigitalphotographshowing(a)Gumtearsofgrade1quality,(b)gumpowdersievedto38μmparticlesize,and(c)centrifugedgumsolutionof0.
5%(w/v).
Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page4of10http://www.
orgmedchemlett.
com/content/2/1/17cubic(fcc)silver.
Thecrystallinityofthesynthesizednano-particleswasalsosupportedfromtheobservedclearlatticefringesinhigh-resolutionimages(Figures4cand5c).
Inter-estinglyat0.
1%gumand1mMofAgNO3concentrationwith30minofautoclaving,nearly70%ofthenanoparticlesformedwereinthesizeof5.
7nm(Figure4e).
Whentheconcentrationofgumwasdecreasedfrom0.
5to0.
1%,theaverageparticlesizeofthesilvernanoparticlesformedFigure3TEMimagesofsilvernanoparticlessynthesizedwith0.
5%(w/v)gumghattiand1mMAgNO3,autoclavedfor30min,at(a)125nm,(b)143nmscale,and(c)histogramshowingtheparticlesizedistribution.
Figure4TEMimagesofsilvernanoparticlessynthesizedwith0.
1%(w/v)gumghattiand1mMAgNO3,autoclavedfor30min,at(a)50nm,(b)20nm,and(c)5nmscale.
(d)CorrespondingSAEDpatternand(e)histogramshowingtheparticlesizedistribution.
Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page5of10http://www.
orgmedchemlett.
com/content/2/1/17decreased.
Thiswasalsoconfirmedinapreviousstudyonsizecontrollablesynthesisofsilvernanoparticleswithtannicacid,inwhichtheconcentrationofthepolyphenoldecreasedfrom23.
5to1.
8μM[3].
ThedecreaseinpolydispersitywithdecreaseintheconcentrationofgumwasalsoevidentfromtheTEMimages(Figures3and4).
Itisworthnotingthattheshapeoftheparticleschangedfromspherestoaniso-tropicnanostructures,whenthereactiontimewasincreasedto60minat0.
1%ofgumconcentration(Figures4and5).
Thisismostlikelyduetothecontinualgrowthofnanoparti-clesduringlongerperiodofautoclaving.
Thisstudyindicatesthattheparticlesizeofthesilvernanoparticlescanbecon-trolledbyvaryingtheconcentrationofgumandreactiontime.
Asaresult,nanoparticleswithnearmonodispersitywereobtainedwith0.
1%gumand30minofreactiontimeat1mMofsilvernitrateconcentration.
X-raydiffractionTheXRDtechniquewasusedtodetermineandconfirmthecrystalstructureofsilvernanoparticles.
TheXRDpatternofthesilvernanoparticlesisshowninFigure6.
Therewerefivewell-definedcharacteristicdiffractionpeaksat38.
3°,44.
6°,64.
8°,77.
6°,and81.
9°,respectively,correspondingto(111),(200),(220),(311),and(222)planesoffcccrystalstructureofmetallicsilver.
Theinterplanarspacing(dhkl)values(2.
348,2.
030,1.
437,1.
229,and1.
175)calculatedfromtheXRDspectrumofsilvernanoparticleswasinagreementwiththestand-ardsilvervalues.
Thus,theXRDpatternfurthercorrobo-ratesthehighlycrystallinenatureofnanoparticlesobservedfromSAEDpatternsandhigh-resolutionTEMimages(Figures4and5).
Thelatticeconstantcalculatedfromthispatternwas4.
061,avaluewhichisinagree-mentwiththevaluereportedinliteratureforsilver(JCPDSPDFcard04–0783).
Also,thebroadeningofthediffractionpeakswasobservedowingtotheeffectofnano-sizedparticles.
Asthenanoparticlesarecappedbythemoietiesofgum,thebackgroundobservedwashigh.
Fouriertransforminfraredspectroscopy(FTIR)TheFTIRspectraofthegumandnanoparticleswererecordedinordertoidentifythefunctionalgroupsofguminvolvedinthereductionandcapping/stabilizationofthesynthesizednanoparticles.
Figure7showstheFTIRspectraofthelyophilizedgumandsilvernanopar-ticles.
ThemajorabsorbancebandspresentintheFigure5TEMimagesofsilvernanoparticlessynthesizedwith0.
1%(w/v)gumghattiand1mMAgNO3,autoclavedfor60min,at(a)50nm,(b)20nm,and(c)5nmscale.
(d)CorrespondingSAEDpatternand(e)histogramshowingtheparticlesizedistribution.
Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page6of10http://www.
orgmedchemlett.
com/content/2/1/17spectrumofgumghattiwereat3425,2928,2368,2341,2122,1635,1406,1311,1234,1068,and1028cm1.
Thebroadbandobservedat3425cm1couldbeassignedtostretchingvibrationsofO–Hgroupsingumghatti.
Thebandsat2928,1406,and1234cm1correspondtoasym-metricstretching,scissoring;andtwistingandrockingvibrationsofmethylenegroups,respectively.
Thebroadbandat2122cm1onlyappearedinthespectrumofgumcouldbeassignedtovariouscarbonylspecies.
Thestrongerbandfoundat1635cm1couldbeassignedtocharacteristicasymmetricalstretchofcarboxylategroup.
Thesymmetricalstretchofcarboxylategroupcanbeattributedtothebandpresentat1311cm1.
Thepeaksat1068and1028cm1wereduetotheC–Ostretchingvibrationofetherandalcoholicgroups,respectively[28].
While,thespectrumoflyophilizednanoparticlesshowedcharacteristicabsorbancebandsat3431,2964,2345,2304,1728,1632,1385,1260,and1024cm1.
IntheIRspectrumofnanoparticles,ashiftintheabsorbancepeakswasobservedfrom3425to3431cm1and1635to1632cm1,and1311to1385cm1,suggestingthebindingofsilverionswithhydroxylandcarboxylategroups,re-spectively.
Itispertinenttonotethatnanoparticlesshowsanewbandat1728cm1correspondingtocarbonylstretchingvibrationsinaldehydes,ketones,andcarboxylicacids[2].
Further,theoccurrenceofthepeakat1728cm1anddisappearanceofthepeakat2122cm1confirmthatthereductionofthesilverionsiscoupledtotheoxi-dationofthehydroxylandcarbonylgroups,indicativeofmoreextensivelyoxidizednatureofthegum.
Basedonthebandshiftinthehydroxylandcarbonylgroupsandthelossofexistingcarbonylsandappearanceofanewcarbonylpeak,itcanbeinferredthatbothhydroxylandcarbonylgroupsofgumareinvolvedinthesynthesisofsilvernanoparticles.
Thevariationsintheshapeandpeakpositionofthehydroxylandcarboxylategroupshavebeenreported,wheresilvernanoparticlesweresynthesizedusinganotherpolysaccharide,gumAcacia[4].
RamanspectroscopyInordertofindoutthepossiblefunctionalgroupsofcap-pingagentsassociatedinthestabilizationofsilvernano-particles,Ramanspectrumofthenanoparticleswasrecorded.
Figure8givestheselectiveenhancementofRamanbandsoftheorganiccappingagentsboundtothenanoparticles.
Thespectrumshowsastrongandsharpbandat240cm1,whichcanbeattributedtothestretch-ingvibrationsofAg–N[30,31]andAg–Obonds[32].
ThisFigure7FTIRspectraoffreezedried(a)gumghattiand(b)silvernanoparticles.
Figure6XRDpatternofthesilvernanoparticles,indicatingfcccrystalstructure.
Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page7of10http://www.
orgmedchemlett.
com/content/2/1/17peakindicatestheformationofachemicalbondbetweensilverandaminonitrogen[31];andsilverandcarboxylategroups[32]ofgummolecules.
Itconfirmsthatthegumisboundtothesilvernanoparticlesurfaceeitherthroughaminoorcarboxylategrouporboth.
ItisknowntohaveclosefrequenciesfortheAg–NandAg–OstretchingvibrationsandtheinvolvementofbothNandOatomsinbindingresultinsurface-enhancedRamanscattering(SERS)bandbroadening[30].
Thebroadonesat1351and1523cm–1correspondtosymmetricandasymmetricC=Ostretchingvibrationsofcarboxylategroup,respect-ively[31].
TheenhancementintheintensityoftheCO2stretchingvibrationsuggeststhedirectbindingoftheCOOgroupwiththesilversurface[32].
Thebroadbandat1040andasharppeakat1123cm–1;theoneat827cm–1comesfromtheC–Hinplanebendingandoutofplanewag,respectively[30],fromthesaccharidestruc-tureofgum.
Thus,fromthepreferentialenhancementofthesebands;itcanbeconcludedthatbothaminoandcarboxylategroupsofthegumareinvolvedinthecappingofthesilvernanoparticles.
Theseresultsareinconcur-rencewithearlierbiosynthesisofsilvernanoparticlescar-riedoutwithnon-pathogenicfungusTrichodermaasperellum[31].
Itwasreportedearlierthatthecarboxyl-ategroupsofglycoproteinofgumAcaciawereinvolvedinbindingofsilvernanoparticles[4].
Itisknownthatpro-teinscanbindtonanoparticleseitherthroughfreeaminogroupsorbyelectrostaticinteractionofnegativelychargedcarboxylategroups[33].
Thegumghattiisknowntocon-tainproteinandtheproteincontentwasreportedtobeintherangeof2.
8–3.
7%[13-17].
Thisobservationisfurthersubstantiatedbythemeasuredproteinconcentrationof2.
7%forthegumandtheUV–Visabsorptionspectrumofthe0.
5%gumsolutionagainstwaterblank,autoclavedfor30min,giveninFigure8a.
Anabsorptionpeakat280nmisclearlyvisibleandisattributedtoelectronicexcitationsintryptophanandtyrosineresiduesintheproteins[1,33],whicharepresentinthegum.
Thestabilizationofnano-particlesbycappingagentsisalsovalidatedfromtheTEMimageshowingasinglenanoparticlethatissurroundedbyalayeroforganicmatrix(Figure8b).
Thus,onecancon-cludethatoncethesilverionsarereducedtosilvernano-particlesbythepolyhydroxylatedgum,proteinspresentinthegumsubsequentlyencapsulateandstabilizethesepar-ticlesalongwithsaccharidemolecules.
Basedontheseobservations,thesesilvernanoparticlescanbeusedasapossiblesubstrateforSERS.
AsobservedinIRspectra(Figure7),gumghattiisrichinvariousfunctionalgroups;andtheircappingonsilvernanoparticlesprovidessurfacereactivity.
Itisreportedthatthefunctionalunitusedasacappingagentplaysanimportantroleanddeterminesthetissuedistributionprofileofgoldnanoparticles[34].
Thus,thesefunctionalizednanoparticlesareusefulforvariousapplicationssuchasdrugdelivery[6],targetedbiologicalinteractions[34],andbiologicallabels[35].
AntibacterialassayForcheckingtheantibacterialactivity,silvernanoparticleswithanaveragesizeof5.
7±0.
2nmwereused.
Thesenano-particleswerepreparedwith0.
1%gumsolutioncontainingFigure8Ramanspectrumofaqueoussilvernanoparticlesolution.
(a)UV–Visabsorptionspectrumofthe0.
5%(w/v)gumsolutionagainstwaterblank,autoclavedfor30minand(b)TEMimageofasinglenanoparticle,surroundedbyalayeroforganicmatrix.
Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page8of10http://www.
orgmedchemlett.
com/content/2/1/171mMAgNO3,autoclavedfor30min.
After24hofincuba-tionat37°C,growthsuppressionwasobservedinplatesloadedwith5μgofsilvernanoparticles.
Whereas,thenega-tivecontrolplatesloadedwithautoclavedgumdidnotpro-duceanyZOI.
Gum–silvernanoparticlesshowedgrowthinhibitionaroundthewellsagainstthetestedbacteria.
ZOIofaround12.
25mmdiameterwasobservedfortheGram-positivebacterialstrainS.
aureusATCC25923.
InthecaseofGram-negativebacterialstrainsE.
coliATCC25922,E.
coliATCC35218,andP.
aeruginosaATCC27853,thedetectedZOIwere9.
0,8.
0,and11.
0mm,respectively.
Asexpected,thepositivecontrolplatesloadedwithsilverni-trateexhibitedinhibitionzones(Table1).
TheZOIvaluesnotedfordifferentbacterialstrainswithsilvernanoparticlesarecomparablewiththepositivecontrols.
Basedontheseresults,itcanbeconcludedthatthesynthesizedsilvernano-particleshadsignificantantibacterialactiononboththeGramclassesofbacteria.
ExperimentalSynthesisofsilvernanoparticlesSilvernitrate(AgNO3)(E.
Merck,Mumbai,India)ofanalyticalreagentgradewasusedforthesynthesis.
"Gumghatti"grade-1waspurchasedfromGirijanCo-operativeCorporationLtd.
,Hyderabad,India.
Allthesolutionswerepreparedinultrapurewater.
GumghattiwaspowderedinaPrestigehigh-speedmechanicalblender(Bengaluru,India)andsievedtoobtainameanparticlesizeof38μm.
Then,0.
5%(w/v)ofhomogenousgumstocksolutionwaspreparedbyaddingthispowdertoreagentbottlecontainingultrapurewaterandstirringovernightatroomtemperature.
Thenthissolutionwascentrifugedtoremovetheinsolublematerialsandthesupernatantwasusedforalltheexperiments.
Thepro-teinconcentrationinthegumsolutionwasquantifiedbyLowry'smethodusingaBangaloreGeneiTMproteinesti-mationkit,CatNo105560(Bengaluru,India).
Thesilvernanoparticlesweresynthesizedbyautoclavingthesilvernitratesolutionscontainingvariousconcentrationsofgumghattiat121°Cand103kPaofpressurefordifferentdurationsoftime,underdarkconditions.
Theeffectofconcentrationofgumandreactiontimeonnanoparticlesynthesiswasstudied.
AntibacterialassayThewell-diffusionmethodwasusedtostudytheanti-bacterialactivityofthesynthesizedsilvernanoparticles.
Alltheglassware,media,andreagentsusedweresteri-lizedinanautoclaveat121°C,103kPaofpressurefor20min.
Staphylococcusaureus(ATCC25923);andEscherichiacoli(ATCC25922),E.
coli(ATCC35218),andPseudomonasaeruginosa(ATCC27853)wereusedasmodelteststrainsforGram-positiveandGram-negativebacteria,respectively.
Bacterialsuspensionwaspreparedbygrowingasinglecolonyovernightinnutri-entbrothandbyadjustingtheturbidityto0.
5McFarlandstandard[36].
MuellerHintonagarplateswereinocu-latedwiththisbacterialsuspensionand5μgofsilvernanoparticleswasaddedtothecenterwellwithadiam-eterof6mm.
Thenanoparticlesusedwerepreparedwith0.
1%gumsolutioncontaining1mMAgNO3,auto-clavedfor30min.
Negativecontrolplatesweremain-tainedwithautoclavedgum-loadedwells.
Thecultureplatesloadedwithsilvernitrateatasilverconcentrationof5μgwereincludedaspositivecontrols.
Theseplateswereincubatedat37°Cfor24hinabacteriologicalincu-batorandthezoneofinhibition(ZOI)wasmeasuredbysubtractingthewelldiameterfromthetotalinhibitionzonediameter.
Threeindependentexperimentswerecar-riedoutwitheachbacterialstrain.
ConclusionsThisstudyreportsthefacilesynthesisofsilvernanoparti-clesfromsilvernitrateusinggumghatti.
Theadoptedmethodiscompatiblewithgreenchemistryprinciplesasthegumservesasadualfunctionalreductantandstabilizerforthesynthesisofnanoparticles.
Atagivengumconcentration,theefficiencyofnanoparticlesynthe-sisincreaseswithreactiontime,apropertyattributabletothelargereductioncapacityofthegum.
Asthepar-ticlesizeofthenanoparticlescanbecontrolled,thismethodcanbeimplementedforthelarge-scaleproduc-tionofmonodispersedandsphericalnanoparticlesofaround5.
7nmduetotheavailabilityoflow-costplant-derivedbiopolymer.
Thehydroxylandcarboxylategroupsofthegumfacilitatethecomplexationofsilverionsduringautoclaving.
Subsequently,thesesilverionsarereducedtoelementalsilverpossiblybyinsituoxida-tionofhydroxylgroups;andbytheintrinsiccarbonylgroupsinadditiontothoseproducedbytheairoxida-tion.
ThisproposedmechanismisalsosubstantiatedbytheFTIRdata.
Further,theformedsilvernanoparticleshadsignificantantibacterialactiononboththeGramclassesofbacteria.
ThesurfacereactivityprovidedbyTable1Inhibitionzones(mm)observedwithdifferentbacterialcultureplatesloadedwithsilvernanoparticlesandsilvernitrateatasilverconcentrationof5μgTestcompoundS.
aureus25923E.
coli25922E.
coli35218P.
aeruginosa27853Silvernanoparticles12.
259.
08.
011.
0Silvernitrate13.
511.
07.
612.
0Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page9of10http://www.
orgmedchemlett.
com/content/2/1/17cappingenablesthesefunctionalizednanoparticlesaspromisingcandidatesforvariouspharmaceutical,bio-medical,andenvironmentalapplications.
Notably,these-lectiveenhancementofRamanbandsoftheorganiccappingagentsboundtothesilvercolloidsallowsthesenanoparticlesassuitablesubstratesforSERS.
Inviewofthis,furtherstudiesareenvisagedtoexploretheotherpotentialapplicationsofthisgum-basednanoparticles.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
AcknowledgmentsWethankDr.
S.
V.
Narasimhan,AssociateDirectorandDr.
TulsiMukherjee,Director,ChemistryGroup,BARC,fortheirconstantsupportandencouragementforthisstudy.
Thesupportrenderedforhigh-resolutionTEMmeasurementsbytheDSTunitonNanoscience,SophisticatedAnalyticalInstrumentFacility(SAIF)atIIT-Madras,Chennai,isgratefullyacknowledged.
Authordetails1NationalCentreforCompositionalCharacterisationofMaterials(NCCCM),BhabhaAtomicResearchCentre,ECILPO,Hyderabad500062AP,India.
2DepartmentofBiochemistry,UniversityCollegeofScience,OsmaniaUniversity,Hyderabad500007,AP,India.
Received:17October2011Accepted:9May2012Published:9May2012References1.
VigneshwaranN,NachaneRP,BalasubramanyaRH,VaradarajanPV(2006)Anovelone-pot'green'synthesisofstablesilvernanoparticlesusingsolublestarch.
CarbohydrRes341:2012–20182.
WeiD,QianW(2008)FacilesynthesisofAgandAunanoparticlesutilizingchitosanasamediatoragent.
ColloidsSurfB62:136–1423.
DadoshT(2009)Synthesisofuniformsilvernanoparticleswithacontrollablesize.
MaterLett63:2236–22384.
MohanYM,RajuKM,SambasivuduK,SinghS,SreedharB(2007)Preparationofacacia-stabilizedsilvernanoparticles:agreenapproach.
JApplPolymSci106:3375–33815.
KoraAJ,SashidharRB,ArunachalamJ(2010)Gumkondagogu(Cochlospermumgossypium):atemplateforthegreensynthesisandstabilizationofsilvernanoparticleswithantibacterialapplication.
CarbohydrPolym82:670–6796.
DharS,ReddyEM,ShirasA,PokharkarV,PrasadBLV(2008)Naturalgumreduced/stabilizedgoldnanoparticlesfordrugdeliveryformulations.
ChemEurJ14:10244–102507.
PandaH(2003)Gumghatti.
In:Thecompletetechnologybookonnaturalproducts(Forestbased).
NationalInstituteofIndustrialResearch,Delhi,pp1–98.
NussinovitchA(2010)Miscellaneoususesofplantexudates.
In:Plantgumexudatesoftheworld:sources,distribution,propertiesandapplications.
CRCPress,TaylorandFrancis,BocaRaton,USA,pp347–3689.
SrivastavaVK,RaiRS(1963)Physico-chemicalstudiesongumDhawa(Anogeissuslatifoliawall.
).
ColloidPolymSci190:140–14310.
AspinallGO,BhavanadanVP,ChristensenTB(1965)Gumghatti(Indiangum).
PartV.
Degradationoftheperiodate-oxidisedgum.
JChemSoc2677–268411.
JefferiesM,PassG,PhillipsGO(1977)Viscosityofaqueoussolutionsofgumghatti.
JSciFoodAgric28:173–17912.
TischerCA,IacominiM,WagnerR,GorinPAJ(2002)Newstructuralfeaturesofthepolysaccharidefromgumghatti(Anogeissuslatifola).
CarbohydrRes337:2205–221013.
AmarV,Al-AssafS,PhillipsGO(2006)Anintroductiontogumghatti:anotherproteinaceousgum.
FoodsFoodIngredientsJJpn211:275–28014.
KatayamaT,IdoT,SasakiY,OgasawaraT,Al-AssafS,PhillipsGO(2008)Characteristicsoftheadsorbedcomponentofgumghattiresponsibleforitsoil–waterinterfaceadvantages.
FoodsFoodIngredientsJJpn213:372–37615.
IdoT,OgasawaraT,KatayamaT,SasakiY,Al-AssafS,PhillipsGO(2008)EmulsificationpropertiesofGATIFOLIA(Gumghatti)usedforemulsionsinfoodproducts.
FoodsFoodIngredientsJJpn213:365–37116.
KaurL,SinghJ,SinghH(2009)Characterizationofgumghatti(Anogeissuslatifolia):astructuralandrheologicalapproach.
JFoodSci74:E328–E33217.
CastellaniO,GaillardC,ViéV,Al-AssafS,AxelosM,PhillipsGO,AntonM(2010)Hydrocolloidswithemulsifyingcapacity.
Part3:adsorptionandstructuralpropertiesattheair–watersurface.
FoodHydrocolloids24:131–14118.
ShahaneJ(2006)Dinkacheladu-remembranceofthingspast.
http://thecookscottagetypepad.
com/curry/2006/03/dinkache_ladure.
html.
Accessed17March201019.
MeenaKL,YadavBL(2010)SomeethnomedicinalplantsofsouthernRajasthan.
IndianJTraditKnowl9:169–17220.
CrocianiF,AlessandriniA,MucciMM,BiavatiB(1994)DegradationofcomplexcarbohydratesbyBifidobacteriumspp.
IntJFoodMicrobiol24:199–21021.
HillMJ(1995)Bacterialfermentationofcomplexcarbohydrateinthehumancolon.
EurJCancerPrevent4:353–35822.
RambergJ,GardinerT(2002)Ghattigum—Anogeissuslatifolisstemgum(ghattigum).
GlycoEssential7Ingredients.
http://www.
glyconutrients-center.
org/ghatti-gum.
php.
Accessed17March201023.
MishraA,RaikwarA(2005)Anogeissuslatifolia(ghattigum):theediblegum.
VanikiSandesh29:27–2824.
FahrenbachMJ,RiccardiBA,GrantWC(1966)Hypocholesterolemicactivityofmucilaginouspolysaccharidesinwhiteleghorncockerels.
ProcSocExpBiolMed123:321–32625.
ParvathiKMM,RameshCK,KrishnaV,ParameshaM,KuppastIJ(2009)HypolipidemicactivityofgumghattiofAnogeissuslatifolia.
PhcogMag5:11–1426.
JoshiMG,SettyCM,DeshmukhAS,BhattYA(2010)Gumghatti:anewreleasemodifierforzero-orderreleasein3-layeredtabletsofdiltiazemhydrochloride.
IndianJPharmEducRes44:78–8527.
RaveendranP,FuJ,WallenSL(2003)Completely"green"synthesisandstabilizationofmetalnanoparticles.
JAmChemSoc125:13940–1394128.
EdwardsHGM,FalkMJ,SibleyMG,Alvarez-BenediJ,RullF(1998)FT-Ramanspectroscopyofgumsoftechnologicalsignificance.
SpectrochimActaA54:903–92029.
HuangaNM,LimHN,RadimanS,KhiewPS,ChiuWS,HashimR,ChiaCH(2010)Sucroseestermicellar-mediatedsynthesisofAgnanoparticlesandtheantibacterialproperties.
ColloidsSurfA353:69–7630.
ChowdhuryJ,GhoshM(2004)Concentration-dependentsurface-enhancedRamanscatteringof2-benzoylpyridineadsorbedoncolloidalsilverparticles.
JColloidInterfaceSci277:121–12731.
MukherjeeP,RoyM,MandalBP,DeyGK,MukherjeePK,GhatakJ,TyagiAK,KaleSP(2008)Greensynthesisofhighlystabilizednanocrystallinesilverparticlesbyanon-pathogenicandagriculturallyimportantfungusT.
asperellum.
Nanotechnology19:075103–07510932.
BiswasN,KapoorS,MahalHS,MukherjeeT(2007)AdsorptionofCGAoncolloidalsilverparticles:DFTandSERSstudy.
ChemPhysLett444:338–34533.
VigneshwaranN,AshtaputreNM,VaradarajanPV,NachaneRP,ParalikarKM,BalasubramanyaRH(2007)BiologicalsynthesisofsilvernanoparticlesusingthefungusAspergillusflavus.
MaterLett61:1413–141834.
GenevieveMF,StanWC,DaeYoungK,RaghuramanK,KavitaK,NripenC,KatteshK(2009)Biodistributionofmaltoseandgumarabichybridgoldnanoparticlesafterintravenousinjectioninjuvenileswine.
Nanomed:NanotechBiolMed5:128–13535.
SchrandAM,Braydich-StolleLK,SchlagerJJ,DaiL,HussainSM(2008)CansilvernanoparticlesbeusefulaspotentialbiologicallabelsNanotechnology19:235104–23511636.
KoraAJ,ManjushaR,ArunachalamJ(2009)SuperiorbactericidalactivityofSDScappedsilvernanoparticles:Synthesisandcharacterization.
MaterSciEngC29:2104–2109doi:10.
1186/2191-2858-2-17Citethisarticleas:Koraetal.
:Size-controlledgreensynthesisofsilvernanoparticlesmediatedbygumghatti(Anogeissuslatifolia)anditsbiologicalactivity.
OrganicandMedicinalChemistryLetters20122:17.
Koraetal.
OrganicandMedicinalChemistryLetters2012,2:17Page10of10http://www.
orgmedchemlett.
com/content/2/1/17