Rev. bras. farmacogn. vol.26 número5

w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Spectroscopic

synthetic

optimizations

monitoring

of

silver

nanoparticles

formation

from

Megaphrynium

macrostachyum

leaf

extract

Franc¸

ois

Eya’ane

Meva

_,

_Marcelle

_Loretta

_Segnou

_,

_Cecile

_Okalla

_Ebongue

_,

Agnes

Antoinette

Ntoumba

_,

_Philippe

_Belle

_Ebanda

_Kedi

_,

_Vandi

_Deli

_,

Marie-Annie

Etoh

_,

_Emmanuel

_Mpondo

_Mpondo

a_{DepartmentofPharmaceuticalSciences,FacultyofMedicineandPharmaceuticalSciences,UniversityofDouala,Douala,Cameroon} b_{DepartmentofChemistry,FacultyofSciencesandEngineering,UniversityofHull,Hull,UnitedKingdom}

c_{DepartmentofBiologicalSciences,FacultyofMedicineandPharmaceuticalSciences,UniversityofDouala,Douala,Cameroon} d_{ClinicalBiologyLaboratory,GeneralHospitalofDouala,Douala,Cameroon}

e_{DepartmentofAnimalBiologyandPhysiology,FacultyofScience,UniversityofDouala,Douala,Cameroon} f_{DepartmentofChemistry,FacultyofSciences,UniversityofDouala,Douala,Cameroon}

g_{DepartmentofPharmacotoxicologyandPharmacokinetics,UniversityofYaoundeI,Yaounde,Cameroon}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30July2015

Accepted24June2016

Availableonline20July2016

Keywords:

Silver Nanoparticles

Megaphryniummacrostachyum

UV–visiblespectroscopy

a

b

s

t

r

a

c

t

Nanobiotechnologyisoneofthemostpromisingareasinmodernnanoscienceandtechnology.

Metal-licnanoparticleshavefoundusesinmanyapplicationsindifferentfields,suchascatalysis,photonics,

electronics,medicineandagriculture.Synthesizednanoparticlesthroughchemicalandphysicalmethods

areexpensiveandhavelowbiocompatibility.Inthepresentstudy,silvernanoparticleshavebeen

syn-thesizedfromMegaphryniummacrostachyum(Benth.&Hook.f.)Milne-Redh.,Marantaceae,leafextract.

MegaphryniummacrostachyumisaplantwithlargeleavesfoundintherainforestofWestand

Cen-tralAfrica.Syntheticoptimizationsfollowingfactorssuchasincubationtime,temperature,pH,extract

andsilverion concentrationduringsilver formationarediscussed. UV–visiblespectragavesurface

plasmonresonanceforsynthesizedsilvernanoparticlesbasedMegaphryniummacrostachyumpeaksat

400–450nm.X-raydiffractionrevealedtheaveragesizeofpurecrystallitescomposedfromAgandAgCl.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Nanomaterialswithacharacteristicdimensionintherangeof 1–100nmareattheleadingedgeofnanosciencesand nanotechnol-ogy(Masarovicováetal.,2014).Inrecentyears,nanomaterialsand specificallymetalnanoparticleshavereceivedparticularinterest in diverse fields ranging frommaterial sciences to biotechnol-ogy(Huang et al., 2007).High-density thin films of silver and

copper nanoclusters have been produced in Middle-Age- and

Renaissance-eraglazedpottery, toexploittheirpeculiar optical propertiesfordecorativepurposes(Pérez-Aranteguietal.,2001). Nanometer-sizedmetallicparticlesinmeltglasseshavebeenused for centuries to produced colored glassware (Bamford, 1977). Becauseofextremelysmallsizeandhighsurfacetovolumeratio

∗ _{Correspondingauthor.}

E-mail:mevae@daad-alumni.de(F.Eya’aneMeva).

ofnanoparticles,thephysicochemicalpropertiesof nanoparticles-containingmaterialsarequitedifferenttothoseofthebulkmaterial (El-Sayed,2001).Thus,nanomaterialshavepotentialapplications inelectronics, photonics,information storage,chemicalsensing, imaging,environmentalremediation,drugdelivery,andbiological labeling(Huangetal.,2007).

Thechemicalsynthesisofsilvernanoparticlesemployschemical reducingagentstoconvertAg+_{ionstoAg-nanoparticles.Oneofthe} mostwidelyusedchemicalreducingagentissodiumborohydride. Thisprocessinvolvestheundesiredusedofhazardouschemicals, andthebiocompatibilityoftheresultingAg-nanoparticlesistoo lowforapplicationinbiologicalsystems(Park,2014).Thebiological methodforthesynthesisofnanoparticlesemploysuseofbiological agentslikebacteria,fungi,actinomycetes,yeastandplants provid-ingawiderange ofresourcesfor thesynthesisof nanoparticles (Rai etal.,2008; Thakkaret al.,2010).Therateofreduction of metalionsusingbiologicalagentsisfoundtobemuchfasterand alsoatambienttemperatureandpressureconditions(Raietal.,

http://dx.doi.org/10.1016/j.bjp.2016.06.002

0102-695X/©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

2009).Theprocessofgreen synthesisrequirestheuseofwater asan environmentally friendlysolvent. Plant mediated synthe-sisofmetalnanoparticlesisgainingmore importanceowingto itssimplicity,eco-friendliness,rapidrateofsynthesisof nanopar-ticlesofattractiveand diversemorphologiesand eliminationof elaboratemaintenanceofcellcultures(VadlapudiandKaladhar, 2014).Earlierliteraturesuggestthatextractsfromvariousplants leavessuchasEucalyptushybrid,Myrtaceae(Dubeyetal.,2009), Helianthusannuus,Asteraceae(Gebruetal.,2013),Acalyphaindica, Euphorbiaceae(Krishnarajetal.,2010),Oryzasativa,Poaceae(Leela andVivekanandan,2008),Gliricidiasepium,Fabaceae(Rajeshetal., 2009),Menthapiperita,Lamiaceae(Alietal.,2011),Atrocarpus het-erophyllus,Moraceae(Thombreetal.,2012),Withaniasomnifera, Solanaceae(Rajeshetal.,2013;Gregoryetal.,2014),couldbeused fortheAg-nanoparticlessynthesis.

Megaphryniummacrostachyum(Benth.&Hook.f.)Milne-Redh. belongstofamilyMarantaceae.Theplantisfoundintherainforest ofWestandCentralAfrica(Jenningsetal.,2001).M.macrostachyum isaperennialsemi-woodyherb,rhizomatous,formingextensive clumps,withstemsto2½mhighbearingasinglelargeleaf30–60 (–90)cm long by 12–30 (–40)cm wide. The flowers, borne on thepetiolebelowtheleafarewhitishwithredorpurplecalyx. Theleavesareharvestedfromtheforestandusedfreshin wrap-ping food in order to preserve the food. In Central Africa, for instance,theyareoftenusedforwrappingcassavasticksbefore cooking(Ajayi and Ojelere,2013).No significantanti-microbial activitiesof M.macrostachyumleavesover Escherichiacoli, Kleb-siellapneumonia,Pseudomonasaeruginosa, Staphylococcusaureus andCandidaalbicansinthedosesconsideredwerefound.The phy-tochemicalscreeningoftheleavesshowspresencesofalkaloids, flavonoids,anthocyanins,saponins,reductingsugarsandgallic tan-nins(Malouekietal.,2013).

Sofar,therehavebeennoreportsonthesynthesisof nanopar-ticlesbyusingM.macrostachyum.Theleavesaresuitableforgreen synthesisandwepresentin thisresearchtheeco-friendly, sim-pleandlowcostsynthesisofsilvernanoparticlesandthesynthetic optimizationsrelatetoincubationcontacttime,temperature,pH, extractandsilver ionconcentration.Astudyofthecrystallinity andcompositionofthesilvernanoparticlesusingX-raypowder diffractionispresented.

Materialsandmethods

Materials

Silvernitrate(AgNO3)wasobtainedfromSigma–Aldrich chem-icalsGermany,H2SO498%fromMerckKGaADarmstadtGermany andNaOHfromR.P.NormapurProlaboParisandusedasreceived. De-ionizedwaterwasusedthroughoutthereactions.Freshleaves ofM.macrostachyum(Benth.&Hook.f.)Milne-Redh.,Marantaceae, wereprocuredfromlocalmarket,Douala,Cameroon,andidentified atthenationalherbariumofCameroonbyTadjouteuFulbergunder numberofdeposit10000/SRFCam.Allglasswareswerewashed withdilutenitricacid(HNO3)andde-ionizedwater,andthendried inhotairoven.M.macrostachyumleafwassurfacecleanedwith runningtapwaterfollowedbyde-ionisidedwatertoremoveall thedust and unwantedvisible particles. Aqueousextract ofM. macrostachyumwaspreparedbyboiling10gofM.macrostachyum leafin200mlde-ionizedwaterfor5minat80◦_{C.Theextractwas} filteredtwicethroughWhatmanNo.1filterpapertoremove par-ticulatematter,getclearsolutionsand storedoneweekat4◦_C. SolutionsofAgNO310−3M,10−2Mand10−1Mwerepreparedin de-ionizedwater.Mixturesolutionofplantextractandsilverion washandshakenduring1minbeforeincubation.

Fig.1. Megaphryniummacrostachyumleaf:32cmlarge,17cmwide.

Instrumentation

TheformationofAg-nanoparticleswasobservedbymeasuring theUV–visspectrumof2.5mlofthereactionsuspensionat differ-enttimeintervals.Ifabsorbancehigherthan4.5u.a.,thesample weredissolvebyafactorof½withdistilledwater.AnUV-visible Uviline9100spectrophotometeroperatedatwith1nmresolution withopticallengthof10mm.UV–visibleanalysisofthereaction mixturewasobservedfora periodof300s. XRDmeasurements werecarriedoutusingaPANalyticalEmpyreanSerie2X-ray diffrac-tometer(CuK-Alpha1[ ˚A]1.54060,K-Alpha2[ ˚A]1.54443,K-Beta[ ˚A] 1.39225)bypreparingathinfilmofsilver-macrostachyumpowder onsiliciumsubstrate.

Preparationofaqueousextract

AqueousextractofM.macrostachyumwaspreparedusing10g offreshlycollectedleaves(Fig.1).Theleavesweresurfacecleaned withrunningtapwater,followedbydistilledwaterandboiledwith 100mlofdistilledwaterat80◦_{Cfor5min.Theextractwasfiltered} andstoredat4◦_{Cforfurtheruse,beingusableforoneweekdue} tothegraduallossofplantextractviabilityforprolongedstorage (Eya’aneMevaetal.,2016).

Greensynthesisofsilvernanoparticles

For thesynthesisof thesilvernanoparticles,a volumeofM. macrostachyumleafextract(5,10,15ml)wasaddedto50mlof 10−3_M,10−2_Mor10−1_MaqueousAgNO

3solutionandincubated atroomtemperatureinthedarktominimizethephotoactivation ofsilvernitrate.Thereactionsweremadeunderstaticconditions. Firsthourofreactionwasmonitoredmeasuringtheabsorbanceat 5,10,20,30,40,50and60min.Inadditionofroomtemperature (30◦_{C)thestudywasdoneat50and80}◦_{Ctoinvestigatetheeffect} oftemperaturefollowingthesampleof10mlextractand10−3_M AgNO3during30min.DifferentpHvalues2,4,6,8,10and12were chosenforinvestigationofpHeffectinspeedofsilvernanoparticles formation.ThepHofthesolutionswasadjustedusing0.1NH2SO4 and0.1NNaOHsolutions.Thecontacttimeofincubationforall studiedsampleswasvariedfrom1hto24handthen96h.

Resultsanddiscussion

UV–visibleAg-nanoparticlesformedandincubationcontacttime

Fig.2.Silvernitrate,Megaphryniummacrostachyumleafextract,Agnanoparticles solution.

silvernanoparticlesusing50mlofAgNO3 10−3Mwith10mlof extractconcentrationduringthefirsthourisshowninFig.3.The solutioncolorchangewithinsecondstopaleyellow,andthento yellowishbrow,duetoformationofplasmonsatthecolloidsurface, indicatingthesynthesis ofsilver nanoparticles.The samesharp surfaceplasmonresonanceabsorbancebandhasbeenobtainwith differentextract concentrations(5,10, 15ml)at10−3_MAgNO

3. Then,5mlofextractisenoughtoreducecompletely50mlof sil-verionat10−3_{Mconcentration(Fig.4).Theplasmonresonance} absorbanceincreaseswhena10−2_MAgNO

3solutionisusedwith differentextractsconcentration,thenmoresilverionisavailablefor

400 500 600 700 800 900 0.0

0.5 1.0 1.5 2.0

Absorbance

Wavelength (nm)

5 min 10 min 20 min 30 min 40 min 50 min 60 min

5 10 20 30 40 50 60

Fig.3.Sixtyminutesofreactionbetween10mlofMegaphryniummacrostarchyum

leavesextractand50mlAgNO310−3M.

reduction.At10−1_{Mconcentrationofthesilverionswiththe} dif-ferentM.macrostachyumextractconcentrationsthenanoparticles areaggregatebecauseofthedeficiencyofmoleculesofleafextract toactascappingagents.Thebarrierpotentialdevelopedasaresult ofthecompetitionbetweenweakVanderWaalsforcesofattraction andelectrostaticrepulsionisbroken(Prathnaetal.,2011).As pos-tulatedbyMie’stheory,sphericalnanoparticlesresultsinasingle surfaceplasmonresonance(SPR)bandintheabsorptionspectra. Ontheotherhand,anisotropicparticlesprovidetwoormoreSPR bandsdependingontheparticleshape(Mie,1908).Inthepresent study,reactionmixturesconfirmsingleSPRbandsdisclosing spher-icalshapeofAg-nanoparticleswhichtendtobecomeanisotropic withtime.TheplantleafextractfromM.macrostachyumactas reductantaswellascappingagent,thereforemediatethe synthe-sisaswellasstabilizationofthesilvernanoparticles.Thesharp surfaceplasmonresonancebandincreaseswithsilverion concen-trationas observedfor oliveleafextract (Khalilet al.,2013).It

900 800 700 600 500 400 0.0 0.5 1.0 1.5 2.0

900 800 700 600 500 400 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

900 800 700 600 500 400 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

A

b

so

rb

an

ce

AgNO3

5 ml 10 ml 15 ml

Wavelength (nm)

AgNO3

5 ml 10 ml 15 ml

A

b

so

rb

an

ce

Wavelength (nm)

AgNO3

5 ml 10 ml 15 ml

1) 10-3M AgNO₃ 2) 10-2M AgNO₃

3) 10-1_{M AgNO}

3

900 800 700 600 500 400 0 1 2 3 4

900 800 700 600 500 400 0 1 2 3 4

900 800 700 600 500 400 0 1 2 3 4 5

900 800 700 600 500 400 0 1 2 3 4

A

b

so

rb

an

ce

AgNO3

1H 24H 96H

AgNO3

1H 24H 96H

Absor

b

ance

Wavelength (nm)

AgNO3

1H 24H 96H

Wavelength (nm)

AgNO3

1H 24H 96H

1) 10

M,

5 ml

extra

ct

2)

10

M, 10 ml extract

3) 10

M, 1

5 ml

extract

4)

10

M,

5 ml

extra

ct

900 800 700 600 500 400 0 1 2 3 4

900 800 700 600 500 400 0 1 2 3 4

900 800 700 600 500 400 0,0 0,2 0,4 0,6 0,8 1,0

900 800 700 600 500 400 0,0 0,2 0,4 0,6 0,8 1,0 1,2

Abso

rb

an

ce

AgNO3

1H 24H 96H

AgNO3

1H 24H 96H

5) 10

M, 1

0 ml

extract

6) 10

M,

15 ml e

xtrac

t

7) 10

M, 5

ml extr

act

8) 10

M,

10 ml e

xtrac

t

Abs

o

rban

ce

Wavelength (nm)

AgNO3

1H 24H 96H

Abs

o

rban

ce

Wavelength (nm)

AgNO3

1H 24H 96H

Fig.5. UV–visspectraofdifferentquantitiesofextractandofsilvernitrateinfunctionoftime.

canbeseenthattheabsorbancebandmaximaofAg-nanoparticles usingM.macrostachyumisintherange400–450nmdueto sur-faceplasmonresonance(Mulvaney,1996)ofAg-nanoparticles.The conductionelectronsundergooscillationduetothestrong interac-tionoflightwiththesilvernanoparticles(Thombreetal.,2012).As

900 800 700 600 500 400 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

Absorbance

Wavelength (nm)

30 °C 50 °C 80 °C

30 °C 50 °C 80 °C

Fig.6. UV–visspectraoftemperaturevariationfrom30to80◦_{Cduringsilver}

nanosynthesis.

ofAg-nanoparticlesoccurredrapidlywithinfewminutes indicat-ingthat M.macrostachyumspeedsupthebiosynthesis of silver nanoparticles.Suchofrapidreductionwasobservedforcarobleaf extractwith2minreductiontime(Awwadetal.,2013).

Thereaction between Ag+ _{and the reducing material in the} extractwasfollowedfor96HandUV–visiblemeasurementswere madeat1h,24hand96h.Fig.5showstheUV–visiblespectraof Ag-nanoparticlesasafunctionoftimeafteradditionofdifferent quantitiesofM.macrostachyumleafextract.Reductionaswellas nucleationandgrowingsizeofnanoparticlesincreasesfrom24h to96hbutpoly-dispersionsoccur.Thereactiontimeresultedin gradualincreasingofabsorbancebands.Thecolorintensityofthe solutionchangefromlightyellowtodeep-brownattheendofthe reactionbecauseofincreasingamountofsilver nanoparticlesas wellasaggregation.

Effectoftemperature

Fig.6 showsUV–visiblespectraoftheAg-nanoparticles pre-pared at 30, 50 and 80◦_{C. It can be seen that the absorbance} increaseswithincreasingtemperature.Thisexperimentsuggests thattherateofnanoparticlesynthesisatroomtemperaturecanbe acceleratedbyincreasingtemperatureofthereactionmixture.On theotherhandtheparticlestendtobepolydispersedat80◦_C.

EffectofpH

TheUV–visiblefollowingthepHduringtheformationofsilver nanoparticlesfrompH2to12isshowninFig.7.Thevariationof coloris inFig.8.It canbeseenthatPlasmonabsorbancebands increaseswithincreasingpHfrom2to12,whichcanbeduetothe

Ab

sorbanc

e

0 1 2 3 4

2

400 4

6 8 10 12

500

Wavelength (nm)

600 700 800 AgN pH2 pH4 pH6 pH8 pH1 pH12

900 O3

0 2

Fig.7. UV–visspectraofthevariationofpH.

Fig.8. ColorsofAgnanoparticlessolutionatpH2,4,6and8,10,12.

increaseinproductionofcolloidalsilvernanoparticlesand reduc-tionrate.TheabsorbancedoesnotdecreaseatpHhigherthan8 suchasobserved for oliveoilleafextracts (Khalil etal., 2013). Furthermore,itisobservedthatthebrowncolorofthe nanopar-ticlesappearedshortlyaftermixingtheAgNO3 withtheextract atpH4–12.AsobservedinPinuseldaricabarkextract,pHaffects theamountofnanoparticleproductionandtheirstabilityandis acriticalfactor ofcontrolofsizeandmorphologyof nanoparti-cles(IravaniandZolfaghari,2013).Furthermore,pHinfluencedthe rateofthereductionreaction.Thereactionmixtureturnedbrown whensilverwasreduced,andthereactionmixturecoloring accel-eratedwhenincreasingpH.AtpH10,thesharpsurfaceplasmon resonancebandindicatesthatamonodispersesuspensionoccurs. Inpreviousstudies,itwasshownthatthesizeandshapeof biosyn-thesizednanoparticlescouldbemanipulatedbyvarying thepH ofthereaction mixtures(Khalilet al.,2013).Amajorinfluence ofthereactionpHisitsabilitytochangetheelectricalchargesof biomoleculeswhichmightaffecttheircappingandstabilizing abil-itiesandsubsequentlythegrowthofthenanoparticles(Khaliletal., 2013).Then,highpHenvironmentenhancedthereducingand sta-bilizingcapabilityoftheantioxidantsintheM.macrostachyumleaf extractasituationfoundforoliveleafextracts.Thenumberofnuclei increaseswithelevatedpHmaybeduetopromotedreactivityof theM.macrostachyumleafextractsreductants.Gardea-Torresdey etal.(2003)foundthatpHisanimportantfactorinthe biosyn-thesisofcolloidalgoldusingalfalfabiomassandconcludedthat thesizeofnanoparticlesvariedwiththechangeinpH.Mocketal. (2002)alsohavereachedsimilarconclusionsandreportedthatpH isresponsiblefortheformationofnanoparticlesofvariousshapes andsizeasdifferentplantextractsandeventheextractscoming fromdifferentpartsofthesameplantmayhavedifferentpH val-ueswhichfurtherneedoptimizationfortheefficientsynthesisof nanoparticles.

X-raydiffraction

20 30 40 50 60 70 80 100

150 200 250 300 350

(311) (220)

(222) (311) (220)

(200) (111)

(200)

(111)

In

te

n

sity

c

oun

ts

(a

.u

.)

2 theta (Degree) (2

Fig.9. XRDpatternofthenanoparticlesfromMegaphryniummacrostarchyum,

representAgnanocrystallitesand䊉representAgClnanocrystallites.

TheaveragecrystallitesizeofthesynthesizedNPwas deter-minedusingtheDebye–Scherrerequation:

D

v

ˇcos

where Dv is the average crystalline size; K is a dimensionless shapefactor,withavalueclosetounity(0.9);isthewavelength of Cu K␣; ˇ is thefull width at half-maximum of the diffrac-tionpeaks;andisBragg’sangle.Noothercharacteristicpeaks werefoundintheXRDspectra,indicatingthehighpurityofthe as-preparedAg@AgClnanoparticles.Tocalculatetheaverage crys-tallineparticlesizeofthesynthesizedAg@AgClnanoparticles,we preferredthemostintensepeaksofAgandAgCl(Eya’aneMeva etal.,2016).Wehaveselectedthe(111)and(200)latticeplanes ofAgandAgCltocalculatetheaveragecrystallineparticlesizeof Ag@AgClNPs.Thecalculatedaveragecrystallineparticlesizeofthe Ag@AgCl-Megaphryniumwasfoundtobe33.7nmand44.2nmfor AgandAgCl,respectively.Theintenseandnarrowdiffractionpeaks revealedthecrystallinenatureofthesynthesized nanoparticles (Wangetal.,2010).

Patternidentificationshowstheformationofpurecrystalsof Ag@AgCl.Asimilarobservationwasmadeusingleafextractsof CorchorusolitorusandIpomeabatatasorflowersextractofAlbizia julibrissin(Eya’aneMevaetal.,2016;Awwadetal.,2015).

Conclusions

Wehavedescribedasimplegreenmethodforsilver nanoparti-clessynthesisusingthereducingpropertiesofM.macrostachyum leafaqueousextract. TheextractofM.macrostachyumleavesin contactwithsilverionsiscapableofproducingsilver nanoparti-cleswithin5min.Theextractactasreductantandstabilizerand thenanoparticlescanbepreparedeasily,rapidlyand ina cost-effectivelymanner.Itwasfoundthatsilvernanoparticlessynthetic rateincreasewithextractandsilverionconcentration,incubation contacttimeand temperature.UV–visiblemeasurementsshows thatthesynthesisispromotedathighpHwithpH10beenmore favorable.PowderX-raydiffractionstudiesconfirmthepurenature ofthecrystallitescomposedwithAgandAgClnanocrystalliteswith size33.7nmand44.2nmforAgandAgCl.

Authors’contributions

SML, AAN, and PBEKcontributed in collectingplant sample and identification,confection ofherbarium,runningpartofthe

laboratorywork.EMFandEMMcarryanalysisofthedata,run lab-oratorywork,providechemicalsanddraftedthepaper.Allauthors contributedtodiscussthespectroscopyandpowder diffraction. EMFandEMMdesignedthestudy,supervisedthelaboratorywork andcontributedtocriticalreadingofthemanuscript.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthanktheMultidisciplinaryLaboratoryofthe Fac-ulty of Medicine and Pharmaceutical Sciences, Department of PharmaceuticalSciencesfortechnicalandfinancialsupport. Sup-portofWordUniversityServiceunderAPA2668forprovidingthe equipmentsusedisappreciate.TheauthorsthanktheAssociation ofCommonwealthUniversityforthegenerousAcademic Fellow-shipCMCF-2015-3.SincerethanksareexpressedtoProf.DavidJ. Evans(UniversityofHull)forhiscontinuoussupportofourwork andhelpfuldiscussions.

References

Ajayi,I.A.,Ojelere,O.O.,2013.Phytochemicalscreening,proximateanalysisand

antimicrobial activityof aqueous extractof Megaphryniummacrostachyum seeds.Int.J.Eng.Res.Technol.2,2123–2131.

Ali,D.M.,Thajuddin,N.,Jeganathan,K.,Gunasekaran,M.,2011.Plantextract

medi-atedsynthesisofsilverandgoldnanoparticlesanditsantibacterialactivity againstclinicallyisolatedpathogens.ColloidsSurf.B85,360–365.

Awwad,A.M.,Salem,N.M.,Abdeen,A.O.,2013.Greensynthesisofsilver

nanopar-ticlesusingcarobleafextractanditsantibacterialactivity.Int.J.Ind.Chem.4, 1–6.

Awwad,A.M.,Salem,N.M.,Ibrahim,Q.M.,Abdeen,A.O.,2015.Phytochemical

fabri-cationandcharacterizationofsilver/silverchloridenanoparticlesusingAlbizia julibrissinflowersextract.Adv.MatterLett.6,726–730.

Bamford,C.R.,1977.ColourGenerationandControlinGlass.Elsevier,Amsterdam,

Netherlands.

Dubey,M.,Bhadauria,S.,Kushwah,B.S.,2009.Grennsynthesisofnanosilverparticles

fromextractofEucalyptushybrid(Safeda)leaf.Dig.J.Nanomater.Biostruct.4, 537–543.

El-Sayed,M.A.,2001.Someinterestingpropertiesofmetalsconfinedintimeand

nanometerspaceofdifferentshapes.AccountsChem.Res.34,257–264.

Eya’aneMeva,F.,Segnou,M.L.,OkallaEbongue,C.,Ntoumba,A.A.,DjiopangYadou,

S.,EssombeMalolo,F.A.,LidwineNgah,L.,HarounaMassai,EmmanuelMpondo

Mpondo,E.,2016.Unexploredvegetalgreensynthesisofsilvernanoparticles:a

preliminarystudywithCorchorusolitorusLinnandIpomeabatatas(L.)Lam.Afr. J.Biotechnol.15,341–349.

Gardea-Torresdey,J.L.,Gomez,E.,Peralta-Videa,J.R.,Parsons,J.G.,Troiani,H.,

Jose-Yacaman,M.,2003.Alfalfasprouts:anaturalsourceforthesynthesisofsilver

nanoparticles.Langmuir19,1357–1361.

Gebru,H.,Taddesse,A.,Kaushal,J.,Yadav,O.P.,2013.Greensynthesisofsilver

nanoparticlesandtheirantibacterialactivity.J.Surf.Sci.Technol.29,47–66.

Gregory,M.,Selvakesavan,R.K.,Franklin,G.,Sarmento,B.,Dias,A.C.P.,2014.Green

synthesisofsilvernanoparticlesusingWithaniasomniferaextractandtheir incorporationintoacreamwithantibacterialactivity.PlantaMed.80,SL26.

Huang,C.C.,Yang,Z.,Lee,K.H.,Chang,H.T.,2007.Synthesisofhighlyfluorescentgold

nanoparticlesforsensingmercury(II).Angew.Chem.Int.Ed.46,6824–6828.

Iravani,S.,Zolfaghari,B.,2013.GreensynthesisofsilvernanoparticlesusingPinus

eldaricabarkextract.BioMedRes.Int.,http://dx.doi.org/10.1155/2013/639725.

Jennings,S.B.,Brown,N.D.,Boshier,D.H.,Whitmore,T.C.,Lopes,J.A.,2001.

Ecol-ogyprovidesapragmaticsolutiontothemaintenanceofgeneticdiversityin sustainablymanagedtropicalrainforest.ForestEcol.Manage.154,1–10.

Khalil,M.M.H.,Ismail,E.H.,El-Baghdady,K.Z.,Mohamed,D.,2013.Greensynthesis

ofsilvernanoparticlesusingoliveleafextractanditsantibacterialactivity.Arab. J.Chem.7,1131–1139.

Krishnaraj,C.,Jagan,E.G.,Rajasekar,S.,Selvakumar,P.,Kalaichelvan,P.T.,Mohan,

N.,2010.SynthesisofsilvernanoparticlesusingAcalypticaindicaleafextracts

anditsantibacterialactivityagainstwaterbornepathogens.ColloidsSurf.B76, 50–56.

Leela,A.,Vivekanandan,M.,2008.Tappingtheunexploitedplantresourcesforthe

synthesisofsilvernanoparticles.Afr.J.Biotechnol.7,3162–3165.

Maloueki,U.,Musuyu,M.,Mbomba,N.B.A.,Ndimbo,K.S.P.,Kapetshi,K.J.,Kabena,

N.O.,2013.Activitésantimicrobiennesetantioxydantesdesextraitsaqueux

Masarovicová,E.,Králóvá,K.,Zinjarde,S.S.,2014.Metalnanoparticlesinplants, for-mationandaction.HandbookofPlantandCropPhysiology,vol.33.,3rded.CRC Press,Taylor&FrancisGroup,pp.684–719.

Mie,G.,1908.BeiträgezurOptiktrüberMedien,speziellkolloidalerMetallösungen.

Ann.Phys.330,345–377.

Mock,J.J.,Barbic,M.,Smith,D.R.,Schultz,D.A.,Schultz,S.,2002.Shapeeffectsin

plasmonresonanceofindividualcolloidalsilvernanoparticles.J.Chem.Phys. 116,6755–6759.

Mulvaney,P.,1996.Surfaceplasmonspectroscopyofnanosizedmetalparticles.

Langmuir12,788–800.

Pérez-Arantegui,J.,Molera,J.,Larrea,A.,Pradell,T.,Vendrell-Saz,M.,Borgia,I.,

Brunetti,B.G.,Cariati,F.,Fermo,P.,Mellini,M.,2001.Lusterpotteryfromthe

thirteenthcenturytothesixteenthcentury:ananostructuredthinmetallicfilm. J.Am.Ceram.Soc.84,442–446.

Park,Y.,2014.Newparadigmshiftforthegreensynthesisofantibacterialsilver

nanoparticlesutilizingplantsextracts.Toxicol.Res.30,169–178.

Prathna, T.C., Chandrasekaran, N., Raichur, M.A., Mukherjee, A., 2011.

Biomimeticsynthesisofsilvernanoparticlesbycitruslimon(lemon) aque-ousextractandtheoreticalpredictionofparticlesize.ColloidsSurf.B82, 152–159.

Rai,M.,Yadav,A.,Gade,A.,2008.Currenttrendsinphytosynthesisofmetal

nanopar-ticles.Crit.Rev.Biotechnol.28,277–284.

Rai,M.,Yadav,A.,Gade,A.,2009.Silvernanoparticlesasanewgenerationof

antimi-crobials.Biotechnol.Adv.27,76–83.

Rajesh, R.W.,Lakkakula, J.R., Niranjan, K.S.,Mendhulkar, V.D.,Sahebrao, K.B.,

2009.PhytosynthesisofsilvernanoparticlesusingGliciridasepium(Jacq).Curr.

Nanosci.5,117–122.

Rajesh,P.,Swati,W.,Sandesh,M.,Sangita,J.,Kulkarni,S.,2013.Greensynthesisof

silvernanoparticlesbyWithaniasomniferaandevaluationofitsantimicrobial potential.J.Empir.Biol.1,38–48.

Thakkar, K.N.,Mhatre,S.S., Parikh,R.Y., 2010. Biologicalsynthesisofmetallic

nanoparticles.Nanomedicine-UK6,257–262.

Thombre,R.,Parekh,F.,Lekshminarayanan,P.,Francis,G.,2012.Studieson

antibac-terialandantifungalactivityofsilvernanoparticlessynthesizedusingArtocarpus heterophyllusleafextract.Biotechnol.Bioinf.Bioeng.2,632–637.

Vadlapudi,V.,Kaladhar,D.S.V.G.K.,2014.Review:greensynthesisofsilverandgold

nanoparticles.MiddleEastJ.Sci.Res.19,834–842.

Wang,P.,Huang,B.,Lou,Z.,Zhang,X.,Qin,X.,Dai,Y.,Zheng,Z.,Wang,X.,2010.