Rev. bras. farmacogn. vol.26 número3

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

Original

Article

Dendranthema

grandiflorum

,

a

hybrid

ornamental

plant,

is

a

source

of

larvicidal

compounds

against

Aedes

aegypti

larvae

Kassia

C.V.W.

Spindola

a

_,

_Naomi

_K.

_Simas

b

_,

_Celso

_E.

_dos

_Santos

a

_,

_Ary

_G.

_da

_Silva

c

_,

_Wanderson

_Romão

d

_,

Gabriela

Vanini

d

_,

_Samantha

_R.C.

_da

_Silva

d

_,

_Grazielle

_R.

_Borges

e

_,

_Fernando

_G.

_de

_Souza

e

_,

Ricardo

M. Kuster

a,d,∗

a_Instituto_de_Pesquisas_de_Produtos_Naturais,_Centro_de_Ciências_da_Saúde,_Universidade_Federal_do_Rio_de_Janeiro,_Rio_de_Janeiro,_RJ,_Brazil

b_Faculdade_de_Farmácia,_Centro_de_Ciências_da_Saúde,_Universidade_Federal_do_Rio_de_Janeiro,_Rio_de_Janeiro,_RJ,_Brazil

c_Functional_Ecology_Laboratory,_Universidade_de_Vila_Velha,_Boa_Vista,_ES,_Brazil

d_Núcleo_de_Competência_em_Química_do_Petróleo,_Departamento_de_Química,_Universidade_Federal_do_Espírito_Santo,_Vitória,_ES,_Brazil

e_Biopolymers_and_Sensors_Laboratory,_Instituto_de_{Macromoléculas,}_Universidade_Federal_do_Rio_de_Janeiro,_Rio_de_Janeiro,_RJ,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15October2015 Accepted21January2016 Availableonline9February2016

Keywords:

Aedesaegypti

Caffeoylquinicacids Agribusiness Fattyacids Larvicidalactivity Triterpenoids

a

b

s

t

r

a

c

t

Inhybridcultivatedform,Dendranthemagrandiflorum(Ramat.)Kitam.,Asteraceae,flowers( Chrysan-themummorifoliumRamat.)wereutilizedintheproductionofextracts,whichwereanalyzedforlarvicidal activityagainstAedesaegyptithirdinstarlarvae.MethanolanddichloromethaneextractsshowedLC50

valuesof5.02and5.93ppm,respectively.UsingGC–MS,phytochemicalanalysesofthedichloromethane extractshowedthepresenceoftriterpenoidsandfattyacids,whileflavonoidsandcaffeoylquinicacids wereshowntooccurinthemethanolextractbyESIFourierTransformIonCyclotronResonanceMass Spectrometry(ESI-FT-ICR-MS).Triterpenoidsandfattyacidsarewellknowninsecticidalcompounds. Fromthisstudy,itcanbeconcludedthatD.grandiflorumgrownforfloriculture,asanagribusiness,can haveadditionalapplicationsasrawmaterialfortheproductionofinsecticidalproducts.

Introduction

Knownforover2000years,thechrysanthemumisan

ornamen-talplantnativetoChina.ItbelongstothegenusDendranthema, Asteraceae,andnumerouscultivarshavebeenproduced(Kimetal.,

2015).Thecutchrysanthemumisoneofthemostcommercially

successfulflowersbythediversityofitsinflorescence(Brackmann etal.,2005).Floricultureis arepresentativeactivityin Brazilian agribusinessand, assuch,it hasmadeanoutstandingeconomic and technological contribution. In particular, the

chrysanthe-mumculture (Dendranthema grandiflorum (Ramat.) Kitam.) has

greatimportanceinBrazilianfloriculture,anditscultivationhas increasedthroughoutthecountry(Polanczyketal.,2008).Climate isanimportantfactorthatfavorstheexpansionoffloriculture,as anagribusiness,inBrazil.Specifically,itallowsforthecultivationof temperateandtropicalflowerswithlowcostandhighproduction allyearlong(DaSilvaJunioretal.,2011).

∗ Correspondingauthor.

E-mail:ricardo.m.kuster@ufes.br(R.M.Kuster).

DengueisamajorpublichealthprobleminBrazil.The trans-missionofthedenguevirustohumansoccursthroughthebiteof themosquitoAedes aegypti.Thediseasecanbetemporarily dis-abling,butinitshemorrhagicform,itcanresultindeath.Although publicpoliciesexist formosquitocontrol,resistanceto conven-tionalinsecticidesisconcerningtohealthauthorities(Liu,2015). Therefore,this paperdescribesa naturalproduct,extractsof D. grandiflorum,whichareobtainedfromthecultivar“yellowsheena”, asasourceofcompoundswithlarvicidalactivityagainstA.aegypti

thirdinstarlarvae.Thestudiedplantisorganicallycultivatedinthe StateofRiodeJaneiro,NovaFriburgoCity.Inviewofitslarvicidal potential,thecutflowershouldalsosupporttheestablishmentof floricultureasanimportantagribusinessinBrazil.

Materialsandmethods

Plantmaterial

Dendranthema grandiflorum (Ramat.)Kitam., Asteraceae, yel-lowsheena,ahybridunderorganiccultivation,wasobtainedfrom farmersinNovaFriburgo,StateofRiodeJaneiro,inSeptember2013 andbotanicallyidentifiedbyDr.MarianaMachadoSaavedrafrom

http://dx.doi.org/10.1016/j.bjp.2016.01.003

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theRiodeJaneiroBotanicalGarden(voucherspecimendeposited attheherbariumProf.JorgePedroPereiraCarautaundernumber

HUNI3531).

Preparationofextracts

Theflowersweredriedinanovenat50◦_C_for₇_days₍₂₂₃_g).

Afterthat, they weresubmitted tomethanol extractionover a

periodof15days.Theextractwasfilteredandthesolventremoved byrotativeevaporationundervacuumtoobtainthefirstproduct,a dryMeOHextract(80g).Seventygramsweresuspendedinwater, andaftersolventremoval,thesuspensionwaspartitioned with dichloromethanetoyieldthesecondproduct,adryCH2Cl2extract (29g).

Phytochemicalanalysis

Electron Impact Ionization/Mass Spectrometry (EI/MS) was

obtainedusing a ShimadzuQP5050 GC–MSon a DB-5 column

(30m×0.25mm and film thicknessof 0.25␮m, J&W Scientific) withanionizingenergyof70eV.Programmingoftheoven temper-aturestartedat80◦_C,_which_was_maintained_for₂_min._Then,_the temperaturewasraisedto260◦_C_at_a_rate_of₁₅◦_C/min_and_again increasedto320◦_C_at₅◦_C/min._An_isotherm_during₁₀_min_was performedatthisfinaltemperature.Thetemperaturesofinjector andionsourcewerekeptat250◦_C_and₂₈₀◦_C,_{respectively.}_Helium wasusedasthecarriergaswithaconstantflowof1.1mlmin−1_. Onemicroliterofthesamplewasinjectedwithasplitratioof70:1.

MassspectraofextractcompoundswerecomparedwiththeNIST

MassSpectralDatabase(MSSearch2.0)andwithliteraturedata (AssimoupoulouandPapageorgiou,2005).

Themethanolicextractof D. grandiflorumwasanalyzedin a

mass spectrometer(Model9.4 T Solarix, BrukerDaltonics,

Bre-men,Germany),whichwassettooperateinnegativeionmode,

ESI(-),overamassrangeofm/z200–1300.Theparametersofthe ESI(-)sourcewereasfollows:nebulizergaspressureof0.5–1.0bar, capillaryvoltageof3–3.5kV,andtransfercapillarytemperatureof 250◦_C._The_mass_spectrum_was_processed_using_the_Compass_Data Analysissoftwarepackage(BrukerDaltonics,Bremen,Germany). Aresolvingpower,m/m50%=∼500,000,inwhichm50%isthefull peakwidthathalf-maximumpeakheightofm/z∼₌400andamass accuracyof<1ppm,providedtheunambiguousmolecularformula assignmentsforsinglychargedmolecularions.Elemental compo-sitionsofthecompoundsweredeterminedbymeasuringthem/z

values.Theunsaturationlevelofeachmoleculecouldbededuced directlyfromitsdoublebondequivalent(DBE),followingthe equa-tionDBE=c−h/2+n/2+1,wherec,h,andnarethenumbersof carbon,hydrogen,andnitrogenatoms,respectively(Ferreiraetal., 2014;Costaetal.,2014;DeSáetal.,2015;Nascimentoetal.,2015). Molecularformula,measuredm/zvalues,DBE,andmasserrorare showninTable1.Tandemmassspectrometry(MS2₎_experiments

were also performedon a quadrupole analyzer coupledto the

FT-ICRmassspectrometer,andquadrupoleFouriertransformion

cyclotronresonancemassspectrometry(Q-FT-ICRMS)was

per-formedforionsofm/z353,431,447,449and515.Thefragments produced (m/zvalues) fromESI(-)-MS/MSspectraare shownin Table3.

Larvicidalactivity

A. aegypti larvae that originatedfrom theNPPN strain were rearedinthelaboratoryundercontrolledphotoperiod(12hlight and12hdark)at27◦_C_and₈₀_±_10%_rel._humidity_in_trays_filled withdechlorinatedtapwaterandcaninefood.

Larvicidalactivitywasconductedfollowingthemethodadapted fromWHO(1970).Foreachtreatmentandcontrol,fivelarvaeof thirdstageinstars,orearlyfourthstageinstars,weretransferred into20mlglassesin14.9mlof distilledwater. Thesolutionsof crudeextracts,orfractions,werepreparedinethanolandadded (0.1ml)tothetreatmentglasseswithapipettetogivethefinal

assay concentrations. Controls received aqueous solution with

0.1mlofethanolonly.After24h,thenumberofdeadlarvaeineach glasswascounted.Alltreatmentswerereplicatedthreetimes.The 50%lethalconcentration(LC50)wascalculatedwitha95% confi-dencelimitbyProbitanalysis.

Resultsanddiscussion

Phytochemicalanalysis

As shown in Fig. 1, methanol extract of D. grandiflorum by

ESI(-)FT-ICRMSyielded 11compoundsidentified asfattyacids, phenylpropanoidsandflavonoids(Table1).Allcompoundswere detected as deprotonated molecules, i.e., adduct ion, as repre-sentedby[M−H]− _ion,_formed_by_the_interaction_of_a _molecule withaproton,orhydrogen(Table1).Amongfattyacids,palmitic

acid, m/z 255.2330, was the major compound found in the

crudeextract.Phenylpropanoids,suchasmonocaffeoylquinicacids,

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200 300 400 500 600 700 800

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m/z

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Table1

Fattyacids,flavonoidsandcaffeicacidderivativesofmethanolextractofDendranthemagrandiflorum,asidentifiedfromESI(-)-FT-ICRMS.

Identification m/zmeasured Identifiedcompounds [M−H]− _Fragments_MS/MS _DBE _Error_(ppm)

1 255.2330 Palmiticacid C16H31O2 – 1 0.05

2 269.0456 Apigenin C15H9O5 – 11 0.26

3 279.2330 Linoleicacid C18H31O2 – 3 0.32

4 285.0406 Luteolin C15H9O6 – 11 0.31

5 293.2123 Linolenicacidmethylester C18H29O3 – 4 0.30

6 295.2280 Linoleicacidmethylester C18H31O3 – 3 0.45

7 353.0881 Monocaffeoylquinicacid C16H17O9 191 8 0.77

8 431.0987 Apigenin7-O-glucoside C21H19O10 269 12 0.65

9 447.0937 Luteolin7-O-glucoside C21H19O11 285 12 0.90

10 449.1094 Eriodictyol7-O-glucoside C21H19O11 431,413,387,311,and287 11 1.01

11 515.1201 Dicaffeoylquinicacid C25H23O12 353 14 1.18

Table2

Compositionoffattyacids,steroidsandtriterpenoidsinthedichloromethanepartitionofDendranthemagrandiflorum.

Retentiontime–RT(min) MajorfragmentsfromMSdata Identifiedcompounds Relativeconcentration(%)

12.95 270(M+_),_227,_143,_99,_87,₇₄_(100%),₅₅ _Methyl_palmitate _2.98

13.20 256(M+_),_213,_185,_171,_157,_143,_129,_115,_83,_73,₅₇_(100%) _Palmitic_acid _3.97 13.40 284(M+_),_256,_241,_227,_213,_157,_101,₈₈_(100%),_83,_73,₅₅ _Ethyl_palmitate _0.23

14.15 294(M+_),_263,_95,_81,₆₇_(100%),₅₅ _Linoleic_acid_methyl_ester _0.15

14.20 292(M+_),_264,_149,_135,_122,_108,_93,₇₉_(100%),_67,₅₅ _Linolenic_acid_methyl_ester _0.17

14.36 298(M+_),_255,_199,_143,_87,₇₄_(100%),₅₅ _Methyl_stearate _0.12

Totalrelativeconcentrationoffattyacids 8.42

32.75 412(M+_),₅₅_(100%) _Stigmasterol _3.68

33.79 414(M+_),₅₅_(100%) _Sitosterol _13.47

34.45 426(M+_),₂₁₈_(100%),₂₀₃_(49%),₁₈₉ _␤-Amyrin _13.53

35.20 426(M+_),₂₁₈_(100%),₂₀₃_(18%),₁₈₉ _␣-Amyrin _22.03

35.79 468(M+_),₂₁₈_(100%),₂₀₇_(61%),₂₀₃_(40%),₁₈₉_(20%) _␤-Amyrin_acetate _1.68 36.50 468(M+_),₂₁₈_(100%),₂₀₇_(67%),₂₀₃_(21%),₁₈₉_(40%) _␣-Amyrin_acetate _4.78 36.68 426(M+_),_207,₁₈₉_(100%),_175,_147,_135,_121,_109,_95,_81,_68,₅₅ _Lupeol _5.06

Totalrelativeconcentrationoftriterpenoidsandsteroids 64.23%

m/z 353.0878, and dicaffeoylquinic acids, m/z 515.1201, were detectedasminorcompounds.Amongtheflavonoidsidentified, apigenin7-O-glucoside,m/z431.0987,eriodictyol7-O-glucoside,

m/z449.1093,andluteolin7-O-glucoside,m/z447.0933,werethe maincompoundsoftheMeOHextract.PurificationandNMR iden-tificationoftheflavonoidsweredescribedbySpindola(2015),and allthecompoundsweredescribedtooccurinD.grandiflorum(Lin andHarnly,2010;Ueharaetal.,2012).Table1showstheidentified compoundsforfattyandcaffeicacids,aswellasflavonoidsofD. grandiflorummethanolextract,asidentifiedfromESI(-)-FT-ICRMS data.

GC–MSanalysesofCH2Cl2extractshowsabundantpeaksintwo differentregionsinthechromatogram:(i)from6to18minand(ii) from30to38min.Ingeneral,fourteencompoundswere identi-fied,andtheirproposedstructures,molecularionsandfragments, retentiontime,andabundancerelativevaluesaresummarizedin Table2.

Inthefirstregion(approximatelybetween13.0and15.0min), themajor fatty acids were as follows: methyl palmitate, ethyl palmitate,palmiticacid,stearicacid,ethylstearate,linoleicacid methyl ester and linolenic acid methyl ester (Table 2). How-ever,in thesecond region(32–38min), steroids(sitosterol and

stigmasterol) and triterpenoids (␤-amyrin, ␣-amyrin, ␤-amyrin acetate,␣-amyrinacetateandlupeol)canbeseenaslessvolatile substancesofthechromatogram.Theywerepreviouslyidentified inChrysanthemummorifolium(TakahashiandSato,1979).The rel-ativeabundancesofthetwomainclassesofidentifiedcompounds were8.42%forfattyacidsand64.23%fortriterpenoidsandsteroids. AsshowninTable2,triterpenoidsandsteroidsarethemajor compoundsinthelipophilicextractofD.grandiflorum.Theywere alsodescribedtooccurinotherChrysanthemumspecies,suchasC. indicum,C.macrocarpumaswellasC.morifolium(D.grandiflorum) cultivars(TakahashiandSato,1979;Akihisaetal.,2005;Kumar etal.,2005).

Larvicidalactivity

LC50valuesforMeOHandCH2Cl2extractswere5.02ppmand 5.93ppm,respectively(Table3).

Attheconcentration1␮g/ml,temephos(TradeName:Abate®_), aspositivecontrol,killedallthetestedlarvae.SincetheCH2Cl2 extractwasobtainedasthesecondproductfromthecrudeMeOH extract,itisreasonabletothinkthatthelarvicidalactivityofthe flowersisconcentratedinthemorelipophiliccompoundsofthe

Table3

LarvicidalefficacyofextractsfromDendranthemagrandiflorumflowersagainstAedesaegypti.

Samples LC50(ppm)(LCL/UCL) LC95(ppm)(LCL/UCL) LC99(ppm)(LCL/UCL)

Dichloromethaneextract 5.9322

(5.1476/6.7862)

12.5650 (11.0297/14.9183)

15.3131 (13.3070/18.4474)

Methanolextract 5.0220

(3.6004/6.3780)

14.5585 (12.1309/18.8137)

18.5096 (15.2542/24.3771)

Temephos nd nd 1.0

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plant.Infact,fromotherstudiedplants,ourgrouphasidentified larvicidalcompoundswiththatnonpolarcharacteristic,including terpenesandalkamides(Simasetal.,2004,2007,2013),although more polarcompounds,like saponins(Amin et al.,2011), have alsobeendescribedaslarvicides.Lipophiliccompoundsaremore active larvicidesbased ontheir easiertransport through insect cellwallandcytoplasmicmembrane(MannandKaufman,2012). FromtheCH2Cl2extractofD.grandiflorum,severallipophilic com-poundswerefound,includingfattyacids,steroidsandtriterpenes. Palmiticacidwasthemajorfattyacididentified.Rahumanetal. (2000)testedpalmiticacidasalarvicide againstCulex quinque-fasciatus,Aedes stephensi and A. aegypti.The LC50 values found were129,79and57ppm,respectively.Thesteroidssitosteroland stigmasterol werealso identified.Sitosterol, isolated from Abu-tilonindicum(L.)Sweet,wasactiveatLC5011.5,3.5and26.7ppm, respectively,againstA.aegypti,A.stephensiandC. quinquefascia-tuslarvae(Ghosh,2013;Rahumanetal.,2008).Triterpenesseem tobethemostactiveofallthecompoundsidentified.Oneofthe triterpenesinD.grandiflorum,␣-amyrinacetate,showedeffective activityagainstallfourlarvalgrowthstages(LC500.01–0.02ppm) andpupa(LC500.005ppm)of themosquitoAnophelesstephensi (Kuppusamyetal.,2009).Cholesterolisoneofthemostimportant nutrientsforA.aegyptilarvaldevelopment.TheproteinAeSCP-2 isusedbymosquitoesforthetransportof cholesterol. Inactiva-tionofitkillsthelarvae.Kumaretal.(2010)biocomputationally evaluatedtheabilityoftriterpenestoinactivatethisprotein,and

␣-amyrinwasstructurallyconsideredtobethemosteffective larvi-cidewiththisparticularmechanismofaction.Themostabundant compoundsintheCH2Cl2 extractoftheflowerswere␣-amyrin (22%),alongwith␤-amyrin(13.5%)andsitosterol(13.47%).

Khanetal.(2014)comparedthelarvicidalactivityofextracts of C. morifolium leaves(LC50 1.5ppm) withextracts of Parthe-niumhysterophorusL.leaves(LC50 1.02ppm)and foundslightly betterefficiencyofthelatteragainstAedesalbopictusthirdinstar mosquitolarvae.Unfortunately,theydidnotdescribethe chemi-calcompositionofthecomparedplants.Bothplantsbelongtothe Asteraceaefamily;therefore,someclassesofcompoundsare simi-lar.P.hysterophorusisasourceofsesquiterpenelactones(Dattaand Saxena,2001),compoundswithhighinsecticidal,cytotoxic, antitu-mor,allergenic,antifeedantandphytotoxicactivity,andtheplant isconsidereda noxiousweedthatthreatensbiodiversity(Patel, 2011).Littleevidencesupportsthepresenceofsesquiterpene lac-tonesinC.morifolium,thechrysanthemumofflorists(Schulzetal., 1975),althoughalantolactonewasdescribedtooccurinthisspecies (Sertoli et al., 1985). Also no environmentaldamage hasbeen reportedforC.morifolium.Pyrethrinesterscompriseanother com-poundclasswithhighinsecticidalactivity.Thesecompoundsare formedbycombiningtwoacids(chrysanthemicacidandpyrethric acid)withthreealcohols(pyrethrolone,cineroloneandjasmolone). Theyare,however,restrictedtoChrysanthemumcinerariaefolium

Vis. (Nagar et al., 2015).Using the techniques described, both sesquiterpenelactonesandpyrethrinswereinvestigated,butnot found,inD.grandiflorum.

InaNuclearMagneticResonance(NMR)-basedmetabolomics

study,Leissetal.(2009)wereabletocomparethemetabolome ofthrips-resistantandthrips-susceptiblechrysanthemums.They concludedthattheresistancewasassociatedwithhigheramounts ofchlorogenicacid,amonocaffeoylquinicacid,inthrips-resistant species.Phenylpropanoidsareknownfortheirinhibitoryeffecton herbivoresandpathogens.Thepossibletoxicityofchlorogenicacid toinsectsresultsfromitsoxidationproduct,chloroquine(Felton etal.,1991).Tunónetal.(1994)testedanethanolextractof Achil-lea millefolium L. for antifeedant activity against the mosquito

A.aegypti.Afterfractionationoftheextract,chlorogenicacidat 1.2mg/cm2_was_one_of_the_most_active_compounds_as_a_mosquito repellent,causing100%larvalmortality.Flavonoidsandfattyacids,

atthesameconcentration,wereactive,butwithlower percent-ageofmortality.ThegreaterlarvicidalactivityofMeOHextractof

D.grandiflorum,whencomparedCH2Cl2extract,whichoriginates fromit,could,therefore,resultfromthepresenceofcaffeoylquinic acidsandotherphenolics,apartfromthetriterpenoids,steroids and fattyacids,aspreviously discussed.Basedontheresultsof thepresentstudy,itmaybespeculatedthatthecompounds,when

combined,mightactasapotentphytocomplexwithsynergistic

effect,butthisneedstobeconfirmedinfuturestudies.

D.grandiflorumhybridcanbeasourceoflarvicidalcompounds easilyextractedwithmethanol.Ornamentalplantsareabundantly cultivatedinBrazilianhighlands;however,afteraging,theyare dis-carded.Yet,theresultsofthepresentstudydemonstratethatsuch discardedplantscouldberecycledfortheirmethanolicextractsand usedtoproposeinnovativeproducts,as,forexample,larvicides, thushelpingtodevelopagribusinessinBrazil.

Authors’contributions

KCVWS(PhDstudent)collectedandidentifiedplantsamples,

ranthelaboratorywork,analyzeddata,anddraftedthepaper.NKS, CESandGRBcarriedoutbiologicalstudies.WRandGVperformed massspectrometryanalysisandSRCStochromatographicanalysis. FGSperformedacriticalreadingofthemanuscript.AGSconducted statisticalanalysis.RMKdesignedthestudy,supervisedthe labo-ratoryworkandofferedcriticalcommentsonthemanuscript.All authorshavereadthefinalmanuscriptandapprovedthe submis-sion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthankCAPESandCNPqforfinancialsupportand

Prof. Sandra Zorat Cordeiro, UNIRIO-RJ, for the plant specimen depositattheherbarium(Prof.JorgePedroPereiraCarauta).

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