Rev. bras. farmacogn. vol.26 número4

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RevistaBrasileiradeFarmacognosia26(2016)530–532

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

Short

communication

Larvicidal

activity

of

Ramalina

usnea

lichen

against

Aedes

aegypti

Antônio

S.N.

Moreira

a,b,∗

_,

_Roberta

_O.S.

_Fernandes

c

_,

_Francisco

_J.A.

_Lemos

d

_,

_Raimundo

_Braz-Filho

e

_,

Ivo

J.C.

Vieira

e

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_Naturais,_Universidade_Estadual_do_Norte_Fluminense_Darcy_Ribeiro,_Campos_dos_Goytacazes,_RJ,_Brazil b_Instituto_Federal_Fluminense,_Campus_Guarus,_Campos_dos_Goytacazes,_RJ,_Brazil

c_Departamento_de_Farmácia,_Faculdade_de_Medicina_de_Campos,_Campos_dos_Goytacazes,_RJ,_Brazil

d_Laboratório_de_{Biotecnologia,}_Centro_de_Biociências_e_{Biotecnologia,}_Universidade_Estadual_do_Norte_Fluminense_Darcy_Ribeiro,_Campos_dos_Goytacazes,_RJ,_Brazil e_Laboratório_de_Ciências_Químicas,_Centro_de_Ciência_e_Tecnologia,_Universidade_Estadual_do_Norte_Fluminense_Darcy_Ribeiro,_Campos_dos_Goytacazes,_RJ,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24November2015 Accepted9March2016 Availableonline5April2016

Keywords: Larvicidalactivity Lichenizedfungi Ramalinausnea Aedesaegypti

a

b

s

t

r

a

c

t

The larvicidal activity of the methanol extract, fractions and compounds 2-hydroxy-4-methoxy-6-propyl-methylbenzoateand(+)-usnicacididentifiedfromthelichenRamalinausnea(L.)R.Howe, Ramalinaceae,wastestedagainstthethirdinstarlarvaeoftheAedesaegyptimosquito.Themethanol extractandthreefractionsshowedactivity,killing100%and96.6%ofthelarvaeataconcentrationof 150␮g/mlat24h.Theisolatedcompounds,2-hydroxy-4-methoxy-6-propyl-methylbenzoateandthe

(+)-usnicacidshowedlarvicidalactivity,presentingLC50valuesof4.85and4.48␮g/ml,respectively.

ThisisthefirststudyofitskindreportingthelarvicidalactivityagainsttheA.aegyptimosquitowith compound(1).

Introduction

Mosquitoesarevectorsthattransmitmanydiseases,suchas malaria,encephalitis,denguefeveramongothers.Denguefeveris aneglectedtropicaldisease,currentlyrankedasthemost impor-tantviraldiseasetransmitted bymosquitoes worldwide(WHO, 2012).ThemosquitoAedesaegyptitransmitsdengueandisalso responsibleforthetransmissionofotherdiseases,suchasyellow fever,chikungunyafever(WHO,2015),andthefevercausedbyzika virus(MS,2015).Brazilis currentlyexperiencinga dengue epi-demicinseveralstatesand1,485,397suspectedcasesofdengue werereportedinthecountrywithabout19,380confirmedcases and761deaths(BoletimEpidemiológico,2015).

As there is no specific antiviral drug for the treatment of thesediseases,vectorcontrolis thebeststrategy (WHO,2015). Tosolvethisproblem,manyinsecticidesmaybeusedtocontrol mosquitoes,butmanyofthemarenotselectiveandcanharm ben-eficialinsects(Cetinetal.,2012),increasetheresistanceofthese insects(Benlietal.,2009),andproduceenvironmental contami-nation(Santosetal.,2011).In thesearchforalternativecontrol methodslessornon-toxictothepopulationandtheenvironment,

∗ Correspondingauthor.

E-mail:ansernam@iff.edu.br(A.S.N.Moreira).

severalstudieshavesurveyedplantsasalternativecontrolagents (GovindarajanandSivakumar,2014).Othernaturalsourcesinclude lichens(Cetinetal.,2012)andmarinenaturalproducts(Samidurai andSaravanakumar,2011),asthesenaturalsourceshave arich diversityofbioactivechemicalcompounds.Newmosquitocontrol methodshavebeentestedinBrazil.TheBritishbiotechcompany Oxitec,throughaninnovativeprojectinvolvinggenetic engineer-ing,producessterilemosquitoes,geneticallymodified,inorderto suppressthepopulationbythereleaseofinsectswithdominant lethality.Theresultof96%ofsuppressionwasobservedinJacobina, inthedistrictofBahia,wherethetestshavebeendonesince2013 (www.oxitec.com/).

Lichens consist of symbiosisbetween a fungus and a green algaorcyanobacteria.Theycontainalargenumberofsecondary compounds(HuneckandYoshimura,1996).Consequently,lichens havebeenusedassourcesofmanymetaboliteswithdifferent bio-logicalactivities(Ingólfsdóttir,2002)asantibiotic(Ingólfsdóttir, 2002),antiviral(Laietal.,2013),andlarvicidal(Cetinetal.,2012), among others. In the present study, we evaluated the larvici-dalactivityoftwocompoundsisolatedfromthelichenRamalina usnea(L.)R.Howe,Ramalinaceae, 2-hydroxy-4-methoxy-6-propyl-methylbeazoate(1)and(+)-usnicacid(2).Thisstudyisthefirst reportintheliteratureofthelarvicidalactivityagainstA.aegyptiof 2-hydroxy-4-methoxy-6-propyl-methylbenzoate(1)and MeOH-solubleextractsofthestudiedlichen.

http://dx.doi.org/10.1016/j.bjp.2016.03.006

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A.S.N.Moreiraetal./RevistaBrasileiradeFarmacognosia26(2016)530–532 531

Table1

Larvalmortality(inpercent)ofthemethanolextractofRamalinausnealichen, frac-tionsandcontrolagainstthird-instarofAedesaegyptiafter24h.

Concentration(␮g/ml)

150 30 15 10 5 1

MeOHextract 100 – – – – –

LM6 57 – – – – –

LM7 96.6 – – – – –

LM6-1 100 – – – – –

LM6-2 100 – – – – –

LM6-3 0 – – – – –

LM6-4 0 – – – – –

LM6-5 10 – – – – –

LM6-2-3(1) – 100 88.67 78 35.33 11.33

LM6-2-7(2) – 100 93.33 66.67 33.33 20

DMSO 0 0 0 0 0 0

DMSO/H2O 0 0 0 0 0 0

–,nottestedconcentration.

Materialsandmethods

1_H₍₅₀₀_MHz)_and13_C₍₁₂₅_MHz)_NMR_experiments_were

con-ducted ona Bruker spectrometer.IRanalyseswere made with a Shimadzu IRAffinity-1 instrument using KBr pellets. GC–MS analyzes were performed using a Shimadzu QP-5050A equip-ment.ColumnchromatographywasperformedonMercksilicagel (0.063–0.2mm)andMercksilicagel60F254wasusedforTLC.The lichenR.usnea(H.)L.Howe,Ramalinaceae,wascollectedonthe RestingaofIquipari,coastlineofGrussaíintheSãoJoãodaBarra municipality,RiodeJaneirostate,Brazil.Voucherspecimenswere identifiedbyDr.MichelNavarroBenatti,herbariumcuratorofthe BotanicalInstituteofSãoPauloanddepositedattheherbariumof theUniversidadeEstadualdoNorteFluminenseDarcyRibeiro, Bio-sciencesandBiotechnologyCenter(identificationnumberLI0001). Thelichen stems wasdried at roomtemperature and pow-dered.About550gofthedriedmaterialwasmaceratedwith3lof methanolforthreetimes.Afterfiltration,thesolventwasremoved under reduced pressure and it produced approximately60gof crudeextract.Asamplewastakentoperformpreliminary biologi-caltestsand35gwerefractionatedbycolumnchromatographyon ambientpressureusingsilicagel,andelutedwithhexane:CH2Cl2 withagradientofpolarity(100:0,95:5to0:100v/v),andfinalizing withCH2Cl2:MeOH(8:2v/v)toobtain8fractionsofapproximately 80mleach(LM1toLM8).FractionLM6(2.82g)wasfurther frac-tionatedbysimilarmethodstothosedescribedabove,andeluted withthesamepolaritygradientofhexane:CH2Cl2,obtainingsix newsubfractions(LM6-1toLM6-6).FractionLM6-2(689.6mg)was chromatographedthroughasimilarprocesstothatofLM6 produc-ingtennewsubfractions(LM6-2-1aLM6-2-10).FractionLM6-2-3 (89.5mg)wasanalyzedbyspectroscopicmethodsandidentified as2-hydroxy-4-methoxy-6-propyl-methylbenzoate(1).Fraction LM6-2-7(149mg)wassubjectedtopreparativeTLCelutedwith hexane:CH2Cl2(20:80v/v,threetimes)and,afteranalysisby spec-troscopicmethods,usnicacid(2)wasidentified.

ThemosquitoesA.aegypti(Diptera:Culicidae)wereobtained fromtheinsectaryoftheBiotechnologyLaboratory,Universidade EstadualdoNorteFluminenseDarcyRibeiro.Thelarvicidal toxic-ityassaysweredevelopedaccordingtostandardmethodologyof WorldHealthOrganization(WHO,2005).Theconcentrationofthe stocksolutionandthesolutionstestedwerechosenbasedon pre-liminarytests.ThecompoundsweresolubilizedinDMSO/H2O(1)

orDMSO(2).Fifteen thirdinstarlarvaewereaddedtothepots containingdistilledwaterandtestsolutionsatconcentrationsthat werebetween1and30␮g/mlforatotaloffiveconcentrations,at

roomtemperature.Thetestswereperformedintriplicateandin tworeplicates.Negativecontrolswerewater,DMSOandasolution ofDMSO/H2O(2.5%).Forpositivecontrol,imidaclopridwasusedat

concentrationsbetween0.01and1.0␮g/ml.Assessmentof

mortal-itywasmade24hafterexposureofthelarvaetothetestsolutions. DatawereanalyzedusingtheanalysisprogramProbit/EPAversion 1.5tocalculatethe50%(LC50)and90%(LC90)lethalconcentrations

aswellastheconfidencelimitforeachtreatmentintheconfidence levelof95%(http://www.epa.gov/nerleerd).

Resultsanddiscussion

Preliminarybioassays withcrude methanolextractand frac-tionswereundertakenataconcentrationof150␮g/ml.Thecrude

extract(LM)produced100%ofmortalityinthelarvaeofthethird instarofA. aegyptiafter24h, thefractionLM657%,LM796.6%, and100%forallfractionsLM6-1(97.8mg)andLM6-2(689.6mg). Theotherfractionsofthiscolumnproducedlowmortalityrates. However,subfractionsLM6-2-3(1)andLM6-2-7(2)showed activ-ity(Table1).Themortalitydataatvariousconcentrationsallowed the determination of the LC50 and LC90 values (Table 2) for

purecompounds1and2andshowedthatthisactivityoccurred inaconcentrationdependentmanner. 2-Hydroxy-4-methoxy-6-propyl-methyl benzoate(1)displayedvalues of11.3, 35.3,78.0, 88.7and100%ofmortality,whileforusnicacid(2)wereobtained 20.0,33.3,66.7,93.3and100%,atconcentrationsof1.0,5.0,10.0, 15.0and30.0␮g/ml,respectively,after24h(Table1).Thevaluesfor

LC50were4.85and4.48forcompounds1and2,respectively,while

theLC90were17.5and20.7(Table2).Therefore,thesecompounds

haveapotentialasnewleadsforlarvicidalnaturalproducts. 2-Hydroxy-4-methoxy-6-propyl-methylbenzoate(1):dark yel-lowamorphous solid;mp122.5–123.8◦_C;_IR_(KBr,_cm−1_):_3435,

2924,2852,1726,1624,1373,1192,960;1_H_NMR_(CDCl

3,500MHz,

TMS),ı6.34(1H,s,H-4),6.29(1H,S,H-6),2.82(2H,t,J=7.0Hz, CH2-7),1.54(2H,m,CH2-8),0.95(3H,t,J=7.0,CH3-9),3.80(3H,s,

C5-OCH3),3.92(3H,s,C10-OCH3),11.76(1H,s,C3-OH);13CNMR

(CDCl3,125MHz,TMS),ıC(ppm):104.6(C-1),163.8(C-2),98.7

(C-3),165.5(C-4),110.7(C-5),55.2(OCH3-4),51.8(OCH3-10).NMR

dataandmeltingpointareinagreementwiththosereportedinthe literature(HuneckandYoshimura,1996).

Usnic acid (2): yellow crystalline solid; [˛]D+494.2 (25◦C,

CHCl3,c1.00),mp202.5–204◦C;IR(KBr,cm−1):3662,3088,1695,

1452,1373,1286,1190,1118,1037,956, 817;1_H _NMR_(CDCl 3,

500MHz,TMS),ı6.05(1H,s,H-4),1.79(3H,s,CH3-10),2.68(3H,

s,CH3-12),2.08(3H,s,CH3-13),2.71(3H,s,CH3-15),13.34(1H,s,

C7-OH),11.06(1H,s,C9-OH).13_C_NMR_(CDCl

3,125MHz),ı198.1

(C-1),105.2(C-2),191.8(C-3),179.3(C-4a),155.2(C-5a), 101.5 (C-6),163.9(C-7),109.3(C-8),157.5)(C-9),104(C-9a),59.1(C-9b), 31.9(C-10).NMRdataandmeltingpointareinagreementwith thosereportedintheliterature(Rashidetal.,1999).

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532 A.S.N.Moreiraetal./RevistaBrasileiradeFarmacognosia26(2016)530–532

Table2

TheLC50andLC90values(␮g/ml)compounds2-hydroxy-4-methoxy-6-propyl-methylbenzoate(1),usnicacid(2)andcontrolImidacloprideagainstthird-instarofAedes aegyptimosquitolarvaeafter24h.

Compound LC50(limits) LC90(limits) Slope(±SE)(limits)

1 4.85(2.93–6.96) 17.5(11.7–36.3) 2.30(0.46)(1.4–3.2)

2 4.48(2.51–6.73) 20.7(12.8–51.5) 1.9(0.4)(1.1–2.7)

Control 0.0412(0.02–0.062) 0.095(0.06–0.26) 0.036(0.011)(0.014–0.058)

Conclusion

From this study, the larvicidal activity of the compounds 2-hydroxy-4-methoxy-6-propyl-methyl benzoate (1) and usnic acid(2)wasdemonstrated.Thesecompoundscouldbeleadsforthe developmentofnewsyntheticmoleculeswithlarvicidalactivityfor thecontrolofA.aegypti.

Authors’contributions

ASNM,ROSFandFJALcontributedtotheexperimentalworkand wrotethemanuscript.RBFandIJCVcontributedtotheNMRspectral analysis.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest

Acknowledgments

TheauthorsaregratefultoFAPERJ forgrantsand aresearch fellowship.Thanksaredue toCNPqandCAPES forresearch fel-lowships.WealsothankDr.MichellNavarroBenatti,Institutode BotânicadeSãoPaulo,fortheidentificationofthelichen.

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