Rev. bras. farmacogn. vol.25 número2

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w w w. s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

In

vitro

anti-

Leishmania

infantum

activity

of

essential

oil

from

Piper

angustifolium

Lauriane

S.S.

Bosquiroli

a

_,

_Daniel

_P.

_Demarque

b

_,

_Yasmin

_S.

_Rizk

a

_,

_Marillin

_C.

_Cunha

c

_,

Maria

Carolina

S. Marques

d

_,

_Maria

_de

_Fátima

_C.

_Matos

c

_,

_Mônica

_C.T.

_Kadri

e

_,

Carlos

A. Carollo

b

_,

_Carla

_C.P.

_Arruda

a,∗

a_Laboratório_de_{Parasitologia}_Humana,_Centro_de_Ciências_Biológicas_e_da_Saúde,_Universidade_Federal_de_Mato_Grosso_do_Sul,_Campo_Grande,_Brazil

b_Laboratório_de_{Farmacognosia,}_Centro_de_Ciências_Biológicas_e_da_Saúde,_Universidade_Federal_de_Mato_Grosso_do_Sul,_Campo_Grande,_Brazil

c_Laboratório_de_Biologia_Molecular_e_Culturas_Celulares,_Centro_de_Ciências_Biológicas_e_da_Saúde,_Universidade_Federal_de_Mato_Grosso_do_Sul,_Campo_Grande,_Brazil

d_Laboratório_de_{Microbiologia,}_Centro_de_Ciências_Biológicas_e_da_Saúde,_Universidade_Federal_de_Mato_Grosso_do_Sul,_Campo_Grande,_Brazil

e_Laboratório_de_{Biofisiofarmacologia,}_Centro_de_Ciências_Biológicas_e_da_Saúde,_Universidade_Federal_de_Mato_Grosso_do_Sul,_Campo_Grande,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received27August2014 Accepted15March2015 Availableonline31March2015

Keywords:

Visceralleishmaniasis Chemotherapy Naturalproducts Sesquiterpenes

a

b

s

t

r

a

c

t

PiperangustifoliumLam.,Piperaceae,popularlyknownas“matito”,“pimenta-de-macaco”,

“pimenta-longa” or “jagurandi” in Brazil, has been commonly used in the treatment of cutaneous

leishmaniasis-associatedlesions,buttherearefewstudiesontheactivityagainstvisceral leishmaniasis-associatedspecies.ThisstudydemonstratesthefirstinvitroantileishmanialactivityoftheP.angustifolium essentialoil,ofwhichthephytochemicalprofileshowedthepresenceofsesquiterpenesand monoter-penes.Themaincompoundswerespathulenol(23.8%)andcaryophylleneoxide(13.1%).P.angustifolium essentialoilwashighlyactive[thehalfmaximuminhibitoryconcentration=1.43␮g/ml]against intra-cellularamastigotesofLeishmaniainfantum,theetiologicalagentofvisceralleishmaniasisintheNewand OldWorld.Activitywasobtained24hafteradditionoftheoil(6.25–50␮g/ml),withareductionof100% intheinfectionindexatconcentrationsof25and50␮g/ml.P.angustifoliumessentialoilshowedlow cytotoxicityformammalianfibroblastsandmacrophages(thehalfmaximuminhibitoryconcentration valuesof31.67and48.22␮g/ml,respectively),anditwas33and22timesmoretoxictoamastigotesthan tomammaliancells,asindicatedbyselectivityindexes.TheresultsdemonstratedthatP.angustifolium essentialoilisapromisingalternativeforthestudyofpotentialdrugsforvisceralleishmaniasis.

Introduction

Visceral leishmaniasis(VL)is a severe systemic chronic dis-easecaused bythe Leishmaniadonovani complex inEastAfrica andtheIndiansubcontinent,andLeishmaniainfantuminEurope, northernAfricaandLatinAmerica(Lukesetal.,2007).Itisoneof themostimportantneglecteddiseasestoday,givenitshigh inci-denceandmortality,especiallyamonguntreatedindividualsand malnourishedchildren. It isalsoconsidereda diseaseemerging inimmunocompromisedindividuals(JarvisandLockwood,2013). Evenduringtreatment,acase-fatalityratioof10–20%isestimated (Collinetal.,2004).

Thefirst choice drugs in thetreatment of leishmaniasisare thepentavalentantimonials(WHO,2010).Thesedrugs,however,

∗ Correspondingauthor.

E-mail:carla.arruda@ufms.br(C.C.P.Arruda).

presentsignificantlimitationsconcerningtheirtherapeuticsafety astheyhave ahighleveloftoxicity. Highfrequency ofadverse effectsandteratogenicriskhavebeendescribed(Mirandaetal., 2006).LipidformulationsofamphotericinB,miltefosineand paro-momycinhavebeenapproved,butthecorrectdoseandefficacyof thesedrugshavenotbeenproveninallendemicareasofthe dis-ease(WHO,2010).Combinationsofthesedrugshavebeenrequired inthecasesofinfectionsresistanttoantimony(SeifertandCroft, 2006).Consideringthescenarioofresistancetothemaindrugsin use,researchershave beenstudyingnew,lesstoxicdrugs, with greateravailabilityandwithinreachoftheunderprivileged popula-tionaffectedbythedisease(Freitas-Junioretal.,2012).Inthesearch fornewandbettercompounds,productsofplantoriginhavebeen testedsincetheyareeasilyobtainedatlowcost(LiandVederas, 2009).

Piper is one of thegenera of great ecological and economic importancewithinthePiperaceaefamily(Monzoteetal.,2010). SeveralclassesofcompoundshavebeenisolatedfromPiperspecies,

http://dx.doi.org/10.1016/j.bjp.2015.03.008

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includingthosewithantileishmanialactivitysuchasbenzoicacids (Flores et al., 2007, 2009), dillapiole (Parise-Filho et al., 2012), and adunchalcone(Dal Picoloetal., 2014).Moreover,in recent years itsessential oilshave becomean importanttarget inthe searchfornewtherapeuticoptionsagainstparasites(Antonyetal., 2005).

Piper angustifolium Lam. (sin.=Piper aduncum L.), popularly known as “matito”, “pimenta-de-macaco”, “pimenta-longa” or “jagurandi”inBrazil,isaplantnativetotropicalregionssuchas theSouthandCentralAmericas,Asia,andthePacificOceanislands (Martínez etal., 2003).Itsleavesare usedin folkmedicine for thetreatmentofstomatitis,vaginitis,erysipelasand liver disor-ders(LorenziandMatos,2002)orasanantiseptic,antidiarrheal, tonic,astringent,andantirheumaticmedicine,andalsoasaninsect repellent (Schaus and Desmarchelier, 2000).In addition,it has beencommonlyusedinthetreatmentofcutaneous leishmaniasis-associated lesions(Martínezet al., 2003), which motivatedthe developmentofsomeworksonLeishmaniaspeciesassociatedwith thisclinicalformofthedisease(Bragaetal.,2007).Incontrast,few studieshavebeenconductedontheactivityagainstLV-associated speciesanduptonow,therehasbeennoresearchontheessential oilextractedfromthisplant.Thus,theaimofthisstudywasto char-acterizethechemicalcompositionoftheP.angustifoliumessential oil(PAEO)andevaluateitsactivityagainstL.infantum.

Materialsandmethods

Plantmaterial

PiperangustifoliumLam.,Piperaceae,wascollectedinJanuary 2014fromtheAbobralSubregionofthePantanalofMatoGrosso doSul.AftertheidentificationperformedbyDr.GeraldoAlves Dam-ascenoJunior,abotanicalvoucherwasdepositedintheherbarium ofCampoGrande/MS,Brazil,undernumber20182.

Extractionandanalysisoftheessentialoil

TheP.angustifoliumessentialoil(PAEO)wasextractedfromthe plantleavesbydistillationinaClevengerapparatus(Vidrolex).The oilwasdriedwithanhydroussodiumsulfate(Vetec,RiodeJaneiro, Brazil)withyieldof0.41%(w/w),andstoredat−5◦_C_in_a_sealed

containeruntilthetimeofanalysis.Thechemicalcompositionwas determinedbygaschromatography–massspectrometry(GC–MS) usingaShimadzuQP2010PlussystemwithaRTX5MScapillary col-umn(30m×0.25mm×0.25␮m).Nitrogenwasappliedascarrier gasusingaflowrateof1.13ml/min.Theconstituentswere con-firmedbycomparisonwithlibrariesandcalculationoftheKovats index.

Parasites

Thestandard strainMHOM/BR/1972/BH46 ofL. (Leishmania)

infantumwasusedforinvitroassaysofantileishmanialactivity. Theamastigotes wereroutinely isolatedfrom Goldenhamsters (Mesocricetusauratus)andmaintainedaspromastigotesin Schnei-der’sInsectMedium(Sigma)supplementedwith20%fetalbovine serum(FBS,Sigma)and140␮g/mlgentamicin(Sigma)at26◦_C._On

the7thdayofcultivation,promastigotesfromuptothreeserial passagesafterisolationwereusedintheexperiments.

Animals

BALB/cmiceagedsixweekswereusedtoobtaintheperitoneal cellsusedintheantileishmanialactivityassays.Theanimalswere obtainedfromthecentralanimalfacilityoftheCenterfor Biolog-icalandHealthSciences(CCBS)oftheFederalUniversityofMato

GrossodoSul(UFMS,Brazil)ingoodhealthandfreeofinfections orparasitescommontorodents,maintainedinindividually venti-latedcagesequippedwithmini-isolators,andfedabalancedfeed (NuvilabCR-1,Nuvital®₎_with_free_access_to_water._The_study_was

approvedbytheEthicsCommitteeonAnimalUse–CEUA/UFMS, underprotocol432/2012.

ActivityagainstintracellularamastigotesofL.infantum

PeritonealmacrophagesfromBALB/cmicewereisolatedafter rinsingwithRPMI1640medium(Sigma)andplacedina24-well plate(1×105_cells/well)_in_RPMI ₁₆₄₀_medium_(Sigma)

supple-mentedwith10%FCS(Cultilab)and140␮g/mlgentamicin(Sigma). Afterincubationat37◦_C_for₁_h,_cells_were_infected_with_L._infantum

promastigotes(1×106_cells/well)_and_incubated_at₃₅◦_C _for₄_h.

PAEOwasaddedatconcentrationsof6.25–50␮g/mlinsetsof sex-tuplicateexperiments.Untreatedinfectedcellsandamphotericin B(Sigma)wereusedasnegativeandpositivecontrol,respectively. Thecellswereincubatedat37◦_C_in_5%_CO₂_,_fixed_and_stained_with

Giemsaafter24h.Thepercentageofinfectedmacrophagesandthe totalnumberofamastigotesweredeterminedbycounting200cells sixfold.Theinfectionindexwasdeterminedbymultiplyingthe per-centageofmacrophagesthathadatleastoneintracellularparasite bythemeannumberofamastigotespermacrophage,asdescribed byPaladietal.(2012).Anonlineardose–responseregressioncurve wasusedtocalculatethehalfmaximuminhibitoryconcentration (IC50).Theresultswereexpressedasthemean±standard

devia-tion(SD)andthedatawereanalyzedusingtheStudent’st-test. Differenceswereconsideredsignificantatp<0.05(representedby anasterisk).

Nitricoxide(NO)evaluation

To evaluate the production of NO by the infected periton-eal cells, supernatants of the aforementioned cultures (100␮l) were collected after 24h of treatment and incubated with an equal volume of Griess Reagent (1% sulfanilamide/0.1% (naph-thyl)ethylenediaminein5%phosphoricacid)atroomtemperature for 10min. The accumulation of nitrite was quantified accord-ingtoDingetal.(1988)andtheabsorbancewasdeterminedat 540nm.Absorbancewasconvertedto␮MofNO2−bycomparing

the samples witha standard curve obtainedwith known con-centrations(1–10␮M)ofsodiumnitritedilutedinRPMImedium. Theresultswereexpressedasthemean±standarddeviation(SD). The data were analyzed using the Student’s t-test and differ-enceswereconsideredsignificantatp<0.05(representedbyan asterisk).

Cytotoxicityassay

Fibroblast(NIH/3T3) and murinemacrophage(J774.A1) cells purchasedfromtheRiodeJaneiroCellBank(Brazil)weretreated withPAEO at concentrations of 0.25–250␮g/ml in triplicate to estimateIC50.PAEOwasdissolvedinDMSO(dimethylsulfoxide

sodium)anddilutedincompletemedium(thehighest concentra-tionofDMSOusedinthetestwas0.25%,anddidnotaffectcell viability).Cellsinculturemediumwereusedasnegativecontrol andamphotericinB(0.025–25␮g/ml)aspositivecontrol.Cell via-bilitywasdeterminedusingthesulforhodamineBassay(Skehan etal.,1990).Thepercentageofgrowthofeachtest samplewas calculatedasdescribedbyMonksetal.(1991).TheIC50was

deter-minedbynonlinearregression(MicrocalOriginVersion6.0and MicrosoftOfficeExcel2007).Selectivityindex(SI)wascalculated bythefollowingformula:IC50onmammaliancells/IC50on

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Table1

ChemicalconstitutionoftheessentialoilfromPiperangustifolium.

Compound % Retentiontime TheoreticalKI CalculatedKI

1 ␣-pinene 5.87 4.64 939 932

2 camphene 0.53 5.05 946 947

3 cymene 2.77 7.10 1025 1032

4 limonene 4.27 7.20 1032 1037

5 limoneneoxide 0.64 8.50 1137 1103

6 cis-verbenol 0.32 8.62 1135 1113

7 cryptone 1.75 9.67 1186 1193

8 cuminaldehyde 1.29 10.24 1242 1249

9 p-cymen-7-ol 0.70 10.71 1291 1297

10 ␦-elemene 1.11 11.14 1339 1339

11 ␣-copaene 0.44 11.55 1393 1388

12 ␤-elemene 4.01 11.68 1389 1402

13 trans-caryophyllene 2.24 12.04 1444 1437

14 aromadendrene 1.80 12.25 1469 1455

15 longifolene 4.5 12.59 1487 1405

16 ␣-muurolene 3.56 12.91 1512 1500

17 ␥-cadinene 3.74 13.13 1534 1526

18 cis-calamenene 0.50 13.24 1543 1535

19 nerolidol 5.80 13.63 1562 1561

20 isospathulenol 1.67 14.04 1589 1630

21 spathulenol 23.78 14.15 1578 1598

22 caryophylleneoxide 13.06 14.28 1613 1605

23 viridiflorol 0.51 14.41 1620 1610

24 torreyol 3.81 15.24 1643 1651

25 ␣-cadinol 4.37 15.52 1656 1665

KI,Kovatsindex.

Resultsanddiscussion

Twenty-five constituentswere identified in theessential oil from P. angustifolium, representing 93.04% of the total com-pounds.Themajorityoftheseweresesquiterpenesandthetwo majorcompoundsidentified,spathulenolandcaryophylleneoxide, correspondedto36.8%ofthetotalcompounds(Table1).The phyto-chemicalprofileofPAEOdifferedfromthatfoundbyAlmeidaetal. (2009),whoobservedthepredominanceofaphenylpropanoid (dil-lapiole–86.9%)intheessentialoilfromtheaerialpartsoftheplant collectedinPará,Brazil.Parise-Filhoetal.(2012)alsofound dil-lapioleasthemajorcompoundofessentialoilfromtheleavesof thesameplantcollectedinSãoPaulo,Brazil.Tirillinietal.(1996), whocollectedthesameplantinPeru,nearCuzco,observedthe monoterpenescamphor(25.3%)andcamphene(22.4%)tobethe mainconstituents.

Theantileishmanialactivityofseveralsesquiterpeneshasbeen describedintheliterature(Mikusetal.,2000;Santosetal.,2008; Arrudaetal.,2005).Valadeauetal.(2009)demonstratedthe activ-ity of sesquiterpene-rich ethanolic extract from Piper dennisii;

Monzoteetal.(2010)foundanIC50of22.3␮g/mlfortheessential

oilfromP.auritumagainstL.donovaniamastigotes.Themethanolic eugenol-richextractfromPiperbetlewasactiveagainstL.donovani

amastigotesandpromastigotes(Misraetal.,2009).Other sesquiter-peneswithleishmanicidalactivityhavepreviouslybeenreported, asshownbyMarquesetal.(2011),whoidentifiedE-nerolidolinthe essentialoilfromPiperclaussenianum(83%).Whentestedagainst

Leishmaniaamazonensisarginase,itshowedanenzymeinhibition of62.2%.Oliveiraetal.(2014)testedtheessentialoilfrom Boca-geopsismultiflorawith16.2%ofspathulenolagainstL.amazonensis

promastigotes.

Thisstudypresentsthefirstdescriptionofanti-L.infantum activ-ityassociatedwiththeessentialoilobtainedfromP.angustifolium. Ourresultsdemonstratedadose-dependentincreaseinthe inhi-bitionoftheintracellularamastigoteproliferation24hafterthe oilwasaddedtoinfectedcells.Theinfectionindexdecreased,in arangefrom88.1to100%fromthelowesttothehighest concen-tration,incomparisonwithuntreatedinfectedcells(Fig.1).TheIC thatreduced50%oftheintracellularformsof L.infantum(IC50)

Control 800

Concentration (μg/ml)

*

Inf

ection inde

x ₆₀₀

400

200

0

6.25μg/ml

12.5μg/ml

25μg/ml

50μg/ml

Fig.1.AntileishmanialactivityofPAEOonL.infantumintracellularamastigotes. Barsrepresentthemean±SDofsextuplicates.*p<0.01,forthedifferent concentra-tionscomparedtountreatedcells(control)(Student’st-test).

was1.43␮g/mlwithlowcytotoxicitytomammaliancells com-paredwithamphotericinB(Table2).WithregardtoSI,PAEOwas 33and22timesmorecytotoxictointracellularamastigotesthanto NIH/3T3andJ774.A1cells,respectively(Table2).Although ampho-tericinBwasaboutfourtimesmoreactivethanPAEO,thisreference drugwasnotasselectiveastheoil,sinceitwasmorecytotoxicto mammaliancells(Table2).

Activation of macrophages was investigated by NO release (Fig.2).Asignificantincrease(p<0.05)inNOreleasewasfound aftertreatmentwithPAEOatconcentrationsof6.25and12.5␮g/ml, suggestingthatitsactivitymaybeassociatedwiththis antileishma-nialmechanism(Stengeretal.,1994).Atconcentrationsof25and 50␮g/ml,however,PAEOdidnotinduceasignificantincreasein NOrelease,showinganatypicalresultthatmaybeduetothe pres-enceofcertaincompoundsintheoil.Theliteraturehasreported severalcompoundsinducingthisbiphasicresponse(Nothnickand Soloway,1998;O’Flahertyetal.,1989,1990;Adedapoetal.,2009; Linneretal.,1993;Calabrese,2005).

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Table2

EffectofPAEOonintracellularamastigotesofLeishmaniainfantum,cytotoxicitytomammaliancellsandcorrespondingselectivityindex(SI).

Testsamples Intracellularamastigotes NIH/3T3 J774.A1

IC50(␮g/ml)a IC50(␮g/ml)a SIb IC50(␮g/ml)a SIb

PAEO 1.43 48.22 33.72 31.67 22.15

AmphotericinB 0.33 2.19 6.63 4.32 13.09

a_IC

50,halfmaximuminhibitoryconcentration. b_SI,_selectivity_index:_IC

50onmammaliancells/IC50onintracellularamastigotes.

Control 5

4

3

2

1

0

Concentration (μg/ml)

μ

M NO

2

–

*

6.25μg/ml

12.5μg/ml

25μg/ml

50μg/ml

Fig.2.EffectofadditionofdifferentconcentrationsofPAEOontheproduction ofnitricoxidebyperitonealcellsinfectedwithL.infantum.Infectedcells with-outtreatmentwereusedascontrols.Thedatarepresentmean±standarddeviation ofquadruplicates.*p<0.05forthedifferentconcentrationsofPAEOversuscontrol (Student’st-test).

Authors’contributions

LSSB,YSRand MCC(MScstudents)contributed tobiological studies.DPD(PhDstudent)contributedbycollectingplant sam-ples,andperformingchromatographicanalysis.MCTKcontributed totheNOevaluationtest.MFCMcontributedtothecytotoxicity test.MCSMcontributedtocriticalreadingofthemanuscript.CCPA andCACdesignedthestudy,supervisedthelaboratoryworkand contributedtocriticalreadingofthemanuscript.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthorsthankthe(CNPq)forthefinancialsupportreceived, andDr.GeraldoAlvesJuniorDamasceno,foridentificationofthe species.

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