Rev. bras. farmacogn. vol.25 número3

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

Antigiardial

activity

of

flavonoids

from

leaves

of

Aphelandra

scabra

Gloria

Ivonne

Hernández-Bolio

_,

_Luis

_Wiliunfo

_Torres-Tapia

_,

_Rosa

_Moo-Puc

_,

Sergio

Rubén

Peraza-Sánchez

a_{UnidaddeBiotecnología,CentrodeInvestigaciónCientíficadeYucatán,Col.ChuburnádeHidalgo,Mérida,Yucatán,Mexico}

b_{UnidaddeInvestigaciónMédicaYucatán,UnidadMédicadeAltaespecialidad,CentroMédicoIgnacioGarcíaTéllez,InstitutoMexicanodelSeguroSocial,Col.Industrial,}

Mérida,Yucatán,Mexico

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26January2015 Accepted30April2015 Availableonline18June2015

Keywords: Aphelandrascabra Acanthaceae Anti-giardialactivity Cirsimaritin Sorbifolin Flavonoids

a

b

s

t

r

a

c

t

Aphelandrascabra(Vahl)Sm.,Acanthaceae,isashrubwidelyusedbysomeMayancommunitiesas carmi-native,antidote,andremedyforsomeinfections.Bio-guidedisolationofthemethanolextractofleaves ledustothepurificationoftheanti-giardialmetabolitescirsimaritinandsorbifolin,alongwiththe inac-tivemetabolitescirsimarin,sorbifolin-6-O-␤-glucopyranoside,andsqualene.Cirsimaritindisplayedhigh activityintheanti-giardialbioassaywithanIC50=3.8␮M,beingconsideredasoutstandingwhen

com-paredtopreviousreportedmetabolites,whilesorbifolinshowedalowactivitywithanIC50=75.6␮M.

Additionally,bothcompoundsprovednottobecytotoxicinaninvitrobioassayagainstHEK-293,a nor-malcellline.Thisisthefirstinvestigationonanti-giardialpropertiesofA.scabraanditsphytochemistry aswell,thustheisolatedcompoundsareconsideredasnewfortheplantgenusandforthespecies.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Giardiasis caused by the flagellated protozoan Giardia lam-blia(G.intestinalis,G. duodenalis)isthemostcommonparasitic diseaseaffectinghumansworldwide(Vázquez-Tsujiand

Campos-Rivera,2009).Itcausesanestimated2.8×108_{casesperyear(Lane}

andLloyd,2002).In Mexico,theNationalSystemfor

Epidemio-logicSurveillancereported20,599casesin2010(SINAVE,2010); whereasYucatanshowsanincidenceof15.2%(Mantilla-Morales

etal.,2002).

Symptomsof giardiasis includeabdominal crampsand pain, and diarrhea, which can lead to malabsorption and failure of children to thrive (Tejman-Yarden and Eckmann, 2011). A varietyofchemotherapeuticagentssuchas5-nitroimidazole com-pounds, quinacrine, furazolidone, paromomycin, benzimidazole compounds, and nitazoxanide have been used in the therapy for giardiasis. Nevertheless,mostdrugs used haveconsiderable adverseeffectsandinadditionGiardiaseemstohaveagreatability toresisttheseagents(Bussatietal.,2009).Thus,thediscoveryof new,effective,andsafeantigiardialagentsisimperativefromother sources(Peraza-Sanchezetal.,2005).

Aphelandrascabra(Vahl.)Sm.,Acanthaceae,aplantdistributed inthesoutheastofMexico,CentralandSouthAmerica(Floradigital:

∗ Correspondingauthor.

E-mail:speraza@cicy.mx(S.R.Peraza-Sánchez).

PeninsuladeYucatan,2010),isusedinMayanQ’eqchi’traditional

medicinetotreatseveralconditions,includingcirculatory,mental, andnervoussystemdisorders,poisoning,andinfections(Treyvaud etal., 2005).InHonduras,a beveragemadefrommashedroots and/orleavessoakedincoolwaterisusedasanantiflatulent(Lentz, 1993).Antimicrobialandleishmanicidalactivitiesofthemethanol extractfromthisplanthavebeenreported(Peraza-Sánchezetal.,

2007; Meurer-Grimesetal., 1996), therefore,a study was

con-ductedinordertoinvestigatetheanti-giardialpropertiesofthis species.

Inthisstudy,wereporttheisolationoffourknownflavonoids andatriterpenefromtheleavesofA.scabraandtheiranti-giardial activity.Itisworthmentioningthatthisisthefirstphytochemical investigationofA.scabra.

Materialsandmethods

Generalexperimentalprocedures

MeltingpointsweredeterminedusingaMelt-TempII Appara-tus(LaboratoryDevices,USA).GC–MSdataweredeterminedonan Agilent6890Ngaschromatographcoupledtoa5975Bmass spec-trometer.1_{HNMR(400MHz)and}13_{CNMR(100MHz)datawere} obtainedona BrukerAvance400spectrometer.Chemicalshifts werereferredtoTMS(ı0).IRspectrawererecordedasKBrpellets onaNicoletProtegé460.UVspectrawereperformedonaGenesys 10 UVThermoSpectronic.Vacuumliquid chromatography(VLC)

http://dx.doi.org/10.1016/j.bjp.2015.04.004

Table1

Yieldandanti-giardialactivityofmethanolextractandfractionsofA.scabra.

Sample Yield[g(%)] IC50(␮g/ml)

MeOHextract(ASH-1) 435.0(14.41)a _11.74

±1.12c Hxfraction(ASH-2a) 21.5(4.94)b _NAd DCMfraction(ASH-2b) 4.3(0.99)b _2.19±0.67 EtOAcfraction(ASH-2c) 4.6(1.05)b _NA Aqueousfraction(ASH-2d) 62.0(14.25)b _NA

Metronidazole 0.2±0.02

a_{Yieldbasedoninitialweightofdriedmaterial.}

b _{Yieldbasedonweightofmethanolextract.}

c _{Standarddeviation.}

d _Notactive.

wasperformedundervacuumusingsilicagel60GF254(200–400 mesh,Sigma–Aldrich).Thinlayerchromatography(TLC)was car-riedoutonsilicagelF254 (Merck)andvisualizedunderUVlight andbysprayingwithphosphomolybdicacidreagentfollowedby heating.

Plantmaterial

Aphelandrascabra(Vahl.)Sm., Acanthaceae,leaveswere col-lectedfromits natural habitat,in thelocality of Piste, Yucatan (Mexico),inJuly2010.Avoucherspecimen(PSima-3019), identi-fiedbyaqualifiedtaxonomist,wasdepositedatCICY’sUNajilTikin Xiuherbarium.

Extractionandisolation

Grounddriedleaves(3018g)wereextractedwithmethanolfor 24handthensolventwasevaporatedundervacuum.Thisprocess wasrepeatedonceagain.Themethanolextract(ASH-1,435g)was partitionedwithhexane(Hx),dichloromethane(DCM)andethyl acetate(EtOAc),forthreetimeseachone.Solventwasevaporated undervacuum.Resultingfractionsincludingaqueousphase (ASH-2a–ASH-2d)wereplacedinseparatevialsandweighted(Table1). Thehexane fractionASH-2a (5g) was furtherfractionatedin a vacuumliquid chromatography(VLC)columnusingmixtures of Hx,acetone(An),EtOAc,andMeOHtoobtainfivefractions (ASH-3a–ASH-3e).FractionASH-3awaspurifiedbyachromatographic column(CC)usingHxtoaffordsqualene(1,22.5mg).TheDCM frac-tionASH-2b(4g)wasfurtherfractionatedbyVLCusingmixturesof Hx,Hx/An,andAn/MeOHtoobtainelevenfractions (ASH-4a–ASH-4k).CrystallizationwithMeOHoffractionsASH-4d–ASH-4iyielded cirsimaritin(2,465.6mg).TheEtOAcfractionASH-2c(3.8g)was fractionated by VLC using mixtures of same solvents used to fractionateDCMextractyieldingninefractions(ASH-6a–ASH-6i). CrystallizationwithMeOHoffractionASH-6hgavecirsimarin(3, 55.1mg);meanwhile,crystallizationwithMeOHoffractionASH-6c yieldedsorbifolin(4,11.3mg).FractionASH-6iwaspurifiedbyVLC usingmixturesofHx/DCM,DCM/An,andDCM/MeOHtoobtainten fractions(ASH-23a–ASH-23j).TheMeOHcrystallizationoffraction ASH-23fledtotheisolationofsorbifolin-6-O-␤-glucopyranoside (5,5.3mg).Puritiesoftheisolatedflavonoidswereverifiedthrough TLCanddeterminationoftheirmeltingpoints,whilepurityof com-pound1wasdeterminedbymeansofCG-EM.

Squalene(1):yellowoil;GC–MSRt=13.168min,m/z(%):410 ([M+H]+_{)(1),341(3),273(1),137(14),95(18),41(37),69(100,} basepeak).

Cirsimaritin(2):yellowneedles;Rf(Hx/An;2:1,2×):0.37,m.p. 253.8–256.2◦_{C.UV(An:H2O;99:1)maxnm(logε):209(3.21),330}

(3.39).IRmax(KBr)cm−1:3283,1655,1600,1570,1465,1445, 1356,1037,853.1_{HNMR (400MHz,DMSO-d}

6)ı3.72(3H,s,

6-OCH3),3.90(3H,s,7-OCH3),6.81(1H,s,H-3),6.87(1H,s,H-8),6.92 (2H,d,J=8.8Hz,H-3′_,H-5′_{),7.93(2H,d,J=8.8Hz,H-2}′_,H-6′_),12.91

(1H,s,5-OH).13_{CNMR(100MHz,DMSO-d}

6)ı55.7(6-OCH3),59.7 (7-OCH3),101.4(C-8),103.0(C-3),104.6(C-10),114.7(C-3′),116.3 (C-5′_),120.7(C-1′_),127.3(C-2′_),128.9(C-6′_{),131.4(C-6),151.6}

(C-9),152.2(C-5),158.1(C-7),160.8(C-4′_{),163.6(C-2),181.7(C-4).}

Cirsimarin(3):whiteamorphouspowder;Rf (CH2Cl2/MeOH; 4.5:0.5,3×):0.50,m.p.175.8–176.3◦_{C.UV(MeOH)maxnm(logε):}

213(4.89),277(4.72),323(4.75).IRmax(KBr)cm−1:3350,1660, 1600,1565,1460,1440,1356,1047,833.1_HNMR(400MHz,

DMSO-d6)ı3.20–3.71 (6H,sugarH),3.74(3H,s, 6-OCH3),3.93(3H,s, 7-OCH3),4.65(1H,s,6′′-OH),5.05(1H,d,J=7.1Hz,H-1′′),5.12(1H, s,4′′_{-OH),5.20(1H,s,3}′′_{-OH),5.45(1H,s,2}′′_{-OH),6.97(1H,s,}

H-3),6.98(1H,s, H-8),7.20 (2H,d, J=8.8Hz, H-3′_,H-5′_),8.08(2H,

d,J=8.8Hz,H-2′_,H-6′_{),12.86(1H,s,5-OH).}13_CNMR(100MHz, DMSO-d6)ı55.6(7-OCH3),60.1(6-OCH3),60.7(C-6′′),69.7(C-4′′), 73.2(C-2′′_),76.6(C-5′′_),77.2(C-3′′_{),91.7(C-8),99.8(C-1}′′_),103.7

(C-3),105.2(C-10),116.6(C-3′_,C-5′_),123.9(C-1′_),128.3(C-2′_,

C-6′_{),131.9(C-6),152.1(C-5),152.7(C-9),158.8(C-7),160.4(C-4}′_),

163.4(C-2),182.4(C-4).

Sorbifolin(4):yellowsolid;Rf(Hx/An;3:2,2×):0.52,m.p.235◦C (d).UV(EtOH)maxnm(logε):206(4.55),281(4.24),338(4.38). IRmax(KBr)cm−1:3173,1665,1570,1500,1455,1361,1027,828. 1_{HNMR(400MHz,DMSO-d}

6)ı3.91(3H,s,7-OCH3),6.81(1H,s, H-3),6.91(1H,s,H-8),6.93(2H,d,J=8.8Hz,H-3′_,H-5′_),7.95(2H,

d,H-2′_,H-6′_{),8.74(6-OH),10.39(4}′_{-OH),12.64(5-OH).}13_CNMR (100MHz,DMSO-d6)ı56.3(7-OCH3),91.2(C-8),102.5(C-3),105.0 (C-10),116.0(C-3′_,C-5′_{),121.4(C-1),128.4(C-2}′_,C-6′_{),129.9(C-6),}

146.2(C-5),149.7(C-9),154.4(C-7),161.2(C-4′_{),163.8(C-2),182.3}

(C-4).

Sorbifolin-6-O-␤-glucopyranoside(5):whiteamorphous pow-der;Rf(CH2Cl2/MeOH;4:1):0.62,UV(An:H2O;62.5:1)maxnm (logε):209(3.16),329(3.04).IRmax(KBr)cm−1:3500–3100,1660, 1605,1565,1500,1460,1356,1047.1_{HNMR(400MHz,DMSO-d}

6) ı3.58(1H,dd,J=4.0,11.1Hz,6b′′_{-OH),3.90(3H,s,7-OCH3),4.12}

(1H,dd,J=5.0,10.4Hz,6a′′_{-OH),4.32(1H,t,J=5.5Hz,5}′′_-OH),4.94

(1H,d,J=5.2Hz,H-1′′_{),5.03(1H,d,J=4.8Hz,4}′′_{-OH),5.05(1H,s,}

3′′_{-OH),5.17(1H,d,J=3.3Hz,2}′′_{-OH),6.86(1H,s,H-3),6.92(2H,}

d,J=8.7Hz,H-3′_,H-5′_{),6.93(1H,s,H-8),7.97(2H,d,J=8.7Hz,}

H-2′_,H-6′_{),10.43(1H,brs,4}′_{-OH),13.06(1H,brs,5-OH).}13_{C NMR} (100MHz,DMSO-d6)ı56.6(7-OCH3),60.9(C-6′′),69.9(C-4′′),74.2 (C-2′′_),76.6(C-5′′_),77.3(C-3′′_{),91.7(C-8),102.0(C-3),102.7(C-1}′′_),

104.9(C-10),116.0(C-3′_,C-5′_{),121.1(C-1),128.1(C-6),128.6(C-2}′_,

C-6′_{),151.7(C-5),152.6(C-9),158.6(C-7),161.3(C-4}′_{),164.0(C-2),}

182.3(C-4).

Parasiteandcultureconditions

Inthisstudy,G.lambliaIMSS:0696:1isolate,obtainedfroman individualwithsymptomaticgiardiasis,wasused(Cedillo-Rivera et al., 2003). Trophozoites were cultured in TYI-S-33 modified medium,supplementedwith10%calfserum,andsubculturedtwice aweek;fortheassay,trophozoitesweretestedintheirlogphase ofgrowth(Cedillo-Riveraetal.,1991).

Growthinhibitionassay

InvitrosusceptibilityofG.lambliawasdeterminedaspreviously described (Cedillo-Rivera and Mu ˜noz, 1992). Stock solutions of extracts,fractionsandpurecompoundswerepreparedwithDMSO (5mg/ml),followingserialtwo-folddilutionsin1.5mlvolumesof culturemediuminmicrocentrifugetubestoaffordconcentrations of5,10,20,and50␮g/ml.ThetubeswereinoculatedwithG.lamblia

weredetachedbychillingand50␮lofeachculturetubewas sub-culturedinto1.5mloffreshculturemediumandincubatedfor48h at37◦_{C.Thefinalnumberofparasiteswasdeterminedbycounting}

inahaemocytometer,andthepercentageoftrophozoitesgrowth inhibitionwascalculatedbycomparisonwithcontrols.The50% inhibitoryconcentration(IC50)wasdefinedastheconcentrationof theextract,fractionorpurecompoundthatinhibitedgrowthby 50%ascalculatedbyprobitanalysisusingtheGraphpadPrism6.0 software.Eachexperimentwasdoneinduplicateandwasrepeated atleastthreetimes.

Cytotoxicassayofactivecompounds

Activecompoundswerealsotestedinacytotoxicbioassayin ordertodiscardthat theanti-giardialactivitywasdue totoxic effects.Cytotoxicityassaywasperformedaccordingtothe estab-lishedmethodofRahmanetal.(2001),where1.5×104_viablecells fromHEK-293celllinewereseededina96-wellplate(Costar)and incubatedfor24–48h.Whencellsreached>80%confluence,the mediumwasreplacedandcellswereincubatedwithcompounds atsuccessiveconcentrations(0.97,1.95,3.90,7.81,15.62,31.25, 62.5,125,250,500␮g/ml)anddissolvedinDMSOatamaximum concentrationof0.05%.After72hofincubation,10␮lof 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromidesolution (MTT,Sigma)solution(5mg/ml)wasaddedtoeachwelland incu-batedat37◦_{Cfor4h.Themediumwasremovedandtheformazan,}

aproductgeneratedbytheactivityofdehydrogenasesincells,was dissolvedinacidifiedisopropanol(0.4NHCl).Theamountof MTT-formazanisdirectlyproportionaltothenumberoflivingcellsand wasdeterminedbymeasuringtheopticaldensity(OD)at590nm usingaBio-assayreader(BioRad,USA).Cellsincubatedonlywith 0.05%ofDMSOwereusedasanegativecontrol.Alldeterminations wereperformedintriplicate.TheCC50valueswerecalculatedusing theGraphpadPrism6.0software.

Resultsanddiscussion

ThemethanolextractionoftheA.scabraleavesyielded435gof thecrudeextractASH-1(14.41%,w/w).Liquid–liquidpartitionof thisextractgavefractionsASH-2a–ASH-2d(Table1).These frac-tionstogetherwiththeextractASH-1wereallevaluatedagainst

G.lamblia.Fromthetestedfractions,onlytheASH-1extractand ASH-2bfractionwereactivewithanIC50=11.74and2.19␮g/ml, respectively(Table1)andconsideredhigh,accordingtothe crite-riaestablishedbyAmaraletal.(2006).Tocontinuethebio-assay guidedstudy,aVLCoftheASH-2bfractionwascarriedtoobtain eleven subfractions which were also tested against G. lamblia

(Table2).Theactivesubfractionsshowedacommonmetabolite

Table2

Anti-giardialactivityofVLCsubfractionsoftheDCMfractionofthemethanolextract ofA.scabra.

Sample IC50(␮g/ml)

ASH-4a NAa

ASH-4b NA

ASH-4c NA

ASH-4d 2.5

ASH-4e –b

ASH-4f 1.9

ASH-4g 1.1

ASH-4h 1.2

ASH-4i NA

ASH-4j NA

ASH-4k NA

Metronidazole 0.2±0.02

a_Notactive.

b_Nottested.

whenobservedinTLCplates.Recrystallizationofthe abovemen-tionedfractionsledtothepurificationofacompoundasyellow needles. Such metabolite was identified as cirsimaritin (2) by comparisonofitsspectroscopicdatawiththosereportedinthe literature(Linetal.,2006).Thismetabolitehasbeenpreviously isolated from the species Cirsium rhinoceros (Yim et al., 2003),

Stizolophusbalsamita(Suleimenovetal.,2008),andHelychrisum vis-cosum(Geissmanetal.,1967)belongingtotheAsteraceaefamily;

Teucriumramosissimum(BenSghaieretal.,2011),Dracocephalum multicaule (Oganesyan, 2009), andOcimum sanctumfrom Lami-aceae(Kelmet al.,2000); Microteadebilis(Phytolaccaceae) (Bai etal.,2011);andRuelliatuberosa,aspeciesofAcanthaceae,same family as the genusAphelandra (Lin et al., 2006).According to theliterature,thismoleculepossessesawiderangeofbiological activities,suchasantioxidant(BenSghaieretal.,2011),cytotoxic against KBand GBC-SD cell lines(Lin et al., 2006; Quan etal., 2010),antiproliferativeagainstCOLO-205cells(Baietal.,2011), andantiprotozoalagainstLeishmaniadonovani,Trypanosomabrucei rhodesienseandT.cruzi(Tasdemiretal.,2006).Thephytochemical studyofASH-2afractionledustoobtainthetriterpenesqualene (1),whichwasidentifiedbycomparisonofitsfragmentation pat-ternwiththoseintheNISTlibraryprovidedbytheGC-MS.Studyof ASH-2cfractionallowedtheisolationoftheflavonesorbifolin(4) andtwoglycosylatedflavones:cirsimarin(3)andsorbifolin-6-O-ˇ -glucopyranoside(5),whichwereidentifiedbycomparisonoftheir spectroscopicdatawiththosereportedintheliterature(Fernandes

etal.,2008;Linetal.,2006;Yimetal.,2003).Sorbifolinhasbeen

iso-latedfromthespeciesAstragalusannularis(El-Hawietetal.,2010) andPterogynenitensfromFabaceaefamily(Fernandesetal.,2008);

Heterothecasubaxilaris(Morimotoetal.,2009)andPulicaria

uligi-nosa(Eshbakovaetal.,1996)fromAsteraceae;andR.tuberosafrom

Acanthaceae(Linetal.,2006).Regardingitsbiologicalactivities, thisflavonoidhasbeentestedinantimicrobial,antioxidant,and myeloperoxidasebioassays,resultinginmoderatetonullactivity

(El-Hawietetal.,2010;Fernandesetal.,2008).Meanwhile,isolation

ofcirsimarinhasbeenreportedfromspeciessuchasR.tuberosa, Cir-siumlineare,C.rhinoceros,TeucriumarduiniandM.debilis(Vukovic

etal.,2011;Baietal.,2011;Jeongetal.,2008;Linetal.,2006;Yim

etal.,2003).Intheotherhand,sorbifolin-6-O-ˇ-glucopyranoside has been isolated from Pterogyne nitens (Fernandes et al.,

2008).

2 R₁ = CH₃; R₂ = H

3 R₁ = CH₃; R₂ = Glc

4 R₁ = R₂ = H

5 R₁ = Glc, R₂ = H

OH O

1

O H₃CO

R₁O

OR₂

Compounds2,3,and4weretestedintheanti-giardial bioas-sayand theresultsaredescribed in Table3. Amongthetested compounds, 2 displayed high activity with an IC50=3.8␮M, beingconsideredasoutstanding,whencomparedwithprevious reported secondary metabolites, suchas usambarensine, which showedan IC50=8.99␮M(Wright etal., 1994), (−)-epicatechin

withandIC50=5.82␮M(Calzadaetal.,2005),andtingenone

hav-inganIC50=0.74␮M(Mena-Rejonetal.,2007),while4showedan

Table3

Anti-giardial,cytotoxicactivityandselectiveindex(SI)ofpuresecondary metabo-litesofA.scabraandmetronidazoleaspositivecontrol.

Sample Anti-giardial

activityIC50(␮M)

Hek293cell lineCC50(␮M)

SI

Cirsimaritin(2) 3.8 378.68±3.44b ₃₁₀

Cirsimarin(3) NAa _{NA –}

Sorbifolin(4) 75.6 478.56±6.78 21

Metronidazole 1.2 89.70±1.25 448

a_{NA:notactive.}

b _{Standarddeviation.}

Compound1showedanIC50=241.28␮Minapreviousstudy

(Calzada,2005);therefore,itwasnottestedinthepresentwork,

whilecompound 5 waspurified sparingly,thus it couldnotbe testedagainstG.lamblia.

Noteworthyisthefindingthatbothactiveflavonoidshada typi-calflavonestructure(2,3_{andC-4ketofunctions)andgroupssuch} as4′_{,5-OH.Noactivitywasobservedincompound3,where4}′_-OH

issubstitutedbyglucose,despitethefactthatitsaglicone2wasthe mostactiveinthepresentstudy.Thattendencyallowssuggesting thatinhibitionisconsiderablyreducedbythepresenceofthesugar, inaccordancewithCalzadaandAlanís(2007).

Thebioactivecompoundswerealsotestedinacytotoxic bioas-sayin order todiscardthat the anti-giardialeffect wasdue to cytotoxicity.Inthis bioassay,bothmetabolitesprovedtobenot cytotoxic(Table3).In particular, cirsimaritinshoweda high SI indicatingitselevatedselectivitytoG.lambliacells.Theseresults areinaccordancewithNijveldtetal.(2001),whoconcludedthat flavonoids arenot toxic or less toxic to normal cells, and thus confirmingthepotentialoftheseflavonestobeusedassafe anti-giardialagents,althoughmoreanalysesarenecessarytoestablish ifthesecompoundscanbeusedinhumans.

Inconclusion,wesuggestthatcirsimaritinandsorbifolinarethe metabolitesresponsiblefortheanti-giardialactivityexhibitedby themethanolextractofA.scabraleaves,beingthepresentstudythe firstreportonthephytochemistryofthisspecies,thustheisolated compoundsareconsideredasnewforthegenusandthespecies.

Authors’contribution

GIHBcontributedinrunningthelaboratorywork,analysisof thedata, structure elucidation of the isolated compounds, and draftedthepaper.LWTTcontributedtochromatographicand spec-trophotometricanalysis. RMP supervised biological studies and analysisoftheresults.SRPSdesignedthestudy,supervisedthe lab-oratorywork,andcontributedtocriticalreadingofthemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisresearchwassupportedbytheNationalCouncilofScience andTechnologyofMexico(Conacyt,projectNo.105346).Weare gratefultoPaulinoSima-Polancoforcollectionandidentification ofplantmaterial.

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