Rev. bras. farmacogn. vol.25 número4

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

Chemical

constituents

from

Sidastrum

paniculatum

and

evaluation

of

their

leishmanicidal

activity

Yanna

C.F.

Teles

a

_,

_Otemberg

_S.

_Chaves

b

_,

_Maria

_de

_Fátima

_Agra

b

_,

_Leônia

_Maria

_Batista

b

_,

Aline

C.

de

Queiroz

c

_,

_Morgana

_V.

_de

_Araújo

c

_,

_Magna

_Suzana

_{Alexandre-Moreira}

c

_,

Raimundo

Braz-Filho

d

_,

_Maria

_de

_Fátima

_V.

_de

_Souza

a,b,∗

a_{Pós-graduac¸ãoemDesenvolvimentoeInovac¸ãoTecnológicaemMedicamentos,UniversidadeFederaldaParaíba,JoãoPessoa,PB,Brazil} b_{Pós-graduac¸ãoemProdutosNaturaiseSintéticosBioativos,UniversidadeFederaldaParaíba,JoãoPessoa,PB,Brazil}

c_{LaboratóriodeFarmacologiaeImunidade,InstitutodeCiênciasBiológicasedaSaúde,UniversidadeFederaldeAlagoas,Maceió,AL,Brazil} d_{LaboratóriodeCiênciasQuímicas,UniversidadeEstadualdoNorteFluminenseDarcyRibeiro,CamposdosGoytacazes,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2August2014

Accepted10February2015

Availableonline21March2015

Keywords:

Sidastrumpaniculatum

Malvaceae

Leishmaniabraziliensis

Antileishmanialactivity

a

b

s

t

r

a

c

t

Sidastrumpaniculatum(L.)Fryxell,Malvaceae,ispopularlyknowninBrazilas“malva-roxa”or “mal-vavisco”.ThespeciesisfoundmainlyinNortheastregionwhereitisusedbylocalstotreatspiderbites andbeestings.AimingtoidentifythechemicalcompoundsfromS.paniculatumsecondarymetabolism andtocontributetothechemotaxonomicknowledgeofMalvaceaefamily,aphytochemicalstudyofS. paniculatumwascarriedout.Besidesthat,theisolatedcompoundswereevaluatedforantileishmanial activityagainstpromastigotesofLeishmaniabraziliensis.Byusingchromatographictechniquesthestudy resultedtheisolationofeightcompounds:3-oxo-21␤-H-hop-22(29)-ene;sebifericacid;sitosterol3-O-␤

-d-glucopyranoside/stigmasterol3-O-␤-d-glucopyranoside;phaeophytina;132(S)-hydroxyphaeophytin

a;132_{(S)-hydroxy-(17}3_{)-ethoxyphaeophorbideaand 7,4}′_{-di-O-methylisoescutellarein.Thestructure} ofallisolatedcompoundswaselucidatedbyspectroscopicanalysis,includingtwo-dimensionalNMR techniques.Inaddition,theisolatedcompoundsphaeophytina;132_{(S)-hydroxyphaeophytina;13}2_(S )-hydroxy-(173_{)-ethoxyphaeophorbideaand7,4}′_{-di-O-methylisoescutellareinexhibitedantileishmanial} activityagainstpromastigotesofL.braziliensis.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Sidastrum paniculatum (L.) Fryxell, Malvaceae, is popularly knowninBrazilas“malvaroxa”or“malvavisco”.Thespeciesiswide spreadinNortheastregion,especiallyinParaibaandPernambuco states(Baracho,1995).TheSidastrumgenuscompriseseightspecies withneotropicaldistribution,occurringfromMexicotoArgentina. PreviousphytochemicalstudiesonSidastrumspecieshavereported thepresence of steroids, phenolic acids, flavonoids and phaeo-phytins(Gomesetal.,2011a).FromS.paniculatumwerepreviously isolated steroids, a feruloyl derivativeand a glycosil flavonoid (Cavalcante et al., 2010).Many species from Malvaceae family areusedinfolkmedicinetotreatdiseases,S.paniculatumleaves are traditionally used as topical treatment to spider bites and

∗ Correspondingauthor.

E-mail:mfvanderlei@ltf.ufpb.br(M.d.F.V.d.Souza).

bee stings. Sidastrummicranthum leaves are used toprepare a tea to treat asthma and bronchitis (Gomes et al., 2011b).Sida rhombifoliaisusedinIndianfolkmedicineagainsthypertension, diabetes and gout(Chaveset al., 2013).Hibiscusesculentus and

Malvaneglectaareusedinpopularmedicinetotreatulcerand stom-achache.Infact,theseplantshaveshowedtopossessaneffective gastroprotectiveeffectagainstulcersinducedbyethanolin rats (Gürbüsetal.,2003,2005).The methanolextractof Herissantia crispa, species rich in phenolic compounds, showed antidiar-rhoeal and antiulcerogenicactivity onthe HCl/ethanol-induced gastric lesions(Lima etal., 2009).Other relevant pharmacolog-ical activities of Malvaceae species are well reported such as anti-inflammatory,antioxidantand antibacterialactivities(Silva et al., 2006; Falcão-Silva et al., 2009; Costa et al., 2009; Silva etal.,2005;Karouetal.,2005;Ghosaletal.,1975;Oliveiraetal., 2012).

Leishmanicidal activity against Leishmania major has also been reported for Malvaceae species (Rocha et al., 2005). The

http://dx.doi.org/10.1016/j.bjp.2015.02.002

leishmaniasisis caused by more than 20 species of protozoan parasitethatbelongstoLeishmaniagenusandhasgreat epidemio-logicalandclinicaldiversity(Kamhawi,2006;Mishraetal.,2009).

Leishmania(Viannia)braziliensisisendemicinLatinAmericaand isthecausativeagentofmucocutaneousdiseaseintheAméricas (Brelazetal.,2012).Thecurrent chemotherapeuticarsenal con-sistsofpentavalentantimony,pentamidine,variousformulations oftheantibioticamphotericinB,andrecentlymiltefosine(Croftand Olliaro,2011).Thesedrugsarewidelyprescribeddespitetheir tox-icity,highcostanddifficultadministration(Croftetal.,2006).All thesefactsshowtheurgentneedfortheresearchanddevelopment ofnewleishmanicidalcompounds,includingfromnaturalsources (Santosetal.,2008).

InordertoincreasetheknowledgeaboutS.paniculatum phy-toconstituentsandaimingtocontributetothechemotaxonomic knowledgeofMalvaceaefamily,thespeciesS.paniculatumwas sub-mittedtoaphytochemicalstudy.Inaddition,isolatedcompounds wereevaluatedforantileishmanialactivityagainstpromastigotes ofL.braziliensis.

Materialsandmethods

Generalexperimentalprocedures

Theisolationofchemicalconstituentswasperformedonglass chromatographic columns using Silica (ASTM, 230–400 mesh, Merck)orSephadexLH-20asstationaryphase.TLCwereperformed onsilica gel PF254 plates and the spots were visualized under

UVlight(254and366nm)andbyexposuretoiodinevaporand vanillin–sulphuricacidreagent. Isolatedcompounds were iden-tifiedbyInfrared(IR),Perkin-ElmerFT-IR-1750andShimadzu– Prestige21modelusingKBrdiscs;and1Dand2DNMRanalysis (1_H500MHz,13_{C125MHz–Varianand}1_H400MHz,13_C100MHz

–Bruker)usingdeuteratedchloroform,DMSOorpyridine.Melting pointsweremeasuredinaMQAPF-302apparatus(Microquimica EquipamentosLtda).

Plantmaterial

TheaerialpartsofSidastrumpaniculatum(L.)Fryxell,Malvaceae, were collectedin Pedra da Boca Park, located in Araruna city, Paraiba/Brazil,inJune2008(SISBIOAuthorizationNumber 46923-2).TheplantwasidentifiedbyProf.Dr.MariadeFátimaAgra,anda voucherspecimen(JPB6051)wasauthenticatedanddepositedat LauroPiresXavierHerbarium(CCEN/UFPB).

Extractionandisolation

The plant material was dried in an oven at 40◦_{C for 72h.}

Afterthat,itwasgroundedinamechanical mill,yielding3.7kg of a powder which wassubmitted tomaceration withethanol during three consecutive days. This process was repeated in order to maximize the extraction of chemical constituents. A rotaryevaporatorwasusedtoconcentratetheextractresulting 250g of crude ethanol extract (CEE). The CEE was partitioned with n-hexane, chloroform (CHCl3), ethyl acetate (EtOAc) and n-butanol. From this process were obtained 82g of n-hexane phase(HP), 32gof CHCl3 phase (CP),3gofEtOAcphase (EAP),

10g of n-butanol phase (BP) and 110g of hydroalcohol phase (HAP).

HP (7g) was subjectedto column chromatography with sil-icagel(210gofsilicagelpackedinaglasscolumnwith4cmof diameter)andelutedwithhexane,dichloromethaneandmethanol resulting25fractionswhichwereanalyzedandcombinedby ana-lyticalTLC.The05/06fractionyieldedaprecipitatethatwaswashed withhexane toyield 20mg of colorless crystals(1).The 10/12

fraction,acolorlesssolid,waspurewhenanalyzedbyTLC,being labeledascompound2(12mg).Thefraction20/22showedawhite powdersolubleinpyridine,thepowderwaswashedwithhexane resultingthemixtureofthecompounds3and4(30mg).The frac-tions13/18(900mg)wererechromatographed insilica column (27gofsilicagelinglasscolumnwith2cmofdiameter) result-ingthe isolation ofthecompounds 5 (50mg),6 (15mg)and 7

(19mg).

Analiquot(2g)ofEAPwaschromatographedinSephadex col-umn(Sephadexgellengthof30cm)usingmethanolassolvent.The fractionswereanalyzedbyTLC,joined,andthechromatography wasrepeatedtoisolatethecompound8(20mg).

InvitroactivityagainstL.braziliensis

Promastigotes of L. braziliensis (MHOM/BR/87/BA125) were obtainedfromDr.Valéria deMatosBorges (Centrode Pesquisa Gonc¸alo Moniz/FIOCRUZ/BA). The parasites were maintained

in vitro in Schneider’s medium, supplemented with 10% FBS and 2% human urine. Stock solutions of compounds and pentamidine (reference leishmanicidal drug) were prepared in DMSO immediately before use. The cytotoxicity of com-pounds isolated from S. paniculatum and pentamidine against promastigotes was determined. Stationary phase L. brazilien-sis promastigotes were plated in 96-well vessels (Nunc) at 1×105cellsperwell,inSchneider’smedium,supplementedwith

10% FBS and 2% human urine. Each compound solution was added at the following concentrations:0.01␮M, 0.1␮M,1␮M, 10␮Mand100␮M.

Cellswerealsoculturedinamediumfreeofcompoundsor vehi-cle(basalgrowthcontrol)orwithDMSO0.1%(vehicle control). After48h,extracellularloadofL.braziliensispromastigoteswas estimatedbycountingthepromastigotesinSchneider’smedium ina CELM automaticcellcounter (modelCC530)(Rangeletal., 1996).

Resultsanddiscussion

Structureelucidationofisolatedcompounds

Byusingtypicalchromatographicproceduresthecompounds

1–8wereisolatedfromtheaerialpartsofS.paniculatum.

TheIRspectraof1suggestedthepresenceofolefinic,methyl, methylene,methynichydrogensandcarbonylbyshowingbands at 3050;2943–2862 and 1708cm−1_{, respectively. The} 1_{H NMR}

showedsevenmethylsinglets,oneofthosepresenting characteris-ticchemicalshiftofprotonbondedtosp2_carbon(ı

H1.72).Olefinic

protonswerefoundasabroadsinglet atıH 4.76indicatingthe

presenceofisopropenylgroup.The13_{CNMRshowed30carbons}

and the isopropenyl group and the carbonyl were confirmed by the signals at ıC 148.64, ıC 110.06 and ıC 220.51. HMQC

and HMBCcorrelations showedthat thecarbonsareconsistent with a hopane-type skeleton with a carbonyl at position C-3. Comparisonswiththeliteratureledtoidentifythecompound1

as3-oxo-21␤-H-hop-22(29)-ene(Davidetal.,2004;Sousaetal., 2012).

Compound 2 showed IR bands at 1708, 2943, 2862 and 3435cm−1_{suggestingpresenceofhydroxyl,doublebondand}

car-bonyl groups in the molecule. The 1_{H NMR spectra showed a}

triterpenoidprofileand thepresenceoftwoisopropenylgroups wasindicated bythepresenceofvinylmethyls(ıH 1.77andıH

1.73)and exomethylenes protonsat ıH 4.76(2H),ıH 4.80 (1H)

and ıH 4.87(1H).The 13C NMR of compound 2 confirmedthe

113.00andıC110.09.The2Dcorrelationswerecarefullyanalyzed

thusthecompound2wasidentifiedassebifericacid,a3,4-seco -hopane-typetriterpene(Sousaetal.,2012;Pradhanetal.,1984). TriterpeneswereisolatedfromsomeMalvaceaespecies,suchas

S.micranthum,Herissantiatiubae,SidaacutaandAbutilon pakistan-icum(Gomesetal.,2011b;Silvaetal.,2009b;Chenetal.,2007; Ahmedetal.,1990).Hopanetriterpenesand3,4-seco-triterpenes havebeenrecentlyreportedfromWissadulaperiplocifolia(Teles etal.,2014).

1

23 24 6 5 4 3 2 10 1 25 11 12 28 13 19 20 21 18 17

14 ₁₆ 30 29

O

15 27 9 8 7

2

2 1 25 23 24 4 5 6 7 8 9 11 12 28 13 19 20 21 18 14 27 15 16 17 30 29

HO

₂

C

Thestructuralassignmentofthemixtureofcompounds3and

4wasperformedbasedonspectralanalysisandcomparisonswith theliterature(Kojimaetal.,1990;Rashedetal.,2014).Thedataare ingoodagreementwiththosereportedandthestructureswere identified asthemixture ofglucosyl steroids:sitosterol3-O-␤

-d-glucopyranoside(₃)andstigmasterol3-O-␤-d-glucopyranoside

(4).Thesesteroidsarewidelyspreadinplants,beingimportant componentofvegetablecellwallandmembrane.Theyhavebeen previouslyreportedfrommanyMalvaceae,suchasW. periplocifo-lia,S.rhombifolia,Sidagalheirensis,H.crispaandBakeridesiapickelli

(Telesetal.,2014;Chavesetal.,2013;Silvaetal.,2006;Costaetal., 2007,2009).

3

_R=

β

-

D

-glucopyranosyl

4

_R=

_β

_-

D

-glucopyranosyl;

_Δ

22,23

RO

3 2 1 4 10 18 9 7 5 6 11 12 19 21 20 22 23 24 28 29 27 25 26 17 13 14 16 15

Thesubstances5,6and7wereisolatedasdarkgreenamorphous solid,andshowedquitesimilarIRspectra.The1_Hand13_CNMR

indi-catedthatthecompoundsarechlorophyllderivatives.Comparisons withtheliteraturedataledtoidentifythecompoundsas phaeo-phytina(5),132_{-hydroxy-phaeophytina(6)(Nogueiraetal.,2013)}

and132_{(S)-hydroxy-17}3_{-ethoxyphaeophorbidea (7)(Silvaetal.,} 2009a),previouslyreportedfromMalvaceaespeciesW. periplocifo-lia,S.rhombifoliaandS.galheirensis(Telesetal.,2014;Chavesetal., 2013;Silvaetal.,2006).Thesecompoundsareformedfrom chloro-phylldegradationbyactionofenzymessuchasMg-dechelatase

andchlorophyllase.Additionalstructuralmodificationscanoccur andrecentlynewstructuresofchlorophyllderivativesarebeing reported(Silvaetal.,2010).Researchersareinterestedin under-standingiftheybelongtothesecondarymetabolismofplantsonce theyhaveshowedtopossessmanybiologicalactivities(Telesetal., 2014).

5

R

₁

=H; R

₂

=phytol

6

R

₁

=OH; R

₂

=phytol

7

R

₁

=OH; R

₂

=CH

₂

CH

₃

HN

NH

N

N

O

O

H

H

H

H

H

O

O

171 133 134 132 121 81 71 21 2 1 20 31 32 7 6 5 4 3 82 8 131 13 15 14 16 17 18 19 12 11 10 9

R

₂

R

1

Thecompound 8 wasisolatedasyellowpowder. By analyz-ing its 1_{H and} 13_{C NMR, besides bidimensional NMR spectra,}

it waspossibleidentifythe compound8 as 5,8-dihydroxy-7,4′

-dimethoxy-flavone(7,4′_{-di-O-methylisoescutellarein),previously}

isolated from Sidastrum micrathum (Gomes et al., 2011b). Flavonoidsareconsideredacharacteristicgroupof constituents fromfamily.Flavones,flavonols,theirmethylandsulphur deriva-tives,andglucosylflavonoids,havebeenreportedfromMalvaceae species(Chavesetal.,2013;Gomesetal.,2011a;Silvaetal.,2006; Cavalcanteetal.,2010;Costaetal.,2007,2009;Silvaetal.,2005; NawwarandBuddrus,1981).

8

H

₃

CO

OCH

₃

OH

OH

O

O

5 6 7 8 9 4 10 3

2 1′ 2′

3′ 4′

5′ 6′

3-oxo-21␤-H_{-hop-22(29)-ene(1):mp 168–170}◦_{C;IR: 2943,}

2862,1708cm−1_.1_{HNMR(500MHz,CDCl}

3,ıppm):2.08(m,

H-1),2.24(m,H-2),1.95(m,H-5),1.10(m,H-6),1.10and2.01(m, H-7),1.42(m,H-9),1.10(m,H-11),1.42(m,H-12),1.32(m,H-13), 1.32(m,H-15),1.62(m,H-16),1.39(m,H-17),1.60(m,H-19),1.81 (m,H-20),2.66(m,H-21),1.02(s,H-23),1.00(s,H-24),0.73(s, H-25),1.14(s,H-26),0.86(s,H-27),0.68(s,H-28),4.76(bs,2H-29), 1.72(s,H-30).13_{CNMR(125MHz,CDCl}

3,ı):31.59(C-1),33.78

(C-2),220.51(C-3),46.82(C-4),47.32(C-5),20.57(C-6),34.17(C-7), 41.65(C-8),43.30(C-9),36.17(C-10),21.96(C-11),24.27(C-12), 49.72(C-13),42.66(C-14),33.55(C-15),21.37(C-16),55.14(C-17), 44.82(C-18),41.53(C-19),27.30(C-20),46.24(C-21),148.64 (C-22),29.36(C-23),19.57(C-24),23.29(C-25),22.08(C-26),17.06 (C-27),15.96(C-28),110.06(29),25.04(C-30)(Davidetal.,2004; Sousaetal.,2012).

3,4-seco-21␤-H-hop-22(29)-en-3-oicacid(sebifericacid)(2):

mp178–180◦_C;IR:1708cm−1_;2941cm−1_,3435cm−1_.1_HNMR

(500MHz,CDCl3,ı):2.25(m,H-1),1.85–2.44(m,H-2),2.04(m,

Table1

LeishmanicidaleffectofcompoundsisolatedfromS.paniculatumagainst

promastig-otesofL.braziliensis.

Treatment IC50a(␮M±S.E.M.) Maximumeffectb(%±S.E.M.)

Pentamidine 1.3±0.4 94.8±0.2**

3/4 >100 NA

5 77.2±3.9 59.5±0.3**

6 72.7±6.3 70.8±5.7**

7 89.0±1.9 56.5±1.4*

8 78.1±9.5 55.6±3.3*

H-17),1.60(m,H-19),1.78(m,H-20),2.67(m,H-21),4.87(s,H-23), 1.77(s,H-24),0.82(s,H-25),1.03(s,H-26),0.97(s,H-27),0.71(s, H-28),4.76(bs,H-29),1.73(s,H-30).13_{CNMR(125MHz,CDCl}

3, ı):30.14(C-1),30.07(C-2),180.07(C-3),147.83(C-4),46.88 (C-5),22.83(C-6),26.70(C-7),41.35(C-8),43.21(C-9),37.94(C-10), 22.49(C-11),24.30(C-12),49.72(C-13),42.99(C-14),33.49(C-15), 21.60(C-16),55.86(C-17),44.76(C-18),42.01(C-19),27.31(C-20), 46.41(C-21),148.65(C-22),113.00(C-23),26.78(C-24),25.20 (C-25),17.02(C-26),16.39(C-27),16.21(C-28),110.09(29),25.01 (C-30)(Sousaetal.,2012;Pradhanetal.,1984).

Sitosterol3-O_-␤-d_{-glucopyranoside}(₃)/_{Stigmasterol3-}O_-␤

-d_{-glucopyranoside}(₄): The1H and 13C NMR spectral data are

consistentwithpublisheddata(Kojimaetal.,1990;Rashedetal., 2014).

Phaeophytina(5):The1_Hand13_{CNMRspectraldataare}

con-sistentwithpublisheddata(Nogueiraetal.,2013).

132₍S_{)-hydroxy-phaeophytina(6):The}1_Hand13_CNMR

spec-traldataareconsistentwithpublisheddata(Nogueiraetal.,2013).

132_{(S)-hydroxy-(17}3_{)-ethoxyphaeophorbidea(7):The}1_Hand

13_{CNMR spectraldataareconsistentwithpublisheddata(Silva} etal.,2009a).

5,8-dihydroxy-7,4′_{-dimethoxy-flavone (7,4}′_-Di-O

-Methylisoescutelarein) (8): 1_{H NMR (500MHz, DMSO-d}

6, ı):

6.87(s,H-3),6.56(s,H-6),8.12(d,J=8.6Hz,H-2′_{),7.13(d,J=8.6Hz,}

H-3′_{), 7.13(d, J=8.6Hz, H-5}′_{), 8.12(d, J=8.6Hz, H-6}′_{), 3.90 (s,}

OCH3-7),3.86(s,OCH3-4′), 12.44(s,OH-5).13CNMR (125MHz,

DMSO-d6, ı):164.0 (C-2),103.6 (C-3),182.9(C-4), 153.6(C-5),

96.2(C-6),154.8(C-7),126.8(C-8),145.0(C-9),104.4(C-10),123.5 (C-1′_{), 129.0(C-2}′_),115.1(C-3′_),162.9(C-4′_{), 162.9(C-5}′_),129.0

(C-6′_),56.9(OCH

3-7),56.1(OCH3-4′)(Gomesetal.,2011a).

Leishmanicidalactivity

TheprotozoanparasiteL.braziliensisisthecausativeorganism fortheclinicallyimportantdiseasecutaneousandmucocutaneous leishmaniasis(Gonzalezetal.,2009).Thecurrentdrugsfor Leish-mania infections are inadequate due to low efficacy and high toxicity,andtheproblemisfurthercompoundedbytheincreasing prevalenceofdrug-resistantparasites.Currentbiomedicalresearch alwayshasitsfocusonthesearchfornewerintervention strate-giesandnaturalcompoundshavebeenusedasnoveltreatments forparasiticdiseases(Amatoetal.,2008;Ndjonkaetal.,2013).

Theantileishmanialactivityofcompounds3–8wasassessed againstextracellularpromastigotesofL.braziliensisascausative agentofcutaneousleishmaniasis.Asaparameterfor antileishma-nialactivity,themaximumleishmanicidaleffectand IC50 value

wereused.

Theobtainedresultsshowedthatthemixtureofphytosterols

3/4 was inactive against promastigote forms of L. braziliensis

(Table 1). The obtained result is in agreement with previous reportswhichdemonstratedthatstigmasterolandsitosterol3-O

-ˇ-d-glucosidewereinactiveagainstLeishmaniadonovani(Graziose etal.,2012;Kirmizibekmezetal.,2011;Camachoetal.,2002). Mor-ever,Torres-Santosetal.verifiedthatstigmasterolandsitosterol

werenotactiveagainstpromastigotesandintracellular amastig-otesofLeishmaniaamazonensis(Torres-Santosetal.,2004).

Dataarereportedasmeans±S.E.M.Differenceswith**p<0.01, *p<0.05 were considered significantin relation to DMSO 0.1% group.NA:Whenthecompoundisnotactiveuntil100␮M.(a)IC50

istheconcentrationrequiredtogive50%inhibition,calculatedby linearregressionanalysisfromtheKcvaluesatemployed concen-trations(100,10,1,0.1and0.01␮M).(b)EMisthemaximumeffect oftreatment.

Ontheotherhand,ithasbeenreportedtheuseofporphyrin compoundsfortheirabilitytoinhibitLeishmania(Hörtensteiner et al., 1998; Sakata et al., 1990). Previous works revealed that phaeophytins possess potent cytotoxic activities (Cheng et al., 2001).In this study,thethree phaeophytin-related compounds phaeophytin a (5), 132_-hydroxy-(132_{-S)-phaeophytin a (6) and}

132 _{(S)-hydroxy-17}3_{-ethoxyphaeophorbidea(7)exhibited}

leish-manicidalactivityagainstpromastigotesofL.braziliensis,especially 132_-hydroxy-(132_{-S)-phaeophytin a (6), showingthat the}

addi-tionofhydroxylgroupin132_{-positionincreasedefficacy,butnot}

potency.In addition,theimportance ofheme in Leishmaniasp. metabolismsjustifiesconsideringthepotentialofporphyrinsand theirprecursorsandderivativesaspotentialantiparasiticagents byinterferingwithhememetabolism(Abadaetal.,2013).Heme isone ofmanyindispensablenutrientsthat mustbescavenged fromthephagolysosomalcompartment by Leishmania(Naderer and McConville, 2008). In metazoa, heme biosynthesis occurs through8 highlyconservedchemical reactions, resultinginthe insertionofferrousironintotheprotoporphyrinIXring(Hamza andDailey,2012).However,severalunicellularorganisms,suchas

Leishmanialackseveraloftheenzymesnecessaryforacomplete heme-biosyntheticpathway(Koren ´yetal.,2013).Hemenotonly isacrucialcomponentofcytochromesintherespiratorychain,but alsofunctionsasanessentialcofactorforhemoproteins,suchasthe onesinvolvedinthebiosynthesisofpolyunsaturatedfattyacidsand sterols(Tripodietal.,2011).

Theflavone5,8-dihydroxy-7,4′_{-dimethoxyflavone(8)showed}

activityagainstL.braziliensis.Thestructureantileishmanialactivity relationshipofflavoneshasbeendescribedandithasbeenshown that this class of flavonoids, consists of potentialantiprotozoal agents.ThepresenceofOHgroupsontheflavonebenzochromone skeletonenhancestheleishmanicidalpotential.Particularly impor-tantpositionsareC-5,C-7,andC-8(Silva-Filhoetal.,2009).Onthe otherhand,thereplacementoftheOHgroups,eitheronAorBrings, bymethoxylgroupswasdemonstratedtodecreasethe antiproto-zoalactivity(Tasdemiretal.,2006).Numerousnaturalcompound screenshavesuccessfullyidentifiednoveltreatmentsforparasitic diseases(Ndjonkaetal.,2013).Extractsobtainedfromplantsand purecompounds,suchascertaintypesofflavonoids,havebeen reportedtopossesssignificantantiprotozoalactivitywithnoside effects(Muzitanoetal.,2006;Fonseca-Silvaetal.,2011).Whilethe mechanismofactionofthe5,8-dihydroxy-7,4′_{-dimethoxyflavone}

tokilltheseparasitesisnotyetknown,biochemicalexperiments withotherflavonesprovideinitialinsights.Amongnaturally occur-ringflavonoids,theflavonesquercetin,luteolinandbaicaleinare reportedastopoisomeraseIIinhibitor(Austinetal.,1992;Mittra etal.,2000).Therefore,wewillcontinuethisstudytoevaluatethe inhibitoryeffectsofthesesanalogsontopoisomeraseofLeishmania

aswellasothervalidatedchemotherapeutictarget.

Conclusion

3-O-␤-d-glucopyranoside (_3/4); three chlorophyll derivatives:

phaeophytin a (5); 132_-hydroxy-(132_{-S)-phaeophytin a (6);}

132_-hydroxy-(132_{-S)-phaeophorbidea(7);andaflavone:7,4}′

-di-O-methylisoescutelarein(8).

ThestudiedspeciesS.paniculatumhasdemonstratedquite sim-ilargroupofchemicalconstituentstoS.micranthum,S.galheirensis,

H.tiubaeandW.periplocifolia,showingarelatedchemotaxonomic profileofthesespecies.

The antileishmanial activity against promastigotes of L. braziliensisofcompounds5–8isbeingherereportedforthefirst time.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Authors’contribution

YCFT,OSCandMFVScarriedoutthechromatographyisolation and identificationofcompounds. MFAcontributed in collecting plantsampleplant,identificationandherbariumdeposit.RB-Fand LMBcontributedonanalysesanddiscussiondata.ACQ,MVAand MSAMcarriedoutthebiologicalstudy.Alltheauthorshaveread thefinalmanuscriptandapprovedthesubmission.

Acknowledgments

The authors thank CAPES, MCT, FINEP, FAPEAL (Pronem 20110722-006-0018-0010), CNPQ (479822/2013-1), CNPQ (404344/2012-7), INCT-INOFAR/CNPq (573.564/2008-6) for the joint funding of this research project. The authors thank severalcolleaguesworking atthe FederalUniversity of Paraiba andFederalUniversityofAlagoasfortheirconstructivecriticism andassistanceincarryingoutthisproject.

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