RevistaBrasileiradeFarmacognosia26(2016)180–183
w ww.e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Antileishmanial
metabolites
from
Lantana
balansae
Eliana
M.
Maldonado
a,b,
Efrain
Salamanca
c,
Alberto
Giménez
c,
Olov
Sterner
a,∗aCentreforAnalysisandSynthesis,LundUniversity,Lund,Sweden
bCentrodeTecnologíaAgroindustrial,SanSimónUniversity,Cochabamba,Bolivia
cInstitutodeInvestigacionesFármacoBioquímicas(IIFB),SanAndrésUniversity,LaPaz,Bolivia
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received5March2015 Accepted9November2015 Availableonline27January2016
Keywords:
Flavonoids
Lantanabalansae
Leishmaniaamazonensis
L.braziliensis
12-oxo-phytodienoicacid
a
b
s
t
r
a
c
t
Elevencompounds,12-oxo-phytodienoicacid(1),persicogenin(2),eriodictyol3′,4′,7-trimethylether
(3), phytol (4), spathulenol (5), 4-hydroxycinnamic acid (6), onopordin(7), 5,8,4′-trihydroxy-7,3′
-dimethoxyflavone(8),quercetin(9),jaceosidin(10),and8-hydroxyluteolin(11),wereisolatedfroman ethanolextractofLantanabalansaeBriq.,Verbenaceae,thatwasfoundtopossessantileishmanial activ-ity.ThestructuresofthecompoundsweredeterminedbyNMRspectroscopyandHRmassspectrometry, and1,2,3,7,8and9wereinvestigatedforantiprotozoalactivitytowardpromastigotesofLeishmania amazonensisandLeishmaniabraziliensis.Compound1wasshowntobethemostpotent,withtheIC50
values2.0MtowardL.amazonensisand0.68MtowardL.braziliensis,althoughlesspotentthanthe positivecontrolAmphotericinB.Allcompoundshavebeenreportedpreviously,butthisisthefirstreport oftheisolationofacyclopentenonefattyacid(1)andflavanones(2and3)fromaLantanaspecies.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Theknowledgeof thetraditional usesof plants totreat dif-ferent conditions has not only been helpful in the search for newbiologicallyactivecompounds,butalsocontributedto pre-servetheinformationobtaineddirectlyfromthepeoplelivingin isolatedruralcommunities.Basedonsuchknowledge,extensive phytochemicalstudiesofdifferentLantanaspecies,particularlyL. camara,have ledtotheidentificationoflantadenes(pentacyclic triterpenoids), flavonoids and phenylpropanoids as the charac-teristicsecondarymetabolitesofthisspecies.Thebiologicaland pharmacologicalevaluation ofcrude extracts,essential oils and isolatedcompoundshaveshownthattheypossessabroadrange ofbiologicalactivities,forexampleantiprotozoal(antiplasmodial, antimalarial,leishmanicidal),antiviral,antioxidant, antiprolifera-tiveandcytotoxicactivities(Ghisalberti,2000;Grace-Lynnetal., 2012;SousaandCosta,2012).
LantanabalansaeBriq.,Verbenaceae,isaperennialshrubwitha pungentodorthatgrowsinthemountainregionofCochabamba, Bolivia, where it is locally known as “k’ichita”. An infusion of freshleavesofL.balansae isusedin thetraditionalmedicineto treatdigestivedisordersandmusclespasms(personal communi-cationwithlocalpeoplewheretheplantwascollected).Previous
∗ Correspondingauthor.
E-mail:olov.sterner@science.lu.se(O.Sterner).
studiesofL.balansaehavereportedtheantimicrobialactivityof themethanolextract(Salvatetal.,2004)andthechemical com-positionofitsessentialoil(DeVianaetal.,1973;SenaFilhoetal., 2012).Aspartofoursearchforbioactivesecondarymetabolites fromthenativefloraofBolivia,anethanolextractofL.balansaewas assayedforleishmanicidalactivitytowardLeishmania amazonen-sisandLeishmaniabraziliensis.Astheextractdisplayedsignificant activitytowardbothspeciesofLeishmaniaitwasselectedforamore detailedstudy.Herein,wewishtoreportthesecondarymetabolites isolatedfromL.balansaeaswellastheleishmanicidalactivitiesof sixmetabolites.
Materialsandmethods
General
1Dand2DNMR spectrawererecordedatroomtemperature withaBrukerAvanceII400oraBrukerAvance500 spectrome-ter.Thechemicalshifts(ı)arereportedinppmrelativetosolvent signals(ıH7.16andıC128.0forC6D6,ıH2.05andıC 206.0for acetone-d6,andıH2.49andıC39.5forDMSO-d6),whilethe cou-plingconstants(J)aregiveninHz.HR-ESI-MSexperimentswere performedwithaWatersQ-TOFMicrosystemspectrometer,using H3PO4forcalibrationandasinternalstandard.Vacuumliquid chro-matography(VLC)separationswerecarriedoutonMercksilicagel 60G(Merck),whilecolumnchromatography(CC)wasperformed usingsilicagel60(230–400mesh,Merck)andgelpermeationon
http://dx.doi.org/10.1016/j.bjp.2015.11.007
E.M.Maldonadoetal./RevistaBrasileiradeFarmacognosia26(2016)180–183 181
SephadexLH-20(GE-Healthcare).AnalyticalTLCplateswere visu-alizedwithUVlightat254nmandsprayingwithvanillinfollowed byheating.PreparativeTLC(PTLC)wasrunon20cm×20cm glass-coatedplates(1mmthickness,Analtech).
Plantmaterial
TheaerialpartsofLantanabalansaeBriq.,Verbenaceae,were collectednearIndependencia,Cochabamba,Boliviaatcoordinates 17◦11.17′S66◦43.58′Wandanelevationof2789m.Voucher
spec-imens,taxonomicallyidentifiedbyLic.ModestoZárate,arekeptat “HerbarioForestalMartínCárdenas”,Cochabamba,underaccession numberMZ-3946.
Extractionandisolation
The air-dried and ground leavesand flowers of L. balansae (1308g) wereextractedtwiceatroomtemperatureby macera-tionin95%EtOHfor48h.Afterfiltrationthecombinedsolutions wereconcentratedunderreduced pressuretoyield136.6gof a darkresidue.Thecrudeorganicextractwassuspendedina mix-tureofH2O:MeOH(9:1,v/v,500ml)andextractedfourtimeswith 500mlhexanefollowedbytheextractionwithethylacetate(four times,500ml).Afterevaporationofthesolvents,thetwofractions (23.5and43.6g,respectively)werefractionated.VLC chromatog-raphy(hexane:CH2Cl21:0to0:1)ofthehexaneextractyieldedten majorfractions(A–J).FractionE(3.0g)wassubjectedtoVLC (hep-tane:EtOAc1:0to8:2)whichgaveninesubfractions(E1–E9).E4 (582.5mg)waspurifiedbyCConSephadexLH-20(CHCl3:MeOH 1:1)toyield4(39.3mg)and5(43.0mg).F(1.2g)wasfractionated by Sephadex LH-20CC (CHCl3:MeOH1:1) yielding six subfrac-tions(F1–F6).Compound10(3.3mg)wasobtainedpurefromF5 (53.0mg).G (485.0mg) and H (789.0mg) were fractionatedby SephadexLH-20CC(CHCl3:MeOH1:1)toyieldeleven(G1–G11) andfive(H1–H5)subfractions,respectively.Compound2(9.0mg) wasobtainedfromG8and3(7.7mg)fromG11.Sequential purifi-cationofH3(510.0mg)bySephadexLH-20CC(CHCl3:MeOH1:1) and VLC(PE:EtOAc1:0 to7:3) yielded 1(26.1mg). Purification of theEtOAC extract (8.0g)by VLC (CH2Cl2:Me2CO 1:0 to0:1) yieldedsevenmajorfractions(A–G).C(1.4g)wassubjectedtoCC onSephadexLH-20(MeOH)togivefifteensubfractions(C1–C15), fromwhichcompounds7(6.4mg),11(4.6mg)and9(4.1mg)were obtainedpurefromC10,C14andC15,respectively.C8andC11 werepurifiedbyPTLC(CH2Cl2:Me2CO8:2)togive8(3.3mg)and6 (5.2mg).
12-Oxo-phytodienoicacid(1) Colorlessoil;1H(400MHz,C
6D6)ıH2.14(H-2,2H,t,7.4Hz),1.50 (H-3,2H,tt,7,7Hz),1.10(H-4,2H,m),1.06(H-5,2H,m),1.06(H-6, 2H,m),1.13(H-7,2H,m),1.06(H-8,2H,m),2.26(H-9,1H,m),6.65 (H-10,1H,dd,5.8,2.6Hz),6.00(H-11,1H,dd,5.8,2.2Hz),1.85 (H-13,1H,ddd,7.8,4.5,2.2Hz),2.50(H-14a,H,m),2.34(H-14b,1H,m), 5.33(H-15,1H,dtt,11,7,1Hz),5.24(H-16,1H,dtt,11,7,1Hz),1.97 (H-17,2H,ddq,7,7,1Hz),0.89(H-18,3H,t,7.5Hz);13C(100MHz, C6D6)ıc179.9(C-1),34.1(C-2),24.9(C-3),29.2(C-4),29.7(C-5), 27.5(C-6),29.3(C-7),34.5(C-8),46.9(C-9),166.1(C-10),133.1 (C-11),209.8(C-12),51.5(C-13),28.5(C-14),125.9(C-15),133.8 (C-16),20.9(C-17),14.4(C-18);HR-ESI-MSm/z293.2148[M+H]+ (caldc.forC18H29O3293.2117);[␣D20]+60.7(c0.89,CDCl3).
Persicogenin(2)
Colorlessoil;1H(400MHz,C6D6)ı
H 4.70 (H-2,1H,dd,12.8, 3.1Hz),2.50(H-3a,1H,dd,17.1,12.8Hz),2.30(H-3b,1H,dd,17.1, 3.1Hz),6.21(H-6,1H,d,2.3Hz),6.08(H-8,1H,d,2.3Hz),6.62 (H-2′,1H,d,2.1Hz),6.35(H-5′,1H,d,8.4Hz),7.03(H-6′,1H,dd,8.4,
2.1Hz),3.09(OMe-7,3H,s),3.08(OMe-4′,3H,s),12.84(OH-5,1H,
brs),5.47(OH-3′,1H,s);13C(100MHz,C
6D6)ıc79.0(C-2),43.4 (C-3),196.2(C-4),165.2(C-5),95.3(C-6),168.3(C-7),94.5(C-8), 163.3(C-9),103.8(C-10),132.5(C-1′), 113.3(C-2′),146.5(C-3′),
147.0(C-4′),110.1(C-5′),118.0(C-6′),55.1(OMe-7),55.3(OMe-4′);
HR-ESI-MSm/z317.1046[M+H]+(caldc.forC
17H17O6317.1025).
Eriodictyol3′,4′,7-trimethylether(3) Colorlessoil;1H(400MHz,C
6D6)ıH4.76(H-2,1H,dd,13.1, 2.9Hz),2.62(H-3a,1H,dd,17.1,13.1Hz),2.39(H-3b,1H,dd,17.1, 2.9Hz),6.25(H-6,1H,d,2.2Hz),6.16(H-8,1H,d,2.2Hz),6.69(H-2′,
1H,d,2Hz),6.52(H-5′,1H,d,8.8Hz),6.68(H-6′,1H,dd,9,2Hz),3.07
(OMe-7,3H,s),3.36(OMe-4′,3H,s),3.40(OMe-5′,3H,s),12.91
(OH-5,1H,brs);13C(100MHz,C
6D6)ıc79.7(C-2),43.9(C-3),196.6(C-4), 165.7(C-5),95.6(C-6),168.6(C-7),94.9(C-8),163.7(C-9),104.2 (C-10),131.8(C-1′),111.0(C-2′),150.8(C-3′),150.6(C-4′),112.2
(C-5′),119.4(C-6′), 55.4(OMe-7), 55.9(OMe-4′), 55.9(OMe-5′);
HR-ESI-MSm/z331.1207[M+H]+(caldc.forC
18H19O6331.1182).
Onopordin(7)
Yellowpowder;1H(500MHz,Acetone-d
6)ıH6.58(H-3,1H,s), 6.60(H-6,1H,s),7.50(H-2′,1H,d,2.2Hz),6.99(H-5′,1H,d,8.3Hz),
7.47(H-6′,1H,dd,8.3,2.2Hz),3.87(OMe-8,3H,s),12.25(OH-5,1H,
brs);13C(125MHz,acetone-d
6)ıc165.4(C-2),103.8(C-3),183.6 (C-4),157.7(C-5),94.8(C-6),154.0(C-7),132.2(C-8),154.1(C-9), 105.8(C-10),123.9(C-1′),114.2(C-2′),146.6(C-3′),150.3(C-4′),
116.7(C-5′),120.2(C-6′),60.8(OMe-8);HR-ESI-MSm/z331.0685
[M+H]+(caldc.forC
16H13O7317.0661).
5,8,4′-Trihydroxy-7,3′-dimethoxyflavone(8) Yellowpowder;1H(500MHz,acetone-d
6)ıH6.71(H-3,1H,s), 6.64(H-6,1H,s),7.65(H-2′,1H,d,2.0Hz),7.01(H-5′,1H,d,8.3Hz),
7.62(H-6′,1H,dd,8.3,2.0Hz),3.87(OMe-7,3H,s),3.99(OMe-3′,3H,
s),13.25(OH-5,1H,brs);13C(125MHz,acetone-d
6)ıc165.4(C-2), 103.9(C-3),183.7(C-4),157.6(C-5),94.8(C-6),132.1(C-7),153.9 (C-8),157.0(C-9),105.8(C-10),123.7(C-1′), 110.5(C-2′), 148.9
(C-3′),151.5(C-4′),116.4(C-5′),121.4(C-6′),60.7(OMe-7),56.6
(OMe-3′);HR-ESI-MSm/z331.0826[M+H]+ (caldc.for C17H15O7 331.0818).
Quercetin(9)
Yellowpowder;1H(500MHz,Acetone-d
6)ıH6.26(H-6,1H,d, 2.0Hz),6.53(H-8,1H,d,2.0Hz),7.83(H-2′,1H,d,2.2Hz),6.99
(H-5′,1H,d,8.5Hz),7.69(H-6′,1H,dd,8.5,2.2Hz),12.17(OH-5,1H,
brs);13C(125MHz,acetone-d
6)ıc164.9(C-2),136.7(C-3),176.6 (C-4),162.3(C-5),99.1(C-6),146.9(C-7),94.4(C-8),157.8(C-9), 104.2(C-10),123.8(C-1′),115.8(C-2′),145.8(C-3′),148.3(C-4′),
116.3(C-5′),121.5(C-6′);HR-ESI-MSm/z303.0546[M+H]+(caldc. forC15H11O7303.0505).
Leishmanicidaldetectionassay
Theantileishmanial assaywasperformedusinga colorimet-ricmethod-XXT(Salamancaetal.,2008).Briefly,theactivitywas measuredinvitro oncultures of theLeishmania parasitein the promastigote forms, of complex L. amazonensis (clon 1: Lma, MHOM/BR/76/LTB-012)andcomplexL.braziliensis(strandM2904 C192RJA)thatwerecultivatedat26◦CinSchneidermedium(pH
6.8)supplementedwithinactivated(byheatingto56◦Cfor30min)
bovinecalfserum(10%).Parasitesinlogarithmicphaseofgrowth, ataconcentrationof1×106parasites/ml,wereseededinthewells of96-wellplates.Solutionsofcompoundstobeassessedat con-centrationrangeof0.09–100g/mlwereadded.DMSO(1%)and amphotericinB(0.5g/ml)wereusedasnegativeandpositive con-trolsduringtheevaluations.Allassayswereperformedintriplicate andthemicrowellplateswereincubatedfor72hat26◦C.After
182 E.M.Maldonadoetal./RevistaBrasileiradeFarmacognosia26(2016)180–183
withPMS(Sigma–Aldrich,0.06mg/ml)wasadded(50l/well),and theplateswerekeptat26◦Cforanother4h.Theopticaldensity
ofeachwellwasdeterminedwithaStatFax(Model2100series platereader)at450nm,andtheIC50valueswerecalculatedusing Microsoft’sExcel2000program.
Resultsanddiscussion
Asdescribed in the Experimental partof the crude ethanol extractafterliquid–liquidpartitionyieldedthehexaneandethyl acetate fractions,from which the elevencompounds were iso-latedandidentifiedas:12-oxo-phytodienoicacid(1)(Bohlmann et al., 1983), persicogenin (2) (Pisutthanan et al., 2006), eri-odictyol 3′,4′,7-trimethyl ether (3) (Fernandez et al., 1988),
phytol (4) (Hasan et al., 1991), spathulenol (5) (Vieira et al., 2013), 4-hydroxycinnamic acid (6) (Pan and Lundgren, 1995), onopordin(7)(Reynaud and Raynaud,1984), 5,8,4′
-trihydroxy-7,3′-dimethoxyflavone(8)(WhalenandMabry,1979),quercetin
(9)(Slowingetal.,1994),jaceosidin(10)(Ulubelenetal.,1979), and8-hydroxyluteolin(11)(Taskovaetal.,2008).Thepresenceof terpenes(4and5)andflavonoids(7–11)inthisspeciesisin agree-mentwithpreviousstudiesofthegenus,however,thisisthefirst reportoftheisolationofacyclopentenonefattyacid(1)andthe flavanones(2and3)fromaLantanaspecies.Alargenumberof sec-ondarymetaboliteshavebeenreportedfromthegenusLantana, andthishasbeenexcellentlyreviewedbySousaandCosta(2012). Thestructuresofallisolatedcompoundswereelucidatedbya combinationofhighresolutionmassspectrometryandhighfield NMRspectroscopy.ForallcompoundsCOSY,NOESY,HMQCand HMBC2DNMRspectrawererecordedandanalyzed.Inthecase thattheNMRdatapreviouslywasreportedinthesamesolvent, thecomparisonwiththepublisheddatawasalsousedtoidentify thecompounds.
Phytooxylipins(Blee,1998)maypossesspotentphytohormone activities(Böttcher and Pollmann, 2009;Pollmann, 2009).They arederivedfromoxidizedC16andC18fattyacidprecursorsthat areabundantinthecellularmembranesofhigherplants(Gfeller etal.,2010),bytheoctadecanoidpathway(Mithöferetal.,2004). Phytooxylipinsplayanimportantroleinplantresponseto vari-ousenvironmentalstressconditions,andrecentlyitwasshown that 12-oxo-phytodienoic acid (1) inhibits the proliferation of humanbreast cancercells by targeting cyclinD1 (Altiok et al., 2008).Flavonoidsarethelargestgroupofnaturalphenolic com-poundsinhigherplants,andmanystudieshaveshownthatthey
Table1
InvitroleishmanicidalactivityonpromastigoteformsofLeishmaniassp.
IC50(g/ml)a
L.amazonensis L.braziliensis
EtOHextract 6.0±0.2 4.9±1.2
Hexanefraction 6.1±1.3 1.3±0.3
EtOAcfraction 9.9±0.5 5.3±1.0
1 0.60±0.1(2.0M) 0.20±0.01(0.68M)
2 >100 90±4.2(285M)
3 >100 >100
7 22±4.2(70M) 16±0.46(51M)
8 14±1.81(42M) 2.7±0.2(8.2M)
9 20±0.77(66M) 41±2.7(136M)
Controlb 0.21±0.06(0.23M) 0.08±0.04(0.087M)
aDataareexpressedasmeanstandarddeviationofthreedeterminations. bAmphotericinBwasusedaspositivecontrol.
exhibitabroadrangeofbiologicalactivities.Examplesare anti-inflammatory, antitumor,antibacterial, antileishmanialand free radicalscavengingactivities(Greccoetal.,2010;Mittraetal.,2000; SamuelssonandBohlin,2010).
ThehexaneandethylacetateextractsofL.balansaeboth pos-sessedactivitytowardthepromastigotesofL.amazonensisandL. braziliensis,withIC50valuesbetween1and10g/ml(seeTable1 fordetails).Duetolimitedtestingcapacitywedecidedto evalu-atetheantileishmanialactivityofaselection ofthecompounds obtainedfromtheseextracts,compounds1–3and 7–9,andthe resultsarepresentedinTable1.Themostpotentcompoundwas foundtobe1. Althoughit is less potentcompared tothe pos-itive control amphotericin B, the antileishmanial activity of 1
is neverthelessinteresting withthe IC50 values 2.0M toward L.amazonensisand0.68MtowardL.braziliensis.The cyclopen-tenonemoietyof1possessingan␣,-unsaturatedcarbonylgroup
E.M.Maldonadoetal./RevistaBrasileiradeFarmacognosia26(2016)180–183 183
andflavones/flavonolsisthelackoftheC-2/C-3doublebondinthe former,affectingboththemolecularformaswellasthe conjuga-tion.However,withoutdetailedinformationaboutthemechanism of action it isdifficult torationalize anySAR’s. Compound8 is slightly morepotentcompared to7and 9,especially towardL. braziliensis.Ofthethree,8 isthemostlipophilicwhich maybe importantforitsabilitytoreachthetarget.3-Methoxyquercitin haspreviouslybeenshowntobemorepotenttowardpromastigote formsofL.amazonensisthanquercetin(Taleb-Continietal.,2004), sothisappearstobeatrend.Theinvitroandinvivoantileishmanial activityofquercetin(9)hasbeenextensivelystudy,andtheresults presentedhereareinagreementwithpreviousdata(daSilvaetal., 2012).
Authors’contributions
EMMhavecontributedtoplantcollection,carriedoutthe lab-oratorywork,analysisoftheNMRdataandwrotethemanuscript. ESandAGperformedthebiologicalstudies.OSsupervisedthe lab-oratorywork,interpretedtheNMRdataandwrotethemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
Thefinancial support of theSwedish International Develop-mentAgency(SIDA)inaframeofcollaborationagreementbetween LundUniversity (Sweden)and SanSimónUniversity (Bolivia)is gratefullyacknowledged.WethanktoLic.ModestoZáratefor iden-tifiyingtheplant.
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