Rev. bras. farmacogn. vol.27 número5

RevistaBrasileiradeFarmacognosia27(2017)641–644

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

Short

communication

Iridoids

from

leaf

extract

of

Genipa

americana

Jovelina

S.F.

Alves

_,

_Layane

_A.

_de

_Medeiros

_,

_Matheus

_de

_F.

_{Fernandes-Pedrosa}

_,

_Renata

_M.

_Araújo

_,

Silvana

M.

Zucolotto

a_{LaboratóriodeFarmacognosia,DepartamentodeFarmácia,UniversidadeFederaldoRioGrandedoNorte,Natal,RN,Brazil}

b_{LaboratóriodeTecnologiaeBiotecnologiaFarmacêutica,ProgramadePós-graduac¸ãoemCiênciasFarmacêuticas,UniversidadeFederaldoRioGrandedoNorte,Natal,RN,Brazil} c_{InstitutodeQuímica,UniversidadeFederaldoRioGrandedoNorte,Natal,RN,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19December2016 Accepted24March2017 Availableonline18August2017

Keywords: Genipaamericana

Rubiaceae Jenipapo Leaves Iridoids

a

b

s

t

r

a

c

t

GenipaamericanaL.,Rubiaceae,isaplantnativefromBrazilpopularlyknownas“jenipapo”.Two iri-doids,1-hydroxy-7-(hydroxymethyl)-1,4aH,5H,7aH-cyclopenta[c]pyran-4-carbaldehyde(1),andiridoid 7-(hydroxymethyl)-1-methoxy-1H,4aH,5H,7aH-cyclopenta[c]pyran-4-carbaldehyde (2) were isolated andidentifiedintheleafextractofG.americana.Compounds1and2wereidentifiedforthefirsttime inG.americana,and1hasnotbeenyetdescribedinliterature.Thesesubstanceswereanalyzedby spec-troscopictechniquessuchasinfrared,highresolutionmassspectrometry,1_Hand13_{C1D;aswellas2D} nuclearmagneticresonance.Moreover,thepresenceofflavonoidswasdetectedbyapreliminaryanalysis byThinLayerChromatography.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Genipa americana L., Rubiaceae, is native plant from Brazil (Zappi,2016)andpopularlyknownas“jenipapo”or“jenipapeiro”. ItisfoundinCentralAmerica,SouthAmericaandwidelydistributed inBrazil(LorenziandMatos,2008).Infolkmedicinetheextracts ofleaveshavebeenusedtotreatsyphilis(Corrêa,1978)andliver diseases(Agraetal.,2008).

Themajorityofthephytochemicalandpharmacologicalstudies werecarriedoutwithG.americanafruits.Thechemicalconstituents ofthesefruitsaremainlyiridoids(Djerassiet al.,1960;Tallent, 1964; Ueda and Iwahashi,1991; Hsua et al., 1997; Ono et al., 2005; Ono et al., 2007). These compoundsare not widely dis-tributedin theplant kingdom.Thus, they havebeen identified ina fewfamilies suchasApocynaceae,Loganiaceae,Lamiaceae, ScrophulariaceaeandVerbenaceae(Villase ˜nor,2007).Specifically to leafextracts of G. americana was described the presence of geniposidicacid(Guarnacciaetal.,1972)andgenipatriol(Hossain etal.,2003).Currently,onlyonereportindicatedthepresenceof flavonoidquercetininfruitsbyHPLCanalysis(Omenaetal.,2012) andanotherworkdescribedflavonoidsandtanninsinitsfruitsby colorimetricmethods(Nogueiraetal.,2014).

Some pharmacological trials showed antibacterial (Tallent, 1964), antitumoral (Hsua et al., 1997), anti-inflammatory (Koo

∗ Correspondingauthor.

E-mail:silvanazucolotto@ufrnet.br(S.M.Zucolotto).

etal.,2004)andantioxidant(Omenaetal.,2012)activitiesinthe fruitextracts.TheleafextractofG.americanahasonlytwo stud-iesreported.Ethanolicandhexanicextractsofthatleavesshowed antidiabetic effect by the inhibition of ␣-glucosidase enzyme

(30.44±0.10%and12.44±0.02%,respectively),whichiscompared

withkojicacid,thepositivecontrol(DeSouzaetal.,2012).Inthe secondstudy,theaqueousleafextractshowedanthelminthic activ-ityat100mg/ml.Thelethalconcentrations(LC90)ofthisaqueous extractfor hatchingandL3larvaedevelopmentinhibition were 79.8and28.7mg/ml,respectively.Theextractwasmoreeffective inlarvaldevelopmentinhibitionthaninhatching(Nogueiraetal., 2014).

Therefore, this study aimed to perform a phytochemi-cal study with the leaves extract of G. americana, since as few studies were identified in literature. According to this study, theiridoids 1-hydroxy-7-(hydroxymethyl)-1,4aH,5H,7aH -cyclopenta[c]pyran-4-carbaldehyde (1) and 7-(hydroximethyl)-1-methoxy-1H,4˛H,5H,7˛H-cyclopental[c]pyran-4-carbaldehyde

(2)wereidentifiedforthefirsttimeinthisplant,and1hasnot beenyetdescribedinliterature.

Materialandmethods

Thenuclearmagneticresonance(NMR)spectrawereperformed on a Bruker Avance DRX-500 (500MHz for 1_{H and 125MHz}

for 13_{C) spectrometer equipped with 5mm inverse detection}

z-gradientprobe.Chemicalshiftsaregiveninppmrelativeto resid-ualDMSO-d6(2.5),andtothecentralpeakofthetripletrelatedto

http://dx.doi.org/10.1016/j.bjp.2017.03.006

642 J.S.Alvesetal./RevistaBrasileiradeFarmacognosia27(2017)641–644

Table1

1_Hand13_{CNMRchemicalshiftcompounds1and2(ıinppm,JinHz).}

C 1 2

ıC ıH(mult.,JinHz) ıC ıH(mult.,JinHz)

1 96.89 4.95(d,5.5) 102.1 4.86(d,5.5)

3 163.2 7.58(s) 162.5 7.57(s)

4 123.08 – 123.7 –

5 33.2 2.98(q,8.0) 32.0 2.99(m)

6 37.2 2.68(m)H˛;1.98(m)Hˇ 37.1 2.00(d,14.7)H˛;2.67(m)Hˇ

7 125.54 5.65(s) 126.1 5.65(s)

8 144.83 – 143.6 –

9 46.8 2.53(m) 45.9 2.51(m)

10 59.6 3.98(d,14.8)4.09(d,14.8) 59.2 4.04(d,15.0;d,13.5)

11 190.9 9.26 191.1 9.25(s)

12 – 7.55(OH)(s) 56.5 3.50(OMe)

DMSO-d6carbon(39.5ppm).TheFouriertransforminfrared(FT-IR) spectrawereobtainedona PerkinElmerSpectrum 1000 spec-trometer,usingauniversalattenuatedtotalreflectanceaccessory (UATR).Thehigh-resolutionelectrosprayionizationmassspectra (HRESIMS)wereacquiredusingaShimadzuLCMS-IT-TOF (225-07100-34)spectrometer.Specificrotationsweremeasuredona JascopolarimetermodelP-2000.HPLCpreparativeanalysiswere conductedonShimadzu®

fittedwithLC-10Advppump,UVdetector Shimadzu®

SPD-10AVvp,degasser(DGU-14A),andamanual injec-tionvalve100␮l(Rheodyne®)equippedwithClassVp®version5.0 software.

TheleavesofGenipaamericanaL.,Rubiaceae,werecollectedin May2012inNatal,inBrazilianstateofRioGrandedoNorte(lat: –6.1278long:–35.1115WGS22).Theplantmaterialwas identi-fiedbyAlandeAraújoRoque(UFRN).Avoucherspecimenhasbeen depositedatHerbarium/UFRNunderthereferencenumber12251. Thecollectionoftheplantmaterialwasconductedunder authoriza-tionofBrazilianAuthorizationandBiodiversityInformationSystem (SISBIO)(processnumber35017).

TheleavesofG.americana(1.8kg)wereair-driedat40◦_C, pow-eredand extracted inEtOH70% (v/v)for 7days (plant:solvent, 2:10,w/v),obtainingthehydroethanolicextract(HE).Afterthat, theextractwasfilteredandsubmittedtoaliquid–liquidextraction withpetroleumether(PE)(3×300ml),CH2Cl2(3×300ml),EtOAc (3×300ml),andn-BuOH(3×300ml).Thefractionswere evapo-ratedunderreducedpressure(temperaturebelow45◦_C)andyields were37g(PE),12g(CH2Cl2),19g(EtOAc),56g(BuOH)and145g

(residualaqueousfraction).

In the phytochemical screening the HE and fractions were analyzedby ThinLayerChromatography (TLC)using aluminum sheets,coatedwithsilicagelF254asabsorbentandtoluene:ethyl acetate:formicacid(5:5:0.5,v/v/v)(systemI);ethylacetate:formic acid:water:methanol(10:1.5:1.6:0.6,v/v/v/v)(systemII)andethyl acetate:formicacid:aceticacid:water(10:1.1:1.1:2.6;v/v/v/v) (sys-temIII)asmobilephases.Thechromatogramswereanalyzedunder 254and365nmultraviolet(UV) light andthen sprayedwithi. vanillinsulfuricacid(4%)andii.NaturalProductReagent (0.5%)-NPReagent.Theretentionfactors(Rf)andcolorsofthespotswere comparedwithchromatographicprofilesofstandardsdescribedin literature(WagnerandBladt,2001).

The isolation of the HE compounds from G. americana was started from EtOAc fraction (5g). Thereby, this fraction was submittedto silica gelvacuum liquid column (10×15cm) and elutedwithCH2Cl2:EtOAc(50:50,40:60,40:60,30:70,20:80and

0:100; v/v) and EtOAc:MeOH (90:10, 70:30, 50:50 and 0:100; v/v).Thisprocedureresultedinsevenfractions.Fraction3(40mg) (CH2Cl2:EtOAc,30:70;v/v)waschromatographedfurtheronasilica

gelcolumn(30×2.2cm),elutedwithCHCl3:H2O:MeOH(9:0.1:0.9;

v/v;2.0mlmin−1_{)and100%MeOH,affordingeightfractions(FLC-1}

toFLC-8).FractionFLC-7 (40mg)was submittedtopreparative

HPLC byisocratic elution: 8%MeCN in H2O, 4.5mlmin−1 flow,

using Waters RP 18 column (250×10mm, 10␮m), and UV

254nm,toafford1,8mg(tR 8.8min)and2,5mg(tR 21.5min).

Resultsanddiscussion

Thepresentworkwasconductedinordertoevaluatethe chemi-calcompositionofHEofG.americanaleaves.TLCanalysisofextract andfractionsofG.americanawithspecificsprayreagentsindicated thepresenceofflavonoidsandiridoids.Phytochemicalscreening (systemI)oftheHEandfractions,exposingwithvanillin sulfu-ricacid,showedtwobrownzones(Rf=0.9and0.95).Theseresults

suggestiridoidsinthatsample,whicharecommoninGenipafruits (Djerassietal.,1960;Tallent,1964;UedaandIwahashi,1991;Hsua etal.,1997;Onoetal.,2005;Onoetal.,2007).Furthermore,in liter-aturetwopapersreportedonlygeniposidicacid(Guarnacciaetal., 1972)andgenipatriolintheleaves(Hossainetal.,2003).TLCplate oftheCH2Cl2 fractionwasdevelopedwithsystemIandsprayed

withNP,showinggreenandorangefluorescentspots(Rf=0.46and

0.54),suggestingflavonoids.InTLCplatesanalysisoftheAcOEt frac-tionwasemployedthesystemIIandtoBuOHfraction,systemIII asthemobilephase. Theseplateswereexposed byNPreagent, underUV365nmshowingmanyyellowandorangespots(Rf=0.43

and0.51and Rf=0.22,0.42 and0.47),respectively.Thesecolors

implyflavonoids.Inresidualaqueousfractionwerevisualizedspots withRfandcolorfeaturing flavonoids,exceptbrownishzoneat

thepointofapplicationwithexposureofsulfuricvanillin, prob-ablyindicatingthepresenceofsugars(WagnerandBladt,2001). Asdescribedbefore,moststudiesdemonstratediridoidsinGenipa

genus,especiallytoG.americana.However,onlyonereport indi-catedthepresenceofflavonoidquercetinbyHPLCanalysis(Omena etal.,2012)andanotherdescribedthepresenceofinitsfruitsby colorimetricmethods(Nogueiraetal.,2014).

In thephytochemicalstudy,EtOAc(5g) wasfractionatedby chromatographicprocedurestoafford1(8mg,tR 8.8min)and2

(5mgtR 21.5min).ColorspotsobservedinTLCanalysis,1Hand

13_{C-NMR,ESI-MSandFT-IRconfirmedthecompounds1and2as}

J.S.Alvesetal./RevistaBrasileiradeFarmacognosia27(2017)641–644 643

Fig. 1.Key 1_H–1_{H COSY and HMBC of}

1-hydroxy-7-(hydroxymethyl)-1H,4aH,5H,7aH-cyclopenta[c]pyran-4-carbaldehyde(1).

1-Hydroxy-7-(hydroxymethyl)-1H,4aH,5H,7aH-cyclopenta[c] pyran-4-carbaldehyde (1) [˛]D20 −80.4◦ _{(c 0.013, CHCl3) was} obtained as a brown solid and showed the molecular formula C10H12O4byHR-ESI-MS,basedonthequasi-molecularionatm/z

197.0795[M+H]+ _{(calcd.for C}

10H12O4 m/z197.0808),indicating

fivedegreesofunsaturation.The1_Hand13_{CNMRspectra(Table1)}

showedmostlysinglepeaks,suggestingcompound1asaniridoid monomer. TheIR spectrumdisplayed thehydroxy (3300cm−1₎

andcarbonyl(1670cm−1_)groups.The1_{HNMRspectrumshowed}

twoolefinicprotonsatıH7.58(H-3,s)andıH5.65(H-7,s);two

methyleneprotonsıH1.98(H-␣-6,m)andıH1.98(H-␣-6,m);two

methineprotonsıH2.98(H-5,q,J=8.0Hz)andıH2.53(H-9,m);

oneoxymethyleneıH3.98(H-10,d,J=14.8Hz);one

dioxymethy-lene ıH 4.95 (H-1,d, J=5.5Hz) and one aldehyde proton atıH

9.26(H-11)assignable toaniridoid framework(Joubouhietal., 2015; Lee et al., 2016). These datawere confirmed by the13_C

NMRspectrum,whichexhibitedtencarbonsignals,includingto carbonylcarbonıC190.9(C-11);onehemiacetalcarbonıC163.02

(C-1); four olefinic carbons ıC 163.2 (C-3), ıC 123.08 (C-4), ıC

125.54(C-7)andıC144.83(C-8);twomethylenesıC37.2(C-6)

andıC 59.06(C-10);andtwo methines33.2(C-5)andıC46.08

(C-9). These signals wereattributed after comparison withthe HSQCandDEPT135spectradata.

The1_{HNMRspectrumof1exhibitedasingletatı}

H9.26and

cor-relationswiththecarbonsignal(1_J

CH)atıC190.9(C-11)intheHSQC

spectrum,profilingaconjugatedaldehydegroup,givingsupportby the1670cm−1_{absorptionintheFTIRspectrumandrepresenting}

oneoftheunsaturations.Moretwounsaturationwererelatedfor twotrisubstitutedvinylgroupfromthetypical13_{CNMRsignalsat} ıC163.3(CH-3),123.1(C-4),125.6(CH-7)and144.8(C-8)aswell

as1_{HNMRsignalsatı}

H7.58(H-3,sl)and5.65(H-7,sl)(Raoand Chary,2013).The1_{HNMRspectrumof(1)exhibitedasingletatı}

H

7.55associatedwithahydroxylgroup,confirmedbythe3300cm−1

absorptionintheFTIRspectrum;evenmoreitshowedtwodoublets withgeminalcouplingatıH3.98and4.09(2H-10).Thesesignals

inferredthisstructure(Zengetal.,2007).

IntheCOSYspectrumwaspossibletocharacterizethesequence ofthesehydrogensandconfirmdiastereotopicprotonsatıH3.98

and4.09(2H-10)inthemolecule(Fig.1).NMRdatatogetherwith theadditionaldataspectral(Table1)andliteraturedatawere possi-bletoestablishaniridoidnucleusforcompound1(Zengetal.,2007; RaoandChary,2013;Leeetal.,2016).Thisstructurewasconfirmed bycorrelationsshownintheHMBCspectrumforprotonsatıH9.26

(H-11)withthecarbonsatıC33.2(C-5)and163.2(C-3);atıH2.98

(H-5)withthecarbonsatıC97.0(C-1),125.5(C-7),163.2(C-3)and

190.9(C-11);atıH5.65(H-7)withthecarbonsatıC46.8(C-9)and

59.6(C-10).OtherkeyHMBCcorrelationsareshowninFig.1.Based onliteratureandbiosyntheticgrounds,thisiridoidshowsthesame stereochemistryfortheringfusedcis5S:9SandforC-1-R, indi-catebythenegativeopticalrotation(Chaudhurietal.,1979;Dinda

etal.,2007a,b).Thedatasetobtainedforthiscompoundallowed theelucidationas1-hydroxy-7-(hydroxymethyl)-1H,4aH,5H,7aH -cyclopenta[c]pyran-4-carbaldehyde (1), iridoid not described in literaturebefore.

IridoidsareveryreportedintheGenipagenus,especiallygenipin infruits(3)(Djerassietal.,1960;Dewick,2002).Basedonthese data,thestructure1wasproposedtobeanewgenipinderivative, withareductioninthecarbonylgroupC-11,compatiblewiththe biogenesisproposedbyDewickin2002foriridoids.

Thecompound2[˛]D20−22.2◦(c0.05,CHCl3),exhibited

spec-troscopicdataverysimilartocompound1,every1_Hand13_CNMR

signalsweresimilarexceptforanadditionalsignalatıC56.5in

thecompound2thatdemonstratedcorrelationwithhydrogenat

ıH3.50intheHSQCspectrum,indicatingamethoxylgroupin2

(Table1).Basedonthesefindingsandcomparingwiththeliterature values,thecompound2wascharacterizedastheiridoid garedenal-I,isolatedpreviouslyfromGardeniajasminoidesEllisfruits(Raoand Chary,2013).Detaileddataofcompounds1and2areasfollow (Fig.1).However,theyarethefirsttimereportedatG.americana.

Iridoids are valuable for pharmaceutical applications due to various bioactive properties. Phytochemical studies showed the pharmacological potential of iridoid nucleus molecules in numeroustrials,mainlyanti-inflammatory,antimicrobial, antivi-ral,anticancerandhypoglycemicactivities(Hsuaetal.,1997;Koo etal.,2004;Hanhetal.,2016;Milellaetal.,2016).Additionally, theiridoidsmayberesponsibleforplantdefensebyinsect rela-tions(LohausandSchwerdtfeger,2014),andtheyareprecursors ofmonoterpenoidindolealkaloids,suchasvincristineand vinblas-tineusedinthetreatmentofcancer(VanMoerkerckeaetal.,2015). HerbaldrugscommercializedasHarpagophytumprocumbens coin-tainiridoidsaschemicalmarkers(Mncwangietal.,2012).Genipin isusedascrosslinkinginnanotechnologicalformulationsandother applications(Lietal.,2015).Thus,moleculesofthisclassassemble medicinal,biological,andcommercialvalue.

1-Hydroxy-7-(hydroxymethyl)-1H,4aH,5H,7aH-cyclopenta[c] pyran-4 carbaldehyde (1): brown solid; Brown spot Rf 0.52

aftersprayingtheplatevanillinsulfuricacid;CHCl3:H2O:MeOH,

(9:0.1:0.9;v/v/v);[˛]D20−80.4◦_{;IR(KBr)max/cm}−1 _3350,2930,

2870,1670,1600,1100.1_{HNMR(300MHz,DMSOd)and}13_CNMR

(75MHz,DMSOd)seeTable1.PositiveHRESIMS(positivemode)

m/z,calcd.forC10H12O4[M+H]+:197.0795,found:197.0808.

7-(Hydroxymethyl)-1-methoxy-1H,4aH,5H,7aH-cyclopenta[c] pyran-4-carbaldehyde (2): brown solid; Brown spot Rf 0.62

afterspraying theplatevanillin sulfuricacid, CHCl3:H2O:MeOH

(9:0.1:0.9;v/v/v);[˛]D20−22.2◦(c0.05,CHCl3).1HNMR(500MHz,

DMSOd)and13_{CNMR(1255MHz,DMSOd)seeTable1.}

Conclusion

InG.americanaleaveswaspossibleidentifiedtwoiridoids: 1-hydroxy-7-(hydroxymethyl)-1,4aH,5H,7aH-cyclopenta[c]pyran-4 -carbaldehyde (1) and 7-(hydroxymethyl)-1-methoxy-1H,4aH, 5H,7aH-cyclopenta[c]pyran-4-carbaldehyde (2).Observing, thus, the presence of flavonoids in the leaves extract and fractions byTLC.It is soimportanttocontinue thephytochemical study to identify more secondary metabolites and investigate new biologicalapplicationsforthisextractandthesemolecules.

Author’scontribution

644 J.S.Alvesetal./RevistaBrasileiradeFarmacognosia27(2017)641–644

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthorsacknowledgeallcontributorsfortheirvaluabletime andcommitmenttothestudy.WealsothanktotheCAPESforthe Mastersscholarship;CNPqforthefinancialsupport (478661/2010-0)andUFRN–UniversidadeFederaldoRioGrandedoNorte.

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