Rev. bras. farmacogn. vol.27 número2

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RevistaBrasileiradeFarmacognosia27(2017)175–178

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

Original

Article

Diterpenes

and

a

new

benzaldehyde

from

the

mangrove

plant

Rhizophora

mangle

Jhessica

N. Martins,

Fabiana

S. Figueiredo,

Gabriel

R. Martins,

Gilda

G. Leitão,

Fernanda

N. Costa

∗

UniversidadeFederaldoRiodeJaneiro,InstitutodePesquisasdeProdutosNaturais,CentrodeCiênciasdaSaúde,BlocoH,IlhadoFundão,RiodeJaneiro,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18July2016

Accepted20October2016

Availableonline23November2016

Keywords:

Manool Jhanol Steviol

p-Oxy-2-ethylhexylbenzaldehyde

Rhizophoramangle

Rhizophoraceae

Counter-currentchromatography

a

b

s

t

r

a

c

t

Thisworkdescribestheisolation,byhigh-speedcounter-currentchromatography,ofthediterpenes manool,jhanolandsteviolandthebenzaldehydep-oxy-2-ethylhexylbenzaldehydefromthestiltroots hexaneextractofthemangroveplantRhizophoramangleL.,Rhizophoraceae.Forthis,anon-aqueous biphasicsolventsystemcomposedofhexane–acetonitrile–methanol1:1:0.5(v/v/v)wasapplied.Asfar asweknow,onlysteviolwaspreviouslyisolatedinRhizophoraceaeandthisisthefirsttimethatp -oxy-2-ethylhexylbenzaldehydeisreported.

Introduction

Mangroves are ecosystems with unusual variety of plants adaptedtoconditionsofhighsalinity,frequentfloodsandmuddy anaerobic soil. Brazil has thesecond largestmangrove area in theworldbuthasonlythreegeneraofAngiosperms(Wuetal., 2008).Amongthem,Rhizophoraisthemostfrequentandabundant genus.RhizophoramangleL.,Rhizophoraceae,popularlyknownas ‘redmangrove’, is anativeBrazilian widespreadmangrovetree (Tomlinson,1986),occurringalongallthecoast,fromtheStateof ParáuntiltheStateofSantaCatarina(Schaeffer-Novellietal.,2000).

Several secondary metabolites have been

iso-lated/identified/detected in four of the ten existing Rhizophora species (Wu et al., 2008; Nebulaet al.,2013): ditepenoidsand triterpenoidsinR.mucronata(Misraetal.,1984;Ghoshetal.,1985; AnjaneyuluandRao,2001;Anjaneyuluetal.,2000,2002)andinR. apiculata(Kokpoletal.,1990;Gaoetal.,2011);phenylpropanoids and other volatiles, triterpenes, flavonoids and a flavoglycan polymer in R.stylosa (Neilson et al.,1986; Azuma etal., 2002; Lietal.,2007);flavonoids,condensedtanninsandtriterpenesin R.mangle(Williams,1999;Kochetal.,2003;Kandiletal.,2004; Zhangetal.,2010;Costaetal.,2014).

∗ Correspondingauthor.

E-mail:[email protected](F.N.Costa).

In this study, high-speed counter-current chromatography (HSCCC) wasusedto isolatethree diterpenesand a new benz-aldehyde from the hexane extract of R. mangle stilt roots. Counter-currentchromatography,CCC,isaliquid–liquidpartition techniqueinwhichtheliquidstationaryphaseisretainedinthe apparatususingcentrifugalforceonly(Conway,1990).High-speed counter-currentchromatography,HSCCC,usestworotationaxes inaplanetarymotion,generatingavariablecentrifugalforcefield (Conway,1990).Theseparationisbasedonpartitioningof ana-lytesbetweentwoimmiscibleliquids(generallyasolventmixture) formingabiphasicsystem(MarstonandHostettmann,2006).The useofanall-liquidtechniqueleadstomanyadvantagesover con-ventionalchromatography(Berthod,2002),beingtherecoveryof labilecompoundswithoutchemicalmodificationsonitsstructure crucialtothesuccessofthiswork.

Materialsandmethods

Extractpreparation

Rhizophora mangle L., Rhizophoraceae (Supplementary data), wascollectedatReservaBiológicaeAntropológicadeGuaratiba,Rio deJaneiro,BrazilinOctober2010.Plantwasidentifiedoncampus byDrGustavoDuqueEstrada.AsthisplantistheonlyRhizophora speciesoccurringinBrazilianmangroveareasbeingits morphol-ogytotallydifferentfromanyotherexistingmangroveplant,there wasnoneedofavoucherspecimen.Driedandgroundedstiltroots

http://dx.doi.org/10.1016/j.bjp.2016.10.004

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176 J.N.Martinsetal./RevistaBrasileiradeFarmacognosia27(2017)175–178

weresubmittedtomacerationwithethanol–water(9:1,v/v).55gof thecrudedriedextractwasdissolvedinmethanol–water(50:350, v/v)andpartitionedbetweenhexane,dichloromethaneandethyl acetate,inthisorder,affordingfourdifferentextracts.

Selectionandpreparationofthebiphasicsolventsystemand samplesolution

Solventsystemselectionwasmadebydissolvingasmallamount ofthesampleintesttubecontainingtheequilibratedbiphasic sys-tem.Thetesttubeswereshakenandthecompoundsallowedto partitionbetweenthetwo phases.Equalaliquotsofeachphase werespottedbesideeachotherseparatelyonsilicagelTLCplates (MerckArt.05554,Darmstadt,Germany),developedwiththe sol-ventsystemhexane–ethylacetate9:1.Theresultswerevisualized underUVlight(254nm).Theresultswerevisualizedafterspraying theplatewithH2SO4(10%)andvanillin(5%).

Theselectedsolventsystemwasequilibratedinaseparatory funnelatroomtemperatureandphaseswereseparatedjustbefore use.Theupperlayerwasusedasstationaryphasewhilethelower layerasmobilephase,inheadtotaildirection.Samplesolutionwas preparedbydissolvingthesampleinthesolventsystemusedfor separation(1:1,v/v).

CCCequipmentandseparationprocedure

Semi-preparativeHSCCCwasperformedonaQuattroHT-Prep counter-currentchromatograph(AECS,Bridgend,UnitedKingdom) equippedwithtwobobbinscontainingtwo polytetrafluoroethy-lenemulti-layercoilseach(26ml,1mmi.d.+234ml,3.2mmi.d. and 95ml, 2mm i.d.+98ml, 2mm i.d.). The rotation speed is adjustablefrom0to865rpm.A5mlsampleloopwasusedtoinject thesample.

Intheseparationprocess,the95mlcoilwasfirstentirelyfilled with the upper stationary phase, and then the apparatus was rotatedat865rpm,while thelowermobilephasewaspumped intothecolumn.Theflowrateusedwas2ml/min.Afterthemobile phasefrontemerged andhydrodynamicequilibrium was estab-lishedin thecolumn, thesample solution(300mgin5ml)was injectedintothecolumnthroughtheinjectionvalve.Fractionsof 4mlwerecollected:50duringelutionand30duringextrusion.

Extractandfractionanalysesandcompoundidentification

Crudeextract and eachfraction fromCCC wereanalysed by TLC (Merck Art. 05554, Darmstadt, Germany), developed with

hexane–ethylacetate9:1and/or7:3,accordingtoitspolarity.The resultswerevisualizedaftersprayingtheplatewithH2SO4(10%) andvanillin(5%).

1_H_and13_C_NMR_data_measurements_for_the_isolated_compounds

wererecordedonaVarianVNMRS500(California,USA)at25◦_C, operatingat 500MHz for 1_H_and ₁₂₅_for 13_C, _using_deuterated chloroformandTMSasinternalstandard.

Resultsanddiscussion

R.mangleistheonlyspeciesoccurringalongallBraziliancoastin mangroveareaswithdifferentclimaticandenvironmental condi-tions(Schaeffer-Novellietal.,2000)andverylittleisknownabout itschemicalprofile,especiallytheoneconcerningnon-polar com-pounds.Followingourpreviousworkonphenolic profileofthe EtOAcextractofR.mangleleaves(Costaetal.,2014)whereseveral compoundsweredescribedforthefirsttimeonthespecies,the stiltrootshexaneextractwasinvestigated.

Preliminaryanalysis

Thehexane extractof R. mangle stilt roots wasanalysed by TLCand theplate(not shown)showedacomplexmixture,with compoundsoflowpolarity,possiblyterpenoids,duetothe pur-plecolourdevelopedaftersprayingtheplatewithsulfuricvanillin. Thisfirstinformationoncomplexityandpolaritywasimportantto guidesolventsystemtesting.

Solventsystemselection

Theselectionofthebiphasicsolventsystemisthemost impor-tantstep inCCC, asit meansthechoiceofboth stationaryand mobilephasesatthesametime(Yto,2005)and,withoutconsulting literature,thissearchcanbeverytime-consuming.

More than 150 solvent systems were formerly used to iso-late terpenoids by CCC (Skalicka-Wo ´zniak and Garrard, 2014). From them, non-aqueous solvent systems have been used for the isolation of low-polarity compoundsmainly from essential oils.Themostcommonly usedarehexane:acetonitrile and hex-ane:methanolmodifiedbychlorinatedsolvents(Leitãoetal.,2012). Aftertesting hexane:acetonitrile (1:1,v/v), hexane:methanol (1:1, v/v), solvents were combined and a further system was evaluated: hexane:acetonitrile:methanol (1:1:0.5, v/v/v). When using only acetonitrile as the polar solvent, compounds were slightlymoreconcentratedintheupperlayer(composedmainly byhexane).Ontheotherhand,whenusingonlymethanolasthe

Elution

Hex-EtOAc 7:3 Hex-EtOAc 9:1

5 6

F1 5-10 211.3 mg

F2 11-13 24.5 mg

F3 14-17 21.8 mg

F4 18-20 17.4 mg

F5 21-23 18.8 mg

F6 24-26 53.3 mg

F7 27-30 39.1 mg

F8 31-33 7.6 mg

F9 34-80 73.1 mg 7 8 9 10 11 12 13 14 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 5153 55 57 59 61 63 65 67 69 71 73 75 77 79

Extrusion

Fig.1.AnalysesofCCCfractionsbyTLCandcombinationbysimilarity.F2,F4,F6andF8arehighlightedandcorrespondtocompounds(1),(2),(3)and(4),respectively.

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J.N.Martinsetal./RevistaBrasileiradeFarmacognosia27(2017)175–178 177

polarsolvent,compoundswereheavilyconcentratedinthelower layer(composed mainlyby methanol). Assuming thatthe suit-ablecondition is achievedwhen having distributioncoefficient approximatelyone,addingasmallproportionofmethanolto hex-ane:acetonitrilesystemgavesatisfactoryresults.

CCCseparationandfractionanalyses

Hexane–acetonitrile–methanolatratio1:1:0.5wasemployed forthesemi-preparativefractionationof300mgofR.manglestilt rootshexaneextractaffordingninemainfractionscombinedbyTLC similarity,F1toF9(Fig.1).Elutionprocesstookplaceuntilfraction 50beingsubstitutedbyextrusionuntilfraction80.Compounds1,

2,3and4inF2,F4,F6andF8,respectively,wereisolatedandwere analysedbyNMR.Compoundspurityisnotknown.

Compoundidentification

Compound1wasidentifiedasthelabdanediterpenemanool bycomparisonwithliteraturedata(Ulubelenetal.,1991).Itwas obtainedasayellowamorphoussolidandthemolecularformula wasdeterminedasC20H34OonthebasisofHRESIMS([M−18+H]+

273.2572).1_H_NMR ₍₅₀₀_MHz,_CDCl

3):ı0.68(s,CH3,H18),0.87 (s,CH3,H19),0.80(s,CH3,H20),1.27(s,CH3,H16),1.00–2.50(m, CH2,H1;CH2,H2;CH2,H3;CH,H5;CH2,H6;CH2,H7;CH,H9; CH2,H11;CH2,H12),4.51(d,1H,C17),4.81(d,1H,C17),5.06(dd, 1H,C15),5.22(dd,1H,C15),5.90and5.94(d,1H,C14).APTNMR (125MHz,CDCl3_):_ı_14.43_(CH

3,C18),17.70(CH2,C10),19.38(CH2, C1),21.71(CH3,C20),24.42(CH2,C5),27.63(CH3,C16),33.56(CH3, C19),33.61(C,C3),38.34(CH2,C6),39.06(CH2,C2),39.86(C,C9), 42.19(CH2,C11),55.57(CH, C4),57.31(CH,C8),73.58(C,C12), 106.44(CH2,C17),111.52(CH2,C15),145.26(CH3,C13),148.66(C, C7).

Compound2wasidentifiedasthekaurane diterpenesteviol bycomparisonwithliteraturedata(Subrahmanyametal.,1999;

Geunsetal.,2006).It wasobtainedasa whitepowderandthe

molecularformula wasdeterminedasC20H30O3 onthebasisof HRESIMS([M−18+Na]329.2461).1HNMR(500MHz,CDCl3):ı0.94 (s,CH3,H20),1.22(s,CH3,H18),0.90–2.50(m,CH2,H1;CH2,H2; CH2,H3;CH,H5;CH2,H6;CH2,H7;CH,H9;CH2,H11;CH2,H12; CH2,H14;CH2,H15),4.80and4.74(d,2H,H17).APTNMR(125MHz, CDCl3_):_ı_15.63_(CH

3,C18),18.41(CH2,C11),19.08(CH2,C2),21.81 (CH2,C6),28.96(CH3,C20),37.80(CH2,C3),39.53(C,C10),40.69 (CH2,C7),41.26(CH2,C1),42.39(C,C8),43.70(CH2,C12),43.82(C,

Fig.2.SelectedHMBCcorrelationsof(4)p-oxy-2-ethylhexylbenzaldehyde.

C4),44.20(CH2,C14),48.94(CH2,C15),55.08(CH,C5),57.02(CH, C9),81.54(C,C13),102.97(CH2,C17),155.85(C,C16),183.79(C, C19).

Compound3wasidentifiedasthelabdanediterpenejhanolby comparisonwithliteraturedata(Gonzálezetal.,1977;Stierleetal., 1988;Fragaetal.,1998).Itwasobtainedasayellowoilandthe molecularformulawasdeterminedasC20H34O2 onthebasisof HRESIMS([M−18+H]289.2526).1HNMR(500MHz,CDCl3):ı0.75 (s,CH3,H18),0.83(s,CH3,H20),1.27(s,CH3,H16),1.30(s,CH3, H17),0.85–1.80(m,CH2,H1;CH2,H2;CH2,H3;CH,H5;CH2,H6; CH2,H7;CH,H9;CH2,H11;CH2,H12),3.13and3.40(d,CH2,H19), 5.86and5.89(d,CH,H14),4.93and5.15(dd,CH2,H15).APTNMR (125MHz,CDCl3_):_ı_15.28_(CH

2,C11),15.77(CH3,C20),17.15(CH3, C18),17.83(CH2,C2),19.68(CH2,C6),25.47(CH3,C17),28.50(CH3, C16),35.27(CH2,C12),35.63(CH2,C3),36.89(C,C4),37.57(C,C10), 38.49(CH2,C1),42.88(CH2,C7),49.64(CH,C5),55.57(CH,C9), 72.05(CH2,C19),73.27(C,C8),74.96(C,C13),110.32(CH2,C15), 147.81(CH,C14).

Compound 4 (Fig. 2) was identified as p-oxy-2-ethylhexyl benzaldehyde (Table 1). It was obtained as a colourless oil, [˛]d20=₋19.15(c=0.2,MeOH),UV(MeOH):203,242and282nm. ThemolecularformulawasdeterminedasC15H22O2onthebasis

Table1

1_H_and13_C_NMR_data_of_p_{-oxy-2-ethylhexyl}_benzaldehyde₍₄₎₍₅₀₀_and₁₂₅_MHz;_CDCl

3;ıinppm;JinHz).

p-Oxy-2-ethylhexylbenzaldehyde(4)

ıC ıH HMBC

H(2_J

C H) H(3JC H)

1 204.38,CH

2 132.61,C 7.54(H3) 7.71(H4)

3/3′ _131.02,_CH _7.54_(dd;_3.3,_9.0) _7.71_(H4)

4/4′ _128.95,_CH _7.71_(dd;_3.3,_9.0) _7.54_(H3)

5 167.90,C 7.71(H4) 4.22(H6)

6 68.31,CH2 4.22(ddd;5.7,8.3) 1.68(H7) 1.42/1.41(H12)

7 38.89,CH 1.68(ddd;6.1,12.2,18.4) 4.22(H6) 0.92(H13)

1.42/1.41(12)

8 30.52,CH2 1.35(m) 1.68(H7) 4.22(H6)

1.42(H12)

9 29.08,CH2 1.32(m) 1.35(H8) 1.68(H7)

0.90(H11)

10 23.14,CH2 1.30(m) 0.90(H11) 1.35(H8)

11 14.20,CH3 0.90(d;7.5)

12 23.91,CH2 1.42(m) 1.68(H7) 4.22(H6)

0.92(H13)

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178 J.N.Martinsetal./RevistaBrasileiradeFarmacognosia27(2017)175–178

ofHRESIMS([M+H]+_234.3355)_indicating_five_degrees_of instau-ration. The IR spectrum showedabsorption at 3500–3250 and 1500–1400cm−1_typical_for_aromatic_signals,_at₁₇₀₀_cm−1 indi-catingthepresenceofacarbonylgroupandat1260and1100cm−1 characteristicofanetherfunction.Foursimilarcompounds differ-inginthenumberofcarbonsinthechainhavebeenpreviously synthesizedinstudiesonlipidbilayerintercalants(Cohenetal., 2008).

1_H,_APT,_HSQC_and_HMBC_{(Supplementary}_data)_experiments

wereperformedforthestructureelucidationofcompound4.The APTspectrumshowed13carbonsignals,allowingtodistinguish carbontype: twomethyls, five methylenes(including one con-nectedto electronegative atom), four methines (including two aromaticsignalsandonecarbonilic)andtwoquaternaryaromatic carbons.

Thepresence of thecarbonyl aldehyde was confirmedwith thesignalat204.38ppm.Thearomaticringcouldbeseenin1_H spectraat 7.71and 7.54chemical displacements. These signals havetheAA′_XX′_system,_a_standard_to_para_{-dissubstituted}_aromatic ringwith3.3and9.0Hztypicalcouplingconstant.TheC3/C3′_and C4/C4′_have_the_same_chemical_shift_due_to_symmetry,_131.02_and 128.95ppm,respectively.Thetypicalchemicalshiftfor CH2O groupappearsat4.22ppmin1_H_and_at_68.31_ppm_in_APT experi-ment.StructurewasconfirmedbybidimensionalHSQCandHMBC experiments(Table1andFig.2).

Thenon-aqueoussolventsystemusedinthisworkshowedtobe effectiveontheisolationofthemediumtolowpolarityditerpenes inR.manglestiltrootshexaneextract.Theuseofthissolvent sys-tem,besidesaffordingtheselabilecompoundswithoutchemical modification,additionallyprovidedanewbenzaldehyde deriva-tive,which, as faras weknow, is being described for thefirst timeinnature.Concerningthediterpenes,literaturereportsthe presenceofkauranes,labdanes,pimaranesandbeyeranesbesides severalaromaticcompoundsinRhizophoraceae(Wuetal.,2008; Nebulaetal.,2013)butonlysteviolwaspreviouslyisolatedinthe family.

Authors’contributions

JNM(undergraduatestudent)didthecompoundisolation pro-cedure.FSF(PhDstudent)contributedincompoundidentification byNMRandliteraturesearch.GRM(PhDstudent)didNMR experi-ments.GGLwasFNC’sPhDsupervisor,wherethisworkstarted,and contributedtocriticalreadingthemanuscript.FNCdesignedthe study,supervisedthelaboratoryworkandwrotethemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

JNMartinsisgratefulforPIBIC-UFRJscholarship.FNCostaand GGLeitãoaregratefulforCNPqandFaperjforfinancialsupport.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.10.004.

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