Rev. bras. farmacogn. vol.27 número4

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

Original

Article

Chemical

composition

and

seasonal

variation

of

the

volatile

oils

from

Trembleya

phlogiformis

leaves

Sarah

R. Fernandes

a

_,

_Heleno

_D.

_Ferreira

b

_,

_Stone

_de

_Sá

a

_,

_Leonardo

_L.

_Borges

c,d

_,

Leonice

M.F.

Tresvenzol

a

_,

_Pedro

_H.

_Ferri

e

_,

_Pierre

_Alexandre

_dos

_Santos

a

_,

_José

_R.

_Paula

a,∗

_,

Tatiana

S. Fiuza

b

a_Faculdade_de_Farmácia,_Universidade_Federal_de_Goiás,_Goiânia,_GO,_Brazil b_Instituto_de_Ciências_Biológicas,_Universidade_Federal_de_Goiás,_Goiânia,_GO,_Brazil

c_Escola_de_Ciências_Médicas,_{Farmacêuticas}_e_Biomédicas,_Pontifícia_Universidade_Católica_de_Goiás,_Goiânia,_GO,_Brazil d_Campus_Henrique_Santillo,_Universidade_Estadual_de_Goiás,_Anápolis,_GO,_Brazil

e_Instituto_de_Química,_Universidade_Federal_de_Goiás,_Goiânia,_GO,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19November2016 Accepted24March2017 Availableonline30April2017

Keywords:

Melastomataeae Volatileoils Seasonality

Trembleya n-Heneicosane Oleicalcohol

a

b

s

t

r

a

c

t

TrembleyaphlogiformisDC.,Melastomataceae,isashrubwhoseleavesareusedasadyefordyeingwool

andcotton.Thepresentarticleaimedtocarryoutthemorphologicaldescriptionofthespecies,tostudy

thechemicalcompositionofvolatileoilsfromtheleavesandflowersandtheseasonalvariabilityfrom

theleavesduringayear.Macroscopiccharacterizationwascarriedoutwiththenakedeyeandwith

astereoscopicmicroscope.VolatileoilswereisolatedbyhydrodistillationinClevengerapparatusand

analyzedbygaschromatography/massspectrometry.ThemajorcomponentsofthevolatileoilofT.

phlogiformisflowerswere:n-heneicosane(33.5%),phytol(12.3%),n-tricosane(8.4%)andlinoleicacid

(6.1%).Itwasverifiedtheexistenceofalargechemicalvariabilityofthevolatileoilsfromtheleavesof

T.phlogiformisoverthemonths,withthemajoritycompound(oleicalcohol,rangingfrom5.7to26.8%)

presentinallsamples.AcombinationofClusterAnalysisandPrincipalComponentAnalysisshowedthe

existenceofthreemainclusters,probablyrelatedtotheseasons.Theresultssuggestedthatthevolatile

oilsofT.phlogiformisleavespossesshighchemicalvariability,probablyrelatedtovariationassociated

withrainfallandthevariationinthebehaviorofspecimensthroughouttheyear.Thisresearchprovides

insightsforfuturestudiesonthevolatileoilsobtainedfromtheT.phlogiformisleavesandflowers,mainly

relatedtobiologicalmarkersofapplicationsmonitoredintheleavesandflowersofthisspecies.

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

MelastomataceaeJuss.familyhasaround150–166genera.In Brazilcanbefound66generaandabout1360species(Goldenberg etal.,2012;Baumgratzetal.,2014).Theseplantspresent strate-giesandadaptationsasalargeseedproduction,highgermination ratesandrapidgrowth,withgreatimportanceinenvironmental restoration(Albuquerqueetal.,2013).

The genus Trembleya DC. belongs to the Melastomataceae family, subfamilyMelastomatoideae Seringe and tribe Microli-cieaeTriana.Thistribe consistsof sixgenera:Stenodon Naudin,

ChaetostomaDC.,LavoisieraDC.,MicroliciaD.Don,Rhynchanthera

∗ Correspondingauthor.

E-mail:joserealino@ufg.br(J.R.Paula).

DC.andTrembleyaDC,characterizedbyreniform,ellipsoidsor elon-gatedseeds(Fritschetal., 2004).Themostspeciesof thistribe contains dimorphic stamens, withprolonged connective below antherwithrostrate apex exceptStenodon Naudin(Goldenberg etal.,2015).

TrembleyaisanexclusivelyBraziliangenus,withaboutfourteen species,andmostoftheseoccurinrockformationsinMinasGerais State(Martins,1997;Martins,2009;Goldenbergetal.,2015). Trem-bleyais characterizedbyherbstoerectshrubs,non-imbricated, sessile orpetiolateleaves, withouttranslucent scores,modified dichasiumflowerswithwhiteorpinkpetalsand5-locularovary (Goldenbergetal.,2015).

Cotaetal.(2002)verifiedantimicrobialactivityoftheTrembleya laniflora(D.Don)Cogn.leavesagainstdifferentstrainsof bacte-riaandfungi.Venturaetal.(2007)observedantimicrobialactivity ofthecrudeextractoftheleavesandstemsofT.lanifloraagainst

MicrococcusluteusandStaphylococcusaureus.

http://dx.doi.org/10.1016/j.bjp.2017.03.003

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TrembleyaphlogiformisDC.,popularlyknownas “quaresminha-do-campo”,isashrubwhoseleavesareusedincommunitiesasa naturaldyetowoolandcotton(Sáetal.,2007).Nopapersinthe scientificliteraturehavebeenfoundregardingchemicalprofileand biologicalactivityofthisspecies.

Severalauthorsemployedmultivariatetechniquessuchas prin-cipalcomponentanalysis(PCA)andclusteranalysis(CA)toverify thechemicalvariabilityandtheinterrelationshipsbetween sam-plesanditssimilarities,regardingvarioussecondarymetabolites, includingvolatileoils. Theseapproaches are ordering methods, aimingthereducingthedatasetdimensiontoeasilythe expla-nationthebehaviorofthesystemaccordingthechemicalprofile (Boiraand Blanquer, 1998; Santosetal., 2006;Lei etal., 2010; Sampaioetal.,2016).

Insearchofnewplantswithpharmacologicalpotential,the pur-posesofthisworkwere:studyofthedevelopmentinlocoandthe morphologicalbehaviorofT.phlogiformisspecimensoveraperiod of12months; studyofthechemicalcomposition ofvolatileoil ofleavesandflowersandanalyzetheseasonalvariabilityofthe volatileoilofleavesfor12months.

Materialsandmethods

Localization

Theplantmaterial wascollectedinPirenópolis, Goiás,Brazil (15◦₄₈′₁₅′′_S_to₄₈◦₅₂′₄₈′′_W,_at_an_elevation_of₁₂₉₅_m_above_sea level).Climaticdatafortheperiodwereobtainedfromthe Meteo-rologicalInstitute(INMET,2014).

Plantmaterialandmorphologicalanalysis

ThecharacterizationofTrembleyaphlogiformisDC., Melastom-ataceae,externalmorphologywasmadewiththenakedeye,every monthfor12months,inloco,andinthePlantTaxonomy Labora-toryoftheDepartmentofBotany,InstituteofBiologicalSciences, Federal University of Goiás, using the stereoscopic microscope OlympusSZ-ST.

TostudythevolatileoilT.phlogiformisDC.,theleaveswere col-lectedmonthlyfor12monthsandtheflowersinMarch2014.

During field visits, the external morphological data and the behaviorofspecimenswererecordedinafieldbook.Theimages wereregisteredwithadigitalcameraCanonEOST4i.Thespecimens ofT.phlogiformiswereidentifiedbyProfessorDr.HelenoDias Fer-reiraandavoucherspecimendepositedattheHerbariumofFederal UniversityofGoiás,Brazil,ConservationUnitPRPPG,undercode numberUFG-47868.

Volatileoils

Foranalysisof volatileoils, healthyleavesandflowerswere collectedoftendifferentindividualsofT.phlogiformisanddried atroomtemperature.Theplantmaterial(300g)weretriturated separatelyandsubmittedtohydrodistillationinaClevenger-type apparatusfor2h.Theoilswerecollected,driedwithanhydrous Na2SO4,measured,and transferredtoglassflasks andkeptat a temperatureof−18◦Cforfurtheranalysis.

The volatile oils were analyzed using a Shimadzu GC-MS QP5050A fitted witha fused silica SBP-5 (30m×0.25mm I.D.;

0.25␮mfilmthickness)capillarycolumn(composedof5% phenyl-methylpolysiloxane) and temperature programmed as follow: 60–240◦_C _at₃◦_C/min,_then_to₂₈₀◦_C_at ₁₀◦_C/min,_ending_with 10minat280◦_C._The_carrier_gas_was_He_at_a_flow_rate_of₁_ml/min andthesplitmodehadaratioof1:20.Theinjectionportwasset at225◦_C._Significant_quadrupole_MS_operating_parameters: inter-facetemperature240◦_C;_electron_impact_ionization_at₇₀_eV_with

scanmassrangeof40–350m/zatasamplingrateof1scan/s. Con-stituentswereidentifiedbycomputersearchusingdigitallibraries ofmassspectraldata(NIST,1998)andbycomparisonoftheir reten-tionindicesandauthenticmassspectra(Adams,2007),relativeto C8–C32n-alkaneseriesinatemperature-programmedrun(VanDen DoolandKratz,1963).

PCAwasappliedtoexaminetheinterrelationshipsbetweenthe chemicalconstituentsofthevolatileoilsfromleavescollectedin differentmonthsusingthesoftwareStatistica(StatSoft,2004).A hierarchicalclusteranalysis(HCA)wasusedtostudythe similar-ityofsamplesbasedonthedistributionoftheconstituents,and hierarchicalclusteringwas performedaccordingtothemethod ofminimumvarianceWard(Ward,1963).Tovalidatethecluster analysiswascarriedoutusingthecanonicdiscriminantanalysis (DCA).

Results

Morphologicaldescription

Trembleya phlogiformis is a subshrub or shrub up to 2m tall. Stem cylindrical, woody, slightly exfoliative, sub-ligneous branches,yellowish-green,slightlyalates(angular),withtectores and glandular trichomes. Sticky leaves, simple, opposite cross, short-petiolate,3–4mmlong,ovateblade,2.5–5cmlong,1–1.5cm wide, membranous, margin entire to serrate, apex acute, base cuneateorsubcordiform,acrodromebothsideswithlargeamount ofglandulartrichomes,seroussurface.Inflorescenceterminalor lateralwithflowersarrangedindichasia.5-mereflowers, hypan-thiumcampanulate,green, thickhairwithglandulartrichomes, 3–4mm long,about 1.8mmwide, presence ofa pairof bracts; pedicel3–4mmlong,withglandulartrichomes;calyx5-laciniate, triangularlaciniae,1.2–3mmlongwithglandulartrichomes exter-nally;5-corollapetals,obovate,glabrous,free,whitetopink,about 9mmlong,4.5–5mmwide;10stamens10,free,dimorphic,white anthers, wine color, poricidal, prolonged connective ventrally; ovary glabrous, 5-carpels, gamo-carpels, 5-locular, placentation axillary,terminalstylus,curved,white.Fruitglabrouscapsuletype orwithglandulartrichomes,4–6mmlong,3mmwide,5-locular. Smallseedsnumerous,brown-clear,elongatedandstooped,1mm long,0.2–0.3mmwide.

Behavior

Trembleyaphlogiformisshoweddifferentbehaviorduringtheir developmentcycle,overaperiodof12months.Inthemonthsof DecemberandJanuarymostoftheindividuals wasinthe vege-tativestatewithgreenandlargeleaveswithstickysubstanceon theleafsurfaceandabsenceofinsectsandfungi,probablydueto thepresenceofthisstickysubstance,aswereobservedsomestuck insectsanddeadintheleaves.Theflowerbudsbegantoappearin FebruaryandinearlyMaytheflowersemergegradually,andmay befoundthroughoutthismonth,inflorescenceswithmanybuds andopenflowerswithpetalswhosecolorsrangefromwhiteto pink-dark.Theyfoundindividuals60cmhighwithinflorescences. ThefruitingbeganinMay,green-brownishcapsulescanbefound, whichbecomebrownish whenripeinJune.Duringthedry sea-son,whichusuallygoesfromJulytoOctober,theaerialbranchesof

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Table1

ClimateinformationofcollectionperiodoftheplantmaterialofTrembleyaphlogiformis.

Station Date Rainfalltotal Averagemaximumtemperature(◦_C) _Average_minimum_temperature₍◦_C)

83376 07/31/2013 0 31 14.6

83376 08/31/2013 4.4 32.5 15.8

83376 09/30/2013 14.5 33.7 18.5

83376 10/31/2013 144.2 32 19.4

83376 11/30/2013 264.6 30.6 19.1

83376 12/31/2013 343.9 30 20

83376 01/31/2014 270.1 30.9 18.3

83376 02/28/2014 252.3 30.7 19.2

83376 03/31/2014 277.1 30.2 19.1

83376 04/30/2014 298.5 30.35 19.2

83376 05/31/2014 4.3 30.9 16.1

83376 06/30/2014 0.1 30.1 14.7

Source:INMET(2014).

branches.Intherainyseason,inNovember,observedthepresence ofnewleaves,restartinganothercycle.

Volatileoils

During the collection period the months of highest rain-fallwereOctober (144.2mm),November(264.6mm),December (343.9mm), January (270.1mm), February (252.3mm), March (277.1mm)andApril(298.5mm),withaveragetemperatures ran-gingfrom32◦_C_to_18.3◦_C._The_months_with_less_rainfall_were_May (4.3mm),June(0.1mm),July(0mm),August(4.4mm)and Septem-ber(14.5mm),withtemperaturesrangingfrom14.6◦_C_to_33.7◦_C (Table1).

Theyieldofvolatileoilofflowerswas0.008%(w/v)andleaves varied0.01to0.03%(w/v)(Table2).

In theflowerswereidentified 82.7%of thesubstances:4.8% monoterpenes oxygenates, 0.17% sesquiterpenes hydrocarbons, 0.76% sesquiterpenes oxygenates and 76.9% other compounds. Themajorcompounds foundinvolatileoilsof flowersweren -heneicosane(33.5%),phytol(12.3%),n-tricosane(8.4%)andlinoleic acid(6.1%)(Table2).

Inthemonthlyanalysis,theyidentified68.9–94.2%ofthe chemi-calcompoundsofvolatileoilsofT.phlogiformisleaves.Amongthem, 0.2–2.2%weremonoterpenehydrocarbons,4.8–29.9%oxygenated monoterpenes,2.8–24.5%sesquiterpenehydrocarbons,1.2–12.6% oxygenatedsesquiterpenesand37.9–66.7%ofotherclassesof com-pounds.Themajorcompoundpresentinallleafsampleswasoleic alcoholrangingfrom5.7to26.8%.Thenerylacetonewas major-ityin theleavesin January(9.2%),March(11.4%), April (13.4%) andNovember(7.8%), thehexenal inFebruary(9.7%),butylated hydroxytolueneinFebruary(13.6%),March(20.6%)and Novem-ber(6.7%);acetate␤-ment-1-en-9-olinMarch(7.3%),dictamnolin April(7.5%),heneicosaneinMay(12.6%),the1,1,6-trimethyl-1, 2-dihydronaphthaleneinJune(8.2%),thehexadecanoicacidinJuly (6.4%)and December(8.3%),6,10,14-trimethyl-2-pentadecanone inAugust(8.4%),the2-pentadecanoneinOctober(19.3%),linalool (7.7%) and ␣-terpineol (8.5%) in November, geranyl acetone in December(7.6%)(Table2).

TheresultsobtainedfromthePCAandclusteranalysisshowed theexistenceofchemicalvariabilityamongsamplesofvolatileoils obtainedfromleavesofT.phlogiformis.Fig.1indicatesthatthe rel-ativepositionoftheaxis2DoriginatedinthePCA.Thisanalysis suggeststhattheclusterIisdiscriminatedbycompoundsbutylated hydroxytoluene,demothoxyageratochromeneandnerylacetone, thesamplespresentintheclusterIarecharacterizedbytheperiod withhigherlevelsofrainfall.ClusterIIobservedinFig.1suggests thatoleicacidisthecompoundcapableofdiscriminatethisgroup characterizedinsamplescollectedinmonthsoflowrainfall,except forthesampleobtainedinDecember.Cluster IIIiscomposedof threemonthsalsowithlessprecipitationandwasnot

discrimi-natedbyanyoftheselectedcompounds,butcoincideswiththe periodinwhichtheT.phlogiformisspecimenshavefewleavesand dryfruits.Theclusteranalysis(Fig.2)suggeststheexistenceof threegroups(whichshowedagreementwiththePCA):clusterI (volatileoilsfromleavescollectedinNovember,January,March andApril);clusterII(volatileoilsfromleavescollectedin Octo-ber,December, February,MayandJuly), clusterIII(volatileoils fromleavescollectedinJune,AugustandSeptember).Theresults indicatethattheclassificationproposedbythePCAandHCAwas appropriatefortheclassificationofsamplesasthechemicalprofile ofvolatileoils.

Canonicdiscriminantanalysiswasperformedtohelptopredict thegroupingoftheclusteranalysis,andtwopredictivevariables were employed: oleicalcohol and demothoxyageratochromene, andthetwodiscriminantfunctionsretain100%ofwell– classifi-cationintheoriginalclustersbyacross-validationapproach.Thus, thecanonicdiscriminantanalysisrevealedthattheclassification proposedandtheirvariablesemployedaresuitabletoshowthat thefindingsoftheHCAandthePCAwereconsistent(Table3).

Discussion

ItwasobservedthatTphlogiformishassimple,opposite,cross leaveswithinflorescencesdichasiatypewithporicidalanthers, typ-icalcharacteristicsofMelastomataceae(Cogniaux,1891;Wurdack etal.,1993; Goldenbergand Reginato,2007).It’sa shrubabout twometerstallwithpyramidaspect,withthewiderbaseofthe leavesthatfoundattheapex;theleavesaresimple,short, oppo-site,petiolatecrusades,ovate,withacuteapex,subcondiformbase, palmate-parallelleaf venationand serrated;theflowershave a calyxwith5sepalsandfivepetalsfreewith10 stamens,which featureswithinthegenreTrembleya(Don,1823;Martins,1997).It wasobservedthatT.phlogiformispetalsvaryfromwhitetopink andclassifiedasdialipetalwithacuteapexasdescribedbyMartins (1997).The seedsshowedgreatsimilarities astotheformatof speciesofthegenusbythepresenceofelongatedandcurvedseeds, butwithdifferenceswhencomparedtothesize,sincetheT. phlogi-formisseedshaveabout1mminlengthanddescribedinthegenre haveupto0.57mm(Martins,1997).

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Table2

PercentageofthechemicalconstituentsofthevolatileoilsfromTrembleyaphlogiformisleavesandflowerscollectedinPirenópolis,Goiás.

Leaves Flowers

Constituents KI RI Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec –

Hexenal 855 832 – 9.7 – – – – – – – – – – –

Heptanal 902 888 – 0.6

2-Pentylfuran 988 986 0.8 – – – – – – – – – – – –

Hepta-2,4-dienal 1007 1002 2.6 – – – – – – – – 0.7 – – –

p-Cymene 1024 1014 – 1.2 – – – – – – – 0.2 – – –

Limonene 1029 1019 – 0.7 – – – – – – – – – – –

1,8-cineole 1031 1022 – 1.6 – – – – – – – – – – –

Benzene acetaldehyde

1042 1031 0.7 0.9 – – – – – – – – 1.8 – –

Octen-1-al 1054 1046 0.3 3.7 – 2.2 2.2 3.0 1.1 1.7 2.7 0.9 – – –

n-Octanol 1068 1061 – 1.0 – 2.0 – – – 0.7 2.2 1.0 1.5 – 0.1

LinalooloxideB 1072 1063 0.4 – – 2.4 0.6 – – – 1.7 0.7 1.2 2.2 –

m-Cymene 1085 1079 – – – – – 2.2 – 0.3 – – – – –

Linalool 1096 1091 1.1 4.6 – 5.6 5.2 1.4 1.2 0.9 2.8 2.2 7.7 – 0.5

n-Nonanal 1100 1094 0.9 2.0 – 4.7 2.8 5.4 1.4 1.8 2.8 1.6 2.5 1.2 2.2

Nonen-1-al<(2Z)> 1144 1150 – 0.5 – – – – – – – – – – –

Camphor 1146 1134 – – – – 0.8 – – – – – – – –

2Z-Nonenal 1149 1143 0.6 – – – – 0.9 – 0.63 0.7 0.6 1.3 – –

2E,6Z-Nonadienal 1154 1150 1.4 – – – 0.4 – – – – 0.8 1.9 1.1 –

Terpinen-4-ol 1177 1182 – – – – – – – – – 0.3 – – –

˛-Terpineol 1188 1182 1.6 0.8 – 4.0 3.6 – – 0.9 1.6 2.0 8.5 2.0 0.2

Safranal 1196 1191 0.8 – – – – – – 0.9 1.5 – – – –

n-Decanal 1201 1196 0.5 – – 2.9 0.8 2.2 1.2 1.4 1.5 1.0 – – 0.2

ˇ-Cyclocitral 1219 1211 1.5 1.6 – 2.1 1.0 2.6 – 1.4 2.9 0.9 2.5 1.6 –

2E-decenal 1263 1251 0.5 0.9 – 3.2 1.3 3.5 1.9 1.7 2.0 1.0 1.2 – 0.2

Thymol 1290 1284 – – 0.6 – – – 1.8 – – – – – –

2E,4Z-Decadienal 1293 12844 0.4 – – – – – – – – 0.5 – 1.1 0.2

Undecan-2-one 1294 1284 – – – – – – – 0.5 0.7 – – – –

n-Tridecane 1300 1291 – – – – – – – – – 2.4 – – –

Undecanal 1306 1296 – – – 1.5 – 2.7 1.6 1.3 1.6 0.6 – – –

2E,4E-Decadienal 1316 1307 1.7 – – – – – – – – 0.7 1.9 – –

1,1,6-Trimethyl-

1,2-dihydronaphthalene sno

1355 1342 1.6 1.8 – 1.8 1.2 8.2 2.6 3.4 2.7 1.1 3.6 1.9 –

2E-Undecenal 1360 1353 – – – 1.9 – 1.6 2.0 0.8 0.7 0.5 – – –

␤

-(2Z)-Damascenona

1364 1375 1.1 – – 1.8 – – – 0.7 1.6 0.9 – 1.2 –

␣-Copaene 1376 1368 – – – – – – – 0.8 – – 1.4 – –

Dodecanal 1408 1398 – – – – – – – – – – – – 0.2

␣-Santalene 1417 1409 1.6 1.0 1.2 2.6 1.3 5.9 4.7 3.7 3.7 1.4 2.5 1.1 –

␤-Caryophyllene 1419 1410 – – – 4.0 – 3.6 1.8 5.8 1.6 0.6 2.0 – 0.2

␤-menth-1-en-9-ol

acetate

1423 1420 4.1 – 7.3 – 1.0 – – 2.4 3.1 3.2 2.0 – –

Dictamnol 1429 1423 2.6 – 2.3 7.5 – – – – – – 5.5 5.8 –

Nerylacetone 1436 1442 9.2 1.1 11.4 13.4 – 4.2 – – 3.9 4.0 7.8 – 0.6

Geranylacetone 1455 1442 – – – – 4.7 – 1.8 3.4 – – – 7.6 –

allo-aromadendrene

1460 1451 – – – – – – – 1.4 – – – – –

6-Demothoxy-ageratochromene

1463 1472 3.4 1.5 5.2 5.0 – – – – 0.7 0.9 3.2 – –

trans-cadina-1-(6),4-diene

1476 1467 – – – – – – 0.9 – – – – –

␥-Muurolene 1478 1479 – – – 1.2 – – 2.3 – – – – –

trans-␤-Ionone 1488 1476 3.2 1.6 3.1 3.9 1.9 1.9 – 1.0 1.5 1.5 5.2 3.4 0.2

␤-Selinene 1490 1486 – – – – – 2.0 – 2.8 1.1 – – – –

2-Tridecanone 1496 1485 – – – – – – – – – 1.2 – – –

Tridecanal 1510 1498 2.5 1.2 – 2.8 – 3.7 5.6 4.2 2.4 1.6 1.6 – 0.2

Butylated hydroxytoluene

1515 1502 2.0 13.6 20.6 5.9 – – 2.7 1.0 – 2.2 6.7 – 3.3

␦-Cadinene 1523 1513 0.6 – 2.5 – 1.6 – – 3.5 1.3 0.6 5.8 1.3 –

E-Nerolidol 1563 1554 – – – – – – – – – 4.9 – – 0.4

Dodecanoicacid 1566 1567 1.8 0.6 7.9 – – 4.9 5.4 2.7 4.0 – – – 1.1

n-Hexadecane 1600 1589 – – – – – – – – 0.9 0.5 – – –

Tetradecanal 1612 1600 0.6 – – – – 1.8 3.4 1.6 – 0.9 – – 0.2

Pentadecanone 1697 1686 – – – – – – – – 0.9 19.3 – – –

n-Heptadecane 1700 1689 – – 1.2 – – – 0.8 – 2.0 – – – – n-Octadecane 1800 1788 – – 2.0 – – – 1.6 – 2.1 0.5 – – –

6,10,14-trimethyl-2-pentadecanone

1833 1826 – – 3.2 – 1.9 – – 8.4 – 1.2 3.1 5.8 –

Farnesylacetate 1822 1825 3.8 – 1.3 3.3 – 1.8 – – – 0.8 – 1.6 0.1

n-Nonadecane 1900 1887 0.4 – 1.3 – 1.4 1.2 2.0 – 1.4 0.5 – – 1.2

5E,9E,

13E-Farnesyl-2-one

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Table2(Continued)

Leaves Flowers

Constituents KI RI Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec –

Phytol 1943 1935 2.8 5.3 0.8 – 1.6 – 2.1 1.4 1.2 1.8 – 2.1 12.3

Hexadeconoicacid 1960 1957 5.7 1.0 – 2.4 1.9 6.4 0.6 3.1 2.8 – 8.3 –

ethyl

hexadecanoate

1993 1981 – – – – 0.7 – – – – 0.7 5.6 2.8 0.8

n-Eicosane 2000 1987 – – 1.87 – – – – – – – – – 1.3

E,E-geranyllinalool 2027 2016 – – – – – – – – – – – – 0.10.11

Kaurene 2043 2027 – – – – – – – – – – – – 0.6

Methyllinoleate 2085 2128 – – – – – – – – – – – – 2.7

n-Heneicosane 2100 2091 0.6 1.3 1.0 1.8 12.6 0.7 4.6 0.7 1.0 1.2 – 0.6 33.5

Oleicalcohol 2101 2101 16.8 16.7 7.2 7.9 14.4 9.0 16.4 5.7 7.8 17.0 6.1 26.8 –

Linoleicacid 2133 2131 3.0 – – – 1.4 – 2.0 – – 0.6 – 3.2 6.1

n-Docosane 2200 2186 – 1.2 – – – – – – – – – – 2.8

n-Tricosane 2300 2287 – 1.4 – – – – – – – – – – 8.4

n-Tretacosane 2400 2384 – – – – – – – – – – – – 0.8

n-Pentacosane 2500 2484 – – – – – – – – – – – – 1.7

Hydrocarbonsmonoterpenes – 1.9 – – – 2.2 – 0.3 – 0.20 – – –

Monoterpenesoxygenates 18.6 9.7 19.3 27.5 16.8 8.1 4.8 9.9 17.6 13.3 29.9 13.8 4.8

Hydrocarbonssesquiterpenes 3.8 2.8 3.7 8.4 5.3 19.7 9.1 24.5 10.5 3.1 15.4 4.2 0.2

Sesquiterpenesoxygenates 10.8 0 5.7 12.6 1.2 3.2 2.1 1.9 3.1 8.5 5.5 10.6 0.8

Others 53.3 63.7 56.4 45.7 45.7 44.5 62.2 37.9 44.0 66.7 43.6 56.4 77.0

Totalidentified 86.5 78.1 85.2 94.1 69.0 77.7 78.1 74.5 75.2 91.8 94.2 84.9 82.7

Yield 0.022 0.024 0.028 0.022 0.020 0.015 0.010 0.013 0.016 0.024 0.031 0.033 0.008

(–),notdetected.KI,Kovatsretentionindex(valuesfromliterature).RI,retentionindex.

ThemajorcomponentsofthevolatileoilofT.phlogiformis flow-erswere:n-heneicosane(33.5%),thephytol(12.3%),n-tricosane (8.4%)andlinoleicacid(6.1%).Itwasverifiedtheexistenceofalarge chemicalvariabilityofthevolatileoilsfromtheleavesofT. phlogi-formisoverthemonths,withthemajoritycompound(oleicalcohol) presentinallsamples,whichdifferentiatesitfromotherspecies ofthefamily.StudiesofvolatileoilsofMelastomataceaespecies fromtheAmazonfoundasmajorcompoundsinBellucia grossulari-oides(L.)Trianathepentadecanone(10.0%),palmiticacid(13.1%);in

Miconiaciliata(Rich.)DCthehexylacetate(8.4%),p-cymene(10.3%) (E,E)-␣-farnesene(14.7%);inMiconiaminutiflora(Bonpl.)DCthe ␣-copaene(22.8%),␤-caryophyllene(14.5%),␣-humulene(12.2%);

inMiconiarubiginosa(Bonpl.)DC.Thenonanal(18.5%),␣-copaene (32.9%);inTibouchinastenocarpa(DC.)Cogn.TypeA:terpinen-4-ol (11.2%),palmiticacid(22.9%)typeB:(E)-␤-ionone(17.2%),palmitic acid(22.9%)(Zoghbietal.,2001).StudiescarriedoutbyMayaand Andrade(2009)withMelastomataceaespeciesusingvariousparts ofplants(leaves,wood,bark,fruit,flowers)revealedasmajor com-poundsoftheBelluciagrossularioides(L.)pentadecanone(10.0%), palmiticacid(13.1%);theMiconiaciliata(Rich.)DC-hexylacetate (8.4%),p-cymene(10.3%)(E,E)-␣-farnesene(14.7%);ofthe Mico-nia minutiflora(Bonpl.) DC-␣-copaene (22.8%), ␤-caryophyllene (14.5%),␣-humulene(12.2%),␤-curcumene(7.9%);oftheMiconia rubiginosa(Bonpl.)DC-nonanal(18.5%),␣-copaene(32.9%);ofthe

0 2 4 6 8 10

Euclidean distance August

September June December October July May February March November April January Samples

(6)

April May

July

August December

–4 –3 –2 –1 0 1 2 3 4 5

PC-1 (65.91%)a

1 0

–1

PC-1b

–2.5 –2.0 –1.5 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 2.5

PC-2 (20.21%)

a

–1 0 1

PC-2

b

Oleic acid

Hidroxytoluene butilate

Demothoxyageratochromene

Neryl acetone Cluster II

Cluster III

Cluster I

June

September Frebruary

October

January

November

March

Fig.2. ScatterplotfromPCAofleavesofTrembleyaphlogiformis,samplescollectedfromPirenópolis,GObelongingtotheclustersI,IIandII.a_Axes_refer_to_scores_from_the samples;b_Axes_refer_to_scores_from_discriminant_oil_constituents_represented_as_vectors_from_the_origin.

Tibouchinastenocarpa(DC.)–TypeA:terpinen-4-ol(11.2%), germa-creneD(7.9%),palmiticacid(22.9%);typeB:(E)-␤-ionone(17.2%). Toudahletal.(2012)identifiedinthevolatileoilofMicrolicia grave-olense thirteen compounds, withthe majority trans-pinocarvila acetate(78.9%).Itcanbeobservedtheoccurrenceofconstituents ␤-pinene,limonene,pinocarvone,terpinen-4-ol,trans-␣-terpineol pinocarveoleinthespeciesM.crenulataeM.graveolens(Pereira, 2013).Komalavallietal.(2014)identifiedasmajorcompoundsin

SonerilatinneveliensisFischer(Melastomataceae) tetrahydrospiril-loxanthin(18:50%),ethyliso-allocate(18:27%).

Itwasverifiedarelationshipbetweenthephenologyand vari-ationin chemical compositionof volatileoilsof T.phlogiformis. Thesampleswereclassifiedintothreeclusters:ClusterI(volatile oilsfromleavescollectedin themonthsofNovember,January, MarchandApril),consistingofsamplescollectedintheperiodwith thehighestrainfall.InJanuaryspecimensaremostlyina

vegeta-tivestate.ClusterII(volatileoilsfromleavescollectedinOctober, December,February,MayandJuly).InFebruarytherewerethefirst flowerbudsthatdevelopthroughthemonthofMay,forming inflo-rescenceswithmanyflowerbudsandopenflowers.Themonthof Decemberhadthehighestrainfallrate(343.9mm)andprobably duetothisincreasedrainfall,thesamplescollectedinDecember showedadifferentchemicalprofiletothevolatileoil,with similar-itytoothermonthsofotherseasons.ClusterIII(volatileoilsfrom leavescollectedinJune,AugustandSeptember)istohighlightthe lowlevelofrainfallandduringthisperiodthedryplantandthe leavesfall,averycommonfeatureamongtheplantsoftheCerrado. Seasonaldifferences inthechemical composition oftheleaf volatileoilshavealsobeendescribedforthevolatileoilsofHortia oreadicaleaves(Santosetal.,2015).Despitethevolatileoilchemical compositionisgeneticallydetermined,manyabioticfactorssuch aslight,temperature,seasonality,nutritionandwateravailability

Table3

CanonicaldiscriminantanalysissummaryofTrembleyaphlogiformis.

Canonicaldiscriminanant

Eingenvaluesfunctions CanonicalR Wilk’sLambda p-Level

F1 7.071305 0.936004 0.051284 0.000045

F2 1.415894 0.765555 0.413925 0.006178

Standardizedcoefficients

Oleicacid −0.12459 −1.03697

Demothoxyageratochromene −1.02881 −0.17993

Eigenvalues 7.07130 1.41589

CumulativeProportion 0.83317 1.00000

ClusterI ClusterII ClusterIII

Percentageoftotalwell-classification p=0.33 p=0.42 p=0.25

ClusterI 100 4 0 0

ClusterII 100 0 5 0

ClusterIII 100 0 0 3

(7)

cansignificantlychangetheproductionofsecondarymetabolites.

Environmentalstimulicanalterthemetabolicpathwaysfor

pro-ductionofthesecompounds,leadingtothebiosynthesisofdifferent compounds(Gobbo-NetoandLopes,2007;Limaetal.,2003).

Thus,theresultsreportedhereinmaysuggestthatthevolatile oilsofT.phlogiformisleavespossesshighchemicalvariability, prob-ablyrelatedtovariationassociatedwithrainfallandthevariationin thebehaviorofspecimensthroughouttheyear.Themajority com-poundsofvolatileoilsofT.phlogiformisleavesandflowerswere notfoundinliteratureasmajorcompoundsofthevolatileoilsin otherspeciesofMelastomataceae,maybetypicaloftheirspecies. Thisresearchprovidesinsightsforfuturestudiesonthevolatileoils obtainedfromtheT.phlogiformisleavesandflowers,mainlyrelated tobiologicalmarkersofapplicationsmonitoredintheleavesand flowersofthisspecies.

Authors’contributions

SRFcontributed incollectingplant sample,runningthe lab-oratory work. HDF contributed in collecting plant sample and identification,confectionofherbariumandmorphological descrip-tion of the species. SS contributed in collecting plant sample, running thelaboratory work.LLB contributed tothe statistical analyzes.LMFTcontributedtocriticalreadingofthemanuscript. PHFcontributedtochromatographicanalysis.JRPcontributedto chemicalstudies,chromatographicanalysisandcriticalreadingof themanuscript.TSFdesignedthestudy,supervisedthelaboratory workcontributedtobiological andchemical studies, chromato-graphicanalysisanddraftedthepaperandcontributedtocritical reading of the manuscript. All the authors had read the final manuscriptandapprovedthesubmissioncontributedtobiological studiesrunningthelaboratorywork,analysisofthedata.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authors gratefully acknowledge the financial support obtainedfromCAPES,CNPq,andFundac¸ãodeAmparoàPesquisa doEstadodeGoiás.

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