Rev. bras. farmacogn. vol.26 número6

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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

variability

of

the

essential

oils

of

leaves

and

morphological

analysis

of

Hyptis

carpinifolia

Stone

de

Sá

a

_,

_Tatiana

_S.

_Fiuza

b

_,

_Leonardo

_L.

_Borges

c

_,

_Heleno

_D.

_Ferreira

b

_,

_Leonice

_M.F.

_Tresvenzol

a

_,

Pedro

H. Ferri

d

_,

_Maria

_H.

_Rezende

b

_,

_José

_R.

_Paula

a,∗

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_Instituto_de_Química,_Universidade_Federal_de_Goiás,_Goiânia,_GO,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25January2016 Accepted5May2016 Availableonline28June2016

Keywords:

Hyptiscarpinifolia

Sesquiterpenes Essentialoilcomposition 1,8-Cineole

a

b

s

t

r

a

c

t

HyptiscarpinifoliaBenth.,Lamiaceae,isaspeciesknownpopularlyas“rosmaninho”and“mata-pasto”, andleavesareemployedinBrazilianfolkmedicinetotreatcolds,flu,andrheumatism.Theaimofthis studywastoperformamorphologicaldescriptionofH.carpinifoliaandtoevaluatetheseasonalchemical variabilityoftheleafessentialoilsduring12months.MacroscopiccharacterizationofH.carpinifoliawas carriedoutwiththenakedeyeandwithastereoscopicmicroscope.Essentialoilswereisolatedfromleaves byhydrodistillationinClevengerapparatusandanalyzedbygaschromatography/massspectrometry. Majorcompoundswerefoundtobe1,8-cineole(39.6–61.8%),trans-cadina-1(6),4-diene(2.8–17.5%),␤ -caryophyllene(4.4–10.0%),prenopsan-8-ol(4.2–9.6%)and␤-pinene(2.9–5.3%).Resultsofessentialoils compositionswereprocessedbyclusteranalysisandprincipalcomponentanalysis.Datashowedhigh variabilityintheconcentrationofthecomponents.Besides,therewasaseasonalvariabilityofchemical composition,probablyrelatedmainlytotherainfallregime.

Introduction

Hyptis genus, belongs to the Lamiaceae family, subfamily

Nepetoideae,tribeOcimeaeandsubtribeHyptidinae.Itisoneofthe largestandmostwidelydistributedplantgeneraintheworldwith morethan300species(Harley,1988).Itiscomposedofherbs, sub-shrubs,shrubsandmorerarelysmalltrees.Thestemsofthespecies ofthisgenusareoftensquareincross-section;theleavesareusually opposite,occasionallywhorled,simpleorrarelywithslits, petio-late,shortlypetiolateorsessileandaromatic,hairsgland-headed withessentialsoils,simple,non-glandular,usuallymulticellularor bothmulticellularandunicellular(Bordignon,1990).

Species ofthis genus are commonly usedin traditional folk medicinetotreatavarietyseveraldiseasessuchasgastrointestinal infections,cramps,painsandskininfections(Corrêa,1931).

TheessentialoilsfoundinthegenusHyptishaveagreat impor-tanceasasourceofbioactiveconstituents,especiallyduetoits

∗ Correspondingauthor.

E-mail:[email protected](J.R.Paula).

biologicalpropertiessuchasantimicrobial,cytotoxicand

insecti-cide(Kuhntetal.,1995).

HyptiscarpinifoliaBenth.,commonlyknowninBrazilas “ros-maninho”and“mata-pasto”,hasastrongrosemaryaromaandis usedinfolkmedicinetotreatcolds,flu(Silvaetal.,2000), rheuma-tism(decoctionorinfusionoftheleaves)(RodriguesandCarvalho, 2001)andforbaths(Brandãoetal.,2012).

Itisabranchedshrubupto3mhigh,glabrousleaves,sessile, elliptictooblong-ovatewithcordatebase(Epling,1949;Harley, 2012);inflorescenceslarge,interminalspikewith1–2cmin diam-eter; violetflowers; linear-lanceolatebracts amongthe flowers

(Harley,2012;Pignaletal.,2013).Xylematicraysandprismatic

crystalswerevisualizedinthecalix(RabeiandEl-Gazzar,2007). H.carpinifoliaisdistributedinBrazil(MatoGrosso,MatoGrosso doSul,Piauí,SãoPaulo,Roraima,Tocantins),Bolivia(Harley,2012)

andPeru(Mobot,2016).

Noreferenceswerefoundintheliteratureregardingthe chemi-calprofileandbiologicalactivitiesofH.carpinifolia.Thus,thisstudy aimedtoperformthemorphologicaldescriptionofH.carpinifolia andtoevaluatethechemicalconstituentsoftheessential oilof leavesanditsseasonalvariability.

http://dx.doi.org/10.1016/j.bjp.2016.05.011

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Materialsandmethods

Plantmaterial

Forthemorphologicaldescription,specimensofHyptis carpini-foliaBenth.,Lamiaceae,deposited inthe herbariaof:Município Alvorada do Norte, Goiás, 02.VII. 1964, J.M. Pires 58142 (UB); NovaColina,Goiás,EstradaBelém-Brasília,5–10kmofNovaColina, 31.VII.1964,G.T.Prance&N.T.Silva58495(UB);Mineiros,Goiás, ParqueNacionaldasEmas,03.VII.1983,05.VII.1983,H.D.Ferreira 225(UFG);specimenscollectedinParqueNacionaldasEmas,in MineirosandinNovaAmérica,Goiás,Brazil,wereexamined.

Morphologicalanalysis

MacroscopiccharacterizationofH.carpinifoliawascarriedout withthenakedeyeandwithastereoscopicmicroscopeOlympus SZ-ST.

EssentialoilsisolationandGC–MSanalysis

Forisolationofessentialoils,H.carpinifolialeaveswerecollected fromtendifferentindividuals,monthly,foroneyear(2013),inthe cityofNovaAmérica/Goiás(15◦₀₂′_29.5′′_S_and₄₉◦₅₉′_0.05′′_W,_at_an elevationof1295mabovesealevel).Plantmaterialwasidentified byDr.HelenoDiasFerreiraandavoucherspecimenwasdeposited attheHerbariumoftheFederalUniversityofGoiás,Brazil,under codeUFG43.833.

Theleavesweredriedatroomtemperatureandgroundin a knifemill.Differentbatches(100g)ofpowderedleaveswere sub-mittedtohydrodistillationinaClevenger-typeapparatusfor2h. AfterdriedoveranhydrousNa2SO4,oilswerekeptinglassvialsata temperatureof−18◦_C_prior_to_further_analysis._The_essential_oil vol-umewasmeasuredinthegraduatedtubeoftheapparatusandwas calculatedaspercentagerelativetotheinitialamountofdryplant materialusedintheextraction.Eachexperimentwasperformedin triplicate.

The essential 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 _at₁₀◦_C/min,_ending _with 10minat280◦_C._The_carrier_gas_was_a_flow_rate_of₁_ml/min_and_the splitmodehadaratioof1:20.Theinjectionportwassetat225◦_C. Significantquadrupolemassspectrometeroperatingparameters: interfacetemperature240◦_C;_electron_impact_ionization_at₇₀_eV withscanmassrangeof40–350m/zatasamplingrateof1scan/s. Constituents were identified by computer search using digital librariesofmassspectraldata(NIST,1998)andalsobycomparison

oftheirretentionindices(VanDenDoolandKratz,1963)relative toC8–C32n-alkanesandmassspectrawithliteraturedata(Adams,

2007).

Statisticalanalysis

PrincipalComponentAnalysis(PCA)wasappliedtoexaminethe interrelationshipsbetweenthechemicalconstituentsofthe essen-tialoilsfromleavescollectedindifferentmonthsusingthesoftware Statistica7(StatSoft,2004).Aclusteranalysiswasusedtostudythe similarityofsamplesbasedonthedistributionoftheconstituents, andhierarchicalclusteringwasperformedaccordingtothemethod ofminimumvarianceWard(Ward,1963).Tovalidatethecluster analysiswascarriedoutusingthecanonicdiscriminantanalysis (DCA).

Toverifythepossibleassociationbetweentheessentialoil com-ponentsselectedalongwithclimaticvariables(temperatureand rainfall) was used the Pearson’scorrelation analysis (

Callegari-Jacques,2003).

Results

Essentialoils

Withinthecollectionperiod,thehighestrainfallregimenswere registeredinthemonthsofOctober,NovemberandDecember.In thisperiod,themaximumtemperaturerangedfrom32to35◦_C andtheminimumtemperaturerangedfrom20to22◦_C._Lowest precipitationwasobservedinthemonthsofJune,July,Augustand Septemberandthemaximumtemperaturerangedfrom33to36◦_C andtheminimumtemperaturerangesfrom18to22◦_C₍_INMET,

2014)(Table1).

The loss of weight of leaves after drying was 72%. The yields of the essential oils of H. carpinifolia leaves ranged from 1.2 to2.0%. The oilsare mainly composedof oxygenated monoterpenes (40.4–62.6%), followed by sesquiterpene hydro-carbons (13.6–37.3%), oxygenated sesquiterpenes (11.2–17.1%) and monoterpene hydrocarbons (4.7–9.4%). Major compounds werefoundtobe1,8-cineole (39.6–61.8%),trans -1-cadina-(6),4-diene (2.8–17.5%), ␤-caryophyllene (4.4–10.0%), prenopsan-8-ol (4.2–9.6%),␤-pinene(2.9–5.3%)(Table2,Fig.1).

TheresultsobtainedbythePCAandclusteranalysisshoweda chemicalvariabilitywithintheH.carpinifoliaoils.Fig.2showsthe relativepositionofthe2D-axisoriginatedinthePCA.Cluster analy-sissuggeststhattherearethreegroups:clusterI(essentialoilsfrom leavescollectedinthemonthsofApril,October,Novemberand December)characterizedbypinonicacid(mean=1.05±0.21),as themaincomponentandthehighestrainfallindex(233.6±115.1); cluster II(essentialoils fromleaves collectedinthe monthsof

Table1

ClimateinformationofthecollectperiodoftheplantmaterialofHyptiscarpinifolia.

Station Date(2013) Rainfallnumberof days

Totalrainfall Maximumtemperature average

Minimumtemperature average

Relativehumidity average

83374 01/31 26 376.3 31 21 78.00

83374 02/28 19 117.1 34 21 74.75

83374 03/31 22 131 34 21 77.05

83374 04/30 6 99.4 33 21 72.26

83374 05/31 2 29.1 34 19 61.42

83374 06/30 4 8.3 33 19 64.25

83374 07/31 0 34.11 34 18 51.16

83374 08/31 0 35.78 35 19 42.39

83374 09/30 4 26.9 36 22 44.05

83374 10/31 13 114.9 35 22 65.66

83374 11/30 20 232 33 20 77.50

83374 12/31 27 315.5 32 21 80.36

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Table2

PercentageofthechemicalconstituentsoftheessentialoilsfromHyptiscarpinifolialeavescollectedmonthlyinNovaAmérica,Goiásduringtheyear2013.

Constituents KI Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec Tricyclene 926 1.5 1.8 1.5 1.2 1.5 1.3 0.9 1.2 1.4 1.1 1.1 1.0 6-Methyl-heptan-2-ol 965 0.4 0.4 0.3 0.2 2.2 0.2 – – – 0.1 – – Sabinene 975 1.1 1.2 1.0 0.9 1.2 1.2 0.9 1.1 1.2 1.0 1.0 0.9

␤-Pinene 979 4.5 5.3 4.1 3.1 4.5 4.2 2.9 4.2 5.1 3.1 3.1 3.1 1,8-Cineole 1031 55.5 51.8 43.9 39.6 47.2 52.6 47.7 57.4 61.8 48.1 51.2 54.5

␥-Terpinene 1059 0.1 0.1 – – – – 0.1 0.1 0.1 – 0.1 0.2

cis-Sabinenehydrate 1070 – – – – – – 0.1 0.1 0.1 0.2 0.2 0.1 Pinocarvone 1164 0.7 – 0.1 0.3 0.5 0.8 0.9 0.7 0.4 1.3 1.3 1.4 Terpinene-4-ol 1177 0.7 0.8 0.7 0.5 0.4 0.3 0.1 0.2 0.3 0.4 0.4 0.5

␣-Copaene 1376 3.4 3.7 4.1 4.4 3.6 3.3 3.1 2.7 2.4 3.9 3.6 3.2

␤-Bourbonene 1388 0.4 0.4 0.4 0.4 0.7 0.7 1.0 0.8 0.8 0.8 0.7 0.5

␤-Caryophyllene 1419 6.1 5.9 8.2 10.0 6.7 7.7 9.2 6.7 4.4 9.9 8.4 6.5 Pinonicacid 1442 0.7 0.7 0.9 1.3 0.9 0.8 0.8 0.6 0.4 1.2 1.0 1.0

␣-Humulene 1454 0.4 – 0.2 0.4 0.2 0.5 0.9 0.6 0.2 – 0.4 0.8

trans-Cadina-1(6),4-diene 1476 3.4 2.8 10.6 17.5 5.5 4.6 5.4 3.9 2.9 7.5 6.2 4.5

␤-Selinene 1490 1.1 1.1 1.2 1.0 0.9 0.9 1.1 0.8 0.7 0.9 1.0 1.0

␦-Selinene 1492 1.6 1.7 1.9 2.0 1.5 1.3 1.5 1.2 1.1 1.6 1.4 1.5

␥-Cadinene 1513 1.0 1.0 1.2 1.4 1.1 1.0 1.2 0.9 0.7 0.8 1.0 1.2

cis-Cadineneether 1553 0.3 – – – 0.3 0.4 – – 0.5 – – – Prenopsan-8-ol 1577 7.0 8.5 7.6 6.0 7.5 6.9 8.3 5.7 4.2 9.6 8.5 7.0

allo-Cedrol 1589 0.2 0.3 0.3 0.3 0.4 0.4 0.4 - 0.4 0.2 0.2 0.2

allo-Hedycaryol 1589 1.7 1.7 1.7 1.4 2.2 2.4 3.4 2.9 2.9 1.7 1.9 2.0 Rosifoliol 1600 0.5 0.8 0.6 0.4 0.7 0.5 0.6 0.4 0.3 0.7 0.6 0.5

epi-Cedrol 1619 0.8 0.8 0.6 0.7 0.1 – – – – 0.3 0.8 –

Caryophylla-4(12),8(13)-dien-5␣-ol 1640 0.3 0.5 0.3 0.2 0.5 0.4 0.5 0.3 0.3 0.3 0.2 0.3 Caryophylla-4(12),8(13)-dien-5␤-ol 1640 0.7 1.1 0.9 0.6 – 1.0 1.2 0.9 0.9 0.7 0.6 0.8 Pogostol 1653 0.9 1.2 1.0 0.7 1.3 0.8 1.0 0.9 0.9 0.4 0.7 0.8 Khusinol(valerianol) 1658 1.7 2.5 1.9 1.2 2.1 1.8 1.6 1.7 2.2 1.2 1.3 1.4

Monoterpenehydrocarbons 7.5 8.9 6.9 5.3 9.4 6.9 4.7 6.6 7.9 5.4 5.3 5.1 Oxygenatedmonoterpenes 56.9 52.6 44.7 40.4 48.0 53.6 48.9 58.4 62.6 50.0 53.2 56.4 Sesquiterpenehydrocarbons 17.7 16.8 28.1 37.3 20.6 20.4 23.8 17.6 13.6 25.5 22.9 19.5 Oxygenatedsesquiterpenes 13.9 17.1 13.6 11.2 14.7 14.1 16.6 12.8 12.3 14.8 14.5 12.7 Others 0.7 0.7 0.9 1.3 0.9 0.8 0.8 0.6 0.4 1.2 1.0 1.0 Totalidentified(%) 96.7 96.1 93.4 95.5 93.6 95.8 94.8 96.0 96.8 96.9 96.9 94.7 Yield(%) 1.8 2.0 2.0 1.9 2.0 1.6 1.3 1.3 1.2 1.6 1.6 1.8 KI,Kovatsretentionindex.

January, February, March and May) characterized by pogostol (1.2±0.2)andthekhusinol(2.2±0.3)as maincomponentsand rainfallindex(92.4±55.3);andclusterIII(essentialoilsfromleaves collectedinthemonthsJune,July,AugustandSeptember) char-acterizedby1,8-cineole(54.9±6.1)andthelowestrainfallindex (26.3±12.6)(Figs.3and4).Theresultsindicatedthatthe classifi-cationproposedbythePCAandHCAareinagreement.

Canonicdiscriminantanalysiswasperformedtohelptopredict thegroupingoftheclusteranalysis,andtwopredictivevariables wasemployed:pogostol(p=0.008)andrainfall(p=0.01),andthe discriminantfunctionretain92%ofwell-classificationinthe orig-inalclustersbyacross-validationapproach.

Through the correlation analysis between ␣-copaene and pinonic acid and humidity, there are a significant correlation [R=0.71 (p=0.01); R=0.60 (p=0.04), respectively], that is, the higher thehumidity thegreater theamount ␣-copaene of and pinonicacid.

Morphologicalanalysis

H.carpinifolia is a shrubup to3mhighwith glandular vil-lous branches.Leaves sessile or subsessile, blade oblong-ovate, oblongorsometimesrounded,usually3–6cmlong,2–3cmwide, adaxialsurfacesubglabrous,abaxialsurfaceglandular-pubescent ortomentose,apexroundedorsometimesacute,basesubcordate orrounded,serratemargin,reticulateandprominentveinsinthe abaxialsurface.Verticillasterscompactorslightlyseparated, pau-ciflorus,sessile,arrangedinterminalspike,bracts7–9mmlength, submembranous,ovate,lanceolate,linear-lanceolate,ciliated mar-ginwithbranchedhairs.Flowersbluesorviolet;calyxtubeofthe flower1.5–2mmlength,glandularpuberulentexternally,hirsute orifice, erectand branchedhairs, teeth subulatecalyx, unequal or subequal, about 2mm length, mature tube calyx 4–4.5mm length;corollatubewith8mmlength,slightlyglandular puberu-lentexternally,filamentofthestamenwithhairssuperiorly,3mm

10 1

23 4

7 8

9 10

11 12

13

14 15 16

17 18

19 20

21

23

22 242526 27 28

29 5 6

15 20 25 30 35

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Jan

Feb Mar

Apr

May Jun Jul

Aug

Sep

Oct

Nov Dec

8 6 4 2

0 –2 –4 –6 –8

PC-1 ( 45.47%)a PC-1b

–5 –4 –3 –2 –1 0 1 2 3 4

PC-2 (18.81%)

a

PC-2

b

Pinonic acid

Rainfall

1,8-cineol

Pogostol Khusinol Cluster I

Cluster II

Cluster III

Means and standard deviation of clusters:

Rainfall Khusinol

Pinonic acid Pogostol 1,8-cineole

Cluster I 1.1 ± 0.2 1.3 ± 0.2 233.6 ± 115.0 0.7 ± 0.2 49.8 ± 6.4 Cluster II 0.8 ± 0.1 2.2 ± 0.3 92.4 ± 55.2 1.2 _± 0.2 47.6 _± 3.9 Cluster III 0.6 ± 0.2 1.8 ± 0.2 26.3 ± 12.6 0.9 ± 0.1 54.9 ± 6.1

Fig.2.ScatterplotofPCAoftheessentialoilsfromtheleavesofHyptiscarpinifoliasamplescollectedfromNovaAmérica/GObelongingtotheclusters(I,IIandIII).a_Axes

referringtothescoresofsamples.b_Axes_referring_to_scores_of_volatile_chemicals_whose_discriminant_constituents_are_represented_by_vectors.

length; ovary glabrous, style glabrous, articulated in the ovary base,10mmlength.TheschizocarpfruitofH.carpinifoliapresents were nutlet smooth, light brown, abruptly apiculate, 1.5mm length.

Itwasfoundinthisstudythat,inGoiás,H.carpinifoliaoccurs wetlandsinthegalleryforestmargins,strictosensuCerrado.Itis foundataltitudes between164mand1295mwithan average

elevationof667m.ThefertileindividualswerecollectedfromMay toJuly.

Discussion

The major compounds of the essentials oils of H.

carpini-folia leaves were 1,8-cineole, trans-cadina-1(6),4-diene,

10 9 8 7 6 5 4 3 2 1 0

Euclidean distance September

August July June March May February November October April December January

Cluster I

Cluster II

Cluster III

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Cluster III Cluster II

Cluster I 0

50 100 150 200 250

Pluviometric precipitation (mm)

Cluster

Fig.4.Correlationbetweenrainfallandclusters.

␤-caryophyllene, prenopsan-8-ol and ␤-pinene. There are not dataintheliteratureonthechemicalcompositionoftheoilsof thisspecies.However,the1,8-cineolewasdescribedasoneofthe majorcompoundsoftheleavesofdifferentspeciesofthisgenus, suchas,H.crenataPohlexBenth.(Rebeloetal.,2009),H.suaveolens (L.)Poit.(Moreiraetal.,2010),H.fruticosaSalzm.exBenth.(Franco etal.,2011),H.goyazensisA.St.-Hil.exBenth.(McNeiletal.,2011) andH.martiusiiBenth.(Araújo etal.,2003;Caldasetal.,2014). ␤-Caryophyllene have been found in the essential oils of the leavesofH.glomerataMart.exSchrank(SilvaandMoura,2011), H.martiusii(Araújoet al., 2003), H. marrubioidesEpling (Botrel

etal.,2010;Salesetal.,2009),H.spicigeraLam.,H.pectinata,H.

floribundaBriq.,H.suaveolens(McNeiletal.,2011);while␤-pinene hasbeenidentifiedintheleavesofH.crenata(Rebeloetal.,2009), H.suaveolens(Moreiraetal.,2010)andH.fruticosa(Francoetal.,

2011).

1,8-Cineoleisamonoterpenefoundinmanyaromaticplantsof thegenusEucalyptus,Croton,Hyptis,Pectis,RosmarinusandSalvia

(Araújoetal.,2003;Vilelaetal.,2009;Hussainetal.,2011).

Numer-ousbiologicalandpharmacologicalproprietieswereassignedto 1,8-cineole, including antimicrobial, insecticidal (Balacs, 1997), anti-allergic,anti-inflammatory(Santosand Rao,1998), hepato-protective(Santosetal.,2001),antitumoral(Motekietal.,2002)and gastrointestinalactivity(Santosetal.,2001).Thepresenceof 1,8-cineolecanalsoexplainthepopularuseoftheH.carpinifoliainflu, colds,andrheumatism.Cardiovasculareffectswerealsoassigned to1,8-cineole,in reducingtheblood pressureofbothconscious andanesthetizedratsandshowingvasorelaxanteffectsonisolated rataorta.Thisactivityseemstobedependentontheintegrityof thevascularendotheliumandoxidenitricreleasing(Lahlouetal.,

2003;Pintoetal.,2009;Santosetal.,2011).

Theobservedvariabilityofthechemicalcompositionof essen-tialoilsduringtheyear,couldbecorrelatedwithrainfall.Thisresult isinaccordancewiththereportsofGobbo-NetoandLopes(2007)

andalsoMorais(2009),whichreportedthattheproductionof

sec-ondarymetabolitesbyplantsmaybeinfluencedbyenvironmental factorssuchasseasonalityandthatthesefactorscanchangethe yieldandchemicalcompositionofessentialoils.Barrosetal.(2009)

foundthatclimateconditionscaninfluencetheenzymatic activi-tiesincertainplantspeciesandtheninfluenceinthebiosynthesis ofcertainsecondarymetabolites,includingterpeniccompounds. ExperimentswithH.suaveolensshowedinterferenceof environ-mentalfactorsonthechemical compositionofitsessential oils

(Martinsetal.,2006).Botreletal.(2010)foundthatmonoterpenes

andsesquiterpenescontentofH.marrubioides,inspring,showed verysimilarvalues,however,inwinter,themonoterpenescontent doubledinrelationtothesesquiterpenes.

H.carpinifoliawasplacedbyEpling(1949)inPolydesmida sec-tion,subsectionRigidaewithonlytwospecies:H.carpinifoliaand H.violaceaBenth.H.carpinifolia differsfromH.violaceafor hav-ingabruptlypeakednutlets,whileinH.violaceathenutletsare obtuse.H.carpinifoliahasadaxialsurfacesubglabrous,abaxial sur-faceglandular-pubescentortomentose,while,accordingwiththe sameauthor.H.violaceahasscabrouslimbonthesurfaceadaxial andalmostglabraontheabaxialsurface.

Epling(1949)describesH.carpinifoliaasashrubof3mhigh.

Theanalyzedspecimensoftheherbariaandfieldinthisstudyalso havenotfoundanyspecimenstallerthan3m.Epling(1949)also reportedforH.carpinifoliacalyxtubewith5.5–6mmlengthand corollatube6–8mmlength.Inthisstudy,thecalyxtubehassmaller length(1.5–2mm)andthecorollatubewith8mmlength.

ThefertileindividualsofH.carpinifoliawerecollectedfromMay toJulywhileinH.suaveolensthefloweringandfruitingoccursin OctobertoDecember(JelaniandPrabhakar,1991).

Thisworkcontributedfor themorphologicaldescriptionand for the knowledge on the chemical composition of essential oils of H. carpinifolia. Major compounds found in the essential oils from leaveswere 1,8-cineole, trans-1-cadina(6),4-diene, ␤ -caryophyllene, prenopsan-8-ol,␤-pinene. The resultspresented hereinstronglysuggestthatthereisaseasonalvariabilityof chem-icalcompoundsrelatedtorainfall.

Authors’contributions

SScontributedincollectingplantsample,runningthe labora-torywork.TSFdraftedthepaperandcontributedtocriticalreading ofthemanuscript.HDFcontributedincollectingplantsampleand identification,confectionofherbariumandmorphological descrip-tionofthespecies.LLBcontributedtothestatisticalanalyzes.MHR contributedincollectingplantsampleandidentification.LMFT con-tributedtocriticalreadingofthemanuscript.PHFcontributedto chromatographicanalysis.JRPdesignedthestudy,supervisedthe laboratoryworkcontributedtobiological and chemicalstudies, chromatographicanalysisandcriticalreadingofthemanuscript.All theauthorshadreadthefinalmanuscriptandapprovedthe sub-missioncontributedtobiologicalstudiesrunningthelaboratory work,analysisofthedata

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

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

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