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medicines Article Chemical Composition, Antifungal and Antioxidant Activities of Hedyosmum brasiliense Mart. ex Miq. (Chloranthaceae) Essential Oils CynthiaMurakami1,2,*,InêsCordeiro2,MarcusTulliusScotti3 ID,PauloRobertoH.Moreno4 ID andMariaCláudiaM.Young2 1 ProgramadePós-GraduaçãoemBiodiversidadeVegetaleMeioAmbiente,InstitutodeBotânicade SãoPaulo,SãoPaulo04301-902,Brazil 2 InstitutodeBotânicadeSãoPaulo,SãoPaulo04301-902,Brazil;[email protected](I.C.); [email protected](M.C.M.Y.) 3 LaboratóriodeQuimioinformática,UniversidadeFederaldaParaíba,JoãoPessoa58051-900,Brazil; [email protected] 4 InstitutodeQuímica,UniversidadedeSãoPaulo,SãoPaulo05508-000,Brazil;[email protected] * Correspondence:[email protected];Tel.:+55-11-5067-6174 AcademicEditor:EleniSkaltsa Received:30June2017;Accepted:13July2017;Published:17July2017 Abstract: Background: HedyosmumbrasilienseMart. exMiq. (Chloranthaceae)isadioeciousshrub popularlyusedinBraziltotreatfootfungiandrheumatism. Thisworkinvestigatedthechemical composition,antifungal,andantioxidantactivitiesofflowersandleavesofH.brasilienseessential oils;Methods: H.brasiliensemaleandfemaleflowersandleaveswerecollectedatIlhadoCardoso (SãoPaulo)andtheessentialoilswereextractedbyhydrodistillationandanalyzedbyGC/MSand their similaritycomparedby Principal ComponentAnalysis. Antifungal activity wasperformed bybioautographyandantioxidantpotentialby2,2-diphenyl-2-picrylhydrazylhydrate(DPPH)free radical scavenging and β-carotene/linoleic acid system; Results: The major compounds for all oils were sabinene, curzerene, and carotol, but some differences in their chemical composition werediscriminatedbyPrincipalComponentAnalysis(PCA)analysis. Bioautographyshowedtwo antifungalbandsatRf’s0.67and0.12inallsamples,thefirstonewasidentifiedascurzerene. Theoils presentedstrongerantioxidantpotentialinβ-carotene/linoleicacidbioassay,withIC ’sfrom80to 50 180µg/mL,thaninDPPHassay,withIC ’sfrom2516.18to3783.49µg/mL;Conclusions: These 50 results suggested that curzerene might be responsible for the antifungal activity of H. brasiliense essentialoils. Besides,theseessentialoilsexhibitedpotentialtopreventlipoperoxidation,butthey haveaweakradicalscavengeractivity. Keywords: Hedyosmum brasiliense; essential oil; GC-MS; bioautography; curzerene; antioxidant; AtlanticRainForest 1. Introduction The family Chloranthaceae includes plants grouped in four genera: Sarcandra Gardner, ChloranthusSw.,AscarinaJ.R.Forst.andG.Forst.,andHedyosmumSw.ThegenusHedyosmumcomprises 40speciesofshrubsandsmalltreesdistributedfromMexico,throughoutCentralAmericatoBolivia, east of the Guianas and the Antilles [1]. The name Hedyosmum comes from the Greek “pleasant fragrance” and alludes to one of the most remarkable characteristics of these plants, the pleasant aromaticsmellcomingfromallitsparts[1,2]. AccordingtoTodzia[1],differentHedyosmumspecies havebeenpopularlyusedallaroundCentralandSouthAmericafordifferentpurposes: H.scabrum (Ruiz&Pav.) Solmsforconceptionstimulation;H.scabrumandH.scaberrimunStandl. asstomachpain Medicines2017,4,55;doi:10.3390/medicines4030055 www.mdpi.com/journal/medicines Medicines2017,4,55 2of13 relievers;H.spruceiSolmsforsnakebites;H.cumbalenseH.Karst. asastimulant;andH.racemosum (Ruiz&Pav.) G.Donasatreatmenttojointpain. Theessentialoilcompositionfromsomespeciesof thisgenus(H.sprucei,H.arborescensSw.,H.scabrum,H.colombianumCuatrec.,H.angustifolium(Ruiz& Pav.) Solms,H.mexicanumC.Cordem.,H.bonplandianumMart.,andH.costaricenseC.E.WoodexW.C. Burger)havepreviouslybeendescribed[3–8],aswellastheirantioxidantandcytotoxicproperties[3,4]. HedyosmumbrasilienseMart. exMiq.,popularlyknownas“chádebugre”,“cidreira”,or“cidrão”, is a dioecious shrub distributed throughout central and southern Brazilian regions and western Paraguay[9,10]. Itsleaveshavebeenusedinfolkmedicinetotreatmigraine,ovariumdysfunction, footfungi,rheumatism,stomachpain,andasadiuretic[1,2]. Chemicalandpharmacologicalstudies withtheaerialpartsofH.brasilienseindicatedanalgesic,anxiolytic,antidepressive,andaphrodisiac effects for the crude extracts and for some isolated sesquiterpene lactones [11–16]. There are also somereportsontheantimicrobialactivityfortheethanolextract[17]andtheessentialoils[18,19], howevernoneofthemcouldattributetheobservedbioactivitytoaspecificcompound. Theessential oilpresentedasmajorcompoundsspecimensabinene,(Z)-β-guaieneandpinocarvone,foraSãoPaulo State [18], and pinocarvone, curzerene and carotol, for one from Santa Catarina State [19]. To the bestofourknowledge,thisisthefirstreportonthechemicalcompositionandbiologicalactivitiesof H.brasiliensefloweressentialoils. Inaddition,thisisalsothefirsttimethattheantioxidantactivity isdescribed forthisspecies. Therefore, thisworkaims toinvestigatethe chemicalcompositionof theessentialoilsfromH.brasiliensemaleandfemaleflowersandleavescollectedatIlhadoCardoso (SãoPaulo,Brazil)andtocomparetheirantifungalandantioxidantactivities,aswellastoidentifythe mainantifungalconstituentsintheessentialoils. 2. MaterialsandMethods 2.1. PlantMaterial HedyosmumbrasilienseMart. exMiq. (Chloranthaceae)flowersandleaveswerecollectedfrom maleandfemalespecimensatParqueEstadualdaIlhadoCardoso,SãoPaulo,Brazil(25◦05(cid:48) Sand 47◦55(cid:48) W,14malt)inSeptemberof2015. ThetaxonomicidentitywasconfirmedbyDr. InêsCordeiro (InstitutodeBotânica,SãoPaulo,Brazil). ThevoucherspecimensweredepositedintheHerbariumat theInstitutodeBotânica,SãoPaulo,Brazil,withtheaccessionNo. SP475335. 2.2. ExtractionoftheEssentialOils Ninemaleandfemalespecimenswerecollected. Leavesandflowerswerepooledtoprovide representativehomogeneoussamplesofthepopulation,separatedintothreereplicatesandstored underrefrigeration(−22◦C)untilextraction. Essentialoilwasobtainedbyhydrodistillationfor3.5h inaClevenger-typeapparatus. Thecrudeoilwasseparated,driedoveranhydroussodiumsulfate andstoredinaglassflaskat −22 ◦CuntilGC-MSanalysisandbiologicalactivities. Theoilyields werecalculatedbasedontheoilandfreshplantmaterialweightsasmean±standarddeviationofthe triplicates[18]. 2.3. GC-MSAnalysis Essential oils were solubilized in acetone 1:100 v/v (Merck KGaA, Darmstadt, Germany). Chemical analysis of the essential oils was performed on an Agilent 6890 Series GC (Agilent, SantaClara,CA,USA),interfacedwitha5973SeriesquadrupoleMSdetector(Agilent,SantaClara, CA, USA) and equipped with a DB-5MS column (30 m × 0.25 mm i.d. × 0.25 µm) (Agilent J&W, Santa Clara, CA, USA). Chromatography conditions were as follows; oven temperature program: 40◦Cfor1minwithsubsequenttemperatureincreaseof3◦C/minupto240◦C,keepingitatthis temperaturefor10min.;carriergas:Heataflowrateof1mL/min;injectoranddetectortemperatureof 250◦C;electronionization: 70eV.Thecomponentabundanceswereexpressedasthemean±standard deviationofthetriplicates. Theessentialoilcomponentswereidentifiedbycomparingtheretention Medicines2017,4,55 3of13 indices(evaluatedinrelationtotheretentiontimesofaseriesofn-alkanes)andbycomparisonoftheir massspectrawiththosereportedintheliterature[20,21]. 2.4. PrincipalComponentAnalysisoftheEssentialOils Principal Component Analysis (PCA) was performed using composition of H. brasiliense essentialoils. PriortothePCAanalysis,allthevariableswerestandardizedforanormalizedPCA. TheUnscrambler® Xversion9.7(CAMOSoftware,Oslo,Norway)wasusedtoperformPCAanalysis andgeneratescoreandloadingplot[22]. 2.5. AntifungalActivity The microorganisms used in antifungal assay, Cladosporium cladosporioides (Fresen de Vries (CCIBt140)andC.sphaerospermumPenz(CCIBt491),havebeenmaintainedatInstitutodeBotânica, SãoPaulo,SP,Brazil. Fortheantifungalassay,carriedoutbybioautographyadaptedfromHomans & Fuchs method [23], 10 µL of solutions corresponding to 200, 100, 50, and 25 µg of essential oils inmethanolwereappliedassinglespotsonpre-coatedThinLayerChromatography(TLC)plates (GF silica-gelchromatoplates,MerckKGaA,Darmstadt,Germany). Nystatin,thymol,andcinnamic 254 acid(5µg)wereusedaspositivecontrols. Theplatesweresprayedwithasuspensionofsaltsand glucose (6:1) containing fungus spores (> 2 × 106 spores/mL) of Cladosporium cladosporioides and C.sphaerospermum.Theplateswereincubatedinmoistchambersat27◦Cfor48h,inthedark.Afterthe incubationperiod,clearinhibitionzoneswereobservedasantifungalactivityoftheessentialoils. Thecomponentsresponsiblefortheantifungalactivitiesweredeterminedbyapplying400µgof eachessentialoilontothreeTLCplatesanddevelopedwithn-hexane/acetone,90:10v/v. Inoneplate, theseparatedfractionbandswerevisualizedwithUVlight(254nm)andsprayedwithVanilin-Sulfuric Acid(VS)reagentfollowedbyheatingto110◦Cfor5min[24]. Theothertwoplateswereusedfor thebioautographyassay. Accordingtotheantifungalactivityresult,7mgoffemaleflowerandleaf essentialoilswereappliedonchromatoplates,separately,anddevelopedwiththesamesolventsystem. AttheexactR ’softheactivebands, theplatewascuthorizontally, washedwith1mLofacetone, f filtered(0.22µmfilters,MerckMillipore,Darmstadt,Germany)andfurtheranalyzedbyGC-MS,atthe sameconditionsastheessentialoils[25]. 2.6. InVitroAntioxidantActivities Theantioxidantactivitywasperformedbythe2,2-diphenyl-2-picrylhydrazylhydrate(DPPH) freeradicalscavengingmethod[26],adaptedformicroplates. 178µLoftheessentialoilssolubilizedin methanolatconcentrationsbetween4480–560µg/mLwereaddedtoa96-wellmicroplateand72µL ofDPPH(0.3mmol/L)solubilizedinmethanolwereadded. Blankreadingwasperformedwiththe test sample prior to the DPPH radical incubation. As a negative control, 178 µL of methanol was used. Aspositivecontrols,quercetinfrom20to0.625µg/mLandGinkgobilobaextract(Herbarium Laboratório Botânico S.A., Paraná, Brazil) from 40 to 1.25 µg/mL were used. The microplate was incubated in the dark for 30 min at room temperature. Then, the absorbance was measured at a wavelengthof518nmusingamulti-wellscanningspectrophotometer(BIOTEKKC4,Winooski,VT, USA).Theabsorbancewasconvertedtopercentageofantioxidantactivity(AA)usingtheformula AA%=100−{[(Abs −Abs )×100]/Abs }.Allsamplesweretestedintriplicateandthe sample blank control resultsareexpressedbymean±standarddeviation. Theamountofessentialoilrequiredtoreducethe initialDPPHconcentrationinthereactionby50%isreferredtoastheinhibitoryconcentration(IC ). 50 The antioxidant activity was also evaluated by the β-carotene bleaching test, which is based onspectrophotometricmeasurementsoftheβ-caroteneoxidationinducedbythelipoperoxidation products from linoleic acid [27] to determine the concentration providing 50% inhibition (IC ). 50 Theessentialoil(10µL)solubilizedinmethanolatconcentrationsbetween10,400–325µg/mLwas addedina96-wellmicroplate,togetherwith250µLofareactivesolutioncomposedbyβ-carotene andlinoleicacidinwatersaturatedwithO . Methanol(10µL)wasusedasthenegativecontrolandas 2 Medicines2017,4,55 4of13 thepositivecontrols,butylhydroxytoluene(BHT)andbutylhydroxyanisole(BHA)atconcentrations between130–4.1µg/mL.Themicroplatewasincubatedinthedarkat45◦Candsubsequentlythe absorbancewasreadat450nm(multi-wellscannerBIOTEKKC4,Winooski,VT,USA),immediately afteraddingthereactivesolutionandevery30minfor2h.Theabsorbancewasconvertedtopercentage of antioxidant activity (AA) using the AA% = 100 − {[(Abs − Abs ) × 100]/Abs }. final initial control Allsamplesweretestedintriplicateandtheresultsareexpressedbymean±standarddeviation. 3. Results 3.1. ChemicalCompositionoftheEssentialOil TheessentialoilyieldsformaleandfemaleH.brasilienseflowerswere0.24±0.01and0.38±0.01%, respectively. Theyieldofleafessentialoils,bothmaleandfemale,presentedayieldof0.33±0.01% (Table1). Fifty compounds were identified from all essential oils analyzed, comprising 96–98% of the totalcomponents. ThemajorcompoundsforallH.brasilienseessentialoilsweresabinene,curzerene, and carotol. Leaf essential oils presented mostly monoterpenes (53% and 55% ), followed by sesquiterpenes(39% and41% ), andphenylpropanoids(2% and5♂% ). Flow♀eressentialoils presentedmonoterpen♂es(54% an♀d45% ),sesquiterpenes(40% ♀and50% ♂),andphenylpropanoids (1% and2% ). Theleafmai♀nconstitu♂entsweresabinene(16%♂),β-pinene♀(5%),1,8-cineole(3–7%), meth♂yl eugeno♀l (2–5%), curzerene (17–18%), and carotol (6%), while flowers presented sabinene (8–10%), α-phellandrene (1–8%), 1,8-cineole (2–7%), germacrene D (4–6%), curzerene (11%), and carotol(9%)asmajorcompounds. Principal Component Analysis (PCA) data from Table 1 generated a plot, which condensed scoresandloadingsofthefirsttwocomponents,PC1andPC2,whichexplain61%and34%oftotal variance(Figure1). Leavespresentedhigheramountsofsabineneandcurzerenethanflowers,while flowerspresentedhigheramountsofcarotol,asdiscriminatedbyPCA(Figure1). Furthermore,PCA analysisshowedthatfemaleflowersandleavespresentedhigheramountsof1,8-cineolethanthemale counterparts(Figure1). Table1.ChemicalcompositionofHedyosmumbrasilienseessentialoils. RelativeAmount(%) Compound RI RIref(a) Flowers Flowers Leaves Leaves 4-hydroxy-4-methyl-2-pentanone 841 839 1♂.58±0.02 1♀.44±0.03 1♂.53±0.06 1♀.49±0.02 α-pinene 933 939 2.23±0.02 1.96±0.02 3.16±0.10 3.66±0.11 sabinene 972 975 9.46±0.04 8.05±0.11 15.81±0.19 15.81±0.17 β-pinene 976 979 3.91±0.03 2.73±0.04 4.64±0.13 5.21±0.19 myrcene 990 990 1.75±0.02 1.45±0.02 2.48±0.05 2.79±0.14 α-phellandrene 1003 1002 8.14±0.07 1.10±0.01 2.79±0.44 0.82±0.06 α-terpinene 1016 1017 1.07±0.01 0.91±0.01 1.18±0.12 1.09±0.12 ρ-cymene 1023 1024 1.51±0.01 limonene 1028 1029 2.53±0.01 1.08±0.01 1.68±0.06 1.46±0.05 1,8-cineole 1031 1031 1.65±0.01 7.22±0.07 3.44±0.04 6.86±0.17 (Z)-β-ocymene 1036 1037 2.26±0.01 0.66±0.01 (E)-β-ocymene 1048 1050 1.85±0.01 3.00±0.04 0.90±0.06 1.91±0.01 γ-terpinene 1059 1059 1.76±0.01 1.44±0.01 1.81±0.16 1.68±0.17 terpinolene 1082 1088 0.57±0.00 linalool 1097 1096 1.53±0.01 1.5±0.01 0.99±0.03 1.31±0.03 camphor 1146 1146 0.73±0.01 pinocarvone 1161 1164 3.65±0.02 3.15±0.02 4.49±0.11 3.94±0.07 terpinen-4-ol 1177 1177 3.34±0.02 3.11±0.04 3.30±0.36 3.09±0.32 α-terpineol 1191 1188 1.07±0.01 1.37±0.01 0.61±0.07 0.76±0.06 citronellol 1224 1225 0.60±0.03 chrysanthenylcis-acetate 1255 1265 1.21±0.01 1.03±0.08 1.55±0.03 bornylacetate 1279 1288 0.58±0.00 thymol 1285 1290 3.63±0.05 1.11±0.20 Medicines 2017, 4, 55   5 of 13  thymol  1285 1290  3.63 ±0.05  1.11 ± 0.20  Medicines2017,4,55 5of13 pinocarvyl cis‐acetate  1303 1312  1.16 ± 0.01 0.72 ± 0.03  1.05 ± 0.02  δ‐elemene  1327 1338  1.33 ± 0.01 2.24 ±0.11  2.30 ± 0.07  α‐terpinyl acetate  1344 1349  1.47 ± 0.01 0.75 ± 0.01 0.93 ± 0.04  1.19 ± 0.02  Table1.Cont.   α‐copaene  1370 1376  0.71 ± 0.00 0.66 ± 0.00 β‐elemene  1384 1390  1.27 ± 0.01 1.68 ± 0.01 1.43 ± 0.09  1.58 ± 0.05  RelativeAmount(%) methCyolm epuoguenndol  R1I394 RIre1f4(0a3)  1.41 ± 0.02 1.96 ± 0.02 4.53 ± 0.21  1.85 ± 0.17  Flowers Flowers Leave s Leaves (E)‐caryophyllene  1412 1419  0.65 ± 0.01 0.69 ± 0.00 pinoγc‐aerlveymlecinse-a cetate 13104323 13114236  ♂0.69 ± 0.00 1♀.106.7±2 0±. 001.01 0♂.720.±700 .±0 30.06 1♀.050±.706.0 ±2 0.02  δ-elemene 1327 1338 1.33±0.01 2.24±0.11 2.30±0.07 α‐humulene  1449 1454  0.80 ± 0.02 0.72 ± 1.36 0.59 ± 0.04  0.58 ± 0.00  α-terpinylacetate 1344 1349 1.47±0.01 0.75±0.01 0.93±0.04 1.19±0.02 germacrene D  1477 1485  4.23 ± 0.04 5.76 ± 0.02 3.74 ± 0.21  3.35 ± 0.13  α-copaene 1370 1376 0.71±0.00 0.66±0.00 cuβr-zeelermeneen e 13184489 13194099  11.217.0±9 0±. 001.11 1.1618.6±80 ±.0 01.05 1.4137±.020. 0±9 0.66 1.581±7.07.80 5± 0.30 βm‐beitshaybloeulegneen ol 13195402 14105305  1.041.9±7 ±0 .002.01 1.926.6±6 0±. 002.01 4.53±0.2 1 1.85±0.17 (Eδ)‐-ccaardyionpehnyel lene 14115211 14115923  0.065.9±8 ±0 .001.01 0.619.5±1 0±. 000.01 0.63 ± 0.02  0.71 ± 0.02  γ-elemene 1423 1436 0.69±0.00 0.72±0.01 0.70±0.06 0.76±0.02 (Z)‐α‐bisabolene  1539 1507  0.63 ± 0.01 0.95 ± 1.36 0.80 ± 0.11  1.86 ± 0.04  α-humulene 1449 1454 0.80±0.02 0.72±1.36 0.59±0.04 0.58±0.00   geerlmemacorel neD 14175742 14185549  4.23±0.04 5.706.6±1 0±. 002.00 3.74±0.21 3.35±0.13 cNur.Iz.e 1re ne 14185956 1499 111.0.917± ±0 0.1.101 11.16.86±6 ±0 .00.501 17.002.8±6 0±.6 06.01 17.780±.800. 3±0 0.02  spβa-tbhisualbeonloenl e 15105270 15105578  0.497.5±3 ±0 .001.04 2.636.0±8 0±. 001.01 3.05 ± 0.44  2.23 ± 0.08  δ-cadinene 1511 1523 0.98±0.01 1.51±0.01 0.63±0.0 2 0.71±0.02 viridiflorol  1578 1592  0.81 ± 0.01 0.82 ± 0.00 (Z)-α-bisabolene 1539 1507 0.63±0.01 0.95±1.36 0.80±0.1 1 1.86±0.04 Ne.lIe.m 2o l 15145287 1549 0.67 ± 0.00 0.601.8±6 0±. 000.01 carNo.tIo.l1  15155696 1594  1.917.7±2 ±0 .001.04 1.696.3±6 0±. 001.05 0.866.±480 .±0 10.10 0.805±.901.0 ±2 0.15  spNa.tIh.u 3l enol 15176020 1578 4.53±0.04 3.008.7±8 0±.001.01  3.05±0.4 4 2.230±.705.0 ±8 0.03  epi‐vαir‐icdaifldoinrooll  15176826 15196240  0.081.9±3 ±0 .001.01 0.82±0.00 0.96 ± 0.17  N.I.2 1587 0.67±0.00 0.86±0.01   daucol  1636 1642  0.61 ± 0.00 carotol 1596 1594 9.72±0.04 9.36±0.05 6.48±0.10 5.91±0.15 NN.I.. I4. 3 16126041 0.74 ± 0.01 0.78±0.01 0.84 ± 0.09 0.75±0.03 αe‐pmi-uαu-craodloinlo l 16126643 16146046  0.93±0.01 0.74 ± 0.08 0.96±0.1 7 α‐euddaeuscmolol  16136648 16146253  0.161.6±6 ±0 .000.02 3.08 ± 0.02 1.06 ± 0.04  1.79 ± 0.08  N.I.4 1641 0.74±0.01 0.84±0.0 9 (Z)‐α‐santalol  1661 1675  0.57 ± 0.00 α-muurolol 1643 1646 0.74±0.08 feruαla-e uladcetsomnoe lI  16240801 16159374  1.166.7±5 ±0 .002.02 3.038.7±0 0±. 002.02 1.061.±880 .±0 40.69 1.792±.408.0 ±8 0.70  M(Zo)n-αot-searnpteanloel hydrocarbon1 6(6%1)  1675 38.12 ± 0.09 0.5279.±6 0±. 000.27 38.08 ± 1.44  41.28 ± 0.97 Ofexruylgaelnacattoende mI onoterpene2 (0%01)  1974 11.765.2±7 0±. 002.06 3.1750.0±00 ±.0 02.10 1.8185±.330. 6±9 0.60 2.481±4.03.87 0± 0.46 SesqMuointeortperepneen ehhyyddrrooccaarrbboonn ((%%))  381.102.9±3 ±0 .009.06 291.66.6±80 ±.2 07.05 38.098.9±3 1±.4 04.74 41.281±0.705.9 ±7 0.50 OxyOgxeyngaetneadte sdemsqounioteterrppeennee ((%%))  162.297.0±3 ±0 .006.19 153.030.4±5 0±. 100.31 15.3239.±390 .±6 01.61 14.383±0.108.4 ±6 1.21 Sesquiterpenehydrocarbon(%) 10.93±0.06 16.68±0.05 9.93±0.74 10.75±0.50 Phenylpropanoid (%)  1.41 ± 0.02 1.96 ± 0.02 4.53 ± 0.21  1.85 ± 0.17  Oxygenatedsesquiterpene(%) 29.03±0.19 33.45±0.31 29.39±1.61 30.18±1.21 NPohte nidyelpnrtoipfiaendo (id%()% ) 1.441.2±4 ±0 .002.04 1.936.3±0 0±. 002.01 4.532.±760 .±2 10.13 1.851±.506.1 ±7 0.02  TotNalo itdideenntitfifiieedd ((%%))  49.254.7±5 0±. 004.04 3.9360.6±90 ±.0 01.01 2.7967±.260. 1±3 0.11 1.569±8.04.40 2± 0.02 TotYalieidlden (t%ifi)e d(%) 950.7.524± ±0 0.0.401 96.06.93±8 ±0 .00.101 97.206.3±3 0±.1 01.01 98.440±.303. 0±2 0.01  Yield(%) 0.24±0.01 0.38±0.01 0.33±0.01 0.33±0.01 RI = Retention indices on DB‐5 column; RIref (a) = [20]; ♂ = male; ♀ = female; NI 1: M+ 220: 159(100)  RI=RetentionindicesonDB-5column;RI (a)=[20]; =male; =female;NI1:M+220:159(100)119(99)145(66) 119(99) 145(66) 105(53) 131(42); NI 2:r eMf + 222: 109(100) 161(91) 107(90) 43(88) 105(86); NI 3: M+ 220:  105(53)131(42);NI2:M+222:109(100)161(91)107(90)♂43(88)105♀(86);NI3:M+220:119(100)91(84)105(82)93(75) 116129((5190);0N) I941:(8M4+) 120205:(82208) (9130(07)51)6 11(6923()5199)3; (N86I) 411: 9M(5+1 )212005:( 4290)8.(100) 161(93) 193(86) 119(51) 105(49).    Figure1.Cont. Medicines 2017, 4, 55   6 of 13  Medicines2017,4,55 6of13 Medicines 2017, 4, 55   6 of 13      FFiigguurree 11.. ((AA)) SSccoorreess aanndd ((BB)) LLooaaddiinnggss ooff PPrriinncciippaall CCoommppoonneenntt AAnnaallyyssiiss ((PPCCAA)) ooff tthhee cchheemmiiccaall  ccFooimmgupprooess ii1ttii.oo n(nA oo) ff SHHcoeedrdeyysoo ssammnuudmm ( Bbbrr)aa ssLiiloliieaenndssieen geessss seoennf ttPiiaarlli noociililsps;;a FlF FFC 1o1,m, 22p,, o33n =e=n fftee mmAaanllaeel yflfslooisww e(ePrrsCs;;A FF)MM o f11 ,,t 2h2,e,  33c =h=e mmmaiaclleael   flfclooowwmeeprross;s; LiLtFiFo lnl,,  22o,,f 3 3H ==e ffdeeymmosaamlleeu lmleea abvvreeasss;;i lLLieMMns lel,,  2e2s,, s33e =n= tmmiaaal lloeei lllees;aa vvFeeFss ..1 , 2, 3 = female flowers; FM 1, 2, 3 = male  flowers; LF l, 2, 3 = female leaves; LM l, 2, 3 = male leaves.  33..22.. AAnnttiiffuunnggaall AAccttiivviittyy  3.2. Antifungal Activity  TThhee eesssseennttiiaall ooiillss ooff HH.. bbrraassiilliieennssee pprreesseenntteedd cclleeaarr iinnhhiibbiittiioonn zzoonneess ccoorrrreessppoonnddiinngg ttoo aannttiiffuunnggaall  aaccttiivviitTtyyh ieinn e ttshhseee nlliitmmiaiiltt  oooiffl sdd eeottfee Hcctt.ii oobnnra aasggilaaieiinnnsssett  pCCrllaaeddseoossnpptooerrdiiuu cmmle accllraa ddinoohssppiboorritiiooioiiddnee ssz aoannnddes CC c..o ssrpprhheaaseeprrooossnppdeeirrnmmguu mtmo  ((aFFniiggtiuufurreen g22)a)..l   FFaeecmmtivaaliletey flf ilonoww theeerrs sl ippmrreietss eoenfn tdteeeddte tthchteeio ssnttr raoognnaggieensssttt a aCcclttaiivdvioittsyyp offoroirru bmboo ctthlha dffuousnnpggoiir fiforroiodmmes  22a00n00d µ μCgg.  usupnnhttaiilel r22o55s pµμeggrm oouff meess s(sFeeningttuiiaarlle oo 2iil)l.   ttFeesesttmeedda,l, ebb fuultot wtthheeer oso tpthhreeerrs eooniilltsse dpp rrteehsseee nsntttreeoddn wgweeesaat kkaeecrrti ivinnihthyiib bfioittriioo bnno,,t uuhp pfu ttonog 55i00 f rµμoggm aa g2ga0a0iinn μsstgt C Cu..n sstppilhh 2aae5err oμossgpp eoerrfmm eususmmen aatnniaddl  totooil   22t55e sµμtgegd aa,g gbaauiinnt ssttht CeC ..o cctllhaadedoro ssoppioolrsri iopoiirddeeesss.e. n ted weaker inhibition, up to 50 μg against C. sphaerospermum and to  25 μg against C. cladosporioides.       Figure2.PreliminaryantifungalactivityofHedyosmumbrasilienseessentialoilsagainstCladosporium Figure 2. Preliminary antifungal activity of Hedyosmum brasiliense essential oils against Cladosporium  cladosporioidesandC.sphaerospermum.FwF=femaleflowers;FwM=maleflowers;LeF=femaleleaves; cFlaigdousrpeo r2i.o Pidreesl iamndin Car. ysp ahnaetirfousnpegraml uamct.i vFiwtyF o =f  fHemedayloes fmlouwme brsr;a FsiwlieMns =e  emsaselen ftlioalw oeirlss ; aLgeaFi n=s fte Cmlaadleo slpeaorvieusm;   LeM=maleleaves;N=nystatin;CA=cinnamicacid;T=thymol. LcelaMdo =sp moraioleid leesa avneds; C N.  s=p hnayesrtoastpienr;m CuAm =. F cwinFn =a mfeimc aacleid fl; oTw =e trhs;y FmwoMl.  = male flowers; LeF = female leaves;  LeM = male leaves; N = nystatin; CA = cinnamic acid; T = thymol. Medicines2017,4,55 7of13 Medicines 2017, 4, 55   7 of 13  AftAerftTerL TCLCel ueltuiotinono foft hthee mmoosstt aaccttiivvee ooilisls, ,twtwo oinihnihbiitbioitnio bnanbdasn, dast ,reattenretitoenn tfiaocntofrasc (tRofr’ss) (oRf 0’s.6)7o f f and 0.12, were observed in all samples (Figure 3). After scraping the active bands, the GC‐MS analysis  0.67and0.12,wereobservedinallsamples(Figure3). Afterscrapingtheactivebands,theGC-MS anailnydsiiscaitneddi ctahtaetd ctuhraztecruenrzee r(Tenabel(eT a2b, lFeig2,uFreig 4u)r ew4a)sw tahse thmeamin acinomcopmoupnodu nfdorf othret hRef R0.607.6 7frafrcaticotinosn, s, f comcpomrispinrigsi9n7g% 97a%nd a8n2d% 8f2o%r bfootrh bfloothw eflroawnedr laenafdo lielsa.f Tohiles.b Tanhde abtaRnd 0a.1t 2Rwf a0.s1c2o wmapso sceodmbpyosaecdo mbyp lae x f complex mixture, with predominance of α‐terpineol, α‐eudesmol, and ferula lactone I (Figure 4) in  mixture,withpredominanceofα-terpineol,α-eudesmol,andferulalactoneI(Figure4)indifferent different proportions, not allowing to point only one single active compound, as presented in Table  proportions,notallowingtopointonlyonesingleactivecompound,aspresentedinTable2. 2.    Figure 3. (A) Detection of terpenes of H. brasiliense essential oils by Vanilin-Sulfuric Acid (VS) Figure 3. (A) Detection of terpenes of H. brasiliense essential oils by Vanilin‐Sulfuric Acid (VS) spray  spray reagent followed by heating to 110 ◦C; (B) Antifungal activity of Hedyosmum brasiliense reagent followed by heating to 110 °C; (B) Antifungal activity of Hedyosmum brasiliense essential oils,  essednetviaelloopields, dweivthel opn‐ehdexwaniteh/ance-htoenxea n(e9/0a:1c0e,t onv/ev)(,9 0a:1g0a,invs/t v)C,laadgoasipnosrtiuCml adcolaspdoosrpiuormioicdleasd osapnodr ioiCd.e s andspCh.aesrpohspaeerrmosupmer.m Ruf m0.6.7 R= fst0r.o6n7g= acsttirvoitnyg; Rafc 0t.i1v2i t=y ;wReafk0 a.1c2tiv=ityw; eFawkF a=c tfievmitayle; fFlwowFe=rsf; eFmwaMle =fl omwaleer s; FwMflo=wemrsa;l eLeflFo =w feerms;aLlee Flea=vfeesm; LaelMel e=a mveasle; LleeaMve=s; mNa =l enlyesatavteisn;. N=nystatin. TabTleab2l.eC 2h. eCmhiecmaliccaol mcopmopsiotsioitnioonf otfh tehae catcivtievea nantitfiufunnggaallf frraaccttiioonnss ooff HH.. bbrraassiilliieennssee ffeemmaalele flfloowweresr sanadn d leavleeasveesss eenssteianltioaill soiilsso ilsaotleadtebdy bbyi obaiouatuotgorgarpahpyh-yg‐guuidideeddT TLLCC..  FwF (%) LeF (%)  Compound  RI  RIref (a) FwF(%) LeF(%) Compound RI RIref(a) Rf0.67 Rf0.12 Rf 0.67  Rf 0.12  1,8‐cineole  1031 1031  Rf 0.67 Rf0.12 R1f20.6.697  Rf0 .12 1,t8e-cripnienoolelene  10311082 10130188    2.24  12. 69 4.65  terpinolene 1082 1088 2.24 4.65 dehydro‐sabina ketone  1121 1120    1.25    2.72  dehydro-sabinaketone 1121 1120 1.25 2.72 cisc-iβs-‐tβe‐rtpeirnpeionleol  11431143 11141444    00..8899      2-2(‐1(Z1)Z-p)‐rpoproenpyeln-pyhl‐epnholenol  11461146 11151050    00..7733      cics-icsh‐crhysraynstahnetnhoelnol  11621162 11161464    00..8899      terpinen-4-ol 1177 1177 1.87 terpinen‐4‐ol  1177 1177    1.87      α-terpineol 1191 1188 9.77 10.45 δ-αel‐etmerepnieneol  13271191 13131888    9.77  0.9 9 10.45  β-eδl‐eemleemneene  13841327 13193038      00..9929    germβa‐ecrleenmeeDne  14771384 14183590  2. 98   01..4952    gcuerrzmeraecnreene D  14891477 14194985  29.79.082    812..4058    δ-amorphene 1508 1512 1.50 curzerene  1489 1499  97.02    82.08    elemol 1542 1549 3.80 3.81 spδa‐athmuolernpohlene  15701508 15175812    19..5402    4.9 9 virideiflleomrooll  15781542 15195249    34..8305    63..5871  epis-αp-actahduinleonlol  16361570 16145078    90..4820    4.99  epi-α-muurolol 1640 1642 4.75 5.12 viridiflorol   1578 1592    4.35    6.57  α-eudesmol 1648 1653 20.95 22.53 veaplie‐rαia‐ncaoldinol  16511636 16156840    02..8203       eaptria‐cαt‐ymlouneurolol  16521640 16156842    4.75  1.8 7 5.12  germacra-4(15α),‐5e,1u0d(1e4s)m-troiel n-1-α-ol 16751648 16186653    200..7915    22.53  ferulalactoneI 2001 1974 21.83 28.29 valerianol  1651 1658    2.23      Intensityofaatnrtaifcutynlgoanlea ctivity 1652 1658  stro ng we  ak st1r.o8n7g  wea k Totalidentified(%) 100 87.98 10 0 89.1 3 germacra‐4(15),5,10(14)‐trien‐1‐α‐ol 1675 1686  0.71  RI=RetentionindicesonDB-5column;ref.RI (a)=[20];fractionsR 0.67=strongantifungalactivity;fractionsR ref f f   0.12=weakantifungalactivity. Medicines 2017, 4, 55   8 of 13    ferula lactone I   2001 1974  21.83  28.29  Intensity of antifungal activity strong weak strong  weak  Total identified (%)  100 87.98 100  89.13  MedicinesR2I 0=1 R7,e4t,e5n5tion indices on DB‐5 column; ref. RIref (a) = [20]; fractions Rf 0.67 = strong antifungal activity;  8of13 fractions Rf 0.12 = weak antifungal activity.    Figure4. ChemicalstructuresofthemaincompoundsfoundintheactivefractionsofH.brasiliense Figure 4. Chemical structures of the main compounds found in the active fractions of H. brasiliense  essentialoils:curzerene(A);α-terpineol(B);α-eudesmol(C);andferulalactoneI(D). essential oils: curzerene (A); α‐terpineol (B); α‐eudesmol (C); and ferula lactone I (D).  3.33..3A. nAtniotxioixdiadnatnAt Actcitviivtiyty  AnAtniotixoixdiadnantta actcitvivitiyty wwaass ddeetteerrmmiinneedd bbyy 22,2,2‐d-dipiphheneynly‐l1-‐1p-ipcircyrlyhlyhdyrdarzayzl y(lD(PDPPHP)H f)refer ereadriacdailc al scasvcaevnegningginagn daβnd-c aβro‐ctaernoetebnleea cbhleinacghminegt hmodesth.oTdhse. 5T0%hei n5h0i%bi toinrhyicboitnocreyn tcroanticoennt(rIaCt5io0)nw  (eIrCe50c)a lwcuelraet ed ancdaltchuelaItCed50 avnadlu tehse aIrCe50p rveaslueneste adrein pTreasbelnet3ed. Qinu eTracbeltei n3.a nQdueGricnektigno abnildob Gaienxktgroa cbtilwobea reexetrmacptl owyeerde as poesmitipvleoyceodn tarso lpsosshitoivwei ncognItCro5l0s 2s.h5o±wi0n.g0 9ICan50 d2.153 ±.5 0±.090 a.5nµd g1/3m.5 L±, 0r.e5s μpgec/mtivLe, lrye,sfpoercDtivPePlyH, faosrs aDyP,PwHh ile buatysslahyy,d wrohxilyet obluuteynlhey(dBrHoxTy)taonludenbeu t(yBlHhyTd) raonxdy bauntiysolhleyd(BroHxAya)nwiseorlee t(hBeHpAo)s witievree cthone tproolssitfivoer βco-ncatrrooltse ne assfoary ,βs‐hcaorwotienngeI aCssay0,. 0s8ho±w0in.0g9 IaCn50d 00.0.287 ± ±0.009.0 a2nµdg 0/.2m7 L±, 0r.e0s2p μegc/tmivLel,y r.espectively.  50 TaTbaleble3 .3. AAnnttiiooxxiiddaanntt  aaccttivivitiiteies s(I(CIC50,5 0,memane an±  S±D)S Dby)  bDyPPDHP PfrHee frraedeicraal dsiccaavlesncgaivngen ganindg  βa‐nd β-ccaarrootteennee//lilninoolelieci cacaicdi dmmetehtohdosd osf othfeth eesseesnsteinalt ioaillso firlsomfro fmlowfleorws aenrsda lneadvleesa ovfe Hso. bfrHas.ilbireanssiel.i e nse. IC50(μg/mL) Samples  DPPH β‐IcCa5r0ot(eµnge//mlinLo)leic acid  Samples ♀ Flowers  3162.79D ±P 4P0H.72  180.71β ±- c2a2r.9o5t ene/linoleicacid ♂ Flowers  2516.18 ± 131.87  113.46 ± 11.38  Flowers 3162.79±40.72 180.71±22.95 ♀ Leaves  3542.01 ± 45.31  71.12 ± 9.07  ♀ Flowers 2516.18±131.87 113.46±11.38 ♂ Leaves♂ Leaves  373835.4429. 0±1 1±32.4756. 31 86.47 ± 18.637 1.12±9.07 ♀ LeavesQuercetin  327.8530. 4±9 0±.091 32.76 ‐  86.47±18.63 Q♂uercetGininkgo biloba  13.25.05 0± ±0.500. 09 ‐  - GinkgobilobaBHT  13.5‐0  ±0.50 0.08 ± 0.09  - BHT BHA  ‐  - 0.27 ± 0.02 0.08±0.09 DPPH = 2,2‐dBiHpAhenyl‐1‐picrylhydrazyl; BHT -= butylhydroxytoluene; BHA0 =.2 b7u±ty0lh.0y2droxyanisole;  DP♂P H= m=a2l,e2;- ♀d i=p hfeemnyall-e1.- picrylhydrazyl; BHT=butylhydroxytoluene; BHA=butylhydroxyanisole; =male; =female. ♂ 4. D♀iscussion  4. Discussion Although there were previous studies on the essential oils from H. brasiliense leaves, none of  theAml tdhiosucrgihmtinhaetreedw aemreonpgre mviaolue sanstdu dfeimesaolen pthlaenetss s[e1n8,t1ia9l,2o8i]l sanfrdo,m toH th.eb rbaessilti eonf soeuler akvneosw,nleodngeeo, fthtihse m disisc rtihme infiarstet dreapmorotn wgimtha flleowanedr feesmseanlteiapl loainlst.s I[n1 8o,1u9r ,2st8u]dayn,d t,hteo etshseenbteisatl oofilo yuirelkdnso wwelreed gsiem,tilhairs fiosrt he firsfltorweperosr tawndit hleflaovwese roef sHse.n btiraalsioliielns.seI,n wouitrh sttuhed ym,tahlee efslosewnetrias lporilesyeineltdinsgw tehree sloimwielsatr ofoilr flamowouenrstsa. nd leaRveegsaordfiHn.gb trhaes illeieanf soei,lw coitnhtetnhtes,m thaele reflsouwltes rwseprree slieknet itnhgosteh feoluonwde fsotro ail Paamraonuán Sttsa.tRe esgpaercdiminegn t(hBeralezailf) oil con[2t8e]n, tcso,ltlhecetereds iunl tasnw aererea lnikote ftahro fsreomfo uIlnhda dfoor CaaPrdaorasno á(SSãtoa tPeasupleoc Simtateen, B(Brarazzili)l.) H[2o8w],ecvoelrl,e tchteedsei vnaaluneasr ea were lower than those found for a specimen collected in Santa Catarina State (Brazil), whose leaves  notfarfromIlhadoCardoso(SãoPauloState,Brazil). However,thesevalueswerelowerthanthose yielded 0.5% (v/w) [19], but this place is further south from our collection site.  foundforaspecimencollectedinSantaCatarinaState(Brazil),whoseleavesyielded0.5%(v/w)[19], butthisplaceisfurthersouthfromourcollectionsite. The essential oil analysis for the Parana (Brazil) specimen was not complete, most of the oil   componentswerenotidentified[28]. Nonetheless,withthecompositionpublisheditwaspossible todetect23commoncompoundswiththeSãoPaulo(Brazil)specimen,butonlysabinene(7%)was acommonmajorcompound.However,fortheSantaCatarina(Brazil)studywerefound22compounds incommon[19],havingasmajorcompoundsα-terpineol(10%),curzerene(9%),pinocarvone(8%), β-thujene(7%),andcarotol(6%). Curzerene(Figure4A)wastheonlycommonmajorcompoundwith thepresentanalysis,butwith17–18%intheleavesand11%inflowers. Medicines2017,4,55 9of13 The essential oils from other Hedyosmum species leaves (H. mexicanum, H. bonplandianum, H. arborescens, and H. angustifolium) have also presented high amounts of sabinene in their composition [4,7,8], as well as β-pinene, pinocarvone, and curzerene in the essential oil from H.colombianumleaves[6],similarlytoourstudy. Hedyosmumsppessentialoilsareknowntocontainseveralphenylpropanoids[3,5,6],sometimes inhighamounts,asinessentialoilfromH.scabrumleaves,whichcontained55.8%ofestragoleand 6.6% of anethole [5]. Also, some H. brasiliense specimens from different geographic origins have shownmethylchavicol,eugenolacetate,methyleugenol,andestragole[18,19,28]. However,theonly phenylpropanoiddetectedinourstudywasmethyleugenol,butinsmallamounts. PCAplotshowsthatleaveshaveahigherpercentageofsabineneandcurzerenethanflowers, whichhaveamoresignificantamountofcarotol,responsiblefordifferentiatingthem. Besidesthat, PCAwasalsoabletodiscriminatemaleflowersandleavesfromthefemaleonesbythe1,8-cineole abundance,whichwashigherinthefemalecounterparts(Table1andFigure1). Theseresultsshowed thatitisimportanttoanalyzeseparatelymaleandfemaleindividuals,partofthevariableresultson thechemicalcompositionmightbeexplainedbynotseparatingtheplantgenders. Cladosporium spp. are phytopathogenic filamentous fungi usually chosen for bioautographic assays,astheypresenthighsensitivityandpermitthedetectionoffungitoxicsubstancesbycontrast withtheirdarkcolor[23]. Preliminaryassaysforthedetectionlimitwithbioautographyconfirmed the antifungal activity for the H. brasiliense specimen from São Paulo against C. cladosporioides and C.sphaerospermum,withthehighestactivityforthefemalefloweressentialoil(Figure2).Inaprevious study, the antimicrobial activity of H. brasiliense leaf essential oils has already been confirmed for Gram-positive bacteria and fungi such as the dermatophytes Microsporum canis, M. gypseum, Trichophytonmentagrophytes,andT.rubrumandtheyeastsCandidaalbicansandC.parapsilosis[19]. The bioautography followed by TLC separation has been useful to determine antimicrobial activityofessentialoilsandtoisolatetheactivecompounds.Abioautographystudywithfivearomatic plants(Thymusvulgaris,LavandulaangustifoliaChaix,EucalyptusglobulusLabill.,MenthaspicataL.,and CinnamomumzeylanicumBlume)againstfivebacteria(Pseudomonassyringae,Xanthomonascampestris, Staphylococcusepidermidis,S.saprophyticus,andS.aureus)allowedtoattributetheactivityofThymus vulgarisessentialoiltothymol,comparingtoapurestandard,aswellastheactivityofC.zeylanicum essentialoiltoeugenol,bythesameprinciple[29]. AbioautographyassaywithMenthaxpiperitaL. essentialoilagainstCandidaalbicans,followedbyTLCseparationoftheactivefraction,indicatedthat mentholwasresponsibleforthisactivity[25]. Also,Guerrinietal.[3]detectedα-cadinol,α-muurolol, τ-muurolol,andlinaloolintheactivefractionwithantibacterialactivityagainstS.aureususingHigh PerformanceThinLayerChromatography(HPTLC)platesforseparationofH.spruceiessentialoil. In our study, the bioautography methodology indicated curzerene as the main compound for the activebandfoundatR ’s0.67andα-terpineol,α-eudesmolandferulalactoneIfortheactiveband f atR 0.12(Table2). TheH.brasilienseleafessentialoilcollectedatSantaCatarina(Brazil)presented f antifungalactivityanditwasalsorichincurzerene(8.9%)andα-terpineol(10.2%)(Figure4A,B)[25], supportingourresults. CurzerenehasbeenknownforitstoxiceffectsagainstAnophelessubpictus(LC =4.14µg/mL), 50 Aedes albopictus (LC = 4.57 µg/mL), and Culex tritaeniorhynchus (LC = 5.01 µg/mL) [30]. 50 50 Inaddition,curzerenealsoshowedantiproliferativeeffectsinSPC-A1humanlungcarcinoma,with IC =47µmol/Lin72h(invitro)and135mg/kgdaily(invivo)[31]. EugeniaunifloraL.essential 50 oil, rich in curzerene (42.6%), showed antifungal activity, inhibiting completely Paracoccidioides Braziliensis at 62.5 µg/mL [32]. Moreover, another E. uniflora essential oil composed by 47.3% of curzerene presented anti-Leishmania activity, with IC = 3.04 µg/mL against promastigotes and 50 IC =1.92µg/mLagainstamastigotesofL.amazonensis[33]. 50 The antioxidant activity of essential oils is already well known [34–37]. According to Karadagetal.[38],theantioxidantpotentialisrelatedtocompoundscapableofprotectingabiological systemagainstthepotentiallyharmfuleffectofreactiveoxygenspecies. Theresultsofasingle-assay Medicines2017,4,55 10of13 cangiveonlyanarrowviewoftheessentialoilantioxidantproperties. Therefore, theantioxidant potentialofH.brasilienseessentialoilswasdeterminedbytwodifferentmechanismsofaction: DPPH freeradicalscavenging,thatworksaselectrontransfer,andβ-carotene/linoleicacidassay,measuring the suppression of lipoperoxides. In the DPPH assay, the IC values for all the oils ranged from 50 2516.18 to 3783.49 µg/mL (Table 2), indicating a weak electron transfer capacity for the oils when comparedtothepositivecontrolsquercetinandG.bilobaextract,thatpresentedIC valuesof2.5and 50 13.5µg/mL,respectively. TheseresultsarecomparabletothoseobtainedforSalviatomentosaMill. essentialoils[36],whichwasrichinβ-pinene(39.7%),α-pinene(10.9%)andcamphor(9.7%),andit wasnotconsideredeffectiveasantioxidantbyDPPHandβ-carotene/linoleicacidassay. Similarly,the essentialoilsofsevenArtemisiaspp. weretestedbythesamemethodsandshowedweakactivities,as theircompositionweremarkedlyrichinnon-phenoliccomponents[39]. Theantioxidantcapacityof essentialoils,bytheDPPHmethodnormally,isnotveryhighwhencomparedwiththatobtainedfor extractsandfractionsrichinphenoliccompounds. Themajorantioxidativeplantphenoliccompounds knownareeugenol,carvacrol,thymol,menthol,andsafrole,amongothers[34]. Inatestwith98pure essentialoilcomponentsfortheirantioxidantactivity,phenols,suchasthymolandcarvacrol,werethe mostactivecompounds,followedbyalcoholsmono-andsesquiterpenesandketonesα,β-insaturated, howeverhydrocarbonspresentedverylowantioxidantactivity[40]. Ontheotherhand,ourresults indicatedthattheessentialoilsofH.brasilienseweremuchmoreactiveintheβ-carotenebleaching assay,presentingIC valuesfrom71.12to180.71µg/mL(Table2),indicatingtheirabilityindestroying 50 theconjugateddienehydroperoxides,theendproductsoflinoleicacidperoxidation. Inthisway,the lowradicalscavengingactivityobservedfortheH.brasilienseessentialoilsmightbeexplainedbythe lowamountsofphenoliccompoundspresentintheoil,only1.11to3.63%ofthymol,andthehigh amountsofmono-andsesquiterpenehydrocarbons(Table1). Sinceantioxidantactivityoftheentire oilistheresultoftheinteractionofallconstituents[41],itishardtoattributetheessentialoilsactivity toasinglecompound,asotheroilcomponentsmaycontributeexhibitingsynergisticorantagonistic effects[42]. 5. Conclusions Theleafandfloweressentialoilsweredominatedbysabinene,1,8-cineole,curzerene,andcarotol. However,thecontentsoftheseconstituentsdifferedaccordingtotheplantpartandgender. This was the first report of antifungal activity of curzerene, explaining partially the activity of H. brasiliense essential oils by bioautography. Moreover, the complex mixture of α-terpineol, α-eudesmol,andferulalactoneImayhaveactedsynergisticallytotheantifungalactivityobserved. Bioautographyassayhelpedtodetectantifungalfractionsinacomplexmatrixofessentialoilsand guidedtotheisolationoftarget-directedconstituents. Furtherstudiesarenecessarytoisolatehigher amountsofpurecurzerene,inordertotestitslimitofdetection. Antioxidant activity of essential oils from H. brasiliense was more effective by β-carotene discoloration assay than by DPPH assay, indicating their higher ability in quenching conjugated hydroperoxidesthanfreeradicals. Theinhibitionoflipidperoxidationmayhavebeenaresultofthe interactionofallconstituentsoftheessentialoils,sinceitwasnotpossibletoattributeantioxidant activitytoasinglecompound. Supplementary Materials: The following are available online at www.mdpi.com/2305-6320/4/3/55/s1, FigureS1:MassspectrumofNon-identifiedcompound1(N.I.1)detectedintheessentialoilofH.brasiliensefrom IlhadoCardoso;FigureS2:MassspectrumofNon-identifiedcompound2(N.I.2)detectedintheessentialoilof H.brasiliensefromIlhadoCardoso;FigureS3:MassspectrumofNon-identifiedcompound3(N.I.3)detectedin theessentialoilofH.brasiliensefromIlhadoCardoso;FigureS4:MassspectrumofNon-identifiedcompound4 (N.I.4)detectedintheessentialoilofH.brasiliensefromIlhadoCardoso. Acknowledgments:TheauthorswishtothankConselhoNacionaldeDesenvolvimentoCientíficoeTecnológico (CNPq,Proc.N.309762/2012-0and164913/2013-0)andFundaçãodeAmparoàPesquisadoEstadodeSãoPaulo (FAPESP,Proc.N.2014/15299-4and2014/17561-8)forfinancialsupportandscholarships.

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(Ruiz & Pav.) Solms (Ruiz & Pav.) G. Don as a treatment to joint pain activity was performed by the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) Miguel, M.G. Antioxidant activity of medicinal and aromatic plants—a review.
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medicines Article Chemical Composition, Antifungal and Antioxidant Activities of Hedyosmum brasiliense Mart. ex Miq. (Chloranthaceae) Essential Oils CynthiaMurakami1,2,*,InêsCordeiro2,MarcusTulliusScotti3 ID,PauloRobertoH.Moreno4 ID andMariaCláudiaM.Young2 1 ProgramadePós-GraduaçãoemBiodiversidadeVegetaleMeioAmbiente,InstitutodeBotânicade SãoPaulo,SãoPaulo04301-902,Brazil 2 InstitutodeBotânicadeSãoPaulo,SãoPaulo04301-902,Brazil;[email protected](I.C.); [email protected](M.C.M.Y.) 3 LaboratóriodeQuimioinformática,UniversidadeFederaldaParaíba,JoãoPessoa58051-900,Brazil; [email protected] 4 InstitutodeQuímica,UniversidadedeSãoPaulo,SãoPaulo05508-000,Brazil;[email protected] * Correspondence:[email protected];Tel.:+55-11-5067-6174 AcademicEditor:EleniSkaltsa Received:30June2017;Accepted:13July2017;Published:17July2017 Abstract: Background: HedyosmumbrasilienseMart. exMiq. (Chloranthaceae)isadioeciousshrub popularlyusedinBraziltotreatfootfungiandrheumatism. Thisworkinvestigatedthechemical composition,antifungal,andantioxidantactivitiesofflowersandleavesofH.brasilienseessential oils;Methods: H.brasiliensemaleandfemaleflowersandleaveswerecollectedatIlhadoCardoso (SãoPaulo)andtheessentialoilswereextractedbyhydrodistillationandanalyzedbyGC/MSand their similaritycomparedby Principal ComponentAnalysis. Antifungal activity wasperformed bybioautographyandantioxidantpotentialby2,2-diphenyl-2-picrylhydrazylhydrate(DPPH)free radical scavenging and β-carotene/linoleic acid system; Results: The major compounds for all oils were sabinene, curzerene, and carotol, but some differences in their chemical composition werediscriminatedbyPrincipalComponentAnalysis(PCA)analysis. Bioautographyshowedtwo antifungalbandsatRf’s0.67and0.12inallsamples,thefirstonewasidentifiedascurzerene. Theoils presentedstrongerantioxidantpotentialinβ-carotene/linoleicacidbioassay,withIC ’sfrom80to 50 180µg/mL,thaninDPPHassay,withIC ’sfrom2516.18to3783.49µg/mL;Conclusions: These 50 results suggested that curzerene might be responsible for the antifungal activity of H. brasiliense essentialoils. Besides,theseessentialoilsexhibitedpotentialtopreventlipoperoxidation,butthey haveaweakradicalscavengeractivity. Keywords: Hedyosmum brasiliense; essential oil; GC-MS; bioautography; curzerene; antioxidant; AtlanticRainForest 1. Introduction The family Chloranthaceae includes plants grouped in four genera: Sarcandra Gardner, ChloranthusSw.,AscarinaJ.R.Forst.andG.Forst.,andHedyosmumSw.ThegenusHedyosmumcomprises 40speciesofshrubsandsmalltreesdistributedfromMexico,throughoutCentralAmericatoBolivia, east of the Guianas and the Antilles [1]. The name Hedyosmum comes from the Greek “pleasant fragrance” and alludes to one of the most remarkable characteristics of these plants, the pleasant aromaticsmellcomingfromallitsparts[1,2]. AccordingtoTodzia[1],differentHedyosmumspecies havebeenpopularlyusedallaroundCentralandSouthAmericafordifferentpurposes: H.scabrum (Ruiz&Pav.) Solmsforconceptionstimulation;H.scabrumandH.scaberrimunStandl. asstomachpain Medicines2017,4,55;doi:10.3390/medicines4030055 www.mdpi.com/journal/medicines Medicines2017,4,55 2of13 relievers;H.spruceiSolmsforsnakebites;H.cumbalenseH.Karst. asastimulant;andH.racemosum (Ruiz&Pav.) G.Donasatreatmenttojointpain. Theessentialoilcompositionfromsomespeciesof thisgenus(H.sprucei,H.arborescensSw.,H.scabrum,H.colombianumCuatrec.,H.angustifolium(Ruiz& Pav.) Solms,H.mexicanumC.Cordem.,H.bonplandianumMart.,andH.costaricenseC.E.WoodexW.C. Burger)havepreviouslybeendescribed[3–8],aswellastheirantioxidantandcytotoxicproperties[3,4]. HedyosmumbrasilienseMart. exMiq.,popularlyknownas“chádebugre”,“cidreira”,or“cidrão”, is a dioecious shrub distributed throughout central and southern Brazilian regions and western Paraguay[9,10]. Itsleaveshavebeenusedinfolkmedicinetotreatmigraine,ovariumdysfunction, footfungi,rheumatism,stomachpain,andasadiuretic[1,2]. Chemicalandpharmacologicalstudies withtheaerialpartsofH.brasilienseindicatedanalgesic,anxiolytic,antidepressive,andaphrodisiac effects for the crude extracts and for some isolated sesquiterpene lactones [11–16]. There are also somereportsontheantimicrobialactivityfortheethanolextract[17]andtheessentialoils[18,19], howevernoneofthemcouldattributetheobservedbioactivitytoaspecificcompound. Theessential oilpresentedasmajorcompoundsspecimensabinene,(Z)-β-guaieneandpinocarvone,foraSãoPaulo State [18], and pinocarvone, curzerene and carotol, for one from Santa Catarina State [19]. To the bestofourknowledge,thisisthefirstreportonthechemicalcompositionandbiologicalactivitiesof H.brasiliensefloweressentialoils. Inaddition,thisisalsothefirsttimethattheantioxidantactivity isdescribed forthisspecies. Therefore, thisworkaims toinvestigatethe chemicalcompositionof theessentialoilsfromH.brasiliensemaleandfemaleflowersandleavescollectedatIlhadoCardoso (SãoPaulo,Brazil)andtocomparetheirantifungalandantioxidantactivities,aswellastoidentifythe mainantifungalconstituentsintheessentialoils. 2. MaterialsandMethods 2.1. PlantMaterial HedyosmumbrasilienseMart. exMiq. (Chloranthaceae)flowersandleaveswerecollectedfrom maleandfemalespecimensatParqueEstadualdaIlhadoCardoso,SãoPaulo,Brazil(25◦05(cid:48) Sand 47◦55(cid:48) W,14malt)inSeptemberof2015. ThetaxonomicidentitywasconfirmedbyDr. InêsCordeiro (InstitutodeBotânica,SãoPaulo,Brazil). ThevoucherspecimensweredepositedintheHerbariumat theInstitutodeBotânica,SãoPaulo,Brazil,withtheaccessionNo. SP475335. 2.2. ExtractionoftheEssentialOils Ninemaleandfemalespecimenswerecollected. Leavesandflowerswerepooledtoprovide representativehomogeneoussamplesofthepopulation,separatedintothreereplicatesandstored underrefrigeration(−22◦C)untilextraction. Essentialoilwasobtainedbyhydrodistillationfor3.5h inaClevenger-typeapparatus. Thecrudeoilwasseparated,driedoveranhydroussodiumsulfate andstoredinaglassflaskat −22 ◦CuntilGC-MSanalysisandbiologicalactivities. Theoilyields werecalculatedbasedontheoilandfreshplantmaterialweightsasmean±standarddeviationofthe triplicates[18]. 2.3. GC-MSAnalysis Essential oils were solubilized in acetone 1:100 v/v (Merck KGaA, Darmstadt, Germany). Chemical analysis of the essential oils was performed on an Agilent 6890 Series GC (Agilent, SantaClara,CA,USA),interfacedwitha5973SeriesquadrupoleMSdetector(Agilent,SantaClara, CA, USA) and equipped with a DB-5MS column (30 m × 0.25 mm i.d. × 0.25 µm) (Agilent J&W, Santa Clara, CA, USA). Chromatography conditions were as follows; oven temperature program: 40◦Cfor1minwithsubsequenttemperatureincreaseof3◦C/minupto240◦C,keepingitatthis temperaturefor10min.;carriergas:Heataflowrateof1mL/min;injectoranddetectortemperatureof 250◦C;electronionization: 70eV.Thecomponentabundanceswereexpressedasthemean±standard deviationofthetriplicates. Theessentialoilcomponentswereidentifiedbycomparingtheretention Medicines2017,4,55 3of13 indices(evaluatedinrelationtotheretentiontimesofaseriesofn-alkanes)andbycomparisonoftheir massspectrawiththosereportedintheliterature[20,21]. 2.4. PrincipalComponentAnalysisoftheEssentialOils Principal Component Analysis (PCA) was performed using composition of H. brasiliense essentialoils. PriortothePCAanalysis,allthevariableswerestandardizedforanormalizedPCA. TheUnscrambler® Xversion9.7(CAMOSoftware,Oslo,Norway)wasusedtoperformPCAanalysis andgeneratescoreandloadingplot[22]. 2.5. AntifungalActivity The microorganisms used in antifungal assay, Cladosporium cladosporioides (Fresen de Vries (CCIBt140)andC.sphaerospermumPenz(CCIBt491),havebeenmaintainedatInstitutodeBotânica, SãoPaulo,SP,Brazil. Fortheantifungalassay,carriedoutbybioautographyadaptedfromHomans & Fuchs method [23], 10 µL of solutions corresponding to 200, 100, 50, and 25 µg of essential oils inmethanolwereappliedassinglespotsonpre-coatedThinLayerChromatography(TLC)plates (GF silica-gelchromatoplates,MerckKGaA,Darmstadt,Germany). Nystatin,thymol,andcinnamic 254 acid(5µg)wereusedaspositivecontrols. Theplatesweresprayedwithasuspensionofsaltsand glucose (6:1) containing fungus spores (> 2 × 106 spores/mL) of Cladosporium cladosporioides and C.sphaerospermum.Theplateswereincubatedinmoistchambersat27◦Cfor48h,inthedark.Afterthe incubationperiod,clearinhibitionzoneswereobservedasantifungalactivityoftheessentialoils. Thecomponentsresponsiblefortheantifungalactivitiesweredeterminedbyapplying400µgof eachessentialoilontothreeTLCplatesanddevelopedwithn-hexane/acetone,90:10v/v. Inoneplate, theseparatedfractionbandswerevisualizedwithUVlight(254nm)andsprayedwithVanilin-Sulfuric Acid(VS)reagentfollowedbyheatingto110◦Cfor5min[24]. Theothertwoplateswereusedfor thebioautographyassay. Accordingtotheantifungalactivityresult,7mgoffemaleflowerandleaf essentialoilswereappliedonchromatoplates,separately,anddevelopedwiththesamesolventsystem. AttheexactR ’softheactivebands, theplatewascuthorizontally, washedwith1mLofacetone, f filtered(0.22µmfilters,MerckMillipore,Darmstadt,Germany)andfurtheranalyzedbyGC-MS,atthe sameconditionsastheessentialoils[25]. 2.6. InVitroAntioxidantActivities Theantioxidantactivitywasperformedbythe2,2-diphenyl-2-picrylhydrazylhydrate(DPPH) freeradicalscavengingmethod[26],adaptedformicroplates. 178µLoftheessentialoilssolubilizedin methanolatconcentrationsbetween4480–560µg/mLwereaddedtoa96-wellmicroplateand72µL ofDPPH(0.3mmol/L)solubilizedinmethanolwereadded. Blankreadingwasperformedwiththe test sample prior to the DPPH radical incubation. As a negative control, 178 µL of methanol was used. Aspositivecontrols,quercetinfrom20to0.625µg/mLandGinkgobilobaextract(Herbarium Laboratório Botânico S.A., Paraná, Brazil) from 40 to 1.25 µg/mL were used. The microplate was incubated in the dark for 30 min at room temperature. Then, the absorbance was measured at a wavelengthof518nmusingamulti-wellscanningspectrophotometer(BIOTEKKC4,Winooski,VT, USA).Theabsorbancewasconvertedtopercentageofantioxidantactivity(AA)usingtheformula AA%=100−{[(Abs −Abs )×100]/Abs }.Allsamplesweretestedintriplicateandthe sample blank control resultsareexpressedbymean±standarddeviation. Theamountofessentialoilrequiredtoreducethe initialDPPHconcentrationinthereactionby50%isreferredtoastheinhibitoryconcentration(IC ). 50 The antioxidant activity was also evaluated by the β-carotene bleaching test, which is based onspectrophotometricmeasurementsoftheβ-caroteneoxidationinducedbythelipoperoxidation products from linoleic acid [27] to determine the concentration providing 50% inhibition (IC ). 50 Theessentialoil(10µL)solubilizedinmethanolatconcentrationsbetween10,400–325µg/mLwas addedina96-wellmicroplate,togetherwith250µLofareactivesolutioncomposedbyβ-carotene andlinoleicacidinwatersaturatedwithO . Methanol(10µL)wasusedasthenegativecontrolandas 2 Medicines2017,4,55 4of13 thepositivecontrols,butylhydroxytoluene(BHT)andbutylhydroxyanisole(BHA)atconcentrations between130–4.1µg/mL.Themicroplatewasincubatedinthedarkat45◦Candsubsequentlythe absorbancewasreadat450nm(multi-wellscannerBIOTEKKC4,Winooski,VT,USA),immediately afteraddingthereactivesolutionandevery30minfor2h.Theabsorbancewasconvertedtopercentage of antioxidant activity (AA) using the AA% = 100 − {[(Abs − Abs ) × 100]/Abs }. final initial control Allsamplesweretestedintriplicateandtheresultsareexpressedbymean±standarddeviation. 3. Results 3.1. ChemicalCompositionoftheEssentialOil TheessentialoilyieldsformaleandfemaleH.brasilienseflowerswere0.24±0.01and0.38±0.01%, respectively. Theyieldofleafessentialoils,bothmaleandfemale,presentedayieldof0.33±0.01% (Table1). Fifty compounds were identified from all essential oils analyzed, comprising 96–98% of the totalcomponents. ThemajorcompoundsforallH.brasilienseessentialoilsweresabinene,curzerene, and carotol. Leaf essential oils presented mostly monoterpenes (53% and 55% ), followed by sesquiterpenes(39% and41% ), andphenylpropanoids(2% and5♂% ). Flow♀eressentialoils presentedmonoterpen♂es(54% an♀d45% ),sesquiterpenes(40% ♀and50% ♂),andphenylpropanoids (1% and2% ). Theleafmai♀nconstitu♂entsweresabinene(16%♂),β-pinene♀(5%),1,8-cineole(3–7%), meth♂yl eugeno♀l (2–5%), curzerene (17–18%), and carotol (6%), while flowers presented sabinene (8–10%), α-phellandrene (1–8%), 1,8-cineole (2–7%), germacrene D (4–6%), curzerene (11%), and carotol(9%)asmajorcompounds. Principal Component Analysis (PCA) data from Table 1 generated a plot, which condensed scoresandloadingsofthefirsttwocomponents,PC1andPC2,whichexplain61%and34%oftotal variance(Figure1). Leavespresentedhigheramountsofsabineneandcurzerenethanflowers,while flowerspresentedhigheramountsofcarotol,asdiscriminatedbyPCA(Figure1). Furthermore,PCA analysisshowedthatfemaleflowersandleavespresentedhigheramountsof1,8-cineolethanthemale counterparts(Figure1). Table1.ChemicalcompositionofHedyosmumbrasilienseessentialoils. RelativeAmount(%) Compound RI RIref(a) Flowers Flowers Leaves Leaves 4-hydroxy-4-methyl-2-pentanone 841 839 1♂.58±0.02 1♀.44±0.03 1♂.53±0.06 1♀.49±0.02 α-pinene 933 939 2.23±0.02 1.96±0.02 3.16±0.10 3.66±0.11 sabinene 972 975 9.46±0.04 8.05±0.11 15.81±0.19 15.81±0.17 β-pinene 976 979 3.91±0.03 2.73±0.04 4.64±0.13 5.21±0.19 myrcene 990 990 1.75±0.02 1.45±0.02 2.48±0.05 2.79±0.14 α-phellandrene 1003 1002 8.14±0.07 1.10±0.01 2.79±0.44 0.82±0.06 α-terpinene 1016 1017 1.07±0.01 0.91±0.01 1.18±0.12 1.09±0.12 ρ-cymene 1023 1024 1.51±0.01 limonene 1028 1029 2.53±0.01 1.08±0.01 1.68±0.06 1.46±0.05 1,8-cineole 1031 1031 1.65±0.01 7.22±0.07 3.44±0.04 6.86±0.17 (Z)-β-ocymene 1036 1037 2.26±0.01 0.66±0.01 (E)-β-ocymene 1048 1050 1.85±0.01 3.00±0.04 0.90±0.06 1.91±0.01 γ-terpinene 1059 1059 1.76±0.01 1.44±0.01 1.81±0.16 1.68±0.17 terpinolene 1082 1088 0.57±0.00 linalool 1097 1096 1.53±0.01 1.5±0.01 0.99±0.03 1.31±0.03 camphor 1146 1146 0.73±0.01 pinocarvone 1161 1164 3.65±0.02 3.15±0.02 4.49±0.11 3.94±0.07 terpinen-4-ol 1177 1177 3.34±0.02 3.11±0.04 3.30±0.36 3.09±0.32 α-terpineol 1191 1188 1.07±0.01 1.37±0.01 0.61±0.07 0.76±0.06 citronellol 1224 1225 0.60±0.03 chrysanthenylcis-acetate 1255 1265 1.21±0.01 1.03±0.08 1.55±0.03 bornylacetate 1279 1288 0.58±0.00 thymol 1285 1290 3.63±0.05 1.11±0.20 Medicines 2017, 4, 55   5 of 13  thymol  1285 1290  3.63 ±0.05  1.11 ± 0.20  Medicines2017,4,55 5of13 pinocarvyl cis‐acetate  1303 1312  1.16 ± 0.01 0.72 ± 0.03  1.05 ± 0.02  δ‐elemene  1327 1338  1.33 ± 0.01 2.24 ±0.11  2.30 ± 0.07  α‐terpinyl acetate  1344 1349  1.47 ± 0.01 0.75 ± 0.01 0.93 ± 0.04  1.19 ± 0.02  Table1.Cont.   α‐copaene  1370 1376  0.71 ± 0.00 0.66 ± 0.00 β‐elemene  1384 1390  1.27 ± 0.01 1.68 ± 0.01 1.43 ± 0.09  1.58 ± 0.05  RelativeAmount(%) methCyolm epuoguenndol  R1I394 RIre1f4(0a3)  1.41 ± 0.02 1.96 ± 0.02 4.53 ± 0.21  1.85 ± 0.17  Flowers Flowers Leave s Leaves (E)‐caryophyllene  1412 1419  0.65 ± 0.01 0.69 ± 0.00 pinoγc‐aerlveymlecinse-a cetate 13104323 13114236  ♂0.69 ± 0.00 1♀.106.7±2 0±. 001.01 0♂.720.±700 .±0 30.06 1♀.050±.706.0 ±2 0.02  δ-elemene 1327 1338 1.33±0.01 2.24±0.11 2.30±0.07 α‐humulene  1449 1454  0.80 ± 0.02 0.72 ± 1.36 0.59 ± 0.04  0.58 ± 0.00  α-terpinylacetate 1344 1349 1.47±0.01 0.75±0.01 0.93±0.04 1.19±0.02 germacrene D  1477 1485  4.23 ± 0.04 5.76 ± 0.02 3.74 ± 0.21  3.35 ± 0.13  α-copaene 1370 1376 0.71±0.00 0.66±0.00 cuβr-zeelermeneen e 13184489 13194099  11.217.0±9 0±. 001.11 1.1618.6±80 ±.0 01.05 1.4137±.020. 0±9 0.66 1.581±7.07.80 5± 0.30 βm‐beitshaybloeulegneen ol 13195402 14105305  1.041.9±7 ±0 .002.01 1.926.6±6 0±. 002.01 4.53±0.2 1 1.85±0.17 (Eδ)‐-ccaardyionpehnyel lene 14115211 14115923  0.065.9±8 ±0 .001.01 0.619.5±1 0±. 000.01 0.63 ± 0.02  0.71 ± 0.02  γ-elemene 1423 1436 0.69±0.00 0.72±0.01 0.70±0.06 0.76±0.02 (Z)‐α‐bisabolene  1539 1507  0.63 ± 0.01 0.95 ± 1.36 0.80 ± 0.11  1.86 ± 0.04  α-humulene 1449 1454 0.80±0.02 0.72±1.36 0.59±0.04 0.58±0.00   geerlmemacorel neD 14175742 14185549  4.23±0.04 5.706.6±1 0±. 002.00 3.74±0.21 3.35±0.13 cNur.Iz.e 1re ne 14185956 1499 111.0.917± ±0 0.1.101 11.16.86±6 ±0 .00.501 17.002.8±6 0±.6 06.01 17.780±.800. 3±0 0.02  spβa-tbhisualbeonloenl e 15105270 15105578  0.497.5±3 ±0 .001.04 2.636.0±8 0±. 001.01 3.05 ± 0.44  2.23 ± 0.08  δ-cadinene 1511 1523 0.98±0.01 1.51±0.01 0.63±0.0 2 0.71±0.02 viridiflorol  1578 1592  0.81 ± 0.01 0.82 ± 0.00 (Z)-α-bisabolene 1539 1507 0.63±0.01 0.95±1.36 0.80±0.1 1 1.86±0.04 Ne.lIe.m 2o l 15145287 1549 0.67 ± 0.00 0.601.8±6 0±. 000.01 carNo.tIo.l1  15155696 1594  1.917.7±2 ±0 .001.04 1.696.3±6 0±. 001.05 0.866.±480 .±0 10.10 0.805±.901.0 ±2 0.15  spNa.tIh.u 3l enol 15176020 1578 4.53±0.04 3.008.7±8 0±.001.01  3.05±0.4 4 2.230±.705.0 ±8 0.03  epi‐vαir‐icdaifldoinrooll  15176826 15196240  0.081.9±3 ±0 .001.01 0.82±0.00 0.96 ± 0.17  N.I.2 1587 0.67±0.00 0.86±0.01   daucol  1636 1642  0.61 ± 0.00 carotol 1596 1594 9.72±0.04 9.36±0.05 6.48±0.10 5.91±0.15 NN.I.. I4. 3 16126041 0.74 ± 0.01 0.78±0.01 0.84 ± 0.09 0.75±0.03 αe‐pmi-uαu-craodloinlo l 16126643 16146046  0.93±0.01 0.74 ± 0.08 0.96±0.1 7 α‐euddaeuscmolol  16136648 16146253  0.161.6±6 ±0 .000.02 3.08 ± 0.02 1.06 ± 0.04  1.79 ± 0.08  N.I.4 1641 0.74±0.01 0.84±0.0 9 (Z)‐α‐santalol  1661 1675  0.57 ± 0.00 α-muurolol 1643 1646 0.74±0.08 feruαla-e uladcetsomnoe lI  16240801 16159374  1.166.7±5 ±0 .002.02 3.038.7±0 0±. 002.02 1.061.±880 .±0 40.69 1.792±.408.0 ±8 0.70  M(Zo)n-αot-searnpteanloel hydrocarbon1 6(6%1)  1675 38.12 ± 0.09 0.5279.±6 0±. 000.27 38.08 ± 1.44  41.28 ± 0.97 Ofexruylgaelnacattoende mI onoterpene2 (0%01)  1974 11.765.2±7 0±. 002.06 3.1750.0±00 ±.0 02.10 1.8185±.330. 6±9 0.60 2.481±4.03.87 0± 0.46 SesqMuointeortperepneen ehhyyddrrooccaarrbboonn ((%%))  381.102.9±3 ±0 .009.06 291.66.6±80 ±.2 07.05 38.098.9±3 1±.4 04.74 41.281±0.705.9 ±7 0.50 OxyOgxeyngaetneadte sdemsqounioteterrppeennee ((%%))  162.297.0±3 ±0 .006.19 153.030.4±5 0±. 100.31 15.3239.±390 .±6 01.61 14.383±0.108.4 ±6 1.21 Sesquiterpenehydrocarbon(%) 10.93±0.06 16.68±0.05 9.93±0.74 10.75±0.50 Phenylpropanoid (%)  1.41 ± 0.02 1.96 ± 0.02 4.53 ± 0.21  1.85 ± 0.17  Oxygenatedsesquiterpene(%) 29.03±0.19 33.45±0.31 29.39±1.61 30.18±1.21 NPohte nidyelpnrtoipfiaendo (id%()% ) 1.441.2±4 ±0 .002.04 1.936.3±0 0±. 002.01 4.532.±760 .±2 10.13 1.851±.506.1 ±7 0.02  TotNalo itdideenntitfifiieedd ((%%))  49.254.7±5 0±. 004.04 3.9360.6±90 ±.0 01.01 2.7967±.260. 1±3 0.11 1.569±8.04.40 2± 0.02 TotYalieidlden (t%ifi)e d(%) 950.7.524± ±0 0.0.401 96.06.93±8 ±0 .00.101 97.206.3±3 0±.1 01.01 98.440±.303. 0±2 0.01  Yield(%) 0.24±0.01 0.38±0.01 0.33±0.01 0.33±0.01 RI = Retention indices on DB‐5 column; RIref (a) = [20]; ♂ = male; ♀ = female; NI 1: M+ 220: 159(100)  RI=RetentionindicesonDB-5column;RI (a)=[20]; =male; =female;NI1:M+220:159(100)119(99)145(66) 119(99) 145(66) 105(53) 131(42); NI 2:r eMf + 222: 109(100) 161(91) 107(90) 43(88) 105(86); NI 3: M+ 220:  105(53)131(42);NI2:M+222:109(100)161(91)107(90)♂43(88)105♀(86);NI3:M+220:119(100)91(84)105(82)93(75) 116129((5190);0N) I941:(8M4+) 120205:(82208) (9130(07)51)6 11(6923()5199)3; (N86I) 411: 9M(5+1 )212005:( 4290)8.(100) 161(93) 193(86) 119(51) 105(49).    Figure1.Cont. Medicines 2017, 4, 55   6 of 13  Medicines2017,4,55 6of13 Medicines 2017, 4, 55   6 of 13      FFiigguurree 11.. ((AA)) SSccoorreess aanndd ((BB)) LLooaaddiinnggss ooff PPrriinncciippaall CCoommppoonneenntt AAnnaallyyssiiss ((PPCCAA)) ooff tthhee cchheemmiiccaall  ccFooimmgupprooess ii1ttii.oo n(nA oo) ff SHHcoeedrdeyysoo ssammnuudmm ( Bbbrr)aa ssLiiloliieaenndssieen geessss seoennf ttPiiaarlli noociililsps;;a FlF FFC 1o1,m, 22p,, o33n =e=n fftee mmAaanllaeel yflfslooisww e(ePrrsCs;;A FF)MM o f11 ,,t 2h2,e,  33c =h=e mmmaiaclleael   flfclooowwmeeprross;s; LiLtFiFo lnl,,  22o,,f 3 3H ==e ffdeeymmosaamlleeu lmleea abvvreeasss;;i lLLieMMns lel,,  2e2s,, s33e =n= tmmiaaal lloeei lllees;aa vvFeeFss ..1 , 2, 3 = female flowers; FM 1, 2, 3 = male  flowers; LF l, 2, 3 = female leaves; LM l, 2, 3 = male leaves.  33..22.. AAnnttiiffuunnggaall AAccttiivviittyy  3.2. Antifungal Activity  TThhee eesssseennttiiaall ooiillss ooff HH.. bbrraassiilliieennssee pprreesseenntteedd cclleeaarr iinnhhiibbiittiioonn zzoonneess ccoorrrreessppoonnddiinngg ttoo aannttiiffuunnggaall  aaccttiivviitTtyyh ieinn e ttshhseee nlliitmmiaiiltt  oooiffl sdd eeottfee Hcctt.ii oobnnra aasggilaaieiinnnsssett  pCCrllaaeddseoossnpptooerrdiiuu cmmle accllraa ddinoohssppiboorritiiooioiiddnee ssz aoannnddes CC c..o ssrpprhheaaseeprrooossnppdeeirrnmmguu mtmo  ((aFFniiggtiuufurreen g22)a)..l   FFaeecmmtivaaliletey flf ilonoww theeerrs sl ippmrreietss eoenfn tdteeeddte tthchteeio ssnttr raoognnaggieensssttt a aCcclttaiivdvioittsyyp offoroirru bmboo ctthlha dffuousnnpggoiir fiforroiodmmes  22a00n00d µ μCgg.  usupnnhttaiilel r22o55s pµμeggrm oouff meess s(sFeeningttuiiaarlle oo 2iil)l.   ttFeesesttmeedda,l, ebb fuultot wtthheeer oso tpthhreeerrs eooniilltsse dpp rrteehsseee nsntttreeoddn wgweeesaat kkaeecrrti ivinnihthyiib bfioittriioo bnno,,t uuhp pfu ttonog 55i00 f rµμoggm aa g2ga0a0iinn μsstgt C Cu..n sstppilhh 2aae5err oμossgpp eoerrfmm eususmmen aatnniaddl  totooil   22t55e sµμtgegd aa,g gbaauiinnt ssttht CeC ..o cctllhaadedoro ssoppioolrsri iopoiirddeeesss.e. n ted weaker inhibition, up to 50 μg against C. sphaerospermum and to  25 μg against C. cladosporioides.       Figure2.PreliminaryantifungalactivityofHedyosmumbrasilienseessentialoilsagainstCladosporium Figure 2. Preliminary antifungal activity of Hedyosmum brasiliense essential oils against Cladosporium  cladosporioidesandC.sphaerospermum.FwF=femaleflowers;FwM=maleflowers;LeF=femaleleaves; cFlaigdousrpeo r2i.o Pidreesl iamndin Car. ysp ahnaetirfousnpegraml uamct.i vFiwtyF o =f  fHemedayloes fmlouwme brsr;a FsiwlieMns =e  emsaselen ftlioalw oeirlss ; aLgeaFi n=s fte Cmlaadleo slpeaorvieusm;   LeM=maleleaves;N=nystatin;CA=cinnamicacid;T=thymol. LcelaMdo =sp moraioleid leesa avneds; C N.  s=p hnayesrtoastpienr;m CuAm =. F cwinFn =a mfeimc aacleid fl; oTw =e trhs;y FmwoMl.  = male flowers; LeF = female leaves;  LeM = male leaves; N = nystatin; CA = cinnamic acid; T = thymol. Medicines2017,4,55 7of13 Medicines 2017, 4, 55   7 of 13  AftAerftTerL TCLCel ueltuiotinono foft hthee mmoosstt aaccttiivvee ooilisls, ,twtwo oinihnihbiitbioitnio bnanbdasn, dast ,reattenretitoenn tfiaocntofrasc (tRofr’ss) (oRf 0’s.6)7o f f and 0.12, were observed in all samples (Figure 3). After scraping the active bands, the GC‐MS analysis  0.67and0.12,wereobservedinallsamples(Figure3). Afterscrapingtheactivebands,theGC-MS anailnydsiiscaitneddi ctahtaetd ctuhraztecruenrzee r(Tenabel(eT a2b, lFeig2,uFreig 4u)r ew4a)sw tahse thmeamin acinomcopmoupnodu nfdorf othret hRef R0.607.6 7frafrcaticotinosn, s, f comcpomrispinrigsi9n7g% 97a%nd a8n2d% 8f2o%r bfootrh bfloothw eflroawnedr laenafdo lielsa.f Tohiles.b Tanhde abtaRnd 0a.1t 2Rwf a0.s1c2o wmapso sceodmbpyosaecdo mbyp lae x f complex mixture, with predominance of α‐terpineol, α‐eudesmol, and ferula lactone I (Figure 4) in  mixture,withpredominanceofα-terpineol,α-eudesmol,andferulalactoneI(Figure4)indifferent different proportions, not allowing to point only one single active compound, as presented in Table  proportions,notallowingtopointonlyonesingleactivecompound,aspresentedinTable2. 2.    Figure 3. (A) Detection of terpenes of H. brasiliense essential oils by Vanilin-Sulfuric Acid (VS) Figure 3. (A) Detection of terpenes of H. brasiliense essential oils by Vanilin‐Sulfuric Acid (VS) spray  spray reagent followed by heating to 110 ◦C; (B) Antifungal activity of Hedyosmum brasiliense reagent followed by heating to 110 °C; (B) Antifungal activity of Hedyosmum brasiliense essential oils,  essednetviaelloopields, dweivthel opn‐ehdexwaniteh/ance-htoenxea n(e9/0a:1c0e,t onv/ev)(,9 0a:1g0a,invs/t v)C,laadgoasipnosrtiuCml adcolaspdoosrpiuormioicdleasd osapnodr ioiCd.e s andspCh.aesrpohspaeerrmosupmer.m Ruf m0.6.7 R= fst0r.o6n7g= acsttirvoitnyg; Rafc 0t.i1v2i t=y ;wReafk0 a.1c2tiv=ityw; eFawkF a=c tfievmitayle; fFlwowFe=rsf; eFmwaMle =fl omwaleer s; FwMflo=wemrsa;l eLeflFo =w feerms;aLlee Flea=vfeesm; LaelMel e=a mveasle; LleeaMve=s; mNa =l enlyesatavteisn;. N=nystatin. TabTleab2l.eC 2h. eCmhiecmaliccaol mcopmopsiotsioitnioonf otfh tehae catcivtievea nantitfiufunnggaallf frraaccttiioonnss ooff HH.. bbrraassiilliieennssee ffeemmaalele flfloowweresr sanadn d leavleeasveesss eenssteianltioaill soiilsso ilsaotleadtebdy bbyi obaiouatuotgorgarpahpyh-yg‐guuidideeddT TLLCC..  FwF (%) LeF (%)  Compound  RI  RIref (a) FwF(%) LeF(%) Compound RI RIref(a) Rf0.67 Rf0.12 Rf 0.67  Rf 0.12  1,8‐cineole  1031 1031  Rf 0.67 Rf0.12 R1f20.6.697  Rf0 .12 1,t8e-cripnienoolelene  10311082 10130188    2.24  12. 69 4.65  terpinolene 1082 1088 2.24 4.65 dehydro‐sabina ketone  1121 1120    1.25    2.72  dehydro-sabinaketone 1121 1120 1.25 2.72 cisc-iβs-‐tβe‐rtpeirnpeionleol  11431143 11141444    00..8899      2-2(‐1(Z1)Z-p)‐rpoproenpyeln-pyhl‐epnholenol  11461146 11151050    00..7733      cics-icsh‐crhysraynstahnetnhoelnol  11621162 11161464    00..8899      terpinen-4-ol 1177 1177 1.87 terpinen‐4‐ol  1177 1177    1.87      α-terpineol 1191 1188 9.77 10.45 δ-αel‐etmerepnieneol  13271191 13131888    9.77  0.9 9 10.45  β-eδl‐eemleemneene  13841327 13193038      00..9929    germβa‐ecrleenmeeDne  14771384 14183590  2. 98   01..4952    gcuerrzmeraecnreene D  14891477 14194985  29.79.082    812..4058    δ-amorphene 1508 1512 1.50 curzerene  1489 1499  97.02    82.08    elemol 1542 1549 3.80 3.81 spδa‐athmuolernpohlene  15701508 15175812    19..5402    4.9 9 virideiflleomrooll  15781542 15195249    34..8305    63..5871  epis-αp-actahduinleonlol  16361570 16145078    90..4820    4.99  epi-α-muurolol 1640 1642 4.75 5.12 viridiflorol   1578 1592    4.35    6.57  α-eudesmol 1648 1653 20.95 22.53 veaplie‐rαia‐ncaoldinol  16511636 16156840    02..8203       eaptria‐cαt‐ymlouneurolol  16521640 16156842    4.75  1.8 7 5.12  germacra-4(15α),‐5e,1u0d(1e4s)m-troiel n-1-α-ol 16751648 16186653    200..7915    22.53  ferulalactoneI 2001 1974 21.83 28.29 valerianol  1651 1658    2.23      Intensityofaatnrtaifcutynlgoanlea ctivity 1652 1658  stro ng we  ak st1r.o8n7g  wea k Totalidentified(%) 100 87.98 10 0 89.1 3 germacra‐4(15),5,10(14)‐trien‐1‐α‐ol 1675 1686  0.71  RI=RetentionindicesonDB-5column;ref.RI (a)=[20];fractionsR 0.67=strongantifungalactivity;fractionsR ref f f   0.12=weakantifungalactivity. Medicines 2017, 4, 55   8 of 13    ferula lactone I   2001 1974  21.83  28.29  Intensity of antifungal activity strong weak strong  weak  Total identified (%)  100 87.98 100  89.13  MedicinesR2I 0=1 R7,e4t,e5n5tion indices on DB‐5 column; ref. RIref (a) = [20]; fractions Rf 0.67 = strong antifungal activity;  8of13 fractions Rf 0.12 = weak antifungal activity.    Figure4. ChemicalstructuresofthemaincompoundsfoundintheactivefractionsofH.brasiliense Figure 4. Chemical structures of the main compounds found in the active fractions of H. brasiliense  essentialoils:curzerene(A);α-terpineol(B);α-eudesmol(C);andferulalactoneI(D). essential oils: curzerene (A); α‐terpineol (B); α‐eudesmol (C); and ferula lactone I (D).  3.33..3A. nAtniotxioixdiadnatnAt Actcitviivtiyty  AnAtniotixoixdiadnantta actcitvivitiyty wwaass ddeetteerrmmiinneedd bbyy 22,2,2‐d-dipiphheneynly‐l1-‐1p-ipcircyrlyhlyhdyrdarzayzl y(lD(PDPPHP)H f)refer ereadriacdailc al scasvcaevnegningginagn daβnd-c aβro‐ctaernoetebnleea cbhleinacghminegt hmodesth.oTdhse. 5T0%hei n5h0i%bi toinrhyicboitnocreyn tcroanticoennt(rIaCt5io0)nw  (eIrCe50c)a lwcuelraet ed ancdaltchuelaItCed50 avnadlu tehse aIrCe50p rveaslueneste adrein pTreasbelnet3ed. Qinu eTracbeltei n3.a nQdueGricnektigno abnildob Gaienxktgroa cbtilwobea reexetrmacptl owyeerde as poesmitipvleoyceodn tarso lpsosshitoivwei ncognItCro5l0s 2s.h5o±wi0n.g0 9ICan50 d2.153 ±.5 0±.090 a.5nµd g1/3m.5 L±, 0r.e5s μpgec/mtivLe, lrye,sfpoercDtivPePlyH, faosrs aDyP,PwHh ile buatysslahyy,d wrohxilyet obluuteynlhey(dBrHoxTy)taonludenbeu t(yBlHhyTd) raonxdy bauntiysolhleyd(BroHxAya)nwiseorlee t(hBeHpAo)s witievree cthone tproolssitfivoer βco-ncatrrooltse ne assfoary ,βs‐hcaorwotienngeI aCssay0,. 0s8ho±w0in.0g9 IaCn50d 00.0.287 ± ±0.009.0 a2nµdg 0/.2m7 L±, 0r.e0s2p μegc/tmivLel,y r.espectively.  50 TaTbaleble3 .3. AAnnttiiooxxiiddaanntt  aaccttivivitiiteies s(I(CIC50,5 0,memane an±  S±D)S Dby)  bDyPPDHP PfrHee frraedeicraal dsiccaavlesncgaivngen ganindg  βa‐nd β-ccaarrootteennee//lilninoolelieci cacaicdi dmmetehtohdosd osf othfeth eesseesnsteinalt ioaillso firlsomfro fmlowfleorws aenrsda lneadvleesa ovfe Hso. bfrHas.ilbireanssiel.i e nse. IC50(μg/mL) Samples  DPPH β‐IcCa5r0ot(eµnge//mlinLo)leic acid  Samples ♀ Flowers  3162.79D ±P 4P0H.72  180.71β ±- c2a2r.9o5t ene/linoleicacid ♂ Flowers  2516.18 ± 131.87  113.46 ± 11.38  Flowers 3162.79±40.72 180.71±22.95 ♀ Leaves  3542.01 ± 45.31  71.12 ± 9.07  ♀ Flowers 2516.18±131.87 113.46±11.38 ♂ Leaves♂ Leaves  373835.4429. 0±1 1±32.4756. 31 86.47 ± 18.637 1.12±9.07 ♀ LeavesQuercetin  327.8530. 4±9 0±.091 32.76 ‐  86.47±18.63 Q♂uercetGininkgo biloba  13.25.05 0± ±0.500. 09 ‐  - GinkgobilobaBHT  13.5‐0  ±0.50 0.08 ± 0.09  - BHT BHA  ‐  - 0.27 ± 0.02 0.08±0.09 DPPH = 2,2‐dBiHpAhenyl‐1‐picrylhydrazyl; BHT -= butylhydroxytoluene; BHA0 =.2 b7u±ty0lh.0y2droxyanisole;  DP♂P H= m=a2l,e2;- ♀d i=p hfeemnyall-e1.- picrylhydrazyl; BHT=butylhydroxytoluene; BHA=butylhydroxyanisole; =male; =female. ♂ 4. D♀iscussion  4. Discussion Although there were previous studies on the essential oils from H. brasiliense leaves, none of  theAml tdhiosucrgihmtinhaetreedw aemreonpgre mviaolue sanstdu dfeimesaolen pthlaenetss s[e1n8,t1ia9l,2o8i]l sanfrdo,m toH th.eb rbaessilti eonf soeuler akvneosw,nleodngeeo, fthtihse m disisc rtihme infiarstet dreapmorotn wgimtha flleowanedr feesmseanlteiapl loainlst.s I[n1 8o,1u9r ,2st8u]dayn,d t,hteo etshseenbteisatl oofilo yuirelkdnso wwelreed gsiem,tilhairs fiosrt he firsfltorweperosr tawndit hleflaovwese roef sHse.n btiraalsioliielns.seI,n wouitrh sttuhed ym,tahlee efslosewnetrias lporilesyeineltdinsgw tehree sloimwielsatr ofoilr flamowouenrstsa. nd leaRveegsaordfiHn.gb trhaes illeieanf soei,lw coitnhtetnhtes,m thaele reflsouwltes rwseprree slieknet itnhgosteh feoluonwde fsotro ail Paamraonuán Sttsa.tRe esgpaercdiminegn t(hBeralezailf) oil con[2t8e]n, tcso,ltlhecetereds iunl tasnw aererea lnikote ftahro fsreomfo uIlnhda dfoor CaaPrdaorasno á(SSãtoa tPeasupleoc Simtateen, B(Brarazzili)l.) H[2o8w],ecvoelrl,e tchteedsei vnaaluneasr ea were lower than those found for a specimen collected in Santa Catarina State (Brazil), whose leaves  notfarfromIlhadoCardoso(SãoPauloState,Brazil). However,thesevalueswerelowerthanthose yielded 0.5% (v/w) [19], but this place is further south from our collection site.  foundforaspecimencollectedinSantaCatarinaState(Brazil),whoseleavesyielded0.5%(v/w)[19], butthisplaceisfurthersouthfromourcollectionsite. The essential oil analysis for the Parana (Brazil) specimen was not complete, most of the oil   componentswerenotidentified[28]. Nonetheless,withthecompositionpublisheditwaspossible todetect23commoncompoundswiththeSãoPaulo(Brazil)specimen,butonlysabinene(7%)was acommonmajorcompound.However,fortheSantaCatarina(Brazil)studywerefound22compounds incommon[19],havingasmajorcompoundsα-terpineol(10%),curzerene(9%),pinocarvone(8%), β-thujene(7%),andcarotol(6%). Curzerene(Figure4A)wastheonlycommonmajorcompoundwith thepresentanalysis,butwith17–18%intheleavesand11%inflowers. Medicines2017,4,55 9of13 The essential oils from other Hedyosmum species leaves (H. mexicanum, H. bonplandianum, H. arborescens, and H. angustifolium) have also presented high amounts of sabinene in their composition [4,7,8], as well as β-pinene, pinocarvone, and curzerene in the essential oil from H.colombianumleaves[6],similarlytoourstudy. Hedyosmumsppessentialoilsareknowntocontainseveralphenylpropanoids[3,5,6],sometimes inhighamounts,asinessentialoilfromH.scabrumleaves,whichcontained55.8%ofestragoleand 6.6% of anethole [5]. Also, some H. brasiliense specimens from different geographic origins have shownmethylchavicol,eugenolacetate,methyleugenol,andestragole[18,19,28]. However,theonly phenylpropanoiddetectedinourstudywasmethyleugenol,butinsmallamounts. PCAplotshowsthatleaveshaveahigherpercentageofsabineneandcurzerenethanflowers, whichhaveamoresignificantamountofcarotol,responsiblefordifferentiatingthem. Besidesthat, PCAwasalsoabletodiscriminatemaleflowersandleavesfromthefemaleonesbythe1,8-cineole abundance,whichwashigherinthefemalecounterparts(Table1andFigure1). Theseresultsshowed thatitisimportanttoanalyzeseparatelymaleandfemaleindividuals,partofthevariableresultson thechemicalcompositionmightbeexplainedbynotseparatingtheplantgenders. Cladosporium spp. are phytopathogenic filamentous fungi usually chosen for bioautographic assays,astheypresenthighsensitivityandpermitthedetectionoffungitoxicsubstancesbycontrast withtheirdarkcolor[23]. Preliminaryassaysforthedetectionlimitwithbioautographyconfirmed the antifungal activity for the H. brasiliense specimen from São Paulo against C. cladosporioides and C.sphaerospermum,withthehighestactivityforthefemalefloweressentialoil(Figure2).Inaprevious study, the antimicrobial activity of H. brasiliense leaf essential oils has already been confirmed for Gram-positive bacteria and fungi such as the dermatophytes Microsporum canis, M. gypseum, Trichophytonmentagrophytes,andT.rubrumandtheyeastsCandidaalbicansandC.parapsilosis[19]. The bioautography followed by TLC separation has been useful to determine antimicrobial activityofessentialoilsandtoisolatetheactivecompounds.Abioautographystudywithfivearomatic plants(Thymusvulgaris,LavandulaangustifoliaChaix,EucalyptusglobulusLabill.,MenthaspicataL.,and CinnamomumzeylanicumBlume)againstfivebacteria(Pseudomonassyringae,Xanthomonascampestris, Staphylococcusepidermidis,S.saprophyticus,andS.aureus)allowedtoattributetheactivityofThymus vulgarisessentialoiltothymol,comparingtoapurestandard,aswellastheactivityofC.zeylanicum essentialoiltoeugenol,bythesameprinciple[29]. AbioautographyassaywithMenthaxpiperitaL. essentialoilagainstCandidaalbicans,followedbyTLCseparationoftheactivefraction,indicatedthat mentholwasresponsibleforthisactivity[25]. Also,Guerrinietal.[3]detectedα-cadinol,α-muurolol, τ-muurolol,andlinaloolintheactivefractionwithantibacterialactivityagainstS.aureususingHigh PerformanceThinLayerChromatography(HPTLC)platesforseparationofH.spruceiessentialoil. In our study, the bioautography methodology indicated curzerene as the main compound for the activebandfoundatR ’s0.67andα-terpineol,α-eudesmolandferulalactoneIfortheactiveband f atR 0.12(Table2). TheH.brasilienseleafessentialoilcollectedatSantaCatarina(Brazil)presented f antifungalactivityanditwasalsorichincurzerene(8.9%)andα-terpineol(10.2%)(Figure4A,B)[25], supportingourresults. CurzerenehasbeenknownforitstoxiceffectsagainstAnophelessubpictus(LC =4.14µg/mL), 50 Aedes albopictus (LC = 4.57 µg/mL), and Culex tritaeniorhynchus (LC = 5.01 µg/mL) [30]. 50 50 Inaddition,curzerenealsoshowedantiproliferativeeffectsinSPC-A1humanlungcarcinoma,with IC =47µmol/Lin72h(invitro)and135mg/kgdaily(invivo)[31]. EugeniaunifloraL.essential 50 oil, rich in curzerene (42.6%), showed antifungal activity, inhibiting completely Paracoccidioides Braziliensis at 62.5 µg/mL [32]. Moreover, another E. uniflora essential oil composed by 47.3% of curzerene presented anti-Leishmania activity, with IC = 3.04 µg/mL against promastigotes and 50 IC =1.92µg/mLagainstamastigotesofL.amazonensis[33]. 50 The antioxidant activity of essential oils is already well known [34–37]. According to Karadagetal.[38],theantioxidantpotentialisrelatedtocompoundscapableofprotectingabiological systemagainstthepotentiallyharmfuleffectofreactiveoxygenspecies. Theresultsofasingle-assay Medicines2017,4,55 10of13 cangiveonlyanarrowviewoftheessentialoilantioxidantproperties. Therefore, theantioxidant potentialofH.brasilienseessentialoilswasdeterminedbytwodifferentmechanismsofaction: DPPH freeradicalscavenging,thatworksaselectrontransfer,andβ-carotene/linoleicacidassay,measuring the suppression of lipoperoxides. In the DPPH assay, the IC values for all the oils ranged from 50 2516.18 to 3783.49 µg/mL (Table 2), indicating a weak electron transfer capacity for the oils when comparedtothepositivecontrolsquercetinandG.bilobaextract,thatpresentedIC valuesof2.5and 50 13.5µg/mL,respectively. TheseresultsarecomparabletothoseobtainedforSalviatomentosaMill. essentialoils[36],whichwasrichinβ-pinene(39.7%),α-pinene(10.9%)andcamphor(9.7%),andit wasnotconsideredeffectiveasantioxidantbyDPPHandβ-carotene/linoleicacidassay. Similarly,the essentialoilsofsevenArtemisiaspp. weretestedbythesamemethodsandshowedweakactivities,as theircompositionweremarkedlyrichinnon-phenoliccomponents[39]. Theantioxidantcapacityof essentialoils,bytheDPPHmethodnormally,isnotveryhighwhencomparedwiththatobtainedfor extractsandfractionsrichinphenoliccompounds. Themajorantioxidativeplantphenoliccompounds knownareeugenol,carvacrol,thymol,menthol,andsafrole,amongothers[34]. Inatestwith98pure essentialoilcomponentsfortheirantioxidantactivity,phenols,suchasthymolandcarvacrol,werethe mostactivecompounds,followedbyalcoholsmono-andsesquiterpenesandketonesα,β-insaturated, howeverhydrocarbonspresentedverylowantioxidantactivity[40]. Ontheotherhand,ourresults indicatedthattheessentialoilsofH.brasilienseweremuchmoreactiveintheβ-carotenebleaching assay,presentingIC valuesfrom71.12to180.71µg/mL(Table2),indicatingtheirabilityindestroying 50 theconjugateddienehydroperoxides,theendproductsoflinoleicacidperoxidation. Inthisway,the lowradicalscavengingactivityobservedfortheH.brasilienseessentialoilsmightbeexplainedbythe lowamountsofphenoliccompoundspresentintheoil,only1.11to3.63%ofthymol,andthehigh amountsofmono-andsesquiterpenehydrocarbons(Table1). Sinceantioxidantactivityoftheentire oilistheresultoftheinteractionofallconstituents[41],itishardtoattributetheessentialoilsactivity toasinglecompound,asotheroilcomponentsmaycontributeexhibitingsynergisticorantagonistic effects[42]. 5. Conclusions Theleafandfloweressentialoilsweredominatedbysabinene,1,8-cineole,curzerene,andcarotol. However,thecontentsoftheseconstituentsdifferedaccordingtotheplantpartandgender. This was the first report of antifungal activity of curzerene, explaining partially the activity of H. brasiliense essential oils by bioautography. Moreover, the complex mixture of α-terpineol, α-eudesmol,andferulalactoneImayhaveactedsynergisticallytotheantifungalactivityobserved. Bioautographyassayhelpedtodetectantifungalfractionsinacomplexmatrixofessentialoilsand guidedtotheisolationoftarget-directedconstituents. Furtherstudiesarenecessarytoisolatehigher amountsofpurecurzerene,inordertotestitslimitofdetection. Antioxidant activity of essential oils from H. brasiliense was more effective by β-carotene discoloration assay than by DPPH assay, indicating their higher ability in quenching conjugated hydroperoxidesthanfreeradicals. Theinhibitionoflipidperoxidationmayhavebeenaresultofthe interactionofallconstituentsoftheessentialoils,sinceitwasnotpossibletoattributeantioxidant activitytoasinglecompound. Supplementary Materials: The following are available online at www.mdpi.com/2305-6320/4/3/55/s1, FigureS1:MassspectrumofNon-identifiedcompound1(N.I.1)detectedintheessentialoilofH.brasiliensefrom IlhadoCardoso;FigureS2:MassspectrumofNon-identifiedcompound2(N.I.2)detectedintheessentialoilof H.brasiliensefromIlhadoCardoso;FigureS3:MassspectrumofNon-identifiedcompound3(N.I.3)detectedin theessentialoilofH.brasiliensefromIlhadoCardoso;FigureS4:MassspectrumofNon-identifiedcompound4 (N.I.4)detectedintheessentialoilofH.brasiliensefromIlhadoCardoso. Acknowledgments:TheauthorswishtothankConselhoNacionaldeDesenvolvimentoCientíficoeTecnológico (CNPq,Proc.N.309762/2012-0and164913/2013-0)andFundaçãodeAmparoàPesquisadoEstadodeSãoPaulo (FAPESP,Proc.N.2014/15299-4and2014/17561-8)forfinancialsupportandscholarships.

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(Ruiz & Pav.) Solms (Ruiz & Pav.) G. Don as a treatment to joint pain activity was performed by the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) Miguel, M.G. Antioxidant activity of medicinal and aromatic plants—a review.
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