(Myrtaceae) Essential Oil from Amazonian Ecuador

12 Pages·2017·0.9 MB·English

molecules
Article
Myrcia splendens (Sw.) DC. (syn. M. fallax (Rich.)
DC.) (Myrtaceae) Essential Oil from Amazonian
Ecuador: A Chemical Characterization and
Bioactivity Profile
LauraScalvenzi1,*,AlessandroGrandini2,AntonellaSpagnoletti2,MassimoTacchini2,
DavidNeill3,JoséLuisBallesteros4 ,GianniSacchetti2andAlessandraGuerrini2,*
1 DepartmentofEarthScience,UniversidadEstatalAmazónica,Puyo160106,Ecuador
2 DepartmentofLifeSciencesandBiotechnology(SVeB),UR7Terra&AcquaTech,UniversityofFerrara,
Ferrara44121,Italy;[email protected](A.G.);[email protected](A.S.);
[email protected](M.T.);[email protected](G.S.)
3 DepartmentofLifeScience,UniversidadEstatalAmazónica,Puyo160106,Ecuador;[email protected]
4 DepartmentofLifeSciences,UniversidadPolitécnicaSalesiana,Quito170525,Ecuador;
[email protected]
* Correspondence:[email protected](L.S.);[email protected](A.G.);
Tel.:[phone](L.S.);[phone](A.G.)
Received:30May2017;Accepted:6July2017;Published:12July2017
Abstract: Inthisstudy,weperformedthechemicalcharacterizationofMyrciasplendens(Sw.) DC.
(Myrtaceae)essentialoilfromAmazonianEcuadorandtheassessmentofitsbioactivityinterms
of cytotoxic, antibacterial, and antioxidant activity as starting point for possible applicative uses.
M.splendensessentialoil,obtainedbyhydro-distillation,wasanalyzedbyGasChromatography-Mass
Spectrometry(GC-MS)andGasChromatography-FlameIonizationDetector(GC-FID):themajor
components were found to be trans-nerolidol (67.81%) and α-bisabolol (17.51%). Furthermore,
we assessed the cytotoxic activity against MCF-7 (breast), A549 (lung) human tumor cell lines,
and HaCaT (human keratinocytes) non-tumor cell line through 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2-H-tetrazoliumbromide(MTT)test: promisingresultsintermsofselectivityandefficacy
against the MCF-7 cell line (IC of 5.59 ± 0.13 µg/mL at 48 h) were obtained, mainly due
50
to α-bisabolol. Furthermore, antibacterial activity against Gram positive and negative bacteria
wereperformedthroughHighPerformanceThinLayerChromatography(HPTLC)bioautographic
assay and microdilution method: trans-nerolidol and β-cedren-9-one were the main molecules
responsible for the low antibacterial effects against human pathogens. Nevertheless, interesting
values of Minimum Inhibitory Concentration (MIC) were noticeable against phytopathogen
strains. RadicalscavengingactivityperformedbyHPTLCbioautographicandspectrophotometric
1,1-diphenyl-2-picrylhydrazyl(DPPH)approacheswerenegligible. Inconclusion,theessentialoil
revealedagoodpotentialforplantdefenseandanti-cancerapplications.
Keywords:Myrciasplendens;essentialoil;cytotoxicactivity;antibacterialactivity;antioxidantactivity;
bioautographicassay
1. Introduction
Essentialoilsaremixturesofcomplexandfragrantsubstances,employedinmanyapplicative
fields, from aromatherapy to medicine and pharmacy, as well as the agro-food industry and
phytoiatry[1]. Theapplicativeimportanceofessentialoilsinsuchdiversifiedfieldsreliesmainlyon
theirchemicalcharacterizationasmixturesoflowmolecularweightlipophilicsubstancesandontheir
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Molecules2017,22,1163 2of12
biologicalpropertiesoftenduetosynergisticeffectsoftwoormoreconstituents[2]. Infact, many
researchpapersaboutessentialoilsfocusontheirchemicalportraitandinvitrobiologicalproperties
in terms of antioxidant, antimicrobial, and cytotoxic activities with the aim of detecting the most
performance. Oneofthemostinterestingchemicalaspects,emergingfromalltheseresearches,istheir
high chemodiversity, i.e., different composition, sometimes even dramatic, among phytocomplex
from plants belonging to the same species, but grown in different geographical conditions and
biodiversitycontexts. Thisaspectismoreevidentforthosespeciesfromareascharacterizedbyhigh
biodiversity,wheretheinteractionamongahighernumberofdifferentspeciesresultsinapropulsive
toolforsecondarymetaboliteswhichgiverisetoremarkablemoleculardiversityandabundancein
plants[3,4]. Biologicalactivitiesofessentialoils,instead,independentlyofthemethodologicaltarget,
are particularly focused on the detection of the compounds responsible of the activity and on the
detectionandquantificationofpossiblesynergisticevidences[2]. Amongseveralkindsofbiological
activities,cytotoxicandantioxidantarethemostinterestingsincetheresultscouldleadtodeepened
researchaboutchemicalcompoundsand/orchemicalplatformsfornewpharmaceuticaltreatmentsof
inflammation-relateddiseases,suchascancers[5]. Givenallthesepremises,achemicalandbioactivity
study has been performed on the essential oil of Myrcia splendens (Sw.) DC. (syn. M. fallax (Rich.)
DC.)(Myrtaceae)obtainedfromplantsgrowninAmazonianEcuador. ManyneotropicalMyrtaceae
species are known for their traditional health uses, but some of them are scarcely investigated
under the chemical point of view and as source of bioactive compounds. Fresh aerial parts of
species belonging to Myrcia genus are employed in South American traditional medicine for the
treatment of diabetes, hypertension, diarrhea, hemorrhage as infusion, and as a compress to treat
inflammationandskininfections[6,7]. Myrciasplendensisatreefoundinneotropicregions,distributed
fromEasternMexicototheSouth-EasternBraziliancoastalforests. Crudedrugs, derivatives, and
preparations from Peruvian [8] and Brazilian plants have been studied and interesting cytotoxic
activitieswereevidenced[9]. SincetheAmazonianbasinisoneofthemostimportantbiodiversity
hotspots world-wide, a first report on chemical characterization and biological properties of the
essentialoilofM.splendensfromEcuadorwasperformed,withtheaimtocompareourdatawiththose
alreadyreportedbyrelatedliterature.
2. Results
2.1. ChemicalCompositionofEssentialOil
The distillation of M. splendens leaves gave a colorless essential oil with a yield of 0.11%
(w/v), which was lower than previously reported (0.35%) [10], and its density was 0.906 g/mL.
TheM.splendensessentialoilcompositionisshowninTable1.
Table1.ChemicalcompositionofM.splendensessentialoilanditscharacterizationintermofactive
antioxidantandantibacterialcompoundsisolatedrespectivelyfromDPPH-HPTLC(1,1-diphenyl-2-
picrylhydrazil-highperformancethinlayerchromatography)andHPTLCbioautographyassays.
Antioxi.F.5 Antibact.F.6
No. %Area1 Component2 AIExp3 AILit4 Rf=0.9 Rf=0.45–0.6 Rf=0.7
1 2.08±0.10 α-pinene 928 939
2 0.11±0.01 β-pinene 972 979
3 0.15±0.02 α-cubebene 1350 1351 1.39
4 0.51±0.03 α-copaene 1375 1377 4.21
5 0.11±0.02 β-elemene 1387 1391 4.39
6 4.21±0.15 β-caryophyllene 1409 1419 36.23
7 0.31±0.02 trans-α-bergamotene 1430 1435 3.15
8 0.45±0.03 α-caryophyllene 1449 1455 3.94
9 0.87±0.06 trans-β-farnesene 1453 1457 7.81
10 0.65±0.04 germacreneD 1475 1485 4.62
11 0.10±0.01 cis-β-guaiene 1485 1493 1.54
Molecules2017,22,1163 3of12
Table1.Cont.
Antioxi.F.5 Antibact.F.6
No. %Area1 Component2 AIExp3 AILit4 Rf=0.9 Rf=0.45–0.6 Rf=0.7
12 0.17±0.02 viridiflorene 1490 1497 1.43
13 0.11±0.01 α-muurolene 1497 1499 1.27
14 0.59±0.03 α-bisabolene 1503 1507 1.24
15 0.24±0.02 cis-γ-bisabolene 1506 1515 8.33
16 0.53±0.04 δ-cadinene 1513 1523 4.93
17 0.16±0.01 trans-calamenene 1518 1529 1.66
18 1.03±0.09 trans-γ-bisabolene 1523 1531 10.04
19 67.81±2.10 trans-nerolidol 1562 1563 100
20 0.15±0.01 caryophylleneoxide 1580 1583
21 Traces β-cedren-9-one 1630 1631 87.34
22 17.51±1.01 α-bisabolol 1690 1686
Total
97.84
identified
1Relativepeakareas±SEM(standarderrormedia),calculatedbyGC-FID;2Componentsarelistedinorderof
elutionandtheirnomenclatureisinaccordanceoftheNIST(NationalInstituteofStandardsandTechnology)library;
3AIexp:arithmeticindicescalculatedonaVarianVF-5mscolumn;4AIlit:arithmeticindices[11];5Antioxi.F.:
fractionofcompoundsresponsibleforantioxidantactivity.6Antibact.F.:fractionofcompoundsresponsiblefor
antibacterialactivity.
Twenty-twocompoundswerecharacterized,correspondingto97.84%ofthetotal. Sesquiterpenes
were predominant and particularly oxygenated sesquiterpenes, corresponding to 85.47% of the
total. Monoterpenes, α- and β-pinene, were present in a lower amount (2.19% as total). Among
the sesquiterpenes the most abundant were trans-nerolidol (67.81%), α-bisabolol (17.51%) and
β-caryophyllene(4.21%). ThepredominanceofsesquiterpenesispeculiartotheMyrciagenus[12].
M.splendens(syn. M.fallax)essentialoilexhibitedadifferentcompositioncomparedtorelatedstudies
onthesamespecies. Infact, Henriquesetal.[10]foundα-bisabolol(83%)asthemaincompound,
while Nakamura et al. [13] found it to be α-bisabolene (80%); Alarcón et al. [7] stated that guaiol
(31%) and carotol (9.9%) were the most abundant constituents. β-elemene was found as the main
compoundinthestudyofLimaetal.[14]andα-pinenecharacterizedtheessentialoilobtainedby
Pereiraetal.[15]. trans-nerolidolwasthemaincompound(80.8%)ofM.bracteataessentialoil[16].
To the best of our knowledge, trans-nerolidol was found only in the M. splendens species of the
present study. β-caryophyllene was already detected in M. glabra, M. multiflora, M. cuprea, and
M.tomentosa[17]andα-pineneinM.bombycinaandM.myrtifolia[18]. Adeepinter-andintraspecific
diversityinchemicalcompositionofMyrtaceaeessentialoilshasbeenreportedbyvariousauthors.
DataobtainedonM.splendensessentialoilfromEcuadorseemtoconfirmthishypothesis. Previous
studies suggested that intraspecific diversity of Myrtaceae terpene profiles could be a response of
adaptationtoawiderangeofenvironmentalconditions,inparticularthemonoterpene: sesquiterpene
ratioseemstobeinfluencedbytheefficiencyofterpenesynthasesenzymes[19]. Obtainedresults
indicatethatendogenousfactors, ashighgeneticdiversity, havemoreinfluenceonthevolatileoil
variabilityofMyrtaceaefamilythanenvironmentalaspects[18]. Inparticular,thedifferencesobserved
in chemical composition of M. splendens essential oils could also depend on Amazonian origin of
thestudiedsamples. Ineffect,thehighbiodiversityofAmazonianregioninducesplantsecondary
metabolismtobiosyntheticpathwayscharacterizedbydiversifiedchemicalprofile[3].
2.2. CytotoxicActivity
MTTassaywasusedtoevaluatetheeffectofM.splendensessentialoilanditsmaincompounds,
α-bisabololandtrans-nerolidol, onthecellviabilityofA549(humanlungcancer), MCF-7(human
breastadenocarcinoma)tumorlinesandHaCaT(humankeratinocytes)normalcells,asthereference
toassessoverallcytotoxicactivity. Allresultsshowed(Figure1)adose-dependentinhibitionofcell
growthand,inparticular,α-bisabololwasthemostactivecomponentagainstthethreestudiedcell
lines,inthedilutionsrangingfrom1to200µg/mL.
Molecules2017,22,1163 4of12
Moreover, at the concentration of 10 µg/mL, α-bisabolol decreased the viability of A549,
MCF-7, andHaCaTcelllinesof70%, 10%and50%, respectively, incomparisonwiththenegative
control. The results highlighted that inhibition on cell growth was highly dependent on cell type
with IC values ranging from 1.24 ± 0.03 to 100.99 ± 2.32 µg/mL. In particular, MCF-7 cancer
50
cells showed more growth inhibition than HaCaT cells after 48 h of treatment with α-bisabolol
(IC =1.24±0.03µg/mLvs. IC =10.15±0.35µg/mL)andessentialoil(IC =5.59±0.13µg/mL
50 50 50
Molecules 2017, 22, 1163 4 of 12
vs. IC =21.58±1.26µg/mL) (Table 2). As reported in other studies, IC values lower than
50 50
30µcgh/emmoLpprerveseenntitvae pgootoedntciahle fmoro tphree evsesnetnitviael pooilt e[2n0t]i.a Hlfoowretvheer,e sthsee nHtiaaClaoTil c[e2l0ls] .sHhoowweedv teor ,bteh meHorae CaT
cellssesnhsoiwtiveed, wtoitbhe ImC5o0 rvealsueenss irtainvgei,nwg iftrhomIC 5100.v1a5l u± e0s.3r5a ntog 2in7g.76fr ±o m2.7160 μ.1g5/m±L0, .t3h5atno t2h7e. 7A654±9 2c.e7l6l lµinge/, mL,
thanwtihthe ICA550 4ra9ncgeinllg lfirnoem, w54i.t2h8 I±C 25.309r taon 1g0i0n.g99f ±ro 2m.325 μ4g.2/m8L±. T2h.3er9eftoore1,0 t0h.e9 e9v±alu2a.t3io2nµ ogf /thme Lc.ytTohtoexriecf ore,
theeavctailvuitayt iorenveoafltehde pcryotmotiosxinicg arcetsiuvlittsy inre vteeramlesd opf rsoemlecitsiivnigtyr easnudl tesffiincatceyrm ofs Mof. sseplleecntdievnitsy eassnednteiaffil coailc yof
M.sapgleanindsetn tshees MseCnFti-a7l coeilll alignae irnastthethr ethManC aFg-a7incsetl lthlien Ae5r4a9th (eTrabthlea 2n).a gainsttheA549(Table2).
A) B)
100
100
Cell viability% vs Control) 5705 Cell viability% vs Control) 5705
( 25 ( 25
0 0
0 50 100 150 200 0 50 100 150 200
Concentration (g/mL) Concentration (g/mL)
C)
M. fallax EO
100
-bisabolol
Cell viability% vs Control) 5705 trans-nerolidol
( 25
0
0 50 100 150 200
Concentration (g/mL)
Figure 1. Effects of M. splendens essential oil (EO), α-bisabolol and trans-nerolidol on viability of A549
Figure 1. Effects of M. splendens essential oil (EO), α-bisabolol and trans-nerolidol on viability of
(A), MCF-7 (B) and HaCaT (C) cell lines. Cytotoxicity was assessed by MTT test after 48 h.
A549(A),MCF-7(B)andHaCaT(C)celllines.CytotoxicitywasassessedbyMTTtestafter48h.
Table 2. Cytotoxic activity of M. splendens EO, α-bisabolol and trans-nerolidol on three cell lines after 48 h.
Table2.CytotoxicactivityofM.splendensEO,α-bisabololandtrans-nerolidolonthreecelllinesafter48h.
Cell Line (IC50µg/mL)1
A5492 CellLinMeC(IFC-5703µg/mL)1HaCaT 4
M. splendens EO 100.99 ± 2.32 6d 5.59 ± 0.13 6c 21.58 ± 1.26 6c
A5492 MCF-73 HaCaT4
α-bisabolol 27.63 ± 2.01 6b 1.24 ± 0.03 6a 10.15 ± 0.35 6b
M.splendensEO 100.99±2.326d 5.59±0.136c 21.58±1.266c
trans-nerolidol 54.28 ± 2.39 6c 40.97 ± 5.07 6d 27.76 ± 2.76 6d
α-bisabolol 27.63±2.016b 1.24±0.036a 10.15±0.356b
doxorubicin 5 0.90 ± 0.01 6a 2.10 ±0.42 6b 0.40 ± 0.01 6a
trans-nerolidol 54.28±2.396c 40.97±5.076d 27.76±2.766d
1 IC50: comdpooxuonrdu bcoicnince5ntrations 0t.h90at± af0f.o0r1d6sa a 50% c2e.1ll0 g±ro0w.4th2 6dbecrease 0a.4ft0er± 408. 0h1. 6IaC50 are the
averages of triplicate experiments and represented as mean ± standard deviation; 2 Adenocarcinoma
1exICpe5c0rei:mlclo elminntpes;oa 3un Bndrdercaeopstnr eacsedenentnrtaeotdciaoarnscsimnthoeaamtnaaf± fcoesrltdla slninade5a;0r 4d% Kdceeervlalitagintriooowncy;tht2edAs ecdceerlenla olsicneaear;cf t5ien Droo4m8xaohrc.ueIlCbli5lc0iinnaer ;we3tahBser ueaasvseetdra aadgsee pnsooocsfaittrricivpienl iocmatae
celllcinoen;tr4oKl;e 6r aDtiantoac ayrtee spcreelslelnintee;d5 aDs omxoeraunb ±ic iSnDw, nas =u 3s.e Mdaesanpso siint ievaeccho ncotrloulm;6nD faotlaloawreedp rbesye dntifefderaesnmt leeatnte±r SD,
n=3a.rMe seiagnnsifinicaenactlhyc doilfufmernenfot l(lpo w< 0ed.05b)y. differentletteraresignificantlydifferent(p<0.05).
According to these results, M. splendens essential oil may be considered a promissory product to
Accordingtotheseresults,M.splendensessentialoilmaybeconsideredapromissoryproduct
be used for innovative therapeutic or preventive strategies against breast carcinoma. Its cytotoxic
tobeusedforinnovativetherapeuticorpreventivestrategiesagainstbreastcarcinoma. Itscytotoxic
activaicttyivimtya myadye dpeepnedndo onnt thhee pprreesseennccee oof fα-αb-ibsaisbaoblooll, owl,hiwchh sichhowshedo wa vederya ivnteerryesitnintger IeCs5t0i nvagluIeC aftevra lue
48 h (1.24 ± 0.03 μg/mL). Previous studies on α-bisabolol exhibited cytotoxic activity against glio5m0 a,
pancreatic, ovarian, and kidney carcinoma cells [9,21,22]. Furthermore, it was reported as a
chemopreventive agent in rat mammary carcinogenesis [23]. Thus the high content of α-bisabolol
(17.51%) in the M. splendens essential oil could explain the cytotoxic activity against MCF-7 cells. On
the other hand, data obtained on A549 were less interesting because the IC50 value (27.63 ± 2.01
μg/mL) was very close to 30 μg/mL, the concentration considered as the upper limit for potential
Molecules2017,22,1163 5of12
after48h(1.24±0.03µg/mL). Previousstudiesonα-bisabololexhibitedcytotoxicactivityagainst
glioma, pancreatic, ovarian, andkidneycarcinomacells[9,21,22]. Furthermore, itwasreportedas
achemopreventiveagentinratmammarycarcinogenesis[23].Thusthehighcontentofα-bisabolol
(17.51%)intheM.splendensessentialoilcouldexplainthecytotoxicactivityagainstMCF-7cells.Onthe
otherhand,dataobtainedonA549werelessinterestingbecausetheIC value(27.63±2.01µg/mL)was
50
verycloseto30µg/mL,theconcentrationconsideredastheupperlimitforpotentialchemopreventive
activity. In the present study, trans-nerolidol exhibited a cytotoxic activity less interesting than
α-bisabolol,neverthelessonA549cellsshowedIC valueat48h(54.28±2.39µg/mL)inlinewith
50
theonerecordedbySylvestreetal.[24](66±12µg/mL).Antineoplasticactivitywasalsorecordedon
bowelbyamixtureofcis-andtrans-nerolidol[25],suggestingthatdeeperstudiescouldbecarriedout
inordertoassesscytotoxicactivityagainstotherkindsofcancercelllines.
2.3. AntibacterialActivity: HPTLC-BioautographyandMIC
Antibacterial properties of M. splendens essential oil was assessed against Gram positive
and negative bacteria and MIC (Minimum Inhibitory Concentration) was determined by serial
microdilutionmethod. M.splendensessentialoilexhibitedaweakantimicrobialactivitycomparedto
chloramphenicol,usedasapositivecontrol,againstalltestedbacteria(Table3).
Table3. AntibacterialactivityofM.splendensessentialoilexpressedasMIC(MinimumInhibitory
Concentration)(µg/mL).
Strain M.splendensEO1 Antibiotic2
Gramnegative
Agrobacteriumtumefaciens DSM30207 5003c 6.253c
Agrobacteriumvitis DSM6583 20003e 1.563a
Pseudomonassyringaepv.syringae DSM10604 2503b 3.123b
Escherichiacoli ATCC4350 >20003f 25.003d
Pseudomonasaeruginosa ATCC27853 >20003f 6.253c
Grampositive
Clavibactermichiganensissubsp.nebraskensis DSM20400 1253a 6.253c
Enterococcusfaecalis ATCC29212 20003e 3.123b
Listeriagrayi DSM20601 10003d 5.003c
Staphylococcusaureus ATCC29230 10003d 3.123b
Staphylococcusepidermidis ATCC14990 10003d 3.123b
1EO:EssentialOil;2Chloramphenicolwasusedaspositivecontrol;3Meansineachcolumnfollowedbydifferent
lettersaresignificantlydifferent(p<0.05).
HPTLC-bioautographywasperformedtofindthepossibleantibacterialfractionofessentialoil
(Figure2).
However, results obtained indicated that phytopathogens strains were more sensitive than
human pathogens after treatment with the tested essential oil. In particular, the Gram positive C.
michiganensis subsp. nebraskensis resulted the most sensitivity, evidencing the lowest MIC value
of 125 µg/mL. Gram negative bacteria showed resistance to M. splendens essential oil except for
P.syringaepv. syringaethatshowedthelowestMICvalueof250µg/mL.HPTLC-bioautographyon
S. aureus (Figure 2) exhibited two different areascorresponding to the fractions R 0.45–R 0.6 and
f f
R 0.7. TheR 0.45–R 0.6TLCareaappearedasthemostbioactiveintermsofantibacterialactivity
f f f
anditwastotallycharacterizedbytrans-nerolidol(100%). ThefractionR 0.7wasmainlycomposed
f
byβ-cedren-9-one(87.34%). Dataobtainedsuggestedthattrans-nerolidolismainlyresponsiblefor
theantibacterialactivityofM.splendensessentialoilthatisinaccordancewiththeresultsobtainedby
variousauthors. Simoesetal.[26]foundthattrans-nerolidolincombinationwithsyntheticantibiotics
ciprofloxacin, erythromycin, and gentamicin enhances their antibacterial activity against S. aureus
andE.coli. Essentialoilscontainingtrans-nerolidolhavebeenreportedtoinhibitthegrowthofquite
Molecules 2017, 22, 1163 5 of 12
chemopreventive activity. In the present study, trans-nerolidol exhibited a cytotoxic activity less
interesting than α-bisabolol, nevertheless on A549 cells showed IC50 value at 48h (54.28 ± 2.39 μg/mL)
in line with the one recorded by Sylvestre et al. [24] (66 ± 12 μg/mL). Antineoplastic activity was also
recorded on bowel by a mixture of cis- and trans-nerolidol [25], suggesting that deeper studies could
be carried out in order to assess cytotoxic activity against other kinds of cancer cell lines.
2.3. Antibacterial Activity: HPTLC-Bioautography and MIC
Antibacterial properties of M. splendens essential oil was assessed against Gram positive and
negative bacteria and MIC (Minimum Inhibitory Concentration) was determined by serial
microdilution method. M. splendens essential oil exhibited a weak antimicrobial activity compared to
chloramphenicol, used as a positive control, against all tested bacteria (Table 3).
Table 3. Antibacterial activity of M. splendens essential oil expressed as MIC (Minimum Inhibitory
Concentration) (μg/mL).
Strain M. splendens EO 1 Antibiotic 2
Gram negative
Agrobacterium tumefaciens DSM 30207 500 3c 6.25 3c
Agrobacterium vitis DSM 6583 2000 3e 1.56 3a
Pseudomonas syringae pv. syringae DSM 10604 250 3b 3.12 3b
Escherichia coli ATCC 4350 >2000 3f 25.00 3d
Pseudomonas aeruginosa ATCC 27853 >2000 3f 6.25 3c
Gram positive
Clavibacter michiganensis subsp. nebraskensis DSM 20400 125 3a 6.25 3c
Enterococcus faecalis ATCC 29212 2000 3e 3.12 3b
Molecules2017,22,1163 6of12
Listeria grayi DSM 20601 1000 3d 5.00 3c
Staphylococcus aureus ATCC 29230 1000 3d 3.12 3b
anumberofbacteSrtiaaphinylcolcuodcciunsg eSpi.daeurmreiudsis[ 27–29],S.eApTidCeCrm 14id99is0 [30],E.1c0o0l0i, 3da ndP.aeru3g.1in2 o3sb a[28,29].
Nerolido1 lE(Oa: mEsisxetnutriael oOfilc;i 2s -Cahnlodratmrapnhse-nniecrool lwidaos lu)siesda alsso poussietidvea csoantfroool;d 3 Mflaevanosr iinn geaacghe cnotlu[2m7n] .foClloonwseidd ering
β-cedrenby-9 d-oifnfeere,ntht leetmterasi narceo smignpiofincaenntltyo dfifRfer0en.7t (fpr a<c 0t.i0o5n)., fewliteraturereferencesarereported: tothe
f
bestofoHuPrTkLnCo-wbiloedaugteo,gtrhaipshsye swquasi tpereprfeonrmeiesdt htoe fminadi nthceo pmopssoiublned anotfibAarctteemriiasli afsrapcetrisoinca orfo eostseenstsieanl toiiall
oil[(3F1ig],ubrue t2n).o dataareavailableforitsantibacterialproperties.

FiguFrigeu2r.eH 2P. HTLPCTL(CH i(gHhigPhe rPfeorrfmoramnacnecTe hTihninL aLyaeyreCr Chrhormomataotoggrarapphhyy))b bioioaauuttooggrraapphhiicc aassssaayy ppeerrffoorrmmeedd
forafonrt iabnatcibtearcitaelraiaclt iavcittiyviotyn oSn. aSu. raeuurse,uos,f oMf .Msp. slepnlednednesness essesnetniatilaol iol.ilR. R:f:r reetteennttiioonn ffaaccttoorr:: iitt rreepprreesseennttss tthhee
f
ratiorabtieot wbeetewneethne thmei mgriagtriaotniodni sdtiasntacnecoe foaf asu sbusbtsatnancecea nanddt htheem migigrraattiioonn ddiissttaannccee ooff tthhee ssoollvveenntt ffrroonnt.t .
R cRofm copmospitoisointioisn ries proeprtoerdteidn iTna Tbalebl1e. 1(.A (A)H) HPTPLTCLCd dereirvivaatitzizeeddw wiitthh vvaanniilllliinn--pphhoosspphhoorriicc aacciidd rreeaaggeennt.t .
f
(B) HPTLC performed for antibacterial activity on S. aureus.
(B)HPTLCperformedforantibacterialactivityonS.aureus.

2.4. AntioxidantActivity
M.splendensessentialoilexhibitedantioxidantactivityathigherconcentrationifcomparedto
positivecontrol. Infact,M.splendensessentialoilshowedaDPPHscavengingactivity(IC =43,537±
50
15µg/mL)whichismuchlowerthanvitaminE(IC =7.8±0.5µg/mL)(Table4).
50
Table4.AntioxidantactivityofM.splendensessentialoilcomparedwiththestandard(vitaminE).
Sample IC (µg/mL)1
50
M.splendensEO 43,537.00±153b
vitaminE2 7.8±0.53a
1IC50:DPPHscavengingactivityofEOwasexpressedasIC50(µg/mL)value;2VitaminEwasusedaspositive
control;3Dataarepresentedasmean±SD,n=3.Meansineachcolumnfollowedbydifferentletteraresignificantly
different(p<0.05).
TheDPPH-HPTLC(1,1-diphenyl-2-picrylhydrazil—highperformancethinlayerchromatography)
bioautographicassay(Figure3)indicatedthatβ-caryophyllene(36.23%),trans-γ-bisabolene(10.04%),
cis-γ-bisabolene(8.33%),andtrans-β-farnesene(7.81%)werethemajorcompoundsresponsibleforthe
lowerscavengingactivityatR 0.9(Table1).
f
Thesesesquiterpenesarepresentatlowamountintheessentialoilandthatmaybethereasonfor
thelowantioxidantactivity. Inaddition,otherexplanationcouldbethatantioxidantactivityisreduced
bysomecompoundswhichexhibitsoxidantactivity.Thishypothesisseemstobeconfirmedbyprevious
researchrelatedtotheevaluationofcytotoxicactivityofPipergaudichaudianumKunthessentialoil,
richintrans-nerolidol,againstSaccharomycescerevisiae.Thecytotoxiceffectincreasedaccordingtothe
absenceofthesuperoxidedismutase(SOD),indicatingthatessentialoilandtrans-nerolidoldeveloped
reactiveoxygenspecies(ROS).ROSproductionwasconfirmedby2(cid:48),7(cid:48)-dichlorofluoresceindictate
Molecules2017,22,1163 7of12
(DCF-DA)test,onstrainsthatdonotproducetheSODenzyme. CytotoxicityofP.gaudichaudianum
essentialoilandtrans-nerolidoldependsonROSandDNAsinglestrandbreaksgeneratedbypresence
ofoxidativelesions[32]. Withthesepremises,thehighconcentration(67.81%)oftrans-nerolidolfound
inEcuadorianM.splendensessentialoilcouldexplaintheweakantioxidantactivityintheDPPHassay.
Molecules 2017, 22, 1163 7 of 12

FFigiguurere3 .3. HHPPTTLLCC bbiiooaauuttooggrraapphhiicc aassssaayy ppeerrffoorrmmeedd ffoorr DDPPPPHH aacctitvivitiyty oof fMM. .spslpelnednednesn sesessesnetniatila oliol.i lR.f
composition is reported in Table 1. (A) HPTLC derivatized with vanillin-phosphoric acid reagent;
R compositionisreportedinTable1.(A)HPTLCderivatizedwithvanillin-phosphoricacidreagent;
f
(B) HPTLC derivatized with solution of DPPH radical.
(B)HPTLCderivatizedwithsolutionofDPPHradical.
3. Materials and Methods
3. MaterialsandMethods
3.1. Plant Material
3.1. PlantMaterial
The leaves of M. splendens were collected at the CIPCA (Centre for Research, Postgraduate and
TheleavesofM.splendenswerecollectedattheCIPCA(CentreforResearch,Postgraduateand
CCoonnsesrevrvataitoionno offt htheeA Ammazaoznon))o fotfh teheU UninvievresrisdidadadE sEtsattaatlaAl Ammazaóznóinciaca(U (UEAEA)()0 (10◦11°41(cid:48)41′31”3″S S,,0 07777◦5°533(cid:48)2′255”″W W,,
570 m) in April 2013, from a wild population in the Amazonian region of Napo, Ecuador. Species
570m)inApril2013, fromawildpopulationintheAmazonianregionofNapo, Ecuador. Species
authentication was performed by Dr. David Neill. Voucher specimens were deposited at the
authenticationwasperformedbyDr.DavidNeill.VoucherspecimensweredepositedattheHerbarium
Herbarium ECUAMZ of the UEA, in Ecuador (voucher specimen: David Neill 17351).
ECUAMZoftheUEA,inEcuador(voucherspecimen: DavidNeill17351).
33.2.2..I sIosolalatitoionno offE EsssesnentitailalO Oilil
TThheee sessesenntitaiallo oililw waasso obbtatainineeddf rforommf rfersehshl elaeavveessb byyh hyyddroroddisitsitlillalatitoionnf oforr2 2h hi nina as tsatianinlelsessss tseteelel
ddisitsitlillelerre eqquuipipppeeddw witihtha aC Clelvevenenggere-rt-ytyppeea appppaararatutus.s.E Esssseenntitaiallo oilily yieieldld( 0(0.1.111%%))w waassc acalcluculalateteddo onna a
mmooisitsuturer-ef-rfereeeb basaissisa nanddd deteetremrmininededa sasa vavereargageeo offt hthrereeed disitsitninctctd disitsitlillaltaitoionns.s.T Thheeo oililw waassd drireidedu usisningg
aannhhyyddrorouusss osoddiuiumms usulflaftaetea nandds tsotroerdedin ins esaelaeldeda mambebrerv ivailaslsa ta4t 4◦ C°C,f, ofrorf ufurtrhtehrera naanlaylsyissi.s.
33.3.3..C Chhememiciaclasls
AAllllc hchrorommaatotoggrarapphhicicg graraddeeo orgrgaannicics osolvlveenntsts,,a allllr erefefererennceces tsatannddaardrdssa annddr eraeaggeenntstsu useseddf oforrG GCC
aannaalylysissisa annddf oforrb bioiolologgiciacalla catcitvivitiiteiessw wereerep puurcrhchasaesdedf rformomS iSgimgma-aA-Aldldrircihch( M(Milialnan,,I tIatlayly),),m micircorobbiaiall
cucultluturerem medediaiaf rforommO Oxoxiodid( M(Milialnan,I, tIatlayl)y.).
3.4. GCandGC-MSAnalysisoftheEssentialOil
3.3. GC and GC-MS Analysis of the Essential Oil
Theessentialoilwaschemicallyanalyzedandtherelativepeakareasforindividualcompounds
The essential oil was chemically analyzed and the relative peak areas for individual compounds
wereaveraged. FortheanalysisaThermoQuestGC-Tracegas-chromatograph(ThermoQuestItalia,
were averaged. For the analysis a ThermoQuest GC-Trace gas-chromatograph (ThermoQuest Italia,
Rodano,Italy)equippedwithanFIDdetectorandaVarianFactorFourVF-5mspoly-5%phenyl-95%-
Rodano, Italy) equipped with an FID detector and a Varian FactorFour VF-5ms poly-5% phenyl-95%-
dimethylsiloxanecolumn(internaldiameter,0.25mm; length,30m; filmthickness,0.15µm)were
dimethylsiloxane column (internal diameter, 0.25 mm; length, 30 m; film thickness, 0.15 μm) were
used. Thefollowingconditionswereadopted: injectortemperature300◦C,FIDtemperature300◦C,
used. The following conditions were adopted: injector temperature 300 °C, FID temperature 300 °C,
carrier(Helium)flowrate1mL/minandsplitratio1:50. Oventemperaturewasasfollow: from55
carrier (Helium) flow rate 1 mL/min and split ratio 1:50. Oven temperature was as follow: from 55 to
100 °C at a rate of 1 °C/min, from 100 to 250 °C at a rate of 5 °C/min and then kept constant at 250 °C
for 15 min. The volume injected was 1 μL, previously dissolved in CH2Cl2. The oil percentage
composition was performed by the normalization method from the GC peak areas, without using
correction factors. The chemical characterization of essential oil compounds was performed by a
Varian GC-3800 gas chromatograph (Palo Alto, CA, USA) equipped with a Varian MS-4000 mass
spectrometer (Palo Alto, CA, USA) using electron impact and hooked to the NIST (National Institute
of Standards and Technology) library. The conditions and column were the same described for GC
Molecules2017,22,1163 8of12
to100◦Catarateof1◦C/min,from100to250◦Catarateof5◦C/minandthenkeptconstantat
250◦Cfor15min. Thevolumeinjectedwas1µL,previouslydissolvedinCH Cl . Theoilpercentage
2 2
compositionwasperformedbythenormalizationmethodfromtheGCpeakareas, withoutusing
correctionfactors.ThechemicalcharacterizationofessentialoilcompoundswasperformedbyaVarian
GC-3800gaschromatograph(PaloAlto,CA,USA)equippedwithaVarianMS-4000massspectrometer
(PaloAlto,CA,USA)usingelectronimpactandhookedtotheNIST(NationalInstituteofStandardsand
Technology)library. TheconditionsandcolumnwerethesamedescribedforGCanalysis. Themass
spectroscopyconditionswereasfollows: ionizationvoltage,70eV;emissioncurrent,10µAmp;scan
rate, 1scan/s; massrange, 29–400Da; traptemperature, 150◦C,transferlinetemperature, 300◦C.
Theessentialoilcomponentswerecharacterizedbycomparingtheirrelativeretentiontime(AI)and
theMSfragmentationpatternwiththoseofotherknownessentialoils,withpurecompoundsand
bymatchingtheMSfragmentationspatternsandretentionindiceswiththeabovementionedmass
spectralibraries,andwiththoseintheliterature[11]. TheArithmeticIndexofthecomponentswas
determined adding a C8–C32 n-alkanes (Sigma-Aldrich) to the essential oil before injecting in the
GC-MSequipmentandanalyzedunderthesameconditionsreportedabove[3].
3.5. InVitroCytotoxicActivity
ThecytotoxicactivitywasperformedonA549(humanlungcancercellline),MCF-7(humanbreast
cancercellline),andHaCaT(humankeratinocytes)purchasedat“IstitutoZooprofilatticoSperimentale
dellaLombardiaedell’Emilia-Romagna”(Brescia,Italy)anditwasexpressedastheconcentration
ofsamplethatinhibited50%ofcellgrowth(IC ). ThecelllinesweregrowninDulbecco’smodified
50
Eagle’smediumsupplementedwith10%fetalbovineserum(FBS),2mML-glutamineand100U/mL
penicillin/streptomycin. Theyweregrownin75cm2 flasksinanairatmospherecharacterizedby5%
humidityand95%CO at37◦C,until80%confluencewasreached. Cytotoxicactivitywasdetermined
2
byMTTcolorimetricassay[33]asreflectedbytheactivityofsuccinatedehydrogenase. Cellswere
seededin96-wellplatesatadensityof2 × 104 cells/wellin200µLDMEM(Dulbecco’sModified
Eagle Medium) complete medium; a period of 24 h was given for ensuring the cell attachment.
Then the culture medium was replaced with 200 µL medium containing different concentrations
(from1to200µg/mL)ofM.splendensessentialoil. Inordertounderstandtheroleofα-bisabololand
trans-nerolidolinthecytotoxicactivity,commerciallyavailablestandardsofthosecompoundswere
testedfollowingthesamemethodology. Negativecontrolwasexposedtovehicleonly,corresponding
toamediumcontaining2%FBS.Thepositivecontrolusedwasdoxorubicin. After48h,theculture
mediumwasremovedandwashedwithPBS(phosphate-bufferedsaline)twice. Alatherof20µL
ofMTT(5mg/mLinPBS)wasaddedineachwellandtheplateswereincubatedfor4hat37 ◦C.
Themediumwasremovedandreplacedwith100µLofdimethylsulfoxidetodissolvetheformazan
crystals. The extent of MTT reduction was measured spectrophotometrically at 570 nm using a
microplatereader.
3.6. AntibacterialActivity: HPTLCBioautographicAssay
Bioautographic assay on a high performance thin layer chromatography (HPTLC) plate and
the minimum inhibitory concentration (MIC) were performed in order to evaluate antibacterial
activityofM.splendensessentialoil[34].AntibacterialactivitywasassessedagainstGramnegative
Escherichia coli (ATCC 4350), Pseudomonas aeruginosa (ATCC 27853), Agrobacterium tumefaciens
(DSM 30207), Agrobacterium vitis (DSM 6583), Pseudomonas syringae pv. syringae (DSM 10604) and
GrampositiveListeriagrayi(DSM20601),Staphylococcusaureus(ATCC29230),Staphylococcusepidermidis
(ATCC14990), Enterococcusfaecalis(ATCC29212), andClavibactermichiganensissubsp. nebraskensis
(DSM20400).
TheHPTLCbioautographywascarriedoutonhumanpathogenS.aureus,withtheaimofdefining
particularconstituentsofM.splendensessentialoilthatareabletodeterminetheantibacterialactivity.
ThebacteriumchoicewasledbecauseofitshighperformancefeaturesonHPTLCplateassay.Theassay
Molecules2017,22,1163 9of12
wasperformedasfollows:30µLofasolutionofM.splendensessentialoil(110mg/mLinethanol)were
appliedontwoseparateHPTLCplatesas10mmwidebandswithLinomatV(Camag),while10µLof
amethanolsolutionoftrans-nerolidol(25mg/mL)and10µLofamethanolsolutionofα-bisabolol
(8mg/mL)wereappliedonathirdoneonthesamespot,asa10mmwideband. Then,allplates
were eluted with a solvent solution containing toluene/ethyl acetate/petroleum ether (93/7/20).
Finally, the solvent was completely dried maintaining plates for 30 min at room temperature: the
firstonewasdisposedinPetridishestogetherwithNutrientAgarmediumpreviouslyaddedwith
thebacterialinoculum(105 CFU/50mL)and0.25%ofa2,3,5-triphenyl-tetrazoliumchloridewater
solution(20mg/mL),asagrowthindicator. Incubationoccurredovernightat37◦C.Antibacterial
compounds,responsibleforpossibleantibacterialproperties,lookedlikeyellowspotsagainstared
coloredbackground. Thesecondplatewasusedtoremoveactivespotsonsilicaandtoextractthese
inmethanolsolutionsthatwasdirectlyanalyzedbyGC-MS,whilethethirdonewassprayedwith
vanillin-phosphoricacidreagenttovisualizetrans-nerolidolandα-bisabololasreferencestandards[35].
3.7. DeterminationofMinimumInhibitoryConcentration(MIC)
TheantibacterialactivitywasdeterminedasMICandanalyzedbythemicrodilutionmethod
using96-wellmicrotiterplates[36].Bacterialcultureswereincubatedovernightat37◦C,inTrypticSoy
Broth,whileGramnegativeA.tumefaciensandA.vitisinNutrientBroth. Onehundredmicrolitersof
sterilemediumtogetherwith100µLofserialdilutionsofM.splendensessentialoilpreviouslydissolved
in ethanol (100 mg/mL), were pipetted into all the micro-wells. Serial dilutions were prepared in
ordertoobtainconcentrationrangesfrom27µg/mLto2000µg/mL.OnehundredµLofbacterial
culturestandardizedto2×107CFU/mLwasaddedtothewellsandincubatedat37◦Cfor16hand
at26◦Cfor24h,forhumanandphytopathogens,respectively. Aftertheincubationperiod,40µLof
watersolution(20mg/mL)of2,3,5-triphenyl-tetrazoliumchloridewasaddedtoeachwellandthen
incubated. Microbialgrowthwascheckedbyamicroplatereaderat615nm,after30minofincubation.
Chloramphenicolwasusedasapositivecontrol[34].
3.8. AntioxidantProperties
Antioxidantactivitywasmeasuredthrough1,1-diphenyl-2-picrylhydrazil(DPPH)spectrophotometric
assay(usingUV-Visspectroscopy)andDPPH-HPTLCbioautographicassay[34].
3.8.1. SpectrophotometricDPPHAssay
Ten microliters of M. splendens essential oil was dissolved into 900 µL of ethanol, then serial
dilutionswereassessedinordertoobtaindifferentconcentrations(0.8–6.67×10−4µL/mL).Analiquot
(2.9 mL) of the ethanol solution of DPPH (4 mg/100 mL) was added to the essential oil solution.
Aftera30minincubation,inanorbitalshakerat200rpm,inthedarkatroomtemperature,themixture
wasplacedinanUV-Visspectrophotometerandtheabsorbancewasreadintriplicateagainstablank
at517nm. TheDPPHinhibitioninpercentwasdeterminedbythefollowingformula: IDPPH%=
[1 − (A1/A2)] × 100. Where A1was theDPPHabsorbance withthe essentialoiland A2without
the essential oil. Vitamin E was used as positive control, according to its well-known antioxidant
properties[37]. EssentialoilantioxidantactivitywasexpressedasIC (concentrationprovidingDPPH
50
50%inhibition),calculatedfrominhibitioncurvesobtainedbyplottinginhibitionpercentageagainst
essentialoilconcentration. Allexperimentswereassessedintriplicateandvalueswerereportedas
mean±SD(StandardDeviation).
3.8.2. DPPH-HPTLCBioautography
DPPH-HPTLC-bioautographywasassessedtodeterminetheactivecompoundsoftheessential
oil, responsible for possible radical scavenging activity. Twelve microliters of an ethanol solution
of M. splendens essential oil (110 mg/mL) were applied twice to a silica gel HPTLC plate as
10mmwidebandswithLinomatV.Then,spotswereelutedwithasolventsolution(toluene/ethyl
Molecules2017,22,1163 10of12
acetate/petroleum ether 93/7/20) in a chromatographic chamber. After development, the first
chromatogram was sprayed with the DPPH ethanol solution (20 mg/100 mL) to detect possible
antioxidant fractions. The active compounds appeared as yellow areas on a violet background.
IsolationandidentificationofantioxidantcompoundswerecarriedoutremovingtheTLCareasinthe
secondchromatogramatR correspondingtopositivespotsandthenextractingthemwithmethanol.
f
ThesolutionswereanalyzedbyGC-MS.ThethirdplateobtainedfromHPTLCbioautographicassay
wasusedtoshowtrans-nerolidolandα-bisabololasreferencestandards.
3.9. StatisticalAnalysis
Theexperimentswereperformedintriplicate. IC valuesofanti-proliferativeandantioxidant
50
activitieswereassessedbylogarithmicregressioncurveswith95%confidentlimits. Relativestandard
deviations and statistical significance (Student’s t test; p ≤ 0.05) were calculated using software
STATISTICA6.0(StatSoftItaliasrl,Vigonza,Italy).
4. Conclusions
ThepresentstudycharacterizedthechemicalprofileofM.splendensessentialoilfromEcuadorfor
thefirsttimeandinvestigateditscytotoxic,antibacterial,andantioxidantproperties. Theessentialoil
ofthisplantrevealedagoodpotentialagainsttheMCF-7celllineandsuggestedthatfurtherdeeper
investigationsofinvitroandinvivoanti-cancerstudiesareneeded. M.splendensessentialoilshowed
negligibleantioxidantactivityandlowantibacterialeffectsagainstGrampositiveandnegativehuman
pathogens,andmoderateactivityagainstphytopathogenstrains. Inaddition,itwasfoundthatthe
chemicalcharacterizationofM.splendensessentialoilfromAmazonianEcuadordiffersqualitatively
andquantitativelyfromessentialoilsobtainedbyplantsobtainedofthesamespeciesbutcoming
fromdifferentregions. Moreover,M.splendensessentialoil,asasourceoftrans-nerolidol,shouldhave
pharmacologicalapplicationsbecauseofitsenhancingpropertiesonimprovingskinabsorptionof
adrugwithlowlipophilicfeatures[38].
Acknowledgments: This research has been supported by an internal grant from the Universidad Estatal
Amazónica,Ecuadorandgrant(AcuerdoEspecificodeCooperacionUPS-UNIFE,2015)ofUniversidadPolitecnica
Salesiana,Ecuador.TheauthorsaregratefultoImmacolataMarescaforhercontributiontopreparefiguresforthe
firstrevisionofmanuscript.
AuthorContributions:Thecontributionsofauthorsareasfollow:L.S.,G.S.andA.Gu.conceivedanddesigned
theexperimentsandanalyzedthedata;L.S.collectedtheplantmaterialandperformedhydrodistillation;A.Gr.
andJ.B.performedtheantimicrobialexperimentsandanalyzedthedata;A.S.andM.T.carriedoutthecytotoxic
andantioxidantassays,andanalyzedthedata;D.N.collectedtheplantmaterialandidentifiedtheplant;L.S.and
A.Gu.wrotethepaper.Allauthorsreadandapprovedthemanuscript.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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