Synergistic Antifungal, Allelopatic and Anti-Proliferative Potential of Salvia officinalis L., and

15 Pages·2017·1.48 MB·English

molecules
Article
Synergistic Antifungal, Allelopatic and
Anti-Proliferative Potential of Salvia officinalis L.,
and Thymus vulgaris L. Essential Oils
ErsiliaAlexa1,RenataMariaSumalan2,* ID,CorinaDanciu3,DianaObistioiu4,
MonicaNegrea1 ID,Mariana-AtenaPoiana1,CristianRus1,IsidoraRadulov5,GeorgetaPop5
andCristinaDehelean3
1 FacultyofFoodProcessingTechology,Banat’sUniversityofAgriculturalSciencesandVeterinaryMedicine
“KingMichaelIofRomania”fromTimisoara,CaleaAradului,No.119,Timisoara300645,Romania;
[email protected](E.A.);[email protected](M.N.);[email protected](M.-A.P.);
[email protected](C.R.)
2 FacultyofHorticultureandForestry,Banat’sUniversityofAgriculturalSciencesandVeterinaryMedicine
“KingMichaelIofRomania”fromTimisoara,CaleaAradului,No.119,Timisoara300645,Romania
3 FacultyofPharmacy,“VictorBabes“UniversityofMedicineandPharmacyTimisoara,
EftimieMurguSquare,No.2,Timisoara300041,Romania;[email protected](C.D.);
[email protected](C.D.)
4 InterdisciplinaryResearchPlatform,Banat’sUniversityofAgriculturalSciencesandVeterinaryMedicine
“KingMichaelIofRomania”fromTimisoara,CaleaAradului,No.119,Timisoara300645,Romania;
[email protected]
5 FacultyofAgriculture,Banat’sUniversityofAgriculturalSciencesandVeterinaryMedicine“KingMichaelI
ofRomania”fromTimisoara,CaleaAradului,No.119,Timisoara300645,Romania;
[email protected](I.R.);[email protected](G.P.)
* Correspondence:[email protected];Tel.:[phone]
Received:26November2017;Accepted:13January2018;Published:16January2018
Abstract: The current study aimed to investigate the chemical composition and the synergistic
potential of two essential oils (EOs), as obtained from Salvia officinalis L. (SEO), and Thymus
vulgarisL.(TEO).TheantifungalpotentialwastestedinvitroagainstFusariumgraminearum(Fg06_17),
theherbicidaleffectwasstudiedusingweedseedsofAmaranthusretroflexus(ARET),Chenopodium
album(CALB),Echinochloacrus-galli(EGAL),butalsowheatseeds(WS)oftheLovrinvarietyand
tomatoseedsSaint-Pierreofthevariety. TheGC-MSprofilehighlightsthatthemainscompounds
identifiedinSEOwere: caryophyllene(25.364%),camphene(14.139%),eucalyptol(13.902%),and
β-pinene(11.230%), whileinTEO,thepredominantphytochemicalswere: γ-terpinene(68.415%)
andp-thymol(24.721%). TheresultsindicatedthatthetestedEOsaloneaswellasincombination
haveallelopathiceffectagainstinvestigatedseeds,whilethesynergisticeffectofTEOandSEOin
termsoffungalgrowthwasdemonstratedatalevelof0.06%. ThymeandsageEOsexhibitedinvitro
anti-proliferativeactivityontwomelanomacelllines,namelyA375humanmelanomaandB164A5
mousemelanomaalone,aswellasincombination. SEOwasmosteffectiveintermsofdecreasingthe
cellviabilityofmurineandhumanmelanomacelllineswhencomparedtoTEO.
Keywords: Salviaofficinalis;Thymusvulgaris;Fusariumgraminearum;A375humanmelanoma;B164A5
mousemelanoma
1. Introduction
Sage (Salvia officinalis L.) and thyme (Thymus vulgaris L.) are medicinal aromatic plants of the
LamiaceaefamilygrownonalargescaleinsouthernandeasternEurope. Theimportancegiventothe
Molecules2018,23,185;doi:10.3390/molecules23010185 www.mdpi.com/journal/molecules
Molecules2018,23,185 2of15
twospeciesliesinthemultifunctionalityoftheactiveprinciplesoftheseplants,foundinextractsand
volatileoils. Theseconsiderations,correlatedwiththecurrenttrendsinthereplacementofsynthesis
productswithnaturalactivecompounds,havegeneratedmultipleusesforbothplantsinmedicine,
food,agriculture,horticulture,andplantprotection. Firstofall,themedicinalplantsoftheLamiaceae
familyhavebeenrecognizedsinceancienttimesfortheirtherapeuticalproperties. Sageisusedasa
naturalremedyintreatingandcuringarterialhypertension,bowel,stomachandspinalcorddisorders,
respiratory tract inflammation, physical and mental fatigue, nervousness, skin ulceration, cough,
bronchitis,dentalabscess,andcellulitis[1]. Thymehasprovencalmingactiononcough,possesses
anthelminticandantisepticproperties[2]. Thepreviousstudieshavehighlightedtheantibacterial
effect of sage essential oil (SEO) and thyme essential oil (TEO), tested on multiple gram positive
andnegativebacteria[3–10]. Intherecentyears,aparticularattentionhasbeenpaidtotheinvitro
antitumorandantimutagenicpropertiesofSEOandTEO.TheantiproliferativeeffectofSalviaand
Thymusspeciesweretestedondifferenthumancancercelllines[11–18].
Along with the therapeutic properties, an important role is represented by the possibility of
usingSEOandTEOasviablealternativestosyntheticproductswithherbicidalorantifungalactivity.
TheantifungalpropertiesoftheanalysedEOsandtheirconstituentsagainstAspergillussp.,Fusariumsp.
orPenicilliumsp. havebeendemonstratedpreviouslybyseveralresearchers[19–26]. Biologicalactivity
ofEOsdependsontheirmainchemotypesthataredeterminedbythegenotypeandinfluencedby
environmentalandagronomicconditions[27]. Inthisregard,themaincompoundofTEO-thymol,
is responsible for inhibiting the fungal growth and mycotoxins synthesis. Various mechanisms of
actionforvolatileoilsonmicrobialcellshavebeenproposedovertime. Thus,thevolatileoilscan
affectboththeoutercoatingofthecellandthecytoplasm. Theirhydrophobiccharacterappearsto
beresponsiblefordisturbingthebacterialstructuresthatleadtotheincreasingofthecytoplasmic
membranepermeability[27].
Allelopathyisdefinedastheabilityofaplanttoproducebiomolecules, especiallysecondary
metabolitesthatmayaffectanotherplantbeneficially,orviceversa[28]. Thebiochemicalinteractions
betweenplantscanhavemanypracticalapplicationsinagriculture. Certainallelochimiccompounds
have been shown to be effective as herbicides. EOs of aromatic plants are explored to find out
possible herbicides, since they do not persist in soil or contaminate the ground water and cause
little or no mammalian toxicity [29]. The inhibition ability of EOs belonging to Lamiaceae family
(lavender and peppermint) against weed germination (Amarantus retroflexus L., Sinapis arvensis L.,
LoliumperenneL.)hasbeenpreviouslyreported. Theresultsrevealedthebio-herbicidalpotentialof
EOsandthepossibilitytousethemasnaturalherbicides[30].
Theantifungal,antibacterial,antioxidant,andantitumoralactivityexhibitedbyTEOandSEO
havebeendemonstrated,butthereareonlyfewdataconcerningthesynergismorantagonismofthese
EOsusedtogetherindifferentresearchareas. ItseemsthattheuseofmixedEOsisverybeneficialin
thefieldofmedicine[31]. Also,toourbestknowledge,therearelimitedstudiesonallelopathiceffects
ofSEOandTEO.
ThisstudyisintendedtobeaninnovativeapproachintermsofSEOandTEOapplicabilityas
naturalfunctionalproductsinagricultureandmedicine. Thus,thismultipurposeworkhastakeninto
considerationthefollowingissues: (i)GC/MScharacterizationofSEOandTEO;(ii)evaluationof
cytotoxicandantiproliferativeactivitiesofSEOandTEOagainstA375humanmelanomaandB164A5
mousemelanomacelllines;(iii)invitroassessmentofantifungaleffectofbothindividual(SEO,TEO)
andmixed(EOsM)againstFusariumgraminearum;and,(iv)investigationonallelopathicpotentialof
SEOandTEOinrelationtocropsandweedsseedsgermination.
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2. ResultsandDiscussions
2.1. ChemicalCompositionofSEOandTEO
InTables1and2arepresentedtheresultsregardingthechemicalcompositionofanalysedEOs
andtheirliniarretentionindices(LRI).
Table1.ChemicalcompositionofSalviaofficinalisessentialoils(SEO).
No.Crt. Compounds TypeofCompound LinearRetentionIndices(LRI) %ofTotal
1 α-pinene MH1 1013 4.816
2 β-pinene MH 1092 11.230
3 eucalyptol MO2 1198 13.902
4 o-cymene MH 1287 0.515
5 α-thujone MO 1416 8.871
6 camphor MO 1493 4.028
7 camphene MH 1498 14.139
8 D-carvone MO 1518 0.205
9 m-menthane MH 1573 0.853
10 p-thymol MO 1578 8.073
11 caryophyllene SH3 1581 25.364
12 trans-calamenene MH 1644 1.676
13 valencene MH 1713 5.525
14 terpinolene MH 1774 0.360
Total 97.624
MH 37.387
SH 25.364
MO 34.874
%ofSEOextraction 3.560
1MH-Monoterpenehydrocarbonates;2MO-Monoterpeneoxygenated;3SH-Sesquiterpenehydrocarbonates.
Table2.ChemicalcompositionofThymusvulgarisessentialoils(TEO).
No.crt. Compounds TypeofCompound LinearRetentionIndices(LRI) %ofTotal
1 γ-terpinene MH1 1250 68.415
2 p-thymol MO2 1578 24.721
3 Caryophyllene SH3 1581 5.508
4 Terpine-4-ol MO 1635 0.099
5 o-thymol MO 1745 0.237
6 p-cymene-7-ol MO 1824 0.242
Caryophyllene
7 SO4 2013 0.222
oxide
8 α-murolene SH 2120 0.555
Total 98.644
MH 68.415
SH 6.063
MO 25.299
SO 0.222
%ofTEOextraction 3.040
1MH-Monoterpenehydrocarbonates;2MO-Monoterpeneoxygenated;3SH-Sesquiterpenehydrocarbonates;4
SO-Sesquiterpeneoxygenated.
InSEO,therehavebeenidentified14majorcompounds,inaconcentrationover0.2%. Themain
compounds identified in this essential oil, at a level over 10%, were: caryophyllene (25.364%),
camphene(14.139%),eucalyptol(13.902%),and β-pinene(11.230%),representing64.635%fromall
analysedcomponents,Table1. Also,othercompoundsasthymol(8.073%),camphor(4.028%),and
valancene(5.525%)havebeenfoundinaproportionover4%. Theanalysedcompoundsaredividedin
monoterpenehydrocarbonates(37.387%),sesquiterpenehydrocarbonates(25.364%),andmonoterpene
oxigenated(34.874%).
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Ourpreviousstudyhasidentifiedthepresenceofcamphor(20.4%),eucalyptol(11.7%),camphene
(11.5%),α-pinene(9.5%)asmajorcompoundsoftheSEOthathasbeenisolatedfromsageoriginating
fromwesternRomania[26]. Camphorhasbeenreportedasthemaincompoundindifferentother
studies[31,32],whileotherauthorshaveidentifiedα-thujoneasmainchemicalcompound[32–34].
The major similarity between the studies around the world regarding the SEO composition is
representedbythefactthatα-thujonewasidentifiedasthemajorcompound,followedbycamphorin
apercentageof20–25%ofthetotalamountofchemicalcompounds.
InTEO,theγ-terpineneandthymolwereidentifiedasthemajorcompoundinproportionof68.415%
and 24.721% respectively. These results are in agreement with those previously published by other
authors,whoreportedthatthemajorcomponentofthymeessentialoilisthymol,amonoterpenephenol
derivativeofcymene[32–34].Rusetal.[35]havereportedthato-cymeneandγ-terpinenearethemain
chemotypesofTEO.Regardingthetypeofcompounds,monoterpenehydrocarbonates(MH)represent
68.415%,monoterpeneoxigenated(MO)25.299%,andsesquiterpenehydrocarbonates(SH)6.063%.
Thecontentofvolatilecompoundsandthedominantchemotypevarieswithbotanicalspecie,
geographicalorigin,andtheharvesttime. Therefore,thestudycarriedoutbyImelouaneetal.[36]
haverevealedcamphorasthemajorcompound(39.390%)ofThymusvulgarisL.essentialoilcultivated
inMorocco,whileHudaibetal.[33]haveidentifiedcarvacrol,asthemainchemotypeofthisessential
oil. InanotherstudyconductedbySchmidtetal.[32],linaloolhasbeenfoundasthemajorcompound
ofThymusvulgarisL.essentialoil(72.5%).
2.2. CytotoxicEffect
Amongthetwovolatileoils,SEOwasmoreactiveintermsofinhibitionofproliferationforthetwo
testedmelanomacelllines. Theinhibitoryactivitywaslinearwiththeconcentrationandquitesimilar
forA375humanmelanomaandB164A5mousemelanomacelllines. Thelowesttestedconcentration
ofSEO(50µg/mL)causedaninhibitionratioof34%forthemurinemelanomacelllineand39%for
thehumanmelanomacellline,whileatestedconcentrationof100µg/mLSEOleadtoaninhibition
ratioof50.5%forthemurinemelanomacelllineand47.5%forthehumanmelanomacellline. TEOata
testedconcentrationof50µg/mLinducedaninhibitionratioof19.5%forB164A5celllineand17.5%
forA375cellline,whileaconcentrationof100µg/mLresultedinaninhibitionratioof30%forB164A5
celllineand27%forA375cellline,Figure1. Regardingtheessentialoilsmixture(EOsM),theuseofa
concentrationof50µg/mLconductedtoapercentageofinhibitionof30%forB164A5celland26.5%
forA375cells. TheantiproliferativeeffectincreaseswiththeconcentrationofEOs.
Figure1.AntiproliferativeactivityofSalviaofficinalisessentialoil(SEO),Thymusvulgarisessentialoil
(TEO)andtheiressentialoilsmixture(EOsM)(a)forA375humanmelanomacellline;(b)forB164A5
mousemelanomacellline:*thevalueissignificantdifferentversuscontrolatp<0.05.
Molecules2018,23,185 5of15
InvitroantiproliferativeactivityagainstM14humanmelanomacellswasreportedfortheessential
oil obtained from other species of sage originating from Lebanon namely Salvia bracteata Banks &
Sol and Salvia rubifolia Boiss [11]. In a comprehensive study, Russo et al. [15] have analyzed the
chemicalcomposition,andthegrowth-inhibitoryandpro-apoptoticpropertiesagainstA375,M14,and
A2058humanmelanomacelllinesoftheSEOgrownineighteendifferentenvironmentalconditions.
Thespeciesshowedinadifferentmannerantiproliferativeandpro-apoptoticpropertiesontheselected
melanomacelllines,thus,demonstratingthattheenvironmentalandpedoclimaticconditionshave
a vital role on the chemical composition of volatile oil, and also, on the biological activity. α- and
β-thujoneisomerswhereassignedasresponsiblefortheanti-melanomaactivity,actingsynergicwith
theothercompoundspresentinthevolatileoil[15].
It has been proven that the EO of Salvia libanotica showed invivo chemoprevetive properties
against skin cancer, experimentally induced with 7.12 dimethylbenz[a]anthracene (DMBA) and
12-0-tetradecanoylphorbol-13-acetate(TPA)[37].
ThestudyconductedbyPriviteraetal.[38]investigatedtheantiproliferativeactivityofSEOata
levelof200µg·mL−1for72hofincubationontwohumanlungcancercelllines(A549andNCI-H226),
andsignificantlyinhibitionhasbeendetected[38].
Togetherwiththevolatileoilobtainedfromotheraromaticplants,theantiproliferativeactivity
of the volatile oil of Salvia triloba L. was studied for MCF7 human breast adenocarcinoma cells.
Interestingly,theethanolextractspresentedantiproliferativecapacitybuttheaqueousextractsand
thepurevolatileoil,not[13]. Inabroadstudy,analyzingtheeffectoftheSEO,Loizzoetal.[39],have
concludedthatthecytotoxiceffectswereremarkedforC32amelanoticmelanomacellline,withanIC50
of367µg/mLandforACHNrenaladenocarcinomahumancelllines,withanIC50of108µg·mL−1.
Ontheotherhand,noeffectswerereportedforLNCaPprostatecarcinomacelllinesandMCF-7human
breastcancercells. EvaluatingtheanticancereffectsofSalviaofficinalisL.volatileoilagainstUMSCC1,
thesquamoushumancellcarcinomacelllineoftheoralcavityhavedescribedadualeffect,including
bothstimulationandinhibition,dependingontheconcentration. IC50valuehasbeenreportedtobe
135µg·mL−1 [40]. Itanietal.[41]havenoticedthatthreemajorcomponentsofSalvialibanoticaEO,
namelylinalylacetate,terpeniol,andcamphorcausedgrowthinhibitionandapoptosisforhuman
coloncancerHCT116cellsbothp53+/+andp53−/−.
ThevolatileoilofthymusendemicspeciesfromAlgeriaandMoroccowasfoundtoshowcytotoxic
propertiesagainstA375humanmelanomacelllinewithanIC5046.95µg·mL−1 [42]. Arecentstudy
depictedtheantiproliferativeactivityagainstTHP-1humanmonocyticcelllineofthevolatileoilobtained
fromdifferentspeciesofthymusoriginatingfromPortugal[43]. Thefindingsweresupportedbythe
group of Tefiani et al. [44] that concluded that Thymus munbyanus volatile oil collected from Algeria
presentremarkableantiproliferativeeffectsagainstTHP-1humanacutemonocyticleukemiacellline.
Theessentialoilsoftwothymusspecies,namelyThymuslinearisBenths.,andThymusserpyllumL.
fromthePakistanflora,havebeenscreenedforantiproliferativeeffectsagainsttwohumancancer
(MCF-7-breastandLNCaP-prostatecarcinoma)andonefibroblast(NIH-3T3)celllines. Bothvolatile
oils showed antiproliferative potential on the above mentioned cell lines, but the effectiveness of
thevolatileoilobtainedfromThymuslinearisBenths. hasbeensignificantlyincreasedwithIC50of
80.7µg·mL−1,70.3µg·mL−1and100.2µg·mL−1[45]. Arecentstudyhasshownthecytotoxiceffectof
ThymuscaramanicusJalas,acommonspeciesinIran,againstKBhumanoralepidermoidcarcinoma[46].
2.3. AntifungalActivity
InTable3,theresultsregardingtheantifungalactivityofSEOandTEOareshownonFusarium
graminearummyceliumgrowtharea(MGA).Atacloserlook,itcanbenotedthatthehighestradial
grow increase of mycelium was recorded in sampletreated with SEOat levelof 0.06%, wherethe
MGAvaluewasabout15.9cm2. ByapplyingofSEOandTEOmixtureatalevelof0.06%(inthesame
proportion)wasobtainedonly0.1cm2 valueofMGA.Thisfindingrevealsthesynergisticeffectof
SEOandTEO.Similarly,thepreviousstudiesonthistopichaveshownthattheantifungaleffectis
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notonlycausedbyamajorcompoundasbythesynergyoftheothercompoundsfoundinsmaller
amounts[47,48].
Table3.AntifungalactivityofSEOandTEOonFusariumgraminearummyceliumgrowth.
Fusariumgraminearum
Variants
1RG(mm) 2MGA(cm2)
Control 46.8a±2.4 16.7a±1.8
0.06%SEO 45.8b±2.5 15.9b±1.7
0.12%SEO 32.8c±2.0 7.9c±1.0
0.20%SEO 20.7d±1.8 2.8d±0.6
0.06%TEO 8.0d±0 0.0e
0.12%TEO 8.0d±0 0.0e
0.2%TEO 8.0d±0 0.0e
0.06%EOsM 9.0d±0 0.1e±0
0.12%EOsM 8.0d±0 0.0e
0.2%EOsM 8.0d±0 0.0e
0.1%thiophanatemethyl 8.0d±0 0.0e
1 Radial Grow; 2 Mycelium Growth Area; Within a column the values with different superscript letters are
significantlydifferent(p<0.05).
WithrespecttotheantifungalpotentialofTEO,ourresultsrevealedatotalinhibitionofFusarium
myceliumgrowthatallinvestigatedconcentrations. Thus,themyceliaradialgrowth(RG)insample
with TEO was similar to thiophanate methyl (as negative control), the MGA values being null (in
Table3).
AlthoughbyapplyingtheEOsMat0.06%anincreaseofmyceliumwasrecorded,thedifferences
arenotstatisticallysignificant(p>0.05)versusnegativecontrol. Theantifungaleffectofsageoilhas
beenhighlightedonotherfungalspecies. Mahmoudietal.[49]haveinvestigatedtheeffectofSEOon
Alternariaalternata,andreportedthatalevelof5mg/Lwastheminimuminhibitoryconcentration
againstthefungusgrowth. ThesameeffecthasbeenshownforBotritiscinereaandFusariumsp.[50,51].
Additionally, it has been proved that the SEO showed antifungal activity against dermatophyte
strains[52].
The antifungal potential of TEO has been demonstrated also on other types of fungi. Thus,
the study performed by Tantaoui-Elaraki et al. [53] investigated the effect of TEO on Aspergillus
parasiticusinvitroconditionsandreportedthattheminimuminhibitoryconcentrationforthistype
of fungi was 0.1%. Also, it has been proved that TEO has shown a strong antifungal activity on
Verticilliumdahliae,Fusariumsp.,Penicilliumsp.,andAspergilliussp.[54]. AmongThymus,Rosmarinus,
andEucalyptus,essentialoilsithasbeenfoundthatTEOshowedthelowestinhibitoryconcentration
againstthefungalgrowth[55].
2.4. AllelopaticPotential
TheresultsregardingtheherbicidaleffectoftheinvestigatedEOscomparedtopositivecontrolas
syntheticherbicideDualGold(960gL−1s-metolachlor),(DG)arepresentedintheFigure2. TheDG
completelyinhibitstheweedsgermination,whilethetomatoseedsareinhibitedinpercentageof76.8%
andwheatseedsinproportionof47.2%.
OurresultshaveproventhatSEOshowedherbicidaleffectonAmaranthusretroflexusL.(ARET),
evenwhenitwasusedatlowconcentrations. Thus,whenSEOwasappliedatalevelof0.3%,the
inhibition of germination was 92.1%, while by increasing the level of SEO to 0.6% or 1.0%, total
inhibitionofgerminationofARETseedsoccurred. ControllingthegerminationofChenopodiumalbum
L.(CALB)weedsbyusingDualGoldis100%effectiveatrecommendeddoseof1Lperhectare. The
samevalueofgerminationinhibitionrate(GI)ofCALBweedisalsoachievedbysprayingtheseeds
withSEOatalevelof1.0%. TheherbicidalcapacityofSEOdecreaseswiththedose. Thus,byapplying
SEOatalevelof0.6%resultedof55.5%valueofGI,whileadoseof0.3%SEOledto22.2%valueGI.
Molecules2018,23,185 7of15
TheherbicidaleffectofSEOonEchinochloacrus-galliL.seeds(EGAL)hasreachedamaximum
valueofGIabout75%byapplyingSEOatalevelof0.6%and1.0%,whilealevelof0.3%SEOinduced
thelowestherbicidaleffectof69.1%.
ThestudyoftheallelopathiceffectofEOsonwheatandtomatoseedsshowsthattheSEO,applied
atalevelof0.6%resultedinaGIvalueof88.4%whilealevelof1.0%hastheabilitytoinhibitthe
germinationinapercentageof100%fortomatoseedsand80.5%forwheatseeds.
Using SEO al a level of 0.3% it was noted a decrease in the germination inhibition of tomato
seedsupto14.8%,respectivelyupto8.3%inwheatseeds,ascomparedtothesyntheticherbicidethat
showedabetterabilitytoinhibittheseedsgermination(76.8%fortomatoesand47.2%forwheat).
Thus,therearenosignificantdifferencesversusDGaspositivecontrolbyusingSEOasanherbicidal
agentforARET,CALBseeds(except0.6%SEOwhenthestatisticaldifferencesweresignificant,p<0.05)
andEGAL(except1.0%SEOwhenthestatisticaldifferencesweresignificant,p<0.05). SEOappliedat
alevelof1.0%inducedsignificantdifference(p<0.05)versuspositivecontrolinGIoftomatoseeds,
whilenosignificantdifferencesversuspositivecontrolwererecordedintheGIofwheatseeds.
By applying TEO at different doses, only for a level of 0.3% there were recorded significant
differences(p<0.05)intheGIofARET,EGAL,andtomatoesversusDGasapositivecontrol.
As regards the essential oils mixtures (EOsM), when was applying at a level of 0.3% in the
same proportion, significant differences were recorded in the GI of ARET versus positive control.
Also, the using of 0.6% EOsM induced significant differences in the GI value of EGAL versus the
positivecontrol.
Figure2.Cont.
Molecules2018,23,185 8of15
Figure2.Theeffectof(a)Salviaofficinalisessentialoil—SEO;(b)Thymusvulgarisessentialoil—TEO;
and (c) their mixtures (EOsM) on seeds germination rate (GI) of Amaranthus retroflexus (ARET),
Chenopodiumalbum(CALB),Echinochloacrus-galli(EGAL),tomato(TS),andwheat(WS).*thevalueis
significantdifferentversusthecontrolwithDualGold(DG)atp<0.05;ns—thevalueisnosignificant
versuscontrol.
AlloftheinvestigatedlevelsofEOsMinducednosignificantdifferencesinGIofwheatseeds
versus positive control, while significant differences were noted in the GI of tomato seeds versus
positivecontrol.
The ability of SEO at a level of 0.3% to inhibit the germination of weed seeds and to slightly
affectthegerminationofwheatandtomatoseedscouldrecommendtheuseofthisessentialoilin
plantprotection.
OurresultshaveproventhattheTEOshowsbetterherbicidalpotentialthanSEO.Thus,atTEO
appliedatalevelof0.6%and1.0%hascompletelyinhibitedthegerminationofCALBandEGALseeds
andinaproportionof60.6%inthecaseofARETseeds.
BydecreasingthelevelofTEOat0.3%donotrecordagreatinhibitionoftheseedsgermination
ofARET(6.6%)andCALB(10%). Unfortunately,TEOshowedaninhibitioneffectonorganictomato
andwheatseeds,atover80%,evenusedatlowdoses.
Thesynergisticeffectoftheinvestigatedoilsresultedinincreasingtheallelopathicpotentialof
theirmixtureandinatotalinhibitionofgerminationonalltypesofweeds,whenEOsMwasapplied
atalevelatleast0.6%. Itisworthnotingthattheuseofthemixturedoesnotinhibitthewheatseeds
germination,thusrecommendingtheblendingofthetwoEOsinequalproportion,asbio-effective
herbicidesinthecontrolofweedgraincrops.
Whencomparedtosynthetic herbicideDG,the investigatedEOsmixturesexertan increased
efficiencywithouthavinganegativeeffectonthewheatgermination. However,thesynergisticeffect
ofthetwoEOs,evenappliedatalowlevel,leadstoacompleteinhibitionoftomatoseedsgermination.
The inhibitory capacity of EOs applied both individually and as a mixture, can be strongly
influencedbytheirchemicalcompositionandalsobythegeneratedsynergism.Ithasbeenreportedthat
theterpenoids,particularlythesesquiterpene,evenatalowlevel,exhibitedspecificstructure-activity
relationship,whichcangeneratephytotoxicity[56].
Thepreviousstudieshavehighlightedthebio-herbicidaleffectsofEOsbelongingtoLamiaceae
family(oreganoandrosemary)onweedscontrolandgerminationgrowthofbreadwheatcultivars[31].
Theresultshaveshownthat2–4µLofEOsexhibitsaninhibitioneffectonSinapisarvensisL.,butnot
againstAvenasterilisL.seeds.Moreover,theseseedshavesurvivedatalldosesoforeganoandrosemary
EOs. Otherstudies[57]revealedtheinhibitionpotentialofpeppermint,cinnamon,andlavenderoils
Molecules2018,23,185 9of15
againstredrootpigweed,whileryegrassandwildmustardseedsgermination. Whencomparedwith
thecontrol,peppermintoilhasbeenthemoreeffectiveininhibitingryegrassseedgermination. Study
regardingtheeffectofeucalyptus,camphor,andlemongrassEOsonseedgerminationandseedling
growthofPartheniumhysterophorusshownthatahigherlevelthan12mL/Linducedatotalinhibition
ofweedgermination[56].
OurresultsrevealedsignificantdifferencesintheinhibitoryeffectoftheinvestigatedEOson
germinationcapacityofwheatandtomatoversusweedspecies. Thesefindingscouldbeassigned
to the different rates to metabolize certain monoterpenes [31]. Thus, the volatile compounds may
alsoberesponsiblefortheinhibitionofseedgermination[58]. Wheatandtomatoseedswereless
affectedthantheweedseedsspecies,whichhighlightsthattheEOs,appliedinaproperdosecouldbe
recommendedasabioherbicideforweedcontrol.
3. Materialsandmethods
3.1. PlantMaterial
AerialpartsofsageandthymewerecollectedinJulyandAugustof2015fromtheexperimental
field of Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of
Romania” from Timisoara at the full flowering stage. Voucher specimens were deposited in the
HerbariumofMedicinalPlantsDepartmentofAgricultureFaculty. Theplantsweredriedatambient
temperature and were ground with the Grindomix Retsch GM 2000 laboratory mill. Weed seeds
ofAmaranthusretroflexus(ARET),Chenopodiumalbum(CALB),Echinochloacrus-galli(EGAL)usedin
allelopathictestwereobtainedfromtheSpontaneousPlantSeedsCollectionoftheDepartmentof
HerbologyfromFacultyofAgricultureTimisoara. Tomatoseeds(TS),Saint-Pierrevariety,andwheat
seeds(WS)Lovrinvarietywerepurchasedfromlocalmarket.
3.2. ExtractionofEssentialOils
SEOandTEOwereobtainedfromdriedplantsbyhydrodistillationusingaClevengerequipment.
300gofplantmaterialwasintroducedintotheapparatuswith3000mLtapwater. Themixturewas
heatingtotheevaporationtemperatureoftheoil. Theextractiontimeisconsideredafterthecomplete
distillationoftheoilandtherecoveredofanother500mLofflavoredwater. TheobtainedEOswas
keptat2–4◦Cindarkconditionsuntiltheirusingforinvitroandinvivoexperiments.
3.3. GC-MSAnalysis
Agilent Technology 7820A (AGILENT Scientific, Santa Clara, CA, USA), coupled with mass
spectrometerMSD5975andequippedwithacapillarycolumnDBWAX(30m×250µm×0.25µm)
was used. The carrier gas was helium with a mass flow of 1 mL/min. In order to separate the
compounds,thefollowingGCovenprogramwasused:40◦Cfor1min,5◦Cmin−1to210◦Cfor5min.
Theinjectorandionsourcetemperatureswere250and150◦C,respectively. Theinjectionvolumewas
1µLofeachpureoilormixture,withoutsolventwithasplitratio1:20. Onerunforeachsamplewas
performed. TheNISTspectralibraryhasbeenusedtoidentifythevolatilecompounds. Identification
was made by comparison of their mass spectra with those stored in NIST 02, Wiley 275 libraries.
ThepercentagesofindividualcomponentswerecalculatedbasedonGCpeakareaswithoutusing
correctionfactors. Thelinearretentionindices(LRI)weredeterminedinrelationtoahomologous
seriesofn-alkanes(C8–C24)underthesameoperatingconditionsandcalculatedaccordingtoVanden
DoolandKratz[59]Formula(1):
100×(t −t )
LRI=100×n+ x x (1)
tn+1−tn
wheret andt aretheretentiontimesofthereferencen-alkanehydrocarbonselutingimmediately
n n+1
beforeandafterchemicalcompound“X”andt istheretentiontimeofcompound“X”.
x
Molecules2018,23,185 10of15
3.4. MTTProliferationAssay
The inhibition of proliferation for the screened extracts was assessed for A375 human
melanomaandB164A5mousemelanomacelllines. Theabove-mentionedcelllineswhereacquired
from Sigma-Aldrich Company, Ayrshire, UK. Cells were cultured in complete growth medium
containing Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL, Invitrogen, Carlsbad, CA,
USA), supplemented with 10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany), 1.0%
Penicillin/Streptomycin mixture (Pen/Strep, 10,000 IU·mL−1, FCS; PromoCell), and 2% HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Gibco BRL). Antiproliferative activity was
assessedbyMTTassay. Onthispurpose, thetwomelanomacelllineswereseededontoa96-well
cultureplateatacellulardensityof6000cellsperwellandwereattachedtothebottomofthewell
overnight. After 24 h, 100 µL of new medium containing Dulbecco’s Modified Eagle’s Medium
(DMEM;GibcoBRL,Invitrogen,Carlsbad,CA,USA)and50µg·mL−1respective100µg·mL−1ofthe
testedsamples(dissolvedindimethylsulfoxide—DMSO;Sigma-AldrichCompany)wereaddedand
incubatedfor72h. Themelanomacellswerethenassayedbytheadditionof10µLof5mg·mL−1
MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)solutionfromtheMTT-based
invitrotoxicologyassaykit(Tox-1; Sigma-AldrichCompany). Theintactmitochondrialreductase
convertedandprecipitatedMTTasbluecrystalsduringa4hcontactperiod. Theprecipitatedcrystals
weredissolvedin100µLoflysissolutionprovidedbythemanufacturer(Sigma-AldrichCompany).
Finally,thereducedMTTwasspectrophotometricallyanalyzedat570nm,usingamicroplatereader
Bio-RadMarkMicroplateSpectrophotometer).
3.5. AntifungalActivity
The invitro antifungal potential of SEO and TEO has been evaluated on CYGA medium
(cloraphenicol-yeast-glucose–agar) (Sigma Aldrich Chemie, Madrid, Spain) for mycelium radial
growth(RG)ofFusariumgraminearum(Fg06_17). ThefunguswasacquiredfromMicrobialCollection
ofMicrobiologyDepartment,FacultyofHorticulture,andForestry. Themethodusedwas“poisoned
food technique” which involves the introduction into the culture media of different amounts of
essentialoil[60]. Thus,inourstudy,SEOandTEOwereindividuallyappliedatthreeconcentrations
of0.06%,0.12%and0.2%. Toinvestigatethesynergisticeffectofthetwoinvestigatedoils,theessential
oilsmixture(EOsM)inequalproportion(v/v)hasbeenincludedalthesameconcentrationsof0.06%,
0.12%,and,respectively,0.2%. Also,anegativecontrol(C)withacommercialfungicidewasused
asthiophanatemethylatalevelof0.1%. TheFusariummyceliumasinoculumswasgetfromafour
daysyoungmyceliumobtainedfromsinglesporetechnique,[61]. Plugs,with8mmdiameterwere
harvestedfromedgeofmyceliumandtransferredonCYGAmediaaddedwithEOs. Threeplugs
for each variant were used as replicates. The Petri dishes at 24 ± 30 ◦C in reverse position and a
photoperiodof12h/daywereincubated. Inthe5thday,wasrecordedthemyceliumradialgrowvalue
(RG)expressedinmm,calculatedasarithmeticmeanofthetwoperpendiculardiameters,usinga
ruler. Themyceliumgrowtharea(MGA)wasestimatedusingtheformula:
RG2×3.14 −50.24
MGA(cm2) = 4 (2)
100
wherethevalueof50.24mm2representstheareaoffungalplug(inoculum),calculatedonthebaseof
fungalplugdiameter(8mm).
ForEOconcentrationswherenofungalgrowthwasrecorded,theconfirmationtestwasperformed.
ThisinvolvedthetransferoffungalplugsonfreshCYGAwithoutEOsaddition. Inthecasethatafter
fourdaystherewasnotrecordedanyfungalgrowth,theEOconcentrationfromwhichthetransferhas
beenperformedisconsideredtohaveafungistaticeffect. Contrary,ifitwasnoticedanyfungusrevival,
theEOconcentrationfromthemediumonwhichthetransferwasmadeisconsideredtobefungicidal.