Biological Activities and Chemical Composition of Santolina africana Jord. et Fourr. Aerial Part

15 Pages·2017·1.11 MB·English

molecules
Article
Biological Activities and Chemical Composition of
Santolina africana Jord. et Fourr. Aerial Part Essential
Oil from Algeria: Occurrence of
Polyacetylene Derivatives
CharafEddineWatheqMalti1,ClémentineBaccati2,MagaliMariani2,FaiçalHassani3,
BrahimBabali3,FewziaAtik-Bekkara1,MathieuPaoli2,JacquesMaury2,FélixTomi2,*
andChahrazedBekhechi1
1 LaboratoiredesProduitsNaturels,DépartementdeBiologie,UniversitéAbouBekrBelkaïd,
ImamaTlemcen13000,Algeria;[email protected](C.E.W.M.);[email protected](F.A.-B.);
[email protected](C.B.)
2 UniversitédeCorse-CNRS,UMR6134SPE,RoutedesSanguinaires,20000Ajaccio,France;
[email protected](C.B.);[email protected](M.M.);
[email protected](M.P.);[email protected](J.M.)
3 Laboratoired’EcologieetGestiondesEcosystèmesNaturels,Départementd’EcologieetEnvironnement,
UniversitéAbouBekrBelkaïd,ImamaTlemcen13000,Algeria;[email protected](F.H.);
[email protected](B.B)
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
AcademicEditor:FrancescaMancianti (cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:24November2018;Accepted:29December2018;Published:8January2019
Abstract: Thechemicalcompositionof18oilsamplesofSantolinaafricanaisolatedfromaerialpartsat
fullflowering,collectedinthreelocationsineasternAlgeriawasdeterminedbyGC(RI),GC/MSand
13C-NMRanalysis. Themajorcomponentswere: germacreneD,myrcene,spathulenol,α-bisabolol,
β-pinene, 1,8-cineole, cis-chrysanthenol, capillene, santolina alcohol, camphor, terpinen-4-ol and
lyratol. The chemical composition appeared homogeneous and characterized by the occurrence
of four derivatives which exhibited a conjugated alkene dialkyne moiety. They were identified
forthefirsttimeinanessentialoilfromS.africana. Thecollectiveoilsampleexhibitedmoderate
antimicrobial and antioxidant activities whereas the anti-inflammatory activity presented a real
potential. IC valueofSantolinaafricanaessentialoil(0.065±0.004mg/mL)is5-foldhigherthan
50
IC valueofNDGAusedaspositivecontrol.
50
Keywords: Santolina africana Jord. et Fourr.; Asteraceae; essential oil composition; 13C-NMR;
antimicrobialactivity;antioxidantactivity;anti-inflammatoryactivity
1. Introduction
The Santolina genus belongs to the Asteraceae family and is represented by more than
10 species widely distributed in Mediterranean area [1]. In all Santolina species, Santolina viridis
W.(SouthofFranceandNorthofSpain), S.pectinataLag. (=S.rosmarinifoliaL.)(IberianPeninsula)
andS.chamaecyparissusL.arethemostwidelyspreadspeciesaroundtheworld. S.africanaJord. et
Fourr. is synonym of Ormenis africana (Jord. et Fourr.) Litard. et Maire and S. chamaecyparissus L.
var. africanaB.etT. ItisanendemicspeciestotheNorthAfrica(Morocco,AlgeriaandTunisia)[2,3]
thatgrowsnaturallyinforestsandsteppepastures. Thisspeciesisabushy,greenorashysub-shrub.
Thestemsarewoody,withfloriferousbrancheserectintuft,bareandthickenedattheapex. Thelower
leavesarelinear-cylindricalwithshortandobtusesegments. Thebractsareovate-oblong. Theouter
corollasaretube-styledovary. Theflowerheadsarediscoidal,yellow,homogamous[2].
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Some members of Santolina genus have been known as medicinal plants for a long time.
S. africana is used in Moroccan folk medicine as a stomachic, abortive, anthelmintic, antidiabetic
andemmenagogue[4,5]. InTunisia, itistraditionallyusedforitshypoglycemiceffectandforthe
treatment of stomache pains [6]. Some biological activities have been reported for the extracts or
essential oils of S. africana, such as antioxidant activity [6–8], antimicrobial activity [7], accaricidal
activity[9]andantidiabeticactivity[8].
ThechemicalcompositionoftheessentialoilsofspeciesbelongingtothegenusSantolinahasbeen
widelystudied[10]. S.chamaecyparissusisprobablythemostinvestigatedspeciesofthisgenus[11–22].
ThecompositionofotherspeciessuchasS.corsica[23,24],S.insularis[25,26],S.rosmarinifolia[27–29]
wasalsoreported. Monoterpenessuchas1,8-cineole,camphor,artemisiaketoneandmyrcenewerethe
majorcomponentsofessentialoilsisolatedfromsomeSantolinaspeciesgrowingindifferentregionsof
theword.
Conversely, only five studies have reported on the chemical composition of S. africana oil.
Thechemicalcompositionofthevolatilecompoundsisolatedfromvariouspartsoftheplanthave
beensubstantiallyinvestigated. Fdiletal.[4]comparedthechemicalcompositionofthreeoilsamples
isolatedfromdifferentorgans(stems,leavesandflowers)ofS.africanaplantsharvestedinMarrakech
province (Morocco). The three samples oils exhibited respectively a composition dominated by
oxygenatedmonoterpenes: camphor(69.14%/71.36%/80.44%),borneol(20.33%/18.13%/12.34%)and
bornylacetate(7.08%/8.12%/3.50%). Thestemoilcontainedalsonoticeableamountsofα-humulene
(3.14%)whiletheflowersoilexhibitedanappreciablecontentof1,8-cineole(3.32%).Anotheraerialpart
oilsampleofMoroccanoriginexhibitedasimilarcomposition,withcamphor(54.3%),borneol(17.24%),
bornylacetate(8.61%)and1,8-cineole(5.27%)asmaincomponents[5]. ATunisianoilsample(stems
andleaves)wascharacterizedbyahighcontentofterpinen-4-ol(54.96%),followedbyα-terpineol
(14.06%)andborneol(8.37%)[9]. ConcerningAlgerianS.africana,onlytwostudiesarereportedin
theliterature. Anoilsample(flowers)harvestedinConstantineprovince(Algeria)wasdominatedby
acenaphtane(25.23%),calarene(21.54%)andocimene(17.44%)[7]. Adrasticallydifferentcomposition
hasbeenreportedforanaerialpartsoilsamplecollectedinthesameregion,β-eudesmol(14.58%)and
β-pinene(12.78%)beingthemajorcompounds,followedby1,8-cineole(10.02%),curcumene(7.96%),
myrcene(6.94%)andspathulenol(5.96%)[18].
Itappearsfromliteraturedatathattheessentialoil(EO)fromaerialpartsofS.africanaexhibitsa
tremendouschemicalvariability. Moreover,mostofthepapersreportedontheanalysesofonlyoneor
twooilsamplesandobviously,thereportedcompositionsarenotalwaysrepresentativeofS.africana.
Thus,theaimofthepresentworkistocharacterizetheEOproducedbythisplantgrowingwildinBatna
province(Algeria). Eighteenoilsamplesisolatedfromaerialpartsatfullfloweringstageharvestedin
threelocationshavebeenanalyzedbyGC,GC/MSand13C-NMR.Then,thebiologicalactivitiesofthe
EOhavebeeninvestigatedasantimicrobial,antioxidantandanti-inflammatoryactivities,thislatter
hasneverbeeninvestigatedinanypreviouspapers.
2. Results
AerialpartsofwildS.africanawerecollectedinMayinthreelocations(Figure1)(sixsamplesper
location). YieldsofEOisolatedbyhydrodistillation,calculatedw/wvs. drymaterialvarieddrastically
fromsampletosamplerangingfrom0.03to0.17%evenwithinalocation(TableS1). Asitcouldbe
seenfromTableS1,thehighestyieldswereobtainedfromHamla(0.08–0.14%,samplesH1–6)and
Bouilef(0.15%,sampleB5and0.17%,sampleB6)andthelowerfromBouilefandFesdis(0.03%for
samplesB2,B4andF6).
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2.1. ChemicalCompositionofEssentialOil
2.1. Chemical Composition of Essential Oil
AerialpartsoilssamplesweresubmittedtoGC-FIDanalysis,todeterminetheretentionindices
Aerial parts oils samples were submitted to GC-FID analysis, to determine the retention indices
(RIs)oftheEOcomponentsontwocolumnsofdifferentpolarityandtoGC/MSanalysis. Further
(RIs) of the EO components on two columns of different polarity and to GC/MS analysis. Further
analysisby13C-NMRconfirmedtheidentificationofthemaincomponents. Toallowtheidentification
analysis by 13C-NMR confirmed the identification of the main components. To allow the identification
offourpolyacetylenederivativespresentatmoderateorlowlevels,acompositesample(F1toF6)
of four polyacetylene derivatives present at moderate or low levels, a composite sample (F1 to F6)
was submitted to column chromatography (CC) over silica gel. Nine fractions were obtained and
was submitted to column chromatography (CC) over silica gel. Nine fractions were obtained and
analyzedbyGC-FID,GC/MSand13C-NMR.Intotal,91componentsaccountingfor92.4%and96.1%
analyzed by GC-FID, GC/MS and 13C-NMR. In total, 91 components accounting for 92.4% and 96.1%
of the whole oil chemical composition were identified (Table S1, Figure 2), including forty-three
of the whole oil chemical composition were identified (Table S1, Figure 2), including forty-three
monoterpenes,thirty-onesesquiterpenes,fivephenylpropanoids,sixpolyacetylenederivativesandsix
monoterpenes, thirty-one sesquiterpenes, five phenylpropanoids, six polyacetylene derivatives and
others. ThecompositionofS.africanaEOsisgenerallyhomogeneous;theoilswerefoundtopossess
six others. The composition of S. africana EOs is generally homogeneous; the oils were found to
littledifferencesinthechemicalcompositionbutconsiderablevariationinthelevelsoftheindividual
possess little differences in the chemical composition but considerable variation in the levels of the
components. Allthesampleswerecharacterizedbyhighproportionsofmonoterpenes(51.5–69.7%),
individual components. All the samples were characterized by high proportions of monoterpenes
exceptthreesamples: B4,B5andF6whichweredominatedbysesquiterpenes(44.3–55.9%). Themain
(51.5–69.7%), except three samples: B4, B5 and F6 which were dominated by sesquiterpenes
componentsweregermacreneD(0.1–25.3%),myrcene(4.2–20.9%),spathulenol(2.5–20.7%),α-bisabolol
(44.3–55.9%). The main components were germacrene D (0.1–25.3%), myrcene (4.2–20.9%),
(2.2–20.0%), β-pinene (2.4–18.7%), 1,8-cineole (5.0–16.8%), cis-chrysanthenol (0.7–16.5%), capillene
spathulenol (2.5–20.7%), α-bisabolol (2.2–20.0%), β-pinene (2.4–18.7%), 1,8-cineole (5.0–16.8%),
(0.1–16.9%),santolinaalcohol(0.2–14.0%),camphor(0.2–7.9%),terpinen-4-ol(1.8–7.3%)andlyratol
cis-chrysanthenol (0.7–16.5%), capillene (0.1–16.9%), santolina alcohol (0.2–14.0%), camphor (0.2–
(0.1–6.7%). Othertwooxygenatedmonoterpenes: lyratal(tr-2.7%)andchrysanthenone(tr-4.5%),three
7.9%), terpinen-4-ol (1.8–7.3%) and lyratol (0.1–6.7%). Other two oxygenated monoterpenes: lyratal
sesquiterpenehydrocarbons: α-curcumene(0.3–3.2%),γ-curcumene(0.1–2.6%)andbicyclogermacrene
(tr-2.7%) and chrysanthenone (tr-4.5%), three sesquiterpene hydrocarbons: α-curcumene (0.3–3.2%),
(0.1–6.3%)aswellastwooxygenatedsesquiterpenes: β-elemol(upto3.5%)andβ-eudesmol(tr-3.0%)
γ-curcumene (0.1–2.6%) and bicyclogermacrene (0.1–6.3%) as well as two oxygenated sesquiterpenes:
werepresentinappreciableamounts.Then,onesampleforeachlocation(B1,F5andH1)andfiveother
β-elemol (up to 3.5%) and β-eudesmol (tr-3.0%) were present in appreciable amounts. Then, one
sampleswhichexhibitedvariouscompositions(B3,B4,B5,B6andH6)werepresentedinTableS1.
sample for each location (B1, F5 and H1) and five other samples which exhibited various
Forinstance,thecontentofcapillene(apolyacetylenederivative)reached16.9%insampleH6
compositions (B3, B4, B5, B6 and H6) were presented in Table S1.
vs. (0.2–7.5%)fortheothersamplesofHamlaandvs. (tr-0.4%)forthesamplesofFesdisandBouilef.
ThesamplesB3andB4werecharacterizedahighamountofoxygenatedsesquiterpenes: spathulenol
(15.1%and20.7%,respectively)associatedwithα-bisabolol(13.2%and20.0%,respectively). Inthelast
twoatypicalsamples(B5andB6),asesquiterpenehydrocarbon(germacreneD)waspresentasmain
compound(25.3and20.2%. respectively)vs. (0.0–7.5%)forallothersamples.

Figure 2. Cont.
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ppeeaakkss aarree tthhee iiddeennttiififieedd ccoommppoonneennttss ((sseeee TTaabblleeS S11))..
2.2. IdFeonrt iifincsattainonceo, ftPhoel ycaocnetteynletn oefD cearpivilalteinvees (a polyacetylene derivative) reached 16.9% in sample H6
vs. (0.2–7.5%) for the other samples of Hamla and vs. (tr-0.4%) for the samples of Fesdis and Bouilef.
Inthisstudy, theidentificationoffourcompoundsproposedbytheMSlibrarywasachieved.
The samples B3 and B4 were characterized a high amount of oxygenated sesquiterpenes: spathulenol
These compounds were presumed to be two pairs of stereoisomers (m/z = 200 and m/z = 198)
(15.1% and 20.7%, respectively) associated with α-bisabolol (13.2% and 20.0%, respectively). In the
corresponding to spiroacetalenol derivatives. Indeed, polyacetylene compounds are commonly
last two atypical samples (B5 and B6), a sesquiterpene hydrocarbon (germacrene D) was present as
found in the Asteraceae family [30]. The identification of these compounds was achieved
bmyai1n3 Cco-NmMpoRunsdp e(c2t5r.o3s acnodpy 20a.2ft%er. rfersapcteicotnivaetiloyn) v(sf.r a(0c.t0i–o7n.s5%Fr) 4foarn adll oFtrh6e,rs seaemEpxlpese.r imental part) by
comparison of their spectral data with those reported in the literature. (E)- and (Z)-tonghaosu
2.2. Identification of Polyacetylene Derivatives
(m/z=200)wereidentifiedbycomparisonwithdatapreviouslydescribedbyChanotiyaetal.[31].
(E)-2-I(n2 (cid:48)t,h4(cid:48)i-sH setuxaddyi,y tnhyel iiddeennet)if-i1c,a6t-idoino xoafs pfoiruor[ 4c.o4m]-npoonuan-d3,s7 -pdrioepnoeswedas biyd etnhtei fiMedS alicbcroarrdyi nwgatso alictheireavtuerde.
dTahtease[ 3c2o]m(FpioguunredsS 1w).erIen pthreesu13mCe-Nd MtoR bsep etwctrou mpaiorsf Forf4 sitnerweohisicohmtehres E(m//Zz r=a t2io00w aansdc lmos/ze t=o 189/81)
(c4o6r.r6e%sp/o6n.3d%in)g, atose rsipeisrooafc1e3tapleenaokls cdoerrrievsaptiovneds.i nIgndtoeetdh,e pZoilsyoamceetrylwenaes ocbosmerpvoeudn.dIst iasrteh ecofimrsmttoinmlye
tfhoautntdh einf othuer Aspsitreoraacceetaael efnamolidlye r[3iv0a].t iTvhese [id30e]nwtifeirceatiidoenn otiffi tehdesien caonmEpOoufrnodms wS.aasf raiccahnieav. Tedh ebyco 1n3Cte-nNtMsoRf
(sEp)e-c2t-r(o2(cid:48)s,c4o(cid:48)-phye xaaftdeiry fnryalcitdioennaet)i-o1n,6 -(dfriaocxtaiospnisr oF[r44. 4a]n-dno Fnra6-, 3s,e7e-d Eixenpeeraimnden(Eta)-lt poanrgth) aboys cuormeapcahreisdo7n. 3o%f th(Be3ir)
aspnedc3tr.a8l% d(aBta6 )w,riethsp tehcotsive erleyp.oTrhteedse inco tmhep loituenradtsuwree. r(eE)p-r aenvdio (uZs)l-ytornepgohratoesdui n(mE/Oz =f r2o0m0) Cwherryes aidnethnetmifiuemd
cboyr ocnoamripuamrisLo.n( waeirtiha ldaptaar ptsr)ev[3io3u]salyn ddeisncrsiobmede bsyo Clvheanntoetixytara ectt asl.f r[3o1m]. (CE.)-l2e-u(c2a′,n4t′-hHemexuamdi(yrnoyoltisd)e[n3e4)]-,
C1,.6c-odrioonxaarsiupmiro([a4e.r4i]a-lnpoanrat-s3),7[3-d5]ieanned wSa.sc hiadmenaetcifyipeadr iascscuosr(dleinavge tsoa lnitderbautudrse) d[3a6t]a. [32] (Figure S1). In the
13C-NMR spectrum of Fr4 in which the E/Z ratio was close to 8/1 (46.6%/6.3%), a series of 13 peaks
corresponding to the Z isomer was observed. It is the first time that the four spiroacetalenol
derivatives [30] were identified in an EO from S. africana. The contents of (E)-2-(2′,4′-
hexadiynylidene)-1,6-dioxaspiro[4.4]-nona-3,7-diene and (E)-tonghaosu reached 7.3% (B3) and 3.8%
(B6), respectively. These compounds were previously reported in EO from Chrysanthemum coronarium
Molecules2019,24,204 5of15
2.3. ChemicalVariability
The 18 samples were submitted to statistical analyses: the principal components analysis
(PCA,covariance)(Figure3,TableS1),inwhichtheplandefinedbythetwoaxesF1andF2described
51.05%ofthetotalvarianceofthepopulation(thetwoaxesF1andF2accountedfor31.70%and19.35%.
respectively). It may be noted that the composition of all samples was qualitatively quite similar.
Althoughthecompositionsoftheindividualsamplesvariedsubstantiallyforvariouscomponents,
itwasnotpossibletodistinguishgroupswithintheessentialoilsamples. Therefore,onemaingroup
(16samples)anddifferentiatedtwoatypicalcompositions(B5andB6,Figure3)wereobserved.Indeed,
B5andB6werediscriminatedbyahighpercentageofsesquiterpenehydrocarbons(bicyclogermacrene,
Molecules 2019, 24, x FOR PEER REVIEW 5 of 14
(E)-α-bisabolene,γ-curcumene)andparticularlygermacreneD,25.3%(B5)and20.2%(B6)vs. 0–7.5%
hfoormtohgeeontehoeurs,s awmhpillee sth. eA collmthpeossiatimonp loefs thfreo msamthpeleFse fsrdoims ltohcea tHioanmwlae lroecahtoiomno agpepneeaoruesd, mwuhcilhe ltehses
hcoommpoogseintieoonuos.f tChoensvaemrspelleys, firto ampptheearHedam thlaatl otchaet ioBnouaiplepfe asaremdpmleus,c hlolceastsehdo bmeotwgeeneeno tuhse. Ctwonov oetrhseelrys,
liotcaaptpioenasr e(dFetshdaits tahnedB Hoaumilelaf)s wamerpel edsi,ffleorceantet,d sob etwtwoe seanmtphleestw (Bo3o atnhder Bs4l)o wcaetiroen asg(gFreesgdaitseda ntod tHhoasme loaf)
wtheer eFdeisfdfeisr enlot,casotiotwn,o wsahmerpelaess (tBh3e antwdoB 4s)awmeprleesa gBg1re gaantded Bto2 thwoesree ofquthiteeF essimdiislalro ctaoti otnh,ew Hhearmealas
sthaemtpwleoss (aFmigpulrees 3B)1. andB2werequitesimilartotheHamlasamples(Figure3).

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2.4. AntimicrobialActivity
2.4. Antimicrobial Activity
TheantimicrobialactivityoftheEOofS.africanaisolatedfromtheaerialpartatfullfloweringwas
The antimicrobial activity of the EO of S. africana isolated from the aerial part at full flowering
assayedagainstfourbacteria,twoyeastsandthreefilamentousfungi,usingtheagardiscdiffusion
was assayed against four bacteria, two yeasts and three filamentous fungi, using the agar disc
method(Table1).
diffusion method (Table 1).
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Table1.AntimicrobialactivityofS.africanaessentialoil.
NegativeControl
PositiveControls
Microorganisms EssentialOil (DMSO)
(15µL/disc)
CHL VAN GMN CIP FLU NY 6.0
Escherichiacoli 8.00±0.0 25.0±0.0 6.0±0.0 23.0±0.0 35.5±0.7 — — 6.0
Klebsiellapneumoniae 6.0±0.0 21.3±0.6 6.0±0.0 20.0±0.0 30.5±0.7 — — 6.0
Staphylococcusaureus 19.7±0.6 25.5±0.7 17.0±0.0 21.0±0.0 32.0±0.0 — — 6.0
Bacilluscereus 6.0±0.0 29.5±0.7 6.0±0.0 21.0±0.0 37.0±0.0 — — 6.0
Candidaalbicans
13.0±0.0 — — — — 6.0±0.0 16.0±0.0 6.0
ATCC10231
Candidaalbicans
15.3±1.5 — — — — 15.0±0.0 19.0±1.0 6.0
ATCC26790
20.5±0.7a — — — — 6.0±0.0 22.3±0.6 6.0
Aspergillusflavus
13.5±0.7b — — — — 6.0±0.0 — 6.0
43.0±2.8a — — — — 6.0±0.0 33.7±1.2 6.0
Aspergillusfumigatus
17.5±3.5b — — — — 6.0±0.0 — 6.0
38.5±2.1a — — — — 6.0±0.0 16.0±1.0 6.0
Fusariumoxysporum
15.0±0.0b — — — — 6.0±0.0 — 6.0
CHL:Chloramphenicol,VAN:Vancomycin,GMN:Gentamicin,CIP:Ciprofloxacin,FLU:Fluconazole.NY:Nystatinwereusedaspositivecontrols.Meanvaluesofthegrowthinhibition
zones.inmm.includingthediscdiameterof6mm.—:Nottested.a:After3days.b:After5days.
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Theoilwasconsideredactivewhenthediameterofinhibitionzonewasequaltoorgreaterthan
13mm[24]. TheagardiffusionmethodshowedthattheoilwaseffectiveagainstStaphylococcusaureus,
thetwoyeastsandthethreefilamentousfungiwithdiametersofinhibitionzonerangingfrom13.0mm
to19.7mm. ThemostpotentactivitywasdemonstratedagainstStaphylococcusaureusandAspergillus
fumigatuswithinhibitionzonesof19.7mmand17.5mmrespectively. Incontrast,thegrowthofBacillus
cereus,KlebsiellapneumoniaeandEscherichiacoliwerenotinhibitedbytheEO.Thisassumptionisin
accordancewithpreviousstudiesontheSantolinagenus. Indeed,theS.africanaEOwhichcontained
acenaphtane (25.23%), calarene (21.54%), ocimene (17.44%) as its major components, exhibited a
moderateorlowactivityagainstthesamemicroorganismswithdiametersofinhibitionzoneranging
from6.50mmto20.15mm. BacillussubtilisandStaphylococcusaureuswerethemostsusceptibletothis
EO,withinhibitionzonesof20.15and19.5mm,respectively[7].
2.5. AntioxidantActivity
TheresultsoftheDPPH• freeradicalscavengingtestwerepresentedinTable2andFigureS2.
TheEOoftheaerialpartsofS.africanahaveahighantioxidantactivityatconcentrationsof32,64,
128 and256 mg/mL, withinhibition percentages ranged between85.54 ± 2.17% and100 ± 0.00%
andanIC valueof1.51±0.04mg/mL.However,theantioxidantpotentialofS.africanaEOwas
50
foundtobelowthanthatofascorbicacid(positivecontrol),withapercentageinhibitionof100%,ata
concentrationof2mg/mLandasignificantlylowerIC valueof0.02±0.0005mg/mL.
50
Table2.IC valuesandanti-inflammatoryactivityofSantolinaafricanaessentialoil.
50
Activity Antioxidant Anti-Inflammatory
Essentialoil 1.51±0.04 Essentialoil 0.065±0.004
Ascorbicacid 0.02±0.0005 *NDGA 0.013±0.003
Anti-inflammatoryactivity(percentageinhibitionofLOX)
Concentration# Inhibition(%) Concentration# Inhibition(%)
0.015 23.4±4.1 0.050 37.5±2.8
0.025 28.5±5.5 0.075 57.6±3.5
Valuesaremeansoftriplicates±standarddeviation;*NDGA:NordihydroguaiareticAcid;#mg/mL.
2.6. Anti-InflammatoryActivity
The anti-inflammatory potential of S. africana EO was evaluated by determining its ability to
inhibitlipoxygenases(LOX).Indeed,LOXsareanon-hemeiron-containingdioxygenasesthatconvert
linoleic,arachidonicandotherpolyunsaturatedfattyacidintobiologicallyactivemetabolitesinvolved
intheinflammatoryandimmuneresponses.Severalinflammatoryprocessessuchasarthritis,bronchial
asthmaandcancerareassociatedwithanimportantproductionofleukotrienescatalysedbyLOX
pathwayfromarachidonicacid[37–40]. TheinhibitionoftheLOXpathwaywithinhibitorsofLOX
wouldpreventtheproductionofleukotrienesandthereforecouldconstituteatherapeutictargetfor
treatingofhumaninflammation-relateddiseases. Thus,thesearchfornewLOXinhibitorsappearsus
criticalbecausemanyofwhichexhibitsignificantanti-inflammatoryactivity.
TheabilityofS.africanaEOtoinhibitsoybeanlipoxygenasewasdeterminedasanindication
of potential anti-inflammatory activity. S. africana EO exhibited an inhibition of LOX activity
(Table2). ThepercentageofinhibitionincreaseswiththeconcentrationofS.africanaEO,i.e.,23.4%at
0.015mg/mLto57.6%at0.075mg/mLofEO.NoLOXactivitycouldbedetectedinthepresenceof
0.1mg/mLofS.africanaEO,suggestingalmostcompleteinhibitionofLOXactivity. TheIC values
50
(concentrationatwhich50%ofthelipoxygenasewasinhibited)weredeterminedfortheS.africana
EO and for the non-competitive inhibitor of lipoxygenase, the nordihydroguaiaretic acid (NDGA)
(Table2),usuallyusedasreferenceinanti-inflammatoryassays[38–40]. DatashowedthattheIC
50
Molecules2019,24,204 8of15
value of S. africana essential oil (0.065 ± 0.004 mg/mL) is 5-fold higher than IC value of NDGA
50
(0.013±0.003mg/mL).
3. Discussion
The composition of S. africana aerial part oils isolated from plants growing wild in eastern
Algeria(Batna)wasdifferentfromthosereportedforoilsfromMorocco[4,5],Tunisia[9]andAlgeria
(Constantine) [7,18], but it should be pointed that a similar composition has been reported for
flowerhead oil of S. chamaecyparissus from Tunisia, which also contained 1,8-cineole, β-eudesmol
(10.49%),terpinen-4-ol(6.97%),spathulenol(5.80%),camphor(5.27%)andgermacreneD(5.03%)as
majorcomponents[20]. Othercompoundsalsooccurredasmainconstituents: δ-cadinene(6.55%)and
myrtenol(4.26%)whicharepresentatlowamountsinalloursamples(tr-0.4%).
We can assume that the moderate or low antimicrobial activity of S. africana EO is related to
one or various major components: 1,8-cineole (12.8%), germacrene D (7.2%), spathulenol (6.2%),
cis-chrysanthenol(6.0%),myrcene(5.8%),β-pinene(5.2%),α-bisabolol(5.2%),terpinen-4-ol(3.2%),
santolina alcohol (3.1%), lyratol (2.4%), capillene (2.2%), (E)-α-bisabolene (2.0%), limonene (1.9%),
camphor(1.6%)andβ-elemol(1.4%). 1,8-Cineolewaspreviouslydescribedasantibacterialagainst
S.aureus[41]whilemyrcenewhichaccountedfor57.2%inthefractionFr1[23]wasalreadyreportedas
ineffectiveagainstS.aureus[23,42]. Ithasbeenreportedalsothatβ-pinene,α-pineneandgermacrene
Dhadslightactivityagainstapanelofmicroorganisms. Indeed,theessentialoilofPinusnigrassp.
pallasiana (α-pinene. 42.3%; germacrene D. 30.6%) exhibited a low antimicrobial activity against
the same strains with MICs in the range 10–20 mg/mL [43], so it has been demonstrated in the
literaturethattheinhibitoryactivityofanEOresultsfromacomplexinteractionbetweenitsdifferent
constituents,whichmayproduceadditive,synergisticorantagonisticeffects,evenforthosepresent
atlowconcentrations,i.e.,1,8-cineoleincombinationwithcamphorhasshownhigherantimicrobial
effects[44]. Inparallel,Lemosetal.[45]reportedthattheessentialoilofRosmarinusofficinaliswhich
containedcamphor(24.4–35.9%)asmajorcompoundexhibitedahighantimicrobialactivityagainst
S.aureuswithMICsintherange0.5–2.0µL/mL.Otherwise,aninterestingantimicrobialactivityofa
lyratol-richfraction(84%)wasobservedagainstS.aureus(19mm),suggestingthatlyratolcouldbe
themainresponsiveoftheantimicrobialpropertiesofSantolinacorsica[23]. Ithasbeensummarized
alsothatoxygenatedterpenes,aswellasalcoholswhicharepresentinappreciableamountsinouroil,
areactivebutwithdifferingspecificityandlevelsofactivity[46,47].
In previous studies, Derouiche et al. [7] reported a percentage inhibition of the free radical
DPPHoftheS.africanaflowerEOofabout13.80%ataconcentrationof0.1M,avaluemuchlower
than ascorbic acid (more than 70% of inhibition) used as a positive control. Nouasri et al. [17]
evaluatedtheantioxidantactivityoftheessentialoiloftheaerialpartsofS.chamaecyparissususingtwo
methods,theDPPH• freeradicalscavengingtestandtheβ-carotenebleachingtest. Theyreported
that S. chamaecyparissus EO had low antioxidant capacity to reduce DPPH• radical with an IC
50
of about43.01 ± 8.04 mg/mL,comparedto BHT (IC = 0.072 ± 0.001 mg/mL)and ascorbic acid
50
(IC =0.004±0.001mg/mL). Theβ-carotenebleachingtestrevealedthattheEOhadamoderate
50
activitywithapercentageinhibitionoftheoxidationoflinoleicacidoftheorderof47.00±3.13%,
avaluethatishigherthanthatofascorbicacidtested(11.05%),butmuchlowerthanBHT(96.92%).
ThemeasurementofantioxidantactivityhasrevealedthataerialpartsofS.africanaEOexhibited
anantioxidantactivitythatcouldhaveaneventualpossibilitytobeusedinthefoodindustry,asa
naturalantioxidantagent,forthepreservationoffoodstuffs,orinthefieldofhealth,fortheprevention
ofvariousdiseases.
Concerningtheanti-inflammatoryactivity,thelowratiobetweenthetwovaluesofIC (S.africana
50
EO vs. NDGA) makes it possible to consider the S. africana EO as a high inhibitor of the LOX
activity[48]. Thus,accordingtotheresults,S.africanaEOexhibitsahighinhibitionofLOXactivity,
suggestingananti-inflammatorypotential.
Molecules2019,24,204 9of15
4. MaterialsandMethods
4.1. PlantMaterial
AerialpartsofSantolinaafricanawerecollectedduringthefloweringperiodinMay2016inthree
locations in the Batna province (Eastern Algeria): Fesdis (Fesdis: F1–6; Bouilef: B1–6) and Oued
Chaaba(Hamla: H1–6)(Figure1). IdentificationoftheplantmaterialwasperformedbyDr. Babali
B.,(LaboratoryofEcologyandManagementofNaturalEcosystems,UniversityofTlemcen,Imama
Tlemcen,Algeria). AvoucherspecimenhasbeendepositedattheLaboratoryofNaturalProducts
(DepartmentofBiology,UniversityofTlemcen,Algeria),undertheaccessionn◦ A.2844. Theessential
oilwasobtainedbyhydrodistillationofdriedaerialparts(around150–280g)for2h. Yieldshavebeen
calculatedfromdrymaterial.
4.2. GasChromatography(GC)Analysis
GCanalyseswereperformedonaClarus500FIDgaschromatograph(PerkinElmer,Courtaboeuf,
France)equippedtwofusedsilicagelcapillarycolumns(50m×0.22mm,filmthickness0.25µm),
BP-1(polydimethylsiloxane)andBP-20(polyethyleneglycol). Theoventemperaturewasprogrammed
from60to220◦Cat2◦C/minandthenheldisothermalat220◦Cfor20min,injectortemperature:
250◦C;detectortemperature: 250◦C;carriergas: hydrogen(1.0mL/min);split: 1/60. Therelative
proportionsoftheoilconstituentswereexpressedaspercentagesobtainedbypeakareanormalization,
withoutusingcorrectingfactors. Retentionindices(RIs)weredeterminedrelativetotheretention
timesofaseriesofn-alkaneswithlinearinterpolation(‘TargetCompounds’softwareofPerkinElmer).
4.3. MassSpectrometry
The EOs were analyzed with a PerkinElmer TurboMass detector (quadrupole, PerkinElmer,
Courtaboeuf, France), directlycoupledtoaPerkinElmerAutosystemXL(PerkinElmer), equipped
with a fused silica gel capillary column (50 m × 0.22 mm i.d., film thickness 0.25 µm), BP-1
(dimethylpolysiloxane). Carrier gas, helium at 0.8 mL/min; split: 1/75; injection volume: 0.5 µL;
injectortemperature: 250◦C;oventemperatureprogrammedfrom60to220◦Cat2◦C/minandthen
heldisothermal(20min);ionsourcetemperature: 250◦C;energyionization: 70eV;electronionization
massspectrawereacquiredoverthemassrange40–400Da.
4.4. NMRAnalysis
13C-NMRanalyseswereperformedonanAVANCE400FourierTransformspectrometer(Bruker,
Wissembourg, France) operating at 100.623 MHz for 13C, equipped with a 5 mm probe, in CDCl ,
3
withallshiftsreferredtointernaltetramethylsilane(TMS).13C-NMRspectrawererecordedwiththe
followingparameters: pulsewidth(PW):4µs(flipangle45◦);acquisitiontime: 2.73sfor128Kdata
tablewithaspectralwidth(SW)of220.000Hz(220ppm);CPDmodedecoupling;digitalresolution
0.183Hz/pt. Thenumberofaccumulatedscansranged2000–3000foreachsample(around40mg
ofoilin0.5mLofCDCl ). Exponentiallinebroadeningmultiplication(1.0Hz)ofthefreeinduction
3
decaywasappliedbeforeFouriertransformation.
4.5. IdentificationofIndividualComponents
Identification of the components was based: (i) on comparison of their GC retention indices
(RIs) on polar and apolar columns, determined relative to the retention times of a series of
n-alkanes with linear interpolation (‘Target Compounds’ software of PerkinElmer), with those
of authentic compounds and (ii) on comparison of the signals in the 13C-NMR spectra of
EOs with those of reference spectra compiled in the laboratory spectral library, with the help
of a laboratory-made software [49–51]. In the investigated samples, individual components
were identified by NMR at contents as low as 0.4%. Several compounds were identified by
Molecules2019,24,204 10of15
comparisonof13C-NMRchemicalshiftswiththosereportedintheliterature,forinstancecapillene
and capillin [27,52]; (E)-2-(2(cid:48),4(cid:48)-hexadiynylidene)-1,6-dioxaspiro[4.4]-nona-3,7-diene [32]; (Z) and
(E)-tonghaosu. (2-(2(cid:48),4(cid:48)-hexa-diynyl-idene)-1,6-dioxaspiro[4.4]-non-3-ene[31,53].
4.6. EssentialOilFractionation
A composite oil sample (F1 to F6, Fesdis; 247.9 mg) was submitted to flash chromatography
(silicagel: 35–70µm). Ninefractions(Fr1-Fr9)wereelutedwithamixtureofsolventsofincreasing
polarity(pentane:diethylether,100:0to0:100,andpuremethanol);Fr1(10.3mg)andFr2(12.6mg);
pentane:Et O,98:2;Fr3(9.2mg);pentane:Et O,95:5Fr4(14.4mg);pentane:Et O,90:10Fr5(18.8mg);
2 2 2
pentane:Et O,75:25Fr6(86.5mg);pentane:Et O,50:50Fr7(18.7mg);pentane:Et O,0:100;Fr8(22.3mg)
2 2 2
andpuremethanol,Fr9(15.6mg). AllfractionsofchromatographywereanalyzedbyGC(RI),GC/MS
and13C-NMR.
4.7. AntimicrobialActivityoftheEssentialOil
4.7.1. MicrobialStrains
AntimicrobialactivityoftheaerialpartEO(CollectivesampleBouilef-Hamla)wereevaluated
againsttwoGram-positivebacteria(StaphylococcusaureusATCC6538andBacilluscereusATCC25921)
and two Gram-negative bacteria (Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 700603),
two yeasts (Candida albicans ATCC 26790 and C. albicans ATCC 10231) and three filamentous
fungi(FusariumoxysporumMNHN963917, AspergillusfumigatusMNHN566andAspergillusflavus
MNHN994294).
4.7.2. ScreeningofAntimicrobialActivity
Theagardiffusionmethod[54]wasusedforthedeterminationofantimicrobialactivityofthe
EOs. Briefly,asuspensionofthetestedmicroorganisms(1mLofasuspensionat106 cells/mLfor
bacteriaandyeasts,107cells/mLforS.aureusand104spores/mLforfilamentousfungi)wasspread
onthesolidmediaplates,usingMueller–Hintonagarforbacteria,Sabourauddextroseforyeastsand
PDAforfilamentousfungi. Filterpaperdiscs(6mmindiameter)wereimpregnatedwith15µLofthe
oiland5µLofDMSOandplacedonthesurfaceofinoculatedplates. Theactivitywasdeterminedby
measuringtheinhibitoryzonediameterinmmafterincubationfor24hat37◦Cforbacteria,24–48h
at 30 ◦C for yeasts and 3 to 5 days at 25 ◦C for filamentous fungi. Fluconazole (FLU 25 µg/disc),
nystatin(NY30µg/disc)wereusedasreferenceantifungalagainstyeastsandfilamentousfungiand
chloramphenicol(CHL30µg/disc),ciprofloxacin(CIP10µg/disc),gentamicin(GMN10µg/disc),
vancomycin(VAN30µg/disc)wereusedaspositivecontrolsagainstbacteria. DMSOwasusedas
negativecontrol. Eachtestwasperformedinduplicateorintriplicate.
4.8. DPPHRadicalScavengingActivity
The antioxidant activity was measured on a sample of EO (Collective sample Fesdis F1–6).
The antioxidant activity of S. africana EO was measured on the basis to scavenge of the
2.2-diphenyl-1-picrylhydrazil(DPPH•)freeradical,accordingtotheexperimentalprotocolofBlois[55].
Avolumeof2.5mLwithvariousconcentrations(256,128,64,32,16,8,4,2,1,0.5,0.25,0.125,0.0625,
0.03125 and 0.015625 mg/mL) of the EO in absolute ethanol were added to 1 mL of an ethanolic
solutionofDPPHat0.03mg/mL.Foreachconcentration,ablankwasprepared. Inparallel,anegative
controlispreparedbymixing2.5mLofabsoluteethanolwith1mLofethanolicsolutionofDPPH.
Afterincubationinthedarkfor30minatroomtemperature,theabsorbancewasmeasuredagainsta
blankat517nm. TheactivityoftheEOwascomparedtoascorbicacidasapositivecontrol. DPPHfree
radicalscavengingactivityinpercentage(%)wascalculatedusingthefollowingformula:
DPPHscavengingactivity(%)=[(A −A )/A ]×100 (1)
control sample control