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.:+33-495-52-4122 (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]. Molecules2019,24,204;doi:10.3390/molecules24010204 www.mdpi.com/journal/molecules Molecules2019,24,204 2of15 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). Molecules2019,24,204 3of15 Molecules 2019, 24, x FOR PEER REVIEW 3 of 14 FFiigguurree 11.. SSaammpplliinngg llooccaattiioonnss ooff SSaannttoolliinnaa aaffrriiccaannaa ffrroomm eeaasstteerrnn ooff AAllggeerriiaa.. 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. Molecules2019,24,204 4of15 Molecules 2019, 24, x FOR PEER REVIEW 4 of 14 FFiigguurree 22.. GGaass cchhrroommaattooggrraammss ooff SSaannttoolliinnaa aaffrriiccaannaa EEOO ((ssaammpplleess FF55 uupp aanndd BB55 ddoowwnn)).. TThhee nnuummbbeerreedd 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). FFiigguurree 33.. PPCCAA ooff SSaannttoolliinnaa aaffrriiccaannaa eesssseennttiiaall ooiill ssaammpplleess.. 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). Molecules2019,24,204 6of15 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. Molecules2019,24,204 7of15 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