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RevistaBrasileiradeFarmacognosia28(2018)564–567

w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original Article

Salvastearolide, a new acyl-glyceride, and other constituents from the seeds of Salvadora persica

Samy K. El-Desouky

a,b,∗

, Usama W. Hawas

c,b

, Habib Khemira

d

, Young-Kyoon Kim

e

aChemistryDepartment,FacultyofScience,JazanUniversity,Jazan,SaudiArabia

bPhytochemistryandPlantSystematicDepartment,NationalResearchCentre,Dokki,Cairo,Egypt

cMarineChemistryDepartment,FacultyofMarineSciences,KingAbdulazizUniversity,Jeddah,SaudiArabia

dCenterforEnvironmentalResearch&Studies,JazanUniversity,Jazan,SaudiArabia

eCollegeofForestScience,KookminUniversity,Chongnung-dong,Songbuk-gu,Seoul,SouthKorea

a r t i c l e i n f o

Articlehistory:

Received29March2018 Accepted14May2018 Availableonline3July2018

Keywords:

Salvastearolide Phytosteroids NMR

Cytotoxicactivities

a b s t r a c t

Thechemicalinvestigationofthen-hexanefractionofSalvadorapersicaL.,Salvadoraceae,seedsafforded anewstearicacidester,salvastearolide,togetherwithfiveotherphytosteroidsidentifiedasstigmasterol,

␤-sitosterol,7-campesterol,7-avenasterolandcampesterol.Theirstructureswereestablishedonthe basisofextensivespectroscopicmethodsincluding1Dand2DNMRexperimentsandHRESImassspec- trometry.Inaddition,salvastearolideandtheisolatedfractionsweretestedfortheircytotoxicityagainst humancancercelllinesMCF-7,MDA-MB-231andHT-29.Then-hexanefractionexhibitedsignificant anti-proliferativeeffectagainsthumanbreastcancercelllineMCF-7(IC5050␮g/ml),whilesalvastearolide possessedaweakcytotoxiceffectagainstMCF-7cellswithIC50103.98␮g/ml.

©2018SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Salvadora persica L., known as miswak, is a shrub with a soft whitewood and belongs tothe Salvadoraceae family. It is distributed mainly in tropical and subtropical Asia. The aque- ousextract of S. persicaleavespossesses analgesicactivity and decreasescarrageenan-inducedinflammationinratpaw(Ramadan andAlshamrani,2016).

S.persicahasbeenreportedtohaveantiulceractivity(Sanogo etal.,1999)andantimicrobialactivity(Almas,1999;Sofrataetal., 2008).TheaqueousextractsofS.persicaenhancethegrowthof fibroblastsandinhibitthegrowthofcariogenicbacteria(Darmani etal.,2006).

Thephytochemicalstudy ofS. persicaledtotheisolation of four benzylamides (Khalil, 2006). The characterization of the obtained compounds were confirmed through spectral anal- ysis. Moreover, five different glycosides were isolated from the stems of S. persica and identified as sodium 1-O-benzyl-

␤-D-glucopyranoside-2-sulphate, 5,5-dimethoxylariciresinol

Correspondingauthor.

E-mail:sdesoky@Jazanu.edu.sa(S.K.El-Desouky).

4,4-bis-O-␤-d-glucopyranoside,syringin,liriodendrinand sitos- terol3-O-glucopyranoside.Thisinvestigationwasthefirstreport ofsyringinandlignanglycosidesfromthefamilySalvadoraceae (Kameletal.,1992).

AnewalkaloidnamedsalvadoricinehasbeenisolatedfromS.

persica.Itschemical structure wasestablished byspectroscopic methodsandconfirmedbysynthesis(Maliketal.,1987).

The volatile roots oil of S. persica contain benzyl isothio- cyanate with other constituents such as ␣-pinene, ␤-pinene,

␤-elemene, camphene, myrcene, limonene, ␦-3-carene, benzyl nitrile,benzaldehyde,umbellulone,myristicin,␥-muurolene,ter- pinolene, longifolene and ␤-caryophyllene (Bader and Flamini, 2002).

GC-MSanalysisofthevolatileoilfromtheleavesofS.persica resultedintheisolationofthymoland␤-caryophyllene,eucalyptol, isoterpinolene,benzylnitrile,isothymolandeugenol(AlaliandAl- Lafi,2003).

Wereportheretheantiproliferativeeffectsofdifferentextracts fromS. persicaseedsas wellasthe isolation and identification ofseveralknownsteroidalcompoundsandanewmonoglyceride derivativefromthe n-hexanefraction. The isolatedcompounds werecharacterizedbyNMRspectroscopicmethods.Furthermore, thenewisolatedcompoundwastestedforitscytotoxicityagainst breastcancercelllineMCF-7invitro.

https://doi.org/10.1016/j.bjp.2018.05.013

0102-695X/©2018SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

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S.K.El-Desoukyetal./RevistaBrasileiradeFarmacognosia28(2018)564–567 565

Materialsandmethods Plantmaterial

SeedsofSalvadorapersicaL.,Salvadoraceae,werecollectedfrom GabalFiafainJazan,SaudiArabia,onJune2016andidentifiedbyDr.

WaelKassim,DepartmentofBotany,JazanUniversity,SaudiAra- bia.Avoucherspecimen(108-42016)hasbeendepositedinJazan UniversityHerbarium(JAZUH).

Isolationandcharacterizationofcompounds

Thegroundedair-driedseedsofS.persica(370g)wereextracted withhexanetoremovefats,thenwithmethanolthrice(3×2l)at roomtemperature(24heach).Thecombinedmethanolextracts werethenconcentrated underreducedpressure togivea crude residue(80.66g).Theresiduewassuspended inwaterandthen partitionedsuccessivelywithhexane,CH2Cl2,EtOAcandn-BuOH affordingresiduesof48.3,458,150and3.8g,respectively.

Accuratelyweighed5gofthehexaneresiduewassubjectedto chromatographicseparationoversilicagelcolumnchromatogra- phyelutedwithCH2Cl2toafford10fractions(1–10).Fractions6, 7and8werefurthercombinedandpurifiedbypreparativeHPLC columnchromatographytoaffordcompounds2–6.Fraction2was purifiedbysilicagelcolumnchromatographyelutedwithhexane- ethylacetate(15:1)followedbyhexane-ethylacetate(30:1)togive threesubfractions(2A,2Band2C).Subfractions2Band2C(780mg) werecombinedandpurifiedbysilicagelcolumnchromatography withCH2Cl2–MeOH(20:1)toaffordcompound1(100mg).

Generalexperimentalmethods

Silicagel60wasusedforcolumnchromatography.Thinlayer chromatographyanalysiswascarriedoutusingsilicagel60F254 plates.Vacuumliquidchromatographywascarriedoutusingsil- icagel60H.ThechromatogramswerevisualizedunderUVlight andthensprayedwithanisaldehydeandvanillinreagents.High- performanceliquidchromatographyanalysiswasconductedusing Waters600Einstrumentwithadiodearraydetector.Infraredspec- trawererecordedonaBrukerIFSinstrument.ESIMSwasperformed onFinniganMATSSQ7000instrument.NMRspectrafortheisolated metaboliteswereobtainedonBruker400spectrometerusingTMS asaninternalstandard.

Cellculture

Thehumancancercell lines,suchas breastcancercelllines (MCF-7 and MDA-MB-231), colon cancer cell line (HT-29) and nonmalignant human adipose-derivedstem cells (ADSCs)were procuredfromKoreanCellLineBank,Korea.Cancercelllineswere growninDMEMsupplementedwith1%antibioticsand10%FBSat 37Cinahumidifiedatmospherecontaining5%CO2.ADSCswere culturedinMesenPRORSTMmediumsupplementedwithgrowth factors(LifeTechnologies)atthesameatmospherewith5%CO2.

Antiproliferativeassay

Theeffectofcrudeextractsaswellasdifferentfractionslike hexane,dichloromethane,ethylacetateandbutanolonprolifera- tionofbothmalignantandnonmalignantcellswasevaluatedbythe MTTassaymethod(Mosmann,1983).Briefly,thecellswereseeded in96-wellplatesatadensityof5000cells/wellin100␮lrespec- tiveculturemedium.Afterovernightincubation,cellsweretreated withdifferentconcentrationsofcrudeextractsaswellasfractions ofseedsandthenincubatedfor24hat37Cwith5%CO2.After

Table1

1Hand13CNMRspectraldataofcompound1inCDCl3inppm,JinHz).a

Position ıH(multiplicity) ıC

1 11.0(br.,OH) 173.50

2 5.25(m,1H),1.57(br.,OH) 69.05

3 4.26(dd,1H,4.4,16.4Hz),4.12(dd,1H,6.0,18.0Hz) 62.30

1 173.09

2 2.28(m,2H) 34.42

3 1.55b 25.06

4 1.22b 29.28

5 1.22b 29.32

6 1.22b 29.55

7 1.22b 29.57

8 1.22b 29.71

9 1.22b 29.87

10 1.22b 29.90

11 1.22b 32.12

12 1.22b 29.83

13 1.22b 29.68

14 1.22b 29.50

15 1.22b 29.48

16 1.22b 34.25

17 1.22b 22.90

18 0.85(t,3H,6.8Hz) 14.32

aTheassignmentswerebasedupon2D-COSY,DEPT,HSQCandHMBCspectra.

bMultiplicityisnotclearforsomeresonancesduetooverlapping.

incubation,themediumwasreplacedwith100␮lofMTT solu- tionwhichwaspreparedfreshlyas0.5mg/mlinphenolredand serum-freeDMEM,filteredthrougha0.22-␮mfilter,wasaddedto eachwell,andthentheplateswereincubatedindarkforadditional 4hat37C.Afterward,themediawereremovedfromthe96-well plates,100␮lofDMSOwasadded toeach wellandabsorbance wasmeasuredat570nmusingamicroplatereader.Theresultsare expressedasthepercentageofcellviabilityincomparisonwiththe controlcells(cellswithoutdrugtreatment).Thecellviabilityofthe controlgroupwithoutexposuretothedrugwasdefinedas100%.

2-Hydroxy-3-(stearoyloxy)propanoicacid(1)

Solidifiedcolourlessoil,IR(KBr):3550,1741,1725cm−1;1H NMRand13CNMR:(CDCl3,400MHzforprotonand100MHzfor carbon):Table1(+)ESI-MS:m/z373[M+H]+,HR-ESI-MS:395.2839 [M+Na]+(calcd.395.2773forC21H40O5Na).

Resultsanddiscussion

Thecytotoxicactivityoftheobtainedfractionsaswellasthe crude methanol extract was evaluated using cancer cell lines suchashumanbreastcancercellsMCF-7andMDA-MB-231,and humancoloncancercellsHT-29byMTTcolorimetricassaymethod (Mosmann,1983).Theadipose-derivedstemcells (ADSCs)were usedasanormalcontrolinordertoassessthetoxicpotentialof theextracts.AsshowninFig.1,thehexanefractioninducedsignif- icantantiproliferativeeffectagainsthumanbreastcancercellline MCF-7,ataminimumconcentrationof50␮g/ml,whereasthesame concentrationofthefractiondidnotinfluencetheproliferationof othercancercelllinesaswellasthenormalstemcells,ADSCs.None oftheotherfractionsshowedsignificantantiproliferativeactivity againstbothnormalandcancercells.

The steroidal compounds(2–6)wereisolated and identified based on their 1H and 13C NMR spectral data compared to thoseintheliteratureasstigmasterol,␤-sitosterol(Chaturvedula and Prakash, 2012; Jain and Bary, 2010), 7-campesterol, 7- avenasterolandcampesterol(Zhangetal.,2016).

Salvastearolide(1)wasisolatedassolidifiedcolourlessoilat roomtemperature.TheESIpositivemodemassspectrumshowed (M+1)+molecularpeakatm/z373,whiletheHR-ESIMSanalysis

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566 S.K.El-Desoukyetal./RevistaBrasileiradeFarmacognosia28(2018)564–567 120

100

80

60

40

20

0

0 10 20 30 40 50 60 70 80 90 100

Concentration µg/ml y=-0.9131x+95.92

Viability (% control)

Figure1. CytotoxicityofhexanefractiononbreastcancerMCF-7cellsasestimated byMTTassay.Dataarerepresentedas%ofcontrol(mean±SE).

resultedin an [M+Na]+ ion at m/z 395.2839, consistentwith a molecularformulaofC21H40O5 (calculated[M+Na]+ ionatm/z 395.2773)andindicativeoftwodegreesofunsaturation.

The IR spectrum of 1 displayed characteristic absorption bandsduetoester(1174and1741cm−1),carbonylofcarboxylic (1725cm1) and hydroxyl (3300cm1) groups. The 13C NMR including DEPT-135 spectra(Table 1) were consistent with its molecularformulaasitshowed21peaks.Seventeenofthembeing sp3methylenesignalsincludingoneoxygenatedmethyleneatı 62.3.Theothercarbonsignalswereduetoonemethylsignalatı 14.32,oneoxygenatedmethinesignalatı69.05andtwocarbonyl carbonsignalsatı173.09and173.5forcarbonylesterandcarbonyl carboxylicacid,respectively.

The1HNMRdatashowedonemethyltripledatı0.85,sixteen overlappedmethylenesignalsaroundı1.22–2.29,onedoubledou- bletatı4.26forH-3aandonedoubledoubletatı4.12forH-3b,and onemultipletsignalatı5.25fortheoxygenatedmethinegroupin agreementwiththesuggestedstructureof1.

TheCOSYNMRspectrumof1showedclearcorrelationsbetween the methylene and methine protons confirming the coupling betweenthetwooxygenatedgroups.Themethyl,theoxygenated methyleneandthemethinecarbonswereunambiguouslycorre- latedtotheattachedprotons in theHMQC spectrum of1.The downfieldshiftofC-3atıC62.30suggestedthatthismethylene carbonmightbelinkedthroughanoxygenbridgewiththecarbonyl carbonofthestearicacid.Thisconnectionwasfurtherconfirmed bythestronglong-rangecorrelationsdetectedintheHMBCspec- trumof1(Fig.2)betweenthemethyleneprotonsatıH4.26(H3a) andıH4.12(H3b)andthetwocarbonylcarbonsatıC173.50(C-1) andıC173.09ppm(C-1).

BasedontheseNMR data,compound 1 wasidentifiedas 2- hydroxy-3-(stearoyloxy) propanoic acid, salvastearolide, a new acyl-glyceridederivative,fromthisplantspecies.

Duetotheinhibitorypropertiesofhexanefractiononbreast cancercells, theeffect of 1 ontheproliferation of MCF-7cells

Figure2.SelectedHMBCcorrelationsof1.

120

100

80

60

40

20

0

0 10 20 30 40 50 60 70 80 90 100

Concentration µg/ml y=-0.4655x +97.987

Viability (% control)

Figure3.CytotoxicityofsalvastearolideonbreastcancerMCF-7cellsasestimated byMTTassay.Dataarerepresentedas%ofcontrol(mean±SE).

wasstudiedafter48hofincubation.Incontrast,theresultsindi- catedthat1exhibitedaweakcytotoxiceffectwithIC50valueof 103.98␮g/ml(Fig.3).

Authorcontributions

S.K.El.-D.and U.H.carried outthelaboratoryworkfor isola- tionandcharacterizationoftheisolatedcompounds.Y.-K.K.and

H.K.carriedoutthecytotoxicityassaysandobtainedtheNMRand MSdata.Alltheauthorshavepreparedthefinalmanuscriptand approvedthesubmission.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

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S.K.El-Desoukyetal./RevistaBrasileiradeFarmacognosia28(2018)564–567 567

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwasfundedbyagrantNo.CERS8/2012fromtheCen- terforEnvironmentalResearch&Studies(CERS),JazanUniversity.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2018.05.013.

References

Alali,F.,Al-Lafi,T.,2003.GC-MSanalysisandbioactivitytestingofthevolatileoil fromtheleavesofthetoothbrushtreeSalvadorapersicaL.Nat.Prod.Res.17, 189–194.

Almas,K.,1999.TheantimicrobialeffectsofextractsofAzadirachtaindica(Neem) andSalvadorapersica(Arak)chewingsticks.IndianJ.Dent.Res.10,23–26.

Bader,A.,Flamini,G.,2002.ThecompositionoftherootoilofSalvadorapersicaLinn.

J.Essent.OilRes.14,128–129.

Chaturvedula,V.S.P.,Prakash,I.,2012.Isolationofstigmasteroland␤-sitosterolfrom thedichloromethaneextractofRubussuavissimus.Int.Curr.Pharm.J.1,239–242.

Darmani,H.,Nusayr,T.,Al-Hiyasat,A.S.,2006.Effectsofextractsofmiswakand derumonproliferationofBalb/C3T3fibroblastsandviabilityofcariogenicbacte- ria.Int.J.Dent.Hyg.4,62–66.

Jain,P.S.,Bary,S.B.,2010.Isolationoflupeol,stigmasterolandcampesterolfrom petroleumetherextractofwoodystemofWrightiatinctoria.AsianJ.PlantSci.

3,163–167.

Kamel,M.S.,Ohtani,K.,Assaf,M.H.,Kasai,R.,El-Shanawani,M.A.,Yamasaki,K., Ali,A.A.,Tanaka,O.,1992.LignanglycosidesfromstemsofSalvadorapersica.

Phytochemistry31,2469–2471.

Khalil,A.T., 2006.Benzylamidesfrom Salvadora persica.Arch.Pharm. Res. 29, 952–956.

Malik,S.,Ahmad,S.S.,Haider,S.I.,Muzaffar,A.,1987.Salvadoricine—anewindole alkaloidfromtheleavesofSalvadorapersica.TetrahedronLett.28,163–164.

Mosmann,T.,1983. Rapidcolorimetricassayforcellulargrowthand survival:

applicationtoproliferationandcytotoxicityassays.J.Immunol.Methods65, 55–63.

Ramadan,K.S.,Alshamrani,S.A.,2016.Phytochemicalanalysisandantioxidantactiv- ityofSalvadorapersicaextracts.J.BasicAppl.Res.2,390–395.

Sanogo, R., Monforte, M.T.,Daquino, A., Rossitto,A., Maur, D.D., Galati, E.M., 1999.AntiulceractivityofSalvadorapersicaL.:structuralmodifications.Phy- tomedicine6,363–366.

Sofrata,A.H.,Claesson,R.L.,Lingström,P.K.,Gustafsson,A.K.,2008.Strongantibacte- rialeffectofmiswakagainstoralmicroorganismsassociatedwithperiodontitis andcaries.J.Periodontol.79,1474–1479.

Zhang,X.,Cambral,A.,Miesch,M.,Roussi,S.,Raul,F.,Aoude-Werner,D.,Marchioni, E.,2016.Separationof5-and7-phytosterolsbyadsorptionchromatography andsemipreparativereversedphasehigh-performanceliquidchromatogra- phyforquantitativeanalysisofphytosterolsinfoods.J.Agric.FoodChem.5, 1196–1202.

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