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

Original

Article

Profile

of

bioactive

compounds

of

Capparis

spinosa

var.

aegyptiaca

growing

in

Egypt

Riham

Omar

Bakr

,

Mahitab

Helmy

El

Bishbishy

PharmacognosyDepartment,FacultyofPharmacy,OctoberUniversityforModernSciencesandArts(MSA),Giza,Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16December2015

Accepted26April2016

Availableonline21May2016

Keywords: Capparisspinosa

LC-HRESI-MS–MS Flavonoids Isothiocyanate Cytotoxicity

a

b

s

t

r

a

c

t

Thepresentstudywasdesignedtoinvestigatepolyphenolicandsulphurcontentsoftheaerialpartsof Capparisspinosavar.aegyptia(Lam.)Boiss.,Capparaceae,wildlygrowinginEgypt.Thechemical com-positionsofthewaterdistilledessentialoilwereinvestigated byGC/MSanalysiswherethemajor constituentoftheoilwasmethylisothiocyanate(24.66%).Hydroethanolicextractwasevaluatedby LC-HRESI-MS–MSinbothpositiveandnegativemodes.Forty-twocompoundswereidentifiedincluding quercetin,kaempferolandisorhamnetinderivativesinadditiontomyricetin,eriodictyol,cirsimaritin andgallocatechinderivatives.Quercetintetrahexosidedirhamnosideaswellaskaempferol dihexo-sidedirhamnosidehavenotbeenidentifiedbeforeingenusCapparis.Phenolicacids,suchasquinic acid,p-coumaroylquinicacidandchlorogenicacidwerealsoidentified.Evaluationofcytotoxicactivity ofhydroethanolicextractagainstthreehumancancercelllines(MCF-7;breastadenocarcinomacells, Hep-G2;hepatocellularcarcinomacellsandHCT-116;coloncarcinoma)using 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazoliumbromide(MTT)assayshowedsignificanteffectwithIC50values24.5,24.4 and11␮g/ml,comparedtoDoxorubicinasastandardcytotoxicdrug.C.spinosarevealeditselfasa promisingcandidatefornutraceuticalresearches.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Capparaceaeis a closely related family tothemustard fam-ily(Cruciferae)withabundanceofglucosinolatesandflavonoids (Täckholm,1974;Inocencioetal.,2000;Kiddleetal.,2001).The genusCapparisisrepresentedinEgyptbysixspecies(Täckholm, 1974).Capparisspinosavar.aegyptia(Lam.)Boiss,(thecaper) grow-ingintheEgyptiandeserts,isaperennialwinter-deciduousplant thatbearsrounded,fleshy,alternativeleavesandthick,shiny,large whitetopinkish-whitecompleteflowers.Theplantisbestknown fortheediblebudandfruit(caperberry).InGreco-ArabandIslamic medicine,thedecoctionof rootbarkis prescribedas deobstru-entto liverand spleen, asanthelmintic and anti-inflammatory agents.Decoctionsfromtherootbarkhavebeenusedintraditional medicinesfordropsy,anemia,arthritis,andgout.Thestembarkis diuretic(SaadandSaid,2011).Thestrongflavorofcapersisusually duetotheverypungentmethylisothiocyanatethatisreleasedafter anenzymaticreactionwithamustardoilglycosidenamed gluco-capparin(methylglucosinolate)(Brevardetal.,1992;Romeoetal., 2007;Sozzietal.,2012).

∗ Correspondingauthor.

E-mail:romar@msa.eun.eg(R.O.Bakr).

C. spinosa is considered as a very important source of medicine for antifungal (Ali-Shtayeh and Abu Ghdeib, 1999) anti-inflammatory(Al-Saidetal.,1988;Zhouetal.,2010), antidia-betic,antihyperlipidemic(Eddouksetal.,2005),antihypertensive (Baytop,1984),antihepatotoxic(GadgoliandMishra,1999), poten-tialinhibitorofNF-kappaB(Zhouetal.,2011),andanticarcinogenic (Kulisic-Bilusicetal.,2012).

Quantitation of flavonoid content in Capers revealed it as a very rich source of the flavonols (Inocencio et al., 2000). C. spinosa has been an interesting field of study. Estima-tion of phenolic compounds in the Croatian species revealed the presence of isorhamnetin-3-O-rutinoside besides chloro-genic acid derivatives and cinnamoyl-quinic acid derivatives (Siracusa et al., 2011). While in China, flavonoids identified in the fruits were isoginkgetin, and ginkgetin and Sakuranetin (Zhao et al., 2013). Egyptian species have been investigated a long time ago. Six glucosinolates were identified, such as glucoiberin,glucocapparin,sinigrin,glucocleomin,glucobrassicin and glucocapangulin. Also, four flavonoids were isolated from C. cartilaginea and C. deserti and identified as kaempferol-3-O-rutinoside,quercetin-3-O-rutinoside,quercetin-7-O-rutinoside and quercetin-3-O-glucoside-7-O-rhamnoside (Ahmed et al., 1972).

Besides the previously identified flavonoids, Quercetin-3-O -glucose-7-O-rhamnoside,quercetin3-O-glucoside andquercetin

http://dx.doi.org/10.1016/j.bjp.2016.04.001

0102-695X/©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

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3-O-[6′′′-␣-l-rhamnosyl-6′′-␤-d-glucosyl]-␤-d-glucoside have beenidentified(Sharafetal.,1997,2000).

StudiesconcerningthesulphurcontentofC.spinosa,revealed thepresenceofbutylisothiocyanate,methylisothiocyanate, iso-propylisothiocyanate,andsec-butylisothiocyanate(Afsharypuor andJazy,1999;Hamedetal.,2007).Nowadays,C.spinosaisalso commerciallycultivatedinseveralcountriesforitsfruits(Gulletal., 2015).

In the present study, the phenolic composition of the hydroethanolicextract(HEE)wascharacterizedusing LC-HRESI-MS–MS (liquid chromatography-high resolution electrospray ionization/mass spectrometry) and X calibur software. While theessentialoilwasdescribed usingGC/MS(Gas chromatogra-phy/massspectrometry).Inaddition,cytotoxicactivityoftheHEE wasevaluatedagainstdifferentcancercelllines.

Materialandmethods

Chemicals

ReagentsforHPLCanalysis:aceticacidandmethanolwereof HPLCgradeandpurchasedfromSigma–Aldrich(Steinheim, Ger-many).

Plantmaterial

Freshplantmaterial(Capparisspinosavar.aegyptia(Lam.)Boiss., Capparaceae)wascollectedfromDahab,SouthSinai,Egypt.The plantwasidentifiedbyAss.Prof.Dr.MonaMarzouk,Department ofPhytochemistryandPlantSystematics,NationalResearch Cen-ter,Egyptandavoucherspecimenoftheaerialpartswerekept attheherbariumofPharmacognosyDepartment,Facultyof Phar-macy,OctoberUniversityforModernSciencesandArts(no.RS014). Theplantsampleswereairdriedintheabsenceofdirectsunlight andgroundjustbeforeextraction.

Extractionofglucosinolates

Powdered air-dried aerial parts (100g) were subjected to hydrodistillation for 3h using Clevenger apparatus. A yellow volatileoilwascollected0.1ml.Thesampleoilwascollectedand freezedtillGC/MSanalysis(AfsharypuorandJazy,1999).

GC–MSanalysisofoil

GC–MSanalysisofthevolatileoilwasperformedusing Hewlett-Packard(HP)6890series(Agilent)GasChromatographySystem, interfacedtoHP5973series(Agilent)massspectrometer,equipped with an auto-sampler and a single capillary injector. TR-FAME (Thermo 260 M142P) (70% cyanopropyl-polysilphenylene sil-oxane)capillaryGCcolumn(30m×0.25mm,i.d.,×0.25␮mfilm thickness)wasused.Samplesizewas1␮l,oventemperature pro-grammedfrom50to230◦Cat5C/min,injectorporttemperature

200◦C,CarriergasHelium,Flowratewas1.5mlHe/min.

Identifica-tionofthevolatileoilconstituentswasbasedoncomparingtheir retentiontimes,andmassfragmentationpatternswiththoseofthe availablereferencesand/orwithpublisheddata(Adams,2004)as wellasthroughNIST-MSdatabaselibrarysearch.Thequantitative estimationwascarriedoutbyrelativepeakareameasurement.

PreparationoftheextractforLC-HRESI-MS–MS

Plant material (100g) was exhaustively extracted with 80% ethanol.Thecombinedhydroethanolicextracts(HEE)werefiltered,

concentratedinvacuumat50◦C,driedandleftforHPLC-MS–MS

analysisandcytotoxicityevaluation.

LC-HRESI-MS–MSapparatus

TheanalysiswasperformedonaBrukermicro-TOF-QDaltonics (API)Time-of-Flightmassspectrometer(Bremen,Germany), cou-pledto1200seriesHPLCsystem(AgilentTechnologies,Waldbronn, Germany),equippedwithahighperformanceautosampler,binary pump,andPDAdetectorG1314C(SL).Chromatographicseparation wasperformedonaSuperspher100RP-18(75×4mmi.d.;4␮m) column(Merck,Darmstadt,Germany).

Identificationofphenoliccompounds

ThemethodwasperformedaccordingtoHassaanetal.(2014). Mobilephaseconsistedoftwosolvents,(A)2%aceticacid(pH2.6) and(B)80%methanol.Theseparationwasperformedusing gradi-entelution,from5%to50%Bat30◦Cataflowrateof100␮l/min.

Theionizationtechniquewasanionspray(pneumaticallyassisted electrospray).Spectrawererecordedinpositiveandnegativeion modebetweenm/z120and1500withcapillaryvoltage,4000V and heated drynitrogen gastemperature,200◦C and flow rate

10l/min,thegasflowtothenebulizerwassetatpressure1.6bar. Forcollision-induceddissociation(CID)MS–MSmeasurements,the voltageoverthecollisioncellvariedfrom20to70eV.Argonwas usedascollisiongas.Dataanalysissoftwarewasusedfordata inter-pretation.Sodiumformatewasusedforcalibrationattheendof LC–MSrun.InterpretationforESI-MSwasperformedbyXcalibur 2.1softwarefromThermoScientific(Berlin,Germany).

Cytotoxicactivity

The cytotoxicity of HEE was assessed using MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay (FotakisandTimbrell,2006)againstthreehumancancercelllines; breast(MCF-7),liver(HEPG-2)andcolon(HCT-116) adenocarci-nomausingDoxorubicin®asreferencestandard.Dosedependent

activitieswerestudiedfrom5 to50␮g/ml,and theIC50 values (concentration which reduced survival to 50%) wereestimated fromgraphicplot.Threeseparateexperimentswereperformedfor eachsample.

Resultsanddiscussion

GC–MSanalysisofthevolatileoil

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Table1

GC–MSanalysisofthevolatileoilconstituentsoftheaerialpartsofC.spinosa.

Peakno. Identifiedcompound RRta Area%

1 Isopropylisothiocyanate 0.72 12.44

2 p-Cymene 0.84 2.93

3 Methylisothiocyanate 1 24.66

4 Butylisothiocyanate 1.17 3.2

5 ␣-Copaene 1.49 4.57

6 Thujone 1.71 1.81

7 Caryophyllene 1.98 2.91

8 Camphor 2.11 1.66

9 Humulene 2.23 4.24

10 1,2,3,5,6,8a-Hexahydro-4,7-dimethyl-1-(1-methylethyl)-naphthalene 2.27 5.56

11 ␥-Muurolene 2.31 2.6

12 Unidentified 2.41 1.17

13 3-Cyclohexen-1-one 2.82 1.21

14 3-Methyl-4-isopropylphenol 3.67 3.98

15 Spathulenol 3.69 1.47

16 Muurolol 3.76 2.42

17 6,10-Dimethyl-2-undecanone 3.84 6.5

18 ␣-Cadinol 3.89 4.26

19 Unidentified 3.91 1.84

20 Unidentified 4.03 0.78

21 Tetracosane 4.22 0.55

22 Eicosane 4.46 1.3

23 Unidentified 4.54 0.75

24 Heptacosane 4.92 2.58

25 1-Naphthalenepropanol 4.99 0.58

26 2-Cyclohexen-1-ol 5.01 0.56

27 1,2-Benzenedicarboxylicacid 5.07 0.91

28 Isobutyl2-methylpent-3-ylester 5.35 1.07

29 Phenanthrene 5.45 1.02

30 Dibutylphthalate 6.24 0.46

Percentageofidentifiedconstituents 95.46%

Percentageofunidentifiedconstituents 4.54%

Majorconstituent Methylisothiocyanate

aRRt,relativeretentiontimeto:methylisothiocyanate=6.99min.

LC-HRESI-MS–MSanalysisofphenoliccompounds

LC–MS analysisdemonstrated thepresence of highly glyco-sylated flavonol in addition toflavone glycosides and phenolic acidderivativesaspreviouslyreportedindifferentcaperextract (Inocencioetal.,2000).Intotal,42compoundsbelongingto differ-entclasseswereidentifiedbyLC–MS/MS-HR-ESIintheHEEofthe aerialpartsofC.spinosa.Dataconcerningtheidentificationofthe peaksareshowninTable2,inwhichwereporttheretentiontime, electrosprayionizationmassspectrometryinnegativeand posi-tiveionmodeforallofthecompoundsdetected.Thestructuresof unknownphenolicacidsorflavonoidswereassessedbasedonthe m/zofbothprecursorionandfragmentionobtained.Moreover,the spectraldatawerecomparedwiththatreportedintheliterature.

Flavonoidswerethe majoridentified components with pre-dominance of flavonol class especially quercetin derivatives (fifteen compounds). New compounds were identified such as quercetintetrahexosideand quercetintetrahexoside dirhamno-side. Kaempferol derivatives have been also identified (seven compounds),isorhamnetinderivatives(sixcompounds)inaddition tomyricetin,eriodictyol,cirsimaritinandgallocatechinderivatives. Sugarmoietiesconsistofhexosides,deoxyhexosidesand pento-sidesasdeducedfromthelossof162,146and132urespectively (Ibrahimetal.,2015).

Quercetin(compound 26)wastentativelyidentified withits basepeakatm/z271.15andcharacteristicpeaksatm/z255,179and 151(Martuccietal.,2014),Quercetintetrahexoside(compound7), tentativelyidentifiedforthefirsttimeinthisgenus,showeda pre-cursorionatm/z949.24[M−H]andtheMS/MSledtoaproduct ionatm/z301.16denotingaquercetinderivative.Inthepositive ionization,precursorionpeakappearedatm/z951.26with prod-uctionsat789.24(M+H-hexose),627.34(M+H-dihexose),465.23

(M+H-trihexose)and303.19(M+H-tetrahexose)anddenotingthe quercetin.

Compound11,wastentativelyidentifiedasquercetin trihexo-siderhamnosidewithaprecursorionpeakat933.25[M−H]and productionpeakatm/z609.28[M−H-dihexose].Positive ioniza-tionshowedaprecursorionat[M+H]atm/z935.26withproduct ionsat789.1[M+H-rhamnose],627.09[M+H-rhamnose-hexose], 465.15 [M+H-rhamnose-dihexose] and 303.13 [M+H-rhamnose-trihexose]. Compound 20, tentatively identified as quercetin dirhamnosidehexosideshowedaprecursorionpeakatm/z755.2 [M−H] with product ions at m/z 593.3 [M−H-hexose], 447.1 [M−H-hexose-rhamnose], 301.2[M−H-hexose-dirhamnose] and acharacteristic peakforquercetinat271.21.Positiveionization showedsimilarfragmentationwith[M+H]atm/z757.22.

Compound34,withaprecursorionpeak[M−H]atm/z1095.25 and[M+H]atm/z1097.32showedproductionpeaksatm/z936.39 [M+H-hexose], 773.31 [M+H-dihexose], 627.19 [M+H-dihexose-rhamnose], 465.11 [M+H-trihexose-rhamnose], 303.07 [M+H-tetrahexose-rhamnose] was tentatively identified as quercetin tetrahexoside rhamnoside. Compound 40 tentatively identified as quercetindihexoside dirhamnoside, showeda precursor ion peak[M−H]atm/z917.23andpositiveionization[M+H]atm/z 919.27withproductionpeakat611.14[M+H-hexose-rhamnose], 465.15[M+H-hexose-dirhamnoside]and303.19 [M+H-dihexose-dirhamnoside].

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Table2

PeakassignmentofmetabolitesinhydroethanolicextractofC.spinosausingLC-HRESI-MS–MSinthenegativeandpositivemodes.

No Rt(min) Compounds [M−H]−

m/z

Negativeionization MS/MS

[M+H]+m/z Positiveionization References

1 1.71 Gluconicacid 195.05 195.05129.07177.1

159.0799.07

197.08 197.08179.09135.10 Felipeetal.(2014)

2 1.89 Quinicacid 191.02 191.02111.08173.04 193.05 193.05175.01161.11

147.04133.03

3 22.23 Chlorogenicacid 353.09 353.09191.11269.21

293.2179.19

355.12 355.10337.19193.06

201.04267.01

Martuccietal.(2014)

4 30.34 Kaempferol-O

-glucoside

447.09 447.09285.14

(M−H-hex)325.21

327.21

449.11 449.11287.11 Rodrigoetal.(1992)

5 45.23 Quercetin-O-glucoside 463.09 463.09301.19

(M−H-hex)343.16

(M−H-120)

465.10 465.10303.10287.13 Martuccietal.(2014)

6 33.31 p-Coumaroylquinic

acid

337.06 337.06191.16179.15

135.22293.2

339.10 339.10320.22293.14

279.11206.18176.16

130.1

Martuccietal.(2014)

7 33.81 Quercetin

tetrahexoside

949.24 949.24625.30

(M−H-dihexose)

301.16

(M−H-tetrahexose)

787.40(M−H-hexose)

463.25

(M−H-trihexose)

951.26 951.26931.43789.24

(−162)627.34(−162)

465.23(−162)303.19

(−162)

8 34.97 Quercetintrihexoside 787.3 787.3621.29625.39

(M−H-hexose)463.31

(M−H-dihexose)

301.24

(M−H-trihexose)

789.23 789.23303.14627.23

(M+H-hex)465.17

(M+H-dihex)

Quetal.(2013)

9 35.43 Epigallocatechin

hexoside

467.12 467.12305.24 469.17 469.17307.02435.91

169.07144.05289.24

10 35.81 Quercetinrhamnoside

hexoside

609.15 609.15301.21

(M−H-Rh-hex)447.28

(M−H-hex)549.28

271.21255.09179.1

611.16 611.16303.12

(M+H-rh-hex)465.06

(M+H-rh)

Faléetal.(2013)

11 36.20 Quercetintrihexoside

rhamnoside

933.25 933.25609.28

(M−H-dihex)301.17

(609-rh-hex)

935.26 935.26789.1(M+H-rh)

627.09(M+H-rh-hex)

465.16(M+H-rh-dihex)

303.13 (M+H-rh-trihex)

12 36.73 Acacetinhexoside 445.21 445.21385.27283.19 447.23 447.23225.04386.94

429.25207.15189.23

LinandHarnly(2010)

13 54.70 Kaempferol 285.04 285.04255.11241.22

229.18213.13163.2

151.1396.97

287.08 287.08241.11269.19

213.07164.99152.99

133.05121.05

Martuccietal.(2014)

14 39.17 Eriodictyolhexoside 449.11 449.11287.15

(M−H-hex)387.28

281.23151.09137.15

451.12 451.12289.15

(M+H-hex)433.22

268.08

Zhaoetal.(2013)

15 39.38 Isorhamnerinhexoside

rutinoside

785.11 785.11623.28

(−M−H-hex)315.12

(M−H-hex-rh-hex)

787.23 787.23317.21

(M+H-rh-dihex)641.13

(M+H-rh)479.15

(M+H-rh-hex)

KimandPark(2009)

16 40.23 Eriodictyol-7-O

-rutinoside

595.17 595.17287.11

(M−H-rutinose)

597.18 597.18435.08

(M+H-hex)289.14

(M+H-hex-rh)577.26

451.07399.2

Zhaoetal.(2013)

17 40.27 Eriodictyol 287.06 287.06151.08125.18

135.07

289.07 289.07163271.13

152.98179.03143.96

Zhaoetal.(2013)

18 41.5 Eriodictyolglucuronide 463.09 463.09287.18301.17

403.23311.26175.09

151.09

Lietal.(2012)

19 43.21 Quercetin

glucoside-O-rutinoside

771.2 771.2609.15(-hex)

463.21(-rh)301.26

(-rh)

773.21 773.21465.18611.10

303.14627.15

Sharafetal.(2000)

20 43.91 Quercetin

dirhamnosidehexoside

755.2 755.2593.3(-hex)

447.1(-rh)301.2(-rh)

271.21

757.22 757.22624.16595.37

611.16449.24303.14

287.21

21 44.62 Myricetinrutinoside 625.14 625.14316.1317.16

(M−H-rh-hex)285.12

271.20479.20

(M−H-rh)607.26

179.08

62.16 627.16319.12

(M+H-rh-hex)464.22

481.11(M+H-rh)

Sójkaetal.(2009)

22 44.82 Cirsimaritinhexoside

rhamnoside

621.15 621.15313.15

(M−H-rh-hex)285.2

257.3

623.16 623.16477.09

(M+H-rh)315.11

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Table2(Continued)

No Rt(min) Compounds [M−H]−

m/z

Negativeionization MS/MS

[M+H]+m/z Positiveionization References

23 59.85 Isorhamnetinhexoside 477.27 477.27315.34

(M−H-hex)

479.12 479.12317.09

(M+H-hex)301.15 360.86419.93459.27

24 45.53 Isorhamnetin

dihexoside dirhamnoside

931.27 931.27767.38

(M−H-hex)621.35 (M−H-hex-rh)315.19 300.08357.35785.40

933.29 933.29317.21

(M+H-dirh-dihex) 914.45771.29 (M+H-hex)769.29 625.22(M+H-hex-rh) 463.2(M+H-rh-dihex)

25 46.75 Gallocatechin 305.07 305.07290.2397.01

225.13275.22259.21

307.11 307.11130.06289.18 262.15242.16204 307.13265.16289.20 247.17178.12144.11

Hossainetal.(2010)

26 47.64 Quercetin 301.04 301.04271.15255.15

257.22179.15151.1

229.93

303.04 303.04285.05302.85 Martuccietal.(2014)

27 47.9 Quercetin-3-O

-hexose-O-pentoside

595.13 595.13301.16

(M−H-hex-pen)285.14

271.22433.43

(M−H-hex)463.27

191.16179.13

597.15 597.15303.12

(M+H-pen-hex)287.12

465.10(M+H-pen)

449.11

Martuccietal.(2014)

28 48.19 Kaempferolhexoside

dirhamnoside

739.21 739.21575.3649.43

429.29284.18255.1

741.22 741.22595.05

(M+H-rh)449.07

(M+H-dirh)287.13

(M+H-dirh-hex)

Rodrigoetal.(1992)

29 48.50 Apigenin8C-glucoside 431.1 431.1311.22

(M−H-120)341.18

(M−H-90)413.21

312.18

433.11 433.11415.23313.16

(M+H-120)397.23

367.18271.16

(M+H-hex)

Abad-Garcíaetal.(2008)

30 49.05 Kaempferolhexoside

rhamnoside

593.15 593.15285.21

(M−H-308)473.24

(M−H-120)503.24

(M−H-90)

595.16 595.16287.05

(M+H-rh-hex)449.11

(M+H-rh)576.38

431.24

Ferreresetal.(2011)

31 49.36 Isorhamnetin3-O

rutinoside

623.16 623.16315.22

(-rutinose)314.15

459.2460.38503.14

(−120)

625.18 625.18317.12

(M+H-rh-hex)479.02

(M+H-rh)

Rorizetal.(2014)

32 50.00 Quercetindihexoside 625.14 625.14301.2

(M−H-dihex)463.14

(M−H-hex)285.26

271.2

627.16 627.16465.11

(M+H-hex)303.10

(M+H-dihex)

Tedescoetal.(2015)

33 51.22 Kaempferol

glucuronide

461.07 461.07285.18381.27 463.09 463.09343.14427.21

299.11268.23

Martuccietal.(2014)

34 51.90 Quercetin

tetrahexoside rhamnoside

1095.29 1095.29609.3(rutin)

787.28(M−H-hex-rh)

483.2933.44

(M−H-hex)302.33

1097.32 1097.32627.19

(M+H-rh-dihex)936.39

(M+H-hex)773.31

(M+H-dihex)465.11

(M+H-trihex-rh) 303.07

(M+H-tetrahex-rh)

449.19(Q-rh)611.09

(rutin)787.39

35 54.13 Isorhamnetin 315.1 315.1271.2285.18

287.11300.11

317.07 317.07302.09285.1

271.13257.12165.07

139.02

Sánchez-Rabanedaetal.(2004)

36 54.24 Kaempferolrutinoside

hexoside

755.2 755.2285.15

(M−H-hex-rh)593.32

(M−H-hex)

757.22 757.22449.12

(M+H-hex-rh)595.15

(M+H-hex)611.10

(M+H-rh)287.09

(M+H-trihex-rh)

37 54.49 Myricetinhexoside

rutinoside

787.17 787.17625.24

(M−H-hex)479.23

(M−H-hex-rh)317.21

(M−H-dihex-rh)

789.19 789.19481.14

(M+H-hex-rh)319.2

(M+H-dihex-rh)

38 55.18 Isorhamnetin

glucuronide

491.08 491.08315.20 493.1 493.1317.16 Due ˜nasetal.(2008)

39 55.47 Quercetinacetyl

hexoside

505.13 505.13353.29459.29

485.31323.24301.20

151.03179.12191.09

Navarro-Gonzálezetal.(2015)

40 58.05 Quercetindihexoside

dirhamnoside

917.23 917.23609.31

(M−H-308)301.16

(M-H-308)

919.27 919.27611.14

(M+H-308)465.15

(M+H-rh)449.16

(6)

Table2(Continued)

No Rt(min) Compounds [M−H]−

m/z

Negativeionization MS/MS

[M+H]+m/z Positiveionization References

41 60.68 Myricetinhexoside 479.16 479.16317.18

(M−H-hex)273.18 258.29447.31461.34 389.12(M−H-90) 359.28(M−H-120)

481.19 481.19319.17

(M+H-hex)464.25 302.14285.21191.14

Raaletal.(2015)

42 62.14 Kaempferoldihexoside

dirhamnoside

901.24 901.24593.23

(M−H-rh-hex)285.2

(M−H-dirh-dihex)

739.44(M−H-hex)

903.27 903.27884.47

(M+H-H2O)287.16

(M+H-dirh-hex)757.16

(M+H-hex)595.23

(M+H-rh-hex)449.08

(M+H-dirh-hex)

rh,rhamnose;hex,hexose;Q,quercetin;pen,pentose.

Fragmentionsarelistedinorderofrelativeabundances.Ionsinboldfaceindicatethemostintenseproduction(100%relativeintensity).

Table3

CytotoxicactivityoftheHEEoftheaerialpartsofCapparisspinosaandDoxorubicinstandardagainsthumanbreastadenocarcinomacells(MCF-7),hepatocellularcarcinoma

cells(Hep-G2)andcoloncarcinomacells(HCT-116).

Cellline MCF-7 Hep-G2 HCT-116

C.spinosaHEE Doxorubicin C.spinosaHEE Doxorubicin C.spinosaHEE Doxorubicin

IC50(␮g/ml) 24.5±2.23 0.426±0.35 24.4±1.87 0.467±0.2 11±1.54 0.23±0.17

Dataarepresentedasmean±SDofIC50(␮g/ml)from3independentexperiments,triplicateforeach.

withproductionpeaksat611.10[M+H-rhamnose],449.12 [M+H-rhamnose-hexose]and287.09[M+H-rhamnose-dihexose].

Compound42,identifiedaskaempferoldihexoside dirhamno-side,showedaprecursorionpeak[M-H]atm/z901.24andpositive ionization[M+H]atm/z903.27withproductionsatm/z757.16 [M+H-rhamnose],595.23[M+H-rhamnose-hexose],449.08 [M+H-dirhamnose-hexose]and287.16[M+H-dirhamnose-dihexose].

IsorhamnetinhasbeenpreviouslyidentifiedinCapparis, how-ever, its derivatives have not. Compound 15, witha precursor ion[M-H]atm/z785.11and[M+H]atm/z787.23anddaughter ion peak at 641.13 [M+H-rhamnose], 479.15 [M+H-rhamnose-hexose]andbasepeakatm/z317.21[M+H-rhamnose-dihexose] wasidentified as Isorhamnetin hexoside rutinoside.Compound 23 was tentatively identified as isorhamnetin hexoside with [M−H]atm/z477.27whilecompound 24,tentativelyidentified as isorhamnetindihexoside dirhamnosidewith a precursor ion peak[M−H]atm/z931.27and[M+H]atm/z933.29,productions inthepositiveionizationat771.29[M+H-hexose],625.22 [M+H-hexose-rhamnose],463.2[M+H-dihexose-rhamnose] and317.21 [M+H-dihexose-dirhamnose].

TheseresultswereinagreementwithGulletal. (2015)and

Behnazetal.(2013)whoreportedC.spinosaleavestobeagood sourceof kaempferol,quercetin, rutin andisorhamnetin. While

Siracusaetal.(2011)reportedrutin,kaempferol,3-O-rutinoside, andisorhamnetin3-O-rutinosideasmajorflavonoidsinC.spinosa. Myricetinanditsderivativeshavenotbeenpreviouslyidentified inCapparis,however,itwastentativelyidentifiedwithits charac-teristicpeakatm/z317inthenegativeionizationandm/z319in thepositiveionization.

Flavoneswerealsopresent.Compound29,wasidentifiedas api-genin8-C-glucosidewithprecursorionatm/z433[M+H]inthe positiveionizationmode.Theproductionsobtainedwith cleav-ageofsugarringhavebeenproposedasdiagnosticions,where m/z313isobservedand m/z283diagnosticof6-C-glucoside is absent(Abad-Garcíaetal.,2008).C-glycosylflavonesproducesMS fragmentationpatternincludingdehydrationandcrossring cleav-ageoftheglucosemoiety thatproduces0,2 crossringcleavage [M−H-120]and0,3crossringcleavage[M−H-90](Martuccietal., 2014).

Phenolicacidswerealsoidentifiedasquinicacid,p-coumaroyl quinicacidandchlorogenicacid.

CytotoxicactivityoftheHEE

HEEofC.spinosashowedpromisingcytotoxicactivity.IntheUS NationalCancerInstitutePlantScreeningProgram,acrudeextract isgenerallyconsideredtohaveinvitrocytotoxicactivityiftheIC50 valueincarcinomacells,followingincubationbetween48and72h, islessthan20␮g/ml(Boik,2001).HCT-116wasfoundtobethe mostsensitivetoHEEwithcellviabilitylessthan50%atthe con-centrationof11±1.54␮g/ml.Investigationshoweddifferencein sensitivityofdifferentcancerouscells totheCapparisextractas hasbeenobservedwithmanyotherspecies(Foucheetal.,2008). PotencyagainstdifferentcancercelllineswaspresentedinTable3. Flavonoidshavebeenreportedtoexhibitprooxidantcytotoxicity againstcancercellsthroughtheROS-triggeredmitochondrial apop-toticpathway,therefore,responsibleforthepromisingcytotoxicity ofC.spinosaagainstvariouscancercelllines(Zhangetal.,2015).

Conclusion

C.spinosaisarichsource,notonlyofsulphurcompounds,but also withphenolic and flavonoid glycosides contributing toits powerfulcytotoxicactivity.C.spinosaissuggested tobeagood candidateforuseinnaturalmedicinewithhistoricalbackground.

Author’scontributions

RO suggested the point, wrote the manuscript, carried out extractionproceduresand interpretedLCMSanalysis.MH inter-pretedtheGC/MSdataofessentialoilandcytotoxicactivityofHEE andrevisedthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

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