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 and11g/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://
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.,×0.25mfilm thickness)wasused.Samplesizewas1l,oventemperature pro-grammedfrom50to230◦Cat5◦C/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.;4m) column(Merck,Darmstadt,Germany).
Identificationofphenoliccompounds
ThemethodwasperformedaccordingtoHassaanetal.(2014). Mobilephaseconsistedoftwosolvents,(A)2%aceticacid(pH2.6) and(B)80%methanol.Theseparationwasperformedusing gradi-entelution,from5%to50%Bat30◦Cataflowrateof100l/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 to50g/ml,and theIC50 values (concentration which reduced survival to 50%) wereestimated fromgraphicplot.Threeseparateexperimentswereperformedfor eachsample.
Resultsanddiscussion
GC–MSanalysisofthevolatileoil
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].
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
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
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, islessthan20g/ml(Boik,2001).HCT-116wasfoundtobethe mostsensitivetoHEEwithcellviabilitylessthan50%atthe con-centrationof11±1.54g/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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