Rev. bras. farmacogn. vol.27 número5

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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

Gamma

radiation

treatment

activates

glucomoringin

synthesis

in

Moringa

oleifera

Tsifhiwa

Ramabulana

a

_,

_Risimati

_D.

_Mavunda

b,c

,

Paul

A. Steenkamp

a,d

,

Lizelle

A. Piater

a

,

Ian

A. Dubery

a

_,

_Ashwell

_R.

_Ndhlala

e

_,

_Ntakadzeni

_E.

_Madala

a,∗

a_Department_of_{Biochemistry,}_University_of_{Johannesburg,}_Auckland_Park,_South_Africa b_Department_of_Physics,_University_of_{Johannesburg,}_Auckland_Park,_South_Africa c_South_African_Nuclear_Energy_Corporation,_Pretoria,_South_Africa

d_Council_of_Scientific_and_Industrial_Research,_Biosciences,_Natural_Products_and_{Agro-processing}_Group,_Pretoria,_South_Africa e_Agricultural_Research_Council,_Vegetable_and_Ornamental_Plants,_Pretoria,_South_Africa

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13February2017 Accepted14May2017 Availableonline24August2017

Keywords: Gammaradiation Glucosinolates Metabolitefingerprinting Oxidativestress UHPLC-qTOF-MS

a

b

s

t

r

a

c

t

Plantsareaveryrichsourceofpharmacologicallyrelevantmetabolites.However,therelative concentra-tionsofthesecompoundsaresubjecttothegeneticmake-up,thephysiologicalstateoftheplantaswellas environmentaleffects.Recently,metabolicperturbationsthroughtheuseofabioticstressorshaveproven tobeavaluablestrategyforincreasingthelevelsofthesecompounds.Oxidativestress-associated stress-ors,includingionizingradiation,havealsobeenreportedtoinducemetaboliteswithvariousbiological activitiesinplants.Hence,theaimofthecurrentstudywastoinvestigatetheeffectofgammaradiation ontheinductionofpurportedanti-cancerousmetabolites,glucomoringinanditsderivatives,inMoringa

oleiferaLam.,Moringaceae.Here,anUHPLC-qTOF-MS-basedtargetedmetabolicfingerprintingapproach

wasusedtoevaluatetheeffectofgammaradiationtreatmentontheafore-mentionedhealth-beneficial secondarymetabolitesofM.oleifera.Followingradiation,anincreaseinglucomoringinandthree acyl-atedderivativeswasnoted.Assuch,thesemoleculescanberegardedascomponentsoftheinducible defensemechanismofM.oleiferaasopposedtobeingconstitutivecomponentsasithaspreviouslybeen assumed.Thismightbeanindicationofapossible,yetunexploredroleofmoringinagainsttheeffects ofoxidativestressinM.oleiferaplants.Theresultsalsosuggestthatplantsundergoingphoto-oxidative stresscouldaccumulatehigheramountsofglucomoringinandrelatedmolecules.

Introduction

Glucosinolates(GS)aresecondarymetabolitesfoundinalmost

all plants of the order Brassicales (Fahey et al., 2001; Mithen,

2001).These compoundsarediversein origin,side chain

mod-ification,degradation and final biological functions (Grubb and

Abel,2006), and comprise short- and long-chain aliphatic

glu-cosinolates (Ile, Leu, Val, Ala and Met), indolic glucosinolates

(Trp) and aromatic glucosinolates (Tyr and Phe)(Brown et al.,

2003;Clarke,2010;AgerbirkandOlsen,2012;Leoneetal.,2015).

Undernormalconditions,GSarechemicallystable,however,

dur-ingplantwoundresponsesthesecompoundsarehydrolyzedby

theenzymemyrosinasetoproduceisothiocyanates,nitriles,

thio-cyanates,epithionitrilesand oxazolidineswhichareresponsible

∗ Correspondingauthor.

E-mail:emadala@uj.ac.za(N.E.Madala).

forthereportedbiologicalactivitiesthereof(BonesandRossiter, 2006; Zandalinasetal., 2012).GS-derivedmolecules arehighly

water-solubleduetothehydroxyl-aminosulfategroupanda

␤-thioglucosylresidueattachedtothevariableR-groupontheGS

skeletal structure (Clarke, 2010; Vo et al., 2013; Förster et al., 2015a; Leone et al., 2015), thereby contributing to a high

bio-availabilityfollowinghumanconsumption.Inplants,GSareknown

toberesponsivetobothbioticandabioticstresses,andhavebeen

shown tobeinduced byvarious environmentalfactorssuchas

solarradiation,temperaturevariationandclimatechanges(Bones

andRossiter,2006;Zandalinasetal.,2012).Almostallthe

afore-mentionedstressorsofplantsareassociatedwithoxidativestress

(Bajguzand Hayat,2009; Demidchik,2015), therebysuggesting

a possible role of these compounds in mitigating the damages

imposedasaresultofsuchstress,aphenomenonwhichhasalso

been extended tohuman-related diseases (Tumer et al., 2015;

Williamsonetal.,1998).

http://dx.doi.org/10.1016/j.bjp.2017.05.012

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T.Ramabulanaetal./RevistaBrasileiradeFarmacognosia27(2017)569–575

Recently, the GS components (4-(␣-l

-rhamnopyranosyloxy)-benzyl glucosinolate followed by three isomeric acetyl-4-(␣-l

-rhamnopyranosyloxy)-benzylglucosinolate(Ac-isomer-GSI,II,III)

ofMoringa oleiferaLam.have beenreportedtopossessindirect anti-oxidantactivityduetotheabilitytoregulateananti-oxidant

enzymaticprocessesinmammaliansystems(Tumeretal.,2015).

GShavealsobeenreportedtocontrolthedamagesofother

physio-logicalconditionsassociatedwithoxidativestresssuchasreducing

therisks of several cancers (colon, bladder and breast cancer)

(Björkmanetal.,2011).IthasbeenshownthatGShavetheability

toinactivatephaseIenzymes(cytochromeP-450)ortostimulate

phaseIIenzymes(glutathione-S-transferase),therebyeliminating

carcinogenicmetabolites(ZhangandTalalay,1994).Morerecently,

consumptionof GS derivedcompounds suchas isothiocyanates

(ITC)hasbeenshowntobebeneficialformammals,sincetheylead

totheup-regulationofxenobioticmetabolism(phaseIImetabolic

enzymes),associatedwithanincreasesintheantioxidant

capac-ity,thusleadingtoimprovedprotectionagainstvariouschronic

physiologicalconditions(TrakaandMithen,2009).

Aspreviouslymentioned,thelevelsofGSinplantsaresubject

toseveralenvironmentalfactorsand,assuch, createconditions

favoringtheproductionofthesecompoundsbyplants,ashasbeen

investigated(Försteretal.,2015a).AnincreaseinaGS,

glucotropae-olin,duetoUV-Bradiationtreatmentofnasturtium(Tropaeolum

majusL.)plantshasbeenreported(Schreineretal.,2009). Differ-entformsofradiationareknowntoinduceoxidativestressinplants (Esnaultetal.,2010;Hollósy,2002;KovácsandKeresztes,2002),

andthustheinvolvementofGScouldbetocontrolthedamagesof

radiation-inducedoxidativestress.

Moringaoleiferaisaversatileandwidelycultivatedspeciesin

themonogenericfamilyofMoringaceae,andisknowntocontain

GSmolecules(Fahey,2005;Försteretal.,2015a;Moyoetal.,2011; PopoolaandObembe,2013).AlmostallpartsoftheM.oleiferaplant

containvaryingamountofaromaticGSs,withtheleavescontaining

thehighestlevels(Clarke,2010;Moyoetal.,2011).Someofthe

reportedpharmacologicalpotencyoftheplanthavebeendirectly

correlatedtothepresenceoftheseGSs(Clarke,2010).

Recently,wehaveshownthatM.oleiferadoesnotproduceother

highlysoughtafterpharmacologicallyrelevantmetabolites(rutin

foranexample)incomparisontootherrelatedspecies,M.

oval-ifolia(Makitaetal.,2016).Moreover,wefurtherspeculatedthat

productionofsuchmetabolitescouldbeinfluencedbyvarious

fac-torssuchasenvironmentalconditionsand thegeneticmake-up

oftheplants(Makitaetal.,2016).Elsewhere,thelevelsof

health-promotingmetaboliteshavebeenshowntobeaffectedbyionizing

radiation(Ramabulana etal.,2015,2016).Radiationis apotent

inducerofoxidativestress,andithasbeenusedtoidentify metabo-liteswithanti-oxidativepropertiesinvariousplants(Mittler,2002).

Withincreasingevidenceontheanti-oxidativepropertiesofGS

molecules(Guerrero-Beltránetal.,2012)andaspotentialagents

for ameliorating oxidative stress-associated diseases (

Dinkova-KostovaandKostov,2012),itisimportanttostudybioticandabiotic

factorswithpotentialofenhancingthelevelsofthesecompounds.

Assuch,inthecurrentstudy,apotentformofradiation,namely

gammaradiationwasusedtotriggeroxidativestressinM.oleifera

leaves.SubsequentperturbationsinthelevelsofGSswere

moni-toredusingUHPLC-ESI-qTOF-MS-basedfingerprinting.

Materialsandmethods

Plantmaterial

TwomontholdMoringaoleiferaLam.,Moringaceae,plantswere

obtainedfrom thePatience Wellness Centre farm in

Lebowak-gomo,South Africa.The plantspecieswasauthenticated, and a

voucherspecimen(withvouchernumberNEM001)wasprepared

anddepositedattheDepartmentofBotany,Universityof

Johan-nesburg,SouthAfrica.

Gammaradiationprocedure

Plants were irradiatedas previously described (Ramabulana

etal.,2015,2016).IrradiationwasperformedatNuclearEnergy

CooperationofSouthAfrica(NECSA)(Phelindaba,Pretoria,South

Africa). Briefly, fifteen plants were irradiated with a Cobalt-60

source(atadoserateof22kGy/h)insideawell-protectedchamber, alongwithfifteennon-irradiatedcontrolplants.Variousradiation

doses(0.1–8kGy)weretestedand2kGydosewasfoundtobemore

potentasshownpreviously(Ramabulanaetal.,2015).Total

radi-ationdoseabsorbedbyplantswasfurtherconfirmedbyHarwell

PerspexPolyMethylMethacrylateAmber(PMMA)3042

dosime-ters(HarwellCo,UnitedKingdom).

Metaboliteextraction

From the optimization results achieved with our preceding

studies, plant leaf material was harvested a day (24h)

post-radiation and dried at 50◦C for 72h(Ramabulana et al., 2015,

2016).Thedried plantmaterial wasground andextracted with

80%aqueousmethanolasdescribedbyRamabulanaetal.(2015,

2016).Theextractswereconcentrated,reconstitutedin50% aque-ousmethanolandstoredat−20◦Cuntilanalyzed.

Chromatographyandmassspectrometryanalyses

Threetechnicalrepeatsofthehydromethanolicextracts(5␮l)

were analyzed using an Acquity UHPLC equipped with an

AcquityBEHC18reversephasecolumn(150mm×2.1mm,1.7␮m)

(WatersCorporation,MA,USA).ThemobilephaseAconsistedof

0.1%formic acidin deionizedwater, while themobile phase B

consistedof0.1%formicacidinacetonitrile(RomilPureChemistry,

Cambridge,UK).Theelutiongradientstartedat98%Auntil5%at

26minfor2min,andthenreturnedtoinitialconditionsof98%Aat

28minfor2minwitharuntimeof30minataconstantflowrate

of0.4ml/min.Chromatographicseparation/elutionwasmonitored

usinga photodiode-arraydetector(PDA)collecting20 spectra/s

betweenthe200and500nmrange.Inaseconddetection,aSynapt

G1high-definitionmassspectrometer(MS) wasusedoperating

inbothpositiveandnegativeelectrosprayionization(ESI)modes.

Briefly,thefollowingMSconditionswereusedasoptimal

exper-imentalconditions:thecapillaryvoltageof2.5kV,multichannel

platedetectorpotentialof1600V,sampleconepotentialof30V,

desolvationtemperatureof450◦C,sourcetemperatureof120◦C,

conegasflow of 50l/hand desolvation gasflow of550l/h.For

MSfragmentationexperiments,theMSacquisitionmethodwith

lowcollisionenergyrampof10–30eVandahighcollisionenergy

rampof15–60eVwasusedtogeneratetypicalMSE

fragmenta-tionpatterns.MassLynxTM_and _MarkerLynxTM _software_(Waters

Corporation,MA, USA) were used tovisualize and analyzethe

UHPLC-qTOF-MSrawdatasoastogeneratedatamatrixforfurther

statisticalmodeling.

Metaboliteidentificationandstatisticalanalyses

TheUHPLC-ESI-MSdatacollectedinnegativeionizationmode

wasanalyzedusingMarkerLynxTM_XS_software_for_peak_alignment,

peak finding, peak integration and retention time (Rt)

correc-tionwiththefollowingparameters:Rt rangeof1–27min,mass

rangeof 100–1000Da, masstoleranceof0.05Da,Rt windowof

0.2min.Datawasnormalizedtototalintensity(area).Theacquired

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Sweden)forPrincipalcomponentanalysis(PCA)andOrthogonal

projection to latent structures-discriminant analysis (OPLS-DA)

computation(Ramabulanaetal.,2015)and,usingthesemodels,

possiblebio-markersshowingdifferentialaccumulationacross

dif-ferenttreatmentswereidentified(Madalaetal.,2012;Ramabulana etal.,2015).ThedatamatrixwasalsoexportedtoMicrosoftExcel

and,usingtheareaunderthepeakcorrespondingtothe

respec-tivemasses(m/z)ofknownGSmoleculesfromM.oleifera(Förster

etal.,2015a,b),weresearchedforandfurtherusedtocreate

box-and-whiskersplotsusingSPSSversion22 software(IBM,United

StatesofAmerica,www.ibm.com/SPSSStatistics).Furthermore,GS

moleculeswithstatisticalsignificancewerecomputedusingthe

studentt-testinMicrosoftExcel.Here,ap-valueof<0.01indicates thatthefoldincreasesoftheidentifiedmetabolitesarestatistically significant.

Tofurtherconfirmtheidentificationofmetabolites,the

frag-mentationpatternsgeneratedwiththeuseofdifferentcollision

energies were compared with the already existing knowledge.

Briefly,themolecularformulaeofallthepeakscorrespondingto

GSmoleculeswerecomputedandselectedbasedonthecriterion

thatthesearewithin5mDamassaccuracywhencomparedtothe

calculatedmassofthecorrespondingmolecules.Metaboliteswere

thusannotatedaccordingtotheMetabolomicStandardsInitiatives,

level2identification(Sumneretal.,2007).

Resultsanddiscussion

Gammaradiation isan inducerof oxidative stressthat

sub-sequently activates complicated defense mechanisms in plants

(Ahujaetal.,2014;Esnaultetal.,2010).M.oleiferaisableto

syn-thesizeGSaspartofitssecondarymetabolites.Thepredominant

GSmoleculeinthisplantspeciesis4-(␣-l

-rhamnopyranosyloxy)-benzylglucosinolate(1),knownasglucomoringin(Clarke,2010;

deGraafetal.,2015;Tumeretal.,2015).Thestructuraluniqueness

oftheseGSderivesfromthepresenceofasecondglycosylresidue

inadditiontothealreadyglycosylatedsidechain(Amagloetal.,

2010).Otherstructuralderivativesofglucomoringinsuchasthe

acylatedformsthereofhavealsobeenreportedinthisplant(Fig.1) (Försteretal.,2015a),makingthesemoleculesinterestingtostudy.

Moreremarkably,glucomoringinhasalwaysbeenthoughttoexist

onlyinM.oleifera.However,ithasalsobeenrecentlyreportedin Noccaeacaerulescensbuttheauthorscouldnotidentifythe acety-latedforms(deGraafetal.,2015).Thissuggeststheacylationof

glucomoringintobeanexclusivephenomenonofM.oleifera.

Inthecurrentstudy,gammaradiation-inducedoxidativestress

resultedinchangestothemetabolomeinM.oleiferaplants(Fig.2, Table1).UsinganUHPLC-ESI-qTOF-MS-basedtargetedmetabolite

fingerprinting approach, increasedlevels of GS molecules were

foundinplantsirradiatedwitha2kGydoseofgammaradiation

as compared to the control plants (Fig. 2; Table 1). Here, the

box-and-whiskers plots display an increase in the

concentra-tionsof glucomoringin and related GS molecules in M. oleifera

followinggammaradiationtreatment(Fig.2).Theaboveresults

provideasemi-quantitativeoverviewoftheamountofGSandits

derivativessince there arenocommerciallyavailablestandards

ofthesemoleculestoachieveabsolutequantification.Moreover,

the results indicate that the fold increase in the identified GS

werestatisticallysignificant,withalmostallhavingp-valuesof

less than 0.01 as shown in Table 1. Interestingly, it should be

re-emphasizedthatadoseof2kGywasfoundtobemorepotent

and non-lethal, thus inducing the highest levels of GS and its

derivatives.Preliminaryoptimizationshowedlowerdoses(0.1,0.5

and1.0kGy)tominimallyaffectthelevelsofGSanditsderivatives

butthelevelsabove2kGysuchas4kGand8kGywerefoundto

belethal,thuskillingtheplantsimmediatelyafterradiation.The

above phenomenon was also highlighted in studies conducted

withanotherplant,Phaseolusvulgaris(Ramabulanaetal.,2015).

Furthermore, the characterization of these metabolites was

achievedbymeansofaccuratemassMSresults(asshowninFig.3)

withtheuseoffragmentationpatternsandcomparisontoalready

publisheddata.Brieflymolecule1withprecursorion([M−H]−)at m/z570.0927(C20H29NO14S2)andRtof3.17minwasidentifiedas

4-(␣-l-rhamnosyloxy)-benzylglucosinolate(glucomoringin).The

acylatedformsofglucomoringin(2–4)producedisobaricprecursor ionsatm/z612.102(C22H31NO15S2).Interestingly,thesemolecules

elutedatdifferentRtand,inaccordancewithalreadypublished

results(Försteretal.,2015a;Tumeretal.,2015),thesethreeisomers

wereidentifiedasacetyl4-(␣-l-rhamnopyranosyloxy)-benzylGS

isomerI(2),II(3)andIII(4)elutingatRtof5.60min,6.46minand 9.63minrespectivey(Bennettetal.,2003;Försteretal.,2015a,b) (Table1).

R₃O

R₂O

CH₂OH OH OH OH

S

S N

OR₁

1 R₁=R₂=R₃=H

2 R₁=Ac; R₂=R₃=H

3 R₁=R₃=H; R₂=Ac

4 R₁=R₂=H; R₃=Ac O

O

O O

O

O O

Thepresenceoftheseacetylatedisomersposeanother

interest-ingbutchallengingdimensiontoourresultssincethefunctionof

100

0

3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00Time

9.60 612.1011

Ac-Isomer-GS III

Ac-Isomer-GS II Ac-Isomer-GS I

α-Isomer-GS

9.46 612.1042 5.60

612.1013 3.17

570.0936

%

Fig.1. UHPLC-ESI-qTOF-MSanalysesinnegativeionizationmodeofhydromethanolicextractsfrom2kGygammairradiatedMoringaoleiferashowingbasepeakintensity(BPI) chromatogramsof4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolate(␣-rhamnoGS),acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolateisomerI(Ac-isomer-GSI),

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800

A

B

C

D

600

Peak intensity

P

eak intensity

400

200

0

200

100

50

0

Control Treated Control Treated

120

100

80

60

40

20

0

500

400

300

200

100

0

Fig.2.Box-and-whiskersplotsshowingrelativecompositionoffouridentifiedglucosinolates(moringinderivatives),increaseddueto2kGygammaradiationtreatment ofM.oleiferawithstatisticalsignificanceofp<0.01.(A)4-(␣-l-Rhamnopyranosyloxy)-benzylglucosinolate;(B)acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolate

isomerI;(C)acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolateisomerII;and(D)acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolate,isomerIII.

Table1

Gammaradiation-inducedglucomoringinmoleculesinMoringaoleifera.

Peak Rt(min) Compoundname Mass(m/z) Elementalcomposition p-values Foldchange

1 3.09 4-(␣-l-Rhamnopyranosyloxy)-benzylglucosinolate(1) 570.0922 C₂₀H₂₉NO₁₄S₂ 1.7_×10−5 22

2 5.60 Acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolateisomerI(2) 612.1029 C₂₂H₃₁NO₁₅S₂ 1.4_×10−5 30

3 6.46 Acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolateisomerII(3) 612.1004 C22H31NO15S2 3.7_×10−5 51

4 9.54 Acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolateisomerIII(4) 612.1044 C22H31NO15S2 1.5_×10−8 10

thismodificationandtheeffectonthebiologicalactivityof glu-comoringinarenotknown.Thepresence ofstructurallyrelated (isomeric) metabolitesin plants is a knownphenomenon with aclassicalexamplebeingpositionalisomersofchlorogenicacids (Ncubeetal.,2014,2016).However,thepresenceofpositional iso-mersofchlorogenicacidsinplantsisalsonotfullyunderstood;but recentlyithasbeenspeculatedtobeastrategydeployedbyplants

toincreasetheconcentrationofthesemoleculesthrough

diversi-fication,soastocreatearichreservetobeutilizedwhenneeded (Karaköseetal.,2015).Assuch,thesamephenomenoncouldbetrue forthecaseofM.oleiferabutmoreresearchisneededtovalidatethis

hypothesis.Althoughalltheseisomersincreasedconcomitantly,

therelativeabundancelevelsinirradiatedplantsdiffered(Fig.1),

suggestingvaryingstabilityamongstthesecompounds.However,

elsewheretheseacetylisomerswerefoundtobeaffectedbythe

typeof extractionmethodand significantrearrangementswere

noted,withastandardofacetyl-isomer-GSIIIbeingconvertedto

acetyl-isomers-GSIandIIinabufferedsystemduetoanapparent

acetylmigration(Försteretal.,2015a).Thus,itcanbepostulated thatthediversityofGSmoleculesinM.oleiferacouldbetheresultof

bothenzymaticandnon-enzymaticreactionsinabiologicalsystem

respondingtoanoxidativestressenvironment.

EventhoughtheMSdatawasacquiredusingbothpositiveand

negativeESImodes,onlytheESInegativedatawasfoundtobe

suitableforidentificationoftheGSmoleculesandthiscouldbedue tothefactthatthesemoleculesareinherentlynegativelycharged).

TheaccurateMSspectraofthesemoleculescollectedatelevated

collisonenergy(30eV)areshowninFig.3.

Usingacombinationofmultivariateandunivariatestatistical

models(datanotshown),underlyingdifferencesinpeak

intensi-tiesoftheextractsobtainedfrombothcontrolandirradiatedplants

werenoted.ThesedifferencesinthelevelsofGSmoleculesisan

indicationofinduction oftheglucomoringinbiosynthesis

path-wayinreponsetotheoxidativestresstriggeredbytheradiation

treatment.Aspreviouslystated,GSmoleculeshavebeenshown

toaccumulatein plantsirradiatedwithUV-radiation(Schreiner

etal.,2009),whileotherresearchhasreportedthesemolecules

to be constitutively present in M. oleifera leaf extracts (Fahey,

2005;Försteretal.,2015b; Jansenetal.,2008;Vo etal.,2013).

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100

100 200 300 400 500 600 700 800 900 1000

0

%

100

100 200 300 400 500 600 700 800 900 1000

0

%

100

100 200 300 400 500 600 700 800 900 1000

0

%

100

100 200 300 400 500 600 700 800 900 1000

0

%

259.0064 _328.0791 424.0306

570.0905

A

B

C

D

571.0988

650.0465 716.1448 _952.1398

958.2131 708.1854

613.1076 612.0959

533.1224 323.1260

191.0526

193.0438

367.0981

612.0970

533.1294

613.1055

708.1782 966.2234

858.1284 693.0696

613.1198 612.1079

549.2479 259.0061 370.0934

Fig.3. SpectraofidentifiedGSsinM.oleiferaleafextractsofplantsirradiatedwith2kGydoseofgammaradiation.(A)4-(␣-l-Rhamnopyranosyloxy)-benzylglucosinolate;

(B)acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolateisomerI;(C)acetyl-4-(␣-l-rhamnopyranosyloxy)-benzylglucosinolateisomerII;and(D)acetyl-4-(␣-l

-rhamnopyranosyloxy)-benzylglucosinolateisomerIII.

induciblecomponentsofthisplantspeciesasthesewerefound

toincreaseuponradiationtreatment.Generally,GSmoleculesare

knownto respondagainst plantwounding (Bodnaryk, 1992), a

phenomenonwhichisinevitableduringleafharvestingandcould

furtherexplainwhythesecompoundsarereportedinnon-induced

leaveselsewhere(Rodríguez-Pérez et al.,2015).Previously,the

distributionand presence of these molecules inM. oleifera has

alsobeenreportedwithmixedoutcomes.Forinstance,onlyone

glucomoringinmoleculewasidentifiedinthecurrentstudybut

three distinct glucomoringin molecules were identified in M.

oleiferafromMadagascar(Rodríguez-Pérez etal.,2015).As

pre-viouslystated, glucomoringinwas alsorecentlyidentified in N.

caerulescens plants, but the distribution was only limited to a

fewsamplesanalyzedandabsentinotheraccessions/cultivars(de

Graafetal.,2015).Thesameauthorsconcludedthatthese differ-encesareduetoregionalgeneticvariationratherthantheinitially

thoughtenvironmentalfactorssuchas metaltoxicity (deGraaf

et al.,2015).Geneticvariation was furtherusedtojustify why

glucomoringinwasneverdetectedinsomespeciesrelatedtoN.

caerulescens(Tolràetal.,2000;Asadetal.,2013).Recently,

acetyl-(4-␣-l-rhamnopyranosyloxy)-benzyl GS isomers were foundto

onlyaccumulateinsome,butnotallM.oleiferaplantsofthesame ecotype(Försteretal.,2015b).Takentogether,alltheaboveresults

areanindicationthatthepresenceandrelativeconcentrationof

these compounds are subject tounderlying cellular conditions

or geneticmakeup of plants. Assuch, not allGS-containing M.

oleiferaplantswillhavesimilarGS-mediatedbio-activities. There-fore,studiesofconditionswiththeabilitytoincreasethelevelsof GSmoleculesinplantscapableofGSsynthesisareimportant.Inthis

regard,thedistributionofGSmoleculesinM.oleiferahasbeen stud-iedbyvaryingthecultivationconditionssuchassulfurfertilization

andwateravailability,andithasbeenshownthattheGScontent

increasedunderawater-deficientregiment,withtheeffectmore

pronouncedinselectedecotypes(Försteretal.,2015b).Thisagain

highlightstheimportanceofgeneticvariationandabioticstress

conditions.

Inthecurrentstudy,anincreaseinGScontentduetogamma

radiationwasnotedand,moreimportantly,alltheirradiatedplants exhibitedaconsistentresponse.Ingeneral,theinvolvementofGSs

againstoxidativestresscausedbybioticandabioticstresseshas

beenreportedelsewhere(Björkmanet al.,2011; Sardansetal.,

2011;Zhangetal.,2011;Zandalinasetal.,2012).Accumulation

oftheGScontentinplantstreatedwithradiation(i.e.UVlight)

hasbeenreportedinT.majus(Schreineretal.,2009), Arabidop-sisthaliana(Wangetal.,2011)andbroccoli(Pérez-Balibreaetal., 2008;Mewisetal.,2012).Therefore,takentogether,theincreasein

GSmoleculesinresponsetoamorepotentstimulatorofoxidative

stressintheformof gammaradiationisanindicationof

possi-bleanti-oxidativepropertiesofthesemoleculesinplants.Hitherto, thereareverylimitedreportsonthedirectanti-oxidantactivityof

GSmoleculesandwhetherthesecompoundsfunctionas

indepen-dententitiesorsynergistically(Försteretal.,2015a,b).Thoughthe

currentresultshasindicatedgammaradiationasapotentinducer

ofmedicinallyimportantmetabolites,careneedstobetakensince

thistypeofradiationisknowntocauseirreversibledamagesto

foodvitaminssuchasvitaminC(Dionísioetal.,2009).Assuch,

pro-longedexposuretomilderformsofradiationcanbeusedinstead

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Conclusion

Thestudyrepresentsaproofonconceptmanipulationof

health-beneficialneutraceuticalsinamedicinalplantwheretheinducer

leavesnochemicalresidue.Here,thetargetedmetabolite

profil-ingconfirmsthepresenceof structurallydiverseglucomoringin

moleculesinM.oleiferaand demonstrates therelative

accumu-lationpost-gammaradiationtreatment.Thecurrent resultsalso

showtheGSmoleculesofM.oleiferatobepartoftheinducible

defensemechanismofplantsratherthanconstitutivecomponents

aspreviously perceived.Our resultssupportsan inplanta

anti-oxidativeroleforglucomoringinandacylatedderivativesfromM.

oleifera, and by extension in thehuman body when consumed

asherbalsupplement. Assuch, consumptionof non-inducedM.

oleiferaleafmaterialdoesnotnecessarilyguaranteethereported

activitiesassociated withthesemolecules.However, theuseof

radiationmayprovideanattractivewaytoenhanceGScontent

and,as such, Moringa plants grown underlight intensive

envi-ronmentscontributingtophoto-oxidativestress,areexpectedto

containahighercontentthereof.Moreover,irradiatedplantsare

alsoexpectedtoexhibitenhancedpharmacologicalpropertiesand,

assuch,futurestudiesshouldfocusonevaluationandbiological

testingofextractspreparedfromirradiatedplants.

Authors’contributions

NEM,RDMandIADconceivedofthestudy,TRconductedthe

experiments.TR,ARN,NEMandPASanalyzedtheMSdata.NEM,

RDM,LAPandIADsupervisedtheprojectandLAPparticipatedin

criticalreadingofthemanuscript.Allauthorsreadandapproved

thefinalmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

SouthAfricanNationalResearchFoundation(NRF),University

ofJohannesburgandNuclearEnergyCorporationofSouthAfrica

(NECSA)arethankedforfinancialsupport.MrManfredRellingis

thankedforhisassistancewithradiationexperiments.

References

Agerbirk,N.,Olsen,C.E.,2012.Glucosinolatestructuresinevolution.Phytochemistry 77,16–45.

Ahuja,S.,Kumar,M.,Kumar,P.,Gupta,V.K.,Singhal,R.K.,Yadav,A.,Singh,B.,2014. Metabolicandbiochemicalchangescausedbygammairradiationinplants.J. Radioanal.Nucl.Chem.300,199–212.

Amaglo,N.K.,Bennett,R.N.,LoCurto,R.B.,Rosa,E.S.,LoTurco,V.,Giuffrida,A.,Curto, A.,Lo,Crea,F.,Timpo,G.M.,2010.Profilingselectedphytochemicalsand nutri-entsindifferenttissuesofthemultipurposetreeMoringaoleiferaL.,grownin Ghana.FoodChem.122,1047–1054.

Asad,S.A.,Young,S.,West,H.,2013.Effectofnickelandcadmiumonglucosinolate productioninThlaspicaerulescens.PakistanJ.Bot.45,495–500.

Bajguz,A.,Hayat,S.,2009.Effectsofbrassinosteroidsontheplantresponsesto environmentalstresses.PlantPhysiol.Biochem.47,1–8.

Bennett,R.N.,Mellon,F.,Foidl,N.,Pratt,J.H.,Dupont,M.S.,Perkins,L.,Kroon,P.,2003. Profilingglucosinolatesandphenolicsinvegetativeandreproductivetissues ofthemulti-purposetreesMoringaoleiferaL.(Horseradishtree)andMoringa stenopetalaL.J.Agric.FoodChem.51,3546–3553.

Björkman,M.,Klingen,I.,Birch,A.N.E.,Bones,A.M.,Bruce, T.J.A.,Johansen,T.J., Meadow,R.,Mølmann,J.,Seljåsen,R.,Smart,L.E.,Stewart,D.,2011. Phytochemi-calsofBrassicaceaeinplantprotectionandhumanhealth–influencesofclimate, environmentandagronomicpractice.Phytochemistry72,538–556.

Bodnaryk,R.P.,1992.Effectsofwoundingonglucosinolatesinthecotyledonsof oilseedrapeandmustard.Phytochemistry31,2671–2677.

Bones,A.M.,Rossiter,J.T.,2006.Theenzymicandchemicallyinduceddecomposition ofglucosinolates.Phytochemistry67,1053–1067.

Brown,P.D.,Tokuhisa,J.G.,Reichelt,M.,Gershenzon,J.,2003.Variationof glu-cosinolateaccumulationamongdifferentorgansanddevelopmentalstagesof Arabidopsisthaliana.Phytochemistry62,471–481.

Clarke,D.B.,2010.Glucosinolates,structuresandanalysisinfood.Anal.Chem.9660, 310–325.

deGraaf,R.M.,Krosse,S.,Swolfs,A.E.M.,teBrinke,E.,Prill,N.,Leimu,R.,vanGalen, P.M.,Wang,Y.,Aarts,M.G.M.,vanDam,N.M.,2015.Isolationand identifica-tionof4-␣-rhamnosyloxybenzylglucosinolateinNoccaeacaerulescensshowing intraspecificvariation.Phytochemistry110,166–171.

Demidchik,V.,2015.Mechanismsofoxidativestressinplants:fromclassical chem-istrytocellbiology.Environ.Exp.Bot.109,212–228.

Dinkova-Kostova,A.T.,Kostov,R.V.,2012.Glucosinolatesandisothiocyanatesin healthanddisease.TrendsMol.Med.18,337–347.

Dionísio,A.P.,Gomes,R.T.,Oetterer,M.,2009.Ionizingradiationeffectsonfood vitamins:areview.Braz.Arch.Biol.Technol.52,1267–2127.

Esnault,M.,Legue,F.,Chenal,C.,2010.Ionizingradiation:advancesinplantresponse. Environ.Exp.Bot.68,231–237.

Fahey,J.,2005.Moringaoleifera:areviewofthemedicalevidenceforits nutri-tional,therapeuticand prophylacticproperties,Part1. TreesLife J. 1,15, http://www.tfljournal.org/article.php/20051201124931586.

Fahey,J.W.,Zalcmann,A.T.,Talalay,P.,2001.Thechemicaldiversityanddistribution ofglucosinolatesandisothiocyanatesamongplants.Phytochemistry56,5–51. Förster,N.,Ulrichs,C.,Schreiner,M.,Müller,C.T.,Mewis,I.,2015a.Developmentof

areliableextractionandquantificationmethodforglucosinolatesinMoringa oleifera.FoodChem.166,456–464.

Förster,N.,Ulrichs,C.,Schreiner,M.,Arndt,N.,Schmidt,R.,Mewis,I.,2015b. Eco-typevariabilityingrowthandsecondarymetaboliteprofileinMoringaoleifera: impactofsulfurandwatervailability.J.Agric.FoodChem.63,2852–2861. Guerrero-Beltrán,C.E.,Calderón-Oliver,M.,Pedraza-Chaverri,J.,Chirino,Y.I.,2012.

Protectiveeffectofsulforaphaneagainstoxidativestress:recentadvances.Exp. Toxicol.Pathol.64,503–508.

Grubb,C.D.,Abel,S.,2006.Glucosinolatemetabolismanditscontrol.TrendsPlant Sci.11,89–100.

Hollósy,F.,2002.Effectsofultravioletradiationonplantcells.Micron33,179–197. Jansen,J.J.,Allwood,J.W.,Marsden-Edwards,E.,vanderPutten,W.H.,Goodacre,R., vanDam,N.M.,2008.Metabolomicanalysisoftheinteractionbetweenplants andherbivores.Metabolomics5,150–161.

Karaköse,H.,Jaiswal,R.,Deshpande,S.,Kuhnert,N.,2015.Investigationofthe pho-tochemicalchangesofchlorogenicacidsinducedbyultravioletlightinmodel systemsandinagriculturalpracticewithSteviarebaudianacultivationasan example.J.Agric.FoodChem.63,3338–3347.

Kovács,E.,Keresztes,Á.,2002.EffectofgammaandUV-B/Cradiationonplantcells. Micron33,199–210.

Leone,A.,Spada,A.,Battezzati,A.,Schiraldi,A.,Aristil,J.,Bertoli,S.,2015.Cultivation, genetic,ethnopharmacology,phytochemistryandpharmacologyofMoringa oleiferaleaves:anoverview.Int.J.Mol.Sci.16,12791–12835.

Madala,N.E.,Steenkamp,P.A.,Piater,L.A.,Dubery,I.A.,2012.Collisionenergy alter-ationduringmassspectrometricacquisitionisessentialtoensureunbiased metabolomicanalysis.Anal.Bioanal.Chem.404,367–372.

Makita,C.,Chimuka,L.,Steenkamp,P.,Cukrowska,E.,Madala,E.,2016.Comparative analysesofflavonoidcontentinMoringaoleiferaandMoringaovalifoliawiththe aidofUHPLC-qTOF-MSfingerprinting.SouthAfricanJ.Bot.105,116–122. Mewis,I.,Schreiner,M.,Nguyen,C.N.,Krumbein,A.,Ulrichs,C.,Lohse,M.,Zrenner,

R.,2012.UV-Birradiationchangesspecificallythesecondarymetaboliteprofile inbroccolisprouts:inducedsignalingoverlapswithdefenseresponsetobiotic stressors.PlantCellPhysiol.53,1546–1560.

Mithen,R.,2001.Glucosinolates–biochemistry, geneticsandbiological activity. PlantGrowthRegul.34,91–103.

Mittler,R.,2002.Oxidativestress,antioxidantsandstresstolerance.TrendsPlant Sci.7,405–410.

Moyo,B.,Masika,P.J.,Hugo,A.,Muchenje,V.,2011.Nutritionalcharacterizationof Moringa(MoringaoleiferaLam.)leaves.AfricanJ.Biotechnol.10,12925–12933. Ncube,E.N.,Mhlongo,M.I.,Piater,L.,Steenkamp,P.,Dubery,I.,Madala,N.E.,2014. Analysesofchlorogenicacidsandrelatedcinnamicacidderivativesfrom Nico-tianatabacumtissueswiththeaidofUPLC-QTOF-MS/MSbasedonthein-source collision-induceddissociationmethod.Chem.Cent.J.8,1–10.

Ncube,E.N.,Steenkamp,P.A.,Madala,N.E.,Dubery,I.A.,2016.Chlorogenicacids biosynthesisinCentellaasiaticacellsisnotstimulatedbysalicylicacid manipu-lation.Appl.Biochem.Biotechnol.179,685–696.

Pérez-Balibrea,S.,Moreno,D.A.,Garcia-Viguera,C.,2008.Influence oflighton health-promotingphytochemicalsofbroccolisprouts.J.Food.Agric.Environ. 88,904–910.

Popoola,J.O.,Obembe,O.O.,2013.Localknowledge,usepatternandgeographical distributionofMoringaoleiferaLam.(Moringaceae)inNigeria.J. Ethnopharma-col.150,682–691.

Ramabulana,T.,Mavunda,R.D.,Steenkamp,P.A.,Piater,L.A.,Dubery,I.A.,Madala, N.E.,2015.SecondarymetaboliteperturbationsinPhaseolusvulgarisleavesdue togammaradiation.PlantPhysiol.Biochem.97,287–295.

Ramabulana,T.,Mavunda,R.D.,Steenkamp,P.A.,Piater,L.A.,Dubery,I.A.,Madala, N.E.,2016.Perturbationofpharmacologicallyrelevantpolyphenoliccompounds inMoringaoleiferaagainstphoto-oxidativedamagesimposedbygamma radia-tion.J.Photochem.Photobiol.B:Biol.156,79–86.

(7)

Sardans,J.,Pe˜nuelas,J.,Rivas-Ubach,A.,2011.Ecologicalmetabolomics:overview ofcurrentdevelopmentsandfuturechallenges.Chemoecology21,191–225. Schreiner,M.,Krumbein,A.,Mewis,I.,Ulrichs,C.,Huyskens-Keil,S.,2009.Short-term

andmoderateUV-Bradiationeffectsonsecondaryplantmetabolismindifferent organsofnasturtium(TropaeolummajusL.).Innov.FoodSci.Emerg.Technol.10, 93–96.

Sumner,L.W.,Amberg,A.,Barrett,D.,Beale,M.H.,Beger,R.,Daykin,C.A.,Fan, T.W.-M.,Fiehn,O.,Goodacre,R.,Griffin,J.L.,Hankemeier,T.,Hardy,N.,Harnly,J., Higashi,R.,Kopka,J.,Lane,A.N.,Lindon,J.C.,Marriott,P.,Nicholls,A.W.,Reily, M.D.,Thaden,J.J.,Viant,M.R.,2007.Proposedminimumreportingstandards forchemicalanalysisChemicalAnalysisWorkingGroup(CAWG)Metabolomics StandardsInitiative(MSI).Metabolomics3,211–221.

Tolrà,R.P.,Alonso,R.,Poschenrieder,C.,Barceló,D.,Barceló,J.,2000. Determi-nationofglucosinolatesinrapeseedandThlaspicaerulescensplantsbyliquid chromatography-atmosphericpressurechemicalionizationmassspectrometry. J.Chromatogr.889,75–81.

Traka,M.,Mithen,R.,2009.Glucosinolates,isothiocyanatesandhumanhealth. Phy-tochem.Rev.8,269–282.

Tumer,T.B.,Rojas-Silva,P.,Poulev,A.,Raskin,I.,Waterman,C.,2015.Directand indi-rectantioxidantactivityofpolyphenol-andisothiocyanate-enrichedfractions fromMoringaoleifera.J.Agric.FoodChem.63,1505–1513.

Vo,Q.V.,Trenerry,C.,Rochfort,S.,Wadeson,J.,Leyton,C.,Hughes,A.B.,2013. Synthe-sisandanti-inflammatoryactivityofaromaticglucosinolates.BioorganicMed. Chem.21,5945–5954.

Wang,Y.,Xu,W.-J.,Yan,X.-F.,Wang,Y.,2011.Glucosinolatecontentandrelated geneexpressioninresponsetoenhancedUV-BradiationinArabidopsis.African J.Biotechnol.10,6481–6491.

Williamson,G.,Faulkner,K.,Plumb,G.W.,1998.Glucosinolatesandphenolicsas antioxidantsfromplantfoods.Eur.J.CancerPrev.7,17–21.

Zandalinas,S.I.,Vives-Peris,V.,Gómez-Cadenas,A.,Arbona,V.,2012.Afastand precisemethodtoidentifyindolicglucosinolatesandcamalexininplantsby combiningmassspectrometricandbiologicalinformation.J.Agric.FoodChem. 60,8648–8658.

Zhang,W.J.,Björn,L.O.,2009.Theeffectofultravioletradiationontheaccumulation ofmedicinalcompoundsinplants.Fitoterapia80,207–218.

Zhang,J.,Sun,X.,Zhang,Z.,Ni,Y.,Zhang,Q.,Liang,X.,Xiao,H.,Chen,J.,Tokuhisa, J.G.,2011. Phytochemistrymetaboliteprofilingof Arabidopsisseedlingsin response to exogenoussinalbin and sulfurdeficiency. Phytochemistry 72, 1767–1778.