molecules Article Is Gamma Radiation Suitable to Preserve Phenolic Compounds and to Decontaminate Mycotoxins in Aromatic Plants? A Case-Study with Aloysia citrodora Paláu ElianaPereira1,2,LillianBarros1,3,AmilcarL.Antonio1,SandraCaboVerde4, CelestinoSantos-Buelga2,IsabelC.F.R.Ferreira1,*andPaulaRodrigues1,* 1 MountainResearchCentre(CIMO),ESA,PolytechnicInstituteofBragança,CampusdeSantaApolónia, 1172,5300-253Bragança,Portugal;[email protected](E.P.);[email protected](L.B.);[email protected](A.L.A.) 2 GrupodeInvestigaciónenPolifenoles(GIP-USAL),FacultaddeFarmacia,UniversidaddeSalamanca, CampusMigueldeUnamunos/n,37007Salamanca,España;[email protected] 3 LaboratoryofSeparationandReactionEngineering(LSRE),AssociateLaboratoryLSRE/LCM, PolytechnicInstituteofBragança,CampusdeSantaApolónia,1134,5301-857Bragança,Portugal 4 CentrodeCiênciaseTecnologiasNucleares(C2TN),IST,UniversidadedeLisboa, EstradaNacional10(km139.7),2695-066BobadelaLRS,Portugal;[email protected] * Correspondence:[email protected](I.C.F.R.F.);[email protected](P.R.); Tel.:+351-273-303-219(I.C.F.R.F.);+351-273-303-332(P.R.); Fax:+351-273-325-405(I.C.F.R.F.);+351-273-325-405(P.R.) AcademicEditor:ThomasJ.Schmidt Received:4January2017;Accepted:20February2017;Published:23February2017 Abstract:Thisstudyaimedtodeterminetheeffectofgammaradiationonthepreservationofphenolic compounds and on decontamination of dry herbs in terms of ochratoxin A (OTA) and aflatoxin B1(AFB1),usingAloysiacitrodoraPaláuasacasestudy. Forthispurpose,artificiallycontaminated dryleavesweresubmittedtogammaradiationatdifferentdoses(1,5,and10kGy;atdoserateof 1.7kGy/h). PhenoliccompoundswereanalysedbyHPLC-DAD-ESI/MSandmycotoxinlevelswere determinedbyHPLC-fluorescence. Elevenphenoliccompoundswereidentifiedinthesamplesand despitetheapparentdegradationofsomecompounds(namelyverbasoside),1and10kGydoses pointtoapreservationofthemajorityofthecompounds. Themeanmycotoxinreductionvaried between5.3%and9.6%forOTAandfrom4.9%to5.2%forAFB1. Itwasnotobservedasignificant effectof theirradiationtreatmentsonmycotoxinlevels, and aslightdegradation ofthephenolic compoundsintheirradiatedsampleswasobserved. Keywords:gammaradiation;herbs;phenoliccompounds;aflatoxinB1;ochratoxinA;chromatography 1. Introduction There is currently a high demand for medicinal and aromatic plants, due to their combined aromaticandbioactiveproperties[1,2].Oneofthemostsignificantcompoundswhichconferbioactive potential to plants are the phenolic compounds. These have been largely studied based on their therapeuticpropertiesrelatedtothepreventionofchronicinflammation,cardiovascularproblems, cancer, and diabetes [2]. Some studies claim that the absorption of these compounds in the body, occurindifferentrouteslinkedtothegastrointestinaltract,wheremicroorganisms,enzymes,andeven glucosetransportersareinvolved. Thepartialreleaseofpolyphenolsoccursinthegastrointestinal lumen, where they are metabolized and rendered absorbable, so that they can exert their health benefits[3]. Nevertheless,theuseofthesemedicinalandaromaticplantsdonotalwaysadheretothe Molecules2017,22,347;doi:10.3390/molecules22030347 www.mdpi.com/journal/molecules Molecules2017,22,347 2of13 industrialandcommercialtoughrequirementsofqualityandsafety. Naturalcontaminationofplant materialbyfungiandassociatedtoxinsduringgrowth,harvesting,storageanddryingprocesses,in general,presentsathreattopublichealth[4]. Medicinalandaromaticplantsarenoexception,asthey arefrequentlycontaminatedwithnumeroustoxigenicfungi. There are more than 400 compounds classified as mycotoxins and, among them, aflatoxins (AF), and ochratoxin A (OTA) are the best studied. AF are produced by Aspergillus flavus and some closely related species. AFB1 is the most common aflatoxin contaminating food products; it is reported as the most toxic and carcinogenic compound naturally produced, being classified as Group1carcinogen[5,6].ThemutagenicandcarcinogeniceffectsofAFB1invariousanimalshave beendocumented,anddifferentepidemiologicalstudiesshowedtheexistenceofacorrelationbetween human liver cancer and the levels of this mycotoxin in the diet [7,8]. OTA is produced by several AspergillusandPenicilliumspeciesandknownasanephrotoxic,hepatotoxic,neurotoxic,teratogenic andimmunotoxicagent. Itspresenceinthediethasbeenassociatedwithafatalhumankidneydisease, referredtoasBalkanEndemicNephropathy(BEN),andwithanincreasedincidenceoftumorsofthe upperurinarytract[9–11]. ItisclassifiedasGroup2Bpotentiallycarcinogen[5]. Thenaturaloccurrenceofmycotoxinsinplantshasbeenfrequentlyreported,someexamples beingtraditionalmedicinalandaromaticherbsfromseveralAsianandAfricancountriesreported tocontainexceedinglevelsofaflatoxinsandOTA[11–13].Althoughthereisnospecificlegislation regulating mycotoxin levels in these herbs, European regulations set maximum levels of 5 µg/kg ofAFB1and15µg/kgofOTAforseveralspices(CENo. 165/2010andCENo. 594/2012).Various techniques have been applied in the decontamination as well as in the preservation of bioactive compoundsinmedicinalandaromaticplants,includingirradiation[14]. Thisisaphysicalprocessin whichplantsareexposedtohigh-energyionizingradiationwiththeaimofimprovingfoodsafetyand shelflife[15–17]. Irradiationisbeingincreasinglyrecognizedasasafeandefficientfoodprocessing methodduetoitspositiveeffectsinpreservation,reductionofnaturallossescausedbyphysiological processes (budding, maturation, and aging), and elimination or reduction of microorganisms and their toxins, parasites and pests, without causing chemical changes to the food [17]. Additionally, irradiationisconsideredasafeprocesssinceithasnotbeenassociatedwithunsaferesidues,aswell asitreducesthedependenceonchemicalfumigantsandpreservativestraditionallyusedinthefood industry [14,16,18]. Irradiation is currently approved by national legislations in over 55 countries worldwide[19].Europeanlegislation[20]establishesashortlistoffoodstuffsauthorizedforirradiation treatmentwhichincludesdriedaromaticherbs,spices,andvegetableseasonings,withapermitted maximumaverageabsorbeddoseof10kGy. Ourresearchgrouppreviouslydemonstratedthatgammaradiationdoesnotsignificantlychange thechemicalprofileofdriedmedicinalandaromaticplants,whenappliedattheauthorizeddoses[21]. Takingintoaccountpreviousreports,whereirradiationisdescribedasanexcellentmethodologyto processanddecontaminateproducts[14],theaimofthepresentstudywastoevaluatetheeffectofthis technologyinthepreservationofphenoliccompoundsandalsoonthedecontaminationofAFB1and OTAindriedherbs,usingAloysiacitrodoraPaláuasacase-study. 2. ResultsandDiscussion DataonphenoliccompoundsidentificationbyHPLC-DAD-ESI/MS(Highperformanceliquid chromatography coupled with a diode array detector and electrospray ionization tandem mass spectrometry) recorded in the negative ion mode (retention time, λ in the visible region, max deprotonated molecules m/z values), the low-energy collision induced dissociation tandem mass spectrometric(CID)fragmentationpathwaysanalysis,andtentativeproductionsidentificationare presentedinTable1.Thephenolicprofileofthecontrolsample,recordedat280nm,isshowninFigure1. Uptoelevenphenoliccompoundsweredetectedandtentativelyidentifiedinthesamples. Fiveofthe identifiedcompoundscorrespondedtocaffeoylphenylethanoidderivatives(peaks5,7,8,10,and11), threetoflavonederivatives(peaks2,4,and6)andtheremainingonestoaphenylethanoidglycoside Molecules2017,22,347 3of13 (peak 1), a hydroxycinnamic acid (peak 3) and a flavonol (peak 9). Compounds were identified basedontheirmassandUV-VISspectraandretentioncharacteristics. Themajorityofthedetected compounds (verbasoside, luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide, verbascoside, Molecules 2017, 22, 347 3 of 13 chrysoeriol-7-O-diglucuronide, isoverbascoside, forsythoside, eukovoside, and martinoside) have alreadybeenreportedinA.citrodora(Table1;[22–24],whichhasbeenusedtosupportcompounds (Table 1; [22–24], which has been used to support compounds identities. Compounds 3 (p-coumaric identities. Compounds 3 (p-coumaric acid) and 9 (isorhamnetin-3-O-glucuronide) were identified acid) and 9 (isorhamnetin-3-O-glucuronide) were identified by comparison with authentic standards. bycomparisonwithauthenticstandards. Asfarasweknow,thesetwocompoundshavenotbeen As far as we know, these two compounds have not been previously reported in A. citrodora. previouslyreportedinA.citrodora. 5 mAU 2000 1750 1500 1250 1000 750 500 2 6 7 8 9 10 11 1 3 4 250 0 0 10 20 30 Time (min) FFiigguurree 11.. AAllooyyssiiaa cciittrrooddoorraa pphheennoolliicc pprrooffiillee rreeccoorrddeedd aatt 228800 nnmm.. PPeeaakk nnuummbbeerriinngg iiss tthhee ssaammee aass iinn TTaabblleess 11 aanndd 22.. The most abundant compound present in all samples was verbascoside (compound 5, Table 2), a Themostabundantcompoundpresentinallsampleswasverbascoside(compound5,Table2),a caffeoyl-phenylethanoid glycoside with antioxidant, anti-inflammatory, and antimicrobial activities, as caffeoyl-phenylethanoidglycosidewithantioxidant,anti-inflammatory,andantimicrobialactivities,as well as wound healing and neuroprotective properties claimed to be beneficial in human health [25,26]. wellaswoundhealingandneuroprotectivepropertiesclaimedtobebeneficialinhumanhealth[25,26]. The effect of gamma radiation on the phenolic compounds of the studied samples presented statistically Theeffectofgammaradiationonthephenoliccompoundsofthestudiedsamplespresentedstatistically significant differences for some compounds (p < 0.05). The results showed a slight decrease of the major significantdifferencesforsomecompounds(p<0.05).Theresultsshowedaslightdecreaseofthemajor compound (verbascoside) at all the applied doses, as well as in the levels of total flavonoids (TF) and compound(verbascoside)atalltheapplieddoses,aswellasinthelevelsoftotalflavonoids(TF)and total phenolic compounds (TPC), as determined by HPLC-DAD (Table 2). However, the concentrations totalphenoliccompounds(TPC),asdeterminedbyHPLC-DAD(Table2). However,theconcentrations of verbasoside, p-coumaric acid, isoverbascoside, forsythoside, eukovoside, and martinoside did not ofverbasoside,p-coumaricacid,isoverbascoside,forsythoside,eukovoside,andmartinosidedidnot change significantly (p > 0.05) when the maximum dose (10 kGy) was applied. The dose of 1 kGy stood changesignificantly(p>0.05)whenthemaximumdose(10kGy)wasapplied. Thedoseof1kGy out from the other applied doses, because it slightly preserved more of the phenolic compounds, in which stoodoutfromtheotherapplieddoses,becauseitslightlypreservedmoreofthephenoliccompounds, three out of the eleven identified phenolic compounds (compounds 2, 6, and 9), showed a significant inwhichthreeoutoftheelevenidentifiedphenoliccompounds(compounds2,6,and9),showeda increase in quantity (p < 0.05) and five of the remaining eight (compounds 1, 3, 5, 8, and 11) showed significantincreaseinquantity(p<0.05)andfiveoftheremainingeight(compounds1,3,5,8,and11) a decrease, with compounds 3 and 5 having a significant decrease (p < 0.05). The decrease induced by showedadecrease,withcompounds3and5havingasignificantdecrease(p<0.05). Thedecrease gamma radiation in the levels of these compounds may be attributed to the possible formation of induced by gamma radiation in the levels of these compounds may be attributed to the possible irradiation-induced degradation products and/or free radicals [15]. The same decreasing effect in the formationofirradiation-induceddegradationproductsand/orfreeradicals[15]. Thesamedecreasing compounds happened in a previous study by Pereira et al. [27], where the effects of gamma radiation effectinthecompoundshappenedinapreviousstudybyPereiraetal.[27],wheretheeffectsofgamma on the phenolic profile of the infusions of Thymus vulgaris L. were analyzed. Several studies were radiationonthephenolicprofileoftheinfusionsofThymusvulgarisL.wereanalyzed. Severalstudies performed and the results diverge according to several factors, such as the plant species studied, type wereperformedandtheresultsdivergeaccordingtoseveralfactors,suchastheplantspeciesstudied, of irradiation, and applied doses [28,29]. typeofirradiation,andapplieddoses[28,29]. Molecules2017,22,347 4of13 Table1.Retentiontime(Rt),wavelengthsofmaximumabsorptioninthevisibleregion(λmax),massspectraldata(MSandMS2)andtentativeidentificationofphenolic compoundsinAloysiacitrodora. Peak Rt(min) λmax(nm) MolecularIon[M−H]−(m/z) MS2(m/z) TentativeIdentification References 1 4.5 280 461 315(8),135(28) Verbasoside [23,24] 2 15.1 344 637 351(100),285(89) Luteolin-7-O-diglucuronide [22–24] 3 16.8 314 163 119(100) p-Coumaricacid - 4 17.7 338 621 351(100),269(20) Apigenin-7-O-diglucuronide [22] 5 18.2 330 623 461(18),315(5) Verbascoside [22–24] 6 20.3 350 651 351(100),299(5) Chrysoeriol-7-O-diglucuronide [23,24] 7 20.6 330 623 461(18),315(5) Isoverbascoside [22] 8 21.3 330 623 461(15),315(10) Forsythoside [23] 9 21.8 350 491 315(100),300(23) Isorhamnetin-3-O-glucuronide - 10 23.2 330 637 491(5),461(60),315(13) Eukovoside [22–24] 11 29.2 330 651 505(7),475(22) Martinoside [23,24] Molecules2017,22,347 5of13 Table2. Tentativeidentificationofphenoliccompounds(mg/gextract)inA.citrodorasubmittedtoirradiationtreatmentsat1, 5, and10kGy, comparedwith non-irradiatedsamples(0kGy). Peak PhenolicCompounds 0kGy 1kGy 5kGy 10kGy 1 Verbasoside1 0.118±0.001a 0.110±0.01a 0.125±0.02a 0.140±0.03a 2 Luteolin-7-O-diglucuronide2 18.9±0.08b 19.1±0.02a 18.6±0.05c 18.1±0.8d 3 p-Coumaricacid3 1.14±0.01b 1.07±0.03c 1.13±0.03b 1.20±0.04a 4 Apigenin-7-O-diglucuronide4 1.79±0.03ab 1.81±0.04a 1.71±0.04b 1.61±0.05c 5 Verbascoside1 71.6±0.24a 69±0.95b 69±0.71b 69.5±0.47b 6 Chrysoeriol-7-O-diglucuronide4 2.93±0.01c 3.27±0.05a 3.04±0.04b 2.80±0.04d 7 Isoverbascoside1 0.74±0.03a 0.79±0.04a 0.73±0.04a 0.67±0.02a 8 Forsythoside1 1.67±0.03a 1.65±0.20a 1.71±0.15a 1.76±0.10a 9 Isorhamnetin-3-O-glucuronide5 1.63±0.03b 1.75±0.06a 1.51±0.05c 1.27±0.04d 10 Eukovoside1 1.00±0.03a 1.00±0.04a 1.05±0.06a 1.11±0.09a 11 Martinoside1 0.57±0.01a 0.56±0.04a 0.62±0.08a 0.67±0.11a TCP 75.8±0.2a 73±1b 73±1b 73.8±0.2b TPA 1.14±0.01b 1.07±0.03c 1.13±0.03b 1.20±0.04a TF 25.22±0.03b 25.96±0.09a 24.9±0.1c 23.7±0.1d TPC 102.1±0.2a 100±1b 99.3±0.6b 98.8±0.2b Theresultsarepresentedasthemean±SD;(n=12).TCP—totalcaffeoylphenylethanoidderivatives(includingverbasoside);TPA—totalhydroxycinnamicacids;TF—totalflavonoids; TPC—totalphenoliccompounds.Calibrationcurves:1caffeicacid(y=359x+488.4;R2=0.997);2Luteolin-7-O-glucoside(y=334.2x−261.39;R2=0.999);3p-coumaricacid(y=706.09x +1228.1;R2=0.9994);4apigenina-7-O-glucoside(y=214.33x−165.38;R2=0.999);5isorhametin-3-O-rutinoside(y=284.12x+67.055;R2=0.999).Ineachrowdifferentlettersmean significantdifferences(p<0.05). Molecules2017,22,347 6of13 It should be highlighted that, at up to 400 µg/mL, none of the irradiated samples showed hepatotoxicity, evaluated in PLP2 cells, contrarily to the toxicity observed for the positive control ellipticine (concentration responsible for 50% of inhibition of the net cell growth—GI = 3.22 ± 50 0.67µg/mL).TheseresultsareinagreementwithpreviousresultsofirradiatedsamplesofT.vulgaris andMenthaxpiperitaL.[30]. Thecalibrationparametersofinstrumentation(linearrange,correlationcoefficient(R2),equations oflinearregression,limitsofdetection(LOD)andlimitsofquantification(LOQ)forAFB1andOTA areshowninTable3. Theanalyticalmethodsforquantificationofthetwomycotoxinsinsamplesof driedaromaticplantswerefurthervalidated. Table4displaystheaccuracyandprecisionoftheOTA andAFB1analysismethodsRecovery,aswellasrepeatabilityrelativestandarddeviation(RSD ),and r reproducibilityrelativestandarddeviation(RDS ),arewithinrecommendedranges[31]. R Table3.CalibrationparametersofinstrumentationforaflatoxinB1andochratoxinAdetectionand quantification. Standard AFB OTA 1 Min 6.79 2.20 Rt(retentiontime) CV,%(n=11) 0.76 2.45 Calibrationcurve y=312.36x−27.24 y=362.40x−31.13 Correlationcoefficient(R2) 0.999 0.999 Linearityrange(ng/mL) 20to0.05 20to0.05 LODa(ng/mL) 0.6 0.5 Limits LOQb(ng/mL) 1.9 1.7 R2:Correlationcoefficient;CV:coefficientofvariation;aLOD:limitofdetectionofthechromatographicmethod; bLOQ:limitofquantificationofthechromatographicmethod. Table4.AccuracyandprecisionoftheanalyticalmethodsforaflatoxinB andochratoxinAforspiking 1 levelsof10ng/gand30ng/g. AFB OTA 1 10ng/g 30ng/g 10ng/g 30ng/g MeanRecovery(%) 88.3 88.9 76.4 92.0 RSDr(%)a 8.3–14.4 0.1 2.5–9.3 5.1 RSD (%)b 3.3 - 5.6 - R RecommendedRange(EuropeanRegulationNo.401/2006) Recovery(%) 70–110 RSDr(%) <21 <22 <21 <22 RSD (%) <32 <34 <32 <34 R aRSDr:Repeatabilityrelativestandarddeviation;bRSDR:Reproducibilityrelativestandarddeviation. DatapresentedinTable5showtheeffectofgammaradiationdoses(1,5,and10kGy)onthe reduction of AFB1 and OTA in dried leaves of A. citrodora. Assays were carried out in powdered samplesspikedwith30ng/gofAFB1andOTA.Thisconcentrationwasselectedbecauseitisanaverage valuecommonlyusedinthistypeofstudies. Whencomparedwithnon-irradiatedsamples(0kGy), ratesofmycotoxinreductionatdifferentirradiationdoses(1,5,and10kGy)rangedbetween21.2and 22.6ng/gforOTA,and19.8to21,9ng/gforAFB1,withnostatisticallysignificantdifferences(p>0.05) between irradiated and non-irradiated samples, independently of the applied dose. No apparent dose-dependenteffectwasdetectedontherateofmycotoxinsdecrease,either. Theseresultssuggest thatirradiationatthetesteddoses,includingthemaximumalloweddoseof10kGy,isnotaneffective treatmentforAFB1andOTAdecontaminationofdriedplants. Molecules2017,22,347 7of13 Table5.Reduction(ng/g;mean±SD;n=6)ofaflatoxinB1andochratoxinAinspikeddriedsamples (30ng/gofeachmycotoxin)ofAloysiacitrodorasubmittedtoirradiationtreatmentsat1,5,and10kGy, incomparisonwithnon-irradiatedsamples(0kGy). MycotoxinDecrease(ng/g) IrradiationDose AFB OTA 1 0kGy 21.9±3.5a 22.6±0.8a 1kGy 20.7±0.4a 21.5±1.0a 5kGy 19.8±1.2a 21.2±1.5a 10kGy 20.4±1.4a 21.4±0.7a aNosignificantdifferences(p<0.05)betweenanyoftheresultswereobserved. Theeffectofgammaradiationonmycotoxindecontaminationhasbeeninvestigatedinseveral foodproducts(spices,feedstuff,coffeebeans,fruits,seeds,vegetables,curedmeat,andothers),but divergent results have been reported. Some studies report high effectiveness of gamma radiation onthereductionofmycotoxinlevelsinvariouslowmoisturefoods[32–36],althoughinsomecases thiseffectisonlyobservedatirradiationdosesof30to60kGy[34,35],higherthanthealloweddose of 10 kGy. In general, for the admissible dose by EU regulations, most reports conclude that no significantpositiveeffectsonmycotoxindecontaminationareobtainedforlowmoisturecontentfoods orfeeds[34,35,37–41]. InastudyperformedbyJalilietal.[35],gammaradiationwasappliedtoblack andwhitepepperandtheyfoundsignificantAFandOTAreductionsonlyatirradiationdosesof30 kGyorhigherand,evenat60kGy,gammarayswerenotcompletelyeffectiveindestroyingthose mycotoxins. At10kGy,mycotoxinreductionvariedbetween1.4%inOTAto7.2%inAFB1forsamples with12%ofmoisturecontent. Thereducedeffectofthistechniqueinlowmoisturematricesseemstobeadirectresultofthe reducedwatercontent. ThepresenceofwaterisanimportantfactorinthedestructionofAFandOTA bygammaradiation,sincewaterradiolysisleadstotheformationofhighlyreactivefreeradicalsthat degradethemycotoxins[42,43]. ThiseffecthasbeendemonstratedinastudybyKumaretal.[40], wheretheeliminationofOTAincoffeegrainswithdifferentmoisturecontents(9%,10%,12%,and 23%)wastested. OTAdegradationinthelowestmoisturecontentgrainswas5%at10kGy,similar totheoneobtainedinourstudy,and90%forthehighestmoisturecontentsamples. Inthepresent study,mycotoxindegradationbygammaradiationhasbeentestedinherbsafterdrying,atthestageof ready-to-useproduct. Toavoidthelimitedeffectofthetreatmentgenerallyobservedforlowmoisture products(namelydriedherbs),futurestudiesonmycotoxindetoxificationofherbsbygammaradiation should contemplate the fresh product, before drying. As previously stated, the treatment of high moisturematrices(freshherbs)shouldresultinhigherdetoxificationeffects. Asconcludedbyseveral studies(e.g.,[44–46]),theapplicationofirradiationtreatmentstoherbswhile,inthefreshstage,should notnegativelyinfluencetheirnutritionalvalue. 3. MaterialsandMethods 3.1. SafetyConsiderations AllrecommendedsecurityconsiderationsweretakenintoaccountwhenhandlingAFandOTA, duetothetoxicityofthesesubstances[47]. Protectiveequipmentwasusedwhenhandlingsolutions andallmaterialsweredecontaminatedbyautoclavingbeforedisposal. Thereusablematerialswere decontaminatedduring12h,immersedinableachsolutionof10%,thenimmersedinacetonesolution of5%,during1handfinallywashedwithdistilledwaterseveraltimes. 3.2. SamplesandSamplePreparation DryleavesofA.citrodora(1000g),whosecommonnameislemonverbena,andbelongingtothe familyVerbenaceae,wereprovidedbyalocalproducer(PragmáticoAromaLda,AlfândegadaFé, Molecules2017,22,347 8of13 Bragança,Portugal). Wateractivity(aw)oftheleaveswasmeasuredusingaRotronicHygroPalmAW1 equipment(RotronicInstrumentsLtd.,Crawley,WestSussex,UK)andrevealedtobe0.51. Leaves werethenreducedtoafinepowderwiththeaidofakitchenblender,fullyhomogenized,anddivided intwosets,oneforirradiationtestsandtheotherforin-housemethodvalidation. Thematerialwas preservedinsealedbagsat20◦Cuntilfurtheruse. 3.3. SpikingwithMycotoxins Totesttheeffectofirradiationonmycotoxinreduction, driedpowderedmaterialwasspiked with 30 ng/g of AFB1 and OTA, carefully homogenized, divided into 5 g aliquots and packaged in appropriate bags (polyethylene, 63 µm thickness). Aliquots were submitted to three different irradiationtreatments: 1,5,and10kGy. Eachirradiationdosewasappliedtothreealiquotsintwo independenttreatments,foratotalofsixreplicatesforeachdose. Non-irradiatedsamples(n=6)were usedascontrol(0kGy). 3.4. IrradiationTreatment Irradiationwasperformedina60Coexperimentalchamber(Precisa22,GravinerManufacturing Company Ltd., London, UK), following a procedure previously described by Pereira et al. [21]. Theestimateddoseratewas1.7kGy/handtheabsorbedgammaradiationdoseswere1.2±0.1kGy, 5.2±0.2kGyand10.4±0.4kGy. Thedoseuniformityratios(D /D )was1.2. Inordertosimplify max min thevalues,0wasconsideredfornon-irradiatedsampleand1,5and10kGywereconsideredirradiated samples. Sampleswerestoredat−18◦Cuntilfurtheranalysiswereperformed. 3.5. PhenolicCompoundsAnalysis Samples(1g)wereextractedbymacerationwith25mLofmethanol/H O(80:20)during1hat 2 25◦Cand150rpmfollowingtheproceduredescribedbyPereiraetal.[48]. The extracts were analyzed using an HPLC-DAD (Agilent Technologies, Santa Clara, CA, USA), connected to a mass spectrometer (MS) equipped with an ESI source and a hybrid triple quadrupole/linear ion trap mass analyzer (API 3200 Qtrap, Applied Biosystems, Darmstadt, Germany)[49].Thephenoliccompoundswereidentifiedbycomparingtheirretentiontimes,UV-VIS and mass spectra with those obtained with standard compounds, when available. Otherwise, compounds were tentatively identified comparing the obtained information with available data reportedintheliterature. Forquantitativeanalysis,acalibrationcurveforeachavailablephenolic standardwasconstructedbasedontheUVsignal. Fortheidentifiedphenoliccompoundsforwhich acommercialstandardwasnotavailable,thequantificationwasperformedthroughthecalibration curveofanothercompoundfromthesamephenolicgroup. Resultswereexpressedasmgpergram ofextract. 3.6. CytotoxicityEvaluationinPorcineLiverCells Cytotoxicity of the extracts was evaluated in porcine liver cells (PLP2) using the SRB assay, previouslydescribedbyAbreuetal.[50]. Theextractsdescribedabove(Section3.5)werere-dissolved inwatertoafinalconcentrationof8mg/mL.Ellipticinewasusedasapositivecontrol. 3.7. MycotoxinAnalysis 3.7.1. AflatoxinExtractionandQuantification ForAFB1extraction,halfoftheirradiatedsamples(2.5g)wereextractedbymaceration(25◦Cat 150rpm)withsodiumchloride(0.5g)andmethanol/water(20mL,80:20,v/v)for30min. Themixture wasthenfilteredbygravitythroughaWhatmanNo. 4filterpaper(Sigma-AldrichCo.,St.Louis,MO, USA)andanaliquot(10mL)ofthefiltratewasdilutedwithaportionofwater(40mL).Theextractwas homogenizedandfurtherfilteredthroughaWhatmanglassmicrofiberfilter(934-AH).Subsequently, Molecules2017,22,347 9of13 the filtered extract (20 mL) was purified through immunoaffinity column (AflaTest WB, VICAM, Watertown,MA,USA)bygravity,atarateofapproximately1–2drops/s. Thecolumnwaswasheda firsttimewithphosphate-bufferedsalinewithTween(PBS-T:NaCl(8g),Na HPO (12g),KH PO 2 4 2 4 (0.2g),KCl(0.2g),Tween20(0,1mL)madeupto1000mLwithdeionizedwaterandthepHvalue wasadjustedto7.0withNaOH),followedbyasecondwashwithultra-purewater(1mL).AFB1was elutedwith2mLofmethanol,collectedinaglassvial,filteredthrough0.2µmnylonfilters(Whatman) andanalyzedbyHPLCwithfluorescencedetection(FLD). SampleswereanalyzedusingaHPLCsystem(Smartline,Knauer,Berlin,Germany)coupledtoa photochemicalpost-columnderivatizationreactor(PHREDunit,AuraIndustries,NewYork,NY,USA), afluorescencedetector(FP-2020,Jasco,Easton,MD,USA)settoλ 365nmandλ 435nmandusing ex em theClarity2.4Software(DataApex,Prague,CzechRepublic). Thecompoundswereseparatedusingan isocraticelutionwithareverse-phaseC18column(100mm×4.6mm,MerckChromolithPerformance, Darmstadt,Germany)at35◦C(7971RGraceoven). Themobilephaseconsistedofamixturewith acetonitrile/methanol/water(10:30:60,v/v/v)withaflowrateof1mL/minandtheinjectionvolume was0.01mL.AFB1wasidentifiedbychromatographiccomparisonwiththestandard(AflatoxinB1, Biopure,Tulln,Austria)andquantificationwasbasedonthefluorescencesignalresponse. 3.7.2. OchratoxinAExtractionandDetermination OTAextractionfollowedtheproceduredescribedbyZhaoetal.[51]withsomemodifications. Briefly,theotherhalfoftheirradiatedsamples(2.5g)wereextractedbystirring(25◦Cat150rpm) withMeOH/1%NaHCO solution(12.5mL,70:30,v/v)for30minandsubsequentlyfilteredthrough 3 WhatmanNo. 4filterpaper. Afterwards,theextract(10mL)wasdilutedwithPBS-T(40mL)and furtherfilteredthroughaWhatmanglassmicrofiberfilter(934-AH).Thefilteredextract(20mL)was purifiedthroughanOchratestWBimmunoaffinitycolumn(VICAM,Watertown,MA,USA)andthe columnwaswashedfirstwithPBS-T(10mL)andthenwithultra-purewater(10mL).Afterwards OTAwaselutedwithmethanol(2mL),collectedinaglassvial,filteredthrough0.2µmnylonfilters (Whatman)andanalysedbyHPLC-FLD. OTAsampleswereanalysedusingtheHPLCsystemandcolumndescribedaboveforAFanalysis, butwithoutthederivatizationprocess.Thefluorescencedetectorwassettoλ 330nmandλem463nm, ex mobilephaseconsistedofamixturewithacetonitrile/water/aceticacid(70:29.5:0.5,v/v/v),witha flowrateof0.8mL/min,andtheinjectionvolumewas10µL.OTAwasidentifiedbychromatographic comparisonwiththestandard(OTAstandardsolutions,SigmaAldrichCo.) andquantificationwas basedonthefluorescencesignalresponse. 3.7.3. In-HouseMethodValidation StocksolutionsofAFB1(5µg/mL)andOTA(1mg/mL)werepreparedandstoredat−20◦C. Workingstandardsolutionsofeachmycotoxin(100ng/mL)werepreparedfromstocksolutionsdaily. Precisionandrecoverywereperformedbyspikingtheblanksamplewithtwodifferentmycotoxin concentrations: 10ng/gand30ng/gofAFB1andOTA,andonesetofunspikedsamplewasusedasa blank. Eachsamplesetwascomposedofsixreplicatesandtestedintwodifferentdays(threereplicates eachday). Instrumentationcalibrationparametersweredeterminedfollowingthemethodologypreviously describedbyAritaetal.[52]andtherecoveryratesweredeterminedfromthesixreplicatesofthe twospikinglevels,bycalculationoftheratioofrecoveredAFB1andOTAconcentrationrelativeto theknownspikedconcentration. Precisionwascalculatedintermsofintradayrepeatability(n=3) andintermediateprecision(interdaywithinlaboratoryreproducibility;twodifferentdays)foreach mycotoxin,atthetwocontaminationlevelsinspikedsamples. Linearity,limitofdetection(LOD),andlimitofquantification(LOQ)weredeterminedbythree series of analyses, using 11 standard solutions with concentrations ranging from 0.05 ng/mL to 20ng/mLofAFB1andOTA.LODandLOQwerecalculatedaccordingtothefollowingequations[52]: Molecules2017,22,347 10of13 LOD=3 × (sa/b)andLOQ=10 × (sa/b), wheresaisthestandarddeviationoftheinterceptofthe regressionlineobtainedfromthecalibrationcurve,andbistheslopeoftheline. 3.8. StatisticalAnalysis Dataanalysiswasperformedusingaone-wayanalysisofvariance(ANOVA)followedbyTukey’s HSDtest(p=0.05)usingaSPSSv. 23.0software(IBMCorp.,Armonk,NY,USA). 4. Conclusions Gammaradiationwastestedasapreservationanddecontaminationtechniqueindriedleaves ofA.citrodora. Regardingphenoliccomposition,doses1and10kGyshowedsomedifferencesinthe results,beingthat,atthelowestdose(1kGy),threeofthephenoliccompounds(compounds2,6,and 9)outoftheelevenidentifiedcompounds,showedasignificantincreaseinquantity(p<0.05). Fiveof theremainingeight(compounds1,3,5,8,and11)showedadecrease,withcompounds3and5having significantdecrease(p<0.05). Ontheotherhand, atthehighestdose(10kGy), onlycompound3 significantlyincreased, whilecompounds2,4,5,6,and9decreased,significantly. Thesametrend followsthroughwiththeTCP,TPA,TF,andTPC.Therefore,irradiationdemonstratedaslightdecrease insomeoftheidentifiedphenoliccompounds. Attheassayeddoses,theirradiationprocess,didnotinducedetectablehepatotoxicity. Treatments at 1, 5, and 10 kGy doses were not effective in significantly decreasing OTA and AFB1. Thus, the legislatedmaximumdoseof10kGyisineffectivetodecreasedecontaminationusingthestudiedmycotoxins. Inthecasewheremycotoxindecontaminationofherbsistheprimarygoalofirradiation,and consideringwatercontentasanimportantparameterinthedestructionofmycotoxinsbygamma rays,futureresearchshouldcontemplatetestingtheeffectivenessofgammaradiationinherbsprior todrying. Forthismatter,otherfeaturessuchasphysicalandchemicalcharacteristicsshouldalso bestudied. Acknowledgments: The authors are grateful to Ministry of Agriculture, Portugal (Project AROMAP— PRODER/FEADER/EU),forfinancialsupportoftheworkandE.Pereiragrant.Theauthorsaregratefultothe FoundationforScienceandTechnology(FCT,Portugal)forfinancialsupporttoCIMO(Pest-OE/AGR/UI0690/2015) andL.Barrosgrant(SFRH/BPD/107855/2015).Also,toPOCI-01-0145-FEDER-006984(LALSRE-LCM),funded byFEDER,throughPOCI-COMPETE2020andFCT.C2TN/ISTarealsogratefullytoFCTforfinancialsupport throughtheRECI/AAG-TEC/0400/2012andUID/Multi/04349/2013projects.Theauthorsarealsogratefulto “MaisErvas—AromáticaseMedicinais”forsamplesproviding.TheGIP-USALisfinanciallysupportedbythe SpanishGovernmentthroughtheprojectAGL2015-64522-C2-2-R. AuthorContributions:P.R.andI.C.F.R.F.designedtheexperiments;E.P.performedalltheexperimentalassays with collaboration of S.C.V. and A.L.A. in the irradiation procedure, and C.S.-B. and L.B. in the phenolic compoundsanalysis. ConflictsofInterest:Theauthorsdeclarenoconflictofinterest. 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