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Determination

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Determination

    Determination

    December2000JoumdofNortheastAgriculturalUmver~ityVo1.7,No.2149~157 ArticleID:1006--8014(2000)02--0149--09

    DeterminationOfThiOglucOsidein

    CabbagebyUsingtheUltraViOletVisible

    LightSpectrophotometer

    WANGJing,WANGQing,ZHENGYi

    (NortheastAlzriculturalUniversity,Harbin,Heilonffiianff150030.PRChlna) ABRACT

    Theultraviolet--visiblelightspectrophotometermethodwasadoptedtOdeterminethioglucosideincab

    bagewiththeseeds.Individualplantofcabbagewasusedastestmaterials,palladiumchlorideaseomplexing

    agentandsodiumcelluloseglycolateasdispersingagent.Theresultsshowedthatpalladiumdthioglucoside

    methodcouldbetakenasaquick,easyandprecisequantitativeanalysismethodtodeterminethic'gluo~sidein

    cabbage.

    KEYWORDS:Ultravioletvisiblelightspeetrophotometermethod:cabbage;

    thioglucoside;determinationmethod

    CLCnumber:S635,Q33,Q591.4Documentcode:A

    1Introduction

    Cabbagevegetableanditskindareonecategoryofthecommonlycultivatedcropsin bothChinaandtheworld.Thereexistsglucosidecompound

    thioglucosideandmeanwhileal

    SOhydrolyticenzyme(alsocalledasmustardenzyme)inastablestateincabbageseedsandits plant,bothofwhicharcnotCO

existentinasametissue(Gelanying1984).Whenonecrushes

    theseedsinwater,thethioglucosidecanbecatalyzedandhydrolyzedintoglUCOSe,bisulfate,

    isothiocyanate,oxazolidinethione,nitrile.Thesetoxicsubstanceswillaffectitsediblequality.

    Thebreedingworkershothathomeandabroadalwaysmaketheireffortsinraisinglow thioglucosidevarieties,butthherecentresearchbelievesthatthecompoundscontainedin cabbagevegetablehaveananti

    cancereffect.Suchcompoundscanblockthecarcinogenicel-

    feetinchemistryandarecalledas"cancerinhibitor.Ithasagreatsignificancetostudythe quickmethodsfordeterminationofthioglucosideincabbage.

    Asregardsthemethodsfordeterminationofthioglucosideincabbage,thefollowing threecategoriesofmethodscanbechosentodeterminethetotalquantityofthioglueosideand variousingredients:totalglucoseandsulfatedeterminationmethods,totalthinglucosidede. terminationmethodsanddegradationproducedeterminationmethods.Fordeterminationof Receiveddatet2000l13O

150?JournalofNortheastAgriculturalUnivers[tyVo1.7

    thioglucosideinthecabbageseedsanditsindividualplantbodyduringitsgrowthperiod,the firstcategoryofmethodssuchasglucosetestpapermethodanddinitrosalicylieacidspec

    trophotometermayCRUSeacertainerrorduetoaninfluenceofglucoseproducedby metabolismofplantbody.Itisdifficultforbariumsulfateweightmethod(WuMoucheng 1985)andatomicabsorptionspectrummethodtosidesteptheinterferenceofSO?

    absorbed

    byplantbodyandthatdegradedbythioglueoside.Tothiscontrary,thesecondcategoryof methodshasahighaccuracywithpreciousinstrument(gasandliquidphaseehromatogram (WuMouchons1988)tobenecessaryandittakesalongtimetopreparethesamplewith trivialdetailstobecopedwith,thusitisimtpractica1.Butthethirdcategoryofmethodssuch asWetterthioureaultravioletspectrummethodcansolvetheexistingproblemasmentioned above,havingsuchspecificfeaturesasquick,simple,exactandrepeatable.Italsohasits

    shortcomingthatitisnecessarytoextractandpreparethemustardenzyme(PcngLiangcaiet a1.1989).Thepresentresearchtakesthecabbageseedsanditsplantbodyasthetestingob

    jectives,puttingforwardwithamethodfordeterminationofpalladiumthioglucoside.Addi

    tionofthedispersingagentsodiumcelluloseglyeolatacanmakethecolorimetriesolution containingpalladiumthioglucosidedepositsintoclearsolsolutionsoastoeliminatetheinflu

    enceofpalladiumthioglucosidedepositsonluminosityanalysis.Atthesametime,astudyof theconditionsfordevelopmentreactionofpelladiumthioglucosideisalsomadewithabetter testingconditionbeingchosen,makingthepalladiumthioglucosidemethodasaqualitativand

    semi--quantitativemethodbecomeaquick,simpleandexactquantitativeanalysismethod. Thiscanlightenandexpeditetoacertainextentthebreedingprocessandstudyofthetoxin incabbageplantbodiesandextractionofanti--cancerpreparation.

    2Materialsandmethods

    2.1Material

    CabbageseedsandcabbagewereprovidedbythehorticultureteststationofNortheast AgrieultureUniversity.

    ColeseedshavingaknownthioglucosidecontentwereprovidedbyChinaNationalIol CropsResearchInstituteCropsQualityImprovementcenter.

    2.2Reagent

    2.2.1PdC1zcolorsolation

    Weigh177mgPdCI2andputitintoa150mLbeaker,thenadd2mLof2mol?Lchlo

    rideacidand2OmLwater.Afterheatinganddissolving,pourintowatertodiluteto250mL. 2.2.20.1%?dtIlmcdIe|I~celatesolutloa

    Weigh1gsodiumcelluloseglycolatetodissolveinalittlewater.Afterheatingandcom

    pletelydissolved,diluteto1000mLbyaddingwater.TakeOuttheclearliquidforfutureuse afterstorageoveranight.

    Thereagentusedinthetestshouldhavethepurityforthepurposeofanalysisandthe

No.2JournaloNortheastAgricuhuralUniversity151?

    waterusedshallbede--ionizedwater.

    2.3Method

    2.3?1Deterndnstienofcabbageseeds

    Preciselyweighthepulverizedcabbageseeds100mgandputintoa10mLtesttube,dry for10mininboiledwatertodeactivateitsmustardenzyme,add810mLhotwaterata

    temperatureofabout90?,thensteamformorethan20mininboiledwatertoextract,take outtokeepmotionlessandCOOldown.dilutewithwatertothescaleandshakeup.Filtrate theliquidattheupperlayertogetclearsolution.take1.0mI,clearsolutionandputintoan- other10mLtesttube.Add2mL0.1sodiumcelluloseglycolatesolutionandshakeupful

    ly,add1mLPdCI2developerandcoverwithaplug,thenshakeupandkeepmotionlessfor1 hourtomakeitbefullydeveloped.Under"theconditionofthewavelengthat540nm,useul

    traviolet--visiblelightspectrophotomcterand1cmcolorimetriccuptodeterminetheabsorp

    tivitybytakingpalladiumchloride--sodiumcelluloseglycolatesolutionblanksolutionasthe contro1.Byconsultingthestandardcurve,calculatethethioglucosidecontent(theabovecon- ditionshallberegardedastheoptimumconditionchosenthroughthetests). 2.3?2Determinationofcabbageplant

    Ch0oseaheadoffreshcabbage,takesamplewiththehelpofsamplingdeviceandexactly weigh20gcabbageandthenputintoabeaker.Steamfor30mininboiledwater,pourintoa mortarandgrindintoamash.Add20mLhotwateratatemperatureofabout90"Candthen pourintoabeaker.Steamfor30mininboiledwater,takeOutandkeepmotionless.A[terit coolsdown.conductafiltrationandcentrLfugalseparationatalowtemperature(2500r? minandatatemperaturebelow10?).Taketheclearsolution15mLsuchobtainedto

    freezeanddryuntilthesolutionisdriedthoroughlybywayoffreezing.Adddeveloperto measuretheabsorptivityandcalculatethethioglueosidecontent.

    3Resultsanddiscussion

    3?1?1Vr*wingoflhestarerdcun0

    Takeandweigh100mgcoleseedshavingaknowncontent.Throughtheprocedures asmentionedabovein2.4.i,extractthioglucosideandtakeout0.50mL,0.80mL,

    l_10mL.1.50mLandz.00mLrespectivelytoputjnto10mLtesttubesafterafihration. Afterdevelopmentbyusingthemethodabovementioned,makeeveryabsorptlvitiesmeasured

    incorrespondingwiththeconcentrationsoftheirsolutionundertesttodrawastandard

    cUrve.

    Tbeknownthioglueosidecontentofcoleseedsis:128.77lamol/gandtheabsorptivities measuredaregiveninTabk1.

    Table1Albsorlptl'~tiesOfcele-e|d|kavlngakaewathioglecosidecontent 怒一"0.220,1289~2086:._462813o3-3863

?152?JournalofNortheastAgriculturalUniversityV01.7

    Accordingtothevaluesmeasured,aregressibnanalysisisconductedwithacurveob

    rained:y=O.O41+1.40x7=0.99154.5V-.ThestandardcurveisshowninFig.1.

    00.t0.20.30.40.50.6

    psol/ml

    Fig.1Standardcrveofthechangeofthethioglucosldecontentwithab~rptivlty 3.1.2Determinationofcabbageseeds

    Calculationformula:Thioglucosidecontent(,umol/g)=MXLXN/m Where:M--ThioglucosideconcentrationbyconsultingthestandardcurveaccordingtO theabsorptivity

    L——amountofdevelopmentsolutioninmL(5mL)

    ?——Theproportionofamountofthesolutionundertestintotalamountofthsolution extracted(10)

    ,,I——amountoftestingmaterialingram(0.10g)

    Table2ThIog|ue~idecontentincabbageseeds

    3.1.3Determlnationofcab~geplant

    Throughthemeasurement,theabsorptivityofcabbageplantis0.2744andtheconcert

    trationis1.667vmol/mLbyconsultingthestandardcurve.Thus,

    Thioglucosidecontent(~mol/g)1.667X3X80/15/20=0?033vmol/g

    3.2Influeneeofreactionconditions

3.2.1Iaflue.eeattheselectioninwavelengthotcolorcomplexonmeasu-t?

    Fig.2isanabsorptivespectrumobtainedbyscanningpalladiumthioglucosidecomplex compouadsolutionfromawavelengthof350nmto600nm.Itcanheseenthatacompara

    7e5432l0??o

Nm2Journa[0fNortheastAgrieutturatUniversity?153?

    tivelygreatabsorptivitywillbeobtainedupondeterminationofpalladiumthioglucosidecolor

    complexinarangefrom540llmto420am.Inordertoselectanappropriatewavelengthfor determination,drawanabsorptivecurveforpalladiumchloride——

    sodiumcelluloseglycolate

    blanksolution.AsshowninFig.3,thesaidsolutionhasacomparativelygreatabsorptivity. Intheprocessofdeterminationofthioglucosidecontent,thecomplexingofpalladiumwith thioglucosidecertainlyconsumespalladiumsolution,thuswhenthedeterminationisconduct

    edat420nm,thepalladiumcontentinthesolutionmayhaveagreateffeCttotheresult.But whenthedeterminationisconductedat540nm,theinfluenceoftheconsumptionofpalladi

    u?chlorideCallbeneglected.Theforegoingindicatesthatitisbettertoselectthewave

    lengthof540nmindeterminationofpalladiumthioglucosidecomplexthantoselectthe wavelengthof420nm.

    1

    

    0.8

    O.e

    O.'

    O.2

    O

    400450500550CO0

    Fig?2Palladiumthloglucosldeal~rptivespectrum

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    Fig?3Palladium~hlorlde--sedlumcellulcc~egly~olateal~orptivespectrum 3?2-2Uniformdistributionofpaliodlumtldoglucosidecomplexanditsstabnlty Palladiumthioglucosidecomplexisacolloida1sedimentandhasaneffecttothedetermi

    nationofluminosity,SOthecolorimetrlcdeterminationcanbeperformeddirectlybyadding thedispersingagentsodiumcelluloseglycolatetomakethecolorimetricsolutionclearSOthat

    itcandissolvethecolloid.Table3indicatesthatbeforeaddingthesodiumceUuloseglycolate. ,??啦0

?154'?JournalofNortheastAgricuLturaLUniversityV01.7

    thepalladiumthioglueosidesolutionproducessedimentanditisdifficulttocarryoutcol

    orimetry.Butwhenthesolutioncontainsoveronethirdof0.10sodiumcelluloseglycolate, thecolorimetricsolutionbecomesbrightandclear.Nosedimentisfoundafterstoragefor10 days.Afterstoragefor2days,theabsorptivitymeasuredisunchanged.Duringthesample tests,itwasalsoseenthatwhenaddinginsufficientsodiumcelluloseglycolate,fewsediment occurstotheindividualsamples.Afterstorageforoneortwodays,thesedimentdisappears andthesolutionbecomesbrightandclear.Thisindicatesthatsodiumcelluloseglycolate playsnotonlytheroleasadispersingagentbutalsoarolefordissolvingthecolloid. Table3InfluenceofsodiumcelluloseglycoluleOIBpalladium--thioglucosldesediment. Theabsorptivitiesinabovetablearetheresultsobtainedafterstoragefor1hourandthe absorptivitiesmarkedwithhretheresultsobtainedafterstoragefor2daysbytheuseofcob orimetry.

    3?2.3Stabilityofcoloredpalladiumthioglueosldesolution

    Undertheconditionsformeasurementaslistedinthebasicoperationprocedures,the palladiumthioglucosidesolutionisinacomparativelystablestateandifitisair--tight,the absorptivitiesmeasuredwillbealmostthesameafterstoragefor-morethan3days.(please seeTable4).

    Table4Stabilitytestofcoloredpalladium--thioglueosidesolution

    3.2.4AcidityusedlapullodiumthloglueosldecolortestsanddevelopmentgOIBpIetlontime

    Theaciditiesusedinpalladiumthioglucosidecolortestscanbewithinarangefrom 0.0028mo1?L'to0.5m01?LpreparedbytheuseofHCIsolutionwiththedelustering valuebeingnodifference.Theresultsofluminositymeasuredafterstoragefor12hoursfol

    lowingthedevelopmentwereshowninFig.4.

    Acidityhasnoobviouseffecttocolorreactionbutaconsiderableeffecttothecompletion timeofdevelopmentprocess.Whendevelopedin0.003mol?L-.to0.05mol?LHCISO-

    Iution.thedevelopmentprocessshalIbecompletedaftermorethan1hour.Butwhenthe acidityisincreased,alongerperiodoftimeshouldbenecessary(morethan12hours)(see

r

    NO.2JournatolNortheastAg6eu[turalUniversity

    Fig.5).Toquickenthecolorreaction,ithadbettertoselectalowacidity-Inthepresenttest, theHCIsolutionhavinganaciditywithinarangefrom0.0O3toolto0-05toolischosenand thedeterminationcanbeconductedafterstoragefor1hourbytheuseofcolorimetry. n6

    n4

    Q2

    0.5

    0.4

    O.3

    O.2

    0

    0nl2345

    Fig.4lnflaence0faciditytocolorreaction

    023

    蛐吼

    t(hr)

    Fig.5Influenceofacidnytothecompletiontimeofdevelopmentprocess 3.2.5Testonconsumptionofpalladiumchloride

    Whenthec0nsumptionofpalladiumis2.50to10timesasmuchastheconsumptionOf

    thioglucoside,thecompleiontimeofdevelopmentprocessisstablebutwhenlowerthn2?50 timesasmuch,thecompletiontimeofdevelopmentprocessshouldbeprolongedwithabad reeatabilitv.Whenm0rethantentimesasmuch,thecolorofpalladiumshallbetoodarkand thedeterminationtestshallalsobeaffectedwithfllargequantityofreagentbeingwasted. Therefore.itisbettertousepalladium2.50to10timesasmuchaStheconsumptionof thioglucoside.

    3.2.6Deactivationofenzyme

    Deactivatl0nofmustardenzymeinthepresenttestwaschoosentosteamdryinboiled wateratatemperatureof100'Cfortenminutesandbyaddingbioledw~tertoex. tract

    thioglucoside.Thesamplingsolutionsuchpreparedhasnotfoundanythloglucosidezymoly

    sisafterstoragefortwodays.ItfollowsfromTable5thatthe~amplingsolutionisstable,in

?156?JournalofNo~heutAgrlcuhuralUniversityVoL7

    dicatingthatthedeactivatingconditionsforenzymearereliable

    TableSInfluenceofthestoragetimeofsolutionoRthiogluc~cddtzymolysls 2-7Studyoftheextractionconditionsforthieglacoslde

    Inordertothoroughlyextractthioghcoside,thetestsonextractiontimeandwater amountconsumedweeconducted.Table6showstheresultsmeasuredatdifferentextraction timesinboiledwaterbyadding10mLboiledwaterafter10minutesofsteaming.Fromthis tableitcanbeseenthatafteraddingboiledwater,morethan15minutesoftheextraction timeshal1beenough.butover20minuteswil1bebetter.

    TaMe6InfluenceofextractiontimeoRahsorpflvlty

    ByaddingdiifferentamountsOfboiledwaterandextractingthioglucosidefar20minutes afatemperatureofIO0"C,theresultsobtainedaredifferent.Thisshowsthattheamountof wateraddedhasaneffecttottheextractionofthioglucoside.Undertheaboveconditions, morethan8mLboiledwatershouldbenecessarytomaketheresultsbecomealmostthe same(Table7).

    Table7Eff~tofamountofboiledwaterwedtoabsorptivity

    Adding8to10mLboiledwaterto100mgtestsampleandextractingfor2Ominutesale suitable.

    3?2-8Effectoftemperature?determinationofcabbagepIlnts

    Whendeterminingtheabsorptivitybytheuseofuhraviolet——visiblelightspectropho

    tometerandthetsbeingwithinarangefrom0.2to0.7,itisbelievedthatsuch tsareauthentic.Butthethioglucosidecontentincabageisverylow.soitisnec essarytoconcentratesoastoincreasetheconcentration.Ascabbagecontainssaccharidesub- stances,toavoidhappeningMillardreactionatahightemperatureandproducingcoloring mutterstoaffectthetestresults,acentrifugalseparationanddryingbywayoffreezingare conductedatalowtemperatureuntildriedthoroughlybywayoffreezing.Thenthemeasure- mentisconductedbytheuseofadevelopmentprocess.'

No.2JoumalofNortheastAgrkultutalUniversity?157?

    4Conclusion

    Onthebasisoftheabovestudyofconditionsformeasurement,theoptimummethodto determinethioglucosidecontentincabbageshall?

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