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..ChemicalEngineeringandProcessing:ProcessIntensificationVolume47,Issue12,November2008,Pages2256–2261ExtractionofisoflavonoidsfromPuerariabycombiningultrasoundwithmicrowavevacuumYangHua,TaoWang\o"Affiliation:a"a,MingxiaoWangb,SufangHan\o"Affiliation:a"a,PingyuWana,MaohongFan\o"Affiliation:c"c,aBeijingUniversityofChemicalTechnology,Beijing100029,bGeneralHospitalofChinaNationalCoalGroupCorp.,Beijing110013,ChinacSchoolofMaterialsScienceandEngineering,GeorgiaInstituteofTechnology,Atlanta,GA30332-0245,USAReceived24December2006.Revised30September2007.Accepted12December2007.Availableonline11January2008.,HowtoCiteorLinkUsingDOIPermissions&ReprintsAbstractThisstudyproposesanewmethodtoquicklyextractanddryisoflavonoidsfromPuerariaLobataOhwi<Pueraria>bycombiningultrasoundandmicrowavetechnologies.Thetimerequiredtoextractisoflavonoidsatcomparablelevelsofproductionbyultrasounddisruptionwas20timesshorterthanbyconventionalrefluxextraction.Moreover,testresultsfordryingtheextractedsubstancefromPuerariashowthatthemicrowave-vacuummethodis10timesfasterthantheconventionaltwo-stepvacuumapproach.Finally,differentinstrumentalanalysesofisoflavonoidsobtainedfromPuerariausingtheproposednewmethodshowthatextractionbyultrasounddisruptionandmicrowave-vacuumdryingaffectsneitherthestructurenorthecompositionoftheextractedsubstance.Keywords：Extraction;Isoflavonoids;Microwavedrying;Ultrasound1.IntroductionUltrasoundhasbeenusedtoextractcompoundsfromthevariouspartsofplantsformorethanthreedecades[1].Duetothedisruptionofcellwallsandenhancedmasstransferofcellcontents,ultrasoundisabletoacceleratetheextractionoforganiccompoundsfromthebodiesofplants[2]

and

[3].Comparedwithconventionalsolventextraction,theuseofultrasoundmakestheextractionofvaluablecompoundsmoreefficientbymeansofshortertimeframesandlowerextractiontemperatures.Ultrasoundiscurrentlyemployedtoextractsuchpharmacologicallyactivecompoundsaspolysaccharides,cellulose,flavonoids,saturatedhydrocarbons,fattyacidesters,andsteroidsfromplantmaterials[4].Microwavedryingisoftenusedtoevaporatewaterinwood,foodstuffs,drugs,andores,amongothercommodities[5]

and

[6];itcanalsobeusedfordistillation[7]andextraction[8],[9]

and

[10].Combiningtheapplicationofmicrowaveswithvacuumtechniquesfordryingofferstwomajoradvantages,namely,rapiddryingduetotheabilityofmicrowavestoheatsolventsinstantaneouslyandhomogeneously,andenhancedrateandextentofmasstransferatsub-atmosphericpressureandlowtemperatures,whichisessentialforthermo-labileproducts.Suchdryingtechnologiesarethereforeimportantforindustriessuchaspharmaceuticals[11].ThepresentstudyoftheextractionofisoflavonoidsfromPuerariabyultrasounddisruption,togetherwiththeirdryingbymeansofcombinedmicrowaveandvacuumtechnologies,offersanalternativetoconventionaltechnologiesforextractinganddryingisoflavonoidsfromPueraria.2.Experimental2.1.PlantmaterialsandchemicalsPuerariacollectedinChina'sHenanprovincewaswashed,dried,andcutinto5

mmsegments.ThestandardsampleofPuerarinwassuppliedbyChina'sNationalInstitutefortheControlofPharmaceuticalandBiologicalProducts.Analyticalreagent-gradeethanolandethylacetatewereusedinallexperiments.2.2.ExtractionFig.1showsthecombinedmicrowave-ultrasoundexperimentalsetupusedforextraction.Theapparatusconsistsmainlyofanextractioncomponent<a>,amicrowavedryingcomponent<b>andasolventrecyclingcomponent<c>.UltrasoundextractionwascarriedoutusingaCF-152050

kHz1200

Wultrasounddisintegrator.AGALANZWP700L17microwaveovenwithaTeflon®evaporatingdishwasusedfordrying.Fig.1.

Schematicdrawingoftheapparatususedforextraction<a:extractingpart;b:microwavedryingpart;c:solventrecyclingpart;1:bracket;2:ultrasoundtransducer;3:beaker;4:sealinggasket;5:teflonevaporatingdish;6:turningplate;7:microwaveoven;8:teflontube;9:latextube;10:inlet;11:outlet;12:vacuumsucker>.\o"Viewthumbnailimages"TwentygramsofPuerariawereputintoa1000

mlbeakerforultrasoundextraction.After400

mlof70%ethanoladded,thebeakerwasputontheultrasounddisintegrator,followedbyextractionundertheultrasoundhornfor30

minatroomtemperature.SamplestakenperiodicallywereanalyzedusinganUV-2102PCUltraviolet–Visible<UV–VIS>spectrophotometer.Arefluxextractionexperimentwasalsoperformedforpurposesofcomparison.After20

gofPuerariaweretransferredintoa1000

mlthree-neckflask,400

mlof70%ethanolwasadded.Themixturewasstirredat600

rpmandrefluxedfor120

min,duringwhichsamplesweretakenperiodicallyforanalysisbyUVspectrophotometer.2.3.DryingofextractedproductAGALANZWP700L17microwaveovenwithaTeflon®evaporatingdishwasusedfordryingtheextractedproduct.Forrecyclingthesolventandensuringsafety,themicrowaveovenwasmadeairproofbydrillingaholethroughitsroofandpassingaTeflon®tubethroughthehole.Thetubewasconnectedtoavacuumsystemtowithdrawsteamandvolatilechemicals,allowingwaterandorganicsolventstobeevaporatedbythemicrowave-vacuumapparatus.Toinitiatedrying,200

mlofultrasound-extractedresultantwasputinanevaporatingdishandplacedinthemicrowaveoven.Theextractedresultantbecamesolidinabout10

min.Another200

mlofextractedresultantwasdriedusingarotatingevaporatoruntilnodistillatecameout,followedbydryingitintopowdersforabout80

mininanoven.2.4.AnalyticalmethodsTheextractedPuerariaisoflavonoidswereanalyzedbyUVat250

nmwavelength[12].ThestandardcurveofabsorbanceversusPuerarinconcentrationwasobtainedinthefollowingmanner:tenmilligramsstandardsampleofPuerarinwasfirstputintoa50

mlvolumetricflask;then95%ethanolwasaddedtothescale,followedbyshakingthemixtureuntilitwashomogeneous.Aliquotsofsolutions<0.2,0.4,0.6,0.8,and1.0

ml>weretakenfromtheflaskandplacedinto10

mlvolumetricflasks.1.0

mlofethanolwasthenaddedtoeachflaskanddeionizedwaterwasaddedtotheflaskscalestocompletethepreparationofstandardsolutions.Blanksampleswerepreparedusingthesameprocedurespreviouslymentionedbutwithouttheadditionofextractedisoflavonoids.TheUVabsorbanceofblankandeachstandardsamplewasmeasuredata250

nmwavelength.Theextractresultantwasanalyzedasfollows;onemilliliterofextractwastransferredintoa50

mlvolumetricflaskfollowedbytheadditionof95%ethanoltothescaleandmixing.Thesolutionwasleftovernight.Onemilliliterofsupernatantwasthentransferredintoa25

mlvolumetricflask;deionizedwaterwasaddedtothescale,followedbymeasuringtheabsorbanceat250

nm.Theconcentrationofisoflavonoidsinthesolutionwasobtainedfromthestandardcurve.Thelevelofisoflavonoidsinthesampleiscalculatedas<1>wherec1istheconcentrationofisoflavonoidsintheextractresultant,V1isthevolumeofextractresultant,andmpuerariaistheweightofthePueraria.Twentymilligramsofextractedproduct<powder>and30

mlof95%ethanolweremixedina50

mlvolumetricflasktoinitiateanalysisoftheextract.Themixturewasthenheatedinahotwaterbathtodissolvetheproduct.Aftercooling,theflaskwasfilledwith95%ethanoltothescale.Afterabout12

h,1.0

mlsupernatantwasplacedina25

mlvolumetricflaskanddilutedwithdeionizedwatertothescale.Theabsorbanceofthesolutionwasthenmeasuredat250

nm.Theyieldofpowder,contentoftotalisoflavonoidsinpowder,andtotalyieldofisoflavonoidsaredefinedasfollows:<2><3><4>wherempowderandmpuerariaaretheweightsofthepowderandPueraria,respectively,c2isconcentrationofthesamplesolution,andV2isthedilutionvolume.AnS-250scanningelectronmicroscopewasusedtoprovethecavitationsandhittingeffectofultrasounddisruptiononthecellsofPueraria.ToexaminetheeffectofultrasounddisruptionandmicrowavedryingontheextractedisoflavonoidsfromPueraria,theextractedresultantwasanalyzedwithaVECIDR22Infrared<IR>SpectrometerandbyLC5500HighPerformanceLiquidChromatography<HPLC>.TheHPLCwasoperatedwithaUVdetector<wavelengthsetat250

nm>andaC18chromatographycolumn.Methanol–water<3:7,v/v>wasusedasthemobilephaseattheflowrateof1

ml

min−1anda0.02

mlsamplewasinjected.3.Resultsanddiscussion3.1.ChoiceofextractingconditionsofultrasoundDifferentsolvents,includingwater,methanol,ethanol,andethylacetate,werecomparedfortheirextractionabilities.Thesamesolventvolumesandextractiontimeswereusedintheexperimentsforcomparativeextractiontests.ThetestresultsareshowninFig.2.Asshown,theextractionefficiencyforisoflavonoidsfromPuerariawassignificantlyaffectedbythetypeofsolventused.Althoughwatercanbeusedtoachievethehighestextractionefficiency,toomuchstarchwasdissolvedinwater,whichmadetheextractedresultantturbid,ropy,anddifficultinfiltration.Sincemethanolistoxicandethylacetateiscostly,amixtureofethanolandwaterwaschosenasanextractionsolvent.Amongthesolventstested,anethanol–watermixtureata7:3volumeratioprovedtohavethehighestextractionefficiency,asshowninFig.2.Fig.2.

Theeffectofdifferentsolventsonultrasoundbasedextractingefficiencyofisoflavonoids<solventvolume:400

ml;liquid/solidratio:20

ml/1

g;extractingtime:20

min>.Theeffectoftheratiothevolumeofsolvent<i.e.amixtureofethanolandwaterata7:3volumeratio>totheweightofPueraria<liquid/solidratio>ontheextractionefficiencyofisoflavonoidsusingultrasoundwasstudied,withtheresultslistedinTable1.Asshown,theincreasingliquid/solidratioledtoaconsiderableincreaseinextractionefficiencyuntiltheratioreached20:1,whichisconsideredoptimalforthemostefficientuseofsolventandenergy.Table1.EffectoftheratioofsolventvolumetoweightofPueraria<liquid/solidratio>ontheultrasoundbasedextractingefficiencyforisoflavonoidsLiquid/solidratio101520253040Contentoftotalisoflavonoids<%>11.715.517.217.517.918.13.2.Comparisonbetweenultrasound-disruptingextractionandrefluxingextractionTheeffectoftimeontheefficiencyofisoflavonoidextractionfromPuerariausingbothultrasounddisruptionandrefluxextractionwasstudied.TherelationshipbetweenthelevelsoftotalisoflavonoidsandextractiontimeisdisplayedinFig.3.Asshown,thelevelsofisoflavonoidsextractedfromPuerariabyultrasounddisruptionreachedabout19%over20

min,whereasthelevelsofisoflavonoidsextractedthroughrefluxwereonly14.3%and16.5%,after1and2

h,respectively.Bycontrast,ultrasounddisruptiontookonly5

mintoreachacomparable16.0%levelofextractionefficiency<Fig.3>,aboutone-twentieththetimerequiredbyrefluxextraction,clearlydemonstratingthatultrasounddisruptionissuperiortorefluxextraction.Fig.3.

Comparisonbetweenultrasound-disruptingandrefluxingextractionforisoflavonoids<weightofPueraria:20

g;extractionsolvent:400

mlof70%ethanol;liquid/solidratio:20

ml/1

g>.Itiswellknownthatmostbiologicallyactivecompoundsofplantsexistintheircellwalls.Toextractthesecompounds,thecellwallscanbedisruptedbyultrasoundextraction.Ultrasoundcausesintenseshaking,highacceleration,intensecavitations,andstirring,allofwhichcanacceleratethedissolutionofpharmacologicalagents.Furthermore,enhancementoftheextractionrateshortensextractiontime,therebyconservingsolventandmitigatingtheeffectsofhightemperaturesontheeffectivenessofpharmacologicalagents.InordertoconfirmthecavitationsandhittingeffectofultrasounddisruptiononcellsofPueraria,scanningelectronmicroscopy<SEM>wasusedtocharacterizePuerariacellsbeforeandafterbothultrasounddisruptionandrefluxextraction.Fig.4showstheprofilesofcells,arrangedinorder,withsolidsubstancesapparentindryPuerariacells<Fig.4a>.SomesmallcrannieswereobservedinafewcellsandnosolidsubstanceswereshowninPuerariacellsafter2

hofrefluxextraction<Fig.4b>.Thiscanbeexplainedbythefactthatthesolventusedduringextractionenteredthecellsthroughtheirgapsandcranniesandcontactedthesolidsubstances,consequentlymovingtheirpharmacologicalcompoundsintothesolvent.Bycontrast,after20

minofultrasound,thecellscouldnotbedistinguishedandtheirwallswerealmostcrackedanddisrupted,asseeninFig.4c,whichresultedfromthehittingandcavitatingofultrasound'sintenseshaking.ThehugeinstantaneousenergygeneratedbytheultrasoundsystemcanleadtothedisruptionofPuerariacellsandthequickdissolutionoftheisoflavonoidsinPuerariacellsinsolventwithoutapermeationprocess.Fig.4.

Scanningelectronmicroscopy<SEM>photosofPuerariacells<a:dryPuerariacells;b:Puerariacellsobtainedthroughrefluxingfor2

h;c:Puerariacellsobtainedthroughtheactionofultrasoundfor20

min;amplificationmagnitude:×200,000;electronacceleratingvoltage:19

kV>.IsoflavonoidsinPuerariaarecomposedprimarilyofpuerarin,daidzin,anddaidzein[13].ThestructuresofthethreecomponentsareshowninFig.5.ToexaminetheeffectsofultrasounddisruptionontheextractedisoflavonoidsfromPueraria,theextractedresultantwasanalyzedbyHPLC,UVSpec,andIRSpec,withtheresultspresentedinFig.6.Fig.6aandbare,respectively,theIRspectraofthepowderextractedbyultrasounddisruptionfor20

minandbyrefluxingfor2

hwith70%ethanolassolvent.Fig.6showsthatthelocationsofcharacteristicfunctionalgroupsofthetwosamplesareidenticalintheirIRspectra,whichdemonstratesthatextractionbyultrasounddisruptiongeneratesnonegativeeffectsonthestructureandcomponentsofextractedresultantscomparedtotheconventionalrefluxextractionmethod.TheUVspectraforthestandardPuerarinsolutionsandextractedresultantsfromultrasounddisruptionandrefluxingareshowninFig.7.Nosignificantdifferenceswereshownamongthethreecurves,exceptfortheheightsofabsorptionpeaksat250

nm.TheretentiontimesinHPLCchromatogramsfortheresultantsfromstandardPuerarinsamplesextractedbyultrasounddisruptionandrefluxingwere11.38,11.29,and11.30

min,respectively.Thecontentsofextractedresultantsfromultrasounddisruptionandrefluxingunderthegivenconditionswere,correspondingly,3.6%and3.7%,whichindicatesthatPuerarinwasnotdestroyedbyultrasound.Fig.5.

ThemolecularstructuresofisoflavonoidsinPueraria.\o"Viewthumbnailimages"Fig.6.

IRspectraofresultantextractedbyultrasound<a>andbyrefluxing<b><refluxingextractiontime:2

h;ultrasounddisruptiontime:20

min;wavenumberscanrange:4000–500

cm−1;weightofPueraria:20

g;extractionsolvent:400

mlof70%ethanol;liquid/solidratio:20

ml/1

g;dryingmethod:vacuum>.\o"Viewthumbnailimages"Fig.7.

UVspectraofstandardsample<a>,resultantextractedbyultrasound<b>andresultantextractedbyrefluxing<c><wavelengthscanrange:200–600

nm;ultrasounddisruptiontime:20

min;refluxingextractiontime:2

h;weightofPueraria:20

g;extractionsolvent:400

mlof70%ethanol;liquid/solidratio:20

ml/1

g>.\o"Viewthumbnailimages"3.3.ComparisonbetweenmicrowaveandconventionaldryingmethodsTheresultsofmicrowaveandconventionaldryingof200

mlofultrasound-extractedresultantsarelistedinTable2.Itcanbeseenthattherateofmicrowave-vacuumdryingis11timesfasterthanthatofconventionalvacuumdrying,eventhoughtheyieldsoftotalisoflavonoidsweresimilar.Table2.ComparisonbetweenmicrowaveandconventionaldryingmethodsDryingmethodTimeofdrying<min>Yieldofthepowder<%>Contentoftotalisoflavonoidsinthepowder<%>Yieldoftotalisoflavonoids<%>Conventionalvacuumdrying14033.348.216.0Microwavevacuumdrying1330.850.115.4Extractionmethod:ultrasoundextraction;extractingtemperature:roomtemperature;extractionsolvent:400

mlof70%ethanol;extractingtime:20

min.Theproductsobtainedfrom5.1%Puerarinwithultrasound-disruptingextractionandmicrowave-vacuumdrying,andthosefrom4.8%Puerarinobtainedwithultrasound-disruptingextractionandconventionalvacuumdryingwerecharacterizedbyHPLC.Thesimilarityofretentiontimes<11.33and11.20

min,respectively>demonstratesthatPuerarinisoflavonoidswerenotdestroyedbymicrowave-vacuumdrying,whichcanbefurtherprovenbythesimilarityoftheirIRspectraasshowninFig.8.Fig.8.

IRspectraofproductsobtainedbyultrasound-disruptingextractionandmicrowavevacuum<a>andultrasound-disruptingextractionaswellasconventionalvacuumdrying<b><dryingtimeofmicrowavevacuum:10

min;dryingtimeofconventionalvacuum:80

min;wavenumberscanrange:4000–500

cm−1>.\o"Viewthumbnailimages"4.ConclusionsThecellwallsofPuerariacanbedisruptedbyultrasound.TheextractiontimeofisoflavonoidsfromPuerariaisshorterthanthatusingconventionalrefluxextraction.Theultrasound-basedmethodincreasesextractionefficiencywithoutdestroyingthepharmacologicalagentsofPueraria.Also,microwave-vacuumtechnologyissuperiortoconventionalvacuumtechnologyfordryingtheproductextractedfromPuerariabyultrasound.Finally,thestructureandcomponentsofPuerariaisoflavonoidsobtainedusingultrasoundandmicrowave-vacuumtechnologiesaresimilartothoseproducedusingconventionalmethods.References[1]M.Salisova,S.Toma,T.J.MasonComparisonofconventionalandultrasonicallyassistedextractionsofpharmaceuticallyactivecompoundsfromSalviaofficinalisUltrason.Sonochem.,4<1997>,pp.131–134[2]H.M.Waksmundzka,A.Petruczynik,A.Dragan,D.WianowskaEffectofextractionmethodontheyieldoffuranocoumarinsfromfruitsofArchangelicaofficinalisHoffmPhytochem.Anal.,15<2004>,pp.313–319[3]Z.Hromadkov,A.Ebringerov,P.ValachovicComparisonof