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1、Chemical Engineering and Processing: ProcessIntensificationVolume 47, Issue 12, November 2008, Pages 22562261Extraction of isoflavonoids from Pueraria by combiningultrasound with microwave vacuum?Yang Hu a,?Tao Wanga,?MingxiaoWang b,?Sufang Han a,.?Pingyu Wan a,?Maohong Fan c,?a Beijing University o

2、f Chemical Technology, Beijing 100029, China?b General Hospital of China National Coal Group Corp., Beijing 110013, China?c School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA30332-0245, USA?Received 24 December 2006. Revised 30 September 2007. Accepted 12 Decem

3、ber 2007. Available online11 January 2008.?/10.1016/j.cep.2007.12.013,How to Cite or Link Using DOI?Permissions & ReprintsAbstractThis studyproposes a new method to quicklyextract and dryisoflavonoidsfrom PuerariaLobata Ohwi (Pueraria) by combining ultrasound and microwave technologi

4、es. The timerequired to extract isoflavonoids at comparable levels of production by ultrasounddisruptionwas 20timesshorterthanby conventionalrefluxextraction.Moreover, testresultsfordryingtheextractedsubstancefrom Pueraria show thatthe microwave-vacuummethod is 10 times faster than the conventional

5、two-step vacuum approach. Finally,differentinstrumentalanalysesofisoflavonoidsobtained from Puerariausing theproposednew method show that extractionby ultrasounddisruptionand microwave-vacuumdrying affects neither the structure nor the composition of the extracted substance.Keywords：Extraction; Isof

6、lavonoids; Microwave drying; Ultrasound1. IntroductionUltrasoundhas been used toextractcompounds from the variouspartsofplantsformorethan three decades1 . Due to the disruption of cell walls and enhanced mass transferof cell contents, ultrasound is able to accelerate the extraction of organic compou

7、ndsfrom the bodiesof plants2and3 . Compared withconventionalsolventextraction,the use of ultrasoundmakes the extractionof valuablecompounds more efficientby meansof shorter time frames and lower extraction temperatures. Ultrasound is currently.employedtoextractsuchpharmacologicallyactivecompoundsasp

8、olysaccharides,cellulose, flavonoids, saturated hydrocarbons, fatty acid esters, and steroids fromplant materials4 .Microwave dryingis oftenused toevaporatewaterinwood,foodstuffs,drugs,and ores,among other commodities5and6; it can also be used for distillation7andextraction8,9and10 . Combining the a

9、pplication of microwaves with vacuumtechniques for drying offers two major advantages, namely, rapid drying due to theabilityofmicrowavesto heatsolventsinstantaneouslyand homogeneously, and enhancedrateand extentof mass transferatsub-atmosphericpressureand low temperatures,whichis essentialforthermo

10、-labileproducts.Suchdryingtechnologiesarethereforeimportant for industries such as pharmaceuticals11 .ThepresentstudyoftheextractionofisoflavonoidsfromPuerariabyultrasounddisruption,togetherwiththeirdryingbymeans ofcombinedmicrowaveandvacuumtechnologies, offers an alternative to conventional technol

11、ogies for extracting anddrying isoflavonoids from Pueraria.2. Experimental2.1. Plant materials and chemicalsPueraria collected in Chinas Henan province was washed, dried, and cut into 5mmsegments. The standard sample of Puerarin was supplied by Chinas National Institutefor the Control of Pharmaceuti

12、cal and Biological Products. Analytical reagent-gradeethanol and ethyl acetate were used in all experiments.2.2. ExtractionFig.1 shows the combinedmicrowave-ultrasoundexperimentalsetupused forextraction.Theapparatusconsistsmainlyofan extractioncomponent(a),a microwave dryingcomponent(b)and a solvent

13、 recyclingcomponent(c).Ultrasound extractionwas carriedoutusingaCF-1520 50kHz1200W ultrasounddisintegrator.A GALANZ WP700L17microwave oven with a Teflon?evaporating dish was used for drying.Fig. 1.Schematic drawing ofthe apparatus used forextraction(a: extracting part;b:microwavedryingpart;c: solven

14、t recycling part;1: bracket; 2:ultrasoundtransducer;3: beaker;4:sealinggasket;5:teflon evaporatingdish; 6:turning plate;7: microwave oven; 8:teflontube;9: latextube; 10: inlet; 11: outlet; 12: vacuum sucker).Twentygrams of Puerariawere put into a 1000 ml beakerfor ultrasound extraction.After400 ml o

15、f 70% ethanol added, the beaker was put on the ultrasound disintegrator,followedby extractionunder the ultrasound horn for 30min at room temperature.Samplestaken periodically were analyzed using an UV-2102PC Ultraviolet Visible (UV VIS)spectrophotometer.A refluxextractionexperimentwas also performed

16、forpurposes of comparison.After20gof Pueraria were transferred into a 1000ml three-neck flask, 400ml of 70% ethanolwas added. The mixture was stirred at 600rpm and refluxed for 120min, during whichsamples were taken periodically for analysis by UV spectrophotometer.2.3. Drying of extracted productA

17、GALANZ WP700L17 microwave oven with a Teflon? evaporating dish was used for dryingthe extracted product. Forrecyclingthe solventand ensuring safety, themicrowave ovenwas made airproofby drillinga holethrough itsroof and passinga Teflon? tube throughthe hole. The tube was connected to a vacuum system

18、 to withdraw steam and volatilechemicals, allowingwaterand organicsolventsto be evaporatedby the microwave-vacuumapparatus.To initiatedrying,200ml of ultrasound-extractedresultantwas put in an evaporatingdish and placed in the microwave oven. The extracted resultant became solid in about10min. Anoth

19、er 200ml of extracted resultant was dried using a rotating evaporatoruntil no distillate came out, followed by drying it into powders for about 80min inan oven.2.4. Analytical methodsThe extracted Pueraria isoflavonoids were analyzed by UV at 250nm wavelength12 .The standard curve of absorbance vers

20、us Puerarin concentration was obtained in thefollowingmanner: tenmilligramsstandardsampleof Puerarinwas firstput intoa 50mlvolumetricflask;then95%ethanolwas added to thescale,followedby shaking themixtureuntil it was homogeneous. Aliquots of solutions (0.2, 0.4, 0.6, 0.8, and 1.0ml) weretaken from t

21、he flask and placed into 10ml volumetric flasks. 1.0ml of ethanol wasthen added to each flask and deionized water was added to the flask scales to completethe preparation of standard solutions. Blank samples were prepared using the sameprocedures previously mentioned but without the addition of extr

22、acted isoflavonoids.The UV absorbanceof blankand each standardsample was measured ata 250nm wavelength.The extractresultantwas analyzedas follows;one milliliterof extractwas transferredinto a 50mlvolumetricflask followedby the additionof95% ethanoltothescaleandmixing. Thesolutionwas leftovernight.On

23、emilliliterofsupernatantwasthentransferred into a 25ml volumetric flask; deionized water was added to the scale,followedby measuringtheabsorbance at250nm. The concentrationofisoflavonoidsinthe solution was obtained from the standard curve. The level of isoflavonoids in thesample is calculated as(1).

24、where c1 isthe concentrationof isoflavonoidsinthe extractresultant,V1 is the volumeof extract resultant, andmpuerariais the weight of the Pueraria.Twenty milligrams of extracted product (powder) and 30ml of 95% ethanol were mixedin a 50ml volumetric flask to initiate analysis of the extract. The mix

25、ture was thenheatedina hot waterbath todissolvethe product.Aftercooling,the flaskwas filledwith 95% ethanol to the scale. After about 12h, 1.0ml supernatant was placed in a25ml volumetric flask and diluted with deionized water to the scale. The absorbanceof the solution was then measured at 250nm. T

26、he yield of powder, content of totalisoflavonoids in powder, and total yield of isoflavonoids are defined as follows:(2)(3).(4)wherempowder andmpueraria are the weights of the powder and Pueraria, respectively,c2 isconcentration of the sample solution, andV2 is the dilution volume.An S-250 scanning

27、electron microscope was used to prove the cavitations and hittingeffect of ultrasound disruption on the cells of Pueraria. To examine the effect ofultrasounddisruptionand microwave dryingon the extractedisoflavonoidsfrom Pueraria,the extracted resultant wasanalyzedwitha VECIDR22 Infrared(IR)Spectrom

28、eter andbyLC5500 High Performance Liquid Chromatography (HPLC). The HPLC was operated with a UVdetector (wavelength set at 250nm) and a C 18 chromatography column. Methanol water(3:7, v/v) was used as the mobile phase at the flow rate of 1mlmin - 1 and a 0.02mlsample was injected.3. Results and disc

29、ussion.3.1. Choice of extracting conditions of ultrasoundDifferentsolvents,includingwater,methanol,ethanol,and ethylacetate, were comparedfortheirextractionabilities.The same solvent volumes and extractiontimeswere usedinthe experimentsforcomparativeextractiontests.The testresultsareshown in Fig.2.A

30、s shown,theextractionefficiencyfor isoflavonoidsfrom Puerariawas significantlyaffectedbythetype ofsolvent used.Althoughwatercan be usedto achievethe highestextractionefficiency,toomuch starchwas dissolvedinwater,whichmade theextractedresultant turbid,ropy, anddifficult in filtration.Since methanol i

31、s toxic andethylacetate is costly, a mixture of ethanol and water was chosen as an extraction solvent.Among the solvents tested, an ethanol water mixture at a 7:3 volume ratio proved tohave the highest extraction efficiency, as shown inFig. 2 .Fig.2.The effectofdifferentsolventsonultrasoundbasedextr

32、actingefficiencyofisoflavonoids(solventvolume:400ml;liquid/solidratio:20ml/1g;extractingtime:20min).The effect of the ratio the volume of solvent (i.e. a mixture of ethanol and water at a 7:3 volume ratio) to the weight of Pueraria (liquid/solid ratio) on the extraction efficiency of isoflavonoids u

33、sing ultrasound was studied, with the results listed in Table 1 . As shown, the increasing liquid/solid ratio led to a considerable increase inextractionefficiencyuntilthe ratioreached 20:1,which isconsideredoptimalforthemost efficient use of solvent and energy.Table1.Effectoftheratioofsolventvolume

34、toweightofPueraria(liquid/solidratio)ontheultrasoundbasedextractingefficiencyforisoflavonoidsLiquid/solid ratio101520253040Content of total isoflavonoids (%)11.7 15.5 17.2 17.5 17.9 18.13.2. Comparison between ultrasound-disrupting extraction and refluxingextractionThe effectoftimeon theefficiencyof

35、 isoflavonoidextractionfrom Pueraria usingbothultrasound disruption and reflux extraction was studied. The relationship between thelevelsoftotalisoflavonoidsand extractiontime isdisplayedinFig.3. As shown, thelevelsofisoflavonoidsextractedfrom Puerariaby ultrasounddisruptionreachedabout19%over20 min

36、,whereas thelevelsof isoflavonoidsextractedthroughrefluxwere only14.3% and 16.5%,after 1 and 2h, respectively.By contrast,ultrasounddisruptiontookonly 5min to reach a comparable 16.0% level of extraction efficiency (Fig. 3), aboutone-twentieththetimerequiredby refluxextraction,clearlydemonstratingth

37、atultrasound disruption is superior to reflux extraction.Fig. 3.Comparison between ultrasound-disrupting and refluxing extraction for isoflavonoids(weight of Pueraria: 20g; extraction solvent: 400ml of 70% ethanol; liquid/solid ratio:20ml/1g).It is well known that most biologically active compounds

38、of plants exist in their cellwalls.To extractthesecompounds,thecellwallscanbe disruptedbyultrasoundextraction.Ultrasoundcauses intenseshaking,high acceleration,intensecavitations,and stirring, all of which can accelerate the dissolution of pharmacological agents.Furthermore,enhancementof theextracti

39、onrateshortens extraction time,therebyconservingsolventand mitigatingtheeffectsof hightemperatureson theeffectivenessof pharmacological agents. In order to confirm the cavitations and hitting effect ofultrasounddisruptionon cellsofPueraria,scanningelectronmicroscopy(SEM) was usedto characterize Puer

40、aria cells before and after both ultrasound disruption and refluxextraction.Fig.4 shows the profilesofcells,arrangedin order,with solidsubstancesapparent in dry Pueraria cells (Fig. 4 a). Some small crannies were observed in a fewcellsand no solidsubstanceswere shown inPuerariacellsafter 2h ofreflux

41、extraction( Fig. 4 b). This can be explained by the fact that the solvent used during extractionentered the cells through their gaps and crannies and contacted the solid substances,consequentlymoving theirpharmacologicalcompounds intothesolvent.By contrast,after20min ofultrasound,thecellscouldnotbe

42、distinguishedand theirwallswere almostcracked and disrupted,as seen inFig.4c,which resultedfromthehittingand cavitatingof ultrasoundsintenseshaking.Thehugeinstantaneousenergygeneratedby theultrasoundsystemcan leadtothedisruptionofPuerariacellsand thequickdissolutionof the isoflavonoids in Pueraria c

43、ells in solvent without a permeation process.Fig. 4.Scanning electron microscopy (SEM) photos of Pueraria cells (a: dry Pueraria cells; b:Puerariacellsobtained throughrefluxingfor 2h; c: Puerariacellsobtainedthrough the actionof ultrasound for 20min; amplification magnitude:200,000; electron acceler

44、ating voltage:19kV).Isoflavonoidsin Puerariaare composed primarilyof puerarin,daidzin,and daidzein13 .The structures of the three components are shown inFig. 5. To examine the effects ofultrasounddisruption ontheextracted isoflavonoids fromPueraria, theextracted.resultantwas analyzed by HPLC, UV Spe

45、c,and IR Spec,withtheresults presented inFig.6. Fig.6a and b are,respectively,the IR spectra ofthepowder extractedby ultrasounddisruption for 20min and by refluxing for 2h with 70% ethanol as solvent.Fig. 6shows that the locations of characteristic functional groups of the two samples areidenticalin

46、theirIRspectra,whichdemonstratesthatextractionbyultrasounddisruption generates no negative effects on the structure and components of extractedresultants compared to the conventional reflux extraction method. The UV spectra forthe standardPuerarinsolutions and extracted resultantsfromultrasounddisru

47、ptionandrefluxing are shown inFig. 7 . No significant differences were shown among the threecurves, except for the heights of absorption peaks at 250nm. The retention times inHPLC chromatograms for the resultants from standard Puerarin samples extracted byultrasound disruption and refluxing were 11.38, 11.29, and 11.30min, respectively.The contentsofextractedresultantsfromultras