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1、Chemical Engin eeri ng and Process ing: ProcessInten sificati onVolume 47, Issue 12, November 2008, Pages 2256- 2261Extractio n of isoflav on oids from Pueraria by comb ining ultraso und with microwave vacuumYa ng Hu：Tao Wan g：bMingxiaoWang ,Sufang Han :Pin gyu Wana,Maoho ng Fan"a Beijing Unive

2、rsity of 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, USAReceived 24 December 2006. Revised 30 September 2007. Accepted 12

3、 December 2007. Available online11 January 2008.,How to Cite or Link Using DOIPermissions & ReprintsAbstractThis study proposes a new method to quickly extract and dry isoflavonoidsfrom PuerariaLobata Ohwi (Pueraria) by combining ultrasou nd and microwave tech nologies. The time required to extr

4、act isoflav ono ids at comparable levels of producti on by ultrasou nddisruptionwas 20 times shorter than by conventional reflux extraction. Moreover, testresults for drying the extracted substanee from Pueraria show that the microwave-vacuum method is 10 times faster than the conventional two-step

5、vacuum approach. Finally, differentinstrumentalanalyses of isoflavonoidsobtained from Pueraria using theproposed new method show that extraction by ultrasounddisruptionand microwave-vacuumdrying affects n either the structure nor the compositi on of the extracted substa nee.Keywords： Extraction; Iso

6、flavonoids; Microwave drying; Ultrasound1. In troduct ionUltrasoundhas been used to extract compounds from the various parts of plants for morethan three decades 1 . Due to the disruption of cell walls and enhanced mass transferof cell contents, ultrasound is able to accelerate the extraction of org

7、anic compoundsfrom the bodies of pla nts 2 and 3 . Compared with conven ti onalsolve nt extracti on,the use of ultrasoundmakes the extractionof valuable compounds more efficientby meansof shorter time frames and lower extracti on temperatures. Ultrasou nd is curre ntlyemployed to extract such pharma

8、cologically activecompounds as polysaccharides,cellulose, flav ono ids, saturated hydrocarb ons, fatty acid esters, and steroids from plant materials 4.Microwave drying is often used to evaporate water in wood, foodstuffs, drugs, and ores, among other commodities 5 and 6 ; it can also be used for di

9、stillation7 andextraction8 , 9 and 10 . Combining the application of microwaves with vacuumtech ni ques for drying offers two major adva ntages, n amely, rapid drying due to theability of microwaves to heat solventsinstantaneouslyand homogeneously, and enhancedrate and extent of mass transfer at sub

10、-atmospheric pressure and low temperatures, which is esse ntial for thermo-labile products. Such dryi ngtech no logiesare thereforeimportant for industries such as pharmaceuticals11.The presentstudy of the extractionof isoflavonoidsfrom Pueraria by ultrasounddisruption,together with their drying by

11、means of combined microwave and vacuumtech no logies, offers an alter native to conven ti onal tech no logies for extract ing and drying isoflav ono ids from Pueraria.2. Experime ntal2.1. Pla nt materials and chemicalsPueraria collected in Chi na's Henan prov ince was washed, dried, and cut into

12、 5mmsegments. The standard sample of Puerarin was supplied by China's National Institute for the Con trol of Pharmaceutical and Biological Products. An alytical reage nt-grade ethanol and ethyl acetate were used in all experiments.2.2. Extracti onFig. 1 shows the combined microwave-ultrasound ex

13、perimental setup used for extraction. The apparatus consists mainly of an extraction component (a), a microwave drying comp onent (b) and a solve nt recycli ng comp onent (c). Ultrasou nd extracti on was carried out using a CF-1520 50 kHz 1200 W ultrasounddisintegrator.A GALANZ WP700L17microwave ove

14、 n with a Teflon? evaporat ing dish was used for drying.Fig. 1. Schematic drawing of the apparatus used for extraction (a: extracting part; b: microwave drying part; c: solvent recycling part; 1: bracket; 2: ultrasound transducer; 3: beaker; 4: sealing gasket; 5: teflon evaporating dish; 6: turning

15、plate; 7: microwave oven; 8: teflon tube; 9: latex tube; 10: inlet; 11: outlet; 12: vacuum sucker).Twenty grams of Pueraria were put into a 1000 ml beaker for ultrasoundextraction.After400 ml of 70% etha nol added, the beaker was put on the ultrasou nd dis in tegrator,followed by extraction under th

16、e ultrasound horn for 30 min at room temperature. Samples taken periodically were analyzed using an UV-2102PC Ultraviolet- Visible (UV - VIS)spectrophotometer.A reflux extraction experiment was also performed for purposes of comparison. After 20 g of Pueraria were tra nsferred into a 1000ml three-n

17、eck flask, 400ml of 70% etha nolwas added. The mixture was stirred at 600rpm and refluxed for 120min, duri ng whichsamples were take n periodically for an alysis by UV spectrophotometer.2.3. Drying of extracted productA GALANZ WP700L17 microwave ove n with a Teflon ? evaporat ing dish was used for d

18、ryingthe extracted product. For recycli ng the solve nt and en suri ng safety, the microwave ove n was made airproof by drillinga hole through its roof and passing a Teflon ? tube throughthe hole. The tube was connected to a vacuum system to withdraw steam and volatile chemicals, allowing water and

19、organic solvents to be evaporated by the microwave-vacuum apparatus.To initiatedrying, 200 ml of ultrasound-extracted resultant was put in an evaporatingdish and placed in the microwave ove n. The extracted resulta nt became solid in about10 min. Ano ther 200 ml of extracted resulta nt was dried usi

20、ng a rotati ng evaporatorun til no distillate came out, followed by drying it into powders for about 80min inan ove n.2.4. An alytical methodsThe extracted Pueraria isoflavonoids were analyzed by UV at 250nm wavelength 12.The sta ndard curve of absorba nee versus Puerari n concen trati on was obta i

21、ned in thefollowing manner: ten milligrams standard sample of Puerarin was first put into a 50 ml volumetric flask;then 95%ethanol was added to the scale, followed by shaking the mixtureun til it was homoge neous. Aliquots of soluti ons (0.2, 0.4, 0.6, 0.8, and 1.0ml) weretake n from the flask and p

22、laced into 10ml volumetric flasks. 1.0ml of etha nol wasthen added to each flask and dei oni zed water was added to the flask scales to complete the preparati on of sta ndard soluti ons. Bla nk samples were prepared using the same procedures previously mentioned but without the addition of extracted

23、 isoflavonoids.The UV absorbanee of blank and each standard sample was measured at a 250nm wavelength.The extractresultantwas analyzed as follows; one milliliter of extract was transferredinto a 50 ml volumetric flask followed by the additionof 95% ethanol to the scale andmixing. The solutionwas lef

24、tovernight.One milliliter of supernatantwas thentra nsferred into a 25ml volumetric flask; dei oni zed water was added to the scale,followed by measuring the absorbanee at 250 nm. The concentrationof isoflavonoidsinthe solution was obtained from the standard curve. The level of isoflavonoids in thes

25、ample is calculated as(1)where ci is the concentrationof isoflavonoids in the extractresultant, V is the volumeof extract resultant, and購。品 is the weight of the Pueraria.Twenty milligrams of extracted product (powder) and 30ml of 95% etha nol were mixedin a 50 ml volumetric flask to in itiate an aly

26、sis of the extract. The mixture was the nheated in a hot water bath to dissolve the product. After cooling, the flask was filled with 95% etha nol to the scale. After about 12h, 1.0 ml super nata nt was placed in a25 ml volumetric flask and diluted with dei oni zed water to the scale. The absorba ne

27、eof the soluti on was the n measured at 250nm. The yield of powder, content of totalisoflavonoids in powder, and total yield of isoflavonoids are defined as follows:where mow and mug are the weights of the powder and Pueraria, respectively,c2 isconcen trati on of the sample soluti on, andV2 is the d

28、iluti on volume.An S-250 scanning electron microscope was used to prove the cavitations and hittingeffect of ultrasound disruption on the cells of Pueraria. To examine the effect of ultrasound disruptionand microwave drying on the extractedisoflavonoidsfrom Pueraria,the extracted resultant wasanalyz

29、ed with a VECIDR22 Infrared (IR) Spectrometer and byLC5500 High Performa nee Liquid Chromatography (HPLC). The HPLC was operated with a UV detector (wavelength set at 250nm) and a Cchromatography column. Methanol water(3:7, v/v) was used as the mobile phase at the flow rate of 1ml min-1 and a 0.02 m

30、lsample was injected.3. Results and discussi on3.1. Choice of extract ing con diti ons of ultraso undDiffere nt solve nts, in cludi ng water, metha nol, etha nol, and ethyl acetate, were compared for their extracti on abilities. The same solve nt volumes and extracti on times were usedin the experim

31、entsfor comparative extraction tests. The test results are shown in Fig.2. As shown, the extraction efficiencyfor isoflavonoids from Pueraria was significantlyaffected by the type of solve nt used. Although water can be used to achieve the highest extractionefficiency,too much starch was dissolved i

32、n water, which made the extractedresultant turbid,ropy, and difficult in filtration.Since methanol is toxic and ethylacetate is costly, a mixture of etha nol and water was chose n as an extracti on solve nt.Among the solve nts tested, an etha nol water mixture at a 7:3 volume ratio proved tohave the

33、 highest extraction efficiency, as shown inFig. 2 .Fig. 2. The effectof differentsolvents on ultrasoundbased extractingefficiencyofisoflavonoids(solventvolume: 400ml; liquid/solidratio:20ml/1 g;extracting time:20 min).The effect of the ratio the volume of solve nt (i.e. a mixture of etha nol and wat

34、er at a 7:3 volume ratio) to the weight of Pueraria (liquid/solid ratio) on the extracti on efficiency of isoflavonoids using ultrasound was studied, with the results listed inTable 1 . As show n, the in creas ing liquid/solid ratio led to a con siderable in crease in extraction efficiency until the

35、 ratio reached 20:1, which is considered optimal for the most efficie nt use of solve nt and en ergy.Table 1. Effect of the ratio of solve nt volume to weight of Pueraria (liquid/solid ratio) on the ultrasoundbased extractingefficiency forisoflav ono idsLiquid/solid ratio10 15 20253040Co ntent of to

36、tal isoflavo noids (%)11.7 15.5 17.2 17.5 17.9 18.13.2. Comparis on betwee n ultraso un d-disrupti ng extracti on and reflux ing extracti onThe effect of time on the efficiencyof isoflavonoidextractionfrom Pueraria using bothultrasou nd disrupti on and reflux extracti on was studied. The relati on s

37、hip betwee n the levels of total isoflavonoids and extraction time is displayedin Fig. 3. As shown, thelevels of isoflavonoidsextracted from Pueraria by ultrasound disruption reached about19%over 20 min, whereas the levels of isoflavonoids extracted through reflux were only 14.3% and 16.5%, after 1

38、and 2 h, respectively. By contrast,ultrasounddisruption tookonly 5 min to reach a comparable 16.0% level of extraction efficiency (Fig. 3 ), abouton e-twe ntieththe time required by refluxextracti on,clearlydem on strati ngthatultrasound disruption is superior to reflux extraction.30 josn 1®FxT

39、rjclioti riinc irmiHiitilf/plollrnmifi- PKQ*=Ewn-7Fig. 3.Comparison between ultrasound-disrupting and refluxing extraction for isoflavonoids(weight of Pueraria: 20g; extraction solvent: 400ml of 70% ethanol; liquid/solid ratio:20 ml/1 g).It is well known that most biologically active compounds of pl

40、ants exist in their cell walls. To extract these compounds, the cell walls can be disrupted by ultrasoundextraction.Ultrasoundcauses intense shaking, high acceleration, intense cavitations, and stirring, all of which can accelerate the dissolution of pharmacological agents.Furthermore,enhancement of

41、 theextractionrate shortens extraction time,therebycon serv ing solve nt and mitigati ng the effects of high temperatures on the effective ness of pharmacological agents. In order to confirm the cavitations and hitting effect of ultrasound disruption on cells of Pueraria, scanning electronmicroscopy

42、 (SEM) was usedto characterize Pueraria cells before and after both ultrasound disruption and refluxextraction.Fig. 4 shows the profiles of cells, arranged in order, with solidsubstancesapparent in dry Pueraria cells (Fig. 4 a). Some small crannies were observed in a fewcells and no solid substances

43、 were shown in Pueraria cells after 2 h of reflux extraction (Fig. 4 b). This can be expla ined by the fact that the solve nt used duri ng extracti on en tered the cells through their gaps and cra nnies and con tacted the solid substa nces,con seque ntlymovi ng their pharmacological compou nds into

44、the solve nt. By con trast, after20 min of ultrasound, the cells could not be distinguished and their walls were almost cracked and disrupted, as seen in Fig. 4c, which resulted from the hittingand cavitatingof ultrasou nd's intenseshak ing.The huge in sta ntan eousen ergy gen eratedby theultras

45、ound system can lead to the disruptionof Pueraria cells and the quick dissolutionof the isoflav ono ids in Pueraria cells in solve nt without a permeati on process.Fig. 4.Scanning electron microscopy (SEM) photos of Pueraria cells (a: dry Pueraria cells; b:Puerariacells obtained through refluxing fo

46、r 2 h;c: Pueraria cells obtained through the actionof ultrasound for 20min; amplification magnitude:x 200,000; electron accelerating voltage:19 kV).Isoflavonoidsin Puerariaare composed primarilyof puerarin,daidz in,and daidze in 13The structures of the three comp onents are show n inFig. 5.To exam i

47、ne the effects ofultrasou nddisrupti on onthe extracted isoflav ono ids fromPueraria, theextractedresultant was analyzed by HPLC, UV Spec, and IR Spec, with the results presented in Fig. 6. Fig. 6a and b are, respectively, the IR spectra of the powder extracted by ultrasound disrupti on for 20min an

48、d by reflux ing for 2h with 70% etha nol as solve nt.Fig. 6shows that the locati ons of characteristic fun cti onal groups of the two samples areiden tical in their IR spectra, which dem on stratesthat extracti on by ultrasou nddisrupti on gen erates no n egative effects on the structure and comp on

49、ents of extracted resulta nts compared to the conven ti onal reflux extracti on method. The UV spectra for the standard Puerarin solutionsand extractedresultantsfrom ultrasounddisruptionandrefluxing are shown inFig. 7 . No significant differences were shown among the threecurves, except for the heig

50、hts of absorpti on peaks at 250nm. The rete nti on times inHPLC chromatograms for the resulta nts from sta ndard Puerari n samples extracted byultrasou nd disrupti on and reflux ing were 11.38, 11.29, and 11.30min, respectively.The contents of extracted resultants from ultrasound disruptionand reflu

51、xing under thegive n con diti ons were, corresp ondin gly, 3.6% and 3.7%, which in dicates that Puerar inwas not destroyed by ultrasou nd.Fig. 5. The molecular structures of isoflavonoids in Pueraria. Fig. 6. IR spectra of resultant extracted by ultrasound (a) and by refluxing (b) (refluxingextracti

52、on time: 2 h; ultrasound disruption time: 20 min; wavenumber scan range:4000- 500 cm： weight of Pueraria: 20 g; extraction solvent: 400 ml of 70% ethanol; liquid/solid ratio: 20ml/1 g; drying method: vacuum).Fig. 7. UV spectra of standard sample (a), resultant extracted by ultrasound (b) and resulta

53、ntextracted by refluxing (c) (wavelength scan range: 200- 600 nm; ultrasound disruption time:20 min; refluxing extraction time: 2 h; weight of Pueraria: 20 g; extraction solvent: 400 ml of 70% ethanol; liquid/solid ratio: 20ml/1 g).3.3. Comparis on betwee n microwave and conven ti onal drying method

54、sThe results of microwave and conven ti onal drying of 200ml of ultrasou nd-extractedresultants are listed in Table 2. It can be seen that the rate of microwave-vacuum drying is 11 times faster than that of conventional vacuum drying, even though the yields of total isoflavonoids were similar.Table

55、2. Comparis on betwee n microwave and conven ti onal drying methodsTime ofYield ofDrying methoddryi ngthe powder(mi n)(%)Conven ti onal14033.3vacuum dryingMicrowave1330.8vacuum dryingContent of total Yield of total isoflav ono ids in theisoflav ono ids (%) powder (%)48.216.050.115.4Extractio nmethod

56、:ultrasou ndextracti on;extract ingtemperature:roomtemperature; extracti on solve nt: 400ml of 70% etha nol; extract ing time:20 min.The products obtained from 5.1% Puerarin with ultrasound-disrupting extraction andmicrowave-vacuumdryi ng,and those from 4.8% Puerari n obta ined withultrasou nd-disru

57、pti ng extracti on and conven ti onal vacuum dryingwere characterized byHPLC. The similarity of retentiontimes (11.33 and 11.20 min, respectively)dem on strates that Puerari n isoflav ono idswere not destroyed by microwave-vacuum drying,which can be further prove n by the similarity of their IR spectra as show n inFig. 8Fig. 8. IR spectra of products obtained by ultrasound-disrupting extraction and microwave vacuum(a) and ultrasound-disrupting extraction as well as conventional vacuum drying (b) (drying ti