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Available online at www.sciencedirect.com

Chemical Engineering and Processing 47 (2008) 2256–2261

Extraction of isoflavonoids from Pueraria by combiningultrasound with microwave vacuum

Yang Hu a, Tao Wang a, MingxiaoWang b, Sufang Han a, Pingyu Wan a,∗, Maohong Fan c,∗∗a Beijing University of Chemical Technology, Beijing 100029, China

b General Hospital of China National Coal Group Corp., Beijing 110013, Chinac School of Materials Science and Engineering, Georgia Institute of Technology,

Atlanta, GA 30332-0245, USA

Received 24 December 2006; received in revised form 30 September 2007; accepted 12 December 2007Available online 11 January 2008

bstract

This study proposes a new method to quickly extract and dry isoflavonoids from Pueraria Lobata Ohwi (Pueraria) by combining ultrasound andicrowave technologies. The time required to extract isoflavonoids at comparable levels of production by ultrasound disruption was 20 times shorter

han by conventional reflux extraction. Moreover, test results for drying the extracted substance from Pueraria show that the microwave-vacuum

ethod is 10 times faster than the conventional two-step vacuum approach. Finally, different instrumental analyses of isoflavonoids obtained fromueraria using the proposed new method show that extraction by ultrasound disruption and microwave-vacuum drying affects neither the structureor the composition of the extracted substance.

2008 Published by Elsevier B.V.

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eywords: Extraction; Isoflavonoids; Microwave drying; Ultrasound

. Introduction

Ultrasound has been used to extract compounds from the var-ous parts of plants for more than three decades [1]. Due to theisruption of cell walls and enhanced mass transfer of cell con-ents, ultrasound is able to accelerate the extraction of organicompounds from the bodies of plants [2,3]. Compared with con-entional solvent extraction, the use of ultrasound makes thextraction of valuable compounds more efficient by means ofhorter time frames and lower extraction temperatures. Ultra-ound is currently employed to extract such pharmacologicallyctive compounds as polysaccharides, cellulose, flavonoids, sat-rated hydrocarbons, fatty acid esters, and steroids from plant

aterials [4].Microwave drying is often used to evaporate water in wood,

oodstuffs, drugs, and ores, among other commodities [5,6]; it

∗ Corresponding author. Tel.: +86 10 6444 5917; fax: +86 10 6444 5917.∗∗ Corresponding author. Tel.: +1 404 385 6725; fax: +1 404 894 9140.

E-mail addresses: pywan@mail.buct.edu.cn (P. Wan),fan3@mail.gateche.edu (M. Fan).

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255-2701/$ – see front matter © 2008 Published by Elsevier B.V.oi:10.1016/j.cep.2007.12.013

an also be used for distillation [7] and extraction [8–10]. Com-ining the application of microwaves with vacuum techniquesor drying offers two major advantages, namely, rapid dryingue to the ability of microwaves to heat solvents instantaneouslynd homogeneously, and enhanced rate and extent of mass trans-er at sub-atmospheric pressure and low temperatures, which isssential for thermo-labile products. Such drying technologiesre therefore important for industries such as pharmaceuticals11].

The present study of the extraction of isoflavonoids fromueraria by ultrasound disruption, together with their dryingy means of combined microwave and vacuum technologies,ffers an alternative to conventional technologies for extractingnd drying isoflavonoids from Pueraria.

. Experimental

.1. Plant materials and chemicals

Pueraria collected in China’s Henan province was washed,ried, and cut into 5 mm segments. The standard sample of Puer-

Y. Hu et al. / Chemical Engineering and Processing 47 (2008) 2256–2261 2257

Fig. 1. Schematic drawing of the apparatus used for extraction (a: extracting part; b: microwave drying part; c: solvent recycling part; 1: bracket; 2: ultrasoundt ate; 7:s

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ransducer; 3: beaker; 4: sealing gasket; 5: teflon evaporating dish; 6: turning plucker).

rin was supplied by China’s National Institute for the Controlf Pharmaceutical and Biological Products. Analytical reagent-rade ethanol and ethyl acetate were used in all experiments.

.2. Extraction

Fig. 1 shows the combined microwave-ultrasound experi-ental setup used for extraction. The apparatus consists mainly

f an extraction component (a), a microwave drying componentb) and a solvent recycling component (c). Ultrasound extrac-ion was carried out using a CF-1520 50 kHz 1200 W ultrasoundisintegrator. A GALANZ WP700L17 microwave oven with aeflon® evaporating dish was used for drying.

Twenty grams of Pueraria were put into a 1000 ml beakeror ultrasound extraction. After 400 ml of 70% ethanol added,he beaker was put on the ultrasound disintegrator, followedy extraction under the ultrasound horn for 30 min at roomemperature. Samples taken periodically were analyzed usingn UV-2102PC Ultraviolet–Visible (UV–VIS) spectrophoto-eter.A reflux extraction experiment was also performed for pur-

oses of comparison. After 20 g of Pueraria were transferred into1000 ml three-neck flask, 400 ml of 70% ethanol was added.he mixture was stirred at 600 rpm and refluxed for 120 min,uring which samples were taken periodically for analysis byV spectrophotometer.

.3. Drying of extracted product

A GALANZ WP700L17 microwave oven with a Teflon®

vaporating dish was used for drying the extracted product. Forecycling the solvent and ensuring safety, the microwave ovenas made airproof by drilling a hole through its roof and pass-

ng a Teflon® tube through the hole. The tube was connectedo a vacuum system to withdraw steam and volatile chemicals,llowing water and organic solvents to be evaporated by theicrowave-vacuum apparatus.To initiate drying, 200 ml of ultrasound-extracted resultant

as put in an evaporating dish and placed in the microwave oven.

he extracted resultant became solid in about 10 min. Another00 ml of extracted resultant was dried using a rotating evap-rator until no distillate came out, followed by drying it intoowders for about 80 min in an oven.

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microwave oven; 8: teflon tube; 9: latex tube; 10: inlet; 11: outlet; 12: vacuum

.4. Analytical methods

The extracted Pueraria isoflavonoids were analyzed by UVt 250 nm wavelength [12]. The standard curve of absorbanceersus Puerarin concentration was obtained in the followinganner: ten milligrams standard sample of Puerarin was first

ut into a 50 ml volumetric flask; then 95% ethanol was added tohe scale, followed by shaking the mixture until it was homoge-eous. Aliquots of solutions (0.2, 0.4, 0.6, 0.8, and 1.0 ml) wereaken from the flask and placed into 10 ml volumetric flasks..0 ml of ethanol was then added to each flask and deionizedater was added to the flask scales to complete the preparationf standard solutions. Blank samples were prepared using theame procedures previously mentioned but without the addi-ion of extracted isoflavonoids. The UV absorbance of blanknd each standard sample was measured at a 250 nm wave-ength.

The extract resultant was analyzed as follows; one milliliterf extract was transferred into a 50 ml volumetric flask followedy the addition of 95% ethanol to the scale and mixing. Theolution was left overnight. One milliliter of supernatant washen transferred into a 25 ml volumetric flask; deionized wateras added to the scale, followed by measuring the absorbance at50 nm. The concentration of isoflavonoids in the solution wasbtained from the standard curve. The level of isoflavonoids inhe sample is calculated as

(c1 × V1)

mpueraria

]× 100% (1)

here c1 is the concentration of isoflavonoids in the extract resul-ant, V1 is the volume of extract resultant, and mpueraria is theeight of the Pueraria.Twenty milligrams of extracted product (powder) and 30 ml

f 95% ethanol were mixed in a 50 ml volumetric flask to initiatenalysis of the extract. The mixture was then heated in a hotater bath to dissolve the product. After cooling, the flask waslled with 95% ethanol to the scale. After about 12 h, 1.0 ml

upernatant was placed in a 25 ml volumetric flask and dilutedith deionized water to the scale. The absorbance of the solutionas then measured at 250 nm. The yield of powder, content of

otal isoflavonoids in powder, and total yield of isoflavonoids

2258 Y. Hu et al. / Chemical Engineering and Processing 47 (2008) 2256–2261

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Table 1Effect of the ratio of solvent volume to weight of Pueraria (liquid/solid ratio) onthe ultrasound based extracting efficiency for isoflavonoids

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It is well known that most biologically active compounds ofplants exist in their cell walls. To extract these compounds, thecell walls can be disrupted by ultrasound extraction. Ultrasound

ig. 2. The effect of different solvents on ultrasound based extracting efficiencyf isoflavonoids (solvent volume: 400 ml; liquid/solid ratio: 20 ml/1 g; extractingime: 20 min).

re defined as follows:

ield of powder =(

mpowder

mpueraria

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Content of total isoflavonoids in powder

=[

(c2 × V2)

mpowder

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otal yield of isoflavonoids =[

(c2 × V2)

mpueraria

]× 100%, (4)

here mpowder and mpueraria are the weights of the powder andueraria, respectively, c2 is concentration of the sample solution,nd V2 is the dilution volume.

An S-250 scanning electron microscope was used to provehe cavitations and hitting effect of ultrasound disruption on theells of Pueraria. To examine the effect of ultrasound disrup-ion and microwave drying on the extracted isoflavonoids fromueraria, the extracted resultant was analyzed with a VECIDR22nfrared (IR) Spectrometer and by LC5500 High Performanceiquid Chromatography (HPLC). The HPLC was operated withUV detector (wavelength set at 250 nm) and a C18 chromatog-

aphy column. Methanol–water (3:7, v/v) was used as the mobilehase at the flow rate of 1 ml min−1 and a 0.02 ml sample wasnjected.

. Results and discussion

.1. Choice of extracting conditions of ultrasound

Different solvents, including water, methanol, ethanol, andthyl acetate, were compared for their extraction abilities. Theame solvent volumes and extraction times were used in thexperiments for comparative extraction tests. The test resultsre shown in Fig. 2. As shown, the extraction efficiency forsoflavonoids from Pueraria was significantly affected by the

ype of solvent used. Although water can be used to achieve theighest extraction efficiency, too much starch was dissolved inater, which made the extracted resultant turbid, ropy, and dif-cult in filtration. Since methanol is toxic and ethyl acetate is

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iquid/solid ratio 10 15 20 25 30 40ontent of total isoflavonoids (%) 11.7 15.5 17.2 17.5 17.9 18.1

ostly, a mixture of ethanol and water was chosen as an extractionolvent. Among the solvents tested, an ethanol–water mixturet a 7:3 volume ratio proved to have the highest extraction effi-iency, as shown in Fig. 2.

The effect of the ratio the volume of solvent (i.e. a mixture ofthanol and water at a 7:3 volume ratio) to the weight of Puerarialiquid/solid ratio) on the extraction efficiency of isoflavonoidssing ultrasound was studied, with the results listed in Table 1.s shown, the increasing liquid/solid ratio led to a considerable

ncrease in extraction efficiency until the ratio reached 20:1,hich is considered optimal for the most efficient use of solvent

nd energy.

.2. Comparison between ultrasound-disrupting extractionnd refluxing extraction

The effect of time on the efficiency of isoflavonoid extrac-ion from Pueraria using both ultrasound disruption and refluxxtraction was studied. The relationship between the levels ofotal isoflavonoids and extraction time is displayed in Fig. 3.s shown, the levels of isoflavonoids extracted from Puer-

ria by ultrasound disruption reached about 19% over 20 min,hereas the levels of isoflavonoids extracted through refluxere only 14.3% and 16.5%, after 1 and 2 h, respectively. By

ontrast, ultrasound disruption took only 5 min to reach a com-arable 16.0% level of extraction efficiency (Fig. 3), aboutne-twentieth the time required by reflux extraction, clearlyemonstrating that ultrasound disruption is superior to refluxxtraction.

ig. 3. Comparison between ultrasound-disrupting and refluxing extraction forsoflavonoids (weight of Pueraria: 20 g; extraction solvent: 400 ml of 70%thanol; liquid/solid ratio: 20 ml/1 g).

Y. Hu et al. / Chemical Engineering and Processing 47 (2008) 2256–2261 2259

F ueraria cells; b: Pueraria cells obtained through refluxing for 2 h; c: Pueraria cellso ×200,000; electron accelerating voltage: 19 kV).

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Fig. 6. IR spectra of resultant extracted by ultrasound (a) and by refluxing (b)(so

cultrasound disruption on the extracted isoflavonoids from Puer-aria, the extracted resultant was analyzed by HPLC, UV Spec,and IR Spec, with the results presented in Fig. 6. Fig. 6a and

ig. 4. Scanning electron microscopy (SEM) photos of Pueraria cells (a: dry Pbtained through the action of ultrasound for 20 min; amplification magnitude:

auses intense shaking, high acceleration, intense cavitations,nd stirring, all of which can accelerate the dissolution of phar-acological agents. Furthermore, enhancement of the extraction

ate shortens extraction time, thereby conserving solvent anditigating the effects of high temperatures on the effectiveness

f pharmacological agents. In order to confirm the cavitationsnd hitting effect of ultrasound disruption on cells of Pueraria,canning electron microscopy (SEM) was used to characterizeueraria cells before and after both ultrasound disruption andeflux extraction. Fig. 4 shows the profiles of cells, arrangedn order, with solid substances apparent in dry Pueraria cellsFig. 4a). Some small crannies were observed in a few cells ando solid substances were shown in Pueraria cells after 2 h ofeflux extraction (Fig. 4b). This can be explained by the facthat the solvent used during extraction entered the cells throughheir gaps and crannies and contacted the solid substances, con-equently moving their pharmacological compounds into theolvent. By contrast, after 20 min of ultrasound, the cells couldot be distinguished and their walls were almost cracked andisrupted, as seen in Fig. 4c, which resulted from the hittingnd cavitating of ultrasound’s intense shaking. The huge instan-aneous energy generated by the ultrasound system can lead tohe disruption of Pueraria cells and the quick dissolution of thesoflavonoids in Pueraria cells in solvent without a permeation

rocess.

Isoflavonoids in Pueraria are composed primarily of puer-rin, daidzin, and daidzein [13]. The structures of the three

Fig. 5. The molecular structures of isoflavonoids in Pueraria.

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refluxing extraction time: 2 h; ultrasound disruption time: 20 min; wavenumbercan range: 4000–500 cm−1; weight of Pueraria: 20 g; extraction solvent: 400 mlf 70% ethanol; liquid/solid ratio: 20 ml/1 g; drying method: vacuum).

omponents are shown in Fig. 5. To examine the effects of

ig. 7. UV spectra of standard sample (a), resultant extracted by ultrasound (b)nd resultant extracted by refluxing (c) (wavelength scan range: 200–600 nm;ltrasound disruption time: 20 min; refluxing extraction time: 2 h; weight ofueraria: 20 g; extraction solvent: 400 ml of 70% ethanol; liquid/solid ratio:0 ml/1 g).

2260 Y. Hu et al. / Chemical Engineering and Processing 47 (2008) 2256–2261

Table 2Comparison between microwave and conventional drying methods

Drying method Time of drying (min) Yield of the powder (%) Content of total isoflavonoidsin the powder (%)

Yield of total isoflavonoids (%)

Conventional vacuum drying 140 33.3 48.2 16.0Microwave vacuum drying 13 30.8 50.1 15.4

E ature; extraction solvent: 400 ml of 70% ethanol; extracting time: 20 min.

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Fig. 8. IR spectra of products obtained by ultrasound-disrupting extrac-tion and microwave vacuum (a) and ultrasound-disrupting extraction as wella14

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xtraction method: ultrasound extraction; extracting temperature: room temper

are, respectively, the IR spectra of the powder extracted byltrasound disruption for 20 min and by refluxing for 2 h with0% ethanol as solvent. Fig. 6 shows that the locations ofharacteristic functional groups of the two samples are iden-ical in their IR spectra, which demonstrates that extractiony ultrasound disruption generates no negative effects on thetructure and components of extracted resultants compared tohe conventional reflux extraction method. The UV spectra forhe standard Puerarin solutions and extracted resultants fromltrasound disruption and refluxing are shown in Fig. 7. No sig-ificant differences were shown among the three curves, exceptor the heights of absorption peaks at 250 nm. The retentionimes in HPLC chromatograms for the resultants from standarduerarin samples extracted by ultrasound disruption and reflux-

ng were 11.38, 11.29, and 11.30 min, respectively. The contentsf extracted resultants from ultrasound disruption and reflux-ng under the given conditions were, correspondingly, 3.6%nd 3.7%, which indicates that Puerarin was not destroyed byltrasound.

.3. Comparison between microwave and conventionalrying methods

The results of microwave and conventional drying of 200 mlf ultrasound-extracted resultants are listed in Table 2. It can beeen that the rate of microwave-vacuum drying is 11 times fasterhan that of conventional vacuum drying, even though the yieldsf total isoflavonoids were similar.

The products obtained from 5.1% Puerarin with ultrasound-isrupting extraction and microwave-vacuum drying, and thoserom 4.8% Puerarin obtained with ultrasound-disrupting extrac-ion and conventional vacuum drying were characterized byPLC. The similarity of retention times (11.33 and 11.20 min,

espectively) demonstrates that Puerarin isoflavonoids were notestroyed by microwave-vacuum drying, which can be fur-her proven by the similarity of their IR spectra as shownn Fig. 8.

. Conclusions

The cell walls of Pueraria can be disrupted by ultra-ound. The extraction time of isoflavonoids from Pueraria ishorter than that using conventional reflux extraction. The

ltrasound-based method increases extraction efficiency with-ut destroying the pharmacological agents of Pueraria. Also,icrowave-vacuum technology is superior to conventional vac-

um technology for drying the product extracted from Pueraria

s conventional vacuum drying (b) (drying time of microwave vacuum:0 min; drying time of conventional vacuum: 80 min; wavenumber scan range:000–500 cm−1).

y ultrasound. Finally, the structure and components of Puerariasoflavonoids obtained using ultrasound and microwave-vacuumechnologies are similar to those produced using conventional

ethods.

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