Separation and Purication Technology 74 (2010) 261270

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Separation and Purication Technology

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Negative pressure cavitation extraction and antiogenisti ca

Dong-Ya JieYuan Gaoa Key Laborator 15004b Engineering R iversitc Department o , 5509

a r t i c l

Article history:Received 3 MaReceived in reAccepted 20 Ju

Keywords:Negative pressPigeon pea rooIsoavonoidsAntioxidant acResponse surface methodology

vitatiamee ext

onsetion aistinin, patio 4lhydr

notable concentration-dependent antioxidant activity with IC50 value of 0.062mg/ml. 2010 Elsevier B.V. All rights reserved.

1. Introdu

Pigeon pno-eye peausage graintropical devas an annuainal usagesof the literastrated notproperties [lent traditioto the mark[3]. Compartochemicalsfact, pigeonas an alexerary as a folmost of the

CorresponEducation, NoChina. Tel.: +8

E-mail add1 Both autho

1383-5866/$ doi:10.1016/j.ction

ea [Cajanus cajan (L.) Millsp.], also known as red gram,, Congo pea, Gungo pea, etc., is a famous and multi-legume crop widely distributed in semi-tropical andeloping countries. Nowadays, pigeon pea is cultivatedl for both forage and its edible beans [1]. Some medic-of this plant have been recorded in worldwide. Mosttures are focused on pigeon pea leaves, which demon-able anti-inammatory, anti-bacterial and abirritative2]. Especially, pigeon pea leaves are used as an excel-nal Chinese medicine (TCM), which has been broughtet for the therapy of ischemic necrosis of femoral headatively, research on pigeon pea roots is scanty, the phy-and possible medicinal uses are not fully explored. Inpea roots have many medicinal uses. They were useditic, anthelminthic, expectorant, sedative, and vulner-k medicine in some local producing areas. However,m are usually discarded as agricultural wastes or used

ding author at: Key Laboratory of Forest Plant Ecology, Ministry ofrtheast Forestry University, Hexing Road, No. 26, Harbin 150040, PR6 451 82190535; fax: +86 451 82190535.ress: yujie fu2002@yahoo.com (Y.-J. Fu).rs contributed equally to this work.

as rewood by farmers, the total amount is huge. Hence, innova-tive technology concepts for the utilization of pigeon pea roots toprovide the raw material for the manufacture of future-orientedproducts are desirable.

Preliminary chemical investigations and pharmacological stud-ies on pigeon pea roots in our group have demonstrated thepresence of polyphenols, especially isoavonoids, and they werethe major benecial compounds responsible for their bioactivities.Genistein and genistin are a pair of isoavonoid compounds foundas the main constituents present in pigeon pea roots, their struc-tures are shown in Fig. 1. They represent important members ofthe avonoid family and possess a wide spectrum of pharmaco-logical activities [47]. Therefore, genistein and genistin have greatpotentials to be used as clinical therapeutic agents, food additivesor nutraceutical products. The application of low-cost technologyto obtain these two compounds from pigeon pea roots is a rationalstrategy to increase the economic value and expand the utilizationof this plant.

Cavitation is a general uid mechanics phenomenon, which canoccur whenever a liquid used in a machine inducing pressure andvelocity uctuations in the uid (e.g. pumps, turbines, and pro-pellers). Cavitation serves as a means of concentrating the diffuseduid energy locally in very short duration and creating a zone ofintense energy dissipation. Acoustic cavitation and hydrodynamiccavitation are two types sorted by the cause of formation [8]. Thestudies on acoustic cavitation such as ultrasonic cavitation have

see front matter 2010 Elsevier B.V. All rights reserved.seppur.2010.06.015n from the roots of pigeon pea [Cajanus

ng Zhanga,b,1, Su Zhanga,b,1, Yuan-Gang Zua,b, Yu-a,b, Jin-Tong Zhaoa,b, Thomas Efferthc

y of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbinesearch Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry Unf Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz

e i n f o

rch 2010vised form 12 June 2010ne 2010

ure cavitation extractionts

tivity

a b s t r a c t

A new methodnegative pressure caextraction of the main isoavonoids, nextraction time and particle size on thite design (CCD) combined with respnegative pressure, ethanol concentraextraction yields of genistein and genmal conditions: extraction time 45mconcentration 70% and liquid/solid rwas assessed by 2,2-diphenyl-1-picry/ locate /seppur

xidant activity of genistein andjan (L.) Millsp.]

Fua,b,, Yu Konga,b,

0, PR Chinay, Harbin 150040, PR China9 Mainz, Germany

on extraction (NPCE) was proposed and investigated for thely genistein and genistin from pigeon pea roots. The effects ofraction yields were rstly optimized, then a central compos-surface methodology (RSM) was used to study the effects ofnd liquid/solid ratio on the extraction yields. The maximumreached 0.418 and 0.398mg/g, respectively, under the opti-article size 50 mesh, negative pressure 0.05MPa, ethanol4:1. Furthermore, the antioxidant activity of NPCE extract

azyl (DPPH) radical-scavenging assay. NPCE extract possessed

262 D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270

Fig. 1. Structures of genistein (A) and genistin (B).

Fig. 2. Schematic diagram and the area distribution of the NPCE system (A), and the sketch map for the process of the mass transfer among gasliquidsolid (B).

D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270 263

been widespread carried out in the elds of phytochemistry, bio-chemistry, physics and metallurgy [9,10]. As an efcient extractionmethod, ultrasonic extraction has attracted growing interest forthe extraction of various secondary metabolites from plant mate-rials [11,12genistin arehigh tempecavitation smetabolites

Negativeitation is geefcient meintensity isthermore, dintroducedcollision anmatrix. Undwith the expounds outonly the adpreventing

In the prfor the extrThe effectswere inveswith NPCEtional extraroots sampscanning elactivity ofwas determ

2. Materia

2.1. Plant m

PigeonProvince, ChLaboratoryForestry Unin the herbathe shade, pkept away f

2.2. Chemic

Genistei(4,5,7-trihyfrom Flukaobtained fracid (96%) w2,2-Diphensupplied bylytical gradChemical RedbyaMilMA, USA).

2.3. Conven

For macoptimizeda beaker wplaced at rtion solutiowas added

repeated for three times, the extraction solutions were combinedand concentrated to dryness on a rotary evaporator (RE-52AA,Shanghai Huxi Instrument, Shanghai, China) at 50 C. Methanol ofHPLC grade was added to get the samples of appropriate concen-

for Hheatccuraetha

mum00 rpother 3hultr

inaryingl/waor ths waas th

gativ

Devicativeped iin F

xtraced i

essurrfaceion plterremreofbie

latili

Extragramfromwasf o

vice.valvs was wagativextraext

d as f

tion

ass o

Experthecingl comdologthanost, 1yed t]. However, some thermosensitive compounds such aseasily degradable by ultrasonic cavitation due to its

rature and intensity [1315]. Therefore, a new kind ofuitable for the extraction of thermosensitive secondaryis warranted.pressure cavitation is another type in which the cav-

nerated by negative pressure. It is a cheap and energythod. It can keep constant lower temperature and itsnot weaker than that of ultrasonic cavitation. Fur-uring the extraction process, nitrogen is continuouslyinto a liquidsolid system to increase the turbulence,d mass transfer between the extraction solvent ander the integrated action, it is efcient formixing sampletraction solvent as well as migrating the target com-of the sample matrix [16]. Thus, this procedure has notvantages of ultrasonic cavitation but also is good forthe degradation of thermosensitive compounds.esent study, a NPCE method was proposed and appliedaction of genistein and genistin from pigeon pea roots.of main operating parameters on the extraction yieldstigated. The extraction efciency of two isoavonoidswas compared with those obtained by three conven-ction methods. The structural disruption to pigeon peales with different extraction methods was observed byectron microscopy (SEM). Furthermore, the antioxidantextracts, obtained using different extraction methodsined by means of DPPH radical-scavenging assay.

ls and methods

aterial

pea roots were collected in autumn from Hainanina, and identied by Prof. Shao-QuanNie from theKey

of Forest Plant Ecology,Ministry of Education,Northeastiversity, PR China. Voucher specimens were depositedrium of the same laboratory. The samples were dried inowdered and sieved (2080 mesh). At last, they were

romlight inadesiccator at roomtemperatureuntil used.

als and reagents

n (4,5,7-trihydroxyisoavone, 98%) and genistindroxyisoavone-7-glucoside, 98%) were purchased(Buchs, Switzerland). Methanol of HPLC grade was

om J&K Chemical Ltd. (Beijing, China), while formicas from DIMA Technology Inc. (Muskegon, MI, USA).

yl-1-picrylhydrazyl (DPPH) and ascorbic acid (VC) wereSigmaAldrich (Steinheim, Germany). Ethanol of ana-e for extraction was bought from Tianjin Kermeleagent Co. (Tianjin, China). Deionized water was puri-li-Qwater-purication system fromMillipore (Bedford,

tional extraction procedures

eration extraction (ME), according to the preliminaryinvestigation, 10g of pigeon pea roots were put intoith 800ml ethanol/water (70:30, v/v). The beaker wasoom temperature for 12h, then, the ltered extrac-n was collected and another 800ml of 70% ethanolinto the beaker for another 12h. After the process was

trationFor

were a650mlto opti5007and ananothe

Forprelimby addethanosonic fprocescess w

2.4. Ne

2.4.1.Neg

develoshownof an eare addtive pran inteextractvent iscan beperatuwith amThe vo

2.4.2.Ten

devicedevicevalve othe deby theprocesprocessize, neon the

Thedene

Extrac

= M

2.4.3.On

inuencentrametho(X1), eCCD teemploPLC analysis.-reuxing extraction (HRE), 10 g of pigeon pea rootstelyweighed and put into a round-bottomed askwithnol/water (70:30, v/v). The extraction was employedconditions of 80 C for 3h under magnetic stirring at

m. Then, the ltered extraction solution was collectedr 650ml of 70% ethanol was added into the ask for. The subsequent process was the same as ME.asound-assisted extraction (USE), according to the

investigation, target compositions were extracted10g of pigeon pea roots into a ask with 450mlter (70:30, v/v), the samplewas then extracted by ultra-ree times in an ultrasonic bath. The optimum extractions performed at 50 C for 1h. Then, the subsequent pro-e same as ME.

e pressure cavitation extraction

e of negative pressure cavitationpressure cavitation equipment (CN2597047) was

n our laboratory. The schematic diagramof the device isig. 2A. The negative pressure cavitation device consiststionpot (1) and a collectionpot (2).Material and solventnto the extraction pot through the inlet (3). The nega-e of the device is provided by a vacuum pump throughon top of the pot body. Nitrogen is introduced into theot through valve 2. After extraction, the extraction sol-ed through a net (4) into the collection pot. The residueoved from the pot through the discharge lid. The tem-the entire systemcanbekept constant becausenitrogennt temperature is injected into the systemcontinuously.zed solvent is refrigerated by the condenser (6).

ction procedures of pigeon pea roots were introduced into the NPCEthe sample portal. After the solvent was added, the

connected to the vacuum pump. At the same time, thew meter was opened and nitrogen was supplied intoThe negative pressure of the device can be controllede. According to the experimental design, the extractions performed under different conditions. The subsequents the same as ME. The effects of extraction time, particlee pressure, ethanol concentration and liquid/solid ratioction efciency of two isoavonoids were investigated.raction efciency of the compounds in sample wasollows:

yield (mg/g DW)

f the compounds in extraction solutionMass of dried material

imental designbasis of the single factor experimental results, majorfactors were conrmed, and then a 23 factorial portionposite design (CCD) combined with response surfacey (RSM) was used for optimizing negative pressurel concentration (X2) and liquid/solid ratio (X3). In the4 experiments and six replicates at the centre wereo t the full quadratic equation model. The general

264 D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270

Table 1Results of the central composite design (CCD) for the extraction of genistein and genistin.

Runs Factors Extraction yield ofgenistein (mg/g DW)

Extraction yield ofgenistin (mg/g DW)

123456789

1011121314151617181920

a Negative pb Ethanol coc Liquid/sold Experimene Predicted

equation is:

Y = 0 +k

j=

where Y is tsion coefcrespectivelyThe variabl1 and 1 foextractionused accord

X i =Xi X0

Xi

where X iis

real value oindependenof the realindependenin Table 1. Aextraction y

2.4.4. StatisThe data

posite was(Design-ExpAnalysis ofmodelingoof P

D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270 265

taken in triplicate, means and standard deviation values were cal-culated.

2.8. DPPH radical-scavenging assay

The DPPis based onsolution aftdetailed mewasdissolv4mg/ml, thto 1ml of ththe mixturetrophotomeabsorbanceacid (Sigmareference. Tsample was

Inhibition p

where AB aof the testewere averagof three ind

3. Results

3.1. The me

The NPCbubbles forand turbuleuous introdthe action oascend amohighly instathe suspenswith the ouow regionlowing concsheets, so thcan rapidlyliquid dropproduce cavand the solvthis can enrent layer, tinto the solvbetween liqprocess of mmotion effetem createsand interfactransfer encompoundsefciency a

3.2. Optimi

3.2.1. EffectTo increa

sumption, textractionuid/solid rapressure of

ffectsand g

120min, respectively. The results are shown in Fig. 3A. It isnt that the effect of time on the extraction yields of genis-d genistin showed a similar trend. The extraction yields ofavonoids distinctly increasedwith the extension of extrac-e in the rst 45min. After 45min, the extraction time had

pact on the extraction yields, indicating that genistein andn reached their distribution equilibrium at around 45min.ering the increaseof extractionyields and theextractionef-of two isoavonoids, 45min was selected as the appropriateion time and used in the following tests.effect of particle size on the extraction yields was inves-using mean particle sizes of 20, 30, 40, 50, 60, 70 or 80ith a 70% ethanol solution at a liquid/solid ratio of 45:1, ae pressure of 0.05MPa and an extraction time of 45min.

sults are shown in Fig. 3B. It can be seen that the extrac-elds increased when the particle size changed from 20 to 60It iswell known thatmechanical treatment on the rawmate-s a large impact on the extraction yields, since it could breakell walls and augment compound accessibility. Hence, thethe particle size is, the higher the extraction yields can beed. However, it is not recommendable and sometimes it isimpossible to process such ne powders industrially. Con-g thehigher extraction yields and thepracticability, 50meshlected for the following experiments.

Optimization of NPCE operating parametersobjective of the present studywas to optimize the operating

ions to achieve an efcient extraction of two isoavonoidshe roots of pigeon pea. Because various parameters poten-uence the NPCE process, the optimization of the operatingH radical-scavenging activity (anti-free radical activity)the determination by DPPH at a steady state in ethanoler adding the mixture of antioxidants. Following thethod of Liyana-Pathiranan and Shahidi [18], the extracted in10mlof absolute ethanol to give a concentrationofen 2ml of 0.004% DPPH (0.2mM) in ethanol was addede extract solution. After shaking themixture vigorously,was immediately placed in an UNICO UV-2100 spec-ter (UNICO, Shanghai, China) tomonitor thedecrease inat 517nmuntil the reaction reached a plateau. AscorbicAldrich), a stable antioxidant, was used as a synthetiche DPPH radical-scavenging activity in percentage ofcalculated according to the following equation [19]:

ercentage (Ip) = 100(AB AA)ABnd AA were the absorbance values of the blank andd samples, respectively, checked after 70min. All dataes (standard deviations) of triplicate determinationsependent tests.

and discussion

chanism of NPCE

E system can be separated into four areas [20] (Fig. 2A):mation layer, suspension layer, axle air current layer,nt layer. In the bubbles formation layer, the contin-uction of nitrogen into the extraction system, underf negative pressure, small nitrogen bubbles appear andng the liquidsolid phase, results in the formation ofble gasliquidsolid system. When the bubbles enterion layer, the volumes of bubbles change rapidly alongtside pressure. Bubbles grow and collapse at certains with lower pressure. The bubbles grow and the fol-ave jet makes the surrounding liquid drops into liquidat the liquid sheets with different solute concentrationcoalesce. As a result, a mass transfer takes place amongs [21]. On the other hand, the collapse of bubbles canitation so that the surface of solid particles is corrodedent can diffuse into the inside of solid particles (Fig. 2B),hance the intra-particle diffusion. In the axle air cur-he compounds in the material are efciently releasedent due to an intense collision and mass transfer effectuid drops and solid particles. In the turbulent phase, theass transfer is accomplished by an action of turbulent

ct. Thereby, the negative pressure cavitation of this sys-intensive cavitation-collision, turbulence, suspensione effects. These effects combine to formadynamicmasshancing system and favor the rapid transfer of targetfrom the matrix to the solvent, increase the extraction

nd recovery of target compounds.

zation of NPCE procedure

s of extraction time and particle sizese the extraction efciency and decrease the time con-he extraction time was rstly optimized. The effect oftime was studied with 70% ethanol solution at a liq-tio of 45:1, a particle size of 50 mesh and a negative0.05MPa. The extraction time was 10, 20, 30, 45, 60,

Fig. 3. Egenistin

90 andapparetein antwo isotion timless imgenistiConsidciencyextract

Thetigatedmesh wnegativThe retion yimesh.rial haplant clowerobtainalmostsiderinwas se

3.2.2.The

conditfrom ttially inof extraction time (A) and particle size (B) on the extraction yields ofenistein.

266 D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270

Fig. 4. Responvarying extracliquid/solid ra

conditionsmethod. CCparametersliquid/solidAll results odata from trized in Tabregressionoperational

Fig. 4A acentrationrespectivelyfrom 0.03se surfaces for genistein (A)(C) and genistin (D)(F) from pigeon pea roots. (A) and (Dtion pressure and liquid/solid ratio; (C) and (F) varying ethanol concentration and liquid/stio.

plays a critical role in the development of a NPCED combined with RSM was used for optimizing. Threeincludingnegative pressure, ethanol concentration andratio were optimized by a 23 factorial portion CCD.btained from 20 experimental runs and the predictedhe model based on the experimental data are summa-le 1. Three-dimensional proles of multiple non-linearmodels were used to depict the interactive effects ofparameters for two isoavonoids (Fig. 4AF).nd D shows the effects of pressure and ethanol con-on the extraction yields of genistein and genistin,. It was observed that decrease of negative pressureto 0.05MPa with ethanol concentration increasing

from 55 togenistin sima certain vincreased athepressurtion yieldsin negativewhich meawere not emass transrecommendlack of cavitmore difcu) varying extraction pressure and ethanol concentration; (B) and (E)olid ratio. P: extraction pressure, EC: ethanol concentration, and LSR:

75% enhanced the extraction yields of genistein andultaneously. If the given ethanol concentration was

alue, while pressure was falling, the extraction yieldst relative high-pressure levels (above 0.05MPa). Oncee reached low levels (lower than0.05MPa), the extrac-slightly decreased. This can be explained by a decreasepressure resulting from reduction of nitrogen ow rate,ns the decrease in formation of tiny bubbles and therenough nitrogen bubbles to form turbulent motion andfer accordingly. However, high pressure is not alwaysed due to the overfull gas in the liquid resulted in aation effect. The inuence of ethanol concentrationwaslt to predict than that of negative pressure. Firstly, the

D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270 267

formation and burst of tiny bubbles can be affected due to dif-ferent ethanol concentrations resulted in different viscosities ofethanolwater binary system. Secondly, different ethanol concen-trationswere related to compounds of different polarities. In Fig. 4Aand D, an inthe ethanolthe changevent polaritconcentratiin this studmoderate ption limitedSince the sobalance betethanol conduring NPC

Fig. 4B asurface forand liquid/sof ethanol cuid/solid raand ethanoto signican(ml/g). Althincrease inratio up to 5isoavonoid

For fallinincreaseof lyields wereE, it was obincreased wbut slightlyratio impro45:1, ml/g)was observcould be acwas due tobe decreaseMeanwhileples couldliquid/solidin order toincreasing lincrease inyields weretions wererstly increcentrationethanol contwo isoavseen from Fconcentratimaximumof ethanol fgenistein isconcentratirial to solveyields withiisoavonoidwas higherume of solvethanol solvwhen the lithan the thby changing

interactions, all of which are detrimental and advantageous conse-quences, respectively.

As shown in Table 1, the extraction yields of two isoavonoidswere similar to those expected, and the results demonstrated the

cy ofientsnistiand 1oththatgni. (I) aationxtrac

1.75

0.02

104

0.86

0.00

104

Y1 ixtrare (Msolidsolvied. T0.05M1;whl conum.421summPa,

electestigm Eccordtion wespopredgen

5minl conmentions.n weRSDmenthe NPwholordineren1.0nistisenteansial ancrease in extraction yields was observed by increasingconcentration in an early stage of extraction, becauseof solvent viscosity was less effective than that of sol-y. The trend was, however, reversed, when the ethanolon reached a certain value under the ranges of pressurey. This can be explained that genistein and genistin areolar compounds, and much lower ethanol concentra-their solubility, resulting in declined extraction yields.lubility of two isoavonoids depended largely on theween solvent viscosity and polarity of the solution, ancentration in the range from 65 to 75% was practicalE of pigeon pea roots.nd E shows the three-dimensional plot of the responsethe extraction yields as related to extraction pressureolid ratio. Fig. 4C and F describes the interactive effectoncentration and liquid/solid ratio. The inuence of liq-tio was not as signicant as those of extraction pressurel concentration. The extraction yields did not continuetly increase until the liquid/solid ratio was over 45:1ough increasing the solvent volumes led to a slightthe extraction yields, further increasing the liquid/solid0:1 (ml/g) resulted in little change in the yields of twos.g pressure from 0.03 to 0.05MPa together with aniquid/solid ratio from30:1 to50:1 (ml/g), the extractionenhanced in different slopes. As shown in Fig. 4B and

served that the yields of two isoavonoids signicantlyith increasing liquid/solid ratio at a higher pressure,enhanced at lower pressure. Increasing the liquid/solidved the extraction yields up to a certain ratio (about, after which no obviously improved extraction yieldsed. According to the chemical dynamics, the extractioncelerated by increasing the amounts of solvent. Thatsolutes saturation in the high volume of solvent wouldd, this means the solubility of solutes was enhanced., the interfacial area between tiny bubbles and sam-be increased with the liquid/solid ratio. Thereby, theratio used in practical production should be enough,complete the reaction. Fig. 4C and F shows that withiquid/solid ratio from 30:1 to 50:1 (ml/g) along with anethanol concentration from 55 to 75%, the extractionnot further enhanced. When the ethanol concentra-

increased, the amounts of two isoavonoids extractedased, but began to decrease when the ethanol con-surpassed 70%. It was noteworthy that for increasedcentration from 55 to 75%, the extraction yields of

onoids were enhanced in different slopes. As can beig. 4C and F, the extraction yields increased when theon of ethanol solution increased until it reached theat about 75% of ethanol for genistein and about 65%or genistin. This can be explained that the polarity ofweaker than that of genistin. An increase in ethanol

on accelerated the mass transfer ratio from plant mate-nt until a certain value, thus increasing the extractionn a certain range. However, the extraction yields of twos decreased slightly if the given ethanol concentrationthan a certain value (about 75%) with excessive vol-ent. This indicates that under the negative pressure, theent with high concentration can be consumed quickly

quid/solid ratio used in practical production was highereoretical value. The extraction yields can be adjustedall the parameters mentioned above because of their

accuracoefcand ge21.64icant. Bchancehigh si

Eqsthe relwith eratio:

Y1 =

Y2 = +

whereis the epressuliquid/

Byobtainsure43.89:ethanomaximwere 0

In0.05Mwere sthe invues frowere acentrafrom rin theters fortime 4ethanoexpericonditgenistiation (experiAfter tof the

Accin diff0.4053and getheprein soybpotentthe predictive model. According to ANOVA, correlation(R2) of 0.9512 and 0.9435, were obtained for genisteinn with the calculated model. The model F-value was8.54, respectively, which implies the model is signif-of the p-value is less than 0.0001, i.e., there is a 0.01%this error is caused by noise. These results imply a verycance of the regression model [22,23].nd (II) were obtained by statistical analysis, and showship between extraction yields of genistein andgenistintion pressure, ethanol concentration and liquid/solid

08 + 16.9377X1 + 0.0402X2 + 0.0162X3 0.0283X1X250X1X3 + 2.5000 106X2X3 147.6362X21 2.7561

X22 1.7308 104X23 (I)

96 + 12.0975X1 + 0.0216X2 + 0.0123X3 + 0.0117X1X233X1X3 + 2.5000 106X2X3 139.1215X21 1.6807

X22 1.4332 104X23 (II)

s the extraction yield of genistein (mg/g DW) and Y2ction yield of genistin (mg/g DW); X1 is the extractionPa), X2 is the ethanol concentration (%) and X3 is theratio (ml/g).ng Eqs. (I) and (II), the optimum conditions werehe predictive values for genistein were negative pres-Pa, ethanol concentration70.67%and liquid/solid ratio

ile for genistin theywere negative pressure 0.05MPa,centration 66.29% and liquid/solid ratio 44.13:1. Thepredictive extraction yields of genistein and genistinand 0.401mg/g, respectively.ary, the optimal parameters of negative pressure

ethanol concentration 70% and liquid/solid ratio 44:1ed on the basis of response surface. Those were withination ranges. When compared with the predictive val-qs. (I) and (II), the extraction parameters for genisteinedwithpredictive values. For genistin, the ethanol con-as a little higher, because some non-signicant effects

nse surface were eliminated but they were involvedictive equations. Accordingly, the better NPCE parame-istein and genistin were obtained as follows: extraction, particle size 50 mesh, negative pressure 0.05MPa,centration 70%, and liquid/solid ratio 44:1. Verications were carried out for ve times under these optimalThe resulting mean extraction yields of genistein andre 0.418 and 0.398mg/g with relative standard devi-) of 1.06 and 1.53%, respectively. In the verications, 440ml solvent was used for extraction in each time.CE process, there was 35ml solvent lost, which was 8%e solvent volume.g toother studies, thecontentsof genisteinandgenistin

t varieties of soybeans were 0.0370.368mg/g and22mg/g, respectively [2426]. The contents of genisteinn in pigeon pea roots revealed by the NPCE method instudy (0.418 and0.398mg/g)were comparable to those. The above results indicated that pigeon pea roots ared ideal source for obtaining genistein and genistin.

268 D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270

Table 2Conditions and results of different extraction methods.

Methode Time (h) Liquid/solidratio (ml/g)

Temperature (C) Pressure (MPa) Extraction yield ofgenistein (mg/gDW)

Extraction yield ofgenistin (mg/gDW)

Total extractionyield (mg/gDW)

ME .322HRE .388USE .402NPCE .418

a,b,c,dshows theeParticle size ofRoom tempergThese metho

3.3. Compa

When thto be an effor genistei(44:1, ml/g(room temattention foof isoavonyet. Hence,regarding thpigeon pea

ME, USEof extractinoptimizedsummarizeisoavonoidusing ME antion yields boptimized cwas not theyield by NPthat of HREFor the extby NPCE foof USE forMoreover, iusing USE abut for genyields, espetemperaturcoside of geand could b(80 C) procfourmethowasneededest. From thoffers a fascompoundssmaller reqtures. Consthe extractmosensitive

3.4. SEM ob

Pigeon pthe morphomethods, wnism. Fig. 5Asample, HRWe can ndples which

andhymig. 5Asamsurfaventbiliz. It cmpef theSEd orat in

raturn suagesgre

ion ee souidence,nd saatescienrele

nten

urthal phA anderi

rall ae phee raen otiesE exundsin anwn ilic coextraightly12 80:1 RTf g 03 65:1 80 g 01 45:1 50 g 00.75 44:1 RTf 0.05 0

values are signicantly different (P

D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270 269

Fig. 5. Scanning electron micrographs of pigeon pea roots samples after (B) ME, (C) HRE, (D) USE and (scanned at the same accelerating voltage of 15.0 kV (20m, 1000 magnication).

Similar trends were occurred in the DPPH scavenging activ-ity test. The samples were assayed over a range of dilutions.The concentration of sample producing a 50% reduction of theradical absorbance (IC50) was used as an index to compare the

Fig. 6. Compaextractswith dUSE, HRE and

antioxidantnal conceextract fromscavengingof the referextract (0.0(0.345mg/m

froms varcon

gingin 0rangedtrationsamplescavengenisterisons of the contents of genistein, genistin and total phenolic inifferentmethods (A) and the free radical-scavenging activity of NPCE,ME extracts (B) (n=3).

obtained. Tcated that gantioxidantand ME, NPand total pHence, theof pigeon pused in footive elds.

4. Conclus

In the pto achievepigeon peaods were aextracts weon the resunative to soof isoavoninvestigatiomay play ain human dindustry.E) NPCE, (A) was untreated pigeon pea roots sample. Each gure was

activity, and the IC50 was calculated by using thentration of the extract. It was observed that NPCE

pigeon pea roots exhibited notable DPPH radical-activity, with an IC50 value of 0.062mg/ml close to thatence VC (0.044mg/ml), and superior to those of USE80mg/ml), HRE extract (0.306mg/ml) and ME extractl). The free radical-scavengingvalues forNPCEextracts90.242.85 to 32.532.80%, with sample concen-

ying from 1.333 to 0.021mg/ml. The decrease in thecentration resulted in the reduction of its free radical-activity. In the authors previous tests, IC50 values of.058 and genistin higher than 0.091mg/ml have been

hese results combined with the data in this test, indi-enistein andgenistin should be twoof themost efcients in pigeon pea roots. In comparison with USE, HRECE extract with higher contents of target compoundshenolic content showed higher antioxidant activity.present result provides evidence that the NPCE extractea roots possesses better antioxidant activity, could bed industry, pharmaceutical industry and other correla-

ions

resent study, the green NPCE process was optimizedan efcient extraction of genistein and genistin fromroots. The total phenolic contents by different meth-ssessed and the antioxidant activities of the resultingre evaluated by DPPH radical-scavenging assay. Basedlts, we conclude that NPCE represents a valuable alter-me conventional methods for the efcient extractionoids from pigeon pea roots. Meanwhile, the presentn indicates the NPCE extract from pigeon pea rootspotential role as health-promoting antioxidant agentiets with economical potential for the pharmaceutical

270 D.-Y. Zhang et al. / Separation and Purication Technology 74 (2010) 261270

Acknowledgements

The authors gratefully acknowledge the nancial supportsby National Natural Science Foundation of China (30770231),Heilongjiang Province Science Foundation for Excellent Youths(JC200704), Agricultural Science and Technology AchievementsTransformation Fund Program (2009GB23600514), Key Project ofChinese Ministry of Education (108049), Key Program for Scienceand Technology Development of Harbin (2009AA3BS083), Funda-mental Research Funds for the Central Universities (DL09EA04),Project for Distinguished Teacher Abroad, Chinese Ministry of Edu-cation (MS2010DBLY031) and Foundation for Excellent Science andTechnology Innovation Project of Northeast Forestry University(GRAM 09).

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Negative pressure cavitation extraction and antioxidant activity of genistein and genistin from the roots of pigeon pea [C...IntroductionMaterials and methodsPlant materialChemicals and reagentsConventional extraction proceduresNegative pressure cavitation extractionDevice of negative pressure cavitationExtraction procedureExperimental designStatistical analysis

Determination of genistein and genistinScanning electron microscopy (SEM)Determination of total phenolic contentDPPH radical-scavenging assay

Results and discussionThe mechanism of NPCEOptimization of NPCE procedureEffects of extraction time and particle sizeOptimization of NPCE operating parameters

Comparison of different extraction methodsSEM observationContents of total phenolic and antioxidant activity

ConclusionsAcknowledgementsReferences