Separation and Purication Technology 74 (2010) 261270

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Separation and Purication Technology

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Negative pressure cavitation extraction and antioxidant activity of genistein and

genistin from the roots of pigeon pea [Cajanus cajan (L.) Millsp.]
Dong-Yang Zhang a,b,1 , Su Zhang a,b,1 , Yuan-Gang Zu a,b , Yu-Jie Fu a,b, , Yu Kong a,b ,
Yuan Gao a,b , Jin-Tong Zhao a,b , Thomas Efferth c
a
b
c

Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China
Engineering Research Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, 55099 Mainz, Germany

a r t i c l e

i n f o

Article history:
Received 3 March 2010
Received in revised form 12 June 2010
Accepted 20 June 2010
Keywords:
Negative pressure cavitation extraction
Pigeon pea roots
Isoavonoids
Antioxidant activity
Response surface methodology

a b s t r a c t
A new methodnegative pressure cavitation extraction (NPCE) was proposed and investigated for the
extraction of the main isoavonoids, namely genistein and genistin from pigeon pea roots. The effects of
extraction time and particle size on the extraction yields were rstly optimized, then a central composite design (CCD) combined with response surface methodology (RSM) was used to study the effects of
negative pressure, ethanol concentration and liquid/solid ratio on the extraction yields. The maximum
extraction yields of genistein and genistin reached 0.418 and 0.398 mg/g, respectively, under the optimal conditions: extraction time 45 min, particle size 50 mesh, negative pressure 0.05 MPa, ethanol
concentration 70% and liquid/solid ratio 44:1. Furthermore, the antioxidant activity of NPCE extract
was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay. NPCE extract possessed
notable concentration-dependent antioxidant activity with IC50 value of 0.062 mg/ml.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Pigeon pea [Cajanus cajan (L.) Millsp.], also known as red gram,
no-eye pea, Congo pea, Gungo pea, etc., is a famous and multiusage grain legume crop widely distributed in semi-tropical and
tropical developing countries. Nowadays, pigeon pea is cultivated
as an annual for both forage and its edible beans [1]. Some medicinal usages of this plant have been recorded in worldwide. Most
of the literatures are focused on pigeon pea leaves, which demonstrated notable anti-inammatory, anti-bacterial and abirritative
properties [2]. Especially, pigeon pea leaves are used as an excellent traditional Chinese medicine (TCM), which has been brought
to the market for the therapy of ischemic necrosis of femoral head
[3]. Comparatively, research on pigeon pea roots is scanty, the phytochemicals and possible medicinal uses are not fully explored. In
fact, pigeon pea roots have many medicinal uses. They were used
as an alexeritic, anthelminthic, expectorant, sedative, and vulnerary as a folk medicine in some local producing areas. However,
most of them are usually discarded as agricultural wastes or used

Corresponding author at: Key Laboratory of Forest Plant Ecology, Ministry of

Education, Northeast Forestry University, Hexing Road, No. 26, Harbin 150040, PR
China. Tel.: +86 451 82190535; fax: +86 451 82190535.
E-mail address: yujie fu2002@yahoo.com (Y.-J. Fu).
1
Both authors contributed equally to this work.
1383-5866/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2010.06.015

as rewood by farmers, the total amount is huge. Hence, innovative technology concepts for the utilization of pigeon pea roots to
provide the raw material for the manufacture of future-oriented
products are desirable.
Preliminary chemical investigations and pharmacological studies on pigeon pea roots in our group have demonstrated the
presence of polyphenols, especially isoavonoids, and they were
the major benecial compounds responsible for their bioactivities.
Genistein and genistin are a pair of isoavonoid compounds found
as the main constituents present in pigeon pea roots, their structures are shown in Fig. 1. They represent important members of
the avonoid family and possess a wide spectrum of pharmacological activities [47]. Therefore, genistein and genistin have great
potentials to be used as clinical therapeutic agents, food additives
or nutraceutical products. The application of low-cost technology
to obtain these two compounds from pigeon pea roots is a rational
strategy to increase the economic value and expand the utilization
of this plant.
Cavitation is a general uid mechanics phenomenon, which can
occur whenever a liquid used in a machine inducing pressure and
velocity uctuations in the uid (e.g. pumps, turbines, and propellers). Cavitation serves as a means of concentrating the diffused
uid energy locally in very short duration and creating a zone of
intense energy dissipation. Acoustic cavitation and hydrodynamic
cavitation are two types sorted by the cause of formation [8]. The
studies on acoustic cavitation such as ultrasonic cavitation have

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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

been widespread carried out in the elds of phytochemistry, biochemistry, physics and metallurgy [9,10]. As an efcient extraction
method, ultrasonic extraction has attracted growing interest for
the extraction of various secondary metabolites from plant materials [11,12]. However, some thermosensitive compounds such as
genistin are easily degradable by ultrasonic cavitation due to its
high temperature and intensity [1315]. Therefore, a new kind of
cavitation suitable for the extraction of thermosensitive secondary
metabolites is warranted.
Negative pressure cavitation is another type in which the cavitation is generated by negative pressure. It is a cheap and energy
efcient method. It can keep constant lower temperature and its
intensity is not weaker than that of ultrasonic cavitation. Furthermore, during the extraction process, nitrogen is continuously
introduced into a liquidsolid system to increase the turbulence,
collision and mass transfer between the extraction solvent and
matrix. Under the integrated action, it is efcient for mixing sample
with the extraction solvent as well as migrating the target compounds out of the sample matrix [16]. Thus, this procedure has not
only the advantages of ultrasonic cavitation but also is good for
preventing the degradation of thermosensitive compounds.
In the present study, a NPCE method was proposed and applied
for the extraction of genistein and genistin from pigeon pea roots.
The effects of main operating parameters on the extraction yields
were investigated. The extraction efciency of two isoavonoids
with NPCE was compared with those obtained by three conventional extraction methods. The structural disruption to pigeon pea
roots samples with different extraction methods was observed by
scanning electron microscopy (SEM). Furthermore, the antioxidant
activity of extracts, obtained using different extraction methods
was determined by means of DPPH radical-scavenging assay.
2. Materials and methods
2.1. Plant material
Pigeon pea roots were collected in autumn from Hainan
Province, China, and identied by Prof. Shao-Quan Nie from the Key
Laboratory of Forest Plant Ecology, Ministry of Education, Northeast
Forestry University, PR China. Voucher specimens were deposited
in the herbarium of the same laboratory. The samples were dried in
the shade, powdered and sieved (2080 mesh). At last, they were
kept away from light in a desiccator at room temperature until used.
2.2. Chemicals and reagents
Genistein (4 ,5,7-trihydroxyisoavone, 98%) and genistin
(4 ,5,7-trihydroxyisoavone-7-glucoside, 98%) were purchased
from Fluka (Buchs, Switzerland). Methanol of HPLC grade was
obtained from J&K Chemical Ltd. (Beijing, China), while formic
acid (96%) was from DIMA Technology Inc. (Muskegon, MI, USA).
2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid (VC) were
supplied by SigmaAldrich (Steinheim, Germany). Ethanol of analytical grade for extraction was bought from Tianjin Kermel
Chemical Reagent Co. (Tianjin, China). Deionized water was puried by a Milli-Q water-purication system from Millipore (Bedford,
MA, USA).
2.3. Conventional extraction procedures
For maceration extraction (ME), according to the preliminary
optimized investigation, 10 g of pigeon pea roots were put into
a beaker with 800 ml ethanol/water (70:30, v/v). The beaker was
placed at room temperature for 12 h, then, the ltered extraction solution was collected and another 800 ml of 70% ethanol
was added into the beaker for another 12 h. After the process was

263

repeated for three times, the extraction solutions were combined

and concentrated to dryness on a rotary evaporator (RE-52AA,
Shanghai Huxi Instrument, Shanghai, China) at 50 C. Methanol of
HPLC grade was added to get the samples of appropriate concentration for HPLC analysis.
For heat-reuxing extraction (HRE), 10 g of pigeon pea roots
were accurately weighed and put into a round-bottomed ask with
650 ml ethanol/water (70:30, v/v). The extraction was employed
to optimum conditions of 80 C for 3 h under magnetic stirring at
500700 rpm. Then, the ltered extraction solution was collected
and another 650 ml of 70% ethanol was added into the ask for
another 3 h. The subsequent process was the same as ME.
For ultrasound-assisted extraction (USE), according to the
preliminary investigation, target compositions were extracted
by adding 10 g of pigeon pea roots into a ask with 450 ml
ethanol/water (70:30, v/v), the sample was then extracted by ultrasonic for three times in an ultrasonic bath. The optimum extraction
process was performed at 50 C for 1 h. Then, the subsequent process was the same as ME.
2.4. Negative pressure cavitation extraction
2.4.1. Device of negative pressure cavitation
Negative pressure cavitation equipment (CN2597047) was
developed in our laboratory. The schematic diagram of the device is
shown in Fig. 2A. The negative pressure cavitation device consists
of an extraction pot (1) and a collection pot (2). Material and solvent
are added into the extraction pot through the inlet (3). The negative pressure of the device is provided by a vacuum pump through
an interface on top of the pot body. Nitrogen is introduced into the
extraction pot through valve 2. After extraction, the extraction solvent is ltered through a net (4) into the collection pot. The residue
can be removed from the pot through the discharge lid. The temperature of the entire system can be kept constant because nitrogen
with ambient temperature is injected into the system continuously.
The volatilized solvent is refrigerated by the condenser (6).
2.4.2. Extraction procedure
Ten grams of pigeon pea roots were introduced into the NPCE
device from the sample portal. After the solvent was added, the
device was connected to the vacuum pump. At the same time, the
valve of ow meter was opened and nitrogen was supplied into
the device. The negative pressure of the device can be controlled
by the valve. According to the experimental design, the extraction
process was performed under different conditions. The subsequent
process was the same as ME. The effects of extraction time, particle
size, negative pressure, ethanol concentration and liquid/solid ratio
on the extraction efciency of two isoavonoids were investigated.
The extraction efciency of the compounds in sample was
dened as follows:
Extraction yield (mg/g DW)
=

Mass of the compounds in extraction solution

Mass of dried material

2.4.3. Experimental design

On the basis of the single factor experimental results, major
inuencing factors were conrmed, and then a 23 factorial portion
central composite design (CCD) combined with response surface
methodology (RSM) was used for optimizing negative pressure
(X1 ), ethanol concentration (X2 ) and liquid/solid ratio (X3 ). In the
CCD test, 14 experiments and six replicates at the centre were
employed to t the full quadratic equation model. The general

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

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

Factors
X1 (Pa , MPa)

X2 (Ec.b , %)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

1 (0.035)
1 (0.065)
1 (0.035)
1 (0.065)
1 (0.035)
1 (0.065)
1 (0.035)
1 (0.065)
1.682 (0.025)
1.682 (0.075)
0 (0.050)
0 (0.050)
0 (0.050)
0 (0.050)
0 (0.050)
0 (0.050)
0 (0.050)
0 (0.050)
0 (0.050)
0 (0.050)

1 (60)
1 (60)
1 (80)
1 (80)
1 (60)
1 (60)
1 (80)
1 (80)
0 (70)
0 (70)
1.682 (53.18)
1.682 (86.18)
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)

a
b
c
d
e

Extraction yield of
genistein (mg/g DW)

Extraction yield of
genistin (mg/g DW)

X3 (LSR.c , ml/g)

Exp.d

Pred.e

Exp.d

Pred.e

1 (30)
1 (30)
1 (30)
1 (30)
1 (50)
1 (50)
1 (50)
1 (50)
0 (40)
0 (40)
0 (40)
0 (40)
1.682 (23.18)
1.682 (57.82)
0 (40)
0 (40)
0 (40)
0 (40)
0 (40)
0 (40)

0.334
0.332
0.341
0.318
0.353
0.332
0.357
0.323
0.352
0.301
0.328
0.357
0.332
0.411
0.415
0.411
0.412
0.418
0.419
0.417

0.328
0.319
0.341
0.316
0.361
0.337
0.374
0.334
0.343
0.302
0.334
0.343
0.347
0.389
0.417
0.417
0.417
0.417
0.417
0.417

0.348
0.322
0.326
0.306
0.357
0.332
0.335
0.318
0.335
0.282
0.377
0.322
0.321
0.392
0.389
0.395
0.393
0.399
0.391
0.398

0.348
0.318
0.321
0.297
0.371
0.342
0.342
0.321
0.328
0.284
0.368
0.327
0.334
0.374
0.395
0.395
0.395
0.395
0.395
0.395

Negative pressure (MPa).

Ethanol concentration (%).
Liquid/solid ratio (ml/g).
Experimental value.
Predicted value.

equation is:
Y = 0 +

k

j=1

j Xj +

k


jj Xj2 +

j=1



ij Xi Xj (k = 3)

i<j

where Y is the predicted response, 0 , j , jj and ij are the regression coefcients for intercept, linearity, square and interaction,
respectively, while Xi and Xj are the independent coded variables.
The variables of each factor were transferred to a range between
1 and 1 for the appraisals, while the dependent variable was the
extraction yield of genistein and genistin. Coded variables were
used according to the equation:
Xi =

Xi X0i
Xi

(i = 1, 2, 3)

Xi

is the coded value of the independent variable. Xi is the

where
real value of the independent variable. X0i is the real value of the
independent variable at the centre point. Xi is the step change
of the real value of variable i. The actual and coded levels of the
independent variables used in the experimental design are shown
in Table 1. All the experiments were repeated three times, and the
extraction yields were given as average values.
2.4.4. Statistical analysis
The data collected from NPCE tests designed by central composite was analyzed using a response surface analysis procedure
(Design-Expert 7.1.3 Trial, State-Ease, Inc., Minneapolis, MN, USA).
Analysis of variance (ANOVA) was performed for calculations and
modeling of optimal conditions for NPCE of pigeon pea roots. Values
of P < 0.01 were regarded as signicant.
2.5. Determination of genistein and genistin
An Agilent 1200 reversed-phase HPLC system was employed to
determine the contents of genistein and genistin. Chromatographic
separation was carried out on a HiQ Sil C18W reversed-phase
column (250 mm 4.6 mm i.d., 5 m). The mobile phase was

methanolwaterformic acid (35:64.935:0.065, v/v/v) and was ltered through a 0.45 m membrane lter (Millipore, USA) prior to
use. The ow rate was 1 ml/min, the injection volume was 10 l,
and the column temperature was set at 30 C. Genistein at wavelength of 260 nm and genistin at 310 nm were veried and used for
the RP-HPLC quantication. The chromatographic peaks of the analytes were conrmed by comparing their retention times and UV
spectra with those of the reference compounds. Eight experimental points were employed for establishing a calibration curve. The
regression lines for genistein and genistin were Y = 51647X + 228.53
(R2 = 0.9998, n = 8), Y = 22855X + 8.19 (R2 = 0.9995, n = 8), respectively, where Y is the peak area of analyte, and X is the concentration
of analyte (mg/ml).
2.6. Scanning electron microscopy (SEM)
The morphological changes of samples with different extraction methods were observed by SEM. After removing the solvent,
the remaining samples of pigeon pea roots were plunged into liquid nitrogen and cut with a cold knife. The sectioned particles
were xed on a specimen holder with aluminium tape and marked,
then sputtered with gold in a sputter-coater. All the samples were
examined with a Quanta-200 SEM (FEI Company, USA) under high
vacuum and at an accelerating voltage of 15.0 kV.
2.7. Determination of total phenolic content
Total phenolics were determined by using FolinCiocalteu
method according to Singleton and Rossi (1965) with a little modication [17]. Briey, 20 l of samples were mixed with 1.58 ml
water and 100 l FolinCiocalteu reagent (1 mol/l). The mixture
was allowed to stand for 5 min at room temperature, then, the
reaction was neutralized with 300 l sodium carbonate solution (7.07 mol/l). The absorbance of the resulting blue color was
measured at 765 nm with a UVvis spectrophotometer (UNICO,
Shanghai, China) after incubation for 90 min at room temperature.
The results were reported in gallic acids equivalents (GAE) per
gram of sample (mg GAE/g extract). All the measurements were

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

265

taken in triplicate, means and standard deviation values were calculated.

2.8. DPPH radical-scavenging assay
The DPPH radical-scavenging activity (anti-free radical activity)
is based on the determination by DPPH at a steady state in ethanol
solution after adding the mixture of antioxidants. Following the
detailed method of Liyana-Pathiranan and Shahidi [18], the extract
was dissolved in 10 ml of absolute ethanol to give a concentration of
4 mg/ml, then 2 ml of 0.004% DPPH (0.2 mM) in ethanol was added
to 1 ml of the extract solution. After shaking the mixture vigorously,
the mixture was immediately placed in an UNICO UV-2100 spectrophotometer (UNICO, Shanghai, China) to monitor the decrease in
absorbance at 517 nm until the reaction reached a plateau. Ascorbic
acid (SigmaAldrich), a stable antioxidant, was used as a synthetic
reference. The DPPH radical-scavenging activity in percentage of
sample was calculated according to the following equation [19]:
Inhibition percentage (Ip ) =

100(AB AA )
AB

where AB and AA were the absorbance values of the blank and

of the tested samples, respectively, checked after 70 min. All data
were averages (standard deviations) of triplicate determinations
of three independent tests.
3. Results and discussion
3.1. The mechanism of NPCE
The NPCE system can be separated into four areas [20] (Fig. 2A):
bubbles formation layer, suspension layer, axle air current layer,
and turbulent layer. In the bubbles formation layer, the continuous introduction of nitrogen into the extraction system, under
the action of negative pressure, small nitrogen bubbles appear and
ascend among the liquidsolid phase, results in the formation of
highly instable gasliquidsolid system. When the bubbles enter
the suspension layer, the volumes of bubbles change rapidly along
with the outside pressure. Bubbles grow and collapse at certain
ow regions with lower pressure. The bubbles grow and the following concave jet makes the surrounding liquid drops into liquid
sheets, so that the liquid sheets with different solute concentration
can rapidly coalesce. As a result, a mass transfer takes place among
liquid drops [21]. On the other hand, the collapse of bubbles can
produce cavitation so that the surface of solid particles is corroded
and the solvent can diffuse into the inside of solid particles (Fig. 2B),
this can enhance the intra-particle diffusion. In the axle air current layer, the compounds in the material are efciently released
into the solvent due to an intense collision and mass transfer effect
between liquid drops and solid particles. In the turbulent phase, the
process of mass transfer is accomplished by an action of turbulent
motion effect. Thereby, the negative pressure cavitation of this system creates intensive cavitation-collision, turbulence, suspension
and interface effects. These effects combine to form a dynamic mass
transfer enhancing system and favor the rapid transfer of target
compounds from the matrix to the solvent, increase the extraction
efciency and recovery of target compounds.
3.2. Optimization of NPCE procedure
3.2.1. Effects of extraction time and particle size
To increase the extraction efciency and decrease the time consumption, the extraction time was rstly optimized. The effect of
extraction time was studied with 70% ethanol solution at a liquid/solid ratio of 45:1, a particle size of 50 mesh and a negative
pressure of 0.05 MPa. The extraction time was 10, 20, 30, 45, 60,

Fig. 3. Effects of extraction time (A) and particle size (B) on the extraction yields of
genistin and genistein.

90 and 120 min, respectively. The results are shown in Fig. 3A. It is
apparent that the effect of time on the extraction yields of genistein and genistin showed a similar trend. The extraction yields of
two isoavonoids distinctly increased with the extension of extraction time in the rst 45 min. After 45 min, the extraction time had
less impact on the extraction yields, indicating that genistein and
genistin reached their distribution equilibrium at around 45 min.
Considering the increase of extraction yields and the extraction efciency of two isoavonoids, 45 min was selected as the appropriate
extraction time and used in the following tests.
The effect of particle size on the extraction yields was investigated using mean particle sizes of 20, 30, 40, 50, 60, 70 or 80
mesh with a 70% ethanol solution at a liquid/solid ratio of 45:1, a
negative pressure of 0.05 MPa and an extraction time of 45 min.
The results are shown in Fig. 3B. It can be seen that the extraction yields increased when the particle size changed from 20 to 60
mesh. It is well known that mechanical treatment on the raw material has a large impact on the extraction yields, since it could break
plant cell walls and augment compound accessibility. Hence, the
lower the particle size is, the higher the extraction yields can be
obtained. However, it is not recommendable and sometimes it is
almost impossible to process such ne powders industrially. Considering the higher extraction yields and the practicability, 50 mesh
was selected for the following experiments.
3.2.2. Optimization of NPCE operating parameters
The objective of the present study was to optimize the operating
conditions to achieve an efcient extraction of two isoavonoids
from the roots of pigeon pea. Because various parameters potentially inuence the NPCE process, the optimization of the operating

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

Fig. 4. Response surfaces for genistein (A)(C) and genistin (D)(F) from pigeon pea roots. (A) and (D) varying extraction pressure and ethanol concentration; (B) and (E)
varying extraction pressure and liquid/solid ratio; (C) and (F) varying ethanol concentration and liquid/solid ratio. P: extraction pressure, EC: ethanol concentration, and LSR:
liquid/solid ratio.

conditions plays a critical role in the development of a NPCE

method. CCD combined with RSM was used for optimizing. Three
parameters including negative pressure, ethanol concentration and
liquid/solid ratio were optimized by a 23 factorial portion CCD.
All results obtained from 20 experimental runs and the predicted
data from the model based on the experimental data are summarized in Table 1. Three-dimensional proles of multiple non-linear
regression models were used to depict the interactive effects of
operational parameters for two isoavonoids (Fig. 4AF).
Fig. 4A and D shows the effects of pressure and ethanol concentration on the extraction yields of genistein and genistin,
respectively. It was observed that decrease of negative pressure
from 0.03 to 0.05 MPa with ethanol concentration increasing

from 55 to 75% enhanced the extraction yields of genistein and

genistin simultaneously. If the given ethanol concentration was
a certain value, while pressure was falling, the extraction yields
increased at relative high-pressure levels (above 0.05 MPa). Once
the pressure reached low levels (lower than 0.05 MPa), the extraction yields slightly decreased. This can be explained by a decrease
in negative pressure resulting from reduction of nitrogen ow rate,
which means the decrease in formation of tiny bubbles and there
were not enough nitrogen bubbles to form turbulent motion and
mass transfer accordingly. However, high pressure is not always
recommended due to the overfull gas in the liquid resulted in a
lack of cavitation effect. The inuence of ethanol concentration was
more difcult to predict than that of negative pressure. Firstly, the

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

formation and burst of tiny bubbles can be affected due to different ethanol concentrations resulted in different viscosities of
ethanolwater binary system. Secondly, different ethanol concentrations were related to compounds of different polarities. In Fig. 4A
and D, an increase in extraction yields was observed by increasing
the ethanol concentration in an early stage of extraction, because
the change of solvent viscosity was less effective than that of solvent polarity. The trend was, however, reversed, when the ethanol
concentration reached a certain value under the ranges of pressure
in this study. This can be explained that genistein and genistin are
moderate polar compounds, and much lower ethanol concentration limited their solubility, resulting in declined extraction yields.
Since the solubility of two isoavonoids depended largely on the
balance between solvent viscosity and polarity of the solution, an
ethanol concentration in the range from 65 to 75% was practical
during NPCE of pigeon pea roots.
Fig. 4B and E shows the three-dimensional plot of the response
surface for the extraction yields as related to extraction pressure
and liquid/solid ratio. Fig. 4C and F describes the interactive effect
of ethanol concentration and liquid/solid ratio. The inuence of liquid/solid ratio was not as signicant as those of extraction pressure
and ethanol concentration. The extraction yields did not continue
to signicantly increase until the liquid/solid ratio was over 45:1
(ml/g). Although increasing the solvent volumes led to a slight
increase in the extraction yields, further increasing the liquid/solid
ratio up to 50:1 (ml/g) resulted in little change in the yields of two
isoavonoids.
For falling pressure from 0.03 to 0.05 MPa together with an
increase of liquid/solid ratio from 30:1 to 50:1 (ml/g), the extraction
yields were enhanced in different slopes. As shown in Fig. 4B and
E, it was observed that the yields of two isoavonoids signicantly
increased with increasing liquid/solid ratio at a higher pressure,
but slightly enhanced at lower pressure. Increasing the liquid/solid
ratio improved the extraction yields up to a certain ratio (about
45:1, ml/g), after which no obviously improved extraction yields
was observed. According to the chemical dynamics, the extraction
could be accelerated by increasing the amounts of solvent. That
was due to solutes saturation in the high volume of solvent would
be decreased, this means the solubility of solutes was enhanced.
Meanwhile, the interfacial area between tiny bubbles and samples could be increased with the liquid/solid ratio. Thereby, the
liquid/solid ratio used in practical production should be enough,
in order to complete the reaction. Fig. 4C and F shows that with
increasing liquid/solid ratio from 30:1 to 50:1 (ml/g) along with an
increase in ethanol concentration from 55 to 75%, the extraction
yields were not further enhanced. When the ethanol concentrations were increased, the amounts of two isoavonoids extracted
rstly increased, but began to decrease when the ethanol concentration surpassed 70%. It was noteworthy that for increased
ethanol concentration from 55 to 75%, the extraction yields of
two isoavonoids were enhanced in different slopes. As can be
seen from Fig. 4C and F, the extraction yields increased when the
concentration of ethanol solution increased until it reached the
maximum at about 75% of ethanol for genistein and about 65%
of ethanol for genistin. This can be explained that the polarity of
genistein is weaker than that of genistin. An increase in ethanol
concentration accelerated the mass transfer ratio from plant material to solvent until a certain value, thus increasing the extraction
yields within a certain range. However, the extraction yields of two
isoavonoids decreased slightly if the given ethanol concentration
was higher than a certain value (about 75%) with excessive volume of solvent. This indicates that under the negative pressure, the
ethanol solvent with high concentration can be consumed quickly
when the liquid/solid ratio used in practical production was higher
than the theoretical value. The extraction yields can be adjusted
by changing all the parameters mentioned above because of their

267

interactions, all of which are detrimental and advantageous consequences, respectively.

As shown in Table 1, the extraction yields of two isoavonoids
were similar to those expected, and the results demonstrated the
accuracy of the predictive model. According to ANOVA, correlation
coefcients (R2 ) of 0.9512 and 0.9435, were obtained for genistein
and genistin with the calculated model. The model F-value was
21.64 and 18.54, respectively, which implies the model is significant. Both of the p-value is less than 0.0001, i.e., there is a 0.01%
chance that this error is caused by noise. These results imply a very
high signicance of the regression model [22,23].
Eqs. (I) and (II) were obtained by statistical analysis, and show
the relationship between extraction yields of genistein and genistin
with extraction pressure, ethanol concentration and liquid/solid
ratio:
Y1 = 1.7508 + 16.9377X1 + 0.0402X2 + 0.0162X3 0.0283X1 X2
0.0250X1 X3 + 2.5000 106 X2 X3 147.6362X12 2.7561
104 X22 1.7308 104 X32

(I)

Y2 = 0.8696 + 12.0975X1 + 0.0216X2 + 0.0123X3 + 0.0117X1 X2

+ 0.0033X1 X3 + 2.5000 106 X2 X3 139.1215X12 1.6807
104 X22 1.4332 104 X32

(II)

where Y1 is the extraction yield of genistein (mg/g DW) and Y2

is the extraction yield of genistin (mg/g DW); X1 is the extraction
pressure (MPa), X2 is the ethanol concentration (%) and X3 is the
liquid/solid ratio (ml/g).
By solving Eqs. (I) and (II), the optimum conditions were
obtained. The predictive values for genistein were negative pressure 0.05 MPa, ethanol concentration 70.67% and liquid/solid ratio
43.89:1; while for genistin they were negative pressure 0.05 MPa,
ethanol concentration 66.29% and liquid/solid ratio 44.13:1. The
maximum predictive extraction yields of genistein and genistin
were 0.421 and 0.401 mg/g, respectively.
In summary, the optimal parameters of negative pressure
0.05MPa, ethanol concentration 70% and liquid/solid ratio 44:1
were selected on the basis of response surface. Those were within
the investigation ranges. When compared with the predictive values from Eqs. (I) and (II), the extraction parameters for genistein
were accorded with predictive values. For genistin, the ethanol concentration was a little higher, because some non-signicant effects
from response surface were eliminated but they were involved
in the predictive equations. Accordingly, the better NPCE parameters for genistein and genistin were obtained as follows: extraction
time 45 min, particle size 50 mesh, negative pressure 0.05 MPa,
ethanol concentration 70%, and liquid/solid ratio 44:1. Verication
experiments were carried out for ve times under these optimal
conditions. The resulting mean extraction yields of genistein and
genistin were 0.418 and 0.398 mg/g with relative standard deviation (RSD) of 1.06 and 1.53%, respectively. In the verication
experiments, 440 ml solvent was used for extraction in each time.
After the NPCE process, there was 35 ml solvent lost, which was 8%
of the whole solvent volume.
According to other studies, the contents of genistein and genistin
in different varieties of soybeans were 0.0370.368 mg/g and
0.40531.022 mg/g, respectively [2426]. The contents of genistein
and genistin in pigeon pea roots revealed by the NPCE method in
the present study (0.418 and 0.398 mg/g) were comparable to those
in soybeans. The above results indicated that pigeon pea roots are
potential and ideal source for obtaining genistein and genistin.

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

Table 2
Conditions and results of different extraction methods.
Methode

Time (h)

Liquid/solid
ratio (ml/g)

Temperature ( C)

Pressure (MPa)

Extraction yield of
genistein (mg/g DW)

Extraction yield of
genistin (mg/g DW)

Total extraction
yield (mg/g DW)

ME
HRE
USE
NPCE

12
3
1
0.75

80:1
65:1
45:1
44:1

RTf
80
50
RTf

g
g
g
0.05

0.322 0.017a
0.388 0.026b
0.402 0.022bc
0.418 0.029c

0.279 0.019a
0.316 0.027b
0.352 0.021c
0.398 0.030d

115.702 8.732a
174.725 7.043ab
147.085 7.329b
151.541 9.146b

a,b,c,d

shows the values are signicantly different (P < 0.05) (n = 3).

Particle size of 50 mesh and 70% ethanol concentration were used in these methods.
Room temperature.
g
These methods were carried out under the atmospheric pressure.
e
f

3.3. Comparison of different extraction methods

When the optimization tests were completed, NPCE was shown
to be an efcient method with high extraction yields (0.418 mg/g
for genistein and 0.398 mg/g for genistin) at low liquid/solid ratio
(44:1, ml/g), short reaction time (45 min) and low temperature
(room temperature). Although NPCE has received considerable
attention for the extraction of biologically active compounds, NPCE
of isoavonoids from pigeon pea roots has not been reported
yet. Hence, a comparison of NPCE with other extraction methods
regarding their performances of two isoavonoids extraction from
pigeon pea roots is indispensable.
ME, USE, HRE and NPCE were compared for their performances
of extracting genistein and genistin from pigeon pea roots at the
optimized conditions. The extraction conditions and yields are
summarized in Table 2. We can nd the extraction yields of two
isoavonoids using NPCE and USE methods are higher than those
using ME and HRE methods. For both two compounds, the extraction yields by USE reached at least 0.402 and 0.352 mg/g under the
optimized conditions. However, the trend of total extraction yields
was not the same as those of two isoavonoids. The total extraction
yield by NPCE was higher than those of USE and ME but lower than
that of HRE which gave a long-term heating at high temperature.
For the extraction conditions, we found higher extraction yields
by NPCE for 45 min at room temperature as compared to those
of USE for 1 h at 50 C, and less solvent was consumed in NPCE.
Moreover, it is noteworthy that the extraction yields of genistein
using USE and HRE were essentially the same as those using NPCE,
but for genistin, both methods gave signicantly lower extraction
yields, especially for HRE which needed long-term heating at high
temperature. The possible reason was that genistin is a potent glycoside of genistein, which is more susceptible to high temperature
and could be degraded to some extent in the USE (50 C) and HRE
(80 C) procedures. The comparison of the extraction conditions of
four methods showed that more solvent and longer extraction time
was needed for ME. Moreover, its extraction efciency was the lowest. From these results, it was concluded that application of NPCE
offers a fast, environmentally friendly and easy route for active
compounds production. The advantages of NPCE were shorter time,
smaller requirement for solvents as well as relative low temperatures. Consequently, NPCE represents a promising alternative for
the extraction of biologically active compounds, especially thermosensitive compounds from natural products.
3.4. SEM observation
Pigeon pea roots samples were examined by SEM to elucidate
the morphological changes of samples using different extraction
methods, which is helpful in understanding the extraction mechanism. Fig. 5AE shows the micrographs of the untreated sample, ME
sample, HRE sample, USE sample and NPCE sample, respectively.
We can nd some differences on the parenchyma of different samples which was the storage of secondary metabolites. As shown in

Fig. 5A and B, it can be observed there was no destruction on the

parenchyma for the untreated sample and we can see the complete
cells (Fig. 5A). After ME process, little destruction of the microstructure of sample was occurred. However, a little wrinkling took place
on the surface of parenchyma (Fig. 5B). In the HRE process (Fig. 5C),
the solvent transfers into the matrix and extracts the compounds
by solubilization. Hence, more solvent and long extraction time are
needed. It can be found the cellular damage was enhanced by the
high temperature and the continuous reuxing of solvent, some
walls of the parenchyma were also destroyed.
In USE and NPCE, the parenchymas of samples were greatly
changed or destroyed (Fig. 5D and E), which were more obvious
than that in HRE. USE possesses the advantages of high effective
temperatures and pressures at the interphase between the solvent
solution subjected to ultrasonic energy and the solid matrix. These
advantages combine with the mechanical effect of ultrasound provides a greater penetration of solvent into cellular materials, via
cavitation effects, and improves the release of chemical substances
into the solvent [27,28]. In NPCE, nitrogen was introduced into
the liquidsolid system, the resulting tiny bubbles increase the
turbulence, collision and mass transfer between the extraction solvent and sample matrix. Moreover, the intensive cavitation effect
penetrates the surface of the sample and enhances the extraction efciency. Hence, the chemical substances within the cell are
rapidly released into the surrounding solvents.
3.5. Contents of total phenolic and antioxidant activity
To further study the efciency of different extraction methods,
the total phenolic content and antioxidant activity were analyzed
(Fig. 6A and B). As we known, phenolic compounds are hydroxylated derivatives of benzoic and cinnamic acids and contribute
to overall antioxidant activity in the plants. Some reports showed
that the phenolic compounds, especially avonoids exhibit extensive free radical-scavenging activities through their reactivity as
hydrogen or electron-donating agents, and metal ion chelating
properties [29]. Hence, the antioxidant activity of ME, HRE, USE
or NPCE extracts is in accordance with the amount of phenolic
compounds. The amount of total phenolic content and contents of
genistein and genistin in extracts with different extraction methods
are shown in Fig. 6A. It can be clearly observed that the highest total
phenolic content was obtained in NPCE extract (80.529 3.021 mg
GAE/g extract) while USE extract (72.359 2.237 mg GAE/g extract)
was slightly lower. Moreover, it is noteworthy that the total phenolic content of HRE extract (56.866 3.232 mg GAE/g extract) was
essentially the same as that using ME (61.865 2.123 mg GAE/g
extract), both of them were signicantly lower than NPCE. This
was because the amount of ME extract was small, and the content
of total phenolic in ME extract was also lower. Meanwhile, large
quantity of extract could be obtained in HRE, but there were lots
of impurities in the extract. The results were similar to the lower
contents of two isoavonoids in HRE extract, which was obtained
by long-term heating at high temperature.

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 (E) NPCE, (A) was untreated pigeon pea roots sample. Each gure was
scanned at the same accelerating voltage of 15.0 kV (20 m, 1000 magnication).

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

antioxidant activity, and the IC50 was calculated by using the

nal concentration of the extract. It was observed that NPCE
extract from pigeon pea roots exhibited notable DPPH radicalscavenging activity, with an IC50 value of 0.062 mg/ml close to that
of the reference VC (0.044 mg/ml), and superior to those of USE
extract (0.080 mg/ml), HRE extract (0.306 mg/ml) and ME extract
(0.345 mg/ml). The free radical-scavenging values for NPCE extracts
ranged from 90.24 2.85 to 32.53 2.80%, with sample concentrations varying from 1.333 to 0.021 mg/ml. The decrease in the
sample concentration resulted in the reduction of its free radicalscavenging activity. In the authors previous tests, IC50 values of
genistein 0.058 and genistin higher than 0.091 mg/ml have been
obtained. These results combined with the data in this test, indicated that genistein and genistin should be two of the most efcient
antioxidants in pigeon pea roots. In comparison with USE, HRE
and ME, NPCE extract with higher contents of target compounds
and total phenolic content showed higher antioxidant activity.
Hence, the present result provides evidence that the NPCE extract
of pigeon pea roots possesses better antioxidant activity, could be
used in food industry, pharmaceutical industry and other correlative elds.

4. Conclusions

Fig. 6. Comparisons of the contents of genistein, genistin and total phenolic in

extracts with different methods (A) and the free radical-scavenging activity of NPCE,
USE, HRE and ME extracts (B) (n = 3).

In the present study, the green NPCE process was optimized

to achieve an efcient extraction of genistein and genistin from
pigeon pea roots. The total phenolic contents by different methods were assessed and the antioxidant activities of the resulting
extracts were evaluated by DPPH radical-scavenging assay. Based
on the results, we conclude that NPCE represents a valuable alternative to some conventional methods for the efcient extraction
of isoavonoids from pigeon pea roots. Meanwhile, the present
investigation indicates the NPCE extract from pigeon pea roots
may play a potential role as health-promoting antioxidant agent
in human diets with economical potential for the pharmaceutical
industry.

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

Acknowledgements
The authors gratefully acknowledge the nancial supports
by National Natural Science Foundation of China (30770231),
Heilongjiang Province Science Foundation for Excellent Youths
(JC200704), Agricultural Science and Technology Achievements
Transformation Fund Program (2009GB23600514), Key Project of
Chinese Ministry of Education (108049), Key Program for Science
and Technology Development of Harbin (2009AA3BS083), Fundamental Research Funds for the Central Universities (DL09EA04),
Project for Distinguished Teacher Abroad, Chinese Ministry of Education (MS2010DBLY031) and Foundation for Excellent Science and
Technology Innovation Project of Northeast Forestry University
(GRAM 09).
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