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Food Anal.

Methods (2010) 3:9097


DOI 10.1007/s12161-009-9091-2

Analysis of Flavonoids in Portulaca oleracea L. by UVVis


Spectrophotometry with Comparative Study on Different
Extraction Technologies
Hongbin Zhu & Yuzhi Wang & Yuxuan Liu & Yalin Xia &
Tian Tang

Received: 25 March 2009 / Accepted: 26 May 2009 / Published online: 12 June 2009
# Springer Science + Business Media, LLC 2009

Abstract Portulaca oleracea L. is a traditional edible and


medicinal plant in China. Flavonoids are one of the main
active ingredients of this plant. Five extraction technologies
of flavonoids from P. oleracea L. were investigated and
compared, including microwave-assisted extraction, ultrasonic extraction, reflux extraction, Soxhlet extraction, and
marinated extraction. The results showed that microwaveassisted extraction was most suitable for the extraction of
flavonoids from P. oleracea L. because of its high effect
and short extraction time. The found optimum extraction
conditions were that the ethanol concentration was 70%
(v/v), solidliquid ratio was 1:50, extracting temperature
was 50 C and irradiation time was 9 min. Quantification
was performed by means of UVVis spectrophotometry
with chromogenic system of NaNO2Al (NO3)3NaOH.
Under the optimum conditions, the calibration curve for the
analyte was linear with the correlation coefficients greater
than 0.9999. The average recovery was 102.6%, and its
RSD was 1.13%(n=5). Eight types of P. oleracea L.
according to different habits were investigated. The total
content of flavonoids was 7.16, 7.10, 9.38, 6.82, 6.78,
11.36, 5.12, and 1.76 mg g1, respectively.
Keywords Food Analysis . Portulaca oleracea L. .
Flavonoids . Microwave-Assisted Extraction .
UVVis Spectrophotometry
H. Zhu : Y. Wang (*) : Y. Liu : Y. Xia : T. Tang
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering,
Hunan University,
Changsha 410082, Peoples Republic of China
e-mail: wyzss@hnu.cn

Introduction
Portulaca oleracea L. (P. oleracea L., purslane) is a
common, herbaceous succulent annual weed, which is
distributed extensively in temperate and tropical regions
worldwide (Radhakrishnan et al. 2001; Garti et al. 1999). It
has been used as a kind of food and medicinal plant for
thousands of years in China. P. oleracea L. is of abundant
nutrition with contents of proteins, carbohydrates, Ca, K,
Zn, and Na (Aberoumand 2008). As a kind of Chinese
traditional medicine, P. oleracea L. is very important
because of its special medical function, and it has been
used traditionally for the treatment of dysentery with
bloody stools and externally for boils and sores, eczema,
erysipelas, and insect and snake bites (Chen et al. 2003;
Zhang et al. 2002; Yazici et al. 2007; Palaniswamya et al.
2004).
Among the effective ingredients of many Chinese
herbal medicines, flavonoids are one of the most
popular compounds in the plant kingdom. Flavonoids
have received much attention because of their richness
in species numbers and effectiveness in reducing blood
lipid, as an anti-oxidative, in assimilating cholesterol,
inhibiting thrombosis, dilating the coronary artery, etc.
(Deng et al. 2008; De Souza et al. 2008; Su et al. 2008;
Shao et al. 2007; Volpi and Bergonzini 2006; Zhang et al.
2007; Keith et al. 2007).
Recently, the development of flavonoids in medical
and food fields becomes a hot research. Flavonoids and
polysaccharides are the most abundant effective components in P. oleracea L. It is reported that seven kinds
of flavonoids were found in P. oleracea L., including
quercetin, kaempferol, myricetin, apigenin, luteolin,

Food Anal. Methods (2010) 3:9097

genistein, and genistin (Chen et al. 2003; Zhang et al.


2007). The extraction and separation of flavonoids with
high biological activity and high content is very important for the medical and food industry. Therefore, it is of
great significance to study the extraction and
the determination technologies of flavonoids from
P. oleracea L.
In traditional methods, the extraction of flavonoids
from P. oleracea L. is usually carried out by means of
marinated extraction and reflux extraction. Being a newly
arisen extraction method, ultrasonic extraction has also
been used for the extraction of flavonoids (Jin et al. 2008;
Chen et al. 2008a). Unfortunately, flavonoids could not be
extracted sufficiently by the mentioned methods due to the
high impurity content and the long extraction time, which
restrict the development of the medicine at a certain
extent. Therefore, it is necessary to develop a more
effective and reliable extraction method. Microwaveassisted extraction seems promising to fulfill the purpose.
Microwave-assisted extraction has the advantage of strong
penetrating force, high selectivity, high heating ability,
less solvent consumption, short extraction time, high
extraction yield (Ganzler et al. 1990), and as a new kind
of extraction technology it is especially suitable for
extracting heat-sensitivity components and natural active
constituents (Chen et al. 2008b). In this paper, pressure
self-control microwave decomposition system was used to
extract flavonoids from P. oleracea L. The temperature of
the system can be controlled at a certain value. Therefore,
it is effective to avoid the loss of the components that are
heat sensitive. In the present study, microwave-assisted
extraction, a simple and effective method, has been
developed. Meanwhile, in order to demonstrate the
advantages of microwave-assisted extraction, the extraction efficiencies of flavonoids with various methodologies
were compared.
Several methods for the determination of flavonoids
in medicinal plants have been reported, including
thin-layer chromatography, high-performance liquid
chromatography, capillary electrophoresis, and spectrophotometry. Among all these methods, only spectrophotometry can be used to determinate the total amount of
the flavonoids. UVVis spectrophotometry is the most
extensively used method to determine the total content
of flavonoids in plant. In this technology, direct
mensuration and coloration methods are both common,
but coloration method is better than direct mensuration
because coloration method will not be disturbed by
other components such as polysaccharides and saponin.
In this study, we present an easy method that uses UV
Vis spectrophotometry with chromogenic system of
NaNO2Al(NO3)3NaOH to determine the total amount
of the flavonoids from P. oleracea L.

91

Materials and Methods


Collection of Samples
In order to investigate the flavonoids content of P. oleracea
L. samples collected in different months, three different
P. oleracea L. samples were used as experimental materials.
Two of the three samples are traditional Chinese medicine
(TCM) samples purchased from Traditional Chinese Medicine Co. Ltd (Hunan, China); one of the TCM samples was
collected in August 2007 and another TCM sample was
collected in July 2007. We collected the third sample (wild
P. oleracea L.) from the suburb of Changsha (Hunan,
China) in June 2008.
Samples Preparation
Fresh samples were cleaned with water, and some of the
fresh samples were split as roots, stems, and leaves for
study. All of the samples were crushed into powder by a
plant grinder after being dried at 50 C. The powders of the
samples were kept in an airer.
Chemicals and Instruments

1. Chemicals: authentic standard rutin (>97%) was purchased from Guoyao Group of Chemical Reagents Ltd.
(China). All reagents and chemicals used in this work
were of analytical grade. Triple-distilled water was used
throughout the study.
2. Instruments: pressure self-control microwave decomposition system (MDS-2002AT, Shanghai, China),
Auto Science AS-2060B Ultrasonic Cleaner (Tianjin,
China), and Soxhlet extractor (250 mL, Changsha,
China) were used for extraction. High-speed centrifugation (TGL-16C, Shanghai, China) was employed
to accelerate the phase separation process. A
versatile plant pulverizer (FW-100, Beijing, China)
was used to make the plant materials into powder.
A rotary evaporator (RE52CS, Shanghai, China) was
used to concentrate the sample solution. UVVis
spectrophotometer V-530 (JASCO, Japan) was used
for determination.

Extraction of Flavonoids
Before extraction, P. oleracea L. was crushed into powder
by versatile plant pulverizer. The powders of the samples
were degreased by Soxhlet extractor with petroleum ether
until the color of the eluate became colorless. Flavonoids

92

were extracted from P. oleracea L. by five extraction


methods as follows:
1. Microwave-assisted extraction
Powders of the samples (0.5 g) were accurately weighed
and placed in a sealed vessel by adding 25 mL of the
ethanolwater (70:30, v/v) solvent, then the sealed vessel
was placed into the pressure self-control microwave
decomposition system for microwave-assisted extraction.
The extraction temperature was 50 C and the irradiation
time was kept at 9 min. After microwave-assisted extraction, the extract was centrifuged at 11,000 rpm for 30 min;
the supernatant was diluted to 25 mL in a volumetric flask
(25 mL) by the ethanolwater (70:30, v/v) solvent.
2. Ultrasonic Extraction
Powders of the samples (0.5 g) were accurately weighed
and placed in a sealed vessel by adding 25 mL of the
ethanolwater (70:30, v/v) solvent, then the sealed vessel
was placed in the ultrasonic cleaning bath for ultrasonic
extraction for an hour at room temperature (25 C). Then
the following works were done (as the description in
microwave-assisted extraction).
3. Condensing Reflux Extraction
Powders of the sample (0.5 g) were accurately weighed
and placed in a sealed vessel by adding 25 mL of the
ethanolwater (70:30, v/v) solvent, then the sealed vessel
was placed into the reflux device, followed by the
extraction for 2.5 h. Then the following works were done
(as the description in microwave-assisted extraction).
4. Soxhlet Extraction

Food Anal. Methods (2010) 3:9097

6 min. Secondly, 0.5 mL of the Al(NO3)3 (10%) solution


was added to the volumetric flask, shaken, and was left to
stand for 6 min. Finally, 3.0 mL of the NaOH (4.3%)
solution was added to the volumetric flask, followed by
addition of water to the scale, shaken, and left to stand for
15 min before determination. Using the sample solution
without coloration as reference solution and 500 nm as
determination wavelength, the coloration method was used
to determine the content of flavonoids in the sample by
ultravioletvisible detector.
Preparation of Standard Solution
A standard solution (0.16 mg/mL) of rutin was prepared as
follows: rutin (200 mg) was accurately weighed and
dissolved in ethanolwater (70:30, v/v) solution, and then
the solution was diluted to 100 mL in a volumetric flask
(100 mL) by ethanolwater (70:30, v/v) solvent. Two
milliliters of this solution was removed and diluted to
25 mL in a volumetric flask (25 mL) by ethanolwater
(70:30, v/v) solvent.
Standard Curve
Six portions of the rutin solution were accurately removed
(0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 mL, respectively) in six
volumetric flasks (10 mL), then the following works were
done (as the description in determination of flavonoids).
Using the concentration of rutin standard solution as
abscissa and the absorbency as y-coordinate, the linear
chart was constructed and the standard curve is shown in
Fig. 1, the regression equation was A= 0.00171 +
11.58482C (r=0.9999).

Powders of the sample (0.5 g) were accurately weighed


and placed in Soxhlet extractor by adding 80 mL of the
ethanolwater (70:30) solvent, followed by the extraction
for 5 h, and then the extract solution was concentrated.
Then the following works were done (as the description in
microwave-assisted extraction).
5. Marinated Extraction
Powders of the sample (0.5 g) were accurately weighed
and placed in a sealed vessel by adding 25 mL of the
ethanolwater (70:30, v/v) solvent, followed by the extraction for 48 h at room temperature (25 C). Then the
following works were done (as the description in
microwave-assisted extraction).
Determination of Flavonoids
Firstly, 2 mL of the sample solution was accurately
removed in a volumetric flask (10 mL) by adding 0.6 mL
of NaNO2 (5%) solution, shaken up and then standing for

Fig. 1 Standard curve

Food Anal. Methods (2010) 3:9097

93

Fig. 2 The color reaction of


flavonoids and chromogenic
system

Results and Discussion

Selection of Detective Wavelength

The Color Reaction of Flavonoids and Chromogenic


System

Ultraviolet scanning (presented in Fig. 3) was performed to


determine the detective wavelength. The solution of rutin
and the solution of sample were prepared (as the description
in determination of flavonoids). From Fig. 3, we can see
that both rutin and sample showed maximum absorption at
the wavelength of 500 nm in visible range, so the
wavelength of 500 nm can be selected for detection of
target analyte.

The basic structure of flavonoids was presented in Fig. 2a,


and most of the flavonoids in P. oleracea L. have 3,4dihydroxy-substituted structure (as shown in Fig. 2b).
Flavonoids with 3,4-dihydroxy-substituted structure can
show special color by reacting with the system of NaNO2
Al(NO3)3NaOH. The color reaction of flavonoids and
chromogenic system is presented in Fig. 2c. As shown in
Fig. 2, this method is based on the reaction of aluminum
ion with flavonoid at alkaline medium forming red chelates.
By measuring the absorption of such red chelates, it is
possible to determine the flavonoids.

Fig. 3 The UVVis spectra of rutin and sample solution

Comparison of Extraction Efficiency with Different


Solvents
The extraction of the three mentioned solvents was
investigated with microwave-assisted extraction, including

Fig. 4 Effect of the solvents on extracting efficiency

94

Food Anal. Methods (2010) 3:9097

methanolwater, ethanolwater, and acetonewater to


check their efficiency for extraction of flavonoids from P.
oleracea L., and the results are shown in Fig. 4. From
Fig. 4, we can see that extraction of flavonoids from P.
oleracea L. with ethanolwater gave the highest efficiency.
Selection of Extraction Method
Five different extraction methods were investigated, including microwave-assisted extraction, marinated extraction,
Soxhlet extraction, reflux extraction, and ultrasonic extraction, and the flavonoids of these samples were determined
by UVVis spectrophotometry. The results are shown in
Table 1.
From the results shown in Table 1, we can find that
microwave-assisted extraction had the highest extraction
efficiency. Compared with Soxhlet extraction, the extraction time of microwave-assisted extraction was shortened
by 97%, and compared with reflux extraction, 94% of
extraction time was shortened.
Microwave-assisted extraction is a rapid, efficient, and
economic method for extracting flavonoids from P. oleracea L. Unlike classical conductive heating methods,
microwave heats the whole sample simultaneously because
microwave rays are very penetrating. The microwave rays
travel through the microwave-transparent extraction medium and reach the inner vascular bundles and cell systems of
the plant material. The result is a sudden rise in temperature
inside the material; the internal pressure exceeds the
capacity of expansion of the cell walls, so the cell walls
cracked and the substances in the cells are then free to flow
out of the cells into the extract solvent. Therefore,
microwave-assisted extraction had the highest extraction
efficiency and it was most suitable for extracting flavonoids
from P. oleracea L.
Optimization of Microwave-Assisted Extraction Conditions
There are many factors affecting the extraction of flavonoids from P. oleracea L. We worked out the optimum
levels of each factor by the single factor test. However,
because of the interaction among the factors, the combina-

tion effects of the optimum levels of each factor may not be


the optimum extraction conditions. The orthogonal test is a
scientific method of arranging multi-factors. Based on a
single factor test, the optimal extraction conditions can be
worked out by orthogonal test. In this paper, the optimal
extraction conditions of microwave-assisted extraction were
worked out through single factor test and orthogonal test.
1. Single Factor Test
There are many factors affecting the extraction yield,
among which the extraction solvent, solidliquid ratio,
radiating time of microwave, and extracting temperature are
the main factors. Single factor test was performed by one
factor varied with different levels while other factors fixed.
(a) Selection of Extraction Solvent
The extraction by three mentioned solvents of different
strengths was investigated; other conditions of microwave
radiation was kept the same (ratio of material to solution
was 1:30, extracting temperature was 35 C, and extraction
time was 5 min). As shown in Fig. 4, the absorbance was
the highest when extraction solvent was ethanol of 70%
(v/v), so ethanolwater of 70% (v/v) was chosen in the
following experiments.
(b) Selection of Solidliquid Ratio
The extraction by solidliquid ratio of 1:10 (g/mL), 1:15,
1:20, 1:30, 1:40, and 1:50 was investigated, other conditions of microwave radiation was kept same (ethanol
water of 70%, extracting temperature was 35 C, and
extraction time was 5 min). As shown in Fig. 5, the
extraction efficiency increased when the ratio increased
from 1:15 to 1:40 and then tended to remain constant in the
ratio range of 1:401:50. Hence, the solidliquid ratio of
1:40 (g/mL) was chosen in the following experiments.
(c) Selection of Extraction Time
The radiating time of microwave of 5, 6, 7, 8, 9, and
10 min were investigated; other conditions of microwave
radiation was kept the same (ethanolwater of 70%, solid
liquid ratio was 1:40, and extracting temperature was

Table 1 Comparison of different extraction methods (n=5)


Extraction methods

Extraction time
(min)

Numbers of
the samples

Absorbance of
each sample

Microwave-assisted extraction
Ultrasonic extraction
Soxhlet extraction
Condensing reflux extraction
Marinated extraction

9
60
300
150
2880

5
5
5
5
5

0.390,
0.371,
0.391,
0.378,
0.313,

0.393,
0.373,
0.390,
0.374,
0.316,

0.391,
0.373,
0.389,
0.375,
0.312,

0.387,
0.376,
0.390,
0.373,
0.317,

0.388
0.373
0.388
0.374
0.313

Average extraction
efficiency (mg/g)

RSD % (n=5)

7.1
6.7
7.0
6.8
5.6

0.29
0.48
0.29
0.51
0.70

Food Anal. Methods (2010) 3:9097

95
Table 3 Results of L9 (34) orthogonal test

Fig. 5 Effect of microwave-assisted extraction conditions: solid


liquid ratio (1:10, 1:15, 1:20, 1:30, 1:40, and 1:50), radiation time (5,
6, 7, 8, 9, and 10 min), and extraction temperature (30, 35, 40, 45, 50,
and 55 C)

35 C). As shown in Fig. 5, the extraction efficiency


increased when microwave treatment time increased from 4
to 8 min and then tended to remain constant from 8 to
10 min. When other factors are fixed, the extraction
efficiency increased when extraction time increased, and
then it reached equilibrium in a certain time. Hence, the
radiating time of microwave of 8 min was chosen in the
following experiments.

Abs

1
2
3
4
5
6
7
8

60%
60%
60%
70%
70%
70%
80%
80%

1:30
1:40
1:50
1:30
1:40
1:50
1:30
1:40

7
8
9
8
9
7
9
7

40
45
50
50
40
45
45
50

0.289
0.295
0.368
0.373
0.387
0.373
0.310
0.347

9
K1
K2
K3
k1 =K1/3
k2 =K2/3

80%
0.952
1.133
0.964
0.317
0.378

1:50
0.972
1.029
1.048
0.324
0.343

8
1.009
0.975
1.065
0.336
0.325

40
0.983
0.978
1.088
0.328
0.326

0.307
T=3.049

k3 =K3/3
R

0.321
0.061

0.349
0.025

0.355
0.030

0.363
0.037

Orthogonal experiment of four factors and three levels


was adopted to optimize the microwave conditions of the

extraction. The experimental design and the results are


shown in Tables 2 and 3.
The K and R values were calculated and are listed in
Table 3. K value is the average extracting efficiency of
every factor at each level. R value is the range of K value.
Range analysis results of the orthogonal experiment
indicated that ethanol concentration is the most important
factor, followed by extraction temperature, radiating time of
microwave, and solidliquid ratio. We also can see that K2
has the highest K value in factor A, which means that,
comparing with other levels of factor A, level two has the
highest extracting efficiency and it is the most suitable level
for extraction. Based on the same principle, level three of
factor B, level three of factor C, and level three of factor D
were chosen for extraction. In other words, the maximum
yield of the active component was obtained at the following
conditions: 70% ethanol solution, 1:50 ratio of solid to
liquid, 9 min of extraction time, and 50C of extraction
temperature, respectively. In order to validate the optimum
conditions, a sample made under these optimum conditions
was determined, and the result indicated that the extraction
efficiency was higher than any group in the orthogonal
experiment table (the absorbance is 0.390, n=5).

Table 2 Factors and levels of orthogonal test

Recovery Studies

(d) Selection of Extraction Temperature


Microwave conditions of ethanolwater of 70%, solid
liquid ratio of 1:40, and the radiating time of 8 min were
fixed, and extraction temperature varied with different
levels (30, 35, 40, 45, 50, and 55 C) were investigated.
As shown in Fig. 5, the extraction efficiency was the
highest when the extraction temperature was 45 C.
However, when the temperature was higher, the extraction
efficiency decreased. It could speculate that some of the
heat-sensitive components in P. oleracea L. decomposed in
higher temperature. Therefore, the extraction temperature of
45 C was chosen as the best extraction temperature.
2. Orthogonal Design Experiment

A
B
C
D

Ethanol concentration (v/v)


The solidliquid ratio (g mL1)
Radiation time (min)
Extraction temperature (C)

60%
1:30
7
40

70%
1:40
8
45

80%
1:50
9
50

Recovery studies were performed by adding 5.0 mg of rutin


to 0.5 g of powders of the sample, determined by
ultravioletvisible detector after microwave-assisted extraction. The results are shown in Table 4. We can see from the
table that the mean recovery for flavonoids from P.
oleracea L. extracted by microwave-assisted extraction

96

Food Anal. Methods (2010) 3:9097

Table 4 The results of recovery studies


No.
1
2
3
4
5

Sample (g)

Content (mg)

Added (mg)

Found (mg)

Recovery (%)

Mean recovery (%)

RSD (%)

0.5008
0.5002
0.5007
0.5007
0.5005

4.6975
4.6919
4.6966
4.6966
4.6947

5.0
5.0
5.1
5.0
5.2

9.8791
9.8255
9.8887
9.8881
9.9516

103.6
102.7
103.8
101.8
101.1

102.6

1.13

and determined by UVVis spectrophotometry was 102.6%


(RSD=1.13%), which indicates good effectiveness of the
method.

Methodology Studies
Precision Test of the Apparatus
Apparatus precision was evaluated by the analysis of a
solution of the sample for ten consecutive times by
ultravioletvisible detector. RSD obtained was 0.22%,
which showed that the precision of the apparatus was
satisfactory.
Repeatability of Analytical Method
To assess the repeatability of the method, five solutions
made by microwave extraction were determined. RSD
obtained was 0.57%, which showed acceptable
repeatability.
Stability Test of the Solution
Stability was evaluated by the analysis a solution of the
sample extracted by microwave every 15 min. RSD
obtained was 0.32%, which indicated that the stability of
the solution had excellent stability in 2 h.

Sample Analysis
Flavonoids from different kinds of P. oleracea L. extracted
by optimized microwave-assisted extraction conditions
were determined by means of UVVis spectrophotometry,
and the results are shown in Table 5.
We could conclude from Table 5 that the extraction
efficiency of flavonoids in P. oleracea L. were different
from different batches, and the flavonoids content in
different parts of P. oleracea L. (root, stem, and leaf)
indicated that the root was rich in flavonoids, followed by
stem and leaf. A comparison of flavonoids content from
P. oleracea L. between degreased and non-degreased
samples was also investigated (seen in Table 5); from the
table, we can see that lipid material had influence on the
detection results, so the samples used were all degreased in
our study.

Concluding Remarks
In the present study, the microwave-assisted extraction has
been established to extract flavonoids from P. oleracea L.
and quantification was performed by means of UVVis
spectrophotometry. The extraction yield of flavonoids by
microwave-assisted extraction is 0.71%. It is higher than
other extractions, which confirms that microwave-assisted
extraction is very suitable for the sample preparation of

Table 5 The analytical results of flavonoids in different samples (n=5)


Samples

Degreased or not

P. oleracea L. (TCM, July)


P. oleracea L. (TCM, July)
P. oleracea L. (TCM, August)
Wild P. oleracea L. (Yuelu Mountains, Changsha)
Wild P. oleracea L. (Yuelu Mountains, Changsha)
Root of wild P. oleracea L.
Stem of wild P. oleracea L.
Leaf of wild P. oleracea L.

No
Yes
Yes
No
Yes
Yes
Yes
Yes

Flavonoids contents mg/g

RSD %

7.16
7.10
9.38
6.82
6.78
11.36
5.12
1.76

0.29
0.61
0.57
0.39
0.30
0.32
0.81
1.54

Food Anal. Methods (2010) 3:9097

flavonoids from P. oleracea L., and the analytical method


performed in this article shows it was convenient, rapid,
reliable, and useful in the determination of flavonoids.
The effective components in P. oleracea L. are complicated and varied. However, according to our knowledge,
there is no special component in P. oleracea L. that has ever
been reported. Flavonoids are major effective components
of P. oleracea L., and flavonoids from P. oleracea L. have
many special roles in medical and health function.
Therefore, the rapid and practical method established in
our work to extract and determine flavonoids from P.
oleracea L. has great significance for studying the active
ingredients of P. oleracea L. and for its quality control.

Acknowledgements This work was financially supported by the


National Natural Science Foundation of China (No. 20875025, No.
J0830415) and the Hunan Provincial Natural Science Foundation (No.
07JJ3018).

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