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Food Sci. Biotechnol. 23(3): 731-738 (2014)
DOI 10.1007/s10068-014-0099-z
Optimization of High Hydrostatic Pressure Process for the Extraction
of Kirenol from Siegesbeckia orientalis L. Using Response Surface
Methodology
Mi-Bo Kim, Ji-Eun Park, Seon Wook Woo, Sang-Bin Lim, and Jae-Kwan Hwang
Received June 4, 2013; revised November 5, 2013; accepted November 12, 2013; published online June 30, 2014
© KoSFoST and Springer 2014
Abstract High hydrostatic pressure (HHP) extraction
method was optimized to maximize the extraction yield of
kirenol from Siegesbeckia orientalis. Operating parameters
such as extraction pressure (100-600 MPa), pressure
holding time (3-20 min), feed-to-solvent ratio (1:10-1:90
(w/v)), and solvent (ethanol) concentration (0-100%) were
investigated individually by mono-factor experiments.
Then, the optimal extraction conditions were determined
using response surface methodology. Box-Behnken design
was applied to evaluate the effects of three independent
variables (extraction pressure, solvent concentration, and
feed-to-solvent ratio) on the extraction yield of kirenol
from S. orientalis. Extraction pressure, solvent concentration,
and feed-to-solvent ratio affected the extraction yields
significantly, whereas the pressure holding time had no
significant effect. The optimal processing conditions,
which gave a maximum extraction yield of 85.9% kirenol
from the raw material, were as follows: extracting pressure
320 MPa, pressure holding time 5 min, ethanol concentration
18%, and feed-to-solvent ratio 1:76 (w/v).
Keywords: high hydrostatic pressure, kirenol, Siegesbeckia
orientalis L., response surface methodology, Box-Behnken
design
Introduction
Siegesbeckia orientalis L. is an annual herb grown in Jeju
Island, Korea. The herb is also distributed widely throughout
tropical and subtropical regions of the world (1). In
folklore medicine, S. orientalis has been used as an
analgesic and anti-inflammatory agents to treat arthritis,
hypertension, malaria, neurasthenia, and snakebite (1-4).
The herb also exhibits anti-oxidative, anti-allergic, infertile,
and other bioactivities (5). Ent-kaurane and ent-pimarane
diterpenoids, as well as sesquiterpenoids, are the bioactive
compounds of S. orientalis (6). Kirenol [8(14)-pimarene-2,
15, 16, 19-tetrol] is the main ent-pimarane diterpenoid,
with its content in S. orientalis grown in China reported to
be 2.1, 0.3, and 0.8 mg/g dry weight in the leaf, root, and
stem, respectively (7). Recently, it was reported that kirenol
showed anti-photoaging effects on UVB-induced skin
damage in human skin fibroblasts and hairless mice (8).
Extraction is a critical process in the recovery and
purification of bioactive constituents from medicinal plants
(9). There are several extraction methods such as cold
extraction, heat reflux extraction, soxhlet extraction,
microwave-assisted extraction, and ultrasound-assisted
extraction, among others. These methods are largely based
on a suitable choice of solvent and input of agitation and
power to increase the mass transfer rate and the solubility
(10). Most of these methods require long extraction times
and large amounts of toxic organic solvents with relatively
low extraction yields (11,12). These methods also involve
the application of heat, which could easily reduce the
bioactivity of thermolabile ingredients (9). Therefore, the
ideal extraction process should be environment-friendly,
economical, non-destructive, and time-saving (13).
The high hydrostatic pressure (HHP) technique a cold
isostatic pressure processing method that can increase mass
Mi-Bo Kim, Jae-Kwan Hwang (�)Department of Biomaterials Science and Engineering, Yonsei University,Seoul 120-749, KoreaTel: +82-2-2123-5881; Fax: +82-2-362-7265E-mail: [email protected]
Ji-Eun Park, Seon Wook Woo, Jae-Kwan HwangDepartment of Biotechnology, College of Life Science and Biotechnology,Yonsei University, Seoul 120-749, Korea
Sang-Bin LimDepartment of Food Bioengineering, Jeju National University, Jeju 690-756, Korea
RESEARCH ARTICLE
732 Kim et al.
transfer rates and solvent permeability in the raw material,
thereby leading to higher extraction yields and significantly
shorter extraction times (within 10 min) (14-16). Due to
the nonthermal nature of this processing method, it has no
negative effects on the activity and structure of bioactive
components (13). In addition, this technique consumes
electric energy, does not cause exhaust emission, and is
recognized as an environment-friendly technology by the
United States Food and Drug Administration (U.S. FDA
2000) (6,13). The HHP extraction method had been widely
applied for the extraction of anthocyanins from grape skin
(14), polyphenols from green tea (12), flavonoids from
propolis (17), and ginsenosides from panax ginseng (18).
Kirenol, a major bioactive compound in S. orientalis,
has been extracted by a conventional extraction method at
room temperature using ethanol (6), a percolation extraction
method using methanol (5), and a reflux extraction method
using methanol (7). However, the HHP extraction method
has not yet been applied for extracting kirenol from S.
orientalis. The objectives of this study were to investigate
the individual operating parameters such as extraction
pressure, pressure holding time, feed-to-solvent ratio, and
solvent concentration by mono-factor experiments and to
optimize an HHP extraction process of kirenol from S.
orientalis by response surface methodology.
Materials and Methods
Plant material Siegesbeckia orientalis L. was obtained
from Radian Inc. (Chuncheon, Korea). A voucher specimen
was deposited in the Department of Biotechnology at
Yonsei University (Seoul, Korea). Dried aerial parts of S.
orientalis were ground and sieved (40-60 mesh) to produce
samples for the extraction. The samples were stored at 4oC
prior to use.
High hydrostatic pressure extraction Extraction of
kirenol from S. orientalis was performed using a high
hydrostatic pressure food processor (Frescal MFP-7000;
Mitsubishi Heavy Industries, Tokyo, Japan). One gram of
the dried sample was mixed with a given extraction solvent.
The mixture was poured into the polyethylene (PE) bag
and the bag was machine-sealed (Sears Roebuck and Co.,
Buffalo, NY, USA). The pressure transfer medium was
water and a hydraulic pump produced the pressure. The
mixture of the sample and solvent was treated under a
given pressure for a period of time at room temperature.
After that, the pressure was released and the extract was
separated from the mixture by filtering with No. 5A filter
paper (Advantec; Toyo Roshi Kaisha, Ltd., Japan). The
filtrate was evaporated in a rotary vacuum evaporator at
40oC and the residue was dissolved in 20 mL of the
extraction solvent and filtered through a 0.45 µm cellulose
acetate filter (Advantec). The extract was stored at 4oC for
further analysis.
The extraction yield of kirenol was calculated as the
weight (mg) of kirenol in the extract compared to that in
the dried raw material, expressed as a percentage, as shown
in the equation.
Extraction yield (%)
Weight (mg) of kirenol in the extract=
Weight (mg) of kirenol in the dried raw material ×100
The content of kirenol in the dried raw material was
determined by extracting the dried sample (1 g) with 50
mL of 60% ethanol in water for 24 h three times at room
temperature.
Mono-factor experiments The major factors that may
influence HHP extraction are the extraction pressure,
pressure holding time, feed-to-solvent ratio, and solvent
concentration. The effects of individual factors on the
extraction yield of kirenol from S. orientalis were
evaluated by mono-factor experiments. The parameters and
their ranges were as follows: pressure (100-600 MPa),
pressure holding time (3-20 min), feed-to-solvent ratio
[1:10-1:90 (w/v)], and solvent (ethanol) concentration in
water (0-100%).
Response surface methodology (RSM) RSM was used
to optimize the extraction parameters such as extraction
pressure, solvent concentration, and feed-to-solvent ratio
for the HHP extraction of kirenol from S. orientalis. The
optimum extraction conditions were determined using the
experimental design called Box-Behnken design (BBD)
(19). BBD is an experimental design for fitting the second-
order response surface based on the structure of balanced
incomplete block designs (20-22).
For the experimental plan of three independent variables
and three levels, the extraction pressure (X1), solvent
concentration (X2), and feed-to-solvent ratio (X3) were
encoded, while the dependent variable was the extraction
yield of kirenol. The symbols and levels are shown in
Table 1. A model equation by the regression analysis was
predicted by using SAS program (SAS version 9.3; SAS
Institute Inc. Cary, NC, USA).
Determination of kirenol by HPLC The quantity of
kirenol in the extract was determined using HPLC with
slight modification, as described by Song et al. (23). The
chromatographic analyses were performed using Agilent
1200 series HPLC system (Agilent Technologies, Palo
Alto, CA, USA) consisting of a solvent gradient delivery
pump (G1311A), autosampler (G1329A), and a diode-
array detector (G1315D). Separation was performed on a
HHP Extraction of Kirenol from S. orientalis 733
Waters XTerra C18 column (250 mm×4.6 mm, 5 µm;
Milford, MA, USA). The mobile phase consisted of 0.1%
phosphoric acid (A) and acetonitrile (B) with a flow rate of
1 mL/min, and was programmed as follows: 0-15 min, 15-
25% A; 15-30 min, 25-28% A; 30-35 min, 28-15% A. The
detection wavelength was 215 nm. The injection volume
was 20 µL. The concentration of kirenol in the extract was
calculated based on the standard curve using a pure kirenol
(Institute for Korea Traditional Medical Industry, Daegu,
Korea). The stock solution of kirenol was prepared with
methanol at a concentration of 600 ppm and stored at 4oC.
A series of standard solutions of kirenol were prepared
over a range of 50-200 ppm by diluting the stock solution
in methanol.
Statistical analysis Statistical analyses were performed
using SPSS version 18.0 software (SPSS Inc., Chicago, IL,
USA). Significant differences (p<0.05) among treatment
means were determined by Duncan’s test.
Results and Discussion
Mono-factor experiments
Extraction temperature: The major advantage of the HHP
technique is in the nonthermal process as compared to
conventional extraction methods (13). Conventional extraction
methods involve the application of heat, which could easily
reduce the bioactivity of thermolabile ingredients (9). Our
preliminary experiments showed that the extraction yield
of kirenol was decreased by increasing the extraction
temperature from room temperature to 50oC (data not
shown). Thus, in our study we didn’t consider the extraction
temperature as an important parameter for the extraction
yield of kirenol by HHP process.
Extraction pressure: Extraction pressure is a significant
factor in the HHP extraction process because it can
increase the mass transfer rate by enhancing the solvent
penetration into the solid material and the release of intra
cellular product by disrupting cell walls (9,12).
In order to investigate the effect of different extraction
pressures on the extraction yield of kirenol from S.
orientalis, the HHP extraction was performed at five
pressures (100, 200, 300, 400, 500, and 600 MPa) for 5
min with 60% ethanol and a feed-to-solvent ratio of 1:50
(w/v) (Fig. 1A). When the extraction pressure was 100,
200, 300, 400, 500, and 600 MPa, the extraction yield of
kirenol was 71.4, 72.4, 74.4, 76.9, 76.7, and 70.8%,
respectively. The extraction yield of kirenol was increased
slightly with increasing pressure from 100 MPa to 500
MPa. However, the difference in the extraction yield was
not significant at the 5% level by Duncan’s multiple test.
Generally, as the pressure increases, the solubility of
bioactive components is improved (13). However, in this
study the extraction yield was not increased. This finding
may have been due to the fact that the extraction pressure
was not the only factor affecting the extraction of kirenol
from S. orientalis. Other factors such as solvent type and
feed-to-solvent ratio may greatly affect the extraction yield
of kirenol. Prasad et al. (10) also reported that when the
extraction pressure increased from 200 MPa to 500 MPa,
the extraction yield of phenolic compounds from Litchi
chinensis Sonn. increased slightly from 28.0 to 29.8%.
Considering the higher cost and lower safety of the higher
pressure equipment, together with the small difference in
the extraction yields, the pressure of 300 MPa was chosen
for further experiments.
Pressure holding time The effect of pressure holding
time on the extraction yield of kirenol from S. orientalis
was investigated with five holding times (3, 5, 10, 15, and
20 min) at 300 MPa with 60% ethanol and a feed-to-
solvent ratio 1:50 (w/v) (Fig. 1B). The extraction yields of
Fig. 1. The effect of the extraction pressure (A) and pressureholding time (B) on the extraction yield of kirenol fromSiegesbeckia orientalis by high hydrostatic pressure. For eachtreatment, means followed by the same letters are not significantlydifferent at the 5% level by Duncan’s multiple test.
734 Kim et al.
kirenol were 72.8, 74.0, 69.2, 71.3, and 77.3% at pressure
holding times of 3, 5, 10, 15, and 20 min, respectively, and
did not change significantly with the increase of pressure
holding time at the 5% level by Duncan’s multiple test.
Therefore, 3 min was considered sufficient for the extraction
of kirenol from S. orientalis by HHP, a lower time
requirement than was demanded by other extraction
techniques.
Under the high-pressure condition, the diffusion rate of
the solvent is very high, and an equilibrium in the intra-
and extra-cellular solvent concentration can occur in a very
short period of time, thereby allowing full contact of the
bioactive components with the extracting solvent (13).
Thus, the extraction could be accomplished completely in
a shorter amount of time (10,11,13). Similar results were
reported by Prasad et al. (10) and Zhang et al. (17) in the
extraction of flavonoids from lychee and propolis,
respectively.
Feed-to-solvent ratio The contact of the extracting
solvent and the sample matrix is a physical process. With
a higher feed-to-solvent ratio, there should be more
opportunities for the desired bioactive compound to make
contact with, and permeate into, the extracting solvent
(12,18,24).
The effect of the feed-to-solvent ratio on the extraction
yield of kirenol from S. orientalis was evaluated in the
range of 1:10-1:90 (w/v) at 300 MPa for 5 min with 60%
ethanol as an extraction solvent (Fig. 2A). When the feed-
to-solvent ratio was 1:10, 1:30, 1:50, 1:70, and 1:90 (w/v),
the extraction yield of kirenol was 47.1, 63.6, 73.9, 76.6,
and 79.1%, respectively. The extraction yield of kirenol
increased with increasing quantity of the extraction solvent.
When the feed-to-solvent ratio increased from 1:10 to 1:50
(w/v), the extraction yield of kirenol increased from 47.1 to
73.9%. This finding is consistent with another report that
revealed that when the amount of the extraction solvent
increased, the chance of the bioactive components coming
into contact with the extracting solvent was increased and
resulted in higher extraction rates (13). However, when the
feed-to-solvent ratio increased from 1:50 to 1:90 (w/v), the
extraction yield of kirenol was not significantly different at
the 5% level by Duncan’s multiple test.
Meanwhile, higher solvent ratio means that more solvent
would be consumed, which makes it more difficult to
evaporate the solvent. Zhang et al. (18) suggested that the
feed-to-solvent ratio should be limited to below 1:70 (w/v).
Therefore, taking the extraction yield, the solvent, and
processing costs into consideration, the feed-to-solvent
ratio was chosen as 1:50 (w/v) for further experiments.
Solvent concentration The extraction yield of bioactive
compounds depends on the difference in polarity of the
extraction solvents, the solubility of the components in the
solvent, and the rate of mass transfer (12). Methanol is a
popular solvent in the extraction of bioactive compounds.
However, when the extract is used for medicinal or
ingestion purposes, pure ethanol or a mixture of ethanol
and water has typically been used due to the toxicity of
methanol (11).
Kirenol is a strong polar compound that has polyhydroxyl
functional groups and can be easily dissolved in water and
ethanol solutions (25). The effect of the ethanol concentration
in water on the extraction yield of kirenol from S. orientalis
was investigated with five ethanol concentrations (0, 20,
40, 60, 80, and 100%) at 300 MPa for 5 min with a feed-
to-solvent ratio of 1:50 (w/v) (Fig. 2B). The extraction
yields of kirenol were 71.7, 78.4, 77.9, 73.9, 53.4, and
5.3% at ethanol concentrations in water of 0, 20, 40, 60,
80, and 100%, respectively. The extraction yield was 13.5
times higher with only water than with only ethanol. The
yield decreased significantly from 73.9 to 5.3% with
increases in ethanol concentration from 60 to 100%.
Fig. 2. The effect of the feed-to-solvent ratio (A) and ethanolconcentration in water (B) on the extraction yield of kirenolfrom S. orientalis by high hydrostatic pressure. For eachtreatment, means followed by the same letters are not significantlydifferent at the 5% level by Duncan’s multiple test.
HHP Extraction of Kirenol from S. orientalis 735
However, it stayed the same when the ethanol concentration
was lower than 60%.
Prasad et al. (10) recommended that a moderate ethanol
concentration be used in extraction solvents because
different ethanol concentrations contribute to the medium
polarity and polarity of bioactive compounds such as
flavonoids, glycosides, and phenolic acids. Jayaprakasha et
al. (26) also reported that 40-50% ethanol had a greater
effectiveness in extracting bioactive compounds from
natural plants compared to pure ethanol, and that high
extraction yields of flavonoids were obtained from Salvia
officinal at 55-75% ethanol (27) and from propolis at 75%
ethanol (17).
Optimization of HHP extraction using Box-Behnken
design regression modeling Based on the results of
mono-factor experiments, the extraction pressure, the
solvent concentration, and the feed-to-solvent ratio were
the major factors, while the pressure holding time was the
minor factor for HHP extraction of kirenol from S.
orientalis. Therefore, optimization of HHP extraction was
performed with the extraction pressure (X1), solvent
concentration (X2), and feed-to-solvent ratio (X3) as the
independent variables at a fixed pressure with a holding
time of 5 min. A Box-Behnken design with three factors
and three levels consisted of 12 different levels of the
independent variables (points 1 to 12), and three central
point (13 to 15) runs used to fit a second-order response
surface and give a measure of process stability and inherent
variability which was used to analyze the experimental
error (22,27).
The 15 experiments were conducted and the measured
response was defined as the extraction yield of kirenol
from S. orientalis (Table 1). Multiple regression modeling
provides a mathematical relationship between independent
variables and dependent responses. The second-order
polynomial fitted was:
Y=β0+β1X1+β2X2+β3X3+β12X1X2+β13X1X3+β23X2X3
+β11X12+β22X2
2+β33X32
where Y represents the experimental response, β are fixed
constant and regression coefficients of the model, and X
are independent variables in coded value. The whole model
includes linear, quadratic, and cross-product terms (22).
The optimum of the analysis and the regression equation
of variance are presented in Table 2. The coefficient of
determination indicated that the model adequately represents
the real relationship between the parameters chosen (22).
Thus, a high value of R2 (0.9910) reflects good relevance of
the dependent variables in the model, which can fit well
Table 1. Box-Behnken design and the response for the extraction yield of kirenol from Siegesbeckia orientalis by high hydrostaticpressure
Run No.
Coded Uncoded Kirenol
X1 X2 X3Extraction pressure
(MPa)Ethanol conc.
(%)Feed-to- solvent
ratio (w/v)Concentration
(ppm) Extraction yield
(%)
1 -1 -1 0 100 0 1:50 11.6 68.4
2 -1 +1 0 100 80 1:50 9.8 37.4
3 +1 -1 0 500 0 1:50 11.4 42.8
4 +1 +1 0 500 80 1:50 12.9 72.3
5 -1 0 -1 100 40 1:10 11.7 45.8
6 -1 0 +1 100 40 1:90 12.6 78.8
7 +1 0 -1 500 40 1:10 10.2 30.0
8 +1 0 +1 500 40 1:90 11.0 47.6
9 0 -1 -1 300 0 1:10 11.9 70.0
10 0 -1 +1 300 0 1:90 10.3 40.9
11 0 +1 -1 300 80 1:10 12.7 41.8
12 0 +1 +1 300 80 1:90 13.2 73.7
13 0 0 0 300 40 1:50 15.8 76.4
14 0 0 0 300 40 1:50 16.1 80.8
15 0 0 0 300 40 1:50 14.3 76.6
Table 2. Second-order polynomials for high hydrostatic pressure extraction of kirenol from S. orientalis
Response Second-order polynomials1) R2 Regression
(p-value)
Extraction yield of kirenol (%)
Y=17.71231+0.12401 X1+0.49497 X2+1.15882 X3-0.00021 X12-
0.00915 X22-0.00775 X3
3+0.00009 X1X2+0.00019 X1X3-0.00258X2X30.9910 0.0001
1)X1,extraction pressure (MPa); X2, ethanol concentration (%); X3, feed-to-solvent ratio (w/v)
736 Kim et al.
with the actual data. Furthermore, the result of the error
analysis showed a high significant multiple regression
relationship (p<0.05). Therefore, the response is sufficiently
explained by the regression equation, and it is feasible,
using this regression model, to forecast the effects of
various extracting parameters on the extraction yield of
kirenol from S. orientalis by HHP within the experimental
model.
Analysis of the response surface The three dimensional
response surface plots can be illustrated by reporting the
response in the function of two factors and keeping the
other constant at a medium level (22). It was visualized by
the extraction yield of kirenol from S. orientalis in relation
to the extraction pressure, solvent concentration, and feed-
to-solvent ratio. The medium levels of the extraction
pressure, solvent concentration, and feed-to-solvent ratio
Fig. 3. Response surface plots for the effect of the extraction pressure (X1), ethanol concentration in water (X2), and feed-to-solvent ratio (X3) on the extraction yield of kirenol (Y) from S. orientalis by high hydrostatic pressure.
HHP Extraction of Kirenol from S. orientalis 737
were 300 MPa, 40% ethanol, and 1:50 (w/v), respectively.
Figure 3A shows the response surface plot of the extraction
yield of kirenol from S. orientalis with different extraction
pressures and solvent concentrations in water at the
medium level of the feed-to-solvent ratio [1:50 (w/v)]. The
extraction yield of kirenol increased significantly with the
decrease of ethanol concentration at constant extraction
pressure. In contrast, it was increased slightly with increasing
extraction pressure at constant solvent concentration.
However, the interaction between the extraction pressure
and the solvent concentration had no significant effect on
the extraction yield of kirenol from S. orientalis by HHP.
Figure 3B shows the response surface plot of the
extraction yield of kirenol as related to the extraction
pressure and the feed-to-solvent ratio at the medium level
of ethanol concentration in water (40%). The extraction
yield of kirenol increased significantly with increasing
feed-to-solvent ratio, particularly at lower extraction
pressure. However, at higher extraction pressure, it was
increased gradually with increasing feed-to-solvent ratio.
The interaction between the extraction pressure and the
feed-to-solvent ratio was also not significant in the
extraction yield of kirenol from S. orientalis by HHP.
Figure 3C shows the response surface plot of the
extraction yield of kirenol with different feed-to-solvent
ratios and solvent concentration in water and at the
medium level of the extraction pressure (300 MPa). The
extraction yield of kirenol increased significantly with
increasing feed-to-solvent ratio, particularly at lower ethanol
concentration. But at higher ethanol concentration, it
increased gradually with increasing feed-to-solvent ratio.
The interaction between the ethanol concentration and the
feed-to-solvent ratio had a positively synergistic effect on
the extraction yield of kirenol from S. orientalis by HHP.
These results indicate that the solvent concentration and
the feed-to-solvent ratio at the medium extraction pressure
had significant effects on the extraction yield of kirenol
from S. orientalis.
Optimal processing conditions and adequacy of the
model equation As demonstrated in Table 3, optimal
processing conditions for the extraction of kirenol from S.
orientalis by HHP were determined from the second-order
polynomial regression as follows: extracting pressure of
320 MPa, ethanol concentration of 18%, and feed-to-
solvent ratio of 1:76 (w/v). Under these conditions, the
predicted value of the extraction yield of kirenol from S.
orientalis was 85.9%. The ridge analysis was performed
and produced a locus of points, each of which was a point
of maximum response.
Adequacy of the model equation for predicting the
response value was tested using at the optimized extraction
condition of the extraction pressure (320 MPa), ethanol
concentration (18%), and feed-to-solvent ratio 1:76 (w/v).
Predicted and experimental values for the extraction yield
of kirenol from S. orientalis are shown in Table 3. The
experimental value was in fairly good agreement with the
predicted value using the model equations by RSM at the
5% level by Duncan’s multiple test. Therefore, the model
appears to be a good predictor of the response.
Taken together, HHP extraction can be a valuable
alternative technology to the traditional extraction techniques
for extracting kirenol from S. orientalis. In this research,
we found that the HHP extraction method resulted in a
high extraction yield using less solvent, an extremely short
processing time, and moderate pressure at room temperature.
BBD and RSM were effectively employed to estimate the
effects of the extraction pressure, solvent concentration,
feed-to-solvent ratio, and their interaction, as well as to
determine the optimal processing conditions.
Acknowledgments This work was supported by the
Ministry for Food, Agriculture, Forestry and Fisheries,
Republic of Korea (Industrialization Support Program for
Bio-technology of Agriculture and Forestry).
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738 Kim et al.
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