Industrial Crops and Products 49 (2013) 837–843

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Optimization of ultrasonic extraction of phenolic compounds from

Euryale ferox seed shells using response surface methodology
Yong Liu a,∗ , Shoulian Wei a , Miaochan Liao b
a
School of Chemistry and Chemical Engineering, Zhaoqing University, Zhaoqing 526061, PR China
b
Department of Logistics Management, Zhaoqing University, Zhaoqing 526061, PR China

a r t i c l e i n f o a b s t r a c t

Article history: The ultrasound-assisted extraction of phenolic compounds from Euryale ferox seed shells was modeled
Received 18 April 2013 using response surface methodology. A three-level-three-factor Box–Behnken design was employed to
Received in revised form 4 July 2013 optimize three extraction variables, including extraction time (X1 ), ethanol concentration (X2 ), and ratio
Accepted 10 July 2013
of aqueous ethanol to raw material (X3 ), for the achievement of high extraction yield of the phenolic
compounds. The statistical analyses show that the independent variables (X1 , X2 ), the quadratic terms
Keywords:
(X12 , X22 and X32 ), and the interactions of X1 with X2 and X3 have significant effect on the yield (p < 0.01).
Euryale ferox
The optimized conditions are X1 of 21 min, X2 of 52%, and X3 of 31 mL/g. Under these conditions, the
Phenolic compounds
Response surface methodology
experimental yield is 15.69 ± 0.082% (n = 3), which is well matched with the predicted yield of 15.70%.
Antioxidant activity The evaluation of antioxidant activity by DPPH assay indicates that the phenolic compounds from E.
Ultrasonic extraction ferox seed shells possess significant antioxidant activity. HPLC analysis reveals that pyrogallol, gallic acid,
chlorogenic acid and rutin are the major composition in the extracts.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Euryale ferox Salisb. (Nymphaeaceae), an aquatic plant, is the
only species of the genus Euryale native to eastern Asia (Song et al.,
2011). In China, the seeds of E. ferox, locally known as Qianshi, are
a traditional Chinese medicine and nourishing ingredients to treat
some diseases, such as kidney problems, chronic diarrhea, exces-
sive leucorrhea, and hypofunction of the spleen (Han et al., 2012). E.
ferox seed shells (Fig. 1), by-products of the seeds, account approx-
imately 40% of the seed quality, and its annual outputs are over
10,000 tons. The shells are rich in polyphenols and are an ideal raw
material for their extraction and recovery (Zhang et al., 2012). How-
ever, the shells are usually used as a fuel for heating and cooking, or
discarded as waste, resulting in resources loss and environmental
pollution. Currently, researches on E. ferox are few and are mainly
focused on nutritional analysis (Jha et al., 1991), physiological activ-
ity (Lee et al., 2002; Shankar et al., 2010), and processing (Jha and
Prasad, 1996), etc. Moreover, there are few studies on utilization of
these shells. Therefore, reuse of the shells is urgently required to
develop value-added products.
Phenolic compounds are plant secondary metabolites, which are
important determinants in the sensory and nutritional quality of
∗ Corresponding author at: School of Chemistry and Chemical Engineering, Zhao-
qing University, Zhaoqing 526061, PR China. Tel.: +86 758 2716357.
E-mail address: lygdut@163.com (Y. Liu).
fruits, vegetables and other plants (Ignat et al., 2011). Phenolic com-
pounds have a variety of physiological activity, such as antioxidant,
antimutagenic, antiallergenic, antiinflammatory, and antimicrobial
effects (Martins et al., 2011), and thus are now widely used in
the fields of biology, medicine, food, and so on. Currently, many
synthetic antioxidants, such as butylated hydroxyanisole (BHA),
t-butylhydroxyquinone (TBHQ), butylated hydroxytoluene (BHT),
and propyl gallate (PG) have been used to retard the oxidation
process, particularly in food systems (Maqsood et al., 2013). How-
ever, applications of synthetic antioxidants in food products are
under strict regulation due to the potential health hazards (Park
et al., 2001). Consequently, development and utilization of natural
antioxidants as alternatives to synthetic ones have attracted global
interest among researchers.
Ultrasonic-assisted extraction (UAE) is one of the most inex-
pensive, rapid, simple and efficient techniques compared with
conventional extraction (Chen et al., 2012), and has been applied to
extract bioactive compounds from different materials owing to its
high reproducibility at shorter time, simplified manipulation, sig-
nificant reduction in solvent consumption and temperature, and
lower energy input (Fan et al., 2012; Yan et al., 2011). Therefore,
the ultrasound technology has been used in some industries, such
as food industry, chemical industry, and material industry (Leonelli
and Mason, 2010).
Response surface methodology (RSM), an effective statistical
technique for modeling and optimization of complex processes, has
been used increasingly to optimize processing parameters owing to

0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.07.023
838 Y. Liu et al. / Industrial Crops and Products 49 (2013) 837–843
Fig. 1. Image of Euryale ferox seed shells.
more efficient and easier arrangement and interpretation of exper-
iments compared to others (Sheng et al., 2013; Stroescu et al.,
2013; Yan et al., 2011). The advantage of RSM is the reduced num-
ber of experimental trials needed to evaluate multiple parameters
and their interactions. Therefore, it widely used in optimizing the
extraction parameters, such as polysaccharides (Zhu and Liu, 2013),
anthocyanins (Pinho et al., 2011), phenolic compounds (Wang et al.,
2013), and protein (Li and Fu, 2005) from different materials.
In this work, the main objective was to investigate the extraction
variables (extraction time, ethanol concentration, and ratio of aque-
ous ethanol to raw material), and optimize these variables values
by RSM for the phenolic compounds recovery yield maximization
from E. ferox seed shells. Moreover, the antioxidant activity of the
phenolic compounds was evaluated by DPPH assay and the major
composition was analyzed by HPLC.
2. Experimental
2.1. Materials and chemicals
E. ferox seed shells were obtained from a farm in Zhaoqing
City (China). Folin–Ciocalteu phenol reagent, sodium carbonate,
2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox, pyrogallol, rutin,
chlorogenic acid, catechin, epicatechin, gallic acid and vitamin
C (Vc ) were from Aladdin (Shanghai, China). Methanol was
HPLC grade, other reagents were analytical grade and used as
received.
2.2. Extraction of phenolic compounds
The process of phenolic compounds extraction from E. ferox
seed shells by ultrasonic-assisted treatment was performed in an
ultrasonic generator (500 W, 53 kHz, SK8200H, Kedao, China). The
dry E. ferox seed shells were powdered by a pulverizer (XS-10B,
Longxin, China) and then passed through an 80 mesh sieve. One
gram of the shell powders was used for each case in a beaker.
The beaker was held in the ultrasonic generator and exposed to
extract phenolic compounds for different extraction time at various
ethanol concentrations in different ratios of aqueous ethanol to raw
material.
2.3. Determination of total phenolic yield
After ultrasonic treatment, the extracted slurry was centrifuged
at 4000 rpm for 15 min to collect the supernatant. The total phenolic
content in the supernatant was determined using Folin–Ciocalteu
method (Santos et al., 2010) with a slight modification. 2.5 mL
of tenfold diluted Folin–Ciocalteu reagent and 2 mL of aqueous
sodium carbonate (75 g/L) were added to 0.5 mL of the supernatant.
The mixture was kept for 5 min in a water bath at 50 ◦ C and then
cooled to room temperature. The absorbance was measured at
760 nm using a UV–vis spectrophotometer (752, Jinghua, China).
The total phenolic content was calculated as gallic acid equiva-
lent from the calibration curve of gallic acid standard solutions
(0–20 mg/L). The percentage phenolic yield is calculated as follows:
weight of extract phenolics (g)
Extraction yield (%) = × 100 (1)
weight of dry shell powders (g)
2.4. DPPH assay
The antioxidant activity of the phenolic compounds from E. ferox
seed shells was measured by DPPH assay (Li et al., 2012) slightly
modified. Briefly, 0.1 mmol/L solution of DPPH in ethanol was pre-
pared and 1 mL of this solution was added 3 mL of sample solution
at different concentrations. The mixture was vortexed thoroughly
and incubated in the dark at room temperature for 30 min, and then
the absorbance was measured at 517 nm in a UV–vis spectropho-
tometer (752, Jinghua, China). Vc was used as positive control. The
radical scavenging effect is calculated using the following equation:
Acontrol − Asample
Scavenging effect (%) = × 100 (2)
Acontrol
where Acontrol is the absorbance value of control solution without
sample, and Asample is the absorbance to sample solution.
2.5. HPLC analysis
The extract phenolics were analyzed using a HPLC (Agilent
1200 series, Agilent Technologies, USA) equipped with a UV detec-
tor (G1314B, Agilent) and an Eclipse XDB-C18 column (5 ␮m,
250 mm × 4.6 mm, Agilent). Ultrapure water was used as solvent
A and 100% methanol as solvent B (A:B = 7:3). The solutions of
the standards and the extract phenolics were filtered through a
0.45 ␮m syringe filter. The operating conditions were: column tem-
perature, 25 ◦ C; injection volume, 20 ␮L; detection wavelength,
280 nm; flow rate, 1.0 mL/min. The identification and peak assign-
ment of the phenolics was based on comparison of retention times
and spectral data with those of the standards. The identified phe-
nolics were quantified according to respective standard calibration
curves.
2.6. Experimental design
A three-level-three-factor, Box–Behnken design (BBD) was
employed to determine the best combination of extraction vari-
ables for the phenolic compounds based on the results of
preliminary single-factor-test. Extraction time (X1 ), ethanol con-
centration (X2 ), and ratio of aqueous ethanol to raw material (X3 )
were the independent variables, and their coded and uncoded
levels were presented in Table 1. Extraction yield (Y) taken as
the response for the design experiment was given in Table 2.
 Y. Liu et al. / Industrial Crops and Products 49 (2013) 837–843 839

Table 1
Independent variables and their levels for Box–Behnken design.

Independent variables Levels

−1 0 1

Extraction time (X1 ) (min) 15 20 25

Ethanol concentration (X2 ) (%, v/v) 40 50 60
Ratio of aqueous ethanol to raw material (X3 ) (mL/g−1 ) 20:1 30:1 40:1

Experimental data were fitted to a quadratic polynomial model and

the model was explained by the following quadratic equation:


3 
3 
2 
3

Y = A0 + Ai Xi + Aii Xi2 + Aij Xi Xj (3)

i=1 i=1 i=1 j=i+1

where Y is the dependent variable; A0 , Ai , Aii , and Aij are the regres-
sion coefficients for intercept, linearity, square and interaction,
respectively; Xi and Xj are the independent variables.

2.7. Statistical analysis

All the data were determined in triplicate and the results were
averaged. SPSS software version 18 was used to evaluate the DPPH
assay. Design Expert software version 8.0.6 (Stat-Ease, Minneapo-
lis) was employed for the regression analysis and the optimization.

3. Results and discussion Fig. 2. Effect of different extraction variables on extraction yield.

3.1. Effect of extraction time on extraction yield of phenolic

compounds
3.2. Effect of ethanol concentration on extraction yield of
phenolic compounds
Extraction process was carried out using extraction time from 10
to 30 min, while other parameters were as following: ethanol con-
Extraction process was carried out at different ethanol concen-
centration 60% (v/v) and ratio of aqueous ethanol to raw material
tration of 30%, 40%, 50%, 60% and 70%, while other parameters
20:1 mL/g. The effect of extraction time on extraction yield of phe-
were as following: extraction time 20 min and ratio of aqueous
nolic compounds from E. ferox seed shells is shown in Fig. 2A. When
ethanol to raw material 20:1 mL/g. The effect of ethanol concentra-
extraction time increases, the variance of extraction yield is rela-
tion on extraction yield of phenolic compounds is shown in Fig. 2B.
tively rapid and reaches a maximum at 20 min, and then decreases
The variance of extraction yield increases first and then decreases
as the extraction proceeds, possibly due to the structural destruc-
with the increase of ethanol concentration, and peaks at 50%. It
tion and the decomposition of polyphenols during the prolonged
is reported that water is acted as the plant swelling agent, while
extraction time (Carrera et al., 2012; Sun et al., 2011; Makris et al.,
ethanol is believed to disrupt the bonding between the solutes and
2007). Therefore, 20 min is favorable for extracting the phenolic
plant matrices (Şahin and Şamlı, 2013). Moreover, water has a high
compounds.
dielectric constant, which leads to different ethanol concentrations
with different polarities (Spigno and De Faveri, 2009). Therefore,
the results may be related to the solvent polarity and the solubility
Table 2
Box–Behnken design for independent variables and their extraction yield. of polyphenols in E. ferox seed shells, and the ethanol concentration
of 50% is good for extracting the phenolic compounds.
Run X1 (extraction X2 (ethanol X3 (ratio of aqueous Extraction
time, min) concentra- ethanol to raw yield (%)
tion, material, mL/g−1 ) 3.3. Effect of ratio of aqueous ethanol to raw material on
%) extraction yield of phenolic compounds
1 −1 0 1 10.61
2 1 −1 0 13.97 Extraction process was carried out using ratio of aqueous
3 0 0 0 15.56 ethanol to raw material in the range of 10:1 to 50:1 mL/g, while
4 0 −1 −1 12.43
extraction time and ethanol concentration were fixed at 20 min
5 −1 −1 0 11.89
6 0 0 0 15.60 and 60%, respectively. The effect of ratio of aqueous ethanol to
7 1 0 1 13.95 raw material on extraction yield of phenolic compounds is shown
8 0 1 −1 13.34 in Fig. 2C. As ratio of aqueous ethanol to raw material increases,
9 1 1 0 13.51 the extraction yield slowly increases first and a maximum yield
10 1 0 −1 11.87
11 −1 1 0 13.69
achieved at 30:1 mL/g, and then slightly decreases after the ratio
12 0 −1 1 12.71 of aqueous ethanol to raw material exceeds 30:1 mL/g. This phe-
13 −1 0 −1 12.48 nomenon may be attributed to the mass transfer principle and the
14 0 0 0 15.69 distribution of ultrasonic energy density in the extraction solutions
15 0 1 1 13.49
(Şahin and Şamlı, 2013; Zeng et al., 2013). Lower ratio of aqueous
840 Y. Liu et al. / Industrial Crops and Products 49 (2013) 837–843

Table 3
Analysis of variance for fitted quadratic model of extraction of phenolic compounds.

Source Sum of squares Degree of freedom Mean square F-value p-Value (Prob. > F)

Model 30.03 9 3.34 143.42 <0.0001

Residual 0.12 5 0.023
Lack of fit 0.11 3 0.036 8.08 0.1121
Pure error 8.87 × 10−3 2 4.43 × 10−3

Cor. total 30.15 14

R2 = 0.9961; Radj
2
= 0.9892; Rpred
2
= 0.9423; C.V.% = 1.14; adequate precision = 39.145.

Table 4
Regression coefficients estimate and their significance test for quadratic model.

Source Sum of squares Degree of freedom Mean square F-value p-Value (Prob. > F)

X1 2.68 1 2.68 115.16 0.0001

X2 1.15 1 1.15 49.32 0.0009
X3 0.051 1 0.051 2.2 0.1981
X12 8.97 1 8.97 385.35 <0.0001
X22 2.32 1 2.32 99.87 0.0002
X32 12.38 1 12.38 531.9 <0.0001
X1 X2 1.28 1 1.28 54.88 0.0007
X1 X3 3.9 1 3.9 167.64 <0.0001
X2 X3 4.23 × 10−3 1 4.23 × 10−3 0.18 0.6877

ethanol to raw material has higher concentration gradient, lead-
ing to higher diffusion and extraction yield. But when the ratio
is over 30:1 mL/g, the decrease of the distribution of ultrasonic
energy density in the extraction solutions is dominant, and has
a negative effect on the extraction yield. The similar tendency of
this parameter was also acquire for the polysaccharides extraction
from Acanthopanax senticosus (Zhao et al., 2013) and Boletus edulis
mycelia (Chen et al., 2012). Therefore, the ratio of aqueous ethanol
to raw material of 30:1 mL/g is sufficient for extracting the phenolic
compounds.
3.4. Optimization of extraction parameters for phenolic
compounds
Table 2 shows the process variables and experimental data of 15
runs containing 3 replicates at center point. By applying multiple
regression analysis on the experimental data, the model for the
response variable could be expressed by the following quadratic
polynomial equation in the form of coded values:
Y = 15.62 + 0.58X1 + 0.38X2 + 0.08X3 − 1.56X21 − 0.79X22
− 1.83X32 − 0.56X1 X2 + 0.99X1 X3 − 0.033X2 X3
Analysis of variance (ANOVA) for the model is shown in Table 3.
The determination coefficient (R2 = 0.9961) indicates that only
0.39% of the total variations are not explained by the model. For a
2 )
good statistical model, the adjusted determination coefficient (Radj
2 (0.9892) is close to
should be close to R2 . As shown in Table 3, Radj
2
R2 . Moreover, Rpred (0.9423) is in reasonable agreement with Radj 2
and confirms that the model is highly significant. The lack of fit
test determines whether the selected model is adequate to explain
the experimental data, or whether another model should be res-
elected. The value of lack of fit test (0.1121) is higher than 0.05,
which is not significant relative to the pure error and indicates that
the fitting model is adequate to describe the experimental data. An
adequate precision is a measure of the signal to noise ratio, which
greater than 4 is considered to be desirable (Canettieri et al., 2007).
The value of adequate precision is 39.145, demonstrating an ade-
quate signal. At the same time, a relatively low value of coefficient
of variation (CV) (1.14) indicates a better precision and reliability
of the experimental values. Therefore, the model is adequate for
prediction in the range of experimental variables.
The significance of each coefficient measured using p-value and
F-value is listed in Table 4. Smaller p-value and greater F-value
mean the corresponding variables would be more significant. The
p-value of the model is less than 0.0001, which indicates that the
model is significant and can be used to optimize the extraction vari-
ables. The two independent variables (X1 , X2 ) and three quadratic
terms (X12 , X22 and X32 ) significantly affect the extraction yield within
a 99% confidence interval, and the interaction between extraction
time (X1 ) and ethanol concentration (X2 ), as well as extraction time
(X1 ) and ratio of aqueous ethanol to raw material (X3 ) is significant
(p < 0.01). Meanwhile, extraction time (X1 ) is the most significant
factor affecting the extraction yield.
3D response surface and 2D contour plots are the graphical rep-
resentations of regression equation and are very useful to judge
the relationship between independent and dependent variables.
Different shapes of the contour plots indicate whether the mutual
interactions between the variables are significant or not. Circular
contour plot means the interactions between the correspond-
ing variables are negligible, while elliptical contour suggests the
interactions between the corresponding variables are significant
(4) (Muralidhar et al., 2001). The three-dimensional representation of
the response surfaces and two-dimensional contours generated by
the model are shown in Figs. 3–5. In these three variables, when
two variables are depicted in three-dimensional surface plots, the
third variable is fixed at zero level. It is found in Figs. 3–5 that all
the three response surfaces are convex in shape, which indicates
that the ranges of variables were chosen properly.
As shown in Fig. 3, extraction yield increases rapidly when
extraction time (X1 ) and ethanol concentration (X2 ) increase
in the range of 15–20.9 min and 40–51.8%, respectively; but
beyond 20.9 min and 51.8%, extraction yield decreases slightly. This
demonstrates that the effect of extraction time (X1 ) and ethanol
concentration (X2 ) on extraction yield is significant and is in good
agreement with the results in Table 4. Moreover, the elliptical con-
tour plots in Fig. 3 mean that there is a significant interaction
between the two variables, which also agrees with the results in
Table 4. From Fig. 4, both extraction time (X1 ) and ratio of aqueous
ethanol to raw material (X3 ) have quadratic effect on extraction
yield. Extraction yield increases at first and then decreased quickly
with increasing of the two parameters, and a maximum extraction
 Y. Liu et al. / Industrial Crops and Products 49 (2013) 837–843 841

Fig. 3. Response surface and contour plots showing effect of extraction time (X1 ) and ethanol concentration (X2 ).

Fig. 4. Response surface and contour plots showing effect of extraction time (X1 ) and ratio of aqueous ethanol to raw material (X3 ).

Fig. 5. Response surface and contour plots showing effect of ethanol concentration (X2 ) and ratio of aqueous ethanol to raw material (X3 ).
842 Y. Liu et al. / Industrial Crops and Products 49 (2013) 837–843
Fig. 6. DPPH radical scavenging effect of phenolic compounds from Euryale ferox
seed shells as function of concentrations.
yield is achieved when extraction time (X1 ) and ratio of aque-
ous ethanol to raw material (X3 ) are 20.9 min and 30.7 mL/g,
respectively. It can be seen that the mutual interactions between
extraction time (X1 ) and ratio of aqueous ethanol to raw mate-
rial (X3 ) are significant due to the elliptical contour plots shown
in Fig. 4, which is also confirmed by the results in Table 4. It is obvi-
ous in Fig. 5 that extraction yield increases slowly with increasing of
ethanol concentration (X2 ) from 40% to 51.8% and decreases slowly
after 51.8%; while extraction yield increases rapidly with increasing
of ratio of aqueous ethanol to raw material (X3 ) from 20 to 30.7 mL/g
and decreases rapidly after 30.7 mL/g. This suggests that the inter-
actions between the two variables are not significant, which is in
agreement with the contour plots in Fig. 5 and the results in Table 4.
3.5. Verification of the model
The suitability of the model equation for predicting the optimum
response values is tested using the selected optimum conditions.
The optimum conditions are extraction time (X1 ) of 20.9 min,
ethanol concentration (X2 ) of 51.8%, and ratio of aqueous ethanol
to raw material (X3 ) of 30.7 mL/g, under which the predicted yield
is 15.70%. However, considering the operability in actual produc-
tion, the optimum conditions are modified as following: extraction
time (X1 ) of 21 min, ethanol concentration (X2 ) of 52%, and ratio of
aqueous ethanol to raw material (X3 ) of 31 mL/g, under which the
experimental yield is 15.69 ± 0.082% (n = 3), agreeing closely with
the predicted yield and consequently indicating the RSM model is
satisfactory and accurate.
3.6. Antioxidant activity of phenolic compounds from E. ferox
seed shells
DPPH assay was used to evaluate the antioxidant activity of
the phenolic compounds from E. ferox seed shells because DPPH
radicals are the stable free radicals and the model of scavenging
them is a most commonly used method to evaluate antioxidant
activities in a relatively short time compared with other methods
(Gülçin et al., 2004). The influence of antioxidants on scavenging
DPPH radicals is related to the ability of their hydrogen donation.
The decrease in absorbance of the DPPH radicals by antioxidants is
determined at 517 nm. Fig. 6 shows that the effect of scavenging
DPPH radicals increases significantly (p < 0.01) with the increases
in the concentrations of vitamin C (Vc ), phenolic compounds and
Trolox. The scavenging effect of Vc and Trolox increases rapidly with
Fig. 7. HPLC chromatograms of standards and extracts: (1) pyrogallol, (2) gallic acid,
(3) catechin, (4) chlorogenic acid, (5) epicatechin, and (6) rutin.
increasing the concentration from 0.1 to 0.6 mg/mL and almost
reaches it maximum. The correlation of scavenging effect and con-
centration of phenolic compounds is linear and R2 is 0.97 (p < 0.01),
and their scavenging effect at the concentration of 1.0 mg/mL
is higher than those of Vc and Trolox at the concentration of
0.6 mg/mL and is no significant difference (p > 0.05) compared with
those of Vc and Trolox at the concentration of 0.8 mg/mL. These
results indicate that the phenolic compounds from E. ferox seed
shells have a noticeable effect on scavenging DPPH free radicals.
3.7. HPLC analysis of extract composition and polyphenol content
Fig. 7 shows the chromatograms of the standard mixture and
the extracts. The HPLC chromatograms reveal that pyrogallol, gal-
lic acid and chlorogenic acid are the major phenolic compounds in
E. ferox seed shells, while rutin is the major flavonoid compounds.
The content of pyrogallol, gallic acid, chlorogenic acid and rutin
in E. ferox seed shells is calculated from respective standard cali-
bration curves and Eq. (1), and the values are 5.10% for pyrogallol,
6.08% for gallic acid, 1.28% for chlorogenic acid and 1.94% for rutin.
These results indicate that pyrogallol and gallic acid may be mainly
responsible for the antioxidant activity.
4. Conclusions
An ultrasonic-assisted extraction technology was performed for
the extraction of phenolic compounds from E. ferox seed shells and
optimized by RSM. Based on the single-factor-test, Box–Behnken
design was used to evaluate and optimize the extraction vari-
ables (extraction time, ethanol concentration, and ratio of aqueous
ethanol to raw material) for the extraction yield. The results show
that the variables (extraction time and ethanol concentration) are
significant and a high correlation of quadratic model obtained is sat-
isfactory and accurate to predict the extraction yield. The optimized
conditions are as follows: extraction time 21 min, ethanol concen-
tration 52%, and ratio of aqueous ethanol to raw material 31 mL/g.
Under these conditions, the experimental yield is 15.69%, which is
agreed closely with the predicted yield of 15.70%. The HPLC analysis
and the DPPH assay indicate that the extracts are composed of pyro-
gallol, gallic acid, chlorogenic acid and rutin, and have significant
antioxidant activity.
 Y. Liu et al. / Industrial Crops and Products 49 (2013) 837–843
Acknowledgements
This work is financially supported by the Science and Technol-
ogy Innovation Project of Zhaoqing City (No. 2012G14) and the
Natural Science Foundation of Zhaoqing University (No. 201201).
We thank Jianfen Zhao for the chromatographic analysis of the
standards and the extracts.
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