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

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


Fig. 1. Image of Euryale ferox seed shells.
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-
more efficient and easier arrangement and interpretation of exper-
pared and 1 mL of this solution was added 3 mL of sample solution
iments compared to others (Sheng et al., 2013; Stroescu et al.,
at different concentrations. The mixture was vortexed thoroughly
2013; Yan et al., 2011). The advantage of RSM is the reduced num-
and incubated in the dark at room temperature for 30 min, and then
ber of experimental trials needed to evaluate multiple parameters
the absorbance was measured at 517 nm in a UV–vis spectropho-
and their interactions. Therefore, it widely used in optimizing the
tometer (752, Jinghua, China). Vc was used as positive control. The
extraction parameters, such as polysaccharides (Zhu and Liu, 2013),
radical scavenging effect is calculated using the following equation:
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 Acontrol − Asample
variables (extraction time, ethanol concentration, and ratio of aque- Scavenging effect (%) = × 100 (2)
Acontrol
ous ethanol to raw material), and optimize these variables values
by RSM for the phenolic compounds recovery yield maximization where Acontrol is the absorbance value of control solution without
from E. ferox seed shells. Moreover, the antioxidant activity of the sample, and Asample is the absorbance to sample solution.
phenolic compounds was evaluated by DPPH assay and the major
composition was analyzed by HPLC.
2.5. HPLC analysis

2. Experimental The extract phenolics were analyzed using a HPLC (Agilent


1200 series, Agilent Technologies, USA) equipped with a UV detec-
2.1. Materials and chemicals tor (G1314B, Agilent) and an Eclipse XDB-C18 column (5 ␮m,
250 mm × 4.6 mm, Agilent). Ultrapure water was used as solvent
E. ferox seed shells were obtained from a farm in Zhaoqing A and 100% methanol as solvent B (A:B = 7:3). The solutions of
City (China). Folin–Ciocalteu phenol reagent, sodium carbonate, the standards and the extract phenolics were filtered through a
2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox, pyrogallol, rutin, 0.45 ␮m syringe filter. The operating conditions were: column tem-
chlorogenic acid, catechin, epicatechin, gallic acid and vitamin perature, 25 ◦ C; injection volume, 20 ␮L; detection wavelength,
C (Vc ) were from Aladdin (Shanghai, China). Methanol was 280 nm; flow rate, 1.0 mL/min. The identification and peak assign-
HPLC grade, other reagents were analytical grade and used as ment of the phenolics was based on comparison of retention times
received. and spectral data with those of the standards. The identified phe-
nolics were quantified according to respective standard calibration
curves.
2.2. Extraction of phenolic compounds

The process of phenolic compounds extraction from E. ferox 2.6. Experimental design
seed shells by ultrasonic-assisted treatment was performed in an
ultrasonic generator (500 W, 53 kHz, SK8200H, Kedao, China). The A three-level-three-factor, Box–Behnken design (BBD) was
dry E. ferox seed shells were powdered by a pulverizer (XS-10B, employed to determine the best combination of extraction vari-
Longxin, China) and then passed through an 80 mesh sieve. One ables for the phenolic compounds based on the results of
gram of the shell powders was used for each case in a beaker. preliminary single-factor-test. Extraction time (X1 ), ethanol con-
The beaker was held in the ultrasonic generator and exposed to centration (X2 ), and ratio of aqueous ethanol to raw material (X3 )
extract phenolic compounds for different extraction time at various were the independent variables, and their coded and uncoded
ethanol concentrations in different ratios of aqueous ethanol to raw levels were presented in Table 1. Extraction yield (Y) taken as
material. 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- of the experimental values. Therefore, the model is adequate for
ing to higher diffusion and extraction yield. But when the ratio prediction in the range of experimental variables.
is over 30:1 mL/g, the decrease of the distribution of ultrasonic The significance of each coefficient measured using p-value and
energy density in the extraction solutions is dominant, and has F-value is listed in Table 4. Smaller p-value and greater F-value
a negative effect on the extraction yield. The similar tendency of mean the corresponding variables would be more significant. The
this parameter was also acquire for the polysaccharides extraction p-value of the model is less than 0.0001, which indicates that the
from Acanthopanax senticosus (Zhao et al., 2013) and Boletus edulis model is significant and can be used to optimize the extraction vari-
mycelia (Chen et al., 2012). Therefore, the ratio of aqueous ethanol ables. The two independent variables (X1 , X2 ) and three quadratic
to raw material of 30:1 mL/g is sufficient for extracting the phenolic terms (X12 , X22 and X32 ) significantly affect the extraction yield within
compounds. a 99% confidence interval, and the interaction between extraction
time (X1 ) and ethanol concentration (X2 ), as well as extraction time
3.4. Optimization of extraction parameters for phenolic (X1 ) and ratio of aqueous ethanol to raw material (X3 ) is significant
compounds (p < 0.01). Meanwhile, extraction time (X1 ) is the most significant
factor affecting the extraction yield.
Table 2 shows the process variables and experimental data of 15 3D response surface and 2D contour plots are the graphical rep-
runs containing 3 replicates at center point. By applying multiple resentations of regression equation and are very useful to judge
regression analysis on the experimental data, the model for the the relationship between independent and dependent variables.
response variable could be expressed by the following quadratic Different shapes of the contour plots indicate whether the mutual
polynomial equation in the form of coded values: interactions between the variables are significant or not. Circular
contour plot means the interactions between the correspond-
Y = 15.62 + 0.58X1 + 0.38X2 + 0.08X3 − 1.56X21 − 0.79X22 ing variables are negligible, while elliptical contour suggests the
interactions between the corresponding variables are significant
− 1.83X32 − 0.56X1 X2 + 0.99X1 X3 − 0.033X2 X3 (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
Analysis of variance (ANOVA) for the model is shown in Table 3.
two variables are depicted in three-dimensional surface plots, the
The determination coefficient (R2 = 0.9961) indicates that only
third variable is fixed at zero level. It is found in Figs. 3–5 that all
0.39% of the total variations are not explained by the model. For a
2 ) the three response surfaces are convex in shape, which indicates
good statistical model, the adjusted determination coefficient (Radj
2 (0.9892) is close to
that the ranges of variables were chosen properly.
should be close to R2 . As shown in Table 3, Radj As shown in Fig. 3, extraction yield increases rapidly when
2
R2 . Moreover, Rpred (0.9423) is in reasonable agreement with Radj 2
extraction time (X1 ) and ethanol concentration (X2 ) increase
and confirms that the model is highly significant. The lack of fit in the range of 15–20.9 min and 40–51.8%, respectively; but
test determines whether the selected model is adequate to explain beyond 20.9 min and 51.8%, extraction yield decreases slightly. This
the experimental data, or whether another model should be res- demonstrates that the effect of extraction time (X1 ) and ethanol
elected. The value of lack of fit test (0.1121) is higher than 0.05, concentration (X2 ) on extraction yield is significant and is in good
which is not significant relative to the pure error and indicates that agreement with the results in Table 4. Moreover, the elliptical con-
the fitting model is adequate to describe the experimental data. An tour plots in Fig. 3 mean that there is a significant interaction
adequate precision is a measure of the signal to noise ratio, which between the two variables, which also agrees with the results in
greater than 4 is considered to be desirable (Canettieri et al., 2007). Table 4. From Fig. 4, both extraction time (X1 ) and ratio of aqueous
The value of adequate precision is 39.145, demonstrating an ade- ethanol to raw material (X3 ) have quadratic effect on extraction
quate signal. At the same time, a relatively low value of coefficient yield. Extraction yield increases at first and then decreased quickly
of variation (CV) (1.14) indicates a better precision and reliability 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. Fig. 7. HPLC chromatograms of standards and extracts: (1) pyrogallol, (2) gallic acid,
(3) catechin, (4) chlorogenic acid, (5) epicatechin, and (6) rutin.
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 increasing the concentration from 0.1 to 0.6 mg/mL and almost
extraction time (X1 ) and ratio of aqueous ethanol to raw mate- reaches it maximum. The correlation of scavenging effect and con-
rial (X3 ) are significant due to the elliptical contour plots shown centration of phenolic compounds is linear and R2 is 0.97 (p < 0.01),
in Fig. 4, which is also confirmed by the results in Table 4. It is obvi- and their scavenging effect at the concentration of 1.0 mg/mL
ous in Fig. 5 that extraction yield increases slowly with increasing of is higher than those of Vc and Trolox at the concentration of
ethanol concentration (X2 ) from 40% to 51.8% and decreases slowly 0.6 mg/mL and is no significant difference (p > 0.05) compared with
after 51.8%; while extraction yield increases rapidly with increasing those of Vc and Trolox at the concentration of 0.8 mg/mL. These
of ratio of aqueous ethanol to raw material (X3 ) from 20 to 30.7 mL/g results indicate that the phenolic compounds from E. ferox seed
and decreases rapidly after 30.7 mL/g. This suggests that the inter- shells have a noticeable effect on scavenging DPPH free radicals.
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. 3.7. HPLC analysis of extract composition and polyphenol content

3.5. Verification of the model Fig. 7 shows the chromatograms of the standard mixture and
the extracts. The HPLC chromatograms reveal that pyrogallol, gal-
The suitability of the model equation for predicting the optimum lic acid and chlorogenic acid are the major phenolic compounds in
response values is tested using the selected optimum conditions. E. ferox seed shells, while rutin is the major flavonoid compounds.
The optimum conditions are extraction time (X1 ) of 20.9 min, The content of pyrogallol, gallic acid, chlorogenic acid and rutin
ethanol concentration (X2 ) of 51.8%, and ratio of aqueous ethanol in E. ferox seed shells is calculated from respective standard cali-
to raw material (X3 ) of 30.7 mL/g, under which the predicted yield bration curves and Eq. (1), and the values are 5.10% for pyrogallol,
is 15.70%. However, considering the operability in actual produc- 6.08% for gallic acid, 1.28% for chlorogenic acid and 1.94% for rutin.
tion, the optimum conditions are modified as following: extraction These results indicate that pyrogallol and gallic acid may be mainly
time (X1 ) of 21 min, ethanol concentration (X2 ) of 52%, and ratio of responsible for the antioxidant activity.
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 4. Conclusions
satisfactory and accurate.
An ultrasonic-assisted extraction technology was performed for
3.6. Antioxidant activity of phenolic compounds from E. ferox the extraction of phenolic compounds from E. ferox seed shells and
seed shells optimized by RSM. Based on the single-factor-test, Box–Behnken
design was used to evaluate and optimize the extraction vari-
DPPH assay was used to evaluate the antioxidant activity of ables (extraction time, ethanol concentration, and ratio of aqueous
the phenolic compounds from E. ferox seed shells because DPPH ethanol to raw material) for the extraction yield. The results show
radicals are the stable free radicals and the model of scavenging that the variables (extraction time and ethanol concentration) are
them is a most commonly used method to evaluate antioxidant significant and a high correlation of quadratic model obtained is sat-
activities in a relatively short time compared with other methods isfactory and accurate to predict the extraction yield. The optimized
(Gülçin et al., 2004). The influence of antioxidants on scavenging conditions are as follows: extraction time 21 min, ethanol concen-
DPPH radicals is related to the ability of their hydrogen donation. tration 52%, and ratio of aqueous ethanol to raw material 31 mL/g.
The decrease in absorbance of the DPPH radicals by antioxidants is Under these conditions, the experimental yield is 15.69%, which is
determined at 517 nm. Fig. 6 shows that the effect of scavenging agreed closely with the predicted yield of 15.70%. The HPLC analysis
DPPH radicals increases significantly (p < 0.01) with the increases and the DPPH assay indicate that the extracts are composed of pyro-
in the concentrations of vitamin C (Vc ), phenolic compounds and gallol, gallic acid, chlorogenic acid and rutin, and have significant
Trolox. The scavenging effect of Vc and Trolox increases rapidly with antioxidant activity.
Y. Liu et al. / Industrial Crops and Products 49 (2013) 837–843 843

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