Beruflich Dokumente
Kultur Dokumente
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
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.
−1 0 1
3
3
2
3
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.
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.
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)
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)
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
Acknowledgements Martins, S., Mussatto, S.I., Martínez-Avila, G., Montañez-Saenz, J., Aguilar, C.N., Teix-
eira, J.A., 2011. Bioactive phenolic compounds: production and extraction by
solid-state fermentation: a review. Biotechnology Advances 29, 365–373.
This work is financially supported by the Science and Technol- Muralidhar, R.V., Chirumamila, R.R., Marchant, R., Nigam, P., 2001. A response surface
ogy Innovation Project of Zhaoqing City (No. 2012G14) and the approach for the comparison of lipase production by Candida cylindracea using
Natural Science Foundation of Zhaoqing University (No. 201201). two different carbon sources. Biochemical Engineering Journal 9, 17–23.
Park, P.J., Jung, W.K., Nam, K.S., Shahidi, F., Kim, S.K., 2001. Purification and char-
We thank Jianfen Zhao for the chromatographic analysis of the acterization of antioxidative peptides from protein hydrolysate of lecithin-free
standards and the extracts. egg yolk. Journal of the American Oil Chemists Society 78, 651–656.
Pinho, C., Melo, A., Mansilha, C., Ferreira, I.M.P.L.V.O., 2011. Optimization of con-
ditions for anthocyanin hydrolysis from red wine using response surface
References methodology (RSM). Journal of Agricultural and Food Chemistry 59, 50–55.
Şahin, S., Şamlı, R., 2013. Optimization of olive leaf extract obtained by
Canettieri, E.V., Rocha, G.J.M., Carvalho, J.A., Silva, J.B.A., 2007. Optimization of acid ultrasound-assisted extraction with response surface methodology. Ultrasonics
hydrolysis from the hemicellulosic fraction of Eucalyptus grandis residue using Sonochemistry 20, 595–602.
response surface methodology. Bioresource Technology 98, 422–428. Santos, S.A.O., Pinto, P.C.R.O., Silvestre, A.J.D., Neto, C.P., 2010. Chemical composi-
Carrera, C., Ruiz-Rodríguez, A., Palma, M., Barroso, C.G., 2012. Ultrasound assisted tion and antioxidant activity of phenolic extracts of cork from Quercus suber L.
extraction of phenolic compounds from grapes. Analytica Chimica Acta 732, Industrial Crops and Products 31, 521–526.
100–104. Shankar, M., Chaudhary, N., Singh, D., 2010. A review on gorgon nut. International
Chen, W., Wang, W.P., Zhang, H.S., Huang, Q., 2012. Optimization of ultrasonic- Journal of Pharmaceutical and Biological Archive 1, 101–107.
assisted extraction of water-soluble polysaccharides from Boletus edulis Sheng, Z.L., Wan, P.F., Dong, C.L., Li, Y.H., 2013. Optimization of total flavonoids con-
mycelia using response surface methodology. Carbohydrate Polymers 87, tent extracted from Flos Populi using response surface methodology. Industrial
614–619. Crops and Products 43, 778–786.
Fan, J.P., Cao, J., Zhang, X.H., Huang, J.Z., Kong, T., Tong, S., Tian, Z.Y., Xie, Y.L., Xu, R., Song, C.W., Wang, S.M., Zhou, L.L., Hou, F.F., Wang, K.J., Han, Q.B., Li, N., Cheng,
Zhu, J.H., 2012. Optimization of ionic liquid based ultrasonic assisted extraction Y.X., 2011. Isolation and identification of compounds responsible for antioxi-
of puerarin from Radix Puerariae Lobatae by response surface methodology. Food dant capacity of Euryale ferox seeds. Journal of Agricultural and Food Chemistry
Chemistry 135, 2299–2306. 59, 1199–1204.
Gülçin, Ì., Şat, İ.G., Beydemir, Ş., Elmastaş, M., Küfrevioǧlu, Ö.İ., 2004. Comparison Spigno, G., De Faveri, D.M., 2009. Microwave-assisted extraction of tea phenols: a
of antioxidant activity of clove (Eugenia caryophylata Thunb) buds and lavender phenomenological study. Journal of Food Engineering 93, 210–217.
(Lavandula stoechas L.). Food Chemistry 87, 393–400. Stroescu, M., Stoica-Guzun, A., Ghergu, S., Chira, N., Jipa, I., 2013. Optimization of fatty
Han, Z., Luo, J., Kong, L.Y., 2012. Two new tocopherol polymers from the seeds of acids extraction from Portulaca oleracea seed using response surface methodol-
Euryale ferox. Journal of Asian Natural Products Research 14, 743–747. ogy. Industrial Crops and Products 43, 405–411.
Ignat, I., Volf, I., Popa, V.I., 2011. A critical review of methods for characterisa- Sun, Y., Xu, W., Zhang, W., Hu, Q., Zeng, X., 2011. Optimizing the extraction of
tion of polyphenolic compounds in fruits and vegetables. Food Chemistry 126, phenolic antioxidants from kudingcha made from Ilex kudingcha C.J. Tseng by
1821–1835. using response surface methodology. Separation and Purification Technology
Jha, S.N., Prasad, S., 1996. Determination of processing conditions for gorgon nut 78, 311–320.
(Euryale ferox). Journal of Agricultural Engineering Research 63, 103–111. Wang, X., Wu, Y., Chen, G., Yue, W., Liang, Q., Wu, Q., 2013. Optimisation of ultra-
Jha, V., Barat, G.K., Jha, U.N., 1991. Nutritional evaluation of Euryale ferox salisb. sound assisted extraction of phenolic compounds from Sparganii rhizoma with
(Makhana). Journal of Food Science and Technology 28, 326–328. response surface methodology. Ultrasonics Sonochemistry 20, 846–854.
Lee, S.E., Ju, E.M., Kim, J.H., 2002. Antioxidant activity of extracts from Euryale ferox Yan, Y.L., Yu, C.H., Chen, J., Li, X.X., Wang, W., Li, S.Q., 2011. Ultrasonic-assisted extrac-
seed. Experimental and Molecular Medicine 34, 100–106. tion optimized by response surface methodology, chemical composition and
Leonelli, C., Mason, T.J., 2010. Microwave and ultrasonic processing: now antioxidant activity of polysaccharides from Tremella mesenterica. Carbohydrate
a realistic option for industry. Chemical Engineering and Processing 49, Polymers 83, 217–224.
885–900. Zeng, Z.L., Xi, G.X., Ge, X.H., 2013. Study on comparison of two extraction methods
Li, B., Du, W., Jin, J., Du, Q., 2012. Preservation of (−)-epigallocatechin-3-gallate of polysaccharide from Momordica charantia L. vines. Science and Technology of
antioxidant properties loaded in heat treated -lactoglobulin nanoparticles. Food Industry 34, 285–308.
Journal of Agricultural and Food Chemistry 60, 3477–3484. Zhang, C., Li, M., Jia, X.L., 2012. Preliminary separation and identification of the ethyl
Li, Q., Fu, C., 2005. Application of response surface methodology for extraction opti- acetate extract from Gorgon Nut (Euryale ferox Salisb.) seed coat. Science and
mization of germinant pumpkin seeds protein. Food Chemistry 92, 701–706. Technology of Food Industry 33, 223–225.
Makris, D.P., Boskou, G., Andrikopoulos, N.K., 2007. Recovery of antioxidant phe- Zhao, Z., Xu, X., Ye, Q., Dong, L., 2013. Ultrasound extraction optimization of Acan-
nolics from white vinification solid by-products employing water/ethanol thopanax senticosus polysaccharides and its antioxidant activity. International
mixtures. Bioresource Technology 98, 2963–2967. Journal of Biological Macromolecules 59, 290–294.
Maqsood, S., Benjakul, S., Shahidi, F., 2013. Emerging role of phenolic compounds as Zhu, C., Liu, X., 2013. Optimization of extraction process of crude polysaccharides
natural food additives in fish and fish products. Critical Reviews in Food Science from pomegranate peel by response surface methodology. Carbohydrate Poly-
and Nutrition 53, 162–179. mers 92, 1197–1202.