Optimization of ultrasonic extraction of polysaccharides from Ziziphus jujubaMill. by response surface methodology
 Chenling Qu^{1}Email author,
 Songcheng Yu^{2},
 Li Luo^{3},
 Yan Zhao^{1} and
 Yawei Huang^{1}
DOI: 10.1186/1752153X7160
© Qu et al.; licensee Chemistry Central Ltd. 2013
Received: 10 July 2013
Accepted: 10 September 2013
Published: 23 September 2013
Abstract
Background
Ziziphus jujuba Mill. is nutritious and used as food and medicine for more than two thousand years. It has many pharmacological effects, such as elimination of fatigue, dilation of blood vessels, etc. The polysaccharide in it is one of the bioactive substances. In this paper, the ultrasonic extraction effects on the yield and activity of polysaccharide were studied.
Results
The optimum ultrasonic extraction conditions were investigated based on a BoxBehnken statistical experimental design. Response surface methodology (RSM) of three factors (ultrasonic power, extraction time and extraction temperature) and three levels was employed to optimize the yield and the antioxidant activity of the polysaccharides. The experimental data were fitted to quadratic response surface models using multiple regression analysis. The best extraction conditions were 120 W, 15 min. and 55°C for highest yield, and 80 W, 15 min. and 40°C for highest hydroxyl radical scavenging activity.
Conclusion
The study showed that high ultrasonic power was good for obtaining high yield but bad for keeping the antioxidant activity of the polysaccharides.
Keywords
Ultrasonic extraction Response surface methodology (RSM) Polysaccharides Ziziphus jujuba Mill. OptimizationIntroduction
Ziziphus jujuba Mill. is a native plant of China and belongs to the genus Ziziphus Mill. (Rhamnaceae) [1]. Its fruits have been used in traditional Chinese medicine for more than two thousand years. The bioactivities of the polysaccharides in Ziziphus jujuba Mill. have been reported, such as immunobiological activities [1–4] and antioxidant activities [5].
Research reports revealed that the bioactivities of polysaccharides in Ziziphus jujuba Mill. were related to their structures. Chang et al. [5] isolated one neutral polysaccharide fraction (ZJPN) and three acidic polysaccharide fractions (ZJPa1, ZJPa2 and ZJPa3). Gas chromatography (GC) analysis revealed that six monosaccharides, namely, rhamnose, arabinose, xylose, mannose, glucose and galactose were present in the polysaccharide fractions. All four polysaccharide fractions were found to be more effective at scavenging superoxide anion radicals than hydroxyl radicals, while the acidic polysaccharides showed a more pronounced effect at chelating ferrous ion [5]. Zhao et al. [2] obtained a fraction, JuB7, which could stimulate spleen cell proliferation and had a molecular mass of over 2000 kDa. This isolated polysaccaride was mainly composed of α1,4linked dgalactopyranosyluronic acid and 1,2linked lrhamnose at a molar ratio of 8.1:1.
Ultrasonic extraction was widely employed to extract polysaccharides from plant material due to its high extraction efficiency [6–9]. However, ultrasonicaion can change the structures of the polysaccharides to some extent [10]. In this paper, the effects of ultrasonic power, extraction time, extraction temperature on the yield and the antioxidant activity of water soluble polysaccharides of Ziziphus jujuba Mill. were investigated by response surface methodology (RSM).
RSM is an effective statistical technique, which is used to find optimum processing parameters [11–13]. It has been used to optimize the polysaccharides extraction process variables and the interactions of these variables [14–18]. In the present study, a threevariable, threelevel Box–Behnken design (BBD) [19–25] was used to optimize the extraction conditions for ultrasonic extraction of water soluble polysaccharides in Ziziphus jujuba Mill.
Experimental
Chemicals and instruments
Ziziphus jujuba Mill., which grew in Xinjiang province (China) was purchased from a local shop in Zhengzhou, China. All reagents used in this study were of analytical grade. Anhydrous ethanol, 95% ethanol and acetone were obtained from Tianli Corporation (Tianjin, China). Ferrous sulfate (FeSO_{4}), salicylic acid and petroleum ether were purchased from Kermel Corporation (Tianjin, China). Hydrogen peroxide (H_{2}O_{2}) was obtained from Haohua Corporation (Luoyang, China). Deionized water used in the experiments was purified by a MilliQ system (Millipore Corporation, USA).
KQ5200DE ultrasonic cleaner, which can control ultrasonic temperature, power and time, was supplied by Kunshan Corporation (Shanghai, China). RE52A rotary evaporator (Yarong Corporation, Shanghai, China) and 752 UV–vis spectrophotometer (Jinghua Corporation, Shanghai, China) were also employed in the experiments.
Extraction procedure
The fruit of Ziziphus jujuba Mill. was first peeled, then the kernel was removed. The obtained pulp was dried at 40°C. The dried sample was extracted in a Soxhlet apparatus, first with petroleum ether, and afterwards with 80% ethanol twice, to remove some colored materials, monosaccharides, oligosaccharides, and small molecular weight materials. The organic solvent was evaporated to yield a dried extracted powder.
Antioxidant activity
Design of experiments
Box–Behnken design and the response values for yield and hydroxyl radical scavenging activity of polysaccharides
Run  X_{1}: Ultrasonic Power (W)  X_{2}: Extraction time (min)  X_{3}: Extraction temperature (°C)  Yield of polysaccharides (%)  Hydroxyl radical scavenging activity of polysaccharides (%)  

Actual values  Predicted values  Actual values  Predicted values  
1  120 (+1)  15 (+1)  50 (0)  4.44  4.53  35.61  35.66 
2  80 (−1)  5 (−1)  50 (0)  3.26  3.17  49.36  49.31 
3  100 (0)  10 (0)  50 (0)  3.98  4.03  50.14  50.37 
4  100 (0)  10 (0)  50 (0)  3.96  4.03  48.35  50.37 
5  100 (0)  10 (0)  50 (0)  4.06  4.03  51.65  50.37 
6  100 (0)  5 (−1)  60 (+1)  3.56  3.59  38.43  36.73 
7  120 (+1)  5 (−1)  50 (0)  3.80  3.82  39.86  41.42 
8  80 (−1)  10 (0)  40 (−1)  2.96  3.01  65.65  65.51 
9  100 (0)  10 (0)  50 (0)  4.12  4.03  51.26  50.37 
10  100 (0)  15 (+1)  60 (+1)  4.16  4.12  41.65  41.46 
11  120 (+1)  10 (0)  40 (−1)  3.82  3.76  47.52  45.77 
12  80 (−1)  15 (+1)  50 (0)  3.58  3.56  65.16  63.60 
13  100 (0)  10 (0)  50 (0)  4.05  4.03  50.45  50.37 
14  100 (0)  15 (+1)  40 (−1)  3.68  3.65  51.68  53.38 
15  100 (0)  5 (−1)  40 (−1)  3.04  3.08  49.36  49.56 
16  80 (−1)  10 (0)  60 (+1)  3.40  3.45  49.56  51.31 
17  120 (+1)  10 (0)  60 (+1)  4.36  4.31  35.08  35.22 
Statistical analyses
DesignExpert trial version 8.0.5 (Statease Inc., Minneapolis, USA) was used to statistically analyze the experimental data. The significant terms in the model were found by analysis of variance (ANOVA) for each response. The significances of all terms in the polynomial were considered statistically different when P < 0.05. The adequacy of model was checked by accounting for the coefficient of determination (R^{2}) and adjustedR^{2} (R^{2}_{adj}).
Results and discussion
Statistical analysis and the model fitting
In these equations, X_{1}, X_{2} and X_{3} were the values of extraction parameters, ultrasonic power (W), extracting time (min.) and extraction temperature (°C), respectively. The variables, experimental data and predicted data are shown in Table 1.
Analysis of variance for the fitted quadratic polynomial model of polysaccharides yield
Source  Sum of squares  Degree of freedom  Mean square  FValue  Pvalue 

Prob > F  
Model  2.94  9  0.33  47.07  <0.0001 
X_{1}  1.30  1  1.30  186.60  <0.0001 
X_{2}  0.61  1  0.61  87.10  <0.0001 
X_{3}  0.49  1  0.49  70.55  <0.0001 
X_{1} X_{2}  0.026  1  0.026  3.69  0.0964 
X_{1} X_{3}  2.5×10^{3}  1  2.5×10^{3}  0.36  0.5674 
X_{2} X_{3}  4.0×10^{4}  1  4.0×10^{4}  0.058  0.8172 
X_{1}^{2}  0.060  1  0.060  8.66  0.0216 
X_{2}^{2}  0.088  1  0.088  12.66  0.0092 
X_{3}^{2}  0.33  1  0.33  47.36  0.0002 
Residual  0.049  7  6.946×10^{3}  
Lack of Fit  0.032  3  0.011  2.54  0.1944 
Pure Error  0.017  4  4.18×10^{3}  
Cor Total  2.99  16  
R^{2}=0.9837 R^{2}_{adj}=0.9628 CV=2.21% 
Analysis of variance for the fitted quadratic polynomial model of hydroxyl radical scavenging activity of polysaccharides
Source  Sum of squares  Degree of freedom  Mean square  FValue  Pvalue 

Prob > F  
Model  1154.38  9  128.26  38.82  <0.0001 
X_{1}  641.89  1  641.89  191.75  <0.0001 
X_{2}  36.51  1  36.51  10.91  0.0131 
X_{3}  306.16  1  306.16  91.46  <0.0001 
X_{1} X_{2}  100.50  1  100.50  30.02  0.0009 
X_{1} X_{3}  3.33  1  3.33  0.99  0.3518 
X_{2} X_{3}  0.20  1  0.20  0.060  0.8128 
X_{1}^{2}  1.78  1  1.78  0.53  0.4897 
X_{2}^{2}  52.24  1  52.24  15.61  0.0055 
X_{3}^{2}  10.35  1  10.35  3.09  0.1222 
Residual  23.43  7  3.35  
Lack of Fit  16.86  3  5.62  3.42  0.1328 
Pure Error  6.57  4  1.64  
Cor Total  1177.82  16  
R^{2}=0.9801 R^{2}_{adj}=0.9545 CV=3.79% 
Table 3 showed the quadratic regression model of hydroxyl radical scavenging activity of the polysaccharides. It can be seen that R^{2} was 0.9801 and R^{2}_{adj} was 0.9545, indicating a high degree of correlation between the observed and predicted values. The coefficient of variation was low (CV=3.79%), indicating a high degree of precision and reliability of the experimental values. Fvalue and Pvalue of the lackoffit were 3.42 and 0.1328, respectively, which implied that it was not significant; there was a 13.28% chance that this lackoffit was due to noise. It can be seen from Table 3 that the three independent variables (X_{1}, X_{2} and X_{3}), one quadratic term (X_{2}^{2}), and the interaction between X_{1} and X_{2} significantly affected the hydroxyl radical scavenging activity of the polysaccharides.
Analysis of response surface plot
As shown in Figure 1a, when the extraction temperature (X_{3}) was fixed at 0 level, the yield increased as the ultrasonic power (X_{1}) and extraction time (X_{2}) increased. Figure 1b showed the effects of ultrasonic power (X_{1}) and extraction temperature (X_{3}) on the yield of polysaccharides. The yield increased with the increase of ultrasonic power. The yield was positively correlated with the extraction temperature when temperature was lower than 55°C and was negatively correlated when temperature was higher than 55°C. The interactions between extraction time (X_{2}) and extraction temperature (X_{3}), when ultrasonic power (X_{1}) was fixed at 0 level, were displayed in Figure 1c. The yield increased with the extraction time.
Figure 2 showed the ultrasonic parameter variables (ultrasonic power, extraction time and extraction temperature) and their interactions on hydroxyl radical scavenging activity of polysaccharides. Ultrasonic power (X_{1}) and extraction temperature (X_{3}) both had a negative impact on the activity. Nevertheless, longer extraction times led to an increase of the activity. Therefore, low extraction temperature and low ultrasonic power were advantageous to the hydroxyl radical scavenging activity of polysaccharides.
Optimization of extracting parameters and validation of the model
Optimum conditions, and the predicted and experimental values of response
Ultrasonic power (W)  Extraction time (min)  Extraction temperature (°C)  Yield of polysaccharides (%)  Hydroxyl radical scavenging activity of polysaccharides (%)  

Optimum condition for yield (predicted)  120  15  54.69  4.59  32.75 
Modified condition for yield (actual)  120  15  55  4.47  30.94 
Optimum condition for activity (predicted)  80  14.91  40  3.07  68.91 
Modified condition for activity (actual)  80  15  40  2.91  67.30 
The optimal predicted extraction condition for achieving the highest hydroxyl radical scavenging activity of 68.91% was ultrasonic power of 80 W, extraction time of 14.91 min. and extraction temperature of 40°C. For practical implementation, the extraction condition was modified as ultrasonic power of 80 W, extraction time of 15 min. and extraction temperature of 40°C. Using these parameters, the hydroxyl radical scavenging activity was 67.30%, which was close to the maximum predicted by the response surface model (Table 4).
Table 4 also displayed that the hydroxyl radical scavenging activity of the polysaccharides under the optimal condition for highest yield (ultrasonic power of 120 W, extraction time of 15 min. and extraction temperature of 54.69°C) was predicted as 32.75% by the quadratic response surface model (Eq. [5]), and the activity obtained at the experiment condition (ultrasonic power of 120 W, extraction time of 15 min. and extraction temperature of 55°C) was 30.94%. At the same time, the yield of polysaccharides under the optimal condition for best hydroxyl radical scavenging activity of polysaccharides (ultrasonic power of 80 W, extraction time of 14.91 min. and extraction temperature of 40°C) was predicted as 3.07% by Equation [4]. The yield in the modified condition (ultrasonic power of 80 W, extraction time of 15 min. and extraction temperature of 40°C) was 2.91%.
These data suggested that the extraction conditions for obtaining high yield of polysaccharides were not suitable for obtaining good hydroxyl radical scavenging activity, and that the optimal conditions for achieving high hydroxyl radical scavenging activity could not be applied to obtain high yield of polysaccharides. High ultrasonic power was advantageous to yield and adverse to activity, and low extraction temperature was more favorable for high radical scavenging activity. Extraction time 15 min. was good to both the yield and the activity.
Conclusion
The results indicated that the optimum extraction conditions of polysaccharides for obtaining highest yield and highest radical scavenging activity were quite different. Ultrasonic power played an important role in ultrasonic extraction.
Therefore, we should consider not only the high yield but also the sacrificed radical scavenging activity of the polysaccharides during the extraction process.
Abbreviations
 RSM:

Response surface methodology
 GC:

Gas chromatography
 BBD:

Box–Behnken design
 ANOVA:

Analysis of variance
 Yyield:

Polysaccharide yield
 Yactivity:

Hydroxyl radical scavenging activity
 CV:

Coefficient of variation.
Declarations
Acknowledgements
This research was supported by grants from the Doctor Research Fund of Henan University of Technology (No: 2009BS027).
Authors’ Affiliations
References
 Zhao Z, Liu M, Tu P: Characterization of water soluble polysaccharides from organs of Chinese Jujube (Ziziphus jujuba Mill. cv. Dongzao). Eur Food Res Technol. 2008, 226: 985989. 10.1007/s0021700706201.View ArticleGoogle Scholar
 Zhao Z, Dai H, Wu X, Chang H, Gao X, Liu M, Tu P: Characterization of a pectic polysaccharide from the fruit of Ziziphus jujuba. Chem Nat Compd. 2007, 43 (4): 311312.Google Scholar
 Zhao Z, Li J, Wu X, Dai H, Gao X, Liu M, Tu P: Structures and immunological activities of two pectic polysaccharides from the fruits of Ziziphus jujuba Mill. cv. jinsixiaozao Hort. Food Res Int. 2006, 39 (8): 917923. 10.1016/j.foodres.2006.05.006.View ArticleGoogle Scholar
 Li J, Shan L, Liu Y, Fan L, Ai L: Screening of a functional polysaccharide from Zizyphus Jujuba cv. Jinsixiaozao and its property. Int J Biol Macromol. 2011, 49 (3): 255259. 10.1016/j.ijbiomac.2011.04.006.View ArticleGoogle Scholar
 Chang SC, Hsu BY, Chen BH: Structural characterization of polysaccharides from Zizyphus jujuba and evaluation of antioxidant activity. Int J Biol Macromol. 2010, 47 (4): 445453. 10.1016/j.ijbiomac.2010.06.010.View ArticleGoogle Scholar
 Zhong K, Wang Q, He Y, He X: Evaluation of radicals scavenging, immunitymodulatory and antitumor activities of longan polysaccharides with ultrasonic extraction on in S180 tumor mice models. Int J Biol Macromol. 2010, 47 (3): 356360. 10.1016/j.ijbiomac.2010.05.022.View ArticleGoogle Scholar
 Lai F, Wen Q, Li L, Wu H, Li X: Antioxidant activities of watersoluble polysaccharide extracted from mung bean (Vigna radiata L.) hull with ultrasonic assisted treatment. Carbohyd Polym. 2010, 81 (2): 323329. 10.1016/j.carbpol.2010.02.011.View ArticleGoogle Scholar
 Pan Y, Dong S, Hao Y, Zhou Y, Ren X, Wang J, Wang W, Chu T: Ultrasonicassisted extraction process of crude polysaccharides from Yunzhi mushroom and its effect on hydroxyproline and glycosaminoglycan levels. Carbohyd Polym. 2010, 81 (1): 9396. 10.1016/j.carbpol.2010.01.060.View ArticleGoogle Scholar
 Chen X, Tang Q, Chen Y, Wang W, Li S: Simultaneous extraction of polysaccharides from Poria cocos by ultrasonic technique and its inhibitory activities against oxidative injury in rats with cervical cancer. Carbohyd Polym. 2010, 79 (2): 409413. 10.1016/j.carbpol.2009.08.025.View ArticleGoogle Scholar
 Zhou C, Yu X, Zhang Y, He R, Ma H: Ultrasonic degradation, purification and analysis of structure and antioxidant activity of polysaccharide from Porphyra yezoensis Ueda. Carbohyd Polym. 2012, 87 (3): 20462051. 10.1016/j.carbpol.2011.10.026.View ArticleGoogle Scholar
 Galai S, Touhami Y, Marzouki MN: Response surface methodology applied to laccases activities exhibited by stenotrophomonas maltophilia AAP56 in different growth conditions. Bioresources. 2012, 7 (1): 706726.Google Scholar
 Liu J, Hu HR, Xu JF, Wen YB: Optimizing enzymatic pretreatment of recycled fiber to improve its draining ability using response surface methodology. Bioresources. 2012, 7 (2): 21212140.Google Scholar
 Xu P, Bao JS, Gao JJ, Zhou T, Wang YF: Optimization of extraction of phenolic antioxidants from tea (Camellia Sinensis L.) fruit peel biomass using response surface methodology. Bioresources. 2012, 7 (2): 24312443.Google Scholar
 Chen Y, Gu X, Huang S, Li J, Wang X, Tang J: Optimization of ultrasonic/microwave assisted extraction (UMAE) of polysaccharides from Inonotus obliquus and evaluation of its antitumor activities. Int J Biol Macromol. 2010, 46 (4): 429435. 10.1016/j.ijbiomac.2010.02.003.View ArticleGoogle Scholar
 Zhong K, Wang Q: Optimization of ultrasonic extraction of polysaccharides from dried longan pulp using response surface methodology. Carbohyd Polym. 2010, 80 (1): 1025.View ArticleGoogle Scholar
 Huang S, Ning Z: Extraction of polysaccharide from Ganoderma lucidum and its immune enhancement activity. Int J Biol Macromol. 2010, 47 (3): 336341. 10.1016/j.ijbiomac.2010.03.019.View ArticleGoogle Scholar
 Tian Y, Zeng H, Xu Z, Zheng B, Lin Y, Gan C, Lo YM: Ultrasonicassisted extraction and antioxidant activity of polysaccharides recovered from white button mushroom (Agaricus bisporus). Carbohyd Polym. 2012, 88 (2): 522529. 10.1016/j.carbpol.2011.12.042.View ArticleGoogle Scholar
 Zhang B, Yan P, Chen H, He J: Optimization of production conditions for mushroom polysaccharides with high yield and antitumor activity. Carbohyd Polym. 2012, 87 (4): 25692575. 10.1016/j.carbpol.2011.11.042.View ArticleGoogle Scholar
 Chen W, Wang W, Zhang H, Huang Q: Optimization of ultrasonicassisted extraction of watersoluble polysaccharides from Boletus edulis mycelia using response surface methodology. Carbohyd Polym. 2011, 87 (1): 614619.View ArticleGoogle Scholar
 Zhao Q, Kennedy JF, Wang X, Yuan X, Zhao B, Peng Y, Huang Y: Optimization of ultrasonic circulating extraction of polysaccharides from Asparagus officinalis using response surface methodology. Int J Biol Macromol. 2011, 49 (2): 181187. 10.1016/j.ijbiomac.2011.04.012.View ArticleGoogle Scholar
 Yang W, Fang Y, Liang J, Hu Q: Optimization of ultrasonic extraction of Flammulina velutipes polysaccharides and evaluation of its acetylcholinesterase inhibitory activity. Food Res Int. 2011, 44 (5): 12691275. 10.1016/j.foodres.2010.11.027.View ArticleGoogle Scholar
 Zou Y, Chen X, Yang W, Liu S: Response surface methodology for optimization of the ultrasonic extraction of polysaccharides from Codonopsis pilosula Nannf.var.modesta L.T. Shen. Carbohyd Polym. 2011, 84 (1): 503508. 10.1016/j.carbpol.2010.12.013.View ArticleGoogle Scholar
 Yan Y, Yu C, Chen J, Li X, Wang W, Li S: Ultrasonicassisted extraction optimized by response surface methodology, chemical composition and antioxidant activity of polysaccharides from Tremella mesenterica. Carbohyd Polym. 2011, 83 (1): 217224. 10.1016/j.carbpol.2010.07.045.View ArticleGoogle Scholar
 Gan C, Latiff AA: Extraction of antioxidant pecticpolysaccharide from mangosteen (Garcinia mangostana) rind: Optimization using response surface methodology. Carbohyd Polym. 2011, 83 (2): 600607. 10.1016/j.carbpol.2010.08.025.View ArticleGoogle Scholar
 Ma L, Gan D, Wang M, Zhang Z, Jiang C, Zeng X: Optimization of extraction, preliminary characterization and hepatoprotective effects of polysaccharides from Stachys floridana Schuttl. ex Benth. Carbohyd Polym. 2012, 87 (2): 13901398. 10.1016/j.carbpol.2011.09.032.View ArticleGoogle Scholar
 Pan WJ, Liao AM, Zhang JG, Dong Z, Wei ZJ: Supercritical carbon dioxide extraction of the oak silkworm (Antheraea pernyi) pupal oil: process optimization and composition determination. Int J Mol Sci. 2012, 13 (2): 23542367.View ArticleGoogle Scholar
 Wei ZJ, Liao AM, Zhang HX, Liu J, Jiang ST: Optimization of supercritical carbon dioxide extraction of silkworm pupal oil applying the response surface methodology. Biores Technol. 2009, 100 (18): 42144219. 10.1016/j.biortech.2009.04.010.View ArticleGoogle Scholar
 Wei ZJ, Zhou LC, Chen H, Chen GH: Optimization of the fermentation conditions for 1deoxynojirimycin production by streptomyces lawendulae applying the response surface methodology. Int J Food Eng. 2011, 7 (3): 1612 ppView ArticleGoogle Scholar
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