Microwave-assisted extraction and antioxidant activity of vaccarin from the seeds of Vaccaria segetalis

Separation and Purification Technology 133 (2014) 91–98

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Microwave-assisted extraction and antioxidant activity of vaccarin from the seeds of Vaccaria segetalis Xiao-Han Yuan a,b,c,1, Li-Nan Fu a,1, Cheng-Bo Gu a,⇑, Yan-Dong Zhang c, Yu-Jie Fu a a

Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China Life Science and Biotechnique Research Center, Northeast Agricultural University, Harbin 150030, China c College of Forestry, Northeast Forestry University, Harbin 150040, China b

a r t i c l e

i n f o

Article history: Received 15 July 2013 Received in revised form 26 May 2014 Accepted 3 June 2014 Available online 2 July 2014 Keywords: Microwave-assisted extraction (MAE) Vaccarin Vaccaria segetalis Antioxidant activity Response surface methodology

a b s t r a c t Microwave-assisted extraction (MAE) of vaccarin from the seeds of Vaccaria segetalis was optimized in this study. Compared with the conventional extraction methods, maceration extraction (ME), ultrasonic-assisted extraction (UAE) and heat reflux extraction (HRE), MAE possessed higher efficiency for the extraction of vaccarin. The MAE conditions including methanol concentration, temperature, liquid/ solid ratio, and microwave power were studied and optimized. The maximum extraction rate of vaccarin reached 0.52%, under the optimal conditions: methanol concentration 57%, temperature 65 °C, liquid/ solid ratio 50 mL/g, and microwave power 400 W. At these optimal extraction parameters, the maximum extraction rate of vaccarin obtained experimentally was found to be very close to its predicted value. Furthermore, MAE extract of vaccarin exhibited substantial free radical-scavenging activity with an IC50 value of 0.48 mg/mL, which indicated crude extracts possess good potential in food and pharmaceutical industry. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Vaccaria segetalis (Neck) Garcke (Caryophyllaceae) is an annual herb widely distributed in Asia, Europe, North America and other parts of the world [1]. The dried seeds of this plant, well-known as Wang-Bu-Liu-Xing in traditional Chinese medicine, have been frequently used for promoting milk secretion, activating blood flow, relieving carbuncles and treating amenorrhea and breast infections in China [2–5]. Previous studies have demonstrated that a wide range of phytochemical compounds including flavonoids, triterpene saponins, cyclic peptides, alkaloids, phenolic acids and steroids were presented in the seeds of this herb, which have been considered being responsible for the beneficial efficacies on human health [1,6–8]. Among these compounds, flavonoids are a large group of diphenolic plant constituents, which have been reported to possess varieties of biological and pharmacological activities. Flavonoids are always occurring in combination with glucoses as glucosides with glucosidic linkages. Recently, vaccarin (seen in Fig. 1), a naturally occurring flavanoid glycoside from the seeds of V. segetalis has gained ever increasing attention because of its first appearance in ⇑ Corresponding author. Tel./fax: +86 451 82190535. 1

E-mail address: [email protected] (C.-B. Gu). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.seppur.2014.06.002 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.

Chinese Pharmacopoeia 2010 edition [9]. Present researches about V. seglistis are almost focused on botanical research and the quantitation of herbs [10,11]. However, there is little report available about efficient extraction technology of vaccarin from the seeds of V. seglistis. Microwave-assisted extraction (MAE) is a process of utilizing microwave energy to heat solvents in contact with a sample in order to partition analytes from the sample matrix into the solvent [12]. The applications of MAE of phytoconstituents from different plant materials have been considered as a potential alternative to conventional extraction techniques because of its many advantages, such as shorter extraction time, lower energy requirement and higher extraction efficiency [13–15]. Response surface methodology (RSM) is a collection of statistical techniques useful for developing, improving, and optimizing complex processes, the main advantage of which was to reduce number of experimental trials needed to evaluate multiple variables and their interactions [16]. To the best of our knowledge, there were no reports of systematic studies on the use of MAE to extract vaccarin from the seeds of V. seglistis. The objectives of the current study were to investigate the effects of MAE on the extraction efficiency of vaccarin from the seeds of V. seglistis and to optimize the extraction process using RSM. The extraction efficiency of vaccarin with MAE was compared with those obtained by three conventional extraction methods.

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2.3.3. Ultrasonic-assisted extraction (UAE) Based on the preliminary experiments, 1 g of seed powder of V. segetalis was accurately weighted and introduced into a conical flask with 50 mL methanol. The samples were then extracted by ultrasonic in an ultrasonic bath. The optimum extraction process was operated in triplicate at 40 °C for 1.5 h. Then, the subsequent process was the same as ME. 2.4. Microwave-assisted extraction (MAE)

Fig. 1. Chemical structure of vaccarin.

Furthermore, the antioxidant capacity of extracts, obtained under optimized MAE extraction conditions was tested by means of DPPH radical-scavenging assay. Our results would offer scientific reference for promoting the utilization of the seeds of V. seglistis. 2. Materials and methods 2.1. Plant materials The seeds of V. segetalis were purchased at Anguo, Hebei province (China) in 2011. The botanical identification was made by Prof. Shao-Quan Nie from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, PR China. Voucher specimens were deposited in the herbarium of the same laboratory. The samples were dried in the shade, powdered and passed through 60 mesh sieves to obtain fine power. 2.2. Chemicals and reagents Vaccarin was purchased from Mansite Medical Technology Co. Ltd. (Chengdu, China). Purity was >99% as checked by HPLC–UV. HPLC-grade methanol and phosphoric acid used for HPLC analysis were purchased from Dikma Company (Dikma, USA). Analyticalgrade methanol for extraction was bought from Tianjin Kermel Chemical Reagent Co. (Tianjin, China). Deionized water was purified by a Milli-Q water-purification system from Millipore (Bedford, MA, USA). 2.3. Conventional extraction procedures 2.3.1. Maceration extraction (ME) According to the preliminary optimized investigation, 1 g of seed powder of V. segetalis was accurately weighed and put into a beaker with 50 mL methanol, then the extraction was performed in triplicate at room temperature for 24 h to obtain the best extraction efficiency. After the extraction step, the extraction solutions obtained from the above experiments were filtered by a 0.45 lm filter membrane for HPLC analysis. 2.3.2. Heat reflux extraction (HRE) After preliminary optimization experiments, 1 g of seed powder of V. segetalis was accurately weighed and put into a round-bottom flask with 50 mL methanol, then the round-bottom flask was placed into a water bath and linked with a condenser at the joint of flask. The extraction was employed to optimum condition of 60 °C for 1 h with three replications to obtain the best extraction efficiency. Then, the subsequent process was the same as ME.

For MAE, 1 g of seed powder of V. segetalis was placed into the extraction vessel(Shanghai Sineo Chemical Equipment Technology Co. Ltd., China.) with 50 mL methanol. The initial conditions were as follows: temperature 55 °C, extraction time for 15 min, microwave power 500 W, liquid/solid ratio 50 mL/g with three replicates to evaluate the extraction efficiency. Then, the subsequent process was the same as ME. For the further optimization of MAE, the major influence factors were chosen differently according to the experimental design. 2.5. Extraction rate of vaccarin On the basis of the vaccarin content of prepared samples measured by the standard curve, the extraction rate of vaccarin was calculated by the equation. The extraction rate of vaccarin in sample was defined as follows:

Extraction rate of vaccarin ð%Þ ¼

CV  100%D 1000M

where C is the vaccarin concentration of the prepared sample (mg/ mL), V is the total volume of prepared sample (mL), D is total dilution value, M is the quality of raw material (g). 2.6. Determination of vaccarin content HPLC analysis was performed using a Jasco LC system (Jasco Company, Japan) equipped with a Jasco PU-1580 intelligent HPLC pump, a Jasco UV-1575 intelligent UV IS Detector as well as Millennium 32 system software. Chromatographic separation was performed on a HiQ Sil C18V reversed-phase column (250 mm  4.6 mm i.d., 5 lm, Kya Tech, Hachioji City, Japan). The UV detector was set at a wavelength of 332 nm. The mobile phase was methanol–water–phosphoric acid (30:69.4:0.6, v/v/v) at a flow rate of 1 mL/min, the injection volume was 10 lL, and the column temperature was maintained at 30 °C, respectively. Chromatographic peaks of the analytes were confirmed by comparing their retention time and UV spectrum with those of the reference compound. The working calibration curve based on reference compound of vaccarin showed good linearity over the range of 10.00–200.00 lg/ mL. The regression line for vaccarin was Y = 23,174X  79,544 (R2 = 0.9997, n = 3), where Y is the peak area of analyte, and X is the concentration of reference compound (lg/mL). 2.7. Optimization of MAE process After the comparison of extraction rate of vaccarin from seed powder of V. segetalis with different extraction methods, MAE process was further optimized with a Box-Behnken design (BBD) and response surface methodology (RSM). BBD is used to achieve maximal information about the process from a minimal number of possible experiments. It is needed to evaluate the effects and interactions of independent variables. In this design, four factors including methanol concentration (X1), temperature (X2), liquid/solid ration (X3) and microwave power

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Xi  X0 DX

xi ¼

ð1Þ

where xi is the (dimensionless) coded value of the variable Xi, X0 is the value of Xi at the center point, and DX is the step change. Table 1 shows the actual design of experiments. The behavior of the system was explained by the following second degree polynomial equation:

Y¼

X

A0 þ

4 4 3 X 4 X X X Ai X i þ Aii X 2i þ Aij X i X j i¼1

i¼1

ð2Þ

i¼1 j¼iþ1

where Y is the predicted response, namely the dependent variable, representing the extraction rate of vaccarin. Xi and Xj are the independent variables, which influenced the response variable Y representing different levels of each factor. A0, Ai, Aii and Aij are the regression coefficients for intercept, linearity, interaction and square, respectively. 2.8. DPPH radical-scavenging assay The DPPH radical-scavenging activity is based on the determination by DPPH at a steady state in methanol solution after adding the mixture of antioxidants. Following the detailed method of Liyana-Pathiranan and Shahidi [17], the extract was dissolved in 10 mL of absolute methanol to give a concentration of 2 mg/mL, then 1 mL of 0.004% DPPH (0.2 mM) in methanol was added to 0.1 mL of the extract solution. After shaking the mixture vigorously, the mixture was immediately placed in an UNICO UV-2100 spectrophotometer (UNICO, Shanghai, China) to monitor the decrease in absorbance at 517 nm until the reaction reached a plateau. Ascorbic acid (Sigma–Aldrich), a stable antioxidant, was used as a synthetic reference. The DPPH radical-scavenging activity in percentage of sample was calculated according to the following equation [18]:

Ihibiton percentage ðIpÞ ¼

ðAB  AA Þ100 AB

8.0.6’’ software. P values <0.05 were regarded as significant and P values <0.01 as very significant. 3. Results and discussion 3.1. Comparison of different extraction methods ME, HRE, UAE and MAE were, respectively, used to extract vaccarin from the seeds of V. segetalis, and comparison of the extraction rates of vaccarin with different extraction methods was shown in Fig. 2. As shown in Fig. 2, the extraction rate by UAE was obviously low, extraction rates by ME, HRE and MAE were higher and the results of the extraction completeness by ME and HRE depended to some extent on the extraction time, whereas that by MAE was almost independent on the extraction time. Moreover, MAE for 15 min gave the almost same result as HRE for 60 min did. Therefore, in this work, MAE was considered a simpler and more effective method for extraction of vaccarin from the seeds of V. segetalis and further optimized in the following tests. 3.2. Single-factor experiments 3.2.1. Effect of methanol concentration Different concentrations of solvent have an effect on the polarity of the solvent, so it is very important to find a optimal concentration in order to get a higher extraction rate. To study the effects of concentration of methanol on the extraction rate of vaccarin, different methanol concentrations of 50%, 60%,70%, 80%, and 90% were used, accompanied by a liquid/solid ratio 50 mL/g, extraction time 15 min, temperature 55 °C, microwave power 500 W by microwave treatment. Fig. 3A shows that the extraction rate of vaccarin increases when the concentration of methanol increased from 50% to 60%, and then decreases when the concentration of methanol was more than 60%. Therefore, the methanol concentration of 60% was considered to be optimal in the present experiment. 3.2.2. Effect of temperature To study the effect of temperature on the extraction rate of vaccarin, the extraction process was carried out under the condition of different temperature (25, 35, 45, 55, and 65 °C)by microwave treatment, while other extraction conditions were as follows: a liquid/solid ratio 50 mL/g, methanol concentration 60%, extraction time 15 min, microwave power 500 W. As can be seen

where AB and AA were the absorbance values of the blank and of the tested samples, respectively, checked after 70 min.

All experimental results were expressed as mean ± SD of three parallel measurements and all calculations were carried out with the help of the statistical package for the social science SPSS 11.5. Analysis of variance (ANOVA) was performed by ‘‘Design-expert

Table 1 Factors and levels of response surface methodology.

1 0 1

A Methanol concentration (%)

B Temperature (°C)

C Liquid/solid ratio (mL/g)

D Microwave power (W)

50 60 70

45 55 60

30 40 50

400 500 600

Extractional time (%)

2.9. Statistical analysis

1600

0.18

1400

0.16

1200

0.14 0.12

1000

0.1

800 0.08

600

0.06

400

0.04

200

0.02

0

Extraction rate of vaccarin (%)

(X4) were selected as independent variables in BBD based on the results of a series of preliminary experiments, and then RSM was conducted to design experimental project. Design-Expert software package was used to establish a mathematical model and obtain the optimum conditions of technological progress. The variables were coded according to the equation:

0 HRE

UAE

ME Extraction time(min)

MAE

Extraction rate(%) Fig. 2. Comparison of extraction rate of vaccarin from the seeds of V. segetalis with different extraction methods.

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increase of temperature from 25 to 65 °C. As there was no significant difference between the extraction rate at 55 and that at 65 °C, from the lower energy consumption point of view, the optimal extraction temperature was considered to be 55 °C in the present experiment. 3.2.3. Effect of liquid/solid ratio The extraction rate of vaccarin affected by different liquid/solid ratio (10, 20, 30, 40, and 50 mL/g) can be seen in Fig. 3C, while other factors were as follows: methanol concentration 60%, extraction time 15 min, and temperature 55 °C, microwave power 500 W. Fig. 3C shows that when the liquid/solid ratio increased from 10 to 50 mL/g, the extraction rate of vaccarin rose steadily, and then reached the critical value at 40 mL/g, and finally dropped from 40 to 50 mL/g. Therefore, the liquid/solid ratio 40 mL/g was chosen in the present experiment. 3.2.4. Effect of microwave power Different microwave power was set at 300, 400, 500, 600, and 700 W to investigate the influence of microwave power on the extraction rate of vaccarin when the other reaction conditions were set as follows: methanol concentration 60%, extraction time 15 min, temperature 55 °C, liquid/solid ratio 40 mL/g. Fig. 3D illustrates that the extraction rate of vaccarin was positively correlated with the increase of microwave power from 300 to 500 W, and it reached the critical value at 500 W. Therefore, taking into account both high extraction rate and energy consumption, 500 W was used for the extraction. 3.3. Optimization of the extraction progress 3.3.1. Mathematical model and optimization of extraction conditions According to the method of Box-Behnken design (BBD) experiment and the levels of independent variables were chosen based on the values obtained in the single factor experiment, methanol concentration (A, %, V/V), temperature (B, °C), liquid/solid ratio(C, mL/g),and microwave power (D, W) which have a great effect on the extraction rate of vaccarin were selected as design variables in the RSM, the extraction rate of vaccarin (Y, %) was employed as a response value, and a four factors and three levels’ RSM test was designed (Tables 1 and 2). Zero experiment was carried out in duplicate. Through performing in the form of analysis of ANOVA for the quadratic model, it was required to test the significance and adequacy of the model. Table 3 shows that the model of Prob > F (0.01), correlation coefficient (R2 = 0.9190), which means that it was a high precision and applicable model. All the values of Prob > F of B, C, D, AC, BC, A2 were lower than 0.01, indicating that the interaction among temperature, liquid/solid ratio and microwave power had a high significant influence on the extraction rate of vaccarin. The model established by regression equation can replace the experimental real point to explain response results. The regression equation was

Y ¼ 4:60  0:041A þ 0:15B þ 0:14C  0:087D þ 0:063AB  0:18AC  0:012AD þ 0:17BC  0:055BD  0:018CD  0:28A2 þ 0:1B2  081C 2  0:026D2

Fig. 3. Effect of methanol concentration (A), temperature (B), liquid/solid ratio (C), and microwave power (D) on the extraction rate of vaccarin.

in Fig. 3B, temperature significantly affects the extraction rate of vaccarin, and the extraction rate of vaccarin increased with the

The negative coefficients of A and D variables indicated that their negative changes can bring about reduction in response value; the positive coefficients of B and C variables revealed that their positive changes can cause increase in the response value; negative quadratic coefficient concluded that the opening of equation paraboloid was downward, which illuminated that it included maximum point and could carry out optimal analysis.

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X.-H. Yuan et al. / Separation and Purification Technology 133 (2014) 91–98 Table 2 The plan and results for response surface methodology. Experiments

A Methanol concentration (%)

B Temperature (°C)

C Liquid/solid ratio (mL/g)

D Microwave power (W)

Y Extraction rate of vaccarin (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

0 0 1 0 1 0 1 1 0 1 0 1 0 0 1 0 0 0 0 1 0 1 1 1 1 0 0 0 0

0 0 1 1 1 1 0 0 1 0 1 0 1 1 1 0 0 0 1 1 1 0 0 0 0 0 0 0 0

1 1 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 1 1 1 1 1 1 0 0 1

1 1 0 1 0 0 1 1 0 1 1 1 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 1

0.431 0.466 0.465 0.457 0.453 0.500 0.422 0.428 0.447 0.439 0.467 0.407 0.497 0.448 0.419 0.45 0.455 0.453 0.466 0.432 0.432 0.394 0.426 0.474 0.419 0.447 0.473 0.469 0.456

Table 3 Analysis of mean square deviation of regress equation. Source

Sum of squares

Degree of freedom

Mean square

F-value

Prob > F

Significant

Model A – methanol concentration B – temperature C – solid/liquid ratio D – power AB AC AD BC BD CD A2 B2 C2 D2

1.586161 0.02027 0.262151 0.233321 0.090874 0.015983 0.130633 0.000546 0.11658 0.011897 0.001276 0.524579 0.064624 0.042825 0.00452

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.113297 0.02027 0.262151 0.233321 0.090874 0.015983 0.130633 0.000546 0.11658 0.011897 0.001276 0.524579 0.064624 0.042825 0.00452

11.34266 2.029363 26.24502 23.35878 9.097799 1.600081 13.07822 0.054702 11.67129 1.191031 0.12778 52.51778 6.469826 4.287388 0.452514

<0.0001 0.1762 0.0002 0.0003 0.0092 0.2265 0.0028 0.8185 0.0042 0.2935 0.7261 <0.0001 0.0234 0.0574 0.5121

Significant

Residual Lack of fit Pure error Cor total

0.13984 0.099071 0.040769 1.726001

14 10 4 28

0.009989 0.009907 0.010192

0.972028

0.5614

By observing linear and quadratic coefficients, we conclude that the order of factors influencing the response value of the extraction rate of vaccarin was as follows: temperature > liquid/solid ratio > microwave power > methanol concentration. The effects of temperature, liquid/solid ratio and microwave power to response values were very significant (P < 0.01). By carrying out parameter optimization on the basis of the built mathematical model, obtained experimental conditions were: methanol concentration 56.67%, temperature 64.99 °C, liquid/solid ratio 50 mL/g, microwave power 400 W. Under the optimal progress, the predictive value of extraction rate of vaccarin was 0.519%.

Not significant

3.3.2. The interaction between the variables The graph of RSM was a 3D response surface plot consisting of response values of experimental variables (presented in Fig. 4). They can reflect the interaction between the variables (methanol concentration, temperature, liquid/solid ratio, and microwave power). Fig. 4A showed that when methanol concentration was at a certain value, the extraction rate of vaccarin increased with the temperature increased. However, when temperature was unchanged, the extraction rate of vaccarin rose and then declined as the methanol concentration increased. These data were consistent with the conclusion of the single factor test. Therefore, both temperature

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Fig. 4. Correlative effects of methanol concentration and temperature (A), methanol concentration and liquid/solid ratio (B), methanol concentration and microwave power (C), temperature and liquid/solid ratio (D), temperature and microwave power (E), and liquid/solid ratio and microwave power (F), on the extraction rate of vaccarin.

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X.-H. Yuan et al. / Separation and Purification Technology 133 (2014) 91–98 Table 4 The result of experimental verification.

Predict Experiment

Methanol concentration (%)

Temperature (°C)

Liquid/solid ratio (mL/g)

Microwave power (W)

Extraction rate of vaccarin (%)

56.67 57.00

64.99 65.00

50.00 50.00

400.00 400.00

0.519 0.523

of optimal analytical model. Adjusted extraction conditions were shown in Table 4. The result showed that the experimental values were not only consistent with the predictive values, but also were better than any single factor experiments. Therefore, the extraction conditions obtained by response surface methodology were not only accurate and reliable, but also with practical value reflecting the expected optimization.

Free radical-scavenging activity (%)

100 90 80 70 60

VC

50 Methanol extract

40

3.4. Free radical-scavenging activity

30 20 10 0

0

0.5

1

1.5

2

Concentration (mg/ml) Fig. 5. Free radical-scavenging activity of optimal MAE extract of the seeds of V. segetalis. VC: ascorbic acid.

and methanol concentration would have independent optimum condition parameters. It can be seen from Fig. 4B that when methanol concentration was at a certain value, the extraction rate of vaccarin increased with liquid/solid ratio increased. When liquid/solid ratio was unchanged, the extraction rate of vaccarin rose and then declined with the methanol concentration extended. The best point of balance should be sought for the maximum extraction rate of vaccarin between methanol concentration and liquid/solid ratio. From Fig. 4C, it can be seen that when methanol concentration was at a certain value, the extraction rate of vaccarin declined with the microwave power increased from 400 to 600 W. When the microwave power did not vary, the extraction rate of vaccarin rose and then declined with the methanol concentration extended. It can be seen from Fig. 4D that when temperature was 60 °C, the extraction rate of vaccarin obviously increased with the liquid/solid ratio added from 30 to 50 mL/g. In addition, when the liquid/solid ratio was 50 mL/g, the extraction rate of vaccarin was significantly increased with temperature gradually rose from 45 to 65 °C. It was concluded that both temperature and liquid/solid ratio significantly affected on the extraction rate of vaccarin, and the maximum extraction rate of vaccarin would be sought when temperature and liquid/solid ratio were all fixed at high values. Generally speaking, when microwave power was at a certain value, the extraction rate of vaccarin increased with the temperature increased (Fig. 4E). However, when temperature was unchanged, the extraction rate of vaccarin gradually declined with the microwave power increased from 400 to 600 W. Both temperature and microwave power had independent optimum condition parameters. It can be seen from Fig. 4F that when microwave power was at a certain value, the extraction rate of vaccarin obviously increased as the liquid/solid ratio added. However, when the liquid/solid ratio was unchanged, the extraction rate of vaccarin was gradually decreased with the increase of microwave power.

Radical-scavenging activity of the MAE extract was tested using DPPH as the free radical. The reduction of the DPPH absorbance at 517 nm after 70 min incubation was measured (Fig. 5). The concentration of sample producing a 50% reduction of the radical absorbance (IC50) was used as an index to compare the antioxidant activity. The MAE extract exhibited DPPH radical-scavenging activity with an IC50 value of 0.48 mg/mL, which was weaker than ascorbic acid, the positive control (IC50 0.23 mg/mL). The results indicated that the MAE extract of the seed powder of V. segetalis has a certain level of radical scavenging activity and has the potential to be used in food industry, pharmaceutical industry and other related fields.

4. Conclusions MAE was found to be possessed higher efficiency for the extraction of vaccarin from the seeds of V. segetalis as compared with traditional extraction methods. MAE process was optimized with response surface methodology. The maximum extraction rate of vaccarin reached 0.52%, under the optimal conditions: methanol concentration 57%, temperature 65 °C, liquid/solid ratio (50 mL/ g), microwave power 400 W, and under the optimized conditions, the experimental values agreed with the predicted values by analysis of variance. Meanwhile, the MAE extract showed radical-scavenging activity with an IC50 value 0.48 mg/mL. The results indicated that MAE extract of the seeds of V. segetalis may play a potential role as health-promoting antioxidant agent in human diets for the pharmaceutical industry. However, further studies concerning the nutritional and health benefits are required before a large scale utilization of the seeds of V. segetalis can be recommended. Acknowledgements This work was supported by the Fundamental Research Funds for the Central Universities (2572014EA02 and DL13CA02), the Opening Project of Key Laboratory of Agricultural Biological Functional Genes in Heilongjiang Higher Education (NSGJ2012-02) and the Scientific Research Fund of Heilongjiang Provincial Education Department (NO: 12541051). References

3.3.3. Verification of predictive model To further test the reliability of the experimental method, the extraction experiment was carried out by adopting the program

[1] S.M. Sang, Z.H. Xia, A. Lao, L. Cao, Z. Chen, J. Uzawa, Y.A. Fujimono, Studies on the constituents of the seeds of Vaccaria segetalis, Heterocycles 59 (2003) 811– 821.

98

X.-H. Yuan et al. / Separation and Purification Technology 133 (2014) 91–98

[2] G. Mazza, Compositional and morphological characteristics of cow cockle (Saponaria vaccaria) seed, a potential alternative crop, J. Agric. Food Chem. 40 (1992) 1520–1533. [3] S.M. Sang, A.N. Lao, H.C. Wang, Z.L. Chen, J. Uzawa, Y.A. Fujimoto, Phenylpropanoid glycoside from Vaccaria segetalis, Phytochemistry 48 (1998) 569–574. [4] S. Sang, A.N. Lao, H.C. Wang, Z.L. Chen, J. Uzawa, Y.A. Fujimoto, Triterpenoid saponins from Vaccaria segetalis, J. Asian Nat. Prod. Res. 1 (1999) 199–205. [5] S.M. Sang, Z.H. Xia, S.L. Mao, A. Lao, Z.L. Chen, Studies on the flavonol glycosides from the seeds of Vaccaria segetalis, China J. Chin. Mater. Med. 25 (2000) 221–222. [6] H. Morita, Y.S. Yun, K. Takeya, H. Itokawa, K. Yamada, Tetrahedron 51 (1995) 6003. [7] R.P. Zhang, C. Zou, N.H. Tan, J. Zhou, Cyclopeptides from Vaccaria segetalis, Acta Botanica Yunnanica 20 (1998) 105–112. [8] K. Kazuo, Z.H. Jia, N. Tamotsu, Triterpenoid saponins from Vaccaria segetalis, Phytochemistry 47 (1998). 1343-134. [9] Chinese Pharmacopoeia Committee, Chinese Pharmacopoeia, 2010 ed., China Medical Science and Technology Press, Beijing, 2010. [10] H. Meng, Y. Chen, W. Qin, X. Tang, Z. Ye, Determination of vaccarin in Vaccariae Semen by HPLC, Zhong Guo Zhong Yao Za Zhi. 35 (2010) 2072–2074.

[11] H.J. Zhang, Profilling analysis of the seeds of Vaccaria segetalis (Necr.) Gracke by HPLC–ESI–MS, Adv. Mater. Res. 96 (2012) 396–398. [12] H.Y. Li, Z.Y. Deng, T. Wu, R.H. Liu, S. Loewen, R. Tsao, Microwave-assisted extraction of phenolics with maximal antioxidant activities in tomatoes, Food Chem. 130 (2012) 928–936. [13] T.S. Ballard, P. Mallikarjunan, K.Q. Zhou, S. O’Keefe, Microwave-assisted extraction of phenolic antioxidant compounds from peanut skins, Food Chem. 120 (2010) 1185–1192. [14] P.L. Buldini, L. Ricci, J.L. Sharma, Recent applications of sample preparation in food analysis, J. Chromatogr., A 975 (2002) 47–70. [15] C.S. Eskilsson, E. Bjorklund, Analytical-scale microwave-assisted extraction, J. Chromatogr., A 902 (2000) 227–250. [16] A. Dean, D. Voss, Design and Analysis of Experiments, Springer, New York Berlin Heidelberg, 1999. pp. 561–564. [17] C.M. Liyana-Pathirana, F. Shahidi, Antioxidant activity of commercial soft and hard wheat (Triticum aestivum L.) as affected by gastric pH conditions, J. Agric. Food Chem. 53 (2005) 2433–2440. [18] G.C. Yen, P.D. Duh, Scavenging effect of methanolic extracts of peanut hulls on free-radical and active-oxygen species, J. Agric. Food Chem. 42 (1994) 629– 632.