International Journal of Biological Macromolecules 69 (2014) 523–531
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Extraction, antioxidant and antilisterial activities of polysaccharides from the flower of viper’s bugloss Saeed Tahmouzi ∗ Department of Food Science and Technology, Faculty of Animal Science and Food Technology, Ramin Agriculture and Natural Resources University, Mollasani, Ahvaz, Iran
a r t i c l e
i n f o
Article history: Received 13 March 2014 Received in revised form 16 May 2014 Accepted 5 June 2014 Available online 17 June 2014 Keywords: Optimization Echium vulgare Polysaccharide Antioxidant Antilisterial
a b s t r a c t The objective of the present research was to investigate the effect of microwave-assisted extraction (MAE) conditions on the extraction yield, antioxidant properties and antilisterial activities of the polysaccharides from the flowers of viper’s bugloss (Echium vulgare L.). The four extraction variables, time (40–100 min), microwave power (200–800 W), temperature (30–70 ◦ C), and the ratio of water to raw material (10–70), were optimized using response surface methodology. The experimental data were matched to a second-order polynomial equation. The optimal conditions for MAE of polysaccharides (EVFP) were time 73.8 min, microwave power 769.2 W, temperature 42.3 ◦ C and the ratio of water to raw material 61.4, where the actual yield of EVFP 25.11 ± 0.87% was obtained (versus the value predicted by the model 25.36%). The results indicated that EVFP has significant radical (·OH and ·DPPH) scavenging abilities in vitro assay. Moreover, the antilisterial activity was confirmed against four species of Listeria. EVFP, at a concentration of 5 mg/mL, demonstrated great antilisterial properties against Listeria ivanovii and Listeria monocytogenes, with inhibition zones of 10.76 ± 0.32 mm and 8.64 ± 0.47 mm, respectively. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Echium vulgare, commonly known as viper’s bugloss or blueweed, indigenous of North America, Europe, and Asia (the zone of western part of Iran including Azerbaijan and Kermanshah) [1]. The leaves and the flowering stems of Viper’s bugloss are popularly employed for the treatment of fissures on hands, wound healing, snakebites. It is known as a diuretic, pectoral, aphrodisiac, demulcent and vulnerary agent [2]. Blueweed oil from the seeds of E. vulgare has been reported as a natural source of polyunsaturated fatty acids (PUFA). This higher ␥-linolenic/saturated acid ratio has been associated with a decrease of the pathogenesis of many disorders such as cancer, autoimmune disease, inflammatory diseases, and cardiovascular abnormalities [3–8]. There are some reports about medicinal properties of Echium. Rosmarinic acid is significantly found in species of the Echium (e.g. viper’s bugloss). It has good anti-inflammatory, analgesic and antioxidant activities [9–12]. In past decades, the popularity of plant extracts has increased significantly duo to its anti-fungal, anti-bacterial, anti-tumor and potential antioxidant activities. Nowadays more and more
∗ Tel.: +98 6123224341; fax: +98 6123222425. E-mail addresses: [email protected], saeed [email protected] http://dx.doi.org/10.1016/j.ijbiomac.2014.06.008 0141-8130/© 2014 Elsevier B.V. All rights reserved.
attention was cast on polysaccharides for researchers because of its unique biological properties employed in medicine or functional foods, especially antioxidant, anti-microbial, and anti-cancer features [13–18]. The conventional method applied to obtain polysaccharides from plants is a hot reflux technique with the multiple step process and tedious time to achieve the high yield. In recent years, scientists have studied the efficacy of different methods for extraction of polysaccharides from plant parts (leaves, seeds, flowers and roots). The alkaline extraction and ultrasound-assisted extraction of polysaccharides have been evaluated [19,20]. Microwave-assisted extraction (MAE) is an alternative method to extract the polysaccharides from biological materials, which has advantages such as simple operation, shorter extraction time, flexibility, energy consumption and higher efficacy. Previous publications show technical improvements in extraction of polysaccharides from the leaves and flowers of plants due to the use of MAE. Microwave facilitates the mass transfer of polysaccharides from natural plant material and transport to the solvent medium. Thus, it has been widely employed to extract polysaccharides from different materials with highest extraction yield [21]. Some chronic disorders including diabetes, and cardiovascular abnormalities, can be obviously induced by free radicals (·OH and ·DPPH). Crude polysaccharides from various plants are known to scavenge radicals and to play a considerable role against various
524
S. Tahmouzi / International Journal of Biological Macromolecules 69 (2014) 523–531
diseases (cancer, atherosclerosis and aging) [22]. The radical scavenging ability is often used to investigate the activity of antioxidant compounds [23]. Recent studies suggested that the antioxidant activity of polysaccharides was related to their molecular weight, structure and degree of polymerization [24]. Listeria is recognized as a food-borne human pathogen, causing serious infections, and 30% mortality in patients. This genus of bacteria currently contains ten species. In developing countries, listerial infections are commonly observed especially in rural areas because of unhygienic conditions [25,26]. Until now, there has been no information regarding the antilisterial activities of crude polysaccharides from the flowers of E. vulgare. Response surface methodology is a simple, rapid and powerful approach for understanding interactions among several variables using a minimal number of trials. RSM is a collection of mathematical techniques that is helpful for designing experiments, building models, evaluating the influences of various parameters and searching for optimal conditions of studied variables for desirable responses [27]. Response surface methodology has been used to develop and optimize the MAE process of polysaccharides from the leaves, roots and flowers of plants. Up to now, there were no detailed reports available in the literature regarding the optimization of microwave-assisted extraction of crude polysaccharides from the flowers of E. vulgare by RSM. In this paper, the extraction parameters (extraction time, microwave power, extraction temperature, and the ratio of water to raw material) were optimized by employing response surface methodology for maximum extraction yield of EVFP. Antioxidant and antilisterial activities of EVFP were studied by various in vitro assays. 2. Materials and methods 2.1. materials Viper’s bugloss flowers were collected from natural sites located in the western part of Iran, washed, air-dried at ambient temperature and then powdered for this research. DPPH and vitamin C were obtained from the Sigma Chemical Co. (St. Louis, MO, USA). All other reagents and solvents used in this paper were of analytical grade and purchased from the Merck Co., Germany. Ultra-pure water was applied throughout the experiments. 2.2. Microwave-assisted extraction of crude polysaccharides from E. vulgare flowers The extraction of EVFP was conducted by the method of Dubois with minor modifications [28]. Briefly, the flowers of Viper’s bugloss were dried at 70 ◦ C for 12 h, powdered and then refluxed in 75% ethanol for 8 h. The extract was discarded and the residue was treated with purified ethanol and acetone, respectively, to remove interference components including free sugars, amino acids and polyphenols. Protein was carefully removed as reported previously [29]. The residue was dried at ambient temperature (25 ◦ C) for 18 h. Pretreated Viper’s bugloss powders (15 g) were immersed with distilled water in the microwave equipment (NJC 03-2, Microwave Experiment Equipment, Nanjing, China) to be extracted under different MAE conditions. After extraction, the vessel was allowed to cool at room temperature. The suspension was centrifuged (4500 × g, 10 min) and the insoluble residue was treated again for 2 times as mentioned above. The supernatant was concentrated to one-fifth of the initial volume employing a rotary evaporator at 60 ◦ C under vacuum. It was precipitated by the addition of ethanol to a final concentration of 80% (v/v). After being washed three times with ethanol, the precipitate was freez-dried to obtain EVFP. The content of the polysaccharides (composed of 25.1% acid
Table 1 Experimental domain of Box–Behnken design (BBD). Factor levels
Independent variables
Extraction time (min) Microwave power (W) Extraction temperature (◦ C) Ratio of water to raw material
−1
0
+1
40 200 30 10
70 500 50 40
100 800 70 70
sugar, 68.8% neutral sugar and 1.2% protein) was measured by phenol–sulfuric method. The polysaccharides extraction yield (%) is measured as follows: EVFP extraction yield % (w/w) =
CO C
(1)
where CO (g) is the dried EVFP weight; C (g) is the dried powder of E. vulgare flower weight. 2.3. Experimental design A number of variables such as extraction time, microwave power, extraction temperature and the ratio of water to raw material can significantly affect the polysaccharide extraction yield. Therefore, a Box–Behnken design (BBD) was used to identify the relationship between the response function (the yield of EVFP) and the process parameters (time, microwave power, temperature and the ratio of water to raw material). The experimental range of the selected process variables is shown in Table 1. The response variable can be expressed as a function of the independent process factors according to the following response surface quadratic model: Y = ˇ0 + ˇ1 X1 + ˇ2 X2 + ˇ3 X3 + ˇ4 X4 + ˇ11 X12 + ˇ22X22 + ˇ33 X32 + ˇ44 X42 + ˇ12 X12 + ˇ13 X13 + ˇ14 X14 + ˇ23 X23 + ˇ24 X24 + ˇ34 X34 (2) The coefficients of the second-order polynomial model were represented by ˇ0 (constant coefficient), ˇ1 , ˇ2 , ˇ3 and ˇ 4 (linear effects), ˇ11 , ˇ22 , ˇ33 and ˇ44 (quadratic effects), and ˇ12 , ˇ13 , ˇ14 , ˇ23 , ˇ24 and ˇ34 (interaction effects). In this four variable BBD, three levels are attributed to each variable (−1, 0, +1) and a set of 30 trials is carried out (Table 2). 2.4. Assay of antioxidant activity in vitro of EVFP 2.4.1. Assay of scavenging activity on hydroxyl free radical The hydroxyl radical scavenging ability of EVFP was determined by the method of Jin with slight modifications [30]. The hydroxyl radical was produced in a mixture of 1.0 mL of 1 mM 1,10phenanthroline, 4.0 mL of 0.1 M sodium phosphate buffer (pH 7.4), 1.0 mL of 1 mM ferric sulfate and 1.0 mL of hydrogen peroxide. After addition of 1.0 mL sample solution in different concentrations (1, 2, 3, 4, 5 and 6 mg/mL), the mixture was incubated at ambient temperature for 25 min. Then, the absorbance of the mixture was measured at 536 nm. Ascorbic acid (vitamin C) was applied as positive control. The scavenging activity on ·OH was expressed as following: Scavenging effect (%) =
Z0 − Z1 × 100(%) Z0
(3)
where Z0 and Z1 are the absorbance of control (without sample) and EVFP, respectively. 2.4.2. Assay of scavenging activity on DPPH free radical The scavenging ability on DPPH free radical was measured by employing the published method with minor modifications [31].
S. Tahmouzi / International Journal of Biological Macromolecules 69 (2014) 523–531
525
Table 2 BBD with the observed responses and predicted values for yield of EVFP (%). Run
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 30
X1 (time)
70 70 70 100 70 70 40 70 70 40 100 70 70 70 100 70 40 100 70 40 70 70 100 40 70 70 70 100 70 40
X2 (microwave power)
500 800 500 500 200 200 500 800 500 500 200 500 500 800 500 800 500 800 500 500 500 500 500 200 200 500 500 500 200 800
X3 (temperature)
50 30 50 70 70 50 70 50 30 50 50 70 30 70 50 50 30 50 50 50 50 70 50 50 30 50 50 30 50 50
Briefly, 0.5 mL DPPH. solution (250 mol/L in dehydrate alcohol) was added to 4.0 mL of EVFP solution with different concentrations (1, 2, 3, 4, 5 and 6 mg/mL), and the mixture was incubated at 25 ◦ C for 40 min in the dark. Then, the absorbance was recorded at 517 nm. Butylated hydroxytoluene (BHT) was used as the positive control, and the scavenging activity was calculated according to the following equation: Scavenging capacity (%) =
S0 − S1 × 100 (%) S0
(4)
where S0 and S1 are the absorbance of control (without sample) and EVFP, respectively. 2.5. Determination of antilisterial activity of EVFP Four species of Listeria (L. seeligeri, L. monocytogenes, L. ivanovii and L. marthii) were selected as test bacteria for this research. The microorganisms were purchased from the Microbiology Laboratory of Razi institute, Iran. Antilisterial activity was measured by the filter disc diffusion plate method and the agar dilution technique [32,33]. The crude polysaccharides (1, 2, 3, 4, 5 and 6 mg/mL) were dissolved in a minimum amount of dimethyl sulfoxide and added to the nutrient medium. A suspension of an overnight culture of each test bacteria containing 106 cells/mL was added to the medium. The result medium was then stored in an incubator (37 ◦ C; 24 h). The diameter of the inhibition zones by the disc diffusion method was evaluated for the agar dilution test to assay the antilisterial effect more precisely. Nutrient agar served as a control. 2.6. Statistical analysis All the data were expressed as the means ± standard deviations within significance p-values of less than 0.05 after subjecting to an analysis of variance (ANOVA) and processed with SPSS 13.0.
X4 (W/R ratio)
40 40 40 40 40 70 40 10 70 10 40 70 10 40 70 70 40 40 40 70 40 10 10 40 40 40 40 40 10 40
Extraction yield (%) Actual
Predicted
23.11 18.91 24.01 9.10 16.32 19.10 16.41 15.42 21.77 14.02 15.93 14.12 11.82 11.11 18.21 24.32 11.76 16.93 23.14 16.23 23.81 14.89 8.03 15.15 20.44 22.94 23.33 15.55 19.43 20.19
23.56 19.21 23.56 9.34 16.17 19.35 16.49 15.31 21.85 14.44 16.12 14.48 11.60 11.29 17.98 24.66 12.02 16.77 23.56 16.36 23.56 15.12 7.94 15.01 20.79 23.56 23.56 15.11 19.85 20.36
3. Results and discussion 3.1. Extraction yield of EVFP 3.1.1. Effect of different times on extraction yield of EVFP Extraction time will obviously affect the extraction yield of polysaccharides from Viper’s bugloss flower. Therefore, it is very important to choose an optimum time for extraction of EVFP. In the present work, it was set at 30, 40, 50, 60, 70, 80, 90 and 100 min to evaluate the effect of extraction time on the yield of polysaccharide while other extraction variables were fixed as the followings: microwave power 500 W, extraction temperature 50 ◦ C and the ratio of water to raw material 40. It was observed that the polysaccharide yield was increased significantly with increasing extraction time from 30 to 100 min (Fig. 1a), and it reached 21.35 ± 0.6% when the extraction time was 100 min. Therefore, 30–100 min was selected in the present work. The extraction coefficient increased with increasing the extraction time because of the increase of the EVFP solubility. This was matched closely with results of other authors in extracting polysaccharides [34,35].
3.1.2. Effect of different microwave powers on extraction yield of EVFP The extraction yield (%) of EVFP extracted by different microwave powers from 200 to 1000 W was shown in Fig. 1b. The extraction time, extraction temperature and the ratio of water to raw material were fixed at 70 min, 50 ◦ C and 40, respectively. The extraction yields of the polysaccharide significantly increased from 7.2% to 20.3% (w/w) as microwave power increased from 200 to 800 W. However, when the power continued to increase (from 800 to 1000 W), the extraction yield decreased to 18.5%, because of increasing driving force for the mass transfer of other chemicals and thermal degradation of the crude polysaccharides. Similar
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Fig. 1. Effects of different (a) extraction times, (b) microwave powers, (c) extraction temperatures, and (d) the ratio of water to raw material on the extraction yield of EVFP.
results have been reported in other researches [36]. Thus, 800 W was chosen as the optimum microwave power. 3.1.3. Effect of different temperatures on extraction yield of EVFP The use of the various temperatures during the extraction process, helping to obtain good extraction yield. The temperature is an important parameter which affects the efficiency of polysaccharides during hot water extraction [37,38]. The influence of extraction temperature on the yield of EVFP is shown in Fig. 1c. Extraction was carried out at different temperatures (30, 40, 50, 60, 70 and 80 ◦ C) when other extraction variables (extraction time (70 min), microwave power (500 W), and the ratio of water to raw material (40)) were fixed. The extraction yields of EVFP significantly increased from 13.6% to 19.8% as extraction temperature increased from 30 to 70 ◦ C. However, when the temperature continued to increase, the extraction yields declined to 18.4%. The highest extraction yield was obtained when the temperature was 70 ◦ C. Therefore, extraction temperature of 70 ◦ C was adopted in the present study.
According to the single-parameter study, it was employed extraction time 40–100 min, microwave power 200–800 W, extraction temperature 30–70 ◦ C, and the ratio of water to the raw material 10–70 for response surface methodology experiments. 3.2. Data analysis and evaluation of the model Four parameters, including X1 (extraction time), X2 (microwave power), X3 (extraction temperature), and X4 (the ratio of water to raw material) were selected and separately optimized by Box–Behnken design. Table 2 illustrated the BBD matrix together with the responses (extraction yields) obtained. The yield of EVFP ranged from 8.03 to 24.32%, and reached maximum with the extraction time 70 min, microwave power 800 W, temperature of 50 ◦ C and the ratio of water to raw material 70. By employing multiple regression analysis on the experimental data, the test factors and response were found to correlate by the following second-order polynomial equation: Y = 23.56 − 1.47X3 + 2.53X4 − 2.56X1 X3 + 2.03X1 X4 + 2.46X2 X4
3.1.4. Effect of different ratios of water to the raw material on extraction yield of EVFP The effect of different ratios of water to the raw material on the yield of EVFP was presented in Fig. 1d. Extraction was performed at different ratios (10, 20, 30, 40, 50, 60, 70 and 80), while other extraction variables were fixed as follows: extraction time of 70 min, microwave power of 500 W and extraction temperature of 50 ◦ C. The results indicated that the extraction yield of EVFP was significantly increased to the value (21.6%) with an increase in the water to raw material ratios, and the efficiency was declined when the ratio was more than 70. So, the optimum ratio of 70 was finally employed in this study.
− 2.72X3 X4 − 5.69X12 − 5.01X32 − 3.06X42
(5)
where Y is the extraction yield of EVFP (%), and X1 , X2 , X3 and X4 are the coded variables for extraction time, microwave power, extraction temperature, and the ratio of water to raw material, respectively. Table 3 demonstrates the results of the ANOVA, fitness and the adequacy of the model. The F-value of 14.35 and p-value less than 0.0001 represented the response surface quadratic model was significant. The corresponding factors become more significant as the p-value becomes smaller and the F-value becomes larger. Thus, the p-value can be applied to study the interaction strength between each independent parameter [39]. In the present case,
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Table 3 Analysis of variance for the fitted models. Source
Sum of squares
Degree of freedom
Mean square
F value
p-Value Prob > F
Model X1 X2 X3 X4 X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4 X1 2 X2 2 X3 2 X4 2 Residual Lack of fit Pure error Cor total R-squared Adj R-squared Pred R-squared Adeq precision C.V. %
586.10 10.87 80.32 26.08 77.11 5.52 26.21 16.48 2.72 24.26 29.65 222.88 7.84 171.23 64.35 43.77 43.77 0.21 629.87 0.961 0.905 0.882 13.71 6.69
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 10 5 29
41.86 10.87 80.32 26.08 77.11 5.52 26.21 16.48 2.72 24.26 29.65 222.88 7.84 171.23 64.35 2.92 4.38 0.08
14.35 3.72 2.74 8.94 26.42 1.89 8.98 5.65 0.93 8.31 10.16 76.03 2.69 58.67 22.05
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Extraction, antioxidant and antilisterial activities of polysaccharides from the flower of viper’s bugloss Saeed Tahmouzi ∗ Department of Food Science and Technology, Faculty of Animal Science and Food Technology, Ramin Agriculture and Natural Resources University, Mollasani, Ahvaz, Iran
a r t i c l e
i n f o
Article history: Received 13 March 2014 Received in revised form 16 May 2014 Accepted 5 June 2014 Available online 17 June 2014 Keywords: Optimization Echium vulgare Polysaccharide Antioxidant Antilisterial
a b s t r a c t The objective of the present research was to investigate the effect of microwave-assisted extraction (MAE) conditions on the extraction yield, antioxidant properties and antilisterial activities of the polysaccharides from the flowers of viper’s bugloss (Echium vulgare L.). The four extraction variables, time (40–100 min), microwave power (200–800 W), temperature (30–70 ◦ C), and the ratio of water to raw material (10–70), were optimized using response surface methodology. The experimental data were matched to a second-order polynomial equation. The optimal conditions for MAE of polysaccharides (EVFP) were time 73.8 min, microwave power 769.2 W, temperature 42.3 ◦ C and the ratio of water to raw material 61.4, where the actual yield of EVFP 25.11 ± 0.87% was obtained (versus the value predicted by the model 25.36%). The results indicated that EVFP has significant radical (·OH and ·DPPH) scavenging abilities in vitro assay. Moreover, the antilisterial activity was confirmed against four species of Listeria. EVFP, at a concentration of 5 mg/mL, demonstrated great antilisterial properties against Listeria ivanovii and Listeria monocytogenes, with inhibition zones of 10.76 ± 0.32 mm and 8.64 ± 0.47 mm, respectively. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Echium vulgare, commonly known as viper’s bugloss or blueweed, indigenous of North America, Europe, and Asia (the zone of western part of Iran including Azerbaijan and Kermanshah) [1]. The leaves and the flowering stems of Viper’s bugloss are popularly employed for the treatment of fissures on hands, wound healing, snakebites. It is known as a diuretic, pectoral, aphrodisiac, demulcent and vulnerary agent [2]. Blueweed oil from the seeds of E. vulgare has been reported as a natural source of polyunsaturated fatty acids (PUFA). This higher ␥-linolenic/saturated acid ratio has been associated with a decrease of the pathogenesis of many disorders such as cancer, autoimmune disease, inflammatory diseases, and cardiovascular abnormalities [3–8]. There are some reports about medicinal properties of Echium. Rosmarinic acid is significantly found in species of the Echium (e.g. viper’s bugloss). It has good anti-inflammatory, analgesic and antioxidant activities [9–12]. In past decades, the popularity of plant extracts has increased significantly duo to its anti-fungal, anti-bacterial, anti-tumor and potential antioxidant activities. Nowadays more and more
∗ Tel.: +98 6123224341; fax: +98 6123222425. E-mail addresses: [email protected], saeed [email protected] http://dx.doi.org/10.1016/j.ijbiomac.2014.06.008 0141-8130/© 2014 Elsevier B.V. All rights reserved.
attention was cast on polysaccharides for researchers because of its unique biological properties employed in medicine or functional foods, especially antioxidant, anti-microbial, and anti-cancer features [13–18]. The conventional method applied to obtain polysaccharides from plants is a hot reflux technique with the multiple step process and tedious time to achieve the high yield. In recent years, scientists have studied the efficacy of different methods for extraction of polysaccharides from plant parts (leaves, seeds, flowers and roots). The alkaline extraction and ultrasound-assisted extraction of polysaccharides have been evaluated [19,20]. Microwave-assisted extraction (MAE) is an alternative method to extract the polysaccharides from biological materials, which has advantages such as simple operation, shorter extraction time, flexibility, energy consumption and higher efficacy. Previous publications show technical improvements in extraction of polysaccharides from the leaves and flowers of plants due to the use of MAE. Microwave facilitates the mass transfer of polysaccharides from natural plant material and transport to the solvent medium. Thus, it has been widely employed to extract polysaccharides from different materials with highest extraction yield [21]. Some chronic disorders including diabetes, and cardiovascular abnormalities, can be obviously induced by free radicals (·OH and ·DPPH). Crude polysaccharides from various plants are known to scavenge radicals and to play a considerable role against various
524
S. Tahmouzi / International Journal of Biological Macromolecules 69 (2014) 523–531
diseases (cancer, atherosclerosis and aging) [22]. The radical scavenging ability is often used to investigate the activity of antioxidant compounds [23]. Recent studies suggested that the antioxidant activity of polysaccharides was related to their molecular weight, structure and degree of polymerization [24]. Listeria is recognized as a food-borne human pathogen, causing serious infections, and 30% mortality in patients. This genus of bacteria currently contains ten species. In developing countries, listerial infections are commonly observed especially in rural areas because of unhygienic conditions [25,26]. Until now, there has been no information regarding the antilisterial activities of crude polysaccharides from the flowers of E. vulgare. Response surface methodology is a simple, rapid and powerful approach for understanding interactions among several variables using a minimal number of trials. RSM is a collection of mathematical techniques that is helpful for designing experiments, building models, evaluating the influences of various parameters and searching for optimal conditions of studied variables for desirable responses [27]. Response surface methodology has been used to develop and optimize the MAE process of polysaccharides from the leaves, roots and flowers of plants. Up to now, there were no detailed reports available in the literature regarding the optimization of microwave-assisted extraction of crude polysaccharides from the flowers of E. vulgare by RSM. In this paper, the extraction parameters (extraction time, microwave power, extraction temperature, and the ratio of water to raw material) were optimized by employing response surface methodology for maximum extraction yield of EVFP. Antioxidant and antilisterial activities of EVFP were studied by various in vitro assays. 2. Materials and methods 2.1. materials Viper’s bugloss flowers were collected from natural sites located in the western part of Iran, washed, air-dried at ambient temperature and then powdered for this research. DPPH and vitamin C were obtained from the Sigma Chemical Co. (St. Louis, MO, USA). All other reagents and solvents used in this paper were of analytical grade and purchased from the Merck Co., Germany. Ultra-pure water was applied throughout the experiments. 2.2. Microwave-assisted extraction of crude polysaccharides from E. vulgare flowers The extraction of EVFP was conducted by the method of Dubois with minor modifications [28]. Briefly, the flowers of Viper’s bugloss were dried at 70 ◦ C for 12 h, powdered and then refluxed in 75% ethanol for 8 h. The extract was discarded and the residue was treated with purified ethanol and acetone, respectively, to remove interference components including free sugars, amino acids and polyphenols. Protein was carefully removed as reported previously [29]. The residue was dried at ambient temperature (25 ◦ C) for 18 h. Pretreated Viper’s bugloss powders (15 g) were immersed with distilled water in the microwave equipment (NJC 03-2, Microwave Experiment Equipment, Nanjing, China) to be extracted under different MAE conditions. After extraction, the vessel was allowed to cool at room temperature. The suspension was centrifuged (4500 × g, 10 min) and the insoluble residue was treated again for 2 times as mentioned above. The supernatant was concentrated to one-fifth of the initial volume employing a rotary evaporator at 60 ◦ C under vacuum. It was precipitated by the addition of ethanol to a final concentration of 80% (v/v). After being washed three times with ethanol, the precipitate was freez-dried to obtain EVFP. The content of the polysaccharides (composed of 25.1% acid
Table 1 Experimental domain of Box–Behnken design (BBD). Factor levels
Independent variables
Extraction time (min) Microwave power (W) Extraction temperature (◦ C) Ratio of water to raw material
−1
0
+1
40 200 30 10
70 500 50 40
100 800 70 70
sugar, 68.8% neutral sugar and 1.2% protein) was measured by phenol–sulfuric method. The polysaccharides extraction yield (%) is measured as follows: EVFP extraction yield % (w/w) =
CO C
(1)
where CO (g) is the dried EVFP weight; C (g) is the dried powder of E. vulgare flower weight. 2.3. Experimental design A number of variables such as extraction time, microwave power, extraction temperature and the ratio of water to raw material can significantly affect the polysaccharide extraction yield. Therefore, a Box–Behnken design (BBD) was used to identify the relationship between the response function (the yield of EVFP) and the process parameters (time, microwave power, temperature and the ratio of water to raw material). The experimental range of the selected process variables is shown in Table 1. The response variable can be expressed as a function of the independent process factors according to the following response surface quadratic model: Y = ˇ0 + ˇ1 X1 + ˇ2 X2 + ˇ3 X3 + ˇ4 X4 + ˇ11 X12 + ˇ22X22 + ˇ33 X32 + ˇ44 X42 + ˇ12 X12 + ˇ13 X13 + ˇ14 X14 + ˇ23 X23 + ˇ24 X24 + ˇ34 X34 (2) The coefficients of the second-order polynomial model were represented by ˇ0 (constant coefficient), ˇ1 , ˇ2 , ˇ3 and ˇ 4 (linear effects), ˇ11 , ˇ22 , ˇ33 and ˇ44 (quadratic effects), and ˇ12 , ˇ13 , ˇ14 , ˇ23 , ˇ24 and ˇ34 (interaction effects). In this four variable BBD, three levels are attributed to each variable (−1, 0, +1) and a set of 30 trials is carried out (Table 2). 2.4. Assay of antioxidant activity in vitro of EVFP 2.4.1. Assay of scavenging activity on hydroxyl free radical The hydroxyl radical scavenging ability of EVFP was determined by the method of Jin with slight modifications [30]. The hydroxyl radical was produced in a mixture of 1.0 mL of 1 mM 1,10phenanthroline, 4.0 mL of 0.1 M sodium phosphate buffer (pH 7.4), 1.0 mL of 1 mM ferric sulfate and 1.0 mL of hydrogen peroxide. After addition of 1.0 mL sample solution in different concentrations (1, 2, 3, 4, 5 and 6 mg/mL), the mixture was incubated at ambient temperature for 25 min. Then, the absorbance of the mixture was measured at 536 nm. Ascorbic acid (vitamin C) was applied as positive control. The scavenging activity on ·OH was expressed as following: Scavenging effect (%) =
Z0 − Z1 × 100(%) Z0
(3)
where Z0 and Z1 are the absorbance of control (without sample) and EVFP, respectively. 2.4.2. Assay of scavenging activity on DPPH free radical The scavenging ability on DPPH free radical was measured by employing the published method with minor modifications [31].
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Table 2 BBD with the observed responses and predicted values for yield of EVFP (%). Run
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 30
X1 (time)
70 70 70 100 70 70 40 70 70 40 100 70 70 70 100 70 40 100 70 40 70 70 100 40 70 70 70 100 70 40
X2 (microwave power)
500 800 500 500 200 200 500 800 500 500 200 500 500 800 500 800 500 800 500 500 500 500 500 200 200 500 500 500 200 800
X3 (temperature)
50 30 50 70 70 50 70 50 30 50 50 70 30 70 50 50 30 50 50 50 50 70 50 50 30 50 50 30 50 50
Briefly, 0.5 mL DPPH. solution (250 mol/L in dehydrate alcohol) was added to 4.0 mL of EVFP solution with different concentrations (1, 2, 3, 4, 5 and 6 mg/mL), and the mixture was incubated at 25 ◦ C for 40 min in the dark. Then, the absorbance was recorded at 517 nm. Butylated hydroxytoluene (BHT) was used as the positive control, and the scavenging activity was calculated according to the following equation: Scavenging capacity (%) =
S0 − S1 × 100 (%) S0
(4)
where S0 and S1 are the absorbance of control (without sample) and EVFP, respectively. 2.5. Determination of antilisterial activity of EVFP Four species of Listeria (L. seeligeri, L. monocytogenes, L. ivanovii and L. marthii) were selected as test bacteria for this research. The microorganisms were purchased from the Microbiology Laboratory of Razi institute, Iran. Antilisterial activity was measured by the filter disc diffusion plate method and the agar dilution technique [32,33]. The crude polysaccharides (1, 2, 3, 4, 5 and 6 mg/mL) were dissolved in a minimum amount of dimethyl sulfoxide and added to the nutrient medium. A suspension of an overnight culture of each test bacteria containing 106 cells/mL was added to the medium. The result medium was then stored in an incubator (37 ◦ C; 24 h). The diameter of the inhibition zones by the disc diffusion method was evaluated for the agar dilution test to assay the antilisterial effect more precisely. Nutrient agar served as a control. 2.6. Statistical analysis All the data were expressed as the means ± standard deviations within significance p-values of less than 0.05 after subjecting to an analysis of variance (ANOVA) and processed with SPSS 13.0.
X4 (W/R ratio)
40 40 40 40 40 70 40 10 70 10 40 70 10 40 70 70 40 40 40 70 40 10 10 40 40 40 40 40 10 40
Extraction yield (%) Actual
Predicted
23.11 18.91 24.01 9.10 16.32 19.10 16.41 15.42 21.77 14.02 15.93 14.12 11.82 11.11 18.21 24.32 11.76 16.93 23.14 16.23 23.81 14.89 8.03 15.15 20.44 22.94 23.33 15.55 19.43 20.19
23.56 19.21 23.56 9.34 16.17 19.35 16.49 15.31 21.85 14.44 16.12 14.48 11.60 11.29 17.98 24.66 12.02 16.77 23.56 16.36 23.56 15.12 7.94 15.01 20.79 23.56 23.56 15.11 19.85 20.36
3. Results and discussion 3.1. Extraction yield of EVFP 3.1.1. Effect of different times on extraction yield of EVFP Extraction time will obviously affect the extraction yield of polysaccharides from Viper’s bugloss flower. Therefore, it is very important to choose an optimum time for extraction of EVFP. In the present work, it was set at 30, 40, 50, 60, 70, 80, 90 and 100 min to evaluate the effect of extraction time on the yield of polysaccharide while other extraction variables were fixed as the followings: microwave power 500 W, extraction temperature 50 ◦ C and the ratio of water to raw material 40. It was observed that the polysaccharide yield was increased significantly with increasing extraction time from 30 to 100 min (Fig. 1a), and it reached 21.35 ± 0.6% when the extraction time was 100 min. Therefore, 30–100 min was selected in the present work. The extraction coefficient increased with increasing the extraction time because of the increase of the EVFP solubility. This was matched closely with results of other authors in extracting polysaccharides [34,35].
3.1.2. Effect of different microwave powers on extraction yield of EVFP The extraction yield (%) of EVFP extracted by different microwave powers from 200 to 1000 W was shown in Fig. 1b. The extraction time, extraction temperature and the ratio of water to raw material were fixed at 70 min, 50 ◦ C and 40, respectively. The extraction yields of the polysaccharide significantly increased from 7.2% to 20.3% (w/w) as microwave power increased from 200 to 800 W. However, when the power continued to increase (from 800 to 1000 W), the extraction yield decreased to 18.5%, because of increasing driving force for the mass transfer of other chemicals and thermal degradation of the crude polysaccharides. Similar
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Fig. 1. Effects of different (a) extraction times, (b) microwave powers, (c) extraction temperatures, and (d) the ratio of water to raw material on the extraction yield of EVFP.
results have been reported in other researches [36]. Thus, 800 W was chosen as the optimum microwave power. 3.1.3. Effect of different temperatures on extraction yield of EVFP The use of the various temperatures during the extraction process, helping to obtain good extraction yield. The temperature is an important parameter which affects the efficiency of polysaccharides during hot water extraction [37,38]. The influence of extraction temperature on the yield of EVFP is shown in Fig. 1c. Extraction was carried out at different temperatures (30, 40, 50, 60, 70 and 80 ◦ C) when other extraction variables (extraction time (70 min), microwave power (500 W), and the ratio of water to raw material (40)) were fixed. The extraction yields of EVFP significantly increased from 13.6% to 19.8% as extraction temperature increased from 30 to 70 ◦ C. However, when the temperature continued to increase, the extraction yields declined to 18.4%. The highest extraction yield was obtained when the temperature was 70 ◦ C. Therefore, extraction temperature of 70 ◦ C was adopted in the present study.
According to the single-parameter study, it was employed extraction time 40–100 min, microwave power 200–800 W, extraction temperature 30–70 ◦ C, and the ratio of water to the raw material 10–70 for response surface methodology experiments. 3.2. Data analysis and evaluation of the model Four parameters, including X1 (extraction time), X2 (microwave power), X3 (extraction temperature), and X4 (the ratio of water to raw material) were selected and separately optimized by Box–Behnken design. Table 2 illustrated the BBD matrix together with the responses (extraction yields) obtained. The yield of EVFP ranged from 8.03 to 24.32%, and reached maximum with the extraction time 70 min, microwave power 800 W, temperature of 50 ◦ C and the ratio of water to raw material 70. By employing multiple regression analysis on the experimental data, the test factors and response were found to correlate by the following second-order polynomial equation: Y = 23.56 − 1.47X3 + 2.53X4 − 2.56X1 X3 + 2.03X1 X4 + 2.46X2 X4
3.1.4. Effect of different ratios of water to the raw material on extraction yield of EVFP The effect of different ratios of water to the raw material on the yield of EVFP was presented in Fig. 1d. Extraction was performed at different ratios (10, 20, 30, 40, 50, 60, 70 and 80), while other extraction variables were fixed as follows: extraction time of 70 min, microwave power of 500 W and extraction temperature of 50 ◦ C. The results indicated that the extraction yield of EVFP was significantly increased to the value (21.6%) with an increase in the water to raw material ratios, and the efficiency was declined when the ratio was more than 70. So, the optimum ratio of 70 was finally employed in this study.
− 2.72X3 X4 − 5.69X12 − 5.01X32 − 3.06X42
(5)
where Y is the extraction yield of EVFP (%), and X1 , X2 , X3 and X4 are the coded variables for extraction time, microwave power, extraction temperature, and the ratio of water to raw material, respectively. Table 3 demonstrates the results of the ANOVA, fitness and the adequacy of the model. The F-value of 14.35 and p-value less than 0.0001 represented the response surface quadratic model was significant. The corresponding factors become more significant as the p-value becomes smaller and the F-value becomes larger. Thus, the p-value can be applied to study the interaction strength between each independent parameter [39]. In the present case,
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Table 3 Analysis of variance for the fitted models. Source
Sum of squares
Degree of freedom
Mean square
F value
p-Value Prob > F
Model X1 X2 X3 X4 X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4 X1 2 X2 2 X3 2 X4 2 Residual Lack of fit Pure error Cor total R-squared Adj R-squared Pred R-squared Adeq precision C.V. %
586.10 10.87 80.32 26.08 77.11 5.52 26.21 16.48 2.72 24.26 29.65 222.88 7.84 171.23 64.35 43.77 43.77 0.21 629.87 0.961 0.905 0.882 13.71 6.69
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 10 5 29
41.86 10.87 80.32 26.08 77.11 5.52 26.21 16.48 2.72 24.26 29.65 222.88 7.84 171.23 64.35 2.92 4.38 0.08
14.35 3.72 2.74 8.94 26.42 1.89 8.98 5.65 0.93 8.31 10.16 76.03 2.69 58.67 22.05
