2017 Volume 23 Issue 1 Pages 111-118
Sedum aizoon is an edible medicinal plant with various pharmacological activities distributed widely in China. The flavonoids of S. aizoon have been shown to possess a variety of important biological activities. In this work, a microwave-assisted extraction (MAE) method was employed for the efficient extraction of flavonoids from S. aizoon leaves. The influences of MAE conditions including temperature, extraction time, solvent to solid ratio, and ethanol concentration on the yield of flavonoids were systematically investigated by single-factor experiments and response surface methodology (RSM) experimental design. Under the optimal MAE conditions of extraction time 20 min, extraction temperature 57°C, solvent to solid ratio 20 mL/g, and ethanol concentration 80.6%, the flavonoids yield reached 24.87 ± 0.26 mg/g and was much higher than that of conventional Soxhlet extraction (CSE) method (18.67 ± 0.35 mg/g). The obtained flavonoids exhibited strong antioxidant activities with IC50 value of 0.315 mg/mL in the DPPH radical-scavenging experients. The results indicated that MAE was a simple and efficient technology for the extraction of flavonoids from S. aizoon leaves and the flavonoids may serve as new potential natural antioxidant for functional food ingredients and additives.
Sedum aizoon is an endemic plant widespread in oriental countries, known as “Tu San-Qi” in China with light green leaves on thick stems and a yellow flower blooming in the summer (Wang et al., 2015). Its root and the whole plant have traditionally been used as Chinese medicine to cure pain or traumatic, various hemorrhage, and palpitation (Li et al., 2011). Furthermore, it is also an edible plant common to Chinese owing to its biological activities in reducing hematic fat and blood pressure (Wang et al., 2011). A large number of chemical constituents have already been isolated from S. aizoon, including flavonoids, phenolic acid compounds, polysaccharose, and alkaloids (Wang et al., 2015; Li et al., 2011; Wang et al., 2011). Flavonoids are considered to be the main bioactive components in S. aizoon, which exhibited several biological activities, such as preventing hyperglycemia, diabetes mellitus, coronary heart disease and anti aging. (Wang et al., 2011).
Flavonoids, one of the most abundant plant secondary products, are important components of the human diet and have revealed to possess various beneficial activities in the human health (Procházková et al., 2011; Di et al., 1999; Havsteen, 1983). These bioactivities are based first and foremost on its capacity to scavenge free radicals, and its ability to prevent lipid peroxidation (Montoro et al., 2005; Ammar et al., 2009; Cirico et al., 2006; Hubbard et al., 2004; Valen et al., 1996). Nowadays, there have been more and more flavonoids used in pharmaceutical, healthy food and cosmetics (Makino et al., 2006; Mustafa et al., 2011). As reported previously, some known flavonoids including quercetin, myricetin, and kaempferol, were isolated from S. aizoon. More recently, other new flavonoids and flavonoid glucosides have also been reported (Wang et al., 2015; Li et al., 2011). However, the extraction of flavonoids from S. aizoon and its antioxidant activity have seldom been investigated. Apart from this, very little is known about the flavonoids profile of S. aizoon, and whether the reported bioactivities are linked to the flavonoid components. Thus, there is a great need to develop simple and efficient methods for extracting flavonoids from S. aizoon for evaluating its potential health benefits and added values.
The traditional methods for the extraction of flavonoids, such as percolation, heating, and soxhlet extraction method, usually require long extraction time and consume large amounts of solvents. Furthermore, conventional techniques may result in a loss of flavonoids due to hydrolysis, ionisation and oxidation during extraction process (Li et al., 2005; Gao et al., 2006). Microwave-assisted extraction (MAE) is an alternative and advanced method used in flavonoids extraction, owing to its special heating mechanism, less solvent, shorter time, and better extraction efficiency under atmospheric conditions (Winny, 2012; Lu et al., 2013). In MAE, the microwave irradiation causes heating through dipolar rotation and ionic conduction, which would lead to the improvement of extraction efficiency (Beatrice and Philippe, 2002; Kubrakova and Toropchenova, 2008; Lu et al., 2013). On the other hand, microwave irradiation can penetrate plant matrix and result in a pressure increase inside plant cells. The increased pressure could help both break of cell walls and release of phytomolecules, and therefore would significantly reduce the extraction time and save the energy consumption during extraction process (Wu et al., 2012; Winny, 2012; Lu et al., 2013).
To the best of our knowledge, there is no report in the literature on applying MAE for the extraction of flavonoids and other bioactive components from S. aizoon. In this work, the feasibility of employing MAE as an efficient method to extraction of flavonoids from leaves of S. aizoon was investigated. The MAE extraction parameters were optimized using the single factor experimental design and response surface methodology (RSM) to obtain a highest product yield (Sharma et al., 2009; Lu et al., 2013). In addition, antioxidant properties of the extracts obtained from MAE were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay to determine its potential utilization as functional natural products for food ingredients.
Materials and reagents The fresh leaves of S. aizoon were collected in Qingyang County, Anhui Province, China. The leaves were dried in an oven at 50°C to constant weight, and then ground into a fine powder (40 – 60 mesh) using the disintegrator. Methanol and formic acid were HPLC grade and purchased from Shanghai Chemicals and Reagents Co., Ltd, China. Rutin, 2,2-diphenyl-1-1picrylhydrazyl, and ascorbic acid were obtained from Sangon Biotech (Shanghai, China). All other reagents were analytical grade and purchased from Shanghai Chemicals and Reagents Co., Ltd, China.
Flavonoids extraction methodologies Flavonoids extraction was carried out by conventional Soxhlet extraction (CSE) and microwave-assisted extraction (MAE). The powdered samples of S. aizoon (5 g) was put into a 500 mL beaker and extracted with different concentrations of ethanol. For MAE process, the extraction was performed in a microwave extractor (Shanghai Sineo Microwave Technology Co., Ltd) under different sets of conditions of temperature, time and solvent volume. After extraction, the breaker was cooled for several minutes to room temperature (25°C) and the obtained solution was then filtered to yield a clear extract used for flavonoids determination.
Optimization of MAE conditions The Box-Behnken experimental design (BBD) of response surface methodology (RSM) was applied to optimize and investigate the individual and interactive effects of MAE parameters on the flavonoids yield. For each independent variable, an experimental range was based on the results of single-factor experiments. In this design, the effect of three parameters on extraction were investigated at three levels (−1, 0, and +1) with total flavonoids extraction yield treated as the response, and 15 groups of experiments were conducted in a randomized order (Table 1). As shown in Table 1, the independent variables and their levels are ethanol concentration 70% – 90%, extraction temperature 50 – 70°C, and solvent to solid ratio 15 – 25 mL/g respectively. The experimental results were fitted to a second-order polynomial model:
 |
| Run | X1 | X2 | X3 | Yields (mg/g) |
|---|---|---|---|---|
| 1 | 60 (0) | 70 (−1) | 15 (−1) | 18.21 |
| 2 | 60 (0) | 90 (1) | 15 (−1) | 19.04 |
| 3 | 60 (0) | 70 (−1) | 25 (1) | 19.25 |
| 4 | 60 (0) | 90 (1) | 25 (1) | 20.02 |
| 5 | 50 (−1) | 70 (−1) | 20 (0) | 21.76 |
| 6 | 50 (−1) | 90 (1) | 20 (0) | 22.58 |
| 7 | 70 (1) | 70 (−1) | 20 (0) | 20.42 |
| 8 | 70 (1) | 90 (1) | 20 (0) | 21.29 |
| 9 | 50 (−1) | 80 (0) | 15 (−1) | 23.08 |
| 10 | 50 (−1) | 80 (0) | 25 (1) | 21.41 |
| 11 | 70 (1) | 80 (0) | 15 (−1) | 21.72 |
| 12 | 70 (1) | 80 (0) | 25 (1) | 23.22 |
| 13 | 60 (0) | 80 (0) | 20 (0) | 25.61 |
| 14 | 60 (0) | 80 (0) | 20 (0) | 25.65 |
| 15 | 60 (0) | 80 (0) | 20 (0) | 25.59 |
where Y was the response; xi and xj were the coded values of independent variables; a0 was a constant term; ai, aii and aij represented the regression coefficients for linear, quadratic and interaction terms, respectively.
The response (flavonoids yield) obtained from each set of experimental design (Table 1) was subjected to multiple regression to obtain the second-order polynomial model using Design-Expert 8.0.5. The analysis of variance tables was generated, the constant term and regression coefficients of individual linear, quadratic and interaction terms were determined. The values of R2, adjusted-R2 of models were evaluated to check the model adequacies. Additional extraction trials were performed under optimized conditions to verify the validity of the BBD. The p-values of less than 0.05 were considered to be statistically significant. All the experiments were performed in triplicate and results were expressed as the mean value of three measurements.
Determination of total flavonoids The flavonoids content was determined by a colorimetric method with some modifications (Amir et al., 2012). This method consisted in the addition of 1.0 mL of diluted extract solution with 4 mL of 80% (v/v) ethanol and 0.1 mL of 5% (w/w) NaNO2 in a 10 mL test tube. After 5 min of stirring, 0.1 mL of 10% AlCl3 (w/w) was added and mixed and then left stand for 15 min. The absorbance of the mixture solution was measured at 510 nm with a spectrophotometer (UV 1240 Shimadzu, Japan). For flavonoids content analysis, all experiments were conducted at room temperature. The calibration curve was established using rutin as standard: y = 0.5215x + 0.0237, where y is absorbance value of sample, x is rutin/flavonoids concentration (0 – 80 µg/mL) (R2 = 0.9997). Total flavonoids content was expressed as mg of rutin equivalents in 1 g of dry leaf sample.
Determination of antioxidant activity The free radical-scavenging activity of the flavonoids extract was determined according to the method as described elsewhere with some modifications (Xie et al., 2010). Aliquots (0.5 mL) of the sample solution (0.01 – 0.06 mg/mL) were mixed with 3 mL of a freshly prepared DPPH methanol solution (0.1 mM). The mixture was shaken fiercely and then kept in the dark for 30 min at room temperature. The decrease in absorbance of the mixture was measured at 517 nm with a spectrophotometer (UNIC7200). Ethanol solution was set as the blank group, ascorbic acid and BHT composition were as the positive group. The percentage of inhibition of the DPPH• radical (I%) was calculated according to the formula:
 |
Where A0 is the absorbance of the control DPPH solution without any solution, A1 is the absorbance of the sample solution, A2 is the absorbance of the mixture including both sample and DPPH solution. The concentrations leading to 50% activity lost (IC50) were also calculated.
Statistical analysis The experimental data were collected and reported as mean ± standard deviation (SD) (n = 3). The Design-Expert (Version 8.0.5. Stat-Ease) was used to design Box-Behnken experimental design and analyze the experimental data. For multiple comparisons, analysis of variance (ANOVA) was used, and a statistical p value <0.05 was regarded as significant, and p value <0.001 was regarded as very significant.
Single factor experimental analysis of MAE
Effect of temperature on the extraction yield of flavonoids A suitable temperature of the MAE method is very important for the isolation of bioactive molecules from raw plant materials (Xie et al., 2010). To study the effects of temperature on flavonoids extraction, the extraction temperature of the MAE process was performed at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C. Respectively, with other parameters set as follows: ethanol concentration 90%, extraction time 10 min and ratio of solvent to material 10:1. As can be seen in Fig. 1a, the extraction yield increased rapidly with increasing extraction temperature when the temperature was ranged from 30°C to 60°C. This may be due to the increased molecular motion and flavonoids solubility caused by higher temperature (Xie et al., 2015). However, the extraction yield reached maximum at the temperature of 60°C and significantly decreased with the elevated extraction temperature (Fig. 1a), as over-elevated temperatures could always degrade phenolic and flavonoid compounds (Wang et al., 2008). Similar results were also observed in the extraction of flavonoids from mango byproducts, in which extraction efficiency is maximum at the temperature of 25°C and the yield of total flavonoids was reduced when the temperature further increased (Xie et al,. 2010). Therefore, temperature of 60°C was considered as the optimal temperature for the further experiments.
Effects of extraction temperature (a), extraction time (b), solvent to solid ratio (c), and concentration of ethanol (d) on the flavonoids extraction of MAE.
Effect of extraction time on the yield of flavonoids In order to investigate the effect of extraction time on the flavonoids recovery, experiments were performed by extracting for 5, 10, 15, 20, 25, and 30 min, respectively. The extraction temperature was 60°C and other factors were set at fixed levels as described previously. It can be seen from Fig. 1b that yield significantly increased from 10.34 ± 0.5 mg/g to 17.61 ± 0.3 mg/g with the extraction time was increased from 5 minutes to 20 minutes. Nevertheless, the flavonoids yield changed little when increase the extraction time to more than 20 min (Fig. 1b). Importantly, a previous report demonstrated that further increase the extraction time may lead to the degradation of flavonoids with prolonged application of microwave . Taking into account the extraction efficiency, 20 min was adopted for further screening experiments.
Effect of solvent to solid ratio on the extraction yield of flavonoids The extraction yields of total flavonoids obtained by MAE at different solvent to solid ratio were investigated with other experimental conditions set as follows: solvent of 90% ethanol, extraction time of 20 min, and extraction temperature of 60°C. In the MAE trials, the yields of flavonoids were found to increase with the increase of solvent to solid ratio and then fall down at the high ratios Fig. 1c. As reported previously, more flavonoids can permeate into the solvent as more solvent can enter plant cells under the higher ratio of solvent to material condition (Shan et al., 2012;). However, a higher solvent to solid ratio was not efficiency for heat/mass transfer and microwave irradiation, and would probably result in decreased extraction efficiency (Mandal et al., 2007; Wang and Weller, 2006). In Fig. 1c, the yield of flavonoids reached maximum (20.01 ± 0.3 mg/g) with the solvent to solid ratio at a value of 20 mL/g. So the solvent to material ratio of 20 mL/g was selected most suitable for the extraction of total flavonoids.
Effect of ethanol concentration on the extracting yield Solvent is one of the most important factors in MAE process, because microwave irradiation and the solubility of the analytes are significantly influenced the solvent properties (Winny, 2012). Methanol and ethanol are the most frequently used solvents in flavonoids extraction. Given the extraction efficiency and safety, ethanol-water solution (50 – 100%, v/v) was selected as the extraction solvent. As shown in Fig. 1d, the yield of flavonoids had a significant improvement when the ethanol concentration increased from 50% to 80%. However, the flavonoids yield decreased with the further increasement of ethanol concentration (Fig. 1d). These results suggest that the variation in the concentration of ethanol could affect the flavonoids extraction efficiency. Similar phenomenon has also been observed in flavonoids extraction from other plant matrix by MAE, which demonstrated that solvent polarity can be used to regulate the extraction efficiency (Chen et al., 2012; Li et al., 2013; Ying et al., 2011; Wang et al., 2008; Xie et al., 2010). Based on the results of Fig. 1d, maximum extraction yield was obtained at ethanol concentration of 80% with a value of 25.61 ± 0.4 mg/g. Therefore, 80% ethanol was selected as optimal solvent for further experiments.
Optimization of MAE conditions by RSM The optimization of the MAE conditions was further conducted to obtain the maximum flavonoids yield. On the basis of a three-variable Box-Behnken design (BBD), RSM optimizations were performed to determine the effect of extraction temperature (X1), concentration of ethanol (X2), and solvent to solid ratio (X3) on the values of responses (Y). As shown in Table 1, each experiment in the BBD was performed and the experimental data were obtained. The data were treated by multiple regression analysis and statistical analysis of variance (ANOVA) with the Design Expert 8.0.5. software. After analysis, the fitted regression model is presented in the following polynomial equation in terms of coded values.
 |
The F-test and p-value were used to determine the statistical meaning and the significance of each coefficient and results were shown in Table 2. The p-values of less than 0.05 were considered to be statistically significant, and the corresponding variables would be more significant if the p-value becomes smaller and the absolute F-value becomes greater (Muralidhar et al., 2001; Kwon et al., 2003). The analysis of variance (ANOVA) of the regression model revealed that the model was statistically strongly significant with a small p value (0.0013) and a high F-test value (24.37), which suggested that the model is adequate for predicting the flavonoids yield. As shown in Table 2, the variables have significant effect on the flavonoids yield were the quadratic terms (X2 × X2 and X3 × X3) and the interaction between X1 and X3 (p < 0.05). Furthermore, the determinant coefficient (R2) close to 1 (0.9777) and the Radj2 (0.9376) close to R2 in Table 2 indicated that the polynomial model had a good fit to the prediction and reliability for the flavonoids extraction (Haddadi-Guemghar et al., 2014; Ranic et al., 2014).
| Source | Sum of squares | df | Mean square | F value | p-value prob >F |
|---|---|---|---|---|---|
| model | 78.45 | 9 | 8.72 | 24.37 | 0.0013 |
| X1 | 0.59 | 1 | 0.59 | 1.66 | 0.2539 |
| X2 | 1.35 | 1 | 1.35 | 3.78 | 0.1094 |
| X3 | 0.43 | 1 | 0.43 | 1.20 | 0.3239 |
| X1X2 | 0.000625 | 1 | 0.000625 | 0.001748 | 0.9683 |
| X1X3 | 2.51 | 1 | 2.51 | 7.02 | 0.0454 |
| X2X3 | 0.0009 | 1 | 0.0009 | 0.002517 | 0.9619 |
| X12 | 0.71 | 1 | 0.71 | 1.98 | 0.2180 |
| X22 | 49.62 | 1 | 49.62 | 138.74 | <0.0001 |
| X32 | 29.38 | 1 | 29.38 | 82.15 | 0.0003 |
| Lack of fit | 1.79 | 3 | 0.60 | 637.97 | 0.0016 |
| Pure | 0.001867 | 2 | 0.0009333 | ||
| Residual | 1.79 | 5 | 5 | ||
| R2 | 0.9777 | ||||
| Adj. R2 | 0.9376 |
Three-dimensional response surfaces were plotted to understand the interactions between independent and dependent variables and to determine the optimum MAE conditions for flavonoids extraction. As shown in Fig. 2a–c, the effects of two factors on the flavonoids yield were depicted with the other one was kept at zero level. The 3-D response surface in Fig. 2a, which set the solvent to solid ratio at zero level, showed that the flavonoids yield increased with increasing of ethanol concentration and extraction temperature at the initial stage and then slightly decreased. Fig. 2b is the 3-D plot at varying extraction temperature and solvent to solid ratio. The variation trends of flavonoids yield were similar to the results shown in Fig. 2a. It can be seen that the flavonoids yield increased with increasing of solvent to solid ratio ranged from 15 mL/g to 20 mL/g. However, further increasing of solvent to solid ratio and extraction temperature would result in an obvious decrease of the extraction yield. The interactions between solvent to solid ratio and extraction temperature were significant from the 3-D response surface, which are much in accordance with single factor experiment and the ANOVA analysis. The intuitive connection between ethanol concentration and solvent to solid ratio is shown in Fig. 2c. The yield of flavonoids increased when the ethanol concentration increased from 70 to 80 and solvent to solid ratio increaded from 15 mL/g to 20 mL/g. Again, higher ethanol concentration and smaller solvent to solid ratio would decrease the yield of flavonoids. Therefore, a relatively higher ethanol concentration, solvent to solid ratio, and extraction temperature are more suitable for the MAE process.
3-D representation of the response surfaces: extraction temperature and concentration of ethanol (a), extraction temperature and solvent to solid ratio (b), concentration of ethanol and solvent to solid ratio (c).
Optimal MAE conditions and validation of the model The optimal MAE conditions of selected variables were determined on the basis of response surface. The predicted optimal conditions were extraction temperature 56.9°C, solvent to solid ratio 20 mL/g, and ethanol concentration 80.6%, respectively. On considering of convenient operation, the modified MAE conditions were extraction temperature 57°C, solvent to solid ratio 20 mL/g, and ethanol concentration 80.6% with slight modification. Under the modified optimum MAE conditions, the yield of flavonoids was 24.87 ± 0.26 mg/g, which was very much in accordance with the predicted value . The good correlation between experimental and predicted results confirmed that the regression model was accurate and adequate for reflecting the MAE optimization.
Compared with conventional Soxhlet extraction (CSE), the application of microwave irradiation positively affected the extraction yield of flavonoids . The flavonoids yield in CSE was 18.67 ± 0.35 mg/g, which was much lower than that in MAE (24.87 ± 0.26 mg/g). This may be due to the degradation of flavonoids caused by prolonged extraction time and high extraction temperature in the CSE. The comparative data demonstrated that MAE exhibited shorter extraction time and better extraction yields than those of CSE method, which suggested that microwave radiation could accelerate the extracting process and improve the flavonoids extraction yield. The results demonstrated that MAE should be a simple and efficient technique for extraction of flavonoids from S. aizoon leaves.
Antioxidant activity DPPH scavenging ability is a widely used method to evaluate antioxidant activity in relatively short time as compared other methods. To determine the antioxidant capacity of flavonoids from S. aizoon, ascorbic acid and butylated hydroxytoluene (BHT) were used as references and assessed alongside the samples from MAE. The DPPH radical scavenging capacity of flavonoid extracts, ascorbic acid, and BHT are shown in Table 3. The antioxidant activity of flavonoids sample solution increased from 24.91 ± 0.01% to 70.28 ± 0.01% with the sample concentration ranged from 0.1 to 0.7 mg/mL. The DPPH free radical-scavenging of flavonoids extract was less than that of ascorbic acid. However, the antioxidant activity of flavonoid samples at 0.7 mg/mL (70.28 ± 0.01%) was higher than that of BHT (60.38 ± 0.01%) as shown in Table 3. Notably, the DPPH radical-scavenging IC50 value of flavonoid sample was 0.315 mg/mL and smaller than that of BHT, which was a synthetic antioxidant with an IC50 value of 0.541 mg/mL. Till now, little information is available on the antioxidant activity of flavonoids from S. aizoon leaves. The obtained results indicated the flavonoids of S. aizoon leaves may play important role on prevention of oxidative-related diseases such as hyperglycemia, diabetes mellitus, coronary heart disease, and anti aging, as well as providing useful source of natural antioxidants in functional foods.
| Sample | mg/mL | Inhibition (I%) | IC50 (mg/mL) |
|---|---|---|---|
| Flavonoids | 0.1 | 24.91 ± 0.01 | 0.315 ± 0.03 |
| 0.2 | 30.54 ± 0.06 | ||
| 0.3 | 48.72 ± 0.02 | ||
| 0.5 | 59.83 ± 0.05 | ||
| 0.7 | 70.28 ± 0.01 | ||
| Ascorbic acid | 0.1 | 84.97 ± 0.03 | |
| 0.2 | 87.39 ± 0.02 | ||
| 0.3 | 95.86 ± 0.04 | ||
| 0.5 | 96.41 ± 0.01 | ||
| 0.7 | 97.32 ± 0.07 | ||
| Butylated hydroxytolunene (BHT) | 0.1 | 12.03 ± 0.02 | 0.541 ± 0.02 |
| 0.2 | 20.45 ± 0.04 | ||
| 0.3 | 31.67 ± 0.01 | ||
| 0.5 | 50.24 ± 0.02 | ||
| 0.7 | 60.38 ± 0.01 |
A microwave-assisted extraction process has been optimized for effective extraction of flavonoids from S. aizoon leaves. The yield of flavonoids under the optimal MAE condition (extraction time 20 min, extraction temperature 57°C, solvent to solid ratio 20 mL/g, and ethanol concentration 80.6%) was 24.87 ± 0.26 mg/g. Compared to CSE, the extraction time was 327 hugely shortened and the flavonoids yield was greatly increased. The experimental results demonstrated that MAE was a rapid and efficient technique for extraction of flavonoids from S. aizoon leaves. Furthermore, the antioxidant activities of the flavonoids obtained by MAE were evaluated by DPPH scavenging assay. The results revealed the flavonoids exhibited good antioxidant activity with an IC50 of 0.315 mg/mL, which was stronger than that of BHT as a synthetic antioxidant. Hence, the obtained flavonoids may be served as potential functional food ingredients. Further experiments on the flavonoids profile of S. aizoon leaves and their structure-biological activity relationship are in progress.
Acknowledgements This work was supported by the Shanghai Educational Development Foundation and Shanghai Municipal Education Commission, and Teaching Research Project of Shanghai Institute of Technology.
