Corn silk (
Flavonoids are effective components of many Chinese herbal medicine with the function of antihypertensive, reducing blood lipid, being antibacterial and antitumor, enhancing immune, antioxidant, and eliminating free radicals [
However, the literature on efficient extraction of corn silk is limited. Traditional methods (distillation or liquid solvent extraction) for the extraction of flavonoids from plant often need long extraction times, use of large amounts of solvent, and low efficiencies. Moreover, flavonoids are thermally unstable and easily degrade during the extraction. In order to increase the extraction yield, ultrasound-assisted extraction [
UV-1100 spectrophotometer, ultrasonic extractor, rotary evaporator, multiuse recycle water vacuum pump, grinder, and electronic balance.
Corn silk was harvested in Sichuan Agricultural University green nursery garden, Rutin standard was obtained from Sichuan Institute for Food and Drug (99% purity, batch number 130910), ethanol, sodium hydroxide, aluminum nitrate, sodium nitrite, ferrous sulfate, concentrated hydrochloric acid, and sulfuric acid were of analytical grade and purchased from Chengdu Kelong Chemical Reagent Company. TPTZ was purchased from Beijing Solarbio Science and Technology Company for the preparation of the FRAP solution.
A total of Rutin standard solution was prepared by dissolving Rutin reference material in 70% ethanol; Rutin solution (0.0 mL, 1.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL) was accurately pipetted into 25 mL volumetric flasks, respectively. Then, 12 mL of the 70% ethanol and 2 mL of the sodium nitrate solution were added. The mixture was shaken up and placed for 10 min, followed by the addition of 2 mL 10% nitric acid aluminum solution, and was shaken up. After 10 min, 20 mL sodium hydroxide was added to scale. The mixture was deposited for 5 min, and then the absorbance of the solution was measured at 500 nm with the reagent blank as reference. Make the standard curve with the concentration of Rutin standard solution as abscissa and absorbency as vertical; the regression equation was
Fixed extraction conditions are as follows: ultrasonic time 20 min, material solvent ratio 1 : 20, ethanol concentration 30%, ultrasonic temperature 60°C, ultrasonic power 500 W, and one-time extraction. The supernatant was taken after being centrifuged for 10 min under 3000 r/min, and then the absorbance was measured according to 2.1. Extraction time (10 min, 15 min, 20 min, 25 min, and 30 min), material liquid ratio (1 : 5, 1 : 10, 1 : 15, 1 : 20, and 1 : 25), and ethanol concentration (20%, 30%, 40%, 50%, and 60%) were selected as the key variables. Their impact on the yield of the total flavonoids was tested separately: Among them, the flavonoids content
According to single factor experiments, coded level of the three factors for Box-Behnken design of RSM were settled as follows: ethanol concentration (20%, 30%, and 40%), extraction time (15 min, 20 min, and 25 min), and material liquid ratio (1 : 10, 1 : 15, and 1 : 20).
The total flavonoids were vacuum-concentrated and dried after being extracted under the optimal extraction conditions, and then the crude flavonoids were obtained and certain concentration of flavonoids solution was prepared with distilled water. A volume of 0.3 mL sample solution was pipetted, and 2.7 mL FRAP solution (preheated to 38°C, prepared by 10 mmol/L TPTZ working solution, 20 mmol/L FeCl3·6H2O, 0.3 mol/L Hac buffer at 1 : 1 : 10, pH = 3.6) was added. The mixture was shaken up and placed for 15 min. We keeping record of the absorbance values at 593 nm. Absolute ethanol was used as blank control for zeroing. FeSO4 concentration (
A volume of 6.08 mg FeSO4 was dissolved with distilled water, 0.25 mL 18 mol/L H2SO4 was added, and the mixture was diluted with distilled water to the scale of 50 mL, and then an iron nail was put into it. 5 mL of the solution was pipetted into 50 mL volumetric flask and was diluted with distilled water to the scale to make 800
From Figure
Effects of extraction time on yield of total flavonoids.
From Figure
Effects of material solvent ratio on yield of total flavonoids.
From Figure
Effects of ethanol concentration on yield of flavonoids.
Based on single factor experiments, extracting time, material solvent ratio, and ethanol concentration were selected, to study the effects of different combinations of the three factors by Box-Behnken design of RSM adopting Design-Expert V8.0.6 (Stat-Ease, Inc.) (Table
Experimental design and data for response surface analysis.
Run | Factors | Flavonoids extraction yield (%) | |||
---|---|---|---|---|---|
|
|
|
Experimental data | Predictive value | |
1 |
|
|
|
1.01 | 1.08 |
2 |
|
|
|
0.97 | 0.93 |
3 |
|
|
|
0.98 | 1.00 |
4 |
|
|
|
1.06 | 1.08 |
5 |
|
|
|
0.91 | 0.89 |
6 |
|
|
|
1.08 | 1.08 |
7 |
|
|
|
1.01 | 1.08 |
8 |
|
|
|
0.99 | 0.97 |
9 |
|
|
|
1.03 | 1.08 |
10 |
|
|
|
0.94 | 0.91 |
11 |
|
|
|
0.75 | 0.79 |
12 |
|
|
|
1.00 | 1.00 |
13 |
|
|
|
1.07 | 1.09 |
14 |
|
|
|
0.82 | 0.84 |
15 |
|
|
|
1.07 | 1.09 |
16 |
|
|
|
0.91 | 0.91 |
17 |
|
|
|
1.22 | 1.08 |
The regression equation was obtained as
Variance analysis results suggested that the effect of the factors in flavonoids extraction yield were
Variance analysis of the regression model.
Sources of variance | Sum of squares | Degrees of freedom | The mean square |
|
Pr > |
Significant |
---|---|---|---|---|---|---|
Model | 0.15 | 9 | 0.016 | 3.29 | 0.0655 |
|
|
6.125 × 10−4 | 1 | 6.125 × 10−4 | 0.12 | 0.7371 | |
|
0.026 | 1 | 0.026 | 5.27 | 0.0553 |
|
|
0.015 | 1 | 0.015 | 3.05 | 0.1242 | |
|
9.025 × 10−3 | 1 | 9.025 × 10−3 | 1.80 | 0.2218 | |
|
0.026 | 1 | 0.026 | 5.10 | 0.0484 |
|
|
2.500 × 10−5 | 1 | 2.500 × 10−5 | 4.982 × 10−3 | 0.9457 | |
|
0.042 | 1 | 0.042 | 8.39 | 0.0231 |
|
|
0.022 | 1 | 0.022 | 4.41 | 0.0739 |
|
|
1.684 × 10−3 | 1 | 1.684 × 10−3 | 0.34 | 0.5805 | |
The residual error | 0.035 | 7 | 5.018 × 10−3 | |||
Loss of quasi item | 7.725 × 10−3 | 3 | 2.575 × 10−4 | 0.38 | 0.7762 | |
Pure error | 0.027 | 4 | 6.850 × 10−3 | |||
Sum | 0.18 | 16 |
Comparison of Figures
Response surface for effect of the extracting time and ethanol concentration on yield.
Response surface for effect of the material solvent ratio and ethanol concentration on yield.
Response surface for effect of the extracting time and ethanol concentration on yield.
The optimal conditions were determined by Design-Expert as follows: extraction time 21.45 min, material solvent ratio 1 : 20, and ethanol concentration 33.75%. Under the optimum conditions, the extraction yield of total flavonoids was 1.129%, which well fits the theoretical value, proving the reliability of extraction parameters obtained from response surface methodology.
The FRAP value is 467.59
In this study, the optimization of ultrasound-assisted extraction of total flavonoids from corn silk and evaluation of their antioxidant activity were conducted. On the basis of single-factor test and Box-Behnken experimental design, the quadratic regression model was established to fit the experiment data in good effect. Through response surface methodology (RSM) of yield, the optimal conditions were determined as follows: extraction time 20 min, solid-liquid ratio 1 : 20, and ethanol concentration 30%. Under the optimum conditions, the extraction yield of total flavonoids was 1.13%, which allowed higher extraction yields with lower temperature and extraction time when compared with conventional solvent extraction methods. With stable results, this method offered a theoretical basis for industrial and experimental extraction of total flavonoids from corn silk. FRAP value of the total flavonoids extracted from corn silk was determined as 467.59
The authors declare that there is no conflict of interests regarding the publication of this paper.
Ling-Li Zheng and Guan Wen contributed equally.
This research was supported by Sichuan Sci-Tech Support Plan (no. 2015SZ0105) and Department of Sichuan Provincial Health (no. 130388).