0 introduction

Corn is one of the main food crops grown in the north. Corn kernels are the main edible part, and corn silk is mostly discarded or incinerated. Corn silk has many chemical components, such as polyphenols, flavonoids, polysaccharides, organic acids, amino acids, etc. It has pharmacological effects such as anti-oxidation, lowering blood sugar, regulating immunity, protecting liver and gallbladder, and anti-tumor. Our country is rich in corn silk resources, but as an accessory product of corn, products using corn silk as raw materials are scarcely found in the market. At present, there are many studies on corn silk, but most of them focus on the extraction of other effective substances. The research on polyphenols, an important bioactive component in corn silk, is not comprehensive, especially the research on its purification and extraction process still needs to be perfected.

Ionic liquids have many unique properties, such as polarity, hydrophobicity, viscosity, and solvent miscibility, which can be effectively adjust and increase the solubility of compounds by changing cations, anions, and attached substituents. Ionic liquids have very low volatility, and does not cause air pollution, hence is considered a "green" solvent. This paper uses corn silk as raw material, comparing the extraction of polyphenols in corn silk with ionic liquid as solvent, cellulase enzymatic hydrolysis and ultrasonic method, determine the optimal process route by using the response surface method, so as to improve the yield of corn silk polyphenols, laying the foundations for developing natural products and industrialized production. At the same time, the comprehensive utilization of corn has been realized, the waste of corn silk resources and environmental pollution have been reduced, the industrial chain of corn production has been further expanded, the commercial value of auxiliary products has been improved, and the application market of corn silk has been expanded.

01 Resources and Measures

Materials and reagents

Corn silk: commercially sold. Gallic acid standard substance: China Institute for the Control of Pharmaceutical and Biological Products; 1-Ethyl-3-methylimidazolium tetrafluoroborate ([Emim]BF4) (purity 98%): Alpha Reagent Co., Ltd.; other reagents are domestic analysis pure.

1.2 Instruments and Equipment

VGT-2013QT ultrasonic cleaner: Guangzhou Gute Ultrasonic Instrument Co., Ltd.; XV-9200 UV spectrophotometer: Beijing Precision Instrument Factory; DFY-X500 high-speed universal pulverizer: Beijing Kewei Yongxing Instrument Co., Ltd.

1.3.1 Extraction of polyphenols by enzymatic reflux-assisted ultrasonic

Take and dry the corn silk in a blast drying oven at 50°C for 2 h, then crush, and pass through a 60-mesh sieve. Precisely weigh 2.0000 g of corn silk powder into a 50 mL round bottom flask, add acetic acid-sodium acetate buffer solution with a pH value of 4.5 to 5.5 at a ratio of 1:20, then add 0.03 g/mL cellulase, and place it in a water bath at 50°C for a certain period of time for enzymolysis. After enzymolysis, inactivate it in a water bath at 90°C for 10 minutes, extract and filter, keep the filtrate, put the filter residue into a round bottom flask, add 20 mL of 60% ethanol solvent into the flask, extract twice in 200 W ultrasonic, and extract after a certain period of time, then combine the filtrates, place the filtrate in a rotary evaporator to evaporate the filtrate to below 50 mL, and dilute to 50 mL with a certain concentration of ethanol.

1.3.2 Ultrasonic direct extraction

Take corn silk in blask drying oven in 50℃ for 2 hours, then crush, and pass it through 60-mesh sieve. Precisely weigh 2.0000g corn silk powder in a 50ml round-bottom flask, and add distilled water at a ratio of 1:20, then reflux it in a water bath in 50℃ for a while, extract and filter, keep the filtrate, put the filter residue in a round-bottom flask, then follow the steps in 1.3.1.

1.3.3 Lonic Liquid Assited Ultrasonic Extraction

Take corn silk in a blastdrying oven in 50℃ for 2 hours, then crush it, and pass it through 60-mesh sieve. Precisely weigh 2.0000g corn silk powder in a 50ml round-bottom flask, and add distilled water at a ratio of 1:20, then reflux it in a water bath in 50℃ for a while, extract and filter, keep the filtrate, put the filter residue in a round-bottom flask, add 1ml Lonic Liquid([Emin]BF4), then follow the steps in 1.3.1.

1.4 Standard curve drawing

Accurately weigh 0.0100 g of gallic acid standard substance, dissolve it in deionized water, transfer it into a 25 mL volumetric flask, and dilute to the mark to make a standard solution. Take 0.20, 0.40, 0.60, 0.80, 1.00 mL of the above standard solution and transfer them into a 10 mL volumetric flask respectively, add 0.5 mL of Folin's phenol reagent and 2 mL of 10% NaCO3 solution, dilute to the mark with deionized water, leave at room temperature for 2 h, and measure the absorbance at a wavelength of 760 nm. A standard curve was drawn with the absorbance at 260 nm as the y-axis and the x-axis as the concentration of gallic acid solution (μg/mL). Take 1 mL of the stock solution after constant-volume process in a 10 mL volumetric flask, add 0.5 mL of Folin’s phenol reagent and 2 mL of 10% NaCO3 solution, place at room temperature, develop color for 2 h, and measure the maximum absorbance at 760 nm.

02 Results and analysis

2.1 Screening results of corn silk polyphenols extraction methods

It can be seen from Figure 1 that the extraction rate of polyphenols reach the highes point by ionic liquid-assisted ultrasonic method is t, indicating that ionic liquid has a stronger ability to enzymatically destroy the cell wall of corn silk than cellulase. Therefore, in this paper, ionic liquid-assisted ultrasonic method is used to extract corn silk polyphenols. The optimized process will have simple operation, time saving, non-toxicity and high efficiency.

2.2 Analysis of single factor test results

Accurately weigh 2.0000 g of corn silk powder, and use the ionic liquid-ultrasonic method to extract polyphenols, investigating the impact on the extraction rate of polyphenols in corn silk brought by the concentration of ionic liquid A(0.08, 0.16, 0.32, 0.48 mol/L) and ultrasonic time B(15, 20, 25, 30min), solid-liquid ratio C (1:10, 1:15, 1:20, 1:25), ethanol concentration (40%, 50%, 60%, 70%). Measure the absorbance at the ultraviolet wavelength of 760 nm, plug in the standard curve equation y=0.0142x+0.104 (R2=0.999) to calculate the extraction rate, and use the polyphenol extraction rate as an index to determine the level of the response surface design.

2.2.1 Effect of ionic liquid concentration on extraction of polyphenols from corn silk

2 Effect of ionic liquid concentration on the extraction rate of polyphenols

It can be seen from Fig.2 that with the increase of the concentration of ionic liquid [Emim]BF4, the extraction rate of polyphenols gradually increased, and when the concentration of [Emim]BF4 was 0.32 mol/L, the extraction rate of polyphenols reached the maximum; When the concentration exceeds 0.32 mol/L, the extraction rate decreases slightly. The reason may be related to the viscosity of the extraction solvent. If the concentration of the ionic liquid is too high, the viscosity of the extract will continue to increase, and the diffusion ability of the solution will become poor, making it difficult for [Emim]BF4 to penetrate into the corn silk cells, hence the dissolution capacity of polyphenols is reduced. Therefore, the concentration of ionic liquid [Emim]BF4 was chosen to be 0.32 mol/L. 2.2.2 The influence of extraction time on the extraction of polyphenols from corn silk can be seen from Fig.3 that the extraction rate of polyphenols will vary with the extraction time within a certain range. Generally, the longer the extraction time, the more fully the extraction and the better the extraction effect. However, it can be seen from the figure that the maximum extraction rate can be obtained when the extraction time increases to 20 min, and then the extraction rate of polyphenols decreases with the increase of extraction time. The reason may be that polyphenols contain unsaturated bonds, hence have poor stability, excessively long extracting time, and preferability to be oxidized or undergo other reactions during the extraction process, resulting in a decrease in extraction efficiency. Therefore, 20 min is the most appropriate extraction time.

Fig.3 Effect of extraction time on polyphenol extraction rate 

2.2.3 Effect of ethanol concentration on extraction of polyphenols from corn silk

The effect of ethanol concentration on the extraction rate of polyphenols can be seen from Figure 4. In the range from 40% to 70%, with the increase of ethanol concentration, the extraction efficiency of polyphenols first increased steadily and then decreased sharply. The reason may be that polyphenols are water-soluble. As the concentration of ethanol increases, the amount of water decreases, resulting that polyphenols cannot be fully dissolved and be transferred to the liquid phase, and the dissolution of other fat-soluble substances has a negative effect on polyphenols. The dissolution of polyphenols also has a certain influence; if the ethanol concentration is too low, the dissolution of alcohol-soluble substances such as polysaccharides will affect the dissolution of polyphenols. Therefore, the concentration of ethanol should not be too high or too low, and the optimal concentration of ethanol is 50%.

2.2.4 Effect of solid-liquid ratio on extraction of polyphenols from corn silk

Figure 5 Effect of solid-liquid ratio on polyphenol extraction rate

It can be seen from Figure 5 that within the range of 1:10 to 1:25, the extraction rate of polyphenols increases continuously with the increase of the solid-liquid ratio; and when it exceeds this range, the extraction rate will not increase but decrease , the reason may be that the material liquid is relatively small, that is, when the amount of extraction solvent is too small, the polyphenols cannot be fully dissolved. In the process of gradually increasing the material-liquid ratio, the increase in the amount of extraction solvent can effectively dilute the solute and expand the raw material. The concentration difference between internal and external solutes increases the osmotic pressure, which is beneficial to the leaching of solutes; increasing the amount of solvent will cause the concentration of polyphenols contained in the solution to be low, and the rotary evaporation time is long, and the loss of polyphenols will occur, resulting in a low final extraction rate. . Therefore, the extraction effect is the best when the solid-liquid ratio is 1:20.

2.3 Response Surface Result and Analysis of Extraction Process Optimization of Corn Silk Polyphenols

2.3.1 Response Surface Experiment Design and Results

Table 1 Design factor level table of the Rresponse surface test

Taking the extraction rate of polyphenols as an index, the concentration of ionic liquid, extraction time, liquid-solid ratio and ethanol concentration were selected as four independent variables, and a response surface optimization experiment with 4 factors and 3 levels was designed according to the Design-Expert 8.0.6 software combination. The influence of each factor on the extraction rate of polyphenols is shown in Table 1 for the level design of the test factors, and in Table 2 for the design scheme and resultsof the test.

Table 2 Response surface test results of extraction process optimization of polyphenols from corn silk

2.3.2 Response Surface Test Setup

Using DesignExpert 8.0.6 software to analyze the data in Table 2, the quadratic regression fitting equation Y=21.26–2.75A+2.02 B+5.85C-4.48 D+8.06AB+1.43AC–4.57AD+2.73BC+2.03BD+2.99CD–0.4A2–6.96B2–4.48C2+0.93D2. The variance analysis was carried out on the regression equation, and the results are shown in Table 3. It can be seen from Table 3 that the model P<0.0001 indicates that the data model equation has reached a very significant level; the lack of fit value P=0.2311>0.05 shows no significant difference, indicating that the regression equation fits well with the test and the error is small. The variance analysis of the regression equation shows that the primary item C, D and the secondary item AB, B2, C2 of the regression model have a significant impact on the model (P<0.01); the primary item A and the secondary item AD have a significant impact on the model (P< 0.05); the other items had no significant effect on the extraction rate of polyphenols from corn silk (P>0.05). Therefore, this model can be used to analyze and predict the extraction process of polyphenols from corn silk.

Table 3 Analysis of variance of response surface test results

Note: P>0.05 means no significant impact on the results; P<0.05 means significant impact on the results; P<0.01 means extremely significant impact on the results.

2.3.3 response surface analysis

Using Box-Behnken software to analyze the response surface diagram can significantly reflect the strength of the interaction between various factors, and obtain the influence of the interaction of each factor on the response value. The results are shown in Figure 6. If the response surface is steeper, it means that this factor has a greater impact on the extraction rate of corn silk polyphenols, and the response value is more sensitive to the change of extraction conditions; if the response surface is relatively flat, it means that the fluctuation of this factor has little influence on the response value. It can be found from Figure 6 that the response surface of the interaction between extraction time and solid-liquid ratio, ionic liquid concentration and extraction time is relatively steep, indicating that the response value is significantly affected; the influence of other conditions is gradually reduced, and the curve is relatively smooth , there was no significant change in the response value. It can be concluded that the order of interaction factors affecting the extraction rate of corn silk polyphenols is AB>AD>CD>BC>BD>AC. In this experiment, ionic liquid was used as solvent combined with ultrasonic extraction, and the theoretical optimal process conditions obtained by response surface optimization were: ionic liquid concentration 0.48 mol/L, extraction time 23 min, solid-liquid ratio 1:10, and ethanol concentration 57%. The extraction rate of corn silk polyphenols can reach 32.46 μg/m L; the optimal extraction conditions were verified by experiments, and considering the actual operation, the optimal process conditions were corrected as: ionic liquid concentration of 0.50 mol/L, liquid-solid ratio of 1 : 10 (g/m L), extraction time 25 min, ethanol concentration 60%, the extraction rate of corn silk polyphenols measured was 30.26 μg/mL, closing to the predicted value.

Figure 6 Response surface diagram of the effect of pairwise interaction of each factor on the extraction rate of polyphenols from corn silk

03  Conclusion

Corn silk contains many polyphenol compounds. As a natural, healthy and non-toxic extract from plants, it is not only easy to get, but also has pharmacological effects, and is welcome in the market. Using ionic liquid as a solvent can improve the ability to destroy the cell wall of corn silk, driving polyphenols more likely to leak out, and is an efficient and pollution-free method for extracting polyphenols.