2.4.2. Effect of Extraction Temperature

Extraction rates at different temperatures (40, 50, 60, 70, 80 ◦C) were investigated under ultrasonic bath conditions (300 W, 50 Hz, 60 min), a liquid–solid ratio of 10:1, and 30% DES moisture content. Extraction temperature is the key factor affecting extraction rate. The target compound is adsorbed on the sample matrix by physical adsorption and chemical interaction, which would be reduced by high temperatures, thereby increasing the leaching of the target compound in the extraction solvent. In addition, a high extraction temperature can greatly reduce the viscosity of the extraction solvent, increase the diffusion of the extraction solvent, and accelerate the mass transfer of the target compound. Figure 3 shows that the extraction temperature of 50 ◦C provided a higher extraction rate than the other test extraction temperatures. Although a higher extraction temperature may improve extraction yield, it may not be able to avoid the thermal degradation of active ingredients at high temperatures [21]. Therefore, the extraction temperature of 50 ◦C was selected as the optimal extraction temperature in this study.

**Figure 3.** Effect of extraction temperature on the extraction yield of coumarins.

#### 2.4.3. Effect of Extraction Time

To some extent, the extraction rate is always proportional to the extraction time [22]. As shown in Figure 4, different extraction times (30, 40, 50, 60 min) were investigated under the conditions of an ultrasonic bath (300 W, 50 Hz, 60 ◦C), a liquid–solid ratio of 10:1, and a DES moisture content of 30%. The extraction rate showed an upward trend at 30–40 min, and a downward trend at 40–60 min. This may have been because the longer the time, the greater the energy generated by ultrasonic waves, forming a strong cavitation effect in the solution, which will cause more damage to the cell wall of medicinal materials, which means the extraction rate is also greater [23,24]. However, with the extension of the ultrasonic action time, the amount of extraction decreases, which may be because when the sample is under long-term ultrasonic action and a high temperature of 60 ◦C, the system is affected by ultrasonic radiation and thermal effect, which may produce double degradation. Therefore, 40 min was selected as the optimal extraction time at 60 ◦C. The extraction time was 20 min less than that of the traditional extraction method.

**Figure 4.** Effect of extraction time on the extraction yield of coumarins.

#### 2.4.4. Effect of DES Moisture Content

There are extensive hydrogen bond networks among DES components, resulting in high viscosity and weak mobility. Its viscosity and fluidity must be altered to obtain good permeability of the pores in the sample matrix and to facilitate mass transfer from the plant matrix to the solution. Adding water to DES can significantly reduce the viscosity and improve the extraction rate. In addition, the DES–water mixture is more alkaline and has a lower cost of extracting the target compound than the normal DES solvent [10]. Therefore, the extraction rate appears to be strongly dependent on the water content of the DES solution. As can be seen from Figure 5, different DES moisture counts were investigated under the conditions of an ultrasonic bath (300 W, 50 Hz, 60 min, 60 ◦C), and a liquid–solid ratio of 10:1. The extraction rate increased in the range of a 0–50% volume ratio and decreased in the range of 50–60%. It is possible that the excess water content increased the polarity of the mixture and reduced the interaction between the compounds. Therefore, a 50% DES moisture content was identified as the best choice during subsequent extraction.

**Figure 5.** Effect of DES moisture content on the extraction yield of coumarins.

On the basis of the preliminary single-factor experiments, the extraction process parameters were optimized by BBD with the ratio of liquid to solid, extraction time, ultrasonic temperature, and DES moisture content as four independent variables. The total extraction rate of 7 coumarin compounds was used as the response of the design experiment. Regression analysis was performed on the experimental data and additional validation experiments were performed to verify the effectiveness of the statistical experimental strategy. Software Design-Expert 12 was used to generate and evaluate experimental designs.

#### *2.5. HPLC Conditions and Method Validation*

The HPLC system consists of a Waters quad gradient high-performance liquid chromatography (ACQUITY Arc), a 122 Rheodyne injector (20 μL sample loop), and a full wavelength (200–600nm) diode array detector. The chromatographic analysis was performed on a C18 column (4.6 × 250 mm, 5.0 μm). The linear gradient elution was performed used methanol (A) and water (B) as mobile phases. The gradient elution procedure was as follows: 0–5 min: A-B (35:65), 15 min: A-B (65:35), 17 min: A-B (85:15), 30 min: A-B (90:10), 35 min: A-B (35:65), 37 min: A-B (35:65). The detection wavelength was set at 268 nm, the flow rate was 1 mL/min, the column temperature was 30 ◦C, and the injection volume was 10 μL. Sampling was started 10 min after system equilibrium, and all samples were analyzed by HPLC after passing through a 0.45 μm microporous membrane. The HPLC chromatogram of 7 coumarin standard substances and 7 coumarin compounds extracted from *A. dahurica* are shown in Figure 6.

The quantitative analysis of 7 coumarins in *A. dahurica* was verified by method validation. The linearity, precision, repeatability, and stability were verified. The calibration curves of 7 coumarins were performed with 6 standard solutions of different concentrations in triplicate. All calibration curves were well linear with high correlation coefficients (R2 > 0.9948) over the test range. The accuracy of the method was determined by intra-day and inter-day testing. Intra-day testing means the same sample was analyzed 6 times in the same day. Inter-day test was repeated testing of 3 samples obtained by the same extraction method for 3 consecutive days. The relative standard deviations of intra-day and inter-day precision were less than 1.24% and 2.13%, respectively. Six replicates of the same sample were prepared and analyzed for the repeatability test. In stability tests, the same sample was stored at room temperature and analyzed repeatedly at 0, 4, 6, 8, 12, and 24 h. The repeatability expressed by relative standard deviation was less than 1.37%, and the stability was less than 1.23%. The results showed that the established method was accurate and

sensitive for the quantitative analysis of 7 components of *A. dahuricae*. All of the above data are shown in Table 2.

**Figure 6.** The typical HPLC chromatograms: (**a**) 7 coumarin reference standards and (**b**) *A. dahurica* sample (1. xanthotoxol, 13.48 min; 2. psoralen, 16.76 min; 3. byakangelicin, 17.13 min; 4. bergapten, 19.04 min; 5. oxypeucedanin, 19.95 min; 6. imperatorin, 21.02 min; and 7. isoimperatorin, 22.38 min) and separation degree of chromatographic peak: 5.86%, 1.27%, 4.17%, 1.87%, 3.36%, 1.82%.

**Table 2.** Validation of linear regression equation, linear range, precision, repeatability, and stability of 7 coumarins.


#### *2.6. Determination of Antioxidant Activity of Plant Extracts*

Oxidation is one of the most important processes in food spoilage. Antioxidants can protect against the harmful effects of free radicals in the body and prevent the oxidation of fats and other food components [25]. The widely documented 1,1-diphenyl-2pyrylhydrazyl (DPPH) radical was used to assess antioxidant activity, because it is a stable free radical that can accept electrons or hydrogen radicals to form stable chemicals [26].

In this study, the antioxidant activities of two different extraction methods were compared (75% ethanol traditional heating reflux extraction and DES ultrasonic extraction). The concentration of the solution varied from 0.1 to 27 mg/mL for each sample. In addition, the vitamin C (VC) solution of the corresponding concentration was prepared by deionized water as a control test of antioxidant activity. The absorbance was measured at 517 nm relative to the blank. The percentage of scavenging activity was calculated as follows: (1 − (A1 − A2)/A0) × 100%, where A0 is the absorbance of the control, A1 is the absorbance of the sample, and A2 is the absorbance of the blank sample without DPPH free radical. The scavenging activity of the sample is expressed as the IC50 value, which is the concentration required to remove 50% DPPH free radicals [27].

#### *2.7. Microstructure of Plant Material*

In order to study the microscopic effects of different extraction methods on the fragmentation degree of *A. dahuricae* powder, the microstructure changes in the powder before and after extraction by three different extraction methods were observed by scanning electron microscope. The dried powder was secured to the sample table with double-sided tape and gilded. Then the morphology of the powders extracted with different solvents was observed by scanning electron microscope at 10 kV accelerating voltage.

#### **3. Results and Discussion**

#### *3.1. Screening of DESs*

The total yield of coumarins in each extraction solvent was the sum of seven kinds of coumarin compounds. The results showed that the total extraction rate of seven coumarin compounds was affected by different solvents. In order to understand the advantages of DESs in extracting coumarin compounds from *A. dahurica*, the extraction rates of different organic solvents (methanol and 75% ethanol) and different DESs were compared. As shown in Figure 7, compared with the traditional extraction method, the extraction rate of DES-3 was significantly higher than that of other DES solvents and traditional organic solvents.

**Figure 7.** The yield of total coumarins extracted with different organic solvents and DES. Note: The yield of total coumarins was the sum of 7 kinds of coumarins.

The composition of DES could influence the hydrogen bond between the hydrogen bond donor and the hydrogen bond acceptor, thus determining its viscosity and polarity. The high viscosity of DES restricts its application in extraction compared to conventional solvents. High viscosity may reduce extraction efficiency because of slow mass transfer,

whereas low-viscosity DES results in high diffusivity and therefore improved extraction performance. Most DESs showed a low total extraction rate for the seven coumarins, but DES prepared with choline chloride, citric acid, and water at a molar ratio of 1:1:2 produced a higher extraction efficiency. This may be because citric acid has higher hydrogen-bonding capacity and more electrostatic interaction with choline chloride than other hydrogen bond donors. Therefore, this type of DES was chosen as the best extraction solvent for coumarins compounds and applied in further tests based on the initial screening results.

#### *3.2. Optimization of the Extraction Conditions by Response Surface Methodology*

The above extraction studies show that the molar ratio of choline chloride, citric acid, and water of 1:1:2 was the best DES. In order to obtain the best extraction rate, RSM was used to optimize several extraction conditions that affected the extraction rate. Four-factor and three-level BBD were used to investigate the influence of four independent variables liquid–solid ratio (A), extraction temperature (B), extraction time (C), and DES moisture content (D)—on the extraction yield (Y), which is shown in Table 3.


**Table 3.** Factors and levels in response surface analysis.

The extraction process parameters of ultrasonic assisted DES extraction were systematically investigated, and the optimal extraction conditions of coumarin compounds in *A. dahurica* were determined. The solid-liquid ratio, ultrasonic temperature, extraction time and DES moisture content were investigated by single factor test. As shown in Table 4, on the basis of single factor test, RSM and BBD were used to determine the optimal combination of solid-liquid ratio, extraction temperature, extraction time and DES moisture content.

**Table 4.** Experimental order, variable levels, and response values in Box–Behnken designs.



The above tests were performed in three replicates. Design-Expert 12 was used to carry out multiple regression of BBD data to obtain the second-order polynomial model.

#### *3.3. Fitting the Response Surface Model*

The effects of four parameters on the yield of total coumarins in *A. dahurica* were investigated at the levels of −1, 0, and +1. A total of 29 different tests were carried out, and the mean values were fitted using a second-order polynomial model, as follows:

$$\begin{array}{c} \text{Y} = 1.01 - 0.14 \text{A} + 0.086 \text{B} + 0.077 \text{C} + 0.15 \text{D} - 0.050 \text{AB} - 0.055 \text{AC} + 0.17 \text{AD} - 0.055 \text{BC} + 0.038 \text{BD} - 0.030 \text{CD} \\ \qquad \qquad \qquad 0.030 \text{CD} - 0.10 \text{A}^2 - 0.040 \text{B}^2 + 0.12 \text{C}^2 - 0.087 \text{D}^2 \end{array}$$

Analysis of variance was used to evaluate the optimal conditions for ultrasonicassisted DES extraction and the relationship between the reaction and the variables. Table 5 shows the analysis of variance for the quadratic model. The F-value in analysis of variance ANOVA was used to estimate differences between groups. The F-value represents the significance of the whole fitting equation. The larger the F value is, the more significant the equation is and the better the fitting degree is. The *p*-value is a measure of the size of the difference. *p* < 0.05 indicates significant difference. *p* < 0.01 indicates extremely significant difference.

**Table 5.** The analysis of variance (ANOVA) results of the quadratic multiple regression model for total coumarin yield.


According to the F-value of the analysis of variance in Table 3, the influence degree of the four factors on the extraction rate of coumarins of *A. dahurica* was DES moisture content (D) > liquid–solid ratio (A) > extraction temperature (B) > extraction time (C), which is visually expressed by the Pareto diagram in Figure 8. The *p*-value of the model < 0.05, indicating that the regression relationship between the total rate of coumarins of *A. dahuricae* and their respective variables is significant, which can better simulate the real surface. The loss of fitting term *p* = 0.1058 > 0.05, and the influence was not significant, indicating that the experimental data and the model were significantly consistent. This model can be used to analyze and predict the extraction rate of coumarins in *A. dahuricae,* and it is true and reliable. In this model, primary term D had a highly significant effect on the total extraction rate of coumarins (*p* < 0.01), and primary term A had a significant effect on the total extraction rate of coumarins in *A. dahuricae* (*p* < 0.05); the interaction effect was not significant. The correlation coefficient R<sup>2</sup> = 0.974 and the adjusted determination coefficient adjustment R2 (Adj R 2) = 0.817, indicating that this model can be used to explain the change in the extraction rate of coumarin components in 81.7% of *A. dahuricae*.

**Figure 8.** Pareto diagram of different influencing factors.

#### *3.4. Verification of Predictive Model*

Using Design-Expert software, the optimal values of independent and dependent variables were as follows: liquid–solid ratio (A) was 10 mL/g, extraction temperature (B) was 59.85 ◦C, extraction time (C) was 50 min, and DES moisture content (D) was 49.28%. The maximum predicted extraction rate was 1.34%. Under the optimal extraction conditions, three verification experiments were carried out, and the average extraction rate was 1.18%. The results showed that the regression model was appropriate for predicting the extraction yield of coumarins from *A. dahuricae*. In addition, compared with the reported methods, the extraction time and solid–liquid ratio in this study were greatly reduced. The traditional solvent extraction time of coumarins is 4.23 h. Compared to the traditional extraction time, the extraction time used in this study was reduced by 5 times, only 50 min. Therefore, this model can save cost and is suitable for the optimization of the ultrasonic-assisted extraction process based on DESs.
