2.2. Comparison of the Extractability of DESs
The extraction efficiency of five different DES was studied by UPE under the same extraction pressure (500 MPa), extraction time (5 min) and liquid-solid ratio (40 mL/g). From
Figure 1, the DES extraction yield of baicalin varied with the different solvents. Using 70% ethanol, the most common solvent used for baicalin extraction as reference, DES-1 displayed clear superiority over other DESs with an extraction yield of 72 mg/g. The lower extraction yield of other DESs may be due to their high viscosity.
It is well known that the extraction efficiency of target compounds is affected by many factors, such as polarity, diffusion, solubility, viscosity, surface tension and physicochemical interaction with extraction solvents [
34,
35,
36]. In other words, the type of DES plays a decisive role on the baicalin extraction efficiency. DES-1 had a better performance than other DESs, which may be due to a higher ability to form hydrogen bonds and more electrostatic interactions of DES-1 with baicalin than other DESs [
37]. Another important factor for the extraction efficiency is the solvent polarity. Theoretically, the bioactive compounds can be easily extracted with solvents having similar polarity. DES-1 gave the highest extraction yield, and this suggested DES-1 has a similar polarity to baicalin [
33]. The solvent viscosity has a great impact on the dissolution and diffusion of extracts. By visual observation, the viscosity of DES-1 was relatively less than other DESs. The lesser viscosity led to the relatively large diffusivity, and this resulted in a high extraction yield [
31]. In summary, DESs as a mixture have complicated physical and chemical properties, when compared with protic organic solvents as pure substances. Therefore, it is difficult to find the rules for the extraction yield in DESs [
38].
2.3. Optimization of the Extraction Conditions by RSM
From the above results, DES-1 was selected as the most appropriate DES for the process optimization to maximize the extraction yield of baicalin. In order to analyze the effect of various independent variables on the extraction rate, a single factor experiment was conducted to determine the main variables of Box-Behnken design (BBD). Four independent variables—water content (A), extraction time (B), extraction pressure (C) and liquid-solid ratio (D)—were determined and encoded at three levels. The associated negative signs (−1) was for low level, zero (0) represented the central value, and high level was expressed by a plus symbol (+1). The experimental design matrix and horizontal factors are shown in
Table 3.
After the optimization, a BBD with 4-factors, 3-levels and 29 experimental runs was employed in this study. With the maximum extraction yield of baicalin as the response index, all response surface experiments and results are summarized in
Table 4.
The non-linear regression fitting based on the Quadratic model of the Design-Expert software (trial version 8.0.6.1, Stat-Ease Inc., Minneapolis, MN, USA) was employed to express baicalin extraction yield (Y
Baicalin). The equation of dependent variables was shown below:
The analysis of variance (ANOVA) for the quadratic models was listed in
Table 5. The model was accurate and reliable by high coefficient determination (r
2 = 0.9913) and non-significant lack-of-fit (
p value of 0.1223). As shown in
Table 5, the interaction terms BD and CD had a significant effect on the yield of baicalin, and the interaction (
p < 0.0001) were significant. The “Pred R-Squared” of 0.976913 was in reasonable agreement with the “Adj R-Squared” of 0.991326.
Three-dimensional surface plots were used to study the interactions between the different variables of water content (A), extraction time (B), extraction pressure (C) and liquid to solid ratio (D) on the extraction yield of baicalin (
Figure 2a–f). Generally, the relationship between the extraction yield and the single variable is described by a quadratic parabola. The results indicate that the extraction yield gradually increases with increasing water content, extraction time, extraction pressure and liquid to solid ratio, and then decrease after reaching the maximum. In summary, the extraction time, extraction pressure and liquid to solid ratio were more important than the water content.
Figure 2a–c show that the water content has little effect on the extraction efficiency. As shown in
Figure 2b, when the water content was fixed, the extraction yield gradually rose with increasing extraction pressure, reached a maximum at 400 MPa, and then slowly drop down. Therefore, the optimal pressure was considered as 400 MPa.
Higher pressure causes structural changes to the plant cells, and also increases the speed of solvent penetration into the plant material. This dual effect resulted in the intracellular active ingredient release from the disrupted cell wall. Therefore, the increase of extraction yield under increased pressure could be attributed to the acceleration of mass transfer. With pressure greater than 400 MPa, the cell wall and cell membrane were fully destroyed, and the target compounds completely dissolved.
Under this circumstance, the change of extraction yield was little. As shown in
Figure 2d, the combined effect of extraction pressure and extraction time had more significant effect on the extraction yield. In
Figure 2e, both the extraction time and liquid to solid ratio had more influence on the extraction yield. So the extraction yield gradually rose, and reached a maximum at 4 min and 110 mL/g. After reaching the maximum, the decrease of extraction yield was also slow. In
Figure 2f, with the increase of extraction pressure and liquid to solid ratio, the extraction efficiency gradually reached the maximum, and then remained almost constant with slight drop-off.
The extraction conditions of baicalin were optimized by the regression analysis of model equation. The optimum conditions were determined as 39.22 vol% water content, extraction pressure of 418.3 MPa; extraction time of 4.15 min and a liquid-solid ratio of 109.23 mL/g. Under the optimum condition, the maximum extraction yield was 116.448 mg/g.
The validation test was conducted with the fixed optimum condition and the prediction value was determined by regression analysis. The final extraction parameters were determined as 40 vol% water content, extraction pressure of 400 MPa, extraction time of 4 min and a liquid-solid ratio of 110 mL/g, respectively. Under the fixed optimum conditions, parallel tests were repeated three times, and the average extraction yield obtained was 116.8 mg/g, which was consistent with the predicted value. These results confirmed that the response model could be used for the optimization2.4. Comparison of Extraction Methods
The comparison of extraction methods and extraction solvents were conducted and listed in
Table 6. Two different extraction solvents in this study were 40% DESs-containing aqueous solutions and 70% ethanol, and three different methods were MAE, UPE and HRAE. The extraction efficiency from DES-based UPE as 116.8 mg/g is slightly higher than 110.4 mg/g from the Pharmacopoeia procedure. However, the extraction time is dramatically shortened from 3 h to 4 min. When compared with other reported baicalin extraction by DESs [
31,
32], the extraction efficiency from DES-based UPE is still better.
From the above results, the DESs-UPE method had a significant superiority over other methods on the extraction efficiency of baicalin. With the acoustic cavitation effect, the solvent penetration into plant cells was promoted, and the release of intracellular active ingredients from the disrupted cell walls was facilitated in the UPE process. As compared with traditional HRAE and MAE, the high pressure from UPE process increased the rate of dissolution and diffusion of baicalin from the plant cells. Furthermore, the DESs also could accelerate plant cell rupture for the release of the intracellular products [
39,
40,
41,
42]. Therefore, the current results revealed the DES-based UPE was a more rapid and efficient extraction method for the target compounds.
2.4. Microstructure Alteration of Different Extraction Procedures
In order to study the effect of different extraction methods on the microstructure of
Scutellaria baicalensis, SEM was used to observe the raw and extracted
Scutellaria baicalensis Georgi samples (
Figure 3). As shown in
Figure 3a, the external surface of the raw sample was smooth without apparent disruption on the cell surface. The hot reflux during HRAE extraction, only a few cells was slightly ruptured (
Figure 3d,g) by the heat conduction and convection. Thus, the target compounds were extracted mainly through solubilization and permeation and the extraction time was long for HRAE. In the process of MAE, the sample was partially destroyed (
Figure 3c,f). In the process of UPE, the pressure build-up within the plant cells probably exceeded their limit of contraction and caused them ruptured more rapidly. After UPE (
Figure 3b,e), the surface of the cells was seriously destroyed with several holes on the walls, and some cells even broke into small fragments. This indicated that ultrahigh pressure resulted in a severe change in the surface of cells, which made the dissolution and diffusion more readily.
With the same extraction methods, the degree of disruption of plant cells followed the order: DESs-based HRAE (
Figure 3d) > 70% ethanol-based HRAE (
Figure 3g), DESs-based MAE (
Figure 3c) > 70% ethanol-based MAE (
Figure 3f), DESs-based UPE (
Figure 3b) > 70% ethanol-based UPE (
Figure 3e). These results indicated that the plant cells were easily disrupted in DESs conditions. The DESs caused damage may be attributed to the cell wall fiber dissolution in DESs [
43]. Generally speaking, the degree of microstructure changes observed by SEM were consistent with the resultant extraction efficiency. The more ruptured cell resulted in a more efficient extraction. From the microscopic point of view, the severe fragmentation from DES-UPE rendered the dissolution and diffusion more easily, and then more efficient extraction.