Study on the Effect of Deep Eutectic Solvent Liquid Phase Microextraction on Quality Standard, Antitussive, and Expectorant of Sangbaipi Decoction

The SD was extracted with a new green eutectic solvent, and the extraction method of TCM decoction was developed. In the quantitative analysis by HPLC, choline chloride phenol was selected as the eutectic solvent, THF was used as the extractant, and investigation of DES type, DES molar ratio, DES-to-THF ratio, vortex time, and material-to-liquid ratio was carried out. The experimental results showed that the optimal extraction method was as follows: the molar ratio of DES was 1 : 3, and the material-liquid ratio was 5 : 1200 (mL/μL). The volume ratio of DES to THF was 1200 : 800 (μL), the vortex time was 3 min, and the extraction was repeated two times. The eutectic solvent liquid phase microextraction method was adopted to optimize the extraction method of SD and reduce the complicated processing, long time, and low efficiency of traditional methods. At the same time, in the mouse ammonia water inducing cough and phenol red excretion and expectorant experiments, SD high- and medium-dose groups have a significant inhibitory effect on the frequency of antitussive in mice and both can increase the excretion of phenol red to varying degrees, indicating that SD has good cough-relieving and expectorant effect. The present study suggests a scientific basis and basis for the clinical research and quality standard formulation of SD.


Introduction
of solid halide salt with a certain stoichiometric ratio. It usually consists of two or three components, of which the melting point is significantly lower than that of each component [4]. Due to the interaction of hydrogen bonds in DES, charge delocalization makes the mixture formed as a liquid system at room temperature, of which the melting point is lower than that of each component [5][6][7][8][9][10]. As a new green and efficient solvent, DES has advantages of a simple preparation process, low cost and toxicity, environmental protection, complete biodegradation, and biocompatibility [11][12][13]. It is also reported to be applied in electrochemistry, preparation of nanomaterials, catalytic reaction, separation process, and preparation of functional materials [14][15][16]. In recent years, DES has extensively been used for extracting bioactive components from natural plant materials. So far, studies on SD are mainly focused on clinical research, instead of its effective components, extraction methods, antitussive, and expectorant effects. In this study, a new liquidphase microextraction method [17] was employed for the separation of SD through optimizing the sample processing method of SD; in addition, in vivo experiments were also conducted to validate the effect of SD on antitussive and expectorant, so as to provide the basis for its clinical rational drug use and quality standard research.

Instrument.
A Huapu S3000 high-performance liquid chromatography system (including a four-element lowpressure stirring pump, automatic sampler, column box, 1100 diode array detector, and chemical workstation) was used for chromatographic analysis. A KQ-250 ultrasonic cleaning machine produced by Kunshan Ultrasonic Instrument Co., Ltd. was used. An AB135-S electronic balance was purchased from Mettler-Toledo International Ltd. An ATY224 type-1/10000 balance, Shimadzu, Japan, was used.
e Ql-901 vortex instrument was purchased from Haimen City Qilin Bell Instrument Manufacturing Co., Ltd., the HH-S4 digital constant-temperature water bath was purchased from Jiangsu Jinyi Instrument Technology Co., Ltd., the TDL-60B centrifuge was purchased from Shanghai Anting Scientific Instrument Factory, and the YLS-8A multifunction cough-inducing and asthma-inducing instrument was purchased from Jinan Yiyan Technology Development Co., Ltd. and BioTek Instruments, Inc.  Table 2.

Materials. Cortex
SPF ICR mice were provided by Jilin Yisi experimental animal Co., Ltd., production license no. SCXK (Ji)-2016-0003. e abovementioned experimental animals were used by Changchun University of Chinese Medicine with the license number of SCXK (Ji)-2016-0017. e ethics number of laboratory animal was 201908A019. ey were reared in the experimental animal center of Changchun University of Chinese Medicine and divided into male and female cages, with rat pellet feed and free drink. e room temperature was 20-23°C, and the humidity was 44%-57%. e pellet feed of SPF-grade laboratory mice was provided by Jilin Yisi Experimental Animal Co., Ltd. e hydrogen bond donor and hydrogen bond acceptor were mixed according to the molar ratio, which was heated in a water bath at 80°C∼100°C, and fully stirred in a beaker until a clear, transparent, and slightly viscous liquid solvent was obtained for preparing DES.

Deep Eutectic Liquid-Phase Microextraction Process (DES-LPME).
e process of DES-LPME is shown in Figure 1. 1200 μL DES was added into the test tube with 5 mL SD and then vortexed for 5 min to premix. en, 800 μL tetrahydrofuran (THF) was added to the test tube for mixing and centrifuged at 6000 rpm for 10 min. e DES phase was separated from the other phase. e upper phase (DES) was removed from the test tube with a pipette gun to obtain the extract. e step was required to repeat two times. e first and second extractions were removed and placed in a 5 mL volumetric flask with THF at constant volume and mixed evenly, and 0.2 mL, acetonitrile to 5 mL, was taken for HPLC determination.

Study on Antitussive and Antitussive Effects of SD on Mice.
e mice were randomly divided into a model group, Chuanbei Loquat Ointment group (4.55 mL/kg), SD with low-dose group (12.125 mL/kg), SD with medium-dose group (24.25 mL/kg), and SD with high-dose group (48.5 mL/kg), with 10 mice in each group, male and female in half. e mice in experiment groups were administrated by required dose, compared to the model group given the same dose of distilled water by gavage for 5 days. After the last administration of 30 min in the antitussive experiments, the mice were placed in the ammonia water coughing device, which is the YLS-8A multifunction cough-inducing asthma apparatus, as well as the 50% ammonia constant-pressure spray for 30 s. e cough was recorded at the first time in the mice (mice largely opened mouth, accompanied by abdominal contraction and occasional cough), which was cough latency, as well as the cough frequency within 3 min at the same time. After the last administration of 30 min in the phlegm experiment, 0.5% phenol red solution (0.2 mL/10 g) was injected intraperitoneally. After 30 minutes, the rats were killed, and the neck was cut open to expose the trachea. A section of the trachea from the thyroid gland to the trachea branch was cut off and put into the mixture of 2 mL sodium bicarbonate and normal saline (the ratio was 1 : 15). e absorbance (OD value) at 546 nm was detected by using an enzyme reader for calculating the phenol red excretion.

Evaluation of the Method.
Under optimal conditions, system suitability testing, linear range (LR), correlation coefficients (R), limit of detection (LOD), limit of quantification (LOQ), accuracy test and relative standard deviation (RSD), and rate of recovery were investigated to evaluate the proposed HPLC-DAD method.
e system adaptability test showed that the chromatographic peaks of palmatine, berberine, baicalein, and wogonin were well separated, with more than 3000 theoretical plates. e results are shown in Figure 2. e standard calibration curves were constructed using a series of blank samples spiked with the standard substance at different concentrations. e analytical results (Table 3) show that there was a good linear relationship with the peak area in the respective concentration range.
e LOD was calculated based on a signal-to-noise ratio of 3, while LOQ was obtained based on the signal-to-noise ratio of 10. e results are shown in Table 3. e precision was tested by intraday (n � 3) and interday (n � 3) analysis, and the RSDs were within 1.13-1.76% and 1.07-1.88%, respectively.
To determine the accuracy of the developed method, 0.5 mL of palmatine reference solution (251 μg/mL), 1 mL of berberine reference solution (641 μg/mL), and baicalein reference solution were added to the selected samples of SDs (35.7 μg/mL) 0.5 mL and wogonin reference substance solution (30.55 μg/mL) 0.5 mL. After extraction and analysis, the average recovery rate of palmatine, berberine, baicalein, and wogonin is calculated to be in the range of 97.51-98.45%, and the RSD value is in the range of 1.34-1.49%.
By changing the composition of the mobile phase, including methanol-phosphoric acid-water, acetonitrilephosphoric acid-water, methanol-water, and acetonitrilewater, we found that the acetonitrile-phosphoric acid-water system can separate the sample well to achieve good resolution, with good chromatographic peak shape of the components to be determined. Using the HPLC-DAD method to scan the full wavelength of the sample, it is found that the peak shape is at 238 nm, the resolution is better, the number of peaks is higher, and the peak response value is higher. erefore, 238 nm was selected as the detection wavelength of mulberry SD. us, based on good stability, precision, and repeatability, the method could be used for the accurate and rapid determination of palmatine, berberine, baicalein, and wogonin in SD.

Screening on DES Species.
e hydrogen bond donor and acceptor were mixed according to the molar ratio. e mixture was heated in a water bath at 80-100°C and stirred well in a beaker until a clear, transparent, and slightly viscous liquid solvent was obtained for preparing DES.   Table 4 were mixed in a certain molar ratio, and DES was prepared by the "2.5" method and extracted according to the "2.6" experimental steps. e results showed that the two phases of DES formed by choline chloride and phenol could be completely separated by THF when extracting with THF, whereas the other eutectic solvents could not be separated by THF. erefore, THF as an extractant and choline chloride phenol as a eutectic solvent were selected for subsequent experiments.
Due to the hydrophobic nature of DES and THF, as well as good solubility to palmatine, berberine, baicalein, and wogonin, the combination is suitable for extracting some active components from an aqueous solution and aqueous suspension for pretreatment on concentration or determination of active components in the TCM decoction.

Extraction Times.
e times of extraction is the key factor affecting the whole extraction process. Choosing the appropriate times of extraction can not only improve the extraction effect but also save the experimental cost and shorten the experimental time.
According to the extraction rate of palmatine, berberine, baicalein, and wogonin in the solution since each extraction, we had found that the extraction rate of palmatine, berberine, baicalein, and wogonin in SD could be significantly improved by using choline chloride phenol as eutectic solvent and THF as extractant. Wogonin could be completely extracted after the first extraction; moreover, the extraction rate of palmatine, berberine, and baicalein can reach more than 93% after two extractions, so the solution with twice extractions was selected for the study.

Effect of DES Molar Ratio.
In the study, the selection of a suitable extraction solvent played an important role in the most effective separation process. We chose choline chloride and phenol as an effective separation solvent, which were made into five kinds of DES with different molar concentrations (1 : 1, 1 : 2, 1 : 3, 1 : 4, and 1 : 5). e results are shown in Figure 3(a). When the molar ratio of choline chloride to phenol was increased to 1 : 3, the extraction rate of palmatine, berberine, and baicalein was significantly increased, and then, the extraction rate had no significant change. So, the molar ratio of choline chloride and phenol was 1 : 3.

3.5.
e Influence of the Proportion of Extraction. Optimizing the solid-liquid ratio is of great significance in the study of liquid-phase microextraction. Different volumes of eutectic reagents (5 : 0.4, 5 : 0.6, 5 : 0.8, 5 : 1.0, 5 : 1.2, and 5 : 1.4) were added to a 5 mL sample solution of SD. We selected the best ratio of material to liquid through the extraction effect. Results are as shown in Figure 3(b); the extraction rate of palmatine, berberine, and baicalein in SD was the highest when the DES dosage was 1200 μL.
3.6. e Influence of the Ratio of DES to THF. As a solvent, tetrahydrofuran (THF) was used to remove DES from the aqueous phase. In this deep eutectic study, THF was used to separate and enrich the DES phase and aqueous phase. After adding THF, the DES phase and water phase of SD were separated efficiently and thoroughly. e effects of different volume ratios of DES and THF (1200 : 200, 1200 : 400, 1200 : 600, 1200 : 800, 1200 : 1000, and 1200 : 1200) on the extraction rate of SD were compared. e results are shown in Figure 3(c). When the volume ratio of DES to THF is 1200 : 800, the extraction rate of palmatine, berberine, and baicalein is the best.

e Influence of Vortex Time.
In the study of liquidphase extraction, the contact time between the sample solution and extract is particularly important. Our research investigated the effects of a vortex time of 1 min, 3 min, 5 min, 7 min, and 9 min on the extraction rate of palmatine, berberine, and baicalein in SD.
e results are shown in Figure 3(d). e results showed that the extraction rate of palmatine, berberine, and baicalein reached the highest when the vortex time had lasted for 5 min. With the extension of vortex time, the extraction rate did not change significantly. erefore, under the premise of high efficiency and accuracy, the vortex time of SD was 5 min.

Response Surface Design and Results.
Combining the single-factor test, according to the Box-Behnken principle, the Design-expert 8.0.6 software is used to design a threefactor three-level test program. e test contains 5 central points and 17 test points, with palmatine and berberine. e content of baicalein is subjected to dimensionless treatment to obtain the comprehensive evaluation value as the inspection index, and the experiment is carried out. e response surface optimization conditions are shown in Table 5, and the design plan results are shown in Table 6.

Quadratic Regression Model Fitting and Model
Analysis. Using the response surface to perform regression fitting on the experimental data, the multinomial regression simulation equation of the extraction rate of SD is obtained as Y It can be seen from Table 7 that the high F value (23.10) and low P value (0.0002) of this test result indicate that the model has extremely significant differences. e F value of the lack-of-fit term is 2.79 and the P value is 0.1731, indicating that the lack of fit is not significant, that is, the model has a good fit. e R 2 Adj of this model is 0.9256 > 0.80, indicating that this model can explain at least 92.56% of the response value change. R 2 � 0.9674, indicating that the actual value has a good correlation with the predicted value of the equation.
e signal-to-noise ratio Adeq Precision � 12.539 > 4 indicates that the model has a very high degree of fit and reliability. From the F value of each individual item, it can be seen that the degree of influence of the analysis conditions on the extraction rate of SD in descending order is B (molar ratio of DES), C (vortex time), and A (the ratio of    Journal of Analytical Methods in Chemistry DES to THF)). From the F value of the quadratic term, it can be seen that the difference between A 2 , B 2 , and C 2 on the response value is extremely significant, the F value is generally higher, and in the regression equation, the coefficients of these three terms are all negative values. It shows that when the maximum value of this factor is exceeded, as the ratio of DES to THF, molar ratio of DES, and vortex time increase, the extraction rate will decrease. is conclusion is consistent with the single-factor test results. In this model, A 2 , B 2 , and C 2 have extremely significant effects on the extraction rate of SD. It can be seen from Figure 4 that the contour map of the AB interaction is obviously elliptical, indicating that the AB interaction has a significant impact. Optimized by Design-expert 8.0.6 software, the optimal extraction process conditions of SD were as follows: DES: THF was 1200 : 803.28 (v: v), the molar ratio was 1 : 2.84, and the vortex time was 3.12 min. Combining the actual operation situation, the final determination was that the extraction process had DES: THF 1200 : 800 (v: v), the molar ratio 1 : 3, and the vortex time 3 min.

Verification
Test. 5 mL of SD was transferred, and 3 verification experiments were performed according to the optimized extraction process. e average extraction rates of palmatine, berberine, and baicalein were 92.02%, 94.98%, and 92.67%, respectively. e comprehensive evaluation value was 3.138. e relative error with the predicted value was less than 2%. It was suggested that the optimized extraction process was stable and reliable.

Comparison in Determination Results of Samples.
To verify the accuracy of the sample processing method, 10 SD samples were prepared by the deep eutectic solvent liquidphase microextraction method, direct determination method, and freeze-drying extraction method. e deep eutectic solvent liquid phase microextraction method was adopted according to "2.6"; the direct determination method was the SD solution, filtered through a 0.22 μm microporous membrane; the freeze-drying extraction method was to take 5 mL SD to freeze-dry till powder, perform ultrasonic   extraction at 60°C for 30 min after adding 50 mL methanol, then filter and dry the obtained filtrate by vacuum, and fix the volume to 5 mL; in terms of the three methods, contents of palmatine, berberine, baicalein, and wogonin in SD were determined as shown in Table 8. According to the results in Table 9, by comparing the same SD determined by different treatment methods, we had found the results in depth, and accurateness of the eutectic solvent liquid-phase microextraction method and freeze-drying extraction method was higher and more than that of the direct determination method.
e TCM decoction contains a large amount of water, which diluted the content of active ingredients to a low level. It is often necessary to concentrate the decoction due to the difficulties in the determination of TCM decoction for making the concentration of the test sample above the limit of quantification and detection. e concentration of water is often accompanied by heating, which may destroy the content of active ingredients [18], as well as complex treatment. e freeze-drying method can also be used in the concentration of samples, which is also more complex, time consuming, and less efficient. In spite of high active ingredients in some decoctions, the determination method of direct filtration will lose some precipitates with poor solubility in water [19], which makes the determination result lower than the real result.

Study on Antitussive and Expectorant Effects of SD on Mice
3.10.1. Antitussive Effect of SD on Mice. Cough, as a protective respiratory reflex action, is a common symptom of bronchial asthma [20], pneumonia, and other diseases. Ammonia water [21] is used as a chemical stimulator. When the body is attacked by this kind of chemical factor, it stimulates the respiratory receptor and then causes cough. After induced by ammonia, the mice in each group had cough reaction at different times. Compared with the model group, the SD group with high, medium, and low doses, as well as the Chuanbei Loquat Ointment group, could significantly prolong the cough latency of mice (P < 0.05 or P < 0.001); the SD group with high and medium doses, as well as the Chuanbei Loquat Ointment group, could also significantly inhibit the cough frequency of mice (P < 0.05 or P < 0.001). erefore, SD has a good antitussive effect. e results are shown in Table 8.

Study on the Expectorant Effect of SD on Mice.
Sputum is the secretion of the human respiratory tract, which is secreted by the trachea, bronchus, nose, and throat. Under the normal physiological state, the mucus secreted by the respiratory tract is very little, which can keep the Note. Compared with the control group, * p < 0.05, * * p < 0.001.  Note. Compared with the control group, * p < 0.05, * p < 0.01. respiratory tract moist. However, when inflammation occurs, the sputum will accumulate, is viscous, and is difficult to discharge. In the expectorant experiment, phenol red [22,23] was injected intraperitoneally to reach the lung through blood circulation, which could be detected in the trachea wall under the secretion of the trachea. Phenol red excretion and expectorant test in mice: First, the standard curve of phenol red solution was established. A series of phenol red solutions with the concentrations of 0, 0.1, 0.2, 0.4, 0.5, and 1.0 μg/mL were prepared by taking an appropriate amount of phenol red and using 1 mol/L sodium hydroxide solution. e absorbance values were determined according to the instrument. e standard curve equation was Y � 0.0692X + 0.0471 (r � 0.9998) by linear regression with phenol red concentration (X, μg/mL) as the abscissa and absorbance value (Y) as the ordinate.
Compared with the model group, the SD groups with the high, medium, and low doses and Chuanbei Loquat Ointment could increase the excretion of phenol red in different degrees, with a significant effect (P < 0.05 or P < 0.001) in SD groups with the high and medium doses and the Chuanbei Loquat Ointment group, which showed that SD improved the secretion of sputum in the trachea of mice and had an expectorant effect. e results are shown in Table 10.

Conclusions
is study provides a new method for the determination and extraction of active components in SD. According to deep eutectic solvent liquid-phase microextraction (LPME), we selected phenol and choline chloride as the deep eutectic solvent to extract and determine four active components, such as palmatine, berberine, baicalein, and wogonin, so as to establish the quality evaluation method of SD. Compared with other methods, this method has the advantages of simple operation, good repeatability, high accuracy, and efficiency, as well as low pollution. In the other way, it is proved that SD has good antitussive and expectorant effects through the experiments of ammonia-induced cough and phenol red excretion and expectorant in mice.

Data Availability
e main table and figure data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare no conflicts of interest.