# Deep Eutectic Solvents (DESs)-Ultrasonic-Assisted Extraction of Paeoniflorin and Paeonol from Moutan Cortex

Authors: Zhuo Chen; Jinnan Zhang; Fuwei Yang
Journal: Journal of Chemistry
 (2022)

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## Abstract

Deep eutectic solvents (DESs-)-ultrasonic-assisted extraction technology was used to evaluate and optimize the extraction process of paeoniflorin and paeonol in Moutan Cortex. First of all, 10 kinds of choline chloride-based DESs were selected as potential extraction solvents, and the DES extraction effect of choline chloride: lactic acid was determined to be the best. Then, single-factor experiments were conducted to explore the effects of DES composition (molar ratio, water content, and ratio of material to liquid) and ultrasonic treatment (ultrasonic power, extraction time, extraction temperature, and vortex time) on the yield of paeoniflorin and paeonol in Moutan Cortex. On this basis, the response surface method was used to optimize the process of ultrasonic-assisted extraction of paeoniflorin and paeonol from Moutan Cortex with DESs. The results showed that the optimum process parameters of DESs-ultrasonic-assisted extraction of paeoniflorin and paeonol from Moutan Cortex were as follows: molar ratio of choline chloride to lactic acid was 1 : 2, water content was 30%, ratio of material to liquid was 1 : 30 (g/mL), ultrasonic power was 300 W, extraction time was 33 min, extraction temperature was 50°C, and vortex time was 5 min. Under these conditions, the yield sum of paeoniflorin and paeonol in the obtained Moutan Cortex was 9.279 mg/g. The difference between the experimental value and the theoretical value was 0.333 mg/g. The results showed that the response surface method can simulate and predict the yield of paeoniflorin and paeonol in Moutan Cortex under different extraction conditions, and it was feasible to optimize the process parameters. The results of this study show that DES has great potential to replace traditional solvents in the extraction of active components of the Moutan Cortex. The study on the extraction of active components of traditional Chinese medicine by DES is helpful to expand the scope of the application of DES in traditional Chinese medicine.


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## Body

## 1. Introduction

Moutan Cortex is the dry root bark ofPaeonia suffruticosa Andr. It is bitter, pungent, and slightly cold, and has the effect of clearing away heat and cooling blood, activating blood circulation and removing blood stasis, used for heat into camp blood, warm toxin hair spot, hematemesis and bleeding, night heat, and early cool, nonsweating bone steaming, amenorrhea dysmenorrhea, falling pain, bloated sore poison, and other symptoms [1]. Moutan Cortex was first published in the Classic of Shennong Materia Medica, listed as middle-grade, and has a medicinal history of more than 2000 years [2]. Modern studies have shown that Moutan Cortex mainly contains flavonoids, phenols, and phenolic glycosides, monoterpenes, and their glycosides, triterpenes and their glycosides, stilbenes, organic acids, volatile oils, phenylpropanoids, and tannins [3–5]. Among them, paeonol and paeoniflorin have significant biological activities [6, 7]. Pharmacological studies have shown that paeonol has a certain protective effect on cardio-cerebrovascular diseases, relaxing blood vessels and lowering blood pressure [8, 9]. At the same time, paeoniflorin also has a certain effect on reducing blood pressure and dilating blood vessels [10].The traditional extraction methods of Moutan Cortex are steam distillation extraction [11] and organic solvent extraction [12]. These methods have some disadvantages such as low extraction efficiency and serious environmental pollution. In 2003, the concept of DES was proposed for the first time [13]. DES is a new green solvent with an intermolecular hydrogen bond synthesized by combining two naturally derived molecules [14]. DES is considered a green solvent due to its characteristics of easy synthesis, low price, environmental friendliness, good biocompatibility [15], low volatility [16], strong solubility, biodegradable, structurally designable, low toxicity, and even nontoxicity [17, 18]. It is widely used in the extraction of bioactive components such as polysaccharides [19], alkaloids [20], and phenolic acids [21]. The screened DES was used as the extraction medium in order to study the green extraction process of Moutan Cortex. Based on a single-factor experiment, and combined with the Box-Behnken response surface method, a model was established to guide the extraction process, and the optimum extraction conditions of Moutan Cortex effective components were optimized to achieve efficient green extraction. Compared with the traditional extraction method, it has outstanding advantages. It can provide a more green, efficient, economical, and environmentally friendly new extraction method and better promote the in-depth development of Moutan Cortex, to provide a theoretical basis for the comprehensive development and utilization of Moutan Cortex.


## 2. Materials and Methods

### 2.1. Reagents

The Moutan Cortex used in this experiment was produced in Sichuan Province, China, which met the requirements of Chinese Pharmacopoeia (2020 edition). Chromatographic grade methanol (batch number 20200601) and acetonitrile (batch number 20200601) were purchased from Fisher Company in the USA, analytical pure phosphoric acid (batch number 20170408) was purchased from Tianjin Guangfu Technology Development Co., Ltd., and ultrapure water was purchased from Hangzhou Wa Co., Ltd. Paeoniflorin (batch number111642-200301, China Institute for Food and Drug Control), paeonol (batch number 111889-201705, China Institute for Food and Drug Control), and choline chloride (batch number 150120) were purchased from Shanghai Zhanyun Chemical Co., Ltd. Maltose (batch number 117101), fructose (batch number 117001), malic acid (batch number 117101), citric acid (batch number 117101), lactic acid (batch number 117101), and xylitol (batch number 117101) were purchased from Zhengzhou Kangyuan Chemical products Co., Ltd. Acetic acid (batch number 117101) were purchased from Xilong Science Co., Ltd. Propylene glycol (batch number 20180309) was purchased from Tianjin Zhonghe Shengtai Chemical Co., Ltd. Glycerin (batch number 20180406) was purchased from Guangzhou Miya Cosmetics Co., Ltd. Urea (batch number 20170109) was purchased from Tianjin Ruijinte Chemical Co., Ltd.


### 2.2. Instrumentation

Agilent 1260 high-performance liquid chromatography system was purchased from Agilent Technologies Co., Ltd. (including four-element low-pressure stirring pump, automatic injector, column incubator, 1100 diode array detector, and chemical workstation) and was used for chromatographic analysis. QL-901 Vortex instrument was purchased from Haimen Qilinbeier Instrument Manufacturing Co., Ltd. JP-300G ultrasonic extractor was purchased from Wuhan Jiapeng Electronics Co., Ltd. AB135-S electronic balance was purchased from Mettler-Toledo International Co., Ltd. ATY224 type-1/10000 balance was purchased from Shimadzu, Japan. TGL-16 freezing centrifuge was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.


### 2.3. Preparation of DESs

Choline chloride was selected as the DESs type of HBA. Because of its simple preparation and stable properties, choline chloride is widely used [22]. It can efficiently extract and separate alkaloids, flavonoids, polysaccharides, and acids from medicinal materials [23, 24]. HBA choline chloride and the various HBDs (maltose, malic acid, lactic acid, fructose, glycerol, propylene glycol, xylitol, urea, acetic, and acid, citric acid) were weighted at a certain molar ratio (Table 1), and the mixtures were stirred in sealed bottles at 80-100°C until a transparent, colorless liquid is formed after 0.5–3 h.Table 1
Different types of DESs.
No.HBAHBDMolar ratioExtraction efficiency mg/gPaeoniflorinPaeonolTotal1Choline chlorideMaltose1 : 14.0304.0378.0672Malic acid1 : 11.9712.6984.6693Lactic acid1 : 24.4604.7209.1804Fructose1 : 12.5672.6865.2535Glycerol1 : 24.3304.5458.8756Propylene glycol1 : 24.1054.6278.7327Xylitol1 : 11.5002.0763.5768Urea1 : 24.6894.0878.7769Acetic acid1 : 24.1104.9209.03010Citric acid1 : 22.7963.8356.630


### 2.4. Extraction Procedures

Moutan Cortex was dried at 60°C, crushed, sifted through 100 mesh, sealed, and stored at room temperature. Precision weighing Moutan Cortex powder 50 mg was taken, adding 1.5 mL DES (containing 30% water) with 60°C warm bath for 5 min, and vortex oscillation for 5 min. After mixing evenly, ultrasonic treatment was carried out, 30 min was extracted by ultrasonic at 60°C, ultrasonic power was 250 W, vortex mixing, 3000 r/min centrifugal for 5 min, 0.2 mL of the supernatant solution was precisely absorbed into the EP tube, methanol was diluted to 1 mL, and the Moutan Cortex extract was obtained for HPLC analysis.


### 2.5. Chromatographic System and Conditions

HPLC analysis was performed on an HPLC-DAD Agilent 1260 system. The separation was performed on an Agilent TC-C18 chromatography column (5 μm, 4.6x250mm). The mobile phase consisted of 100% acetonitrile (solvent A) and 0.1% phosphoric acid aqueous solution (solvent B). The gradient program was as follows: 0–17 min 14% (A), 17–18 min 14 → 47% (A), and 18–30 min 47% (A). The injection volume was 15 μL, and the flow rate was 1 mL/min-1 at 30°C column temperature. The detection wavelength was set to 238 nm. The identification of paeoniflorin and paeonol from the samples was determined by comparing their retention times with those of standard compounds measured under the same chromatographic conditions. All solvents and solutions were filtered through 0.45-μm polytetrafluoroethylene (PTFE) filters before injection.


### 2.6. Statistical Analysis

All results in the text and the tables were expressed asmeanvalues±standarddeviations. Microsoft Excel was used for data preparation and results in output. The handling of the statistical data was done with SPSS Statistics 13.0.


## 2.1. Reagents

The Moutan Cortex used in this experiment was produced in Sichuan Province, China, which met the requirements of Chinese Pharmacopoeia (2020 edition). Chromatographic grade methanol (batch number 20200601) and acetonitrile (batch number 20200601) were purchased from Fisher Company in the USA, analytical pure phosphoric acid (batch number 20170408) was purchased from Tianjin Guangfu Technology Development Co., Ltd., and ultrapure water was purchased from Hangzhou Wa Co., Ltd. Paeoniflorin (batch number111642-200301, China Institute for Food and Drug Control), paeonol (batch number 111889-201705, China Institute for Food and Drug Control), and choline chloride (batch number 150120) were purchased from Shanghai Zhanyun Chemical Co., Ltd. Maltose (batch number 117101), fructose (batch number 117001), malic acid (batch number 117101), citric acid (batch number 117101), lactic acid (batch number 117101), and xylitol (batch number 117101) were purchased from Zhengzhou Kangyuan Chemical products Co., Ltd. Acetic acid (batch number 117101) were purchased from Xilong Science Co., Ltd. Propylene glycol (batch number 20180309) was purchased from Tianjin Zhonghe Shengtai Chemical Co., Ltd. Glycerin (batch number 20180406) was purchased from Guangzhou Miya Cosmetics Co., Ltd. Urea (batch number 20170109) was purchased from Tianjin Ruijinte Chemical Co., Ltd.


## 2.2. Instrumentation

Agilent 1260 high-performance liquid chromatography system was purchased from Agilent Technologies Co., Ltd. (including four-element low-pressure stirring pump, automatic injector, column incubator, 1100 diode array detector, and chemical workstation) and was used for chromatographic analysis. QL-901 Vortex instrument was purchased from Haimen Qilinbeier Instrument Manufacturing Co., Ltd. JP-300G ultrasonic extractor was purchased from Wuhan Jiapeng Electronics Co., Ltd. AB135-S electronic balance was purchased from Mettler-Toledo International Co., Ltd. ATY224 type-1/10000 balance was purchased from Shimadzu, Japan. TGL-16 freezing centrifuge was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.


## 2.3. Preparation of DESs

Choline chloride was selected as the DESs type of HBA. Because of its simple preparation and stable properties, choline chloride is widely used [22]. It can efficiently extract and separate alkaloids, flavonoids, polysaccharides, and acids from medicinal materials [23, 24]. HBA choline chloride and the various HBDs (maltose, malic acid, lactic acid, fructose, glycerol, propylene glycol, xylitol, urea, acetic, and acid, citric acid) were weighted at a certain molar ratio (Table 1), and the mixtures were stirred in sealed bottles at 80-100°C until a transparent, colorless liquid is formed after 0.5–3 h.Table 1
Different types of DESs.
No.HBAHBDMolar ratioExtraction efficiency mg/gPaeoniflorinPaeonolTotal1Choline chlorideMaltose1 : 14.0304.0378.0672Malic acid1 : 11.9712.6984.6693Lactic acid1 : 24.4604.7209.1804Fructose1 : 12.5672.6865.2535Glycerol1 : 24.3304.5458.8756Propylene glycol1 : 24.1054.6278.7327Xylitol1 : 11.5002.0763.5768Urea1 : 24.6894.0878.7769Acetic acid1 : 24.1104.9209.03010Citric acid1 : 22.7963.8356.630


## 2.4. Extraction Procedures

Moutan Cortex was dried at 60°C, crushed, sifted through 100 mesh, sealed, and stored at room temperature. Precision weighing Moutan Cortex powder 50 mg was taken, adding 1.5 mL DES (containing 30% water) with 60°C warm bath for 5 min, and vortex oscillation for 5 min. After mixing evenly, ultrasonic treatment was carried out, 30 min was extracted by ultrasonic at 60°C, ultrasonic power was 250 W, vortex mixing, 3000 r/min centrifugal for 5 min, 0.2 mL of the supernatant solution was precisely absorbed into the EP tube, methanol was diluted to 1 mL, and the Moutan Cortex extract was obtained for HPLC analysis.


## 2.5. Chromatographic System and Conditions

HPLC analysis was performed on an HPLC-DAD Agilent 1260 system. The separation was performed on an Agilent TC-C18 chromatography column (5 μm, 4.6x250mm). The mobile phase consisted of 100% acetonitrile (solvent A) and 0.1% phosphoric acid aqueous solution (solvent B). The gradient program was as follows: 0–17 min 14% (A), 17–18 min 14 → 47% (A), and 18–30 min 47% (A). The injection volume was 15 μL, and the flow rate was 1 mL/min-1 at 30°C column temperature. The detection wavelength was set to 238 nm. The identification of paeoniflorin and paeonol from the samples was determined by comparing their retention times with those of standard compounds measured under the same chromatographic conditions. All solvents and solutions were filtered through 0.45-μm polytetrafluoroethylene (PTFE) filters before injection.


## 2.6. Statistical Analysis

All results in the text and the tables were expressed asmeanvalues±standarddeviations. Microsoft Excel was used for data preparation and results in output. The handling of the statistical data was done with SPSS Statistics 13.0.


## 3. Results and Analysis

### 3.1. Validation of the HPLC-DAD Method

#### 3.1.1. Linear Range and Detection Limit of the Method

Prepared standard series solutions were injected under the established chromatographic conditions to obtain HPLC peak areas at different concentrations. The concentration of paeoniflorin and paeonol showed a good linear relationship with the peak area in the range of 8.04-402μg/mL and 8.08-404 μg/mL. The results are shown in Table 2.Table 2
Linear range and detection limit.
ComponentConcentration range (μg/mL)Linear curverPaeoniflorin8.04-402y=19.249x−4.85290.9996Paeonol8.08-404y=34.638x+4.02420.9998


#### 3.1.2. Precision and Method Accuracy

According to the above HPLC analysis conditions, the same Moutan Cortex extract was injected continuously 6 times, and the RSD values of paeoniflorin and paeonol peak area were 1.84% and 1.01%, respectively.Based on the above method, 6 samples of Moutan Cortex extract were prepared and analyzed by HPLC. The average contents of paeoniflorin and paeonol were4.548±0.080mg/g and 4.478±0.082mg/g, respectively, and the RSDs were 1.76% and 1.83%, respectively.Taking 6 samples of Moutan Cortex with known content, each was about 25 mg. Accurately removing paeoniflorin reference solution (concentration 0.614 mg/mL) 0.2 mL and paeonol reference solution (concentration 0.605 mg/mL) 0.2 mL in the same centrifuge tube, steaming dry solvent in a water bath, adding well-weighed Moutan Cortex powder, and extracting Moutan Cortex extract according to 2.4method, it was analyzed by HPLC. The average recoveries of paeoniflorin and paeonol were 98.59% (RSD 1.92%) and 99.00% (RSD 1.42%), respectively. The results are shown in Table3.Table 3
Precision and method accuracy.
ComponentsWeigh quantity/gOriginal/μgAddition/μgMeasured/μgRecovery rate/%Average recovery rate/%Paeoniflorin0.0245111.517122.800231.16297.4398.590.0248112.883122.800231.99397.000.0256116.524122.800241.418101.700.0261118.800122.800238.75497.680.0253115.159122.800234.94297.540.0244111.062122.800234.059100.16Paeonol0.0245109.844121.000228.76498.2899.000.0248111.189121.000233.175100.820.0256114.775121.000234.28198.770.0261117.017121.000234.54697.130.0253113.430121.000232.58398.470.0244109.395121.000231.015100.51


### 3.2. DESs Composition

#### 3.2.1. DES Types

DESs with choline chloride as HBA have the following advantages, namely, simple preparation, stable properties, low price, low toxicity, and biodegradability [25]. The extraction amount of paeoniflorin and paeonol is the most important factor that should be considered in optimizing the HBD component. In this study, the effects of 10 kinds of DESs (Table 3) on the extraction amount of paeoniflorin and paeonol were discussed. As shown in Figure 1, DES-3, the combination of choline chloride and lactic acid, was the most efficient in extracting paeoniflorin and paeonol from Moutan Cortex.Figure 1
HPLC chromatogram of Moutan Cortex reference substance (a) and sample (b) ((1) paeoniflorin, (2) paeonol).
(a)(b)


#### 3.2.2. Molar Ratio

The properties of DES (solubility, physical and chemical interactions, polarity, surface tension, and viscosity) are largely determined by HBA and HBD donors. In addition, the change of the molar ratio of HBA to HBD affects the molecular hydrogen bonding force of DESs. The molar ratio of choline chloride to lactic acid was investigated from 1 : 1 to 1 : 4. As shown in Figure2, the extraction amount of paeoniflorin and paeonol in Moutan Cortex increased at first and then decreased with the increase of the molar ratio of choline chloride and lactic acid. When the molar ratio is 1: 2, the extraction amount of paeoniflorin and paeonol is the maximum, so 1 : 2 is the best molar ratio.Figure 2
Effect of molar ratio on Moutan Cortex extraction efficiency.


#### 3.2.3. DES Water Content

The viscosity of DESs is usually very high, and the polarity and viscosity of DESs can be adjusted by water content [26]. However, the best water content is also related to specific compounds. In this study, the water content of 10%-50% was investigated. As shown in Figure 3, the extraction amount of paeoniflorin and paeonol in Moutan Cortex increases significantly with the increase of water content in Moutan Cortex. However, the continued increase of water content affects the intermolecular hydrogen bond of DESs, which may change the structure of DES and decrease the solubility of effective components of the Moutan Cortex. As a consequence, the increasing water content in a certain range is beneficial to paeoniflorin and paeonol in Moutan Cortex. Therefore, DES with 30% water content was selected as the extraction solvent of Moutan Cortex.Figure 3
Effect of DES water content on Moutan Cortex extraction efficiency.


#### 3.2.4. Solid/Liquid Ratio

The amount of extraction solvent has a great influence on the extraction rate. In this study, the volume range of DES was 1.0-2.0 mL. The amount of DES can directly affect the extraction efficiency of paeoniflorin and paeonol in the Moutan Cortex. This is mainly due to the insufficient amount of extraction solvent, which cannot completely extract the target components. However, the excessive amount of DES will cause the mixture which cannot be separated by demulsification and centrifugation. As can be seen from Figure4, when the volume of DES is 1.5 mL, the extraction rates of paeoniflorin and paeonol are relatively the highest. Finally,1.5 mL of DES was chosen as the optimum amount of extraction solvent in this study.Figure 4
Effect of solid/liquid ratio on Moutan Cortex extraction efficiency.


### 3.3. Single-Factor Experimental Analysis of Ultrasonic Treatment

#### 3.3.1. Effect of Ultrasonic Power

Ultrasonic can promote the dissolution of active substances. In addition, the molecular diffusion speed increases with the increase of ultrasonic power. In the course of this study, the change of extraction rate of ultrasonic power in the range of 100-500 W was investigated. It can be seen from Figure5 that the extraction rates of paeoniflorin and paeonol increase with the increase of ultrasonic power. Therefore, it is determined that the ultrasonic power is 300 W.Figure 5
Effect of ultrasonic power on Moutan Cortex extraction efficiency.


#### 3.3.2. Effect of Ultrasonic Time

Ultrasonic time is also one of the important factors affecting the extraction effect. The results (Figure6) showed that there was a positive correlation between the extraction rate of paeoniflorin and paeonol with the increase of ultrasonic time, and the extraction rate decreased when it exceeded 30 min by investigating the ultrasonic time in the range of 20-50 min. The ultrasonic time was consistent with the ultrasonic extraction time of paeonol [27], which may be due to the gradual increase of temperature with the increase of extraction time. In addition, the system may produce double degradation under the action of ultrasonic radiation and thermal effect at the same time [28].Figure 6
Effect of ultrasonic time on Moutan Cortex extraction efficiency.


#### 3.3.3. Effect of Ultrasonic Temperature

The viscosity of DESs decreases at a certain temperature. When it reaches a certain temperature, it can promote solute diffusion. In this study, the effect of temperature in the range of 40-70°C on the extraction rate of paeoniflorin and paeonol from Moutan Cortex was investigated. It can be seen from Figure7 that the extraction rate of paeoniflorin and paeonol was the highest when the ultrasonic temperature was 40°C. With the increase of extraction temperature, the extraction rate of both decreased, which may be due to the destruction of the original structure of some substances. In addition, it was consistent with the ultrasonic extraction temperature of paeonol [27]. Therefore, 50°С was set as the optimal condition in the following study.Figure 7
Effect of ultrasonic temperature on Moutan Cortex extraction efficiency.


#### 3.3.4. Vortex Time

It is not easy to mix the sample with DES because of the high viscosity and poor fluidity of DES. However, the vortex can ensure that the sample is completely mixed with the solvent. It can form a uniform system, which is convenient for the extraction of effective components. In the course of this study, the change of extraction rate of vortex time in the range of 1-9 min was investigated. It can be seen from Figure8 that the extraction rate of paeoniflorin and gallic acid increases with the extension of vortex time, and the best extraction purpose can be achieved by vortex oscillating 5 min. Therefore, 5 min is chosen as the best vortex time.Figure 8
Effect of vortex time on Moutan Cortex extraction efficiency.


### 3.4. Box-Behnken Response Surface Test Design

Based on the results of the single-factor experiment, ultrasonic power (W), ultrasonic time (min), ultrasonic temperature (°C), and vortex time (min) were taken as the influencing factors of the response surface test according to the design principle of Box-Behnken central combination test (Table4). The response surface analysis method of four factors and three levels was adopted by using Design-Expert8.0.6 software. 29 test sites were designed and the best process was verified. The results are shown in Table 5.Table 4
Response surface test factors and horizontal design.
FactorsLevel-101Ultrasonic power/W200300400Ultrasonic time/min203040Ultrasonic temperature/°C405060Vortex time/min357Table 5
Box-Behnken response surface design and results.
No.ABCDExtraction efficiency/(mg/g)PaeoniflorinPaeonolTotal1200306054.1764.1338.3092400306054.1774.0618.2383400305074.5374.4348.9714200305074.4054.3688.7735400405054.6644.5419.2056300305054.7104.6119.3217300306074.1494.0278.1768300404054.2874.1748.4619300205074.5134.3088.82110200405054.7134.4939.20611300306033.8523.6907.54212300304034.1404.1008.24013300305054.6914.5809.27114300305054.6064.5399.14515300305054.7484.6949.44216300206054.2884.1198.40717400305034.1634.0618.22418400205054.7004.5329.23219200305034.1604.1028.26220200205054.5924.5669.15821300204053.9814.3828.36322300305054.4054.7019.10623300205034.1654.3408.50524300405033.9294.2868.21525200304053.9614.4518.41226300304073.8704.2458.11527400304053.8914.2598.15028300405074.2544.5098.76329300406054.1284.2978.425


#### 3.4.1. Model Establishment and Analysis

The test data were analyzed by Design-Expert8.0.6 software, and the results were fitted by multiple regression, and the regression equation wasY=9.26−8.333E−003A−0.018B−0.054C+0.22D−0.019AB+0.048AC+0.059AD−0.02BC+0.058BD+0.19CD−0.12A2−0.041B2−0.78C2−0.56D2. R2 was 0.9367, which means that the fitting degree of the model is good, and the experimental error is small. As a consequence, this model can be used for analysis and prediction. Further analysis of variance of the equation is carried out, and the results are shown in Table 6.Table 6
Analysis of variance of regression model.
SourceSum of squaresdfMean squareF valueP valueModel6.14140.4414.81< 0.0001SignificantA8.333E-00418.333E-0040.0280.8692B3.710E-00313.710E-0030.130.7287C0.03510.0351.170.2983D0.5810.5819.480.0006AB1.406E-00311.406E-0030.0470.8306AC9.120E-00319.120E-0030.310.5877AD0.01410.0140.470.5041BC1.600E-00311.600E-0030.0540.8196BD0.01310.0130.450.5112CD0.1410.144.860.0447A20.09110.0913.060.1021B20.01110.0110.360.5577C23.9613.96133.59< 0.0001D22.0312.0368.65< 0.0001Residual0.41140.030Lack of fit0.34100.0341.850.2911Not significantPure error0.07440.018Cor total6.5528R20.9367R2Adj0.8735The results of Table4 show that when P<0.0001 and assuming P>0.05, the model was significantly high and the fitting degree was good. Besides, the model can be used for the analysis and prediction of the extraction process. There was no significant effect on A, B, and C, but there was a significant influence on D factors (P<0.01). The above results showed that the effect of various factors on the extraction rate of the effective components of the prescription was not a simple linear relationship, and the significant influence on the extraction process should be arranged in the order of D > C > B > A.


#### 3.4.2. The Interaction of Various Factors

The response surface diagram of the quadratic regression equation was obtained by Design-Expert8.0.6 software. In addition, the shape of the response surface graph was investigated, and the effects of ultrasonic power, ultrasonic time, ultrasonic temperature, and vortex time on the extraction rate of paeoniflorin and paeonol in Moutan Cortex were analyzed. Response surface graphs and contours can more directly reflect the interaction between response values and various factors as well as among factors. It can be seen from Figure9 that the slope of the interactive surface of CD was steeper, indicating that the extraction rate of paeoniflorin and paeonol in Moutan Cortex was greatly affected by ultrasonic temperature and vortex time.Figure 9
Response surface drawings of Moutan Cortex analyzed by the interaction of two factors.According to the analysis of Design-Expert8.0.6 software, the optimum preparation parameters were as follows: ultrasonic power was 301.84 W, ultrasonic time was 29.17 min, the ultrasonic temperature was 49.90°C, and vortex time was 5.38 min. According to the actual operation, the ultrasonic power was 300 W, the ultrasonic time was 30 min, the ultrasonic temperature was 50°C, and the vortex time was 5 min. According to the above experimental conditions, three parallel verification experiments were carried out, and the results showed that the sum of the extraction rates of paeoniflorin and Moutan Cortex in Moutan Cortex was 9.279 mg/g. In addition, the deviation was 0.333 mg/g compared with the predicted value 9.612 mg/g. The results showed that the regression model was reasonable and had good predictability.


### 3.5. Comparison of another Extraction Solvent

The results of DESs extraction were compared with those of common extraction solvents such as methanol and ethanol. It can be seen from the experimental process that the extraction process with DES is not only simple and stable, does not need to add a large number of reagents, but also greatly shortens the extraction time and saves the steps of solvent concentration [28]. In addition, it can be seen from Figure 10 that the extraction effect of DES extraction is better than that of conventional solvent extraction.Figure 10
Comparison of different extraction solvents.


## 3.1. Validation of the HPLC-DAD Method

### 3.1.1. Linear Range and Detection Limit of the Method

Prepared standard series solutions were injected under the established chromatographic conditions to obtain HPLC peak areas at different concentrations. The concentration of paeoniflorin and paeonol showed a good linear relationship with the peak area in the range of 8.04-402μg/mL and 8.08-404 μg/mL. The results are shown in Table 2.Table 2
Linear range and detection limit.
ComponentConcentration range (μg/mL)Linear curverPaeoniflorin8.04-402y=19.249x−4.85290.9996Paeonol8.08-404y=34.638x+4.02420.9998


### 3.1.2. Precision and Method Accuracy

According to the above HPLC analysis conditions, the same Moutan Cortex extract was injected continuously 6 times, and the RSD values of paeoniflorin and paeonol peak area were 1.84% and 1.01%, respectively.Based on the above method, 6 samples of Moutan Cortex extract were prepared and analyzed by HPLC. The average contents of paeoniflorin and paeonol were4.548±0.080mg/g and 4.478±0.082mg/g, respectively, and the RSDs were 1.76% and 1.83%, respectively.Taking 6 samples of Moutan Cortex with known content, each was about 25 mg. Accurately removing paeoniflorin reference solution (concentration 0.614 mg/mL) 0.2 mL and paeonol reference solution (concentration 0.605 mg/mL) 0.2 mL in the same centrifuge tube, steaming dry solvent in a water bath, adding well-weighed Moutan Cortex powder, and extracting Moutan Cortex extract according to 2.4method, it was analyzed by HPLC. The average recoveries of paeoniflorin and paeonol were 98.59% (RSD 1.92%) and 99.00% (RSD 1.42%), respectively. The results are shown in Table3.Table 3
Precision and method accuracy.
ComponentsWeigh quantity/gOriginal/μgAddition/μgMeasured/μgRecovery rate/%Average recovery rate/%Paeoniflorin0.0245111.517122.800231.16297.4398.590.0248112.883122.800231.99397.000.0256116.524122.800241.418101.700.0261118.800122.800238.75497.680.0253115.159122.800234.94297.540.0244111.062122.800234.059100.16Paeonol0.0245109.844121.000228.76498.2899.000.0248111.189121.000233.175100.820.0256114.775121.000234.28198.770.0261117.017121.000234.54697.130.0253113.430121.000232.58398.470.0244109.395121.000231.015100.51


## 3.1.1. Linear Range and Detection Limit of the Method

Prepared standard series solutions were injected under the established chromatographic conditions to obtain HPLC peak areas at different concentrations. The concentration of paeoniflorin and paeonol showed a good linear relationship with the peak area in the range of 8.04-402μg/mL and 8.08-404 μg/mL. The results are shown in Table 2.Table 2
Linear range and detection limit.
ComponentConcentration range (μg/mL)Linear curverPaeoniflorin8.04-402y=19.249x−4.85290.9996Paeonol8.08-404y=34.638x+4.02420.9998


## 3.1.2. Precision and Method Accuracy

According to the above HPLC analysis conditions, the same Moutan Cortex extract was injected continuously 6 times, and the RSD values of paeoniflorin and paeonol peak area were 1.84% and 1.01%, respectively.Based on the above method, 6 samples of Moutan Cortex extract were prepared and analyzed by HPLC. The average contents of paeoniflorin and paeonol were4.548±0.080mg/g and 4.478±0.082mg/g, respectively, and the RSDs were 1.76% and 1.83%, respectively.Taking 6 samples of Moutan Cortex with known content, each was about 25 mg. Accurately removing paeoniflorin reference solution (concentration 0.614 mg/mL) 0.2 mL and paeonol reference solution (concentration 0.605 mg/mL) 0.2 mL in the same centrifuge tube, steaming dry solvent in a water bath, adding well-weighed Moutan Cortex powder, and extracting Moutan Cortex extract according to 2.4method, it was analyzed by HPLC. The average recoveries of paeoniflorin and paeonol were 98.59% (RSD 1.92%) and 99.00% (RSD 1.42%), respectively. The results are shown in Table3.Table 3
Precision and method accuracy.
ComponentsWeigh quantity/gOriginal/μgAddition/μgMeasured/μgRecovery rate/%Average recovery rate/%Paeoniflorin0.0245111.517122.800231.16297.4398.590.0248112.883122.800231.99397.000.0256116.524122.800241.418101.700.0261118.800122.800238.75497.680.0253115.159122.800234.94297.540.0244111.062122.800234.059100.16Paeonol0.0245109.844121.000228.76498.2899.000.0248111.189121.000233.175100.820.0256114.775121.000234.28198.770.0261117.017121.000234.54697.130.0253113.430121.000232.58398.470.0244109.395121.000231.015100.51


## 3.2. DESs Composition

### 3.2.1. DES Types

DESs with choline chloride as HBA have the following advantages, namely, simple preparation, stable properties, low price, low toxicity, and biodegradability [25]. The extraction amount of paeoniflorin and paeonol is the most important factor that should be considered in optimizing the HBD component. In this study, the effects of 10 kinds of DESs (Table 3) on the extraction amount of paeoniflorin and paeonol were discussed. As shown in Figure 1, DES-3, the combination of choline chloride and lactic acid, was the most efficient in extracting paeoniflorin and paeonol from Moutan Cortex.Figure 1
HPLC chromatogram of Moutan Cortex reference substance (a) and sample (b) ((1) paeoniflorin, (2) paeonol).
(a)(b)


### 3.2.2. Molar Ratio

The properties of DES (solubility, physical and chemical interactions, polarity, surface tension, and viscosity) are largely determined by HBA and HBD donors. In addition, the change of the molar ratio of HBA to HBD affects the molecular hydrogen bonding force of DESs. The molar ratio of choline chloride to lactic acid was investigated from 1 : 1 to 1 : 4. As shown in Figure2, the extraction amount of paeoniflorin and paeonol in Moutan Cortex increased at first and then decreased with the increase of the molar ratio of choline chloride and lactic acid. When the molar ratio is 1: 2, the extraction amount of paeoniflorin and paeonol is the maximum, so 1 : 2 is the best molar ratio.Figure 2
Effect of molar ratio on Moutan Cortex extraction efficiency.


### 3.2.3. DES Water Content

The viscosity of DESs is usually very high, and the polarity and viscosity of DESs can be adjusted by water content [26]. However, the best water content is also related to specific compounds. In this study, the water content of 10%-50% was investigated. As shown in Figure 3, the extraction amount of paeoniflorin and paeonol in Moutan Cortex increases significantly with the increase of water content in Moutan Cortex. However, the continued increase of water content affects the intermolecular hydrogen bond of DESs, which may change the structure of DES and decrease the solubility of effective components of the Moutan Cortex. As a consequence, the increasing water content in a certain range is beneficial to paeoniflorin and paeonol in Moutan Cortex. Therefore, DES with 30% water content was selected as the extraction solvent of Moutan Cortex.Figure 3
Effect of DES water content on Moutan Cortex extraction efficiency.


### 3.2.4. Solid/Liquid Ratio

The amount of extraction solvent has a great influence on the extraction rate. In this study, the volume range of DES was 1.0-2.0 mL. The amount of DES can directly affect the extraction efficiency of paeoniflorin and paeonol in the Moutan Cortex. This is mainly due to the insufficient amount of extraction solvent, which cannot completely extract the target components. However, the excessive amount of DES will cause the mixture which cannot be separated by demulsification and centrifugation. As can be seen from Figure4, when the volume of DES is 1.5 mL, the extraction rates of paeoniflorin and paeonol are relatively the highest. Finally,1.5 mL of DES was chosen as the optimum amount of extraction solvent in this study.Figure 4
Effect of solid/liquid ratio on Moutan Cortex extraction efficiency.


## 3.2.1. DES Types

DESs with choline chloride as HBA have the following advantages, namely, simple preparation, stable properties, low price, low toxicity, and biodegradability [25]. The extraction amount of paeoniflorin and paeonol is the most important factor that should be considered in optimizing the HBD component. In this study, the effects of 10 kinds of DESs (Table 3) on the extraction amount of paeoniflorin and paeonol were discussed. As shown in Figure 1, DES-3, the combination of choline chloride and lactic acid, was the most efficient in extracting paeoniflorin and paeonol from Moutan Cortex.Figure 1
HPLC chromatogram of Moutan Cortex reference substance (a) and sample (b) ((1) paeoniflorin, (2) paeonol).
(a)(b)


## 3.2.2. Molar Ratio

The properties of DES (solubility, physical and chemical interactions, polarity, surface tension, and viscosity) are largely determined by HBA and HBD donors. In addition, the change of the molar ratio of HBA to HBD affects the molecular hydrogen bonding force of DESs. The molar ratio of choline chloride to lactic acid was investigated from 1 : 1 to 1 : 4. As shown in Figure2, the extraction amount of paeoniflorin and paeonol in Moutan Cortex increased at first and then decreased with the increase of the molar ratio of choline chloride and lactic acid. When the molar ratio is 1: 2, the extraction amount of paeoniflorin and paeonol is the maximum, so 1 : 2 is the best molar ratio.Figure 2
Effect of molar ratio on Moutan Cortex extraction efficiency.


## 3.2.3. DES Water Content

The viscosity of DESs is usually very high, and the polarity and viscosity of DESs can be adjusted by water content [26]. However, the best water content is also related to specific compounds. In this study, the water content of 10%-50% was investigated. As shown in Figure 3, the extraction amount of paeoniflorin and paeonol in Moutan Cortex increases significantly with the increase of water content in Moutan Cortex. However, the continued increase of water content affects the intermolecular hydrogen bond of DESs, which may change the structure of DES and decrease the solubility of effective components of the Moutan Cortex. As a consequence, the increasing water content in a certain range is beneficial to paeoniflorin and paeonol in Moutan Cortex. Therefore, DES with 30% water content was selected as the extraction solvent of Moutan Cortex.Figure 3
Effect of DES water content on Moutan Cortex extraction efficiency.


## 3.2.4. Solid/Liquid Ratio

The amount of extraction solvent has a great influence on the extraction rate. In this study, the volume range of DES was 1.0-2.0 mL. The amount of DES can directly affect the extraction efficiency of paeoniflorin and paeonol in the Moutan Cortex. This is mainly due to the insufficient amount of extraction solvent, which cannot completely extract the target components. However, the excessive amount of DES will cause the mixture which cannot be separated by demulsification and centrifugation. As can be seen from Figure4, when the volume of DES is 1.5 mL, the extraction rates of paeoniflorin and paeonol are relatively the highest. Finally,1.5 mL of DES was chosen as the optimum amount of extraction solvent in this study.Figure 4
Effect of solid/liquid ratio on Moutan Cortex extraction efficiency.


## 3.3. Single-Factor Experimental Analysis of Ultrasonic Treatment

### 3.3.1. Effect of Ultrasonic Power

Ultrasonic can promote the dissolution of active substances. In addition, the molecular diffusion speed increases with the increase of ultrasonic power. In the course of this study, the change of extraction rate of ultrasonic power in the range of 100-500 W was investigated. It can be seen from Figure5 that the extraction rates of paeoniflorin and paeonol increase with the increase of ultrasonic power. Therefore, it is determined that the ultrasonic power is 300 W.Figure 5
Effect of ultrasonic power on Moutan Cortex extraction efficiency.


### 3.3.2. Effect of Ultrasonic Time

Ultrasonic time is also one of the important factors affecting the extraction effect. The results (Figure6) showed that there was a positive correlation between the extraction rate of paeoniflorin and paeonol with the increase of ultrasonic time, and the extraction rate decreased when it exceeded 30 min by investigating the ultrasonic time in the range of 20-50 min. The ultrasonic time was consistent with the ultrasonic extraction time of paeonol [27], which may be due to the gradual increase of temperature with the increase of extraction time. In addition, the system may produce double degradation under the action of ultrasonic radiation and thermal effect at the same time [28].Figure 6
Effect of ultrasonic time on Moutan Cortex extraction efficiency.


### 3.3.3. Effect of Ultrasonic Temperature

The viscosity of DESs decreases at a certain temperature. When it reaches a certain temperature, it can promote solute diffusion. In this study, the effect of temperature in the range of 40-70°C on the extraction rate of paeoniflorin and paeonol from Moutan Cortex was investigated. It can be seen from Figure7 that the extraction rate of paeoniflorin and paeonol was the highest when the ultrasonic temperature was 40°C. With the increase of extraction temperature, the extraction rate of both decreased, which may be due to the destruction of the original structure of some substances. In addition, it was consistent with the ultrasonic extraction temperature of paeonol [27]. Therefore, 50°С was set as the optimal condition in the following study.Figure 7
Effect of ultrasonic temperature on Moutan Cortex extraction efficiency.


### 3.3.4. Vortex Time

It is not easy to mix the sample with DES because of the high viscosity and poor fluidity of DES. However, the vortex can ensure that the sample is completely mixed with the solvent. It can form a uniform system, which is convenient for the extraction of effective components. In the course of this study, the change of extraction rate of vortex time in the range of 1-9 min was investigated. It can be seen from Figure8 that the extraction rate of paeoniflorin and gallic acid increases with the extension of vortex time, and the best extraction purpose can be achieved by vortex oscillating 5 min. Therefore, 5 min is chosen as the best vortex time.Figure 8
Effect of vortex time on Moutan Cortex extraction efficiency.


## 3.3.1. Effect of Ultrasonic Power

Ultrasonic can promote the dissolution of active substances. In addition, the molecular diffusion speed increases with the increase of ultrasonic power. In the course of this study, the change of extraction rate of ultrasonic power in the range of 100-500 W was investigated. It can be seen from Figure5 that the extraction rates of paeoniflorin and paeonol increase with the increase of ultrasonic power. Therefore, it is determined that the ultrasonic power is 300 W.Figure 5
Effect of ultrasonic power on Moutan Cortex extraction efficiency.


## 3.3.2. Effect of Ultrasonic Time

Ultrasonic time is also one of the important factors affecting the extraction effect. The results (Figure6) showed that there was a positive correlation between the extraction rate of paeoniflorin and paeonol with the increase of ultrasonic time, and the extraction rate decreased when it exceeded 30 min by investigating the ultrasonic time in the range of 20-50 min. The ultrasonic time was consistent with the ultrasonic extraction time of paeonol [27], which may be due to the gradual increase of temperature with the increase of extraction time. In addition, the system may produce double degradation under the action of ultrasonic radiation and thermal effect at the same time [28].Figure 6
Effect of ultrasonic time on Moutan Cortex extraction efficiency.


## 3.3.3. Effect of Ultrasonic Temperature

The viscosity of DESs decreases at a certain temperature. When it reaches a certain temperature, it can promote solute diffusion. In this study, the effect of temperature in the range of 40-70°C on the extraction rate of paeoniflorin and paeonol from Moutan Cortex was investigated. It can be seen from Figure7 that the extraction rate of paeoniflorin and paeonol was the highest when the ultrasonic temperature was 40°C. With the increase of extraction temperature, the extraction rate of both decreased, which may be due to the destruction of the original structure of some substances. In addition, it was consistent with the ultrasonic extraction temperature of paeonol [27]. Therefore, 50°С was set as the optimal condition in the following study.Figure 7
Effect of ultrasonic temperature on Moutan Cortex extraction efficiency.


## 3.3.4. Vortex Time

It is not easy to mix the sample with DES because of the high viscosity and poor fluidity of DES. However, the vortex can ensure that the sample is completely mixed with the solvent. It can form a uniform system, which is convenient for the extraction of effective components. In the course of this study, the change of extraction rate of vortex time in the range of 1-9 min was investigated. It can be seen from Figure8 that the extraction rate of paeoniflorin and gallic acid increases with the extension of vortex time, and the best extraction purpose can be achieved by vortex oscillating 5 min. Therefore, 5 min is chosen as the best vortex time.Figure 8
Effect of vortex time on Moutan Cortex extraction efficiency.


## 3.4. Box-Behnken Response Surface Test Design

Based on the results of the single-factor experiment, ultrasonic power (W), ultrasonic time (min), ultrasonic temperature (°C), and vortex time (min) were taken as the influencing factors of the response surface test according to the design principle of Box-Behnken central combination test (Table4). The response surface analysis method of four factors and three levels was adopted by using Design-Expert8.0.6 software. 29 test sites were designed and the best process was verified. The results are shown in Table 5.Table 4
Response surface test factors and horizontal design.
FactorsLevel-101Ultrasonic power/W200300400Ultrasonic time/min203040Ultrasonic temperature/°C405060Vortex time/min357Table 5
Box-Behnken response surface design and results.
No.ABCDExtraction efficiency/(mg/g)PaeoniflorinPaeonolTotal1200306054.1764.1338.3092400306054.1774.0618.2383400305074.5374.4348.9714200305074.4054.3688.7735400405054.6644.5419.2056300305054.7104.6119.3217300306074.1494.0278.1768300404054.2874.1748.4619300205074.5134.3088.82110200405054.7134.4939.20611300306033.8523.6907.54212300304034.1404.1008.24013300305054.6914.5809.27114300305054.6064.5399.14515300305054.7484.6949.44216300206054.2884.1198.40717400305034.1634.0618.22418400205054.7004.5329.23219200305034.1604.1028.26220200205054.5924.5669.15821300204053.9814.3828.36322300305054.4054.7019.10623300205034.1654.3408.50524300405033.9294.2868.21525200304053.9614.4518.41226300304073.8704.2458.11527400304053.8914.2598.15028300405074.2544.5098.76329300406054.1284.2978.425


### 3.4.1. Model Establishment and Analysis

The test data were analyzed by Design-Expert8.0.6 software, and the results were fitted by multiple regression, and the regression equation wasY=9.26−8.333E−003A−0.018B−0.054C+0.22D−0.019AB+0.048AC+0.059AD−0.02BC+0.058BD+0.19CD−0.12A2−0.041B2−0.78C2−0.56D2. R2 was 0.9367, which means that the fitting degree of the model is good, and the experimental error is small. As a consequence, this model can be used for analysis and prediction. Further analysis of variance of the equation is carried out, and the results are shown in Table 6.Table 6
Analysis of variance of regression model.
SourceSum of squaresdfMean squareF valueP valueModel6.14140.4414.81< 0.0001SignificantA8.333E-00418.333E-0040.0280.8692B3.710E-00313.710E-0030.130.7287C0.03510.0351.170.2983D0.5810.5819.480.0006AB1.406E-00311.406E-0030.0470.8306AC9.120E-00319.120E-0030.310.5877AD0.01410.0140.470.5041BC1.600E-00311.600E-0030.0540.8196BD0.01310.0130.450.5112CD0.1410.144.860.0447A20.09110.0913.060.1021B20.01110.0110.360.5577C23.9613.96133.59< 0.0001D22.0312.0368.65< 0.0001Residual0.41140.030Lack of fit0.34100.0341.850.2911Not significantPure error0.07440.018Cor total6.5528R20.9367R2Adj0.8735The results of Table4 show that when P<0.0001 and assuming P>0.05, the model was significantly high and the fitting degree was good. Besides, the model can be used for the analysis and prediction of the extraction process. There was no significant effect on A, B, and C, but there was a significant influence on D factors (P<0.01). The above results showed that the effect of various factors on the extraction rate of the effective components of the prescription was not a simple linear relationship, and the significant influence on the extraction process should be arranged in the order of D > C > B > A.


### 3.4.2. The Interaction of Various Factors

The response surface diagram of the quadratic regression equation was obtained by Design-Expert8.0.6 software. In addition, the shape of the response surface graph was investigated, and the effects of ultrasonic power, ultrasonic time, ultrasonic temperature, and vortex time on the extraction rate of paeoniflorin and paeonol in Moutan Cortex were analyzed. Response surface graphs and contours can more directly reflect the interaction between response values and various factors as well as among factors. It can be seen from Figure9 that the slope of the interactive surface of CD was steeper, indicating that the extraction rate of paeoniflorin and paeonol in Moutan Cortex was greatly affected by ultrasonic temperature and vortex time.Figure 9
Response surface drawings of Moutan Cortex analyzed by the interaction of two factors.According to the analysis of Design-Expert8.0.6 software, the optimum preparation parameters were as follows: ultrasonic power was 301.84 W, ultrasonic time was 29.17 min, the ultrasonic temperature was 49.90°C, and vortex time was 5.38 min. According to the actual operation, the ultrasonic power was 300 W, the ultrasonic time was 30 min, the ultrasonic temperature was 50°C, and the vortex time was 5 min. According to the above experimental conditions, three parallel verification experiments were carried out, and the results showed that the sum of the extraction rates of paeoniflorin and Moutan Cortex in Moutan Cortex was 9.279 mg/g. In addition, the deviation was 0.333 mg/g compared with the predicted value 9.612 mg/g. The results showed that the regression model was reasonable and had good predictability.


## 3.4.1. Model Establishment and Analysis

The test data were analyzed by Design-Expert8.0.6 software, and the results were fitted by multiple regression, and the regression equation wasY=9.26−8.333E−003A−0.018B−0.054C+0.22D−0.019AB+0.048AC+0.059AD−0.02BC+0.058BD+0.19CD−0.12A2−0.041B2−0.78C2−0.56D2. R2 was 0.9367, which means that the fitting degree of the model is good, and the experimental error is small. As a consequence, this model can be used for analysis and prediction. Further analysis of variance of the equation is carried out, and the results are shown in Table 6.Table 6
Analysis of variance of regression model.
SourceSum of squaresdfMean squareF valueP valueModel6.14140.4414.81< 0.0001SignificantA8.333E-00418.333E-0040.0280.8692B3.710E-00313.710E-0030.130.7287C0.03510.0351.170.2983D0.5810.5819.480.0006AB1.406E-00311.406E-0030.0470.8306AC9.120E-00319.120E-0030.310.5877AD0.01410.0140.470.5041BC1.600E-00311.600E-0030.0540.8196BD0.01310.0130.450.5112CD0.1410.144.860.0447A20.09110.0913.060.1021B20.01110.0110.360.5577C23.9613.96133.59< 0.0001D22.0312.0368.65< 0.0001Residual0.41140.030Lack of fit0.34100.0341.850.2911Not significantPure error0.07440.018Cor total6.5528R20.9367R2Adj0.8735The results of Table4 show that when P<0.0001 and assuming P>0.05, the model was significantly high and the fitting degree was good. Besides, the model can be used for the analysis and prediction of the extraction process. There was no significant effect on A, B, and C, but there was a significant influence on D factors (P<0.01). The above results showed that the effect of various factors on the extraction rate of the effective components of the prescription was not a simple linear relationship, and the significant influence on the extraction process should be arranged in the order of D > C > B > A.


## 3.4.2. The Interaction of Various Factors

The response surface diagram of the quadratic regression equation was obtained by Design-Expert8.0.6 software. In addition, the shape of the response surface graph was investigated, and the effects of ultrasonic power, ultrasonic time, ultrasonic temperature, and vortex time on the extraction rate of paeoniflorin and paeonol in Moutan Cortex were analyzed. Response surface graphs and contours can more directly reflect the interaction between response values and various factors as well as among factors. It can be seen from Figure9 that the slope of the interactive surface of CD was steeper, indicating that the extraction rate of paeoniflorin and paeonol in Moutan Cortex was greatly affected by ultrasonic temperature and vortex time.Figure 9
Response surface drawings of Moutan Cortex analyzed by the interaction of two factors.According to the analysis of Design-Expert8.0.6 software, the optimum preparation parameters were as follows: ultrasonic power was 301.84 W, ultrasonic time was 29.17 min, the ultrasonic temperature was 49.90°C, and vortex time was 5.38 min. According to the actual operation, the ultrasonic power was 300 W, the ultrasonic time was 30 min, the ultrasonic temperature was 50°C, and the vortex time was 5 min. According to the above experimental conditions, three parallel verification experiments were carried out, and the results showed that the sum of the extraction rates of paeoniflorin and Moutan Cortex in Moutan Cortex was 9.279 mg/g. In addition, the deviation was 0.333 mg/g compared with the predicted value 9.612 mg/g. The results showed that the regression model was reasonable and had good predictability.


## 3.5. Comparison of another Extraction Solvent

The results of DESs extraction were compared with those of common extraction solvents such as methanol and ethanol. It can be seen from the experimental process that the extraction process with DES is not only simple and stable, does not need to add a large number of reagents, but also greatly shortens the extraction time and saves the steps of solvent concentration [28]. In addition, it can be seen from Figure 10 that the extraction effect of DES extraction is better than that of conventional solvent extraction.Figure 10
Comparison of different extraction solvents.


## 4. Conclusion

The application of DES in the extraction of active components of traditional Chinese medicine was explored. Aiming at the extraction contents of paeonol and paeoniflorin in Moutan Cortex, the effects of ultrasonic-assisted extraction of DES with different components were systematically investigated and compared with those of traditional Chinese medicine extraction solvents (water, methanol, and ethanol). The results showed that DES formed with choline chloride and lactic acid (molar ratio 1 : 2) could effectively extract paeonol and paeoniflorin from Moutan Cortex. To further optimize the extraction conditions, the optimum extraction conditions of paeonol and paeoniflorin from Moutan Cortex were obtained by single-factor test and response surface test: 30% water content of DES (V/V), the ratio of material to liquid was 1 : 30 g/mL, the vortex was 5 min, ultrasonic time was 30 min, the ultrasonic temperature was 50°C, ultrasonic power was 300 W, and the total extraction rate of paeonol and paeoniflorin was 9.612 mg/g. Compared with traditional solvent extraction, the results showed that the extraction effect of DES extraction is better than that of conventional solvent extraction. At the same time, compared with organic solvents, DES has no pungent smell and is easy to biodegrade. Thus, it can be seen that DESs extraction is not only a high extraction efficiency but also a green chemical solvent. It accords with the concept of green chemistry in the new century and provides a certain basis for its application in other medicinal materials in the future.



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*Source: 5904038-2022-04-21.xml*