**1. Introduction**

*Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav.* (*A. dahuricae*) is the dry root belonging to umbelliferaceae. *A. dahuricae* has been frequently used as a food additive and a folk medicinal herb in Asian countries [1]. *A. dahuricae* has a wide range of pharmacological activities, including analgesia, anti-inflammatory, antibacterial, vascular dilation, anti-cancer, and central excitatory properties, and so on [2]. It has been used as an anodyne, and is very effective at relieving neuralgic pain [3]. *A. dahuricae* is known to contain a large number of compounds, including volatile oil, coumarins, and glycosides. Among these compounds, coumarins are generally considered the major components; so far, more than 20 kinds of coumarins have been isolated from this crude drug, such as oxypeucedanin, imperatorin, cnidilin, isoimperatorin, xanthotoxol, byakangelicin, bergapten, etc. [4]. Therefore, the efficient extraction of coumarins is important and desirable.

As known to us, a major disadvantage of conventional methods such as impregnation, percolation, and soxhlet extraction using alcohol or other common organic solvents is the use of a large number of flammable, non-degradable, or toxic organic solvents in the extraction process. In order to solve this problem, a new green natural deep eutectic solvents (NADESs) has been studied [5]. Compared to traditional organic solvents, NADES is a promising alternative to traditional organic solvents because of its green, non-toxic, biodegradable, and recyclable properties [6].

**Citation:** Wang, T.; Li, Q. DES Based Efficient Extraction Method for Bioactive Coumarins from *Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. Separations* **2022**, *9*, 5. https://doi.org/10.3390/separations 9010005

Academic Editor: Ki Hyun Kim

Received: 30 November 2021 Accepted: 21 December 2021 Published: 23 December 2021

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At present, DESs are widely used in science to extract the following types of biologicallyactive substances (BASs): flavonoids, alkaloids, anthocyanins, flavors, saponins, etc. [7]. DESs are mostly composed of primary metabolites such as sugars, sugar alcohols, organic acids, amino acids, and amines and additionally often contain water in certain molar ratios [8]. Compared with traditional extraction solvents, DESs could improve the extraction rate of products, which is an effective method to extract bioactive products [9]. Owing to these unique properties, DESs have been widely used in catalysis, organic synthesis, and analytical chemistry [10,11]. DESs have been widely used in liquid–liquid extraction, solid-phase microextraction, and other fields [12]. Recently, several studies used DESs for the extraction and separation of different types of bioactive compounds, such as phenolic acids, flavonoids, and alkaloids from various plant materials [13]. In general, the extraction capacity of DESs for biologically active natural products is related to their physicochemical properties, including H-bond interactions, polarity, pH, and viscosity. The polarity of DESs is an important factor affecting the extraction efficiency. Due to the high polarity of synthesized DESs, they have been used for the extraction of polar natural compounds like alkaloids, anthocyanins, polysaccharides, and glycosides. In general, as for natural compounds with low polarity, such as anthraquinones, their extraction rate is low [6,14]. In addition, the solid–liquid ratio of the sample to the DES, the extraction time, and even the molecular weight of the DES may affect the extraction effect. A high-efficiency and green extraction approach by DESs prepared by inexpensive and natural components was successfully developed for the extraction of coumarins in Cortex Fraxini by Wang et al. [15].

Ultrasonic-assisted extraction (UAE) UAE can significantly improve the efficiency of the classical extraction process, and the combination of UAE with NADESs can also be effective [16]. Compared with the conventional method using 96% ethanol as solvent, the NADES-UAE method had higher extraction efficiency for trans-cinnamaldehyde and coumarin from C. burmanni by Widya Dwi ARYATI [17,18]. Chen et al. established a liquid chromatography–tandem mass spectrometry (LC-MS /MS) method for simultaneous quantification of nine furanocoumarins, which could separate nine furanocoumarins within 6 min [19]. Pfeifer et al. detected coumarin in *A. dahurica* roots by supercritical fluid chromatography [20]. However, as known to us, the method of extracting bioactive compound coumarins by DESs is still limited, and the efficiency of extracting coumarin from *A. dahuricae* is not clear.

In this study, a series of DESs based on choline chloride was developed to evaluate the extraction effect on coumarin of *A. dahurica*. A Box–Behnken design (BBD) and the response surface method (RSM) were used to optimize the extraction conditions of DESs. Scanning electron microscopy (SEM) was used to study the degree of crushing of medicinal powder by ultrasonic-assisted DES and conventional extraction. Finally, the scavenging effect of DES extract on DPPH free radical was studied.

#### **2. Experimental**

#### *2.1. Materials and Reagents*

*A. dahurica* was purchased from Suining City, Qing Mountain sulphur free food store (Sichuan, China). An appropriate amount of medicinal materials was taken and baked in an oven at 60 ◦C for 24 h. Then, they were crushed through a 40-mesh sieve and the coarse powder of *A. dahurica* root was obtained, which was put into a zipper bag for use.

All the chemicals for the preparation of the DES and coumarins were purchased from companies in several Chinese cities mentioned later. Analytical reagents such as choline chloride, sucrose, xylitol, citric acid, glucose, ethylene glycol, 1,2-propanediol, 1,4 butanediol, 1,3-butanediol, glycerol, fructose, and urea were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); malic acid was purchased from Shanghai Sanpu Chemical Co., Ltd. (Shanghai, China); acetic acid was purchased from Tianjin Beichenfang Reagent., Ltd. (Tianjin, China) and lactic acid was purchased from Yantai Shuangshuang Chemical Co., Ltd. (Yantai, China). All the above reagents are analytically pure.

Oxypeucedanin (lot: Y29S9S65152 ≥ 98%), isoimperatorin (lot: 18062202 ≥ 98%), bergapten (lot: 17120501 ≥ 98%), psoralen (lot: 19101703 ≥ 98%), imperatorin (lot: 1805150 ≥ 98%), and xanthotoxol (lot: 19111602 ≥ 99%) were purchased from Chengdu Pufeide Biological Technology Co., Ltd. (Sichuan, China)Byakangelicin (lot: B-005-180921 ≥ 98%) was purchased from Chengdu Ruifensi Biological Technology Co., Ltd. (Sichuan, China) The deionized water used in this study was obtained from a Milli-Q water purification system (Bedford, NY, USA). All other reagents and chemicals used were of analytical grade. The chemical structures of coumarins used in this study are shown in Figure 1.

**Figure 1.** Chemical structures of xanthotoxol, psoralen, byakangelicin, bergapten, oxypeucedanin, imperatorin, and isoimperatorin.

#### *2.2. Preparation of DESs*

The direct synthesis method of DESs is easy to operate and has few equipment requirements. The mixture with a certain molar ratio was put into a small beaker of 100 mL and stirred in an oil bath at a certain temperature (80–90 ◦C) until a uniform and stable transparent liquid was formed (generally 120–180 min). Because some components have high viscosity and a high melting point, it was difficult to obtain an ideal solvent by the direct heating synthesis method. A certain proportion of water should be added before heating to reduce the viscosity. Five kinds of three-phase DESs and 11 kinds of two-phase DESs were prepared by the above method, which are shown in the following Table 1.




**Table 1.** *Cont.*

### *2.3. Experimental Design*

#### 2.3.1. Preparation of Standard Solution

A certain amount of standard was precisely weighed, placed in a 10 mL volumetric flask, and then filled with methanol to the scale line to obtain 7 standard solutions of varying concentrations. Then, 1 mL of each standard solution was precisely sucked and added to the same volumetric flask to obtain a mixed standard solution. After filtration with 0.45 μm microporous membrane, the mixed standard solution was injected into highperformance liquid chromatography (HPLC) at different volumes (4, 8, 10, 12, 16, 20 μL), and the peak area was recorded for each.

#### 2.3.2. Preparation of Sample Solution

In the initial screening, 1 g *A. dahurica* herb powder was accurately weighted and added to a 150 mL conical flask with a certain proportion of DES solvent. After vortexing, the mixture was put into an ultrasonic bath at 60 ◦C, 300 W power, and 50 Hz for 60 min, then the solution was collected and centrifuged at 6000 RPM for 15 min. A total of 1 mL of the preparation solution was added to a 5 mL volumetric flask and the volume was fixed with methanol to the scale line. Then it was filtered through a 0.45 μm filter and quantified by HPLC analysis. Each experiment was performed three times.

#### 2.3.3. Traditional Extraction Method Comparison

Compared with traditional extraction methods, 75% ethanol and methanol were used for an ultrasonic bath (300 W, 50 Hz, 60 ◦C, 60 min). After filtration with a 0.45 μm microporous membrane, 10 μL of filtrate were injected into HPLC for analysis, and the yield of total coumarins was calculated.
