Natural hydrophobic deep eutectic solvent and method for extracting and purifying triterpenoids and chalcone A in liquorice by using natural hydrophobic deep eutectic solvent

Abstract

The invention provides a natural hydrophobic deep eutectic solvent, which can be used for simultaneously extracting and purifying triterpenoids and chalcone A in plants such as liquorice, and mainly solves the technical problems of environmental pollution and harm to human health in the process of extracting and purifying triterpenoids and chalcone A by using a traditional organic solvent. The hydrophobic deep eutectic solvent mainly takes menthol as a hydrogen bond acceptor (HBA) and fatty acid as a hydrogen bond donor (HBD), the menthol and the fatty acid are mixed according to a certain proportion and react at a certain temperature to prepare the natural hydrophobic deep eutectic solvent, and raw materials of the solvent are natural nontoxic compounds, so that the solvent is an environment-friendly medium. The natural hydrophobic deep eutectic solvent is combined with an ultrasonic-assisted liquid-liquid microextraction method, and can be used for extracting and purifying triterpenoids and flavonoid chalcone A in plants. The method is simple to operate, good in repeatability, high in stability and high in sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product extraction, and particularly relates to a natural hydrophobic deep eutectic solvent and a method for extracting and purifying triterpenoid compounds and chalcone A from licorice by using the same. Background Art

[0002] Licorice is a herbaceous plant of the legume family with important medicinal value and has a long history of use all over the world, enjoying the title of "ancestor of plants". The main components of triterpenoid compounds in licorice are glycyrrhizic acid (GL) and glycyrrhetinic acid (GA), which have medicinal activities such as anti-inflammatory, anti-tumor, antioxidant, antiviral, antibacterial, liver protection and immunomodulatory effects. Licorice chalcone A is a unique flavonoid compound in licorice, which has various effects such as anti-inflammatory, anti-tumor, anti-microbial, anti-HIV and anti-allergic.

[0003] Since triterpenoid compounds and licorice chalcone A are extremely insoluble in water, organic solvents such as methanol are commonly used for extraction. Not only does it consume a large amount of solvents, causing great harm to human health and the social environment, but also it will extract more non-target components at the same time, causing difficulties in subsequent separation and purification; meanwhile, the use of a large amount of low-boiling organic solvents also brings great safety hazards.

[0004] There are great differences in the structures of glycyrrhizic acid, glycyrrhetinic acid and licorice chalcone A, and it is very difficult to extract and purify the three compounds simultaneously. Dilute ammonia water is commonly used as a solvent to assist in the extraction of glycyrrhizic acid and glycyrrhetinic acid. Common extraction methods for triterpenic acid compounds include: reflux extraction method, microwave-assisted extraction method, ultrasonic-assisted extraction method, supercritical CO2 extraction method, etc. The reflux extraction method requires a high extraction temperature and a long time; the supercritical CO2 extraction method has strong extraction ability, high extraction yield and is environmentally friendly and pollution-free, but the equipment is expensive and the cost is high; ultrasonic-assisted extraction has the advantages of a wide extraction range, high efficiency and low price, and is safer compared with conventional microwave-assisted extraction. Therefore, the present invention selects the ultrasonic-assisted extraction method for the simultaneous extraction of triterpenoid compounds and licorice chalcone A from licorice and optimizes the process.

[0005] Deep eutectic solvents (DES) are regarded as a promising extraction solvent because of their advantages such as simple preparation, good solubility, low price and low toxicity. When common hydrophilic eutectic solvents are used as extractants, it is difficult to separate them from the extraction system, while hydrophobic eutectic solvents can well solve this drawback. Based on this, this application is developed. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a natural hydrophobic deep eutectic solvent and a method for extracting and purifying triterpenoid compounds and chalcone A from licorice by using the same. It has the advantages of simple preparation method, environmentally friendly solvent, short extraction time and high extraction efficiency.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A preparation method of a natural hydrophobic deep eutectic solvent, which disperses a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) evenly at a temperature of 50 - 60 °C, and the obtained clear, transparent and homogeneous solution is the natural hydrophobic deep eutectic solvent (DES).

[0008] Specifically, in the above preparation method of the natural hydrophobic deep eutectic solvent, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor can be 1:0.5 - 3.

[0009] Further, the hydrogen bond acceptor can be menthol, etc., and the hydrogen bond donor can be at least one of formic acid, oleic acid, lactic acid, lauric acid, n-butanol, etc.

[0010] The present invention provides a natural hydrophobic deep eutectic solvent prepared by the above preparation method.

[0011] The present invention provides the application of the above natural hydrophobic deep eutectic solvent in extracting and purifying triterpenoid compounds and chalcone A in licorice.

[0012] The present invention also provides a method for extracting and purifying triterpenoid compounds and chalcone A in licorice by using the natural hydrophobic deep eutectic solvent, which includes the following steps: 1) Prepare a licorice extract: 2) After mixing the licorice extract with the natural hydrophobic deep eutectic solvent, perform ultrasonic extraction, and after extraction, perform centrifugal separation, and take the supernatant to obtain the extraction solution of triterpenoid compounds and chalcone A. This method also includes the determination of the contents of triterpenoid compounds and chalcone A, which is filtered through a 0.22 μm organic filter membrane and used for determination by high-performance capillary electrophoresis.

[0013] Specifically, the volume ratio of the natural hydrophobic deep eutectic solvent to the licorice extract in step 2) can be 0.06 - 0.3:1.

[0014] Further, in step 2), the ultrasonic extraction temperature is 30 - 70 °C, the ultrasonic extraction power is 40 - 80 W, and the ultrasonic extraction time is 1 - 20 min. The centrifugal separation in step 2) is carried out at a rotation speed of 8000 - 12000 rpm for 3 - 10 min.

[0015] Further, the preparation of the licorice extract in step 1) is specifically as follows: The dried licorice is crushed and sieved. After adding ammonia water with a concentration of 0.3 - 0.7% (V / V) to the licorice powder, it is heated and stirred at 100 ± 10 °C for 0.5 - 2 h for extraction. The extraction is repeated 0 - 3 times, and the extraction liquids are combined and filtered to obtain the extract, which is stored in a refrigerator at 4 °C in the dark for later use. Specifically, after crushing, the licorice is sieved through a 60 - 120 - mesh sieve; the material - liquid ratio of the licorice powder to ammonia water is 1 g:8 - 15 mL.

[0016] In the present invention, menthol is used as a hydrogen - bond acceptor (HBA), and formic acid, oleic acid, lactic acid, lauric acid, n - butanol, etc. are used as hydrogen - bond donors (HBD). The natural hydrophobic deep - eutectic solvent is synthesized by heating and stirring, and then added to the licorice extract for ultrasonic extraction. After centrifugation, the supernatant is taken for capillary electrophoresis (CE) analysis.

[0017] The present invention provides a method for simultaneously extracting and purifying triterpenoids and chalcone A in licorice using a natural hydrophobic deep - eutectic solvent. The solvent is environmentally friendly, the extraction is efficient, and the method is simple and rapid, providing a new idea and method for the extraction of active ingredients in licorice and the rational utilization of resources.

[0018] The present invention provides a natural hydrophobic deep - eutectic solvent, which can be used for simultaneously extracting and purifying triterpenoids and chalcone A in plants such as licorice. It mainly solves the technical problems of environmental pollution and harm to human health existing in the traditional organic solvent extraction and purification of triterpenoids and chalcone A. The described hydrophobic deep - eutectic solvent mainly uses menthol as a hydrogen - bond acceptor (HBA) and fatty acids as hydrogen - bond donors (HBD). The two are mixed in a certain proportion and reacted at a certain temperature to obtain the natural hydrophobic deep - eutectic solvent. Since the raw materials of this solvent are all natural non - toxic compounds, this solvent is an environmentally friendly medium. The natural hydrophobic deep - eutectic solvent of the present invention combined with the ultrasonic - assisted liquid - liquid micro - extraction method can be used for extracting and purifying triterpenoids and flavonoid chalcone A in plants such as licorice. This method is simple to operate, has good repeatability, high stability, and high sensitivity.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1) The present invention uses licorice powder as a raw material and adopts ultrasonic - assisted hydrophobic deep - eutectic solvent to simultaneously extract and purify triterpenoids in the licorice extract. The extraction and purification process conditions are mild, the time is short, and the purification efficiency is high; 2) The AGREE pictogram of the method greenness can be made by combining the AGREE score table of the method greenness with the AGREE software to evaluate the impact of the analysis method on the environment. The total score is 0.57, indicating that the greenness of this method is relatively high, proving that this method is environmentally friendly; 3) The method of the present invention is simple to operate, requires ordinary equipment, and has mild solvent preparation conditions, low cost, environmental friendliness, and meets the concepts of safety, efficiency, and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] To more clearly illustrate the embodiments of the present invention, the accompanying drawings required for use in the embodiments will be briefly introduced below. The accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 Infrared spectra of hydrogen bond donors, hydrogen bond acceptors, and DES-1 to DES-11, where A is DES-1, DES-2, and DES-3; B is DES-4, DES-5, and DES-6; C is DES-7 and DES-9; D is DES-10; E is DES-11; Figure 2 Thermogravimetric curves and differential scanning calorimetry curves (embedded) of DES-1 to DES-11; where A is DES-1, DES-2, and DES-3; B is DES-4, DES-5, and DES-6; C is DES-7 and DES-9; D is DES-10 and DES-11; Figure 3 Hydrophobic conditions of DES-1 to DES-11 at different times, with DES on the upper layer and secondary water on the lower layer; Figure 4 Electrophoresis spectra of triterpenoid compounds and chalcone A standards (A), electrophoresis spectrum of licorice extract before extraction (B), and electrophoresis spectrum of hydrophobic DES-phase compounds after dilution by 5 times (C); Figure 5 AGREE pictogram of the greenness of the method. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The technical solutions of the present invention will be further described in detail below in combination with the embodiments, but the protection scope of the present invention is not limited thereto.

[0023] In the following embodiments, the raw materials used are all ordinary commercially available products that can be directly purchased, or can be prepared by conventional techniques in the art. The licorice samples were provided by a cooperative enterprise and were identified as Glycyrrhiza glabra.

[0024] Examples 1-11 This example provides a method for extracting and purifying triterpenoid compounds and chalcone A from licorice using a natural hydrophobic deep eutectic solvent, including the following steps: (1)Preparation of licorice extract sample: After drying the licorice, it was crushed with a pulverizer, passed through an 80-mesh sieve, and stored in the dark at room temperature for later use. Accurately weigh 10 g of licorice powder and place it in a beaker. Add 100 mL of 0.5% ammonia water according to the solid-liquid ratio of 1 g:10 mL, heat and stir at 100 °C for 1 h for extraction. Repeat the extraction 2 times, combine the extracts, filter, and store in the dark in a refrigerator at 4 °C for later use.

[0025] (2)Preparation of natural deep eutectic solvents: For the preparation of natural deep eutectic solvents, menthol was weighed as a hydrogen bond acceptor and placed in a beaker. Hydrogen bond donors formic acid, oleic acid, lactic acid, lauric acid, and n-butanol were added according to the molar ratios of 1:1, 1:2, and 2:1, and magnetically stirred at a constant temperature of 50 - 60 °C until the solution became clear and transparent, thus obtaining natural deep eutectic solvents (see Table 1 for details), and then sealed for later use.

[0026] Table 1 Composition and ratio of different solvent types DES is a co - solvent mixture formed by two or more components of HBA and HBD through hydrogen bonds. Comparing the chemical structures of DES components, the hydroxyl group of menthol serves as a hydrogen bond acceptor, and fatty acids containing carboxyl groups are usually used as hydrogen bond donors. At the same time, n - butanol can also be used as a hydrogen bond donor. They form DES under hydrogen bond interactions. Infrared tests were performed on the raw materials and the synthesized DES to prove the formation of hydrogen bonds, as Figure 1 shown. In DES - 1, DES - 2, and DES - 3, the stretching vibration of the O - H bond of menthol changed from the original 3254.3 cm -1 to 3448 cm -1 , 3442 cm -1 , 3446.6 cm -1 , and the stretching vibration of C = O of formic acid changed from 1701.6 cm -1 to 1722.5 cm -1 , 1719.5 cm -1 , 1719.5 cm -1 . The positions of the characteristic peaks changed, proving the generation of hydrogen bonds. In DES - 4, DES - 5, and DES - 6, the stretching vibrations of the O - H bond of menthol were 3415.2 cm -1 , 3421.1 cm -1 , 3409.2 cm -1 , respectively, and the stretching vibration of C = O in oleic acid changed from 1707.6 cm -1 to 1710.5 cm -1 , 1713.5 cm -1 , 1710.5 cm -1, the positions of the characteristic peaks changed, indicating the formation of hydrogen bonds. In DES-7 and DES-9, the stretching vibrations of the O-H bonds of menthol were 3427.1 cm -1 and 3448 cm -1 , respectively. The stretching vibration of the C=O bond of lactic acid changed from 1722.5 cm -1 to 1725.4 cm -1 . The positions of the characteristic peaks changed, indicating the formation of hydrogen bonds. In DES-10, the stretching vibration of the O-H bond of menthol changed to 3409.2 cm -1 , and the stretching vibration of the C=O bond of lauric acid changed from 1698.6 cm -1 to 1713.5 cm -1 . The positions of the characteristic peaks changed, indicating the formation of hydrogen bonds. In DES-11, the stretching vibration of the O-H bond of menthol changed to 3672.9 cm -1 , and the stretching vibration of the O-H bond of n-butanol changed from 3319.8 cm -1 to 3334.7 cm -1 . The positions of the characteristic peaks changed, indicating the formation of hydrogen bonds.

[0027] The thermogravimetric curves and differential scanning calorimetry curves of DES-1~DES-11 are as shown in Figure 2 . It can be seen from the figure that the glass transition temperatures of the synthesized DES-1, DES-2, DES-3, DES-4, DES-5, DES-6, DES-7, DES-9, DES-10, and DES-11 are very low, being -8.211 °C, 3.178 °C, 8.344 °C, -3.576 °C, 2.185 °C, -7.35 °C, -2.86 °C, -8.152 °C, 10.68 °C, and -1.16 °C, respectively, and the decomposition temperatures are relatively high. The infrared and thermogravimetric results indicate the successful synthesis of the 11 DESs.

[0028] Hydrophobic property test of DES-1~DES-11: The prepared DES-1~DES-11 were respectively transferred into centrifuge tubes, and the same volume of secondary water (rhodamine B stained) was added to each. After thorough mixing, they were placed in a centrifuge and centrifuged at a speed of 6000 rpm·min -1 for 5 min. The miscibility of DES with secondary water at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h was observed, and the results are shown in Figure 3 . As can be seen from Figure 3 , after centrifugation, the two phases were separated. After standing for 24 h, except for a slight change in the separation situation of DES-8, the separation situations of the other DESs were still obvious, indicating that the hydrophobic effect of DES-8 was slightly poor, and the 11 synthesized DESs all had good hydrophobic properties.

[0029] (3)Ultrasonic-assisted extraction and purification of triterpenoids and chalcone A from licorice: Pipette 5 mL of the licorice extract prepared in (1) above into a 10 mL centrifuge tube, add 1000 μL of DES-9 prepared in (2) above, and then perform ultrasonic extraction at 70 °C for 4 min at a power of 50 W. After extraction, centrifuge at 10000 rpm for 5 min to separate, and take the supernatant to obtain the extraction solution of triterpenoids and chalcone A. After filtering through a 0.22 μm organic filter membrane, it is used for the determination of high-performance capillary electrophoresis.

[0030] Taking the contents of glycyrrhizic acid, glycyrrhetinic acid and chalcone in the extraction solution as indicators, high-performance capillary electrophoresis analysis was carried out, and the results are as Figure 4 shown. High-performance capillary electrophoresis conditions: The inner diameter of the capillary is 75 μm and the length is 76 cm. The buffer solution is 35 mM borax buffer (15% methanol) with pH = 9.6. Pressure injection (5 s, 50 mbar) is used, the detection wavelength is 254 nm, the separation voltage is 20 KV, and the capillary column temperature is 25 °C.

[0031] Figure 4 Among them, (A) is the electrophoresis chromatogram of the standard products of triterpenoids glycyrrhizic acid (GL), glycyrrhetinic acid (GA) and chalcone A (ChA), which is used for chromatogram comparison and confirmation of the analytes in the sample; (B) is the chromatogram of the original licorice extract obtained in step (1); (C) is the electrophoresis chromatogram of the DES-extracted licorice extract (since DES is hydrophobic when analyzed and detected by a capillary electrophoresis instrument, and the buffer solution of capillary electrophoresis is water, adding hydrophobic DES to the buffer solution is prone to flow interruption and cannot be detected. Moreover, the concentration of analytes in the extraction solution is also relatively high, so it is diluted 5 times with methanol. The extraction method adopted in step (1) of the present invention is for the extraction of glycyrrhizic acid, so the extraction effect of glycyrrhizic acid is better, as Figure 4As shown, the concentration in the glycyrrhiza extract sample before extraction was relatively high, at 6.17 mg / mL. However, the extraction method in step (1) had a poor extraction effect on glycyrrhetinic acid and chalcone A, and the contents of glycyrrhetinic acid and chalcone A in the extract were relatively low, at 23.15 μg / mL and 13.97 μg / mL respectively. Due to the hydrophobicity of triterpenoids and glycyrrhizachalcone A, their solubility in the DES phase was relatively high, so they could be extracted and enriched by DES. After extraction, the contents of glycyrrhizic acid, glycyrrhetinic acid and chalcone A in DES increased significantly, with concentrations of 7.19 mg / mL, 75.74 μg / mL and 60.53 μg / mL respectively. Calculated based on the change in concentration before and after extraction, glycyrrhizic acid was concentrated 1.2 times, glycyrrhetinic acid was concentrated 3.3 times, and chalcone A was enriched and concentrated 4.3 times. From the extraction and enrichment effect of DES, it can be seen that the DES synthesized in the present invention does not cause loss of the triterpenoid glycyrrhizic acid with a relatively high content, while having a good enrichment and concentration effect on glycyrrhetinic acid and glycyrrhizachalcone A with relatively low concentrations. Therefore, it can be used for the simultaneous extraction and purification of triterpenoids and chalcone A in glycyrrhiza.

[0032] Meanwhile, in order to evaluate the impact of this analytical method on the environment, the present invention also used the Analytical Greenness metric (AGREE) to evaluate the greenness of this method, and the results are as Figure 5 shown. The greenness scoring table of AGREE consists of 12 principles of green analytical chemistry. The score is calculated based on specific experimental parameters, and the 12 evaluation criteria of AGREE are transformed into a red-yellow-green color scale, corresponding to a score of 0 to 1, where 0 represents the lowest greenness and 1 represents the highest greenness. The final score is the comprehensive score of all evaluation criteria. The AGREE score table for greenness is shown in Table 2. The total score of this method is 0.57, indicating that the present invention uses fewer toxic reagents or compounds, and less waste is generated during the analysis process. The technology adopted in the present invention is friendly to the environment, human health and human safety, meeting the social needs of green sustainable development.

[0033] Table 2 AGREE score table for method greenness Example 2 This example provides a method for extracting and purifying triterpenoids and chalcone A from glycyrrhiza using a natural hydrophobic deep eutectic solvent, comprising the following steps: (1) Preparation of the glycyrrhiza extract sample: the same as (1) in Example 1.

[0034] (2) Preparation of the natural hydrophobic deep eutectic solvent: the same as (2) in Example 1.

[0035] (3)Ultrasonic-assisted extraction and purification of triterpenoids and chalcone A from licorice: Pipette 5 mL of the licorice extract prepared in (1) above into a 10 mL centrifuge tube, add 1000 μL of DES-7 prepared in (2) above, and then perform ultrasonic extraction at 50 °C for 20 min at a power of 70 W. After extraction, centrifuge at 10000 rpm for 5 min to separate, and take the supernatant to obtain the extraction solution of triterpenoids and chalcone A. After filtering through a 0.22 μm organic filter membrane, it is used for the determination by high-performance capillary electrophoresis.

[0036] (4)Taking the contents of glycyrrhizic acid, glycyrrhetinic acid and chalcone in the extraction solution as indexes, high-performance capillary electrophoresis analysis was carried out. Before extraction, the concentrations of glycyrrhizic acid, glycyrrhetinic acid and chalcone A in the licorice extract prepared in (1) above were 5.95 mg / mL, 21.16 μg / mL and 15.95 μg / mL respectively. After extraction, the concentrations of glycyrrhizic acid, glycyrrhetinic acid and chalcone A in the DES phase were 7.45 mg / mL, 77.37 μg / mL and 66.61 μg / mL respectively. After calculation, the three were enriched 1.3, 3.7 and 4.2 times respectively. High-performance capillary electrophoresis conditions: the inner diameter of the capillary was 75 μm, the length was 76 cm, the buffer solution was 35 mM borax buffer solution (15% methanol) with pH = 9.6, pressure injection (5 s, 50 mbar) was used, the detection wavelength was 254 nm, the separation voltage was 20 KV, and the capillary column temperature was 25 °C.

[0037] Example 3 This example provides a method for extracting and purifying triterpenoids and chalcone A from licorice by natural hydrophobic deep eutectic solvents, including the following steps: (1)Preparation of licorice extract sample: The same as (1) in Example 1.

[0038] (2)Preparation of natural hydrophobic deep eutectic solvents: The same as (2) in Example 1.

[0039] (3)Ultrasonic-assisted extraction and purification of triterpenoids and chalcone A from licorice: Pipette 5 mL of the licorice extract prepared in (1) above into a 10 mL centrifuge tube, add 1000 μL of DES-9 prepared in (2) above, and then perform ultrasonic extraction at 50 °C for 4 min at a power of 50 W. After extraction, centrifuge at 10000 rpm for 5 min to separate, and take the supernatant to obtain the extraction solution of triterpenoids and chalcone A. After filtering through a 0.22 μm organic filter membrane, it is used for the determination by high-performance capillary electrophoresis.

[0040] (4)Taking the contents of glycyrrhizic acid, glycyrrhetinic acid and chalcone in the extract as indexes, high performance capillary electrophoresis analysis was carried out. The glycyrrhiza extract prepared in the above (1) was the solution before extraction, and the concentrations of glycyrrhizic acid, glycyrrhetinic acid and chalcone A were 5.92 mg / mL, 19.81 μg / mL and 14.63 μg / mL respectively. After extraction, the concentrations of glycyrrhizic acid, glycyrrhetinic acid and chalcone A in the DES phase were 7.53 mg / mL, 72.49 μg / mL and 66.08 μg / mL respectively. After calculation, the three were enriched 1.3, 3.3 and 4.5 times respectively. High performance capillary electrophoresis conditions: the inner diameter of the capillary was 75 μm, the length was 76 cm, the buffer solution was 35 mM, pH = 9.6 borax buffer solution (15% methanol), pressure injection was used (5 s, 50 mbar), the detection wavelength was 254 nm, the separation voltage was 20 KV, and the capillary column temperature was 25 °C.

[0041] The above has introduced in detail a ultrasonic extraction process of triterpenoids based on deep eutectic solvents provided by the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments only describe the principle of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of a natural hydrophobic deep eutectic solvent, characterized in that, Disperse the hydrogen bond acceptor and the hydrogen bond donor evenly at a temperature of 50-60 °C, and the obtained clear, transparent and homogeneous solution is the natural hydrophobic deep eutectic solvent.

2. The preparation method of the natural hydrophobic deep eutectic solvent according to claim 1, characterized in that, The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:0.5-3.

3. The preparation method of the natural hydrophobic deep eutectic solvent according to claim 1, wherein The hydrogen bond acceptor is menthol, and the hydrogen bond donor is at least one of formic acid, oleic acid, lactic acid, lauric acid and n-butanol.

4. A natural hydrophobic deep eutectic solvent prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the natural hydrophobic deep eutectic solvent according to claim 4 in extracting and purifying triterpenoids and chalcone A in licorice.

6. A method for extracting and purifying triterpenoids and chalcone A from licorice using the natural hydrophobic deep eutectic solvent described in claim 4, characterized in that, It includes the following steps: 1) Prepare a licorice extract: 2) After mixing the licorice extract with the natural hydrophobic deep eutectic solvent according to claim 4, perform ultrasonic extraction, and after extraction, perform centrifugal separation, and take the supernatant to obtain an extract of triterpenoids and chalcone A.

7. The method for extracting and purifying triterpenoids and chalcone A from licorice according to claim 6, wherein, In step 2), the volume ratio of the natural hydrophobic deep eutectic solvent to the licorice extract is 0.06-0.3:

1.

8. The method for extracting and purifying triterpenoids and chalcone A from licorice as claimed in claim 6, wherein In step 2), the ultrasonic extraction temperature is 30-70 °C, the ultrasonic extraction power is 40-80 W, and the ultrasonic extraction time is 1-20 min.

9. The method for extracting and purifying triterpenoid compounds and chalcone A from licorice according to claim 6, characterized in that, In step 1), the preparation of the licorice extract is specifically as follows: Crush and sieve the dried licorice, add ammonia water with a concentration of 0.3-0.7% to the licorice powder, and heat and stir at 100±10 °C for 0.5-2 h for extraction. Repeat the extraction 0-3 times, combine the extracts, and filter to obtain it.

10. The method for extracting and purifying triterpenoid compounds and chalcone A from licorice as claimed in claim 9, wherein, After crushing, sieve through a 60-120 mesh sieve; the material-liquid ratio of the licorice powder to the ammonia water is 1 g:8-15 mL.

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