Deep eutectic solvent as well as preparation method and application thereof

Abstract

The invention provides a deep eutectic solvent and a preparation method and application thereof, the effective components of the deep eutectic solvent are composed of choline chloride and ethylene glycol, the molar ratio of choline chloride to ethylene glycol is 1: 4, the water content of the effective components is 30%, and the deep eutectic solvent can replace an organic solvent to extract total flavonoids from fructus amomi; chemical components capable of distinguishing the varieties of the fructus amomi are determined through quantitative experiments on the fructus amomi flavonoid compounds, serve as differential markers epicatechin, procyanidine B2 and procyanidine B4 for distinguishing the varieties of the fructus amomi and have very high antioxidant activity, and H2O2-induced GES-1 cell oxidative stress is relieved by removing ROS in cells and up-regulating the level of SOD and GSH-Px.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, in particular to the technical field of a deep eutectic solvent and a preparation method and application thereof. Background Art

[0002] Amomum villosum is the dried mature fruit of the ginger plant Amomum villosum, native to Southeast Asia. With its unique and attractive aroma, it is widely used in food. It not only significantly enhances the aroma of dishes, but also has excellent therapeutic effects. As a traditional medicinal ingredient, Amomum villosum also has significant anti-inflammatory, antioxidant and antibacterial activities, and is widely used to treat various gastrointestinal diseases.

[0003] There are many varieties of Amomum villosum, among which Yangchun Amomum villosum and Green Shell Amomum villosum are the most popular in the market, and the Yangchun Amomum villosum grown in Yangchun City, Guangdong Province is the most famous. However, both fresh and processed Amomum villosum are very similar in appearance, which to a certain extent has caused the phenomenon of mixed varieties and different quality grades in the market.

[0004] At present, in order to distinguish the varieties of Amomum villosum, most studies focus on analyzing its volatile terpenoids, especially the content of bornyl acetate and camphor. However, Amomum villosum not only contains abundant volatile oils, but also includes other important compounds such as quercetin and epicatechin. Pharmacological studies have further shown that the total flavonoids in Amomum villosum have a significant inhibitory effect on the proliferation of MFC gastric cancer cells. Therefore, if the varieties and quality of Amomum villosum are distinguished by volatile components alone, other valuable active ingredients may be ignored, resulting in a waste of Amomum villosum resources.

[0005] In recent years, the harm of traditional organic solvents to the environment has become increasingly prominent, prompting people to pay extensive attention to the development of environmentally friendly chemical solvents. Natural deep eutectic solvents (NADES) are alternatives to traditional reagents and were first proposed as derivatives of deep eutectic solutions in 2011. NADES is a mixture of natural metabolites such as carboxylic acids, polyols, sugars, choline salts and amino acids, which has low toxicity, degradability and biocompatibility. These components act as hydrogen bond donors (HBD) and acceptors (HBA). The different compositions of NADES lead to differences in its physicochemical properties (polarity, density and pH value), making it suitable for the extraction of solutes with different properties. Therefore, NADES, as a new type of green solvent, is highly favored in phytochemical extraction and deserves further development and research. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a deep eutectic solvent.

[0007] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned deep eutectic solvent.

[0008] Another technical problem to be solved by the present invention is to provide application of the above-mentioned deep eutectic solvent.

[0009] The technical solution adopted by the present invention is:

[0010] A deep eutectic solvent, wherein the effective components are composed of choline chloride and ethylene glycol, the molar ratio of the choline chloride to the ethylene glycol is 1:4, and the water content of the effective components is 30% (v / v).

[0011] The preparation method of the above-mentioned deep eutectic solvent comprises the following specific steps: adding choline chloride and glycerol into a sample bottle at a molar ratio of 1:4, stirring evenly with a glass rod, setting the water bath temperature to 80°C, stirring magnetically until a uniform transparent liquid is formed, adding 30% (v / v) ultrapure water, and mixing evenly to obtain.

[0012] Application of the above deep eutectic solvent in extracting total flavonoids from Amomum villosum.

[0013] Preferably, the application of the above deep eutectic solvent, the specific extraction method is as follows:

[0014] (1) extracting the Amomum villosum powder in a water bath at 60° C. for 30 minutes at a solid-liquid ratio of 1:20 to obtain a mixed solution;

[0015] (2) The mixed solution was centrifuged at 14000 rpm for 10 minutes and the supernatant was collected.

[0016] The supernatant was used to determine the total flavonoids content in Amomum villosum.

[0017] Preferably, in the application of the above-mentioned deep eutectic solvent, the component in the supernatant is total flavonoids of Amomum villosum.

[0018] Preferably, the deep eutectic solvent is used, and the identification steps of the total flavonoids in Amomum villosum are as follows: using UHPLC-Orbitrap Exploris MS (Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA), the flavonoids in Amomum villosum are identified; chromatographic separation is performed on a ThermoHypersil GOLD column (2.1×100 mm, 1.9 um, Thermo Fisher, USA) at a flow rate of 0.3 mL / min and a temperature of 35°C; mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, and the solvent gradient was as follows: 0-3 min, 3-12% B; 3-14 min, 12%-13% B; 14-17 min, 13-20% B; 17-25 min, 20-35% B; 25-30 min, 35-75% B; 30-38 min, 75-80% B; 38-40 min, 80-95% B; 40-45 min, 95% B; the injection volume of all samples was 3 uL; full MS / DD-MS was performed separately 2 The mass range was 100-1500 m / z, and the general parameters in the negative ion mode were set as follows: spray voltage 3.0 kV, capillary temperature 320 °C, gas heater temperature 350 °C, and normalized collision energies of 20 V, 40 V, and 60 V.

[0019] Preferably, the deep eutectic solvent is used to quantitatively determine the chemical components of the Amomum villosum flavonoid compounds identified above as the difference markers for distinguishing the Amomum villosum varieties (Amomum villosum and Amomum villosum), and the specific steps are as follows:

[0020] (1) After the qualitative analysis of the flavonoid components of Amomum villosum, the differential markers for distinguishing Amomum villosum varieties were used to quantify the main flavonoid compounds. The samples were analyzed by UHPLC-QQQ-MS. The chromatographic separation was performed on a ThermoHypersil GOLD column (2.1×100 mm, 1.9 μm, Thermo Fisher, USA) at a flow rate of 0.3 mL / min and a temperature of 35°C; mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, and the solvent gradient was as follows: 0-1 min, 3-9% B; 1-8 min, 9-9% B; 8-12 min, 9-20% B; 12-16 min, 20-22% B; 16-18 min, 22-50% B; 18-19 min, 50-95% B; 19-22 min, 95% B; 22-23 min, 95-3% B, 22-23 min, 3-3% B; the injection volume of all samples was 3 μL; multiple reaction monitoring (MRM) was used to optimize the parameters of cone voltage and collision energy, and the general parameters in the negative ion mode were set as follows: capillary voltage 2.0 kV, desolvation gas temperature 350°C, nebulizer gas flow rate 3.0 L / min, drying gas flow rate 4.0 L / min;

[0021] (2) After quantification, the flavonoid content data of each Amomum villosum sample was imported into Simca14.1 software for unsupervised PCA analysis to identify differential markers that distinguish Amomum villosum varieties.

[0022] Preferably, in the application of the above-mentioned deep eutectic solvent, the differential markers are epicatechin, procyanidin B2 and procyanidin B4.

[0023] A total flavonoids extract of Amomum villosum obtained by the application method.

[0024] Application of the Amomum villosum total flavonoids extract in preparing products with antioxidant activity.

[0025] The beneficial effects of the present invention are:

[0026] The deep eutectic solvent is a natural deep eutectic solvent (NADES), which can replace organic solvents to extract total flavonoids from Amomum villosum. The deep eutectic solvent is evaluated by total flavonoid content. The optimal extraction conditions of total flavonoids from Amomum villosum are optimized by response surface optimization method: water content 30%, choline chloride-ethylene glycol as solvent, molar ratio of 1:4, solid-liquid ratio of 1:30g / mL, extraction temperature of 73°C, extraction time of 47 minutes, and extraction rate of 41.38±1.01mg RE / g, which is much higher than the traditional ethanol extraction (17.48±1.23mg RE / g). Under these conditions, 18 batches of Amomum villosum (AMV) and AMVX from different sources were extracted and purified. A total of 26 flavonoids were identified by LC-MS, and flavan-3-ol was the main flavonoid in Amomum villosum, of which 19 were quantified by UHPLC-QQQ-MS. By comparative analysis, epicatechin, proanthocyanidin B2 and proanthocyanidin B4 were identified as differential markers of AMV and AMVX. By establishing differential markers of Amomum villosum, a theoretical basis was provided for further identification of Amomum villosum varieties, measurement of Amomum villosum quality, selective selection of Amomum villosum varieties, rational application of Amomum villosum resources and standardization of the Amomum villosum market. Finally, the antioxidant activity of the three differential markers was determined by flow cytometry fluorescence technology. The results showed that the three differential markers had strong antioxidant activity, which alleviated H by scavenging intracellular ROS and upregulating SOD and GSH-Px levels. 2 O 2 Induced oxidative stress in GES-1 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Different NADES Amomum villosum flavonoids extracts.

[0028] Figure 2 The effects of different extraction parameters on TFC of Amomum villosum. (A) Water content; (B) Solid-liquid ratio; (C) Extraction temperature; (D) Extraction time; (EG) 3D response surface diagram of the interaction of various factors on total flavonoids (TFC).

[0029] Figure 3 Comparison of the flavonoid content in Amomum villosum (AMV) and AMVX. *P<0.05; **P<0.01

[0030] Figure 4 The principal component analysis diagrams of Amomum villosum (AMV) and AMVX. (A) PCA score diagram, (B) OPLS-DA score diagram, (C) OPLS-DA model permutation test diagram, (D) OPLS-DA VIP diagram.

[0031] Figure 5 Cell morphology. (A) Control group, (B) Model group, (C) Different concentrations of H2 O 2 Effects on cell survival rate, (D) Toxic effects of epicatechin, proanthocyanidin B2, and proanthocyanidin B4 on GES-1 cells, (E) Effects of flavonoids on H 2 O 2 Effects of induction on GES-1 cell viability. * P<0.05 compared with the control group. # P<0.05 compared with the model group.

[0032] Figure 6 The effects of high, medium and low doses of different biomarkers on cell oxidative damage markers. (A) Four-color flow cytometry was used to measure the production of intracellular ROS, (B) SOD, and (C) GSH-Px. * P<0.05 compared with the control group. # P<0.05 compared with the model group. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0034] Example 1

[0035] The natural deep eutectic solvent is prepared in the following steps:

[0036] Add the corresponding components into the sample bottle according to the molar ratio in Table 1, stir evenly with a glass rod, set the water bath temperature to 80° C., and stir magnetically until a uniform transparent liquid is formed. In order to reduce the viscosity of the NADES system, 30% (v / v) water was added.

[0037] Table 1 Eight natural deep eutectic solvents

[0038]

[0039] Example 2

[0040] The natural deep eutectic solvent described in Example 1 is used to extract the flavonoid components in Amomum villosum, and the specific steps are as follows:

[0041] The powder of Amomum villosum medicinal material was extracted in a water bath at 60°C for 30 minutes at a solid-liquid ratio of 1:20. The mixture was then centrifuged at 14,000 rpm for 10 minutes, and the supernatant was collected for total flavonoid content determination. For comparison, 1 g of sample was similarly mixed with 20 mL of 70% ethanol and extracted under the same conditions. The total flavonoids (TFC) were measured using the aluminum chloride colorimetric method, and the results were expressed as milligrams of rutin equivalents (RE) per gram of extract (mg RE / g). The results are shown in Figure 2. Figure 1As shown in the figure, choline chloride and ethylene glycol have the best extraction amount. Taking the extraction amount as the quantitative index, the single factor investigation combined with the response surface methodology was used to optimize the optimal extraction conditions of choline chloride and ethylene glycol.

[0042] Single factor optimization: Four factors were optimized based on the selected (choline chloride:ethylene glycol) NADES to improve the extraction efficiency, including water content (10%, 20%, 30%, 40% and 50%), solid-liquid ratio (1:10, 1:20, 1:30, 1:40 and 1:50 g / mL), extraction temperature of 40°C, 50°C, 60°C, 70°C and 80°C, and extraction time of 15, 30, 45, 60 and 75 min.

[0043] Response surface methodology (RSM) optimization: Based on the results of the single factor experiment, a three-factor, three-level Box-Behnken design was used to further optimize the flavonoid extraction. Three independent variables were selected at three levels: solid-liquid ratio (A), extraction temperature (B) and extraction time (C), and the total flavonoid content (Y) was used as the response variable. The response surface experimental design process and results are shown in Tables 2, 3 and 4.

[0044] Table 2 Independent variables and levels of response surface experiment

[0045]

[0046] Table 3 Design of response surface methodology and extraction of total flavonoids from Amomum villosum

[0047]

[0048] Table 4 Results of variance analysis and significance test

[0049]

[0050] Based on the regression model, the optimal extraction conditions for TFC were determined to be a solid-liquid ratio of 1:29 g / mL, an extraction temperature of 73.17 °C, and an extraction time of 46.71 min (see Figure 2 ). These conditions were adjusted to a solid-liquid ratio of 1:29 g / mL, an extraction temperature of 73°C, and an extraction time of 47 min. Through three repetitions, the experimental TFC obtained was 41.38 ± 1.01 mg RE / g, which was very close to the predicted value of 41.51 mg RE / g. These results confirm the accuracy of the RSM method in optimizing the extraction method.

[0051] Purification of flavonoids from Amomum villosum and Amomum villosum extracts: 18 batches (sources see Table 5) of Amomum villosum were extracted using NADES constructed with choline chloride and ethylene glycol (molar ratio: 1:4, water content 30% v / v), with the extraction conditions of solid-liquid ratio 1:29 g / mL, extraction temperature 73°C, and extraction time 47 minutes. After the extraction, the NADES extract was adsorbed using AB-8 macroporous resin. The column was washed with three bed volumes (BV) of water and then eluted with three column volumes of 50% ethanol. The eluate was collected and concentrated to dryness. After freeze-drying, 18 batches of purified total flavonoids were obtained and stored at -20°C until analysis.

[0052] Table 5 Sources of 18 batches of Amomum villosum samples

[0053]

[0054] Example 3

[0055] The freeze-dried powder obtained in Application Example 2 was used to identify the flavonoid components of Amomum villosum, and the specific steps were as follows:

[0056] The flavonoids in Amomum villosum were identified using UHPLC-Orbitrap Exploris MS (Thermo Fisher Scientific Inc., Waltham, MA, USA). Chromatographic separation was performed on a ThermoHypersilGOLD column (2.1×100 mm, 1.9 um, Thermo Fisher, USA) at a flow rate of 0.3 mL / min and a temperature of 35°C. Mobile phase A was 0.1% formic acid in water and mobile phase B was acetonitrile. The solvent gradient was as follows: 0-3 min, 3-12% B; 3-14 min, 12%-13% B; 14-17 min, 13-20% B; 17-25 min, 20-35% B; 25-30 min, 35-75% B; 30-38 min, 75-80% B; 38-40 min, 80-95% B; 40-45 min, 95% B. The injection volume for all samples was 3 uL. Full MS / DD-MS 2 Scanning, mass range 100-1500 m / z, the general parameters in negative ion mode were set as follows: spray voltage 3.0 kV, capillary temperature 320 °C, gas heater temperature 350 °C, normalized collision energy 20 V, 40 V and 60 V. By matching the collected MS data with databases and literature reports, and confirming some compounds with standard references, 26 flavonoids were identified (see Table 6). The flavonoids in Amomum villosum mainly include flavonols with quercetin, rhamnosine and luteolin as aglycones, and proanthocyanidins composed of catechins and epicatechins.

[0057] Table 6 Identification table of flavonoid compounds in Amomum villosum

[0058]

[0059]

[0060]

[0061]

[0062] *Compounds identified by standards.

[0063] Example 4

[0064] The 26 Amomum villosum flavonoids identified in Example 3 were used to quantify 19 of them and to find the differential markers that can distinguish Amomum villosum from Amomum villosum. The specific steps are as follows:

[0065] After the qualitative characterization of the flavonoid components of Amomum villosum, the chemical components that can distinguish the varieties of Amomum villosum were determined by quantitative experiments as differential markers for distinguishing the varieties of Amomum villosum. In order to quantify the main flavonoid compounds, 18 batches of samples were analyzed using UHPLC-QQQ-MS (Xevo-TQ-S, Waters, Milford, MA, USA) as described above. Chromatographic separation was performed on a ThermoHypersilGOLD column (2.1×100 mm, 1.9 μm, Thermo Fisher, USA) at a flow rate of 0.3 mL / min and a temperature of 35°C. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. The solvent gradient was as follows: 0-1 min, 3-9% B; 1-8 min, 9-9% B; 8-12 min, 9-20% B; 12-16 min, 20-22% B; 16-18 min, 22-50% B; 18-19 min, 50-95% B; 19-22 min, 95% B; 22-23 min, 95-3% B, 22-23 min, 3-3% B. The injection volume of all samples was 3 μL. Multiple reaction monitoring (MRM) was used to optimize the parameters of cone voltage and collision energy. The ion pairs and fragmentation parameters used for quantitative analysis are shown in Table 7. The general parameters in the negative ion mode were set as follows: capillary voltage 2.0 kV, desolvation gas temperature 350 ° C, nebulizer gas flow rate 3.0 L / min, and drying gas flow rate 4.0 L / min.

[0066] The results are as follows Figure 3As shown, Amomum villosum mainly contains flavan-3-ols and epicatechins with the highest contents ranging from 4325.36 to 25068.77 μg / g FD, followed by proanthocyanidin B2 and catechins with concentrations exceeding 1618.22 μg / g FD. Quercetin is the most abundant flavonol with a content ranging from 1327.46 to 2689.35 μg / g FD, followed by isoquercetin and its isomers with concentrations of 39.42 μg / g FD or above.

[0067] After quantification, the content data of 19 flavonoid compounds in each Amomum villosum sample were imported into Simca14.1 software for unsupervised PCA analysis to identify differential markers for differentiating Amomum villosum varieties. Figure 4 A The results showed that the distribution of AMV and AMVX showed a high degree of differentiation. There was no clustering of Amomum villosum from the same origin, indicating that the origin had no significant effect on the flavonoid content. OPLS-DA was used to identify potential differential components that affect the differentiation of Amomum villosum species. The dispersion diagram of 18 batches of Amomum villosum samples is shown in Figure 2. Figure 4 (B) shows that AMV and AMVX are clearly divided into two groups. The model R2X = 0.985, R2Y = 0.994, Q2 = 0.96, indicating high reliability and strong fit. The model was tested 200 times, R2 = 0.423, Q2 = -0.434, indicating that the model is not overfitting and has good predictive ability ( Figure 4 (C)). Continue using the VIP diagram of the established model ( Figure 4 (D)) to further analyze the key components that affect the differentiation of Amomum villosum species. The higher the VIP value, the greater the impact on the differentiation of Amomum villosum varieties. According to the criteria of VIP>1 and P<0.05, the potential differential components were identified as anthocyanidin B4, epicatechin and anthocyanidin B2. According to the content determination results, these three components are also the main components of Amomum villosum fruit.

[0068] Table 7 Mass spectrometry conditions - ion pairs

[0069]

[0070]

[0071] Example 5

[0072] Using the differential markers screened in Example 4, a cell-level antioxidant experiment was performed to evaluate the antioxidant activity of the three differential markers. 2 O 2 ) is an endogenous oxidant that is commonly used to induce oxidative damage in cell studies. The cells in the control group were maintained in normal culture medium, while the model group was exposed to H 2 O 2 In. Figure 5As shown in (A, B), compared with the control group, H 2 O 2 The induction of H caused significant damage in the model group, which was manifested by vacuolar degeneration, increased intercellular spaces, and a significant decrease in cell count. 2 O 2 Treat cells for 4 hours ( Figure 5 C), showing that as H 2 O 2 As the concentration increased, the survival rate gradually decreased. The concentration at which the cell survival rate was close to 50% was selected as the optimal condition for simulating cell damage. Therefore, exposure to 70 μM H 2 After 4 hours, the cell survival rate was 56.10%.

[0073] To determine the optimal drug concentration, cytotoxicity assays were performed on the three labeled compounds, and the results are shown in Figure 5 (D) When the concentration was below 50 μM, the markers such as epicatechin, proanthocyanidin B2 or proanthocyanidin B4 showed no obvious cytotoxicity, and the cell viability was maintained at ≥90%. Therefore, the subsequent antioxidant experiments used these compounds at a concentration of ≤50 μM, while the model group only used 70 μM H 2 O 2 Processing. Figure 5 As shown in (E), the cell survival rate in the model group was significantly lower than that in the control group. In the treatment groups, each compound showed a protective effect on GES-1 cells in a concentration-dependent pattern: the cell survival rate initially increased with increasing concentrations but then decreased at higher concentrations.

[0074] To further explore the intracellular oxidative changes, different concentrations of epicatechin (6.25, 12.5, 25 μM), proanthocyanidin B2 (3.125, 6.25, 12.5 M), and proanthocyanidin B4 (3.125, 6.25, 1250 μM) were divided into low (L), medium (M), and high (H) treatment categories. ROS levels were measured by flow cytometry, and the fluorescence intensity was analyzed by FlowJo software, as shown in Figure 2 . Figure 6 (A) As shown. ROS levels were significantly increased in the model group compared with the control group. In the treatment group, all compounds except low concentration of procyanidin B4 significantly reduced ROS levels, indicating that epicatechin, procyanidin B2, and procyanidin B4 contribute to the alleviation of oxidative stress by reducing ROS levels. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are endogenous enzymes that protect cells from ROS damage (Bhattacharyya, Chattopadhyay, Mitra, & Crowe, 2014). Figure 6The results of (B, C) showed that the levels of SOD and GSH-Px were higher in the pretreatment group compared with the model, indicating that these three markers protected GES-1 cells from H by enhancing the cellular antioxidant defense system. 2 O-induced oxidative damage.

[0075] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field shall fall within the protection scope of the claims of the present invention.

Claims

1. A deep eutectic solvent, characterized in that: The effective components consist of choline chloride and ethylene glycol, the molar ratio of the choline chloride to the ethylene glycol is 1:4, and the water content of the effective components is 30%.

2. The method for preparing the deep eutectic solvent according to claim 1, characterized in that: The specific steps are as follows: add choline chloride and glycerol into a sample bottle at a molar ratio of 1:4, stir evenly with a glass rod, set the water bath temperature to 80°C, stir magnetically until a uniform transparent liquid is formed, add 30% ultrapure water, and mix evenly to obtain.

3. Use of the deep eutectic solvent according to claim 1 in extracting total flavonoids from Amomum villosum.

4. The use of the deep eutectic solvent according to claim 3, characterized in that: The specific extraction method is as follows: (1) extracting the Amomum villosum powder in a water bath at 60° C. for 30 minutes at a solid-liquid ratio of 1:20 to obtain a mixed solution; (2) The mixed solution was centrifuged at 14000 rpm for 10 minutes and the supernatant was collected.

5. The use of the deep eutectic solvent according to claim 4, characterized in that: The components in the supernatant are total flavonoids of Amomum villosum.

6. The use of the deep eutectic solvent according to claim 3, characterized in that: The identification steps of the total flavonoids of Amomum villosum are as follows: using UHPLC-Orbitrap Exploris MS to identify the flavonoids in Amomum villosum; chromatographic separation is performed on a ThermoHypersil GOLD column at a flow rate of 0.3 mL / min and a temperature of 35° C.; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile, and the solvent gradient is as follows: 0-3 min, 3-12% B; 3-14 min, 12%-13% B; 14-17 min, 13-20% B; 17-25 min, 20-35% B; 25-30 min, 35-75% B; 30-38 min, 75-80% B; 38-40 min, 80-95% B; 40-45min, 95% B; injection volume of all samples was 3uL; full MS / DD-MS 2 The mass range was 100-1500 m / z, and the general parameters in the negative ion mode were set as follows: spray voltage 3.0 kV, capillary temperature 320 °C, gas heater temperature 350 °C, and normalized collision energies of 20 V, 40 V, and 60 V.

7. The use of the deep eutectic solvent according to claim 6, characterized in that: The identified Amomum villosum flavonoid compounds were quantitatively determined to be chemical components that can distinguish Amomum villosum varieties as differential markers for distinguishing Amomum villosum varieties. The specific steps are as follows: (1) After the qualitative analysis of the flavonoid components of Amomum villosum, the differential markers of Amomum villosum varieties were distinguished and the samples were analyzed using UHPLC-QQQ-MS. The chromatographic separation was performed on a ThermoHypersil The flow rate was 0.3 mL / min and the temperature was 35°C on the GOLD column; the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the solvent gradient was as follows: 0-1 min, 3-9% B; 1-8 min, 9-9% B; 8-12 min, 9-20% B; 12-16 min, 20-22% B; 16-18 min, 22-50% B; 18-19 min, 50-95% B; 19-22 min, 95% B; 22-23 min, 95-3% B, 22-23 min, 3-3% B; the injection volume of all samples was 3 μL; the general parameters in the negative ion mode were set as follows: capillary voltage 2.0 kV, desolvation gas temperature 350°C, nebulizing gas flow rate 3.0 L / min, drying gas flow rate 4.0 L / min; (2) After quantification, the flavonoid content data of each Amomum villosum sample was imported into Simca14.1 software for unsupervised PCA analysis to identify differential markers that distinguish Amomum villosum varieties.

8. The use of the deep eutectic solvent according to claim 7, characterized in that: The differential markers are epicatechin, procyanidin B2 and procyanidin B4.

9. A total flavonoids extract of Amomum villosum obtained by the application method according to any one of claims 3 to 8.

10. Use of the total flavonoids extract of Amomum villosum according to claim 9 in preparing products with antioxidant activity.

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