A deep eutectic solvent and a preparation method and application thereof

Abstract

The application relates to the technical field of natural product extraction, and discloses a eutectic solvent as well as a preparation method and application thereof. The eutectic solvent is formed by the combination of a hydrogen bond acceptor and a hydrogen bond donor through hydrogen bond force; the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3-1:1-3; the hydrogen bond acceptor is citric acid; and the hydrogen bond donor is polypropylene glycol with an average molecular weight of 200-400 or polyethylene glycol with an average molecular weight of 200-800. The eutectic solvent can extract various active substances from tea dregs, improves the extraction rate of active components in the tea dregs, and especially the extraction rate of theanine in the active components in the tea dregs is higher than that of water extraction and methanol extraction.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural product extraction, and particularly relates to a eutectic solvent and a preparation method and application thereof. BACKGROUND

[0002] Tea is a traditional beverage with a long history. With the increasing demand for tea products, a large amount of tea leaf by-products is also produced. The tea leaf by-products mainly refer to stems, leaf fragments, hair, tea ash, tea dregs and the like separated in the tea processing process and cannot be sold as commodities. The tea dregs contain high contents of catechins, theanine and caffeine and the like, but have not been fully utilized.

[0003] At present, many studies have proved that the above-mentioned components have good biological activities. For example, EGCG has the effects of antioxidation, antibiosis, anti-inflammation and cardiovascular protection. Theanine has the effect of protecting the nervous system. Caffeine can improve cognitive ability, enhance the lung function and capacity of the cardiopulmonary system, and can also be used to treat apnea. Therefore, the tea dregs are used as raw materials to extract active components such as catechins, theanine and caffeine, which is beneficial to the comprehensive utilization of the tea dregs and improves the comprehensive value of the tea industry.

[0004] At present, organic solvents such as methanol are commonly used media for extracting chemical components in tea leaves and the like, but these traditional solvents have the disadvantages of toxicity, strong volatility, easy environmental pollution, organic solvent residue and the like, which limits their extraction in the fields of food and medicine. In recent years, a eutectic solvent, abbreviated as DES, has attracted wide attention and application in extraction and separation. Compared with traditional organic solvents, DES has the characteristics and advantages of low melting point, adjustable properties, non-toxicity, low volatility and high thermal stability. At present, DES has been successfully applied to the extraction and separation of flavonoids, phenolic acids, alkaloids, anthraquinones and other plant natural active compounds. DES is mainly divided into five categories, among which the chlorocholine type DES is most widely used in the field of extraction and separation. However, the chlorocholine type DES reported at present is difficult to efficiently extract multiple target substances with different properties from tea or tea dregs matrix at the same time. SUMMARY

[0005] In order to solve the above technical problems, the application provides a eutectic solvent and a preparation method and application thereof.

[0006] A eutectic solvent, which is formed by the combination of a hydrogen bond acceptor and a hydrogen bond donor through hydrogen bond force; the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3-1:1-3;

[0007] The hydrogen bond acceptor is citric acid;

[0008] The hydrogen bond donor is polyethylene glycol with an average molecular weight of 200-400 or polyethylene glycol with an average molecular weight of 200-600; preferably, the hydrogen bond donor is any one of polypropylene glycol with an average molecular weight of 200, polypropylene glycol with an average molecular weight of 400, polyethylene glycol with an average molecular weight of 200, polyethylene glycol with an average molecular weight of 400, or polyethylene glycol with an average molecular weight of 600.

[0009] The hydrogen bond acceptor citric acid in the present application contains carboxyl and hydroxyl groups, and can form intermolecular interactions such as hydrogen bonding and electrostatic interaction with active substances. The polypropylene glycol and polyethylene glycol in the hydrogen bond donor contain hydroxyl groups and methylene structures, and have amphiphilic properties, which have good solubility for active substances with different polarities. Therefore, the hydrogen bond acceptor and the hydrogen bond donor are mixed in a molar ratio of 3-1:1-3, the -COOH bond in citric acid and the -OH bond in the hydrogen bond donor form new hydrogen bonds, which not only ensure the structural stability of the deep eutectic solvent, but also have better extraction effect.

[0010] The second object of the present application is to protect the preparation method of the deep eutectic solvent, which specifically comprises the following steps:

[0011] The raw materials are mixed in a molar ratio, and reacted at 80-100°C for 120-360 min to obtain the deep eutectic solvent. If the reaction temperature is lower than this temperature range, it is difficult to form a deep eutectic solvent through interaction; and if the reaction temperature is higher than this temperature range, the hydrogen bond donor or acceptor may be subjected to thermal cracking or other reactions.

[0012] The third object of the present application is to protect the application of the deep eutectic solvent in extracting active ingredients in tea residue, and the active ingredients are amino acids, alkaloids, catechins, and flavonoids.

[0013] Preferably, the specific extraction method comprises the following steps:

[0014] The tea residue powder is mixed with an aqueous solution of the deep eutectic solvent, and then subjected to microwave-assisted extraction at 100-500 W for 2-15 min, and centrifuged, and the supernatant contains the active ingredients in the tea residue.

[0015] Preferably, the volume percentage of the deep eutectic solvent in the aqueous solution of the deep eutectic solvent is 10-90%. The viscosity of the deep eutectic solvent is relatively large, and adding a certain amount of water can significantly reduce the viscosity, but too much water can reduce the extraction rate, so this range is set.

[0016] Preferably, the power of the microwave-assisted extraction is 100-500 W, and the temperature is 30-80°C. Heating to a certain temperature is beneficial to reduce the viscosity of the deep eutectic solvent and improve the extraction effect, but too high a temperature may damage the stability of the target substance.

[0017] Preferably, the ratio of tea residue powder to the aqueous solution of the eutectic solvent is 1g:10mL-50mL;

[0018] Preferably, the centrifugal separation is performed at a speed of 4000r / min-8000r / min for 3min-5min.

[0019] Preferably, the tea residue powder has a water content of less than 5mg / 100g and a particle size of ≤250μm.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] The eutectic solvent in the present application forms new hydrogen bonds through the -COOH bond in citric acid and the -OH bond in the hydrogen bond donor, and citric acid contains carboxyl and hydroxyl groups, which can interact with active substances through hydrogen bonds and electrostatic interactions, etc. The polypropylene glycol and polyethylene glycol in the hydrogen bond donor contain hydroxyl and methylene structures, and have amphiphilic properties, which have good solubility for active substances of different polarity, so that a plurality of active substances can be extracted from tea residue, the extraction rate of active ingredients in tea residue is improved, and the extraction rate of theanine in tea residue is much higher than that of water extraction and methanol extraction. The antioxidant activity of other active ingredients extracted from tea residue is equivalent to that of methanol extract, and is much higher than that of pure water extract. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The FTIR spectra of citric acid, PEG400 and the eutectic solvent citric acid-PEG400.

[0023] Figure 2 The FTIR spectra of citric acid, PEG200 and the eutectic solvent citric acid-PEG200.

[0024] Figure 3 The FTIR spectra of citric acid, PEG600 and the eutectic solvent citric acid-PEG600.

[0025] Figure 4 The FTIR spectra of citric acid, PEG800 and the eutectic solvent citric acid-PEG800.

[0026] Figure 5 The FTIR spectra of citric acid, PPG200 and the eutectic solvent citric acid-PPG200.

[0027] Figure 6 The FTIR spectra of citric acid, PPG400 and the eutectic solvent citric acid-PPG400.

[0028] Figure 7Figure for extraction effect of different solvents.

[0029] Figure 8 Figure for influence of water content of deep eutectic solvent on extraction effect; wherein a is a figure for extraction effect when water content of deep eutectic solvent is 90%.

[0030] Figure 9 Figure for extraction power curve of four main substances.

[0031] Figure 10 Figure for regression equation of ln[C ∞ / (C ∞ -C)] and extraction time.

[0032] Figure 11 Figure for extraction effect of three reagents.

[0033] Figure 12 Figure for comparison of antioxidant activity of extraction solutions of three reagents.

[0034] Among them, PEG200 is polyethylene glycol with an average molecular weight of 200, PEG400 is polyethylene glycol with an average molecular weight of 400, PEG600 is polyethylene glycol with an average molecular weight of 600, PEG800 is polyethylene glycol with an average molecular weight of 800, PPG200 is polypropylene glycol with an average molecular weight of 200, and PPG400 is polypropylene glycol with an average molecular weight of 400. DETAILED DESCRIPTION

[0035] The technical solutions in the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0036] The methods described in the embodiments of the present application are all conventional methods unless otherwise specified. The materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0037] Citric acid is abbreviated as CA.

[0038] Embodiment 1

[0039] A deep eutectic solvent, the deep eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is citric acid, and the hydrogen bond donor is PPG200; wherein the molar ratio of citric acid to PPG200 is 3:1.

[0040] The preparation method of the above deep eutectic solvent specifically comprises the following steps:

[0041] The raw materials are mixed according to the molar ratio, and then heated at 80 DEG C for 120 min to obtain the eutectic solvent, which is stored in a brown bottle for standby. The viscosity of the prepared eutectic solvent is 3091 mPa s, and the density is 1.241 g / cm 3 .

[0042] The specific process of extracting active ingredients in tea dregs by the eutectic solvent includes the following steps:

[0043] S1, dry the tea dregs to a water content of less than 5 mg / 100 g, crush, and sieve to select a particle size of ≤250 μm to obtain tea dregs powder.

[0044] S2, mix the tea dregs powder with the aqueous solution of the eutectic solvent according to the solid-liquid ratio of 1 g:10 mL to obtain a mixed solution. The volume percentage of the eutectic solvent in the aqueous solution of the eutectic solvent is 70%, that is, the volume percentage of water in the aqueous solution of the eutectic solvent is 30%.

[0045] S3, place the mixed solution into a microwave reactor and microwave-assisted extraction at 30 DEG C for 2 min to obtain an extraction mixture. The microwave power is 100 W.

[0046] S4, centrifuge the extraction mixture at a speed of 4000 r / min for 5 min to obtain a supernatant containing the active ingredients in the tea dregs.

[0047] Example 2

[0048] A eutectic solvent, the eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is citric acid, and the hydrogen bond donor is PPG400; wherein the molar ratio of citric acid and PPG400 is 3:2.

[0049] The preparation method of the eutectic solvent includes the following steps:

[0050] The raw materials are mixed according to the molar ratio, and then heated at 85 DEG C for 180 min to obtain the eutectic solvent, which is stored in a brown bottle for standby. The viscosity of the prepared eutectic solvent is 1948 mPa s, and the density is 1.145 g / cm 3 .

[0051] The specific process of extracting active ingredients in tea dregs by the eutectic solvent includes the following steps:

[0052] S1, dry the tea dregs to a water content of less than 5 mg / 100 g, crush, and sieve to select a particle size of ≤250 μm to obtain tea dregs powder.

[0053] S2, the tea dregs powder and the aqueous solution of the deep eutectic solvent were mixed according to a solid-liquid ratio of 1g:20mL to obtain a mixed solution. The volume percentage of the deep eutectic solvent in the aqueous solution of the deep eutectic solvent was 70%, that is, the volume percentage of water in the aqueous solution of the deep eutectic solvent was 30%.

[0054] S3, the mixed solution was placed into a microwave reactor and subjected to microwave-assisted extraction at 50 DEG C for 4min to obtain an extraction mixture. The power of the microwave-assisted extraction was 300W.

[0055] S4, the extraction mixture was centrifuged at a speed of 8000r / min for 5min to obtain a supernatant containing the active ingredients in the tea dregs.

[0056] Example 3

[0057] A deep eutectic solvent, which is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is citric acid, and the hydrogen bond donor is PEG200; wherein the molar ratio of citric acid to PEG200 is 1:1.

[0058] The preparation method of the above deep eutectic solvent, specifically comprising the following steps:

[0059] After the raw materials were mixed according to the molar ratio, they were heated at 90 DEG C for 240min to obtain the deep eutectic solvent, which was stored in a brown bottle for standby. The viscosity of the prepared deep eutectic solvent was 4687mPa·s, and the density was 1.311g / cm 3 .

[0060] The specific process of extracting active ingredients in tea dregs by using the above deep eutectic solvent includes the following steps:

[0061] S1, the tea dregs were dried to a water content of less than 5mg / 100g, crushed, and sieved to obtain tea dregs powder with a particle size of ≤250μm.

[0062] S2, the tea dregs powder and the aqueous solution of the deep eutectic solvent were mixed according to a solid-liquid ratio of 1g:30mL to obtain a mixed solution. The volume percentage of the deep eutectic solvent in the aqueous solution of the deep eutectic solvent was 30%, that is, the volume percentage of water in the aqueous solution of the deep eutectic solvent was 70%.

[0063] S3, the mixed solution was placed into a microwave reactor and subjected to microwave-assisted extraction at 60 DEG C for 6min to obtain an extraction mixture. The power of the microwave-assisted extraction was 300W.

[0064] S4, the extraction mixture was centrifuged at a speed of 8000r / min for 5min to obtain a supernatant containing the active ingredients in the tea dregs.

[0065] Example 4

[0066] A deep eutectic solvent, the deep eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is citric acid, and the hydrogen bond donor is PEG400; wherein the molar ratio of citric acid, PEG400 is 1:2.

[0067] The preparation method of the above-mentioned deep eutectic solvent, specifically comprising the following steps:

[0068] After mixing the raw materials according to the molar ratio, heating at 95℃ for 300min, the deep eutectic solvent is obtained, and stored in a brown bottle for standby. The viscosity of the prepared deep eutectic solvent is 3278mPa·s, and the density is 1.250g / cm 3 .

[0069] The specific process of extracting active ingredients in tea dregs by the above-mentioned deep eutectic solvent, comprising the following steps:

[0070] S1, dry the tea dregs to a water content of less than 5mg / 100g, crush, and screen to select a particle size of ≤250μm to obtain tea dregs powder.

[0071] S2, mix the tea dregs powder with the aqueous solution of the deep eutectic solvent according to the solid-liquid ratio of 1g:40mL to obtain a mixed solution. Among them, the volume percentage of deep eutectic solvent in the aqueous solution of deep eutectic solvent is 70%, that is, the volume percentage of water in the aqueous solution of deep eutectic solvent is 30%.

[0072] S3, put the mixed solution into a microwave reactor and microwave-assisted extraction at 70℃ for 8min to obtain an extraction mixture; wherein the power of the microwave-assisted extraction is 400W.

[0073] S4, centrifuge the extraction mixture at a speed of 8000r / min for 5min to obtain a supernatant containing the active ingredients in the tea dregs.

[0074] Example 5

[0075] A deep eutectic solvent, the deep eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is citric acid, and the hydrogen bond donor is PEG600; wherein the molar ratio of citric acid, PEG600 is 1:3.

[0076] The preparation method of the above-mentioned deep eutectic solvent, specifically comprising the following steps:

[0077] After mixing the raw materials according to the molar ratio, heating at 100℃ for 360min, the deep eutectic solvent is obtained, and stored in a brown bottle for standby. The viscosity of the prepared deep eutectic solvent is 2863mPa·s, and the density is 1.221g / cm 3 .

[0078] The specific process of extracting active ingredients in tea dregs by the above-mentioned deep eutectic solvent includes the following steps:

[0079] S1, dry the tea dregs to a water content of less than 5 mg / 100 g, crush, and sieve to select tea dreg powder with a particle size of ≤250 μm.

[0080] S2, mix the tea dreg powder with an aqueous solution of the deep eutectic solvent at a solid-liquid ratio of 1 g:50 mL to obtain a mixed solution. The aqueous solution of the deep eutectic solvent contains 70% deep eutectic solvent by volume, i.e., 30% water by volume, i.e., 90% water by volume.

[0081] S3, place the mixed solution into a microwave reactor and perform microwave-assisted extraction at 80℃ for 15 min to obtain an extraction mixture. The power of the microwave-assisted extraction is 500 W.

[0082] S4, centrifuge the extraction mixture at a speed of 8000 r / min for 5 min to obtain a supernatant containing the active ingredients in the tea dregs.

[0083] Example 6

[0084] A deep eutectic solvent, which is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is citric acid, and the hydrogen bond donor is PPG400; wherein the molar ratio of citric acid to PPG400 is 1:1.

[0085] The preparation method of the above-mentioned deep eutectic solvent specifically includes the following steps:

[0086] After mixing the raw materials according to the molar ratio, heat at 85℃ for 180 min to obtain the deep eutectic solvent, which is stored in a brown bottle for standby. The viscosity of the prepared deep eutectic solvent is 1948 mPa·s, and the density is 1.145 g / cm 3 .

[0087] The specific process of extracting active ingredients in tea dregs by the above-mentioned deep eutectic solvent includes the following steps:

[0088] S1, dry the tea dregs to a water content of less than 5 mg / 100 g, crush, and sieve to select tea dreg powder with a particle size of ≤250 μm.

[0089] S2, mix the tea dreg powder with an aqueous solution of the deep eutectic solvent at a solid-liquid ratio of 1 g:20 mL to obtain a mixed solution. The aqueous solution of the deep eutectic solvent contains 50% deep eutectic solvent by volume, i.e., 50% water by volume.

[0090] S3, the mixed solution is placed into a microwave reactor, and microwave-assisted extraction is carried out at 50 DEG C for 4 min, to obtain an extraction mixture. In the microwave-assisted extraction, the power is 200 W.

[0091] S4, the extraction mixture is centrifuged at a speed of 8000 r / min for 5 min, to obtain a supernatant containing the active ingredients in the tea dregs.

[0092] Example 7

[0093] A deep eutectic solvent, which is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is citric acid, and the hydrogen bond donor is PPG400; wherein the molar ratio of citric acid to PPG400 is 1:1.

[0094] The preparation method of the above-mentioned deep eutectic solvent, specifically comprising the following steps:

[0095] After the raw materials are mixed according to the molar ratio, heating is carried out at 85 DEG C for 180 min, to obtain the deep eutectic solvent, which is stored in a brown bottle for standby. The viscosity of the prepared deep eutectic solvent is 1948 mPa·s, and the density is 1.145 g / cm 3 .

[0096] The specific process of extracting active ingredients in tea dregs by using the above-mentioned deep eutectic solvent, comprising the following steps:

[0097] S1, the tea dregs are dried to a water content of less than 5 mg / 100 g, crushed, and sieved to select tea dregs powder with a particle size of ≤250 μm.

[0098] S2, the tea dregs powder is mixed with an aqueous solution of the deep eutectic solvent according to a solid-liquid ratio of 1 g:20 mL, to obtain a mixed solution. In the aqueous solution of the deep eutectic solvent, the volume percentage of the deep eutectic solvent is 30%, i.e. the volume percentage of water in the aqueous solution of the deep eutectic solvent is 70%, and the solid-liquid ratio of the tea dregs powder to the aqueous solution of the deep eutectic solvent is.

[0099] S3, the mixed solution is placed into a microwave reactor, and microwave-assisted extraction is carried out at 30 DEG C for 4 min, to obtain an extraction mixture. In the microwave-assisted extraction, the power is 200 W.

[0100] S4, the extraction mixture is centrifuged at a speed of 8000 r / min for 5 min, to obtain a supernatant containing the active ingredients in the tea dregs.

[0101] Example 8

[0102] A deep eutectic solvent, which is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is citric acid, and the hydrogen bond donor is PPG400; wherein the molar ratio of citric acid to PPG400 is 1:1.

[0103] The preparation method of the above eutectic solvent specifically comprises the following steps:

[0104] After the raw materials are mixed according to the molar ratio, heating is performed at 85℃ for 180min to obtain the eutectic solvent, which is stored in a brown bottle for standby. The viscosity of the prepared eutectic solvent is 1948mPa·s, and the density is 1.145g / cm 3 .

[0105] The specific process of extracting active ingredients in tea dregs by the above eutectic solvent comprises the following steps:

[0106] S1, dry the tea dregs to a water content of less than 5mg / 100g, crush, and sieve to select a particle size of ≤250μm to obtain tea dreg powder.

[0107] S2, mix the tea dreg powder with the aqueous solution of the eutectic solvent according to a solid-liquid ratio of 1g:20mL to obtain a mixed solution. The volume percentage of the eutectic solvent in the aqueous solution of the eutectic solvent is 10%, i.e. the volume percentage of water in the aqueous solution of the eutectic solvent is 90%.

[0108] S3, place the mixed solution into a microwave reactor and perform microwave-assisted extraction at 50℃ for 4min to obtain an extraction mixture. The power of the microwave-assisted extraction is 200W.

[0109] S4, centrifuge the extraction mixture at a speed of 8000r / min for 5min to obtain a supernatant containing the active ingredients in the tea dregs.

[0110] Comparative Example 1

[0111] The difference between this comparative example and Example 2 is that pure water is used instead of 70% eutectic solvent citric acid-PPG200 in S2, the above ratio is a volume ratio, and the rest is consistent with Example 2.

[0112] Comparative Example 2

[0113] The difference between this comparative example and Example 2 is that 70% methanol aqueous solution is used instead of 70% eutectic solvent citric acid-PPG200 aqueous solution in S2, the above ratio is a volume ratio, and the rest is consistent with Example 2.

[0114] Comparative Example 3

[0115] The difference between this comparative example and Example 2 is that the eutectic solvent is composed of citric acid and PEG800; the molar ratio of citric acid to PEG800 is 1:1, and the rest is consistent with Example 2.

[0116] Since the eutectic solvent prepared in Example 2 has the lowest viscosity under the same conditions, the eutectic solvent prepared in Example 2 is taken as a reference below.

[0117] Comparative Example 4

[0118] The difference between this comparative example and Example 2 is only that the volume percentage of water in the aqueous solution of the eutectic solvent is 30%, and the rest is consistent with Example 2.

[0119] Comparative Example 5

[0120] The difference between this comparative example and Example 2 is only that the volume percentage of water in the aqueous solution of the eutectic solvent is 10%, and the rest is consistent with Example 2.

[0121] 1Materials and reagents

[0122] The tea dregs were provided by Kunming Sike Tea Trade Co., Ltd., a local tea company in Yunnan. After being ground into powder, the tea dregs were sieved through a 60-mesh sieve and stored in a sealed bag in a -20°C refrigerator. The main reagents and instruments used in the present application are shown in Tables 1 and 2.

[0123] Table 1 Main reagent information

[0124] Reagent name Use Purity Company Acetonitrile Mobile phase Mass spectrometry pure Merck, Germany Ammonium formate Mobile phase Mass spectrometry pure Thermo Formic acid Mobile phase Mass spectrometry pure Sigma Methanol Preparation Analytically pure Tianjin Damao Chemical Reagent Factory Polyethylene glycol 200, PEG 200 Preparation ＞98％ Ron reagent Polyethylene glycol 400, PEG 400 Preparation ＞98％ Ron reagent Polyethylene glycol 600, PEG 600 Preparation ＞98％ Ron reagent Polyethylene glycol 800, PEG 800 Preparation ＞98％ Ron reagent Polypropylene glycol 200, PPG 200 Preparation ＞98％ Ron reagent Polypropylene glycol 400, PPG 400 Preparation ＞98％ Ron reagent Citric acid Preparation ＞95％ Ron reagent

[0125] Table 2 Main instrument information

[0126]

[0127]

[0128] 2Determination of physicochemical properties

[0129] 2.1 Viscosity determination

[0130] The eutectic solvent stored in a brown bottle was placed in a 40°C water bath for 1 hour. The viscosity was measured using an NDJ-5S viscometer (Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.). The appropriate viscometer rotor was selected, and the rotor was placed in the eutectic solvent until it was submerged to the calibration line. The viscometer was started, and the torque was between 30 and 80, and the viscosity no longer changed. The viscosity of the eutectic solvent was recorded. After measurement, the rotor was removed and cleaned with distilled water. Each eutectic solvent was measured three times, and the average value was taken.

[0131] 2.2 Density

[0132] After the prepared DESs were placed in a 40°C water bath for 1 hour, 10 mL of DES was accurately measured in a graduated cylinder, and the mass of the graduated cylinder and the total mass of the graduated cylinder and the DES were weighed. The density of the used DES was calculated according to the formula, and the specific calculation formula is as follows:

[0133] p = (m 总 -m0) / v

[0134] where p represents the density of DES, g / cm 3 ; m0represents the mass of the cylinder, g; m 总 represents the total mass of the cylinder and DES, g; V represents the volume of DES, cm 3 .

[0135] 2.3 Fourier infrared spectrum

[0136] The absorption functional groups of citric acid, polymer and DES synthesized therefrom were determined by infrared spectrum to characterize the synthesis of DES. The specific operation procedure is as follows. Since these substances do not contain water, the infrared spectrum scanning can be directly determined by KBr tabletting method. The wavelength scanning range is 4000cm -1 ~ 5000cm -1 ; the scanning number is 20; and the apodization function is Happ-Genze.

[0137] 2.4 Optimization of extraction conditions

[0138] 2.4.1 Extraction of active ingredients

[0139] Since the viscosity of the eutectic solvent is high, the high viscosity DES will reduce the mass transfer efficiency. Adding an appropriate amount of water during the experiment helps to reduce the viscosity of the DES, which is conducive to subsequent extraction. In order to preliminarily select the best DES, 0.5 g of tea dregs was weighed, dried and crushed and sieved, and then added to a test tube containing 5 mL of DES aqueous solution with a water content of 30%, vortexed and mixed, and then subjected to microwave-assisted extraction. The extraction conditions were as follows: time 6 min, power 300 W, and temperature 40℃. After extraction, the extraction liquid was centrifuged, and then the supernatant was diluted 100 times with 70% acetonitrile water and filtered through a 0.22 μm organic membrane before being used for UPLC-QQQ-MS / MS analysis.

[0140] 2.4.2 Selection of eutectic solvent

[0141] Comparative Examples 1 to 3, and Examples 1 to 5, as the extraction solvent, except for Comparative Example 1, the rest are 70% water solution by volume. Comparative Examples 8 extraction solvents were compared in terms of the extraction effect on 21 compounds with high content in tea dregs. The best DES was selected for subsequent experiments.

[0142] 2.4.3 Determination of water content of eutectic solvent

[0143] DES has a larger viscosity than organic solvents, and adding a certain amount of water can reduce its viscosity and polarity. The water content has a greater impact on the extraction effect of the eutectic solvent. The extraction effect of DES with different water contents was investigated to determine the optimal water content of DES. The water contents were 10%, Comparative Example 5, Comparative Example 4, 70%, Example 7, and Example 8.

[0144] 2.4.4 Orthogonal experiment

[0145] The main factors affecting microwave-assisted extraction are microwave power, microwave time, and microwave temperature. In the solid-liquid extraction process, the solid-liquid ratio is an important factor that determines the extraction effect. Ultrasonic-assisted extraction mainly investigates the extraction time, microwave power, and extraction temperature. Orthogonal experiments were designed to optimize them, and the specific parameters are shown in Table 3. Each experiment was repeated three times.

[0146] Table 3 Factors and levels of orthogonal experiment

[0147]

[0148] 2.4.5 Extraction kinetics

[0149] The extraction of tea compounds by eutectic solvent is essentially a complex multi-substance non-steady-state diffusion process. Fick's first and second laws are applicable to steady-state or non-steady-state diffusion, and Fick's second law gives the relationship between the concentration of diffusing substances and time. Therefore, this study investigated the diffusion process of caffeine, ECG, EGCG, and theanine in eutectic solvent in tea residue at 50°C based on Fick's second law model. The following assumptions were made: (1) tea residue is a uniform spherical particle; (2) the target substance is uniformly distributed inside the tea residue; (3) the powder particles are uniformly distributed in the solvent.

[0150] In[C∞ / (C∞-C)]=kt+b

[0151] In the formula, C∞is the concentration of each substance in the equilibrium state, g / mL; C is the concentration of each substance at a certain temperature and different microwave times, g / mL; k is the rate constant of extraction, and b is the intercept.

[0152] 2.5 Determination of in vitro antioxidant activity

[0153] 2.5.1 DPPH free radical scavenging activity

[0154] To compare the in vitro activity of tea residue extract by eutectic solvent, methanol, and water, 0.05 mL of extract was taken in a test tube, 0.95 mL of 1,1-diphenyl-2-trinitrobenzene hydrazine radical, abbreviated as DPPH, working solution was added, and vortexed. The mixture was placed in the dark for 30 min. After the reaction was completed, the absorbance value A of the reaction solution was measured at 517 nm on a spectrophotometer. 测定The absorbance value A of 0.05 mL of the tea leaf extract of the deep eutectic solvent, methanol and water mixed with 0.95 mL of methanol was measured 对照 The absorbance value A of 0.05 mL of water mixed with 0.95 mL of methanol was measured 空白 The deep eutectic solvent, methanol and water were used as the extraction reagent control, V C The solution was used as the positive control. The DPPH free radical scavenging capacity was calculated as follows:

[0155] DPPH free radical scavenging rate (%) = [A 对照 - (A 测定 - A 空白 )] / A 空白 x 100

[0156] 2.5.2 ABTS free radical scavenging activity

[0157] To compare the in vitro activity of the tea residue extract of the deep eutectic solvent, methanol and water, 0.05 mL of the extract was taken in a reaction tube, 0.85 mL of 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt free radical, abbreviated as ABTS working solution, 0.1 mL of potassium peroxydisulfate application solution were added, vortexed and mixed, and reacted at room temperature for 6 min. After the reaction was completed, the absorbance value A of the reaction solution was measured at 405 nm by a spectrophotometer 测定 The absorbance value A of 0.05 mL of water mixed with 0.95 mL of ABTS working solution was measured 空白 The absorbance value A of 0.05 mL of the tea leaf extract of the deep eutectic solvent, methanol and water mixed with 0.95 mL of anhydrous ethanol was measured 对照 The deep eutectic solvent, methanol and water were used as the extraction reagent control, V C The solution was used as the positive control. The ABTS free radical scavenging capacity was calculated as follows:

[0158] ABTS free radical scavenging rate (%) = [A 对照 - (A 测定 - A 对照 )] / A 空白 x 100

[0159] 2.5.3 Data analysis

[0160] First, the raw data was integrated by the Labsolution software of UHPLC-QQQ-MS / MS to obtain the peak area of each substance. Then, the standard curve was calculated by using the standard substance, the peak area of the target substance was integrated, and the concentration mg / mL of the substance was obtained by substituting the linear equation. According to the weight of the tea powder, the solid-liquid ratio during extraction, the dilution multiple of the extract, etc., the concentration was converted into the content of the target substance in the tea sample mg / kg, DW.

[0161] The results were analyzed by SPSS software, and the p value was calculated to determine whether there was a significant difference between the indicators of each group, p<0.05.

[0162] 3 Results and discussion

[0163] 3.1 Synthesis of deep eutectic solvent

[0164] The hydrogen bond donor and hydrogen bond acceptor are solid-liquid mixtures, which form stable transparent liquids under the action of hydrogen bonds after heating for a period of time. Citric acid as a hydrogen bond donor forms hydrogen bonds with the hydroxyl groups of the polymer. The prepared DES is placed in the dark at room temperature for a week to observe whether a precipitate is formed. If not, the DES can be used for experiments. The composition of the DES used in the present application and its physicochemical properties are shown in Table 4.

[0165] Table 4 Composition of deep eutectic solvent

[0166]

[0167] 3.2 Fourier infrared analysis

[0168] The hydrogen bond donor and hydrogen bond acceptor combine to form a deep eutectic solvent, which is mainly a strong intermolecular interaction. FT-IR spectra can prove that hydrogen bonds are formed between citric acid and the polymer. The Fourier infrared spectrum of the deep eutectic solvent is as follows Figures 1-6 In the FT-IR spectrum of pure citric acid, the three peaks at 1778 cm -1 -1650 cm -1 belong to the stretching vibration of C=O double bond, the peaks at 1416 cm -1 and 1206 cm -1 belong to the coupling of in-plane bending of -OH and stretching vibration of C-O. In the FT-IR spectrum of pure polyethylene glycol, the peak at 2860 cm -1 is the stretching vibration of C-H, the peaks at 1457 cm -1 and 1350 cm -1 belong to the bending vibration of -CH-, the peak at 1098 cm -1 belongs to the stretching vibration of C-O-C, and the peaks at 950 cm -1 and 840 cm -1 belong to the bending vibration of C-O-C. The FT-IR spectrum of pure polypropylene glycol is basically the same as that of polyethylene glycol, and the former has more absorption peaks at 2500 cm -1 -3000 cm -1 , which are attributed to the -CH3 and -CH2- groups in the structure. By comparing the FT-IR spectra of each group of DES and various pure raw materials, the peaks at 3200 cm -1 -3500 cm -1the stretching vibration of -OH, there is a significant red shift in DES, and the peak shape is broadened, the wider the peak, the more hydrogen bonds formed; the stretching vibration of carbonyl group moves from 1778 cm -1 -1650cm -1 to 1730 cm -1 , indicating that the -COOH bond in citric acid and the -OH bond in polymer may form new hydrogen bonds, which indicates that citric acid and polymer form stable DES.

[0169] 3.3 Screening of deep eutectic solvent

[0170] Water, 70% methanol solution, 70% DES-1 solution, 70% DES-2 solution, 70% DES-3 solution, 70% DES-4 solution, 70% DES-5 solution, and 70% DES-6 solution were used as extraction solvents. The above proportions are all volume ratios. According to the previous experimental results, the extraction effect of 21 components with high content in tea dregs was used as the basis, including four types of substances, amino acids, alkaloids, catechins, and flavonoids. The extraction effect is as follows: Figure 7As shown, the extraction effect of the eutectic solvent prepared by the embodiment of the present application is obviously higher than that of the traditional extraction solvent water and methanol. The viscosity relationship is 70% aqueous solution of CA-PPG400 < 70% aqueous solution of CA-PEG600 < 70% aqueous solution of CA-PPG200 < 70% aqueous solution of CA-PEG400 < 70% aqueous solution of CA-PEG200 < 70% aqueous solution of CA-PEG800. The extraction effect of the 70% aqueous solution of CA-PPG400 is the best, and the total extraction amount is as high as 22760.7 mg / kg, the total extraction amount of the 70% aqueous solution of CA-PPG200 is 17254.6 mg / kg, the total extraction amount of the 70% aqueous solution of CA-PEG600 is 18367.3 mg / kg, and the total extraction amount of the 70% aqueous solution of CA-PEG200 is 19389.2 mg / kg. By comparing the extraction effect and the viscosity, it is not difficult to find that the extraction effect of the DES with high viscosity is lower than that of the DES with low viscosity, that is, the viscosity and the extraction effect are inversely proportional. Among the four types of chemical components investigated in the present application, the extraction effect of the amino acid type is the best, and the content of theanine is the highest. The extraction effect of theanine is in the range of 55016.16 mg / kg-130484.14 mg / kg. Among them, the extraction effect of the 70% aqueous solution of citric acid-PPG400 is 129352.14 mg / kg, and the extraction effect of the 70% aqueous solution of citric acid-PEG200 is 130484.14 mg / kg. The extraction effect of the 70% aqueous solution of citric acid-PPG400 and the 70% aqueous solution of citric acid-PEG200 on theanine is the best, which is 2.4 times the extraction effect of water and methanol. It may be due to the fact that the pKa of theanine is 2.24, which is more easily extracted by an acidic solution. In summary, the aqueous solution of citric acid-PPG400 is finally used as the extraction reagent, and the extraction conditions are optimized.

[0171] 3.3 Extraction condition optimization results

[0172] 3.3.1 Determination of water content of eutectic solvent

[0173] Studies have shown that the presence of water can reduce the viscosity of DES by 10-200 times. The present application investigates the effect of water content of 10%-90% on the extraction efficiency of CA-PPG400. The extraction effects of different water contents are as follows: Figure 8As shown, the extraction effect of the eutectic solvent aqueous solution presents a trend of first increasing and then decreasing with the increase of the water content. The extraction effect is best when the water content is 30%, which is 105992.6 mg / kg; and the extraction effect is second when the water content is 90%, which is 94481.4 mg / kg. When the water content increases from 10% to 30%, the extraction rate increases significantly, and this phenomenon may be that the increase of the water content reduces the viscosity of the DES, accelerates the mass transfer effect of the tested substance, and increases the extraction rate of the tested substance. When the water content increases from 50% to 90%, the extraction rate decreases significantly, and this may be that the high water content may destroy the microstructure of the DES, thereby weakening the interaction between the DES and the solute and reducing the extraction efficiency; or the high water content changes the polarity of the DES, and the dissolution effect of the tested substance under the polarity condition becomes poor, thereby reducing the extraction efficiency. Finally, the eutectic solvent of citric acid-PPG400 with a water content of 30% is used to extract the substances in the tea dregs.

[0174] 3.3.2 Orthogonal experiment optimization

[0175] The present application takes more than twenty compounds in tea dregs as indexes, selects the best microwave-assisted extraction process conditions as the purpose, adopts L9(3 4 ) design method, and takes the total amount of the extracted compounds as the index. Important factors such as microwave power, microwave time, microwave temperature and solid-liquid ratio are optimized. As shown in Table 5, within the parameter range designed in the present study, the influence on the extraction effect of 21 compounds in tea dregs is D > C > B > A, that is, microwave power > solid-liquid ratio > microwave temperature > microwave time, and the corresponding best process is A3B3C1D2, but considering the time cost and the small influence of time on the extraction effect, and referring to the overall effect, the final best process is determined as A2B3C1D2, that is, the time is 15 min, the temperature is 70 DEG C, the power is 300 W, and the solid-liquid ratio is 1 g:30 mL.

[0176] Table 5 Orthogonal experiment results

[0177]

[0178]

[0179] 3.3.3 Extraction kinetics analysis

[0180] Considering the industrialization of the extraction process, the extraction kinetics of the DES on four main compounds is studied. Under the conditions of microwave temperature 50 DEG C, ultrasonic power 300 W and solid-liquid ratio 1:30, the contents of caffeine, catechin gallate, epicatechin gallate and theanine are determined at the microwave extraction time of 5 min-36 min, and the time is taken as the horizontal coordinate, and the content of the substance determined at the time point is taken as the vertical coordinate to plot a graph. Figure 9As shown, the concentration of the substances in the extract increased with the increase of the microwave time, and the extraction rate increased rapidly from 0 min to 15 min, and then the increasing speed became slow, but the time and the substance concentration were not linearly related. The extraction could be divided into three steps, the solvent adhered to the cell wall of the tea leaves, promoted the contact between the solute and the solvent, and thus realized the rapid extraction; the solute diffused into the solvent, the microwave-assisted cavitation released sufficient energy and accelerated the process, and then the extraction reached equilibrium.

[0181] The kinetic behavior of the microwave extraction method for extracting active compounds from tea leaves was evaluated by using the second Fick's law, the concentrations of the four substances were substituted into the formula for kinetic fitting, as shown in the following formula: Figure 10 The relationship between the time and ln(C ∞ / C ∞ -C) was plotted as shown in the following figure, the correlation coefficient R 2 was greater than 0.8, indicating that there was a reliable linear relationship between the time and ln(C ∞ / C ∞ -C), and the kinetic model was consistent with the experimental phenomenon. Among them, the regression equation between the time and ln(C ∞ / C ∞ -C) of caffeine was y=0.0552x+0.2261, R 2 was 0.9435; the regression equation between the time and ln(C ∞ / C ∞ -C) of catechin gallate was y=0.0442x+0.3628, R 2 was 0.99; and the regression equation between the time and ln(C ∞ / C ∞ -C) of theanine was y=0.007x+0.5004.

[0182] 3.3.4 In vitro antioxidant activity

[0183] DPPH free radical scavenging capacity is one of the indicators for measuring the antioxidant activity of substances, DPPH exists in the form of free radicals in organic solution, has a maximum absorption peak at a wavelength of 517 nm, and after the free radical scavenger captures DPPH, the absorbance value of the solution at the maximum absorbance wavelength will decrease, and the lower the absorbance value, the higher the antioxidant activity, so as to evaluate the antioxidant capacity of the solution.

[0184] Figure 11The results showed that the content of catechin in methanol and DES extraction solution was significantly higher, but the content of theanine in DES extraction solution was significantly higher than that in pure water and methanol. The antioxidant capacity of the extraction solution was determined by DPPH method, and the DPPH scavenging capacity of water, methanol and DES tea extraction solution under the optimal process of microwave assisted extraction was compared. When the free radical scavenging rate was greater than 90% or less than 5%, the determination result was not accurate, so the extraction solution was diluted 100 times for the determination of free radical scavenging rate, and the results were shown in Table 2. Figure 12 The DPPH scavenging capacity of DES extraction solution was significantly higher than that of water extraction solution. By comparing the relationship between DPPH free radical scavenging capacity and the content of four substances, it can be seen that the DPPH free radical scavenging capacity and the content of catechin tend to be consistent. It is proved that the structure of DES extract can be well maintained, and the antioxidant activity of phenolic substances can be well played.

[0185] ABTS free radical scavenging capacity can be used to determine the antioxidant capacity of substances. ABTS charged particles have a maximum absorption peak at 405 nm. The addition of antioxidants will inhibit the generation of ABTS charged particles, making the system fade, and the absorbance at 405 nm decreases. The degree of decrease in absorbance can reflect the ability of the sample to scavenge ABTS free radicals. The ABTS scavenging capacity of water, methanol and DES tea extraction solution was compared, as shown in Table 3. Figure 12 Under the condition of diluting the extraction solution 100 times, the ABTS scavenging capacity of DES extraction solution was significantly higher than that of water extraction solution.

[0186] The application establishes a method for efficiently extracting active ingredients in tea dregs by designing a series of DES and combining microwave-assisted extraction. The DES used in the application is structurally characterized by Fourier infrared spectroscopy, and the viscosity and density of the DES are determined, and the viscosity and extraction effect show a negative correlation. By comparing the extraction effect, it is found that the extraction effect of CA-PPG400 with a molar ratio of 1:1 is optimal. Through single factor and orthogonal test optimization of extraction process, under the conditions of DES water content 30%, extraction time 15 min, extraction temperature 70 DEG C, microwave power 300 W, solid-liquid ratio 1:30 g / mL, the extraction effect is the best, and the extraction rates of caffeine, theanine and EGCG are 31340.7 mg / kg, 99352.1 mg / kg and 11289.5 mg / kg respectively. The results of extraction kinetics research show that the process of extracting caffeine, catechin and theanine by DES conforms to Fick's second law. The results of in vitro antioxidant activity test show that the activity of DES extract is equivalent to that of methanol extract, but far higher than that of pure water extract. Therefore, the new eutectic solvent of citric acid-PPG400 is a kind of green solvent for efficiently extracting active ingredients in tea dregs, and under the support of microwave-assisted extraction technology, it can recover the main active ingredients in tea dregs at high yield, and provides a method for the comprehensive utilization of tea dregs.

[0187] Although preferred embodiments of the application have been described, those skilled in the art will be able to make additional changes and modifications without departing from the spirit and scope of the application. Therefore, the appended claims are intended to cover all such changes and modifications that fall within the scope of the application.

[0188] Obviously, those skilled in the art can make various modifications and variations to the application without departing from the spirit and scope of the application. Thus, if these modifications and variations of the application fall within the scope of the claims of the application and their equivalent technologies, the application also intends to include these modifications and variations.

Claims

1. The application of a eutectic solvent in the extraction of active ingredients from tea residue, characterized in that, The eutectic solvent is formed by hydrogen bond acceptors and hydrogen bond donors bonded together by hydrogen bonding forces. The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3~1:1~3; The hydrogen bond acceptor is citric acid; the hydrogen bond donor is polypropylene glycol with an average molecular weight of 200-400 or polyethylene glycol with an average molecular weight of 200-800. The eutectic solvent is prepared according to the following steps: the raw materials are mixed according to the molar ratio and reacted at 80℃~100℃ for 120min~360min to obtain the eutectic solvent; The active ingredients are amino acids, alkaloids, catechins, and flavonoids.

2. The application according to claim 1, characterized in that, The specific process for extracting active ingredients from tea residue is as follows: The tea residue powder was mixed with an aqueous solution of the eutectic solvent and microwave-assisted extraction was performed for 2 to 15 minutes. After centrifugation, a supernatant containing the active ingredients was obtained.

3. The application according to claim 2, characterized in that, The volume percentage of the eutectic solvent in the aqueous solution is 10% to 90%.

4. The application according to claim 2, characterized in that, The ratio of tea residue powder to the aqueous solution of the eutectic solvent is 1g:10mL~50mL.

5. The application according to claim 2, characterized in that, The centrifugation conditions are: centrifugation at a speed of 4000 r / min to 8000 r / min for 3 min to 5 min.

6. The application according to claim 2, characterized in that, The tea residue powder has a moisture content of less than 5 mg / 100g and a particle size of ≤250 μm.

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