Method for screening eutectic solvent based on Hansen solubility parameter and method for extracting triterpene acid in plant

Abstract

The invention provides a method for screening eutectic solvents based on Hansen solubility parameters and a method for extracting triterpene acid in plants, and belongs to the technical field of plant source active ingredient extraction. According to the method, target extract standard substances are placed in multiple groups of organic solvents with different Hansen solubility respectively, solubility data of the target extract standard substances in the organic solvents are obtained, then Hansen three-dimensional solubility parameter balls are obtained, and on the basis, the dissolution radius and the dissolution center of the target extract standard substances can be obtained respectively; therefore, a candidate hydrogen bond donor and a candidate hydrogen bond receptor can be obtained through screening, and a candidate eutectic solvent can be prepared; and then extracting a to-be-extracted sample, and screening by taking the extraction amount of the target extract in the to-be-extracted sample as a reference to obtain the target eutectic solvent. The eutecticevaporate solvent is screened based on Hansen solubility parameters, efficient screening of the eutecticevaporate solvent can be achieved, and low efficiency of a traditional trial and error method is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction of plant-derived active ingredients, and particularly to a method for screening deep eutectic solvents based on Hansen solubility parameters and a method for extracting triterpenic acids from plants. Background Art

[0002] Triterpenic acids, especially pentacyclic triterpenic acids, are important plant-derived active ingredients widely present in herbal medicines. They have been proven to have various biological functions, including antibacterial, anti-tumor, and anti-inflammatory effects, and have broad development and application prospects in the fields of medicine, health foods, and cosmetics. However, the existing triterpenic acid extraction technologies mainly rely on volatile organic solvents represented by ethanol. Although these volatile organic solvents have relatively ideal extraction effects, there are undeniable disadvantages in their use process. On the one hand, volatile organic solvents may remain in the final products, posing a potential threat to food safety; on the other hand, their volatility and toxicity will cause environmental pollution and increase the ecological burden. These problems have prompted researchers to search for more green, safe, and efficient alternative extraction technologies.

[0003] Natural deep eutectic solvents have become a research hotspot in the field of extraction of plant-derived active ingredients in recent years due to their characteristics of being green, environmentally friendly, biodegradable, low toxicity, and easy preparation. Compared with traditional solvents, deep eutectic solvents have high designability and can optimize the dissolution performance through combinations of different components and ratios to meet the extraction requirements of specific target compounds. However, in the development and application of deep eutectic solvents, the traditional trial-and-error method requires preparing a large number of deep eutectic solvents as candidate solvents and then testing each of these large numbers of candidate solvents one by one, which is not only time-consuming and laborious but also wastes a large amount of resources. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for screening deep eutectic solvents based on Hansen solubility parameters and a method for extracting triterpenic acids from plants. The present invention screens deep eutectic solvents based on Hansen solubility parameters, which can achieve efficient screening of deep eutectic solvents and avoid the inefficiency of the traditional trial-and-error method.

[0005] To achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0006] The present invention provides a method for screening deep eutectic solvents based on Hansen solubility parameters, comprising the following steps:

[0007] Respectively placing standard products of target extracts in multiple groups of organic solvents with different Hansen solubilities to obtain solubility data of the standard products of the target extracts in the organic solvents;

[0008] Based on the solubility data, a Hansen three-dimensional solubility parameter sphere is obtained, and according to the Hansen three-dimensional solubility parameter sphere, the dissolution radius and dissolution center of the target extract standard are obtained respectively;

[0009] Candidate hydrogen bond donors and candidate hydrogen bond acceptors are screened according to the dissolution radius and dissolution center;

[0010] Candidate deep eutectic solvents are prepared using the candidate hydrogen bond donors and candidate hydrogen bond acceptors;

[0011] The sample to be extracted is extracted using the candidate deep eutectic solvent, and based on the extraction amount of the target extract in the sample to be extracted, the target deep eutectic solvent is screened.

[0012] Preferably, the Hansen solubility differences between any two groups of organic solvents in the multiple groups of organic solvents satisfy at least one of the following conditions: the dispersion force parameter δ d differs by ≥2, the polarity parameter δ p differs by ≥4, and the hydrogen bond parameter δ h differs by ≥4;

[0013] The solubility data includes the dispersion force parameter δ d , the polarity parameter δ p , the hydrogen bond parameter δ h and the dissolution fraction of the target extract standard in the organic solvent. The acquisition method of the dissolution fraction includes: when the target extract standard is completely dissolved in the organic solvent under predetermined conditions, the dissolution fraction is recorded as 1, otherwise the dissolution fraction is recorded as 0; the preset conditions include: the dosage ratio of the target extract standard to the organic solvent is 1 mg: 3 - 10 mL; the temperature is 30 - 60 °C, the ultrasonic power is 200 - 400 W, and the time is 5 - 20 min.

[0014] Preferably, the acquisition method of the Hansen three-dimensional solubility parameter sphere includes: inputting the solubility data into the HSPiP software to obtain the Hansen three-dimensional solubility parameter sphere.

[0015] Preferably, the sample to be extracted includes a plant sample; the target extract includes triterpenic acid; the candidate hydrogen bond donors and candidate hydrogen bond acceptors are from natural plant components.

[0016] Preferably, the triterpenic acid includes pentacyclic triterpenic acid; the natural plant components include one or several of acidic natural plant components, alcoholic natural plant components, phenolic natural plant components, saccharide natural plant components, aldehyde natural plant components, and ketone natural plant components.

[0017] Preferably, the pentacyclic triterpenic acid includes corosolic acid, maslinic acid or ursolic acid; each of the multiple groups of organic solvents is acetic acid, acetone, acetonitrile, 1,4-butanediol, 1-n-butanol, n-butyl acetate, chloroform, cyclohexane, cyclohexanol, diacetone alcohol, diethyl ether, N,N-dimethylformamide, 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, hexane, methanol, 1-pentene, 2-propanol, acrylate, tetrachloroethylene, tetrahydrofuran; the plant-derived natural components include acetic acid, isoleucine, leucine, proline, methionine, sorbic acid, menthol, phenylalanine, glycine, salicylic acid, pyruvic acid, glutamic acid, lauric acid, nerol, geraniol, thymol, vanillin, linalool, fructose, lactic acid, d-camphor, citral, malonic acid, carvone, citric acid, coumarin, malic acid, glycerol, glycolic acid, oxalic acid, xylitol and tartaric acid.

[0018] Preferably, the dissolution radius R 0 is 10.5 - 11.8, and the dispersion force parameter δ corresponding to the dissolution center d = 15.51 - 16.51, the polarity parameter δ p = 6.51 - 7.92, the hydrogen bond parameter δ h = 12.53 - 14.26; the candidate eutectic solvents include the combination of menthol and thymol, the combination of menthol and geraniol, the combination of menthol and lauric acid, the combination of menthol and linalool, the combination of menthol and pyruvic acid, the combination of menthol and carvone, the combination of menthol and lactic acid, the combination of thymol and geraniol, the combination of thymol and linalool, the combination of thymol and carvone, and the combination of thymol and lactic acid.

[0019] Preferably, the target eutectic solvent is the combination of thymol and linalool, and the molar ratio of thymol to linalool in the target eutectic solvent is 1 - 2:1.

[0020] The present invention provides a method for extracting triterpenic acid from plants, comprising the following steps:

[0021] Screen the target eutectic solvent according to the method described in the above technical solution;

[0022] Introduce nanobubbles into the target eutectic solvent to obtain a eutectic solvent containing nanobubbles;

[0023] Mix the plant sample to be extracted with the eutectic solvent containing nanobubbles, and perform extraction under ultrasonic conditions to obtain the triterpenic acid.

[0024] Preferably, the method for introducing nanobubbles into the target deep eutectic solvent includes: introducing nitrogen gas into the target deep eutectic solvent for pressurization, and then decompression. The pressurization and decompression steps are repeated to form nanobubbles, and the deep eutectic solvent containing nanobubbles is obtained; the pressure of the pressurization is 0.3 - 0.6 MPa, and the number of cycles of pressurization and decompression is 20 - 60 times;

[0025] The extraction conditions include: the temperature is 40 - 60 °C, the time is 30 - 70 min, and the ultrasonic power is 160 - 320 W.

[0026] The present invention provides a method for screening deep eutectic solvents based on Hansen solubility parameters, including the following steps: placing target extract standards in multiple groups of organic solvents with different Hansen solubilities to obtain the solubility data of the target extract standards in the organic solvents; obtaining a Hansen three-dimensional solubility parameter sphere according to the solubility data, and respectively obtaining the dissolution radius and dissolution center of the target extract standards according to the Hansen three-dimensional solubility parameter sphere; screening candidate hydrogen bond donors and candidate hydrogen bond acceptors according to the dissolution radius and dissolution center; preparing candidate deep eutectic solvents using the candidate hydrogen bond donors and candidate hydrogen bond acceptors; extracting the sample to be extracted using the candidate deep eutectic solvents, and screening the target deep eutectic solvent based on the extraction amount of the target extract in the sample to be extracted. The present invention assists in selecting hydrogen bond donors and hydrogen bond acceptors based on Hansen solubility parameters to prepare deep eutectic solvents, which can achieve efficient screening of deep eutectic solvents and avoid the inefficiency of traditional trial-and-error methods. On this basis, the present invention further introduces the synergistic effect of nanobubbles and ultrasonic waves to extract triterpenic acids from plants, which can significantly improve the mass transfer efficiency and extraction efficiency. The results of the examples show that taking the extraction of corosolic acid from the outer seed coat of pecan as an example, the extraction efficiency of the method of the present invention is increased by 278.26% compared with the traditional ultrasonic-assisted ethanol extraction method, and it has both environmental friendliness and high efficiency, and is suitable for the development and utilization of active ingredients (such as plant active ingredients). Description of the Drawings

[0027] Figure 1 It is a diagram of the dissolution situation of corosolic acid in some organic solvents;

[0028] Figure 2 It is a diagram of the results of the Hansen three-dimensional solubility parameter sphere of corosolic acid;

[0029] Figure 3 It is a diagram of the results of the influence of the number of compression cycles and nitrogen pressure on the average size and size intensity of nanobubbles;

[0030] Figure 4It is a graph showing the effects of ultrasonic temperature, ultrasonic power, and ultrasonic time on the extraction amount of corosolic acid in the outer seed coat of pecans. Detailed implementation manners

[0031] The present invention provides a method for screening deep eutectic solvents based on Hansen solubility parameters, comprising the following steps:

[0032] Placing the target extract standard in multiple groups of organic solvents with different Hansen solubilities to obtain the solubility data of the target extract standard in the organic solvents;

[0033] Obtaining a Hansen three-dimensional solubility parameter sphere according to the solubility data, and respectively obtaining the dissolution radius and dissolution center of the target extract standard according to the Hansen three-dimensional solubility parameter sphere;

[0034] Screening candidate hydrogen bond donors and candidate hydrogen bond acceptors according to the dissolution radius and dissolution center;

[0035] Preparing candidate deep eutectic solvents using the candidate hydrogen bond donors and candidate hydrogen bond acceptors;

[0036] Using the candidate deep eutectic solvents to extract the sample to be extracted, and screening the target deep eutectic solvents based on the extraction amount of the target extract in the sample to be extracted.

[0037] Aiming at the inefficiency of screening deep eutectic solvents by the traditional trial-and-error method in the related art, the method provided by the present invention realizes the efficient screening of deep eutectic solvents. The present invention mainly screens natural deep eutectic solvents, and the natural deep eutectic solvents are specifically hydrophobic natural deep eutectic solvents. The method of the present invention will be described in detail below.

[0038] The present invention places the target extract standard in multiple groups of organic solvents with different Hansen solubilities to obtain the solubility data of the target extract standard in the organic solvents. As an embodiment of the present invention, the target extract may include triterpenic acids; the triterpenic acids may include pentacyclic triterpenic acids; the pentacyclic triterpenic acids may include corosolic acid, maslinic acid, or ursolic acid. In the embodiments of the present invention, corosolic acid is specifically used as an example to illustrate the feasibility of the method of the present invention. As an embodiment of the present invention, the Hansen solubility difference between any two groups of organic solvents in the multiple groups of organic solvents satisfies at least one of the following conditions: the dispersion force parameter δ d differs by ≥2, for example, it may differ by 2 - 5, specifically, it may differ by 2, 3, 4, or 5; the polarity parameter δ p differs by ≥4, for example, it may differ by 4 - 10, specifically, it may be 4, 5, 6, 7, 8, 9, or 10; the hydrogen bond parameter δ hThe difference is ≥ 4, for example, it can be 4 - 10, specifically it can be 4, 5, 6, 7, 8, 9 or 10; wherein the dispersion force parameter δ d , the polarity parameter δ p and the hydrogen bond parameter δ h are Hansen solubility parameters. According to the HSP theory, the solubility properties of any substance are defined by a set of values in a 3D solubility space, which can also be called the so-called "Hansen space", and the set of values includes the above three values (i.e., the dispersion force parameter δ d , the polarity parameter δ p and the hydrogen bond parameter δ h ). In a 3D graphical illustration, each substance has a 3D sphere in the Hansen space, where δ d , δ p and δ h are the centers of the 3D sphere and the dissolvable range of each substance is the radius (R 0 ) of the 3D sphere. If the δ d , δ p and δ h of the solvent are located inside the 3D sphere of the solute, the solute has good solubility in the solvent, and the closer to the center of the 3D sphere of the solute, the better the solubility of the solute in it. As an embodiment of the present invention, taking corosolic acid as an example of the target extract (i.e., the target extract standard is the corosolic acid standard), each group of the multiple groups of organic solvents can be acetic acid, acetone, acetonitrile, 1,4-butanediol, 1-butanol, n-butyl acetate, chloroform, cyclohexane, cyclohexanol, diacetone alcohol, diethyl ether, N,N-dimethylformamide, 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, hexane, methanol, 1-pentene, 2-propanol, acrylate, tetrachloroethylene, tetrahydrofuran, that is, a total of 23 groups of organic solvents.

[0039] As an embodiment of the present invention, the solubility data may include the dispersion force parameter δ d , the polarity parameter δ p , the hydrogen bond parameter δ hThe dissolution fraction of the target extract standard in the organic solvent. The method for obtaining the dissolution fraction may include: when the target extract standard is completely dissolved in the organic solvent under predetermined conditions, the dissolution fraction is recorded as 1; otherwise, the dissolution fraction is recorded as 0 (i.e., incomplete dissolution is recorded as 0). As an embodiment of the present invention, the predetermined conditions include: the dosage ratio of the target extract standard to the organic solvent may be 1 mg: 3 - 10 mL, specifically 1 mg: 4 mL, 1 mg: 5 mL or 1 mg: 6 mL; the temperature may be 30 - 60 °C, specifically 45 °C, 50 °C or 55 °C; the ultrasonic power may be 200 - 400 W, specifically 200 W, 250 W or 300 W; the time may be 5 - 20 min, specifically 5 min, 8 min or 10 min. In the embodiment of the present invention, taking the target extract as corosolic acid as an example, specifically, the corosolic acid standard (1 mg) is respectively placed in the above 23 groups of organic solvents (5 mL), ultrasonicated for 5 min at a temperature of 50 °C and a power of 200 W, and then cooled to room temperature. Observe the dissolution status of corosolic acid, where complete dissolution is recorded as 1 and the rest are recorded as 0.

[0040] After obtaining the solubility data of the target extract standard in the organic solvent, the present invention obtains the Hansen three-dimensional solubility parameter sphere according to the solubility data, and obtains the dissolution radius and dissolution center of the target extract standard according to the Hansen three-dimensional solubility parameter sphere. As an embodiment of the present invention, the method for obtaining the Hansen three-dimensional solubility parameter sphere may include: inputting the solubility data into the HSPiP software to obtain the Hansen three-dimensional solubility parameter sphere. In the embodiment of the present invention, the three Hansen solubility parameters of the organic solvent correspond to the three coordinates of the Hansen three-dimensional solubility parameter sphere. When drawing the Hansen three-dimensional solubility parameter sphere of the target substance, the organic solvents with a dissolution fraction recorded as 1 are inside the sphere, and the organic solvents with a dissolution fraction recorded as 0 are outside the sphere. Accordingly, the Hansen three-dimensional solubility parameter sphere of the target substance can be drawn, and then the dissolution radius and dissolution center can be determined. In the embodiment of the present invention, input the name of the organic solvent into the HSPiP software, and the three Hansen solubility parameters of the organic solvent will be automatically displayed in the HSPiP software. At the same time, input the dissolution fraction of the target extract standard in the organic solvent into the HSPiP software, and the Hansen three-dimensional solubility parameter sphere can be obtained. As an embodiment of the present invention, taking the target extract as corosolic acid and the organic solvent as the above 23 groups of organic solvents as an example, the dissolution radius R of corosolic acid 0 may be 10.5 - 11.8, and the dispersion force parameter δ corresponding to the dissolution center d = 15.51 - 16.51, the polar parameter δp = 6.51 to 7.92, hydrogen bond parameter δ h = 12.53 to 14.26; In the embodiments of the present invention, the dissolution radius R of the corosolic acid 0 is 11.4, and the dispersion force parameter δ corresponding to the dissolution center d = 16.49, polar parameter δ p = 7.37, hydrogen bond parameter δ h = 12.85.

[0041] After obtaining the dissolution radius and dissolution center of the target extract standard, the present invention screens for candidate hydrogen bond donors and candidate hydrogen bond acceptors according to the dissolution radius and dissolution center. As an embodiment of the present invention, the candidate hydrogen bond donors and candidate hydrogen bond acceptors are from plant-derived natural components, and the plant-derived natural components may include one or several of acid plant-derived natural components, alcohol plant-derived natural components, phenolic plant-derived natural components, saccharide plant-derived natural components, aldehyde plant-derived natural components, and ketone plant-derived natural components; the plant-derived natural components may specifically include acetic acid, isoleucine, leucine, proline, methionine, sorbic acid, menthol (specifically L-menthol), phenylalanine, glycine, salicylic acid, pyruvic acid, glutamic acid, lauric acid, nerol, geraniol, thymol, vanillin, linalool, fructose, lactic acid, d-camphor, citral, malonic acid, carvone (specifically (-)-carvone), citric acid, coumarin, malic acid, glycerol, glycolic acid, oxalic acid, xylitol, and tartaric acid, that is, a total of 32 plant-derived natural components, that is, candidate hydrogen bond donors and candidate hydrogen bond acceptors are screened from the above 32 plant-derived natural components. In the embodiments of the present invention, using plant-derived natural components to prepare deep eutectic solvents is green, environmentally friendly, biodegradable, and meets the requirements of sustainable development. In the embodiments of the present invention, taking the target extract as corosolic acid as an example, the method of screening candidate hydrogen bond donors and candidate hydrogen bond acceptors from the above 32 plant-derived natural components may include: calculating the Hansen solubility parameters (i.e., dispersion force parameter δ d , polar parameter δ p , and hydrogen bond parameter δ h ) of the 32 plant-derived natural components respectively according to the group contribution method, specifically, it can be obtained by inputting the SMILES formula of the plant-derived natural components into the HSPiP software for auxiliary calculation, and calculating the distance between each plant-derived natural component and the dissolution center of corosolic acid according to Equation I:

[0042] R a 2 = 4(δ d,j - δ d,i ) 2 +(δ p,j - δ p,i )2 +(δ h,j -δ h,i ) 2 Formula I;

[0043] In Formula I, R a is the distance between the dissolution center of the natural plant-derived component and corosolic acid, δ d,j is the δ of the natural plant-derived component d value, δ d,i is the δ of corosolic acid d value, δ p,j is the δ of the natural plant-derived component p value, δ p,i is the δ of corosolic acid p value, δ h,j is the δ of the natural plant-derived component h value, δ h,i is the δ of corosolic acid h value.

[0044] As an embodiment of the present invention, after obtaining the distance between the dissolution center of each natural plant-derived component and corosolic acid (i.e., R a ), specifically, a natural plant-derived component with R a < R 0 = 11.4 can be selected as a candidate hydrogen bond donor and a candidate hydrogen bond acceptor. It should be noted that the hydrogen bond donor referred to in the present invention is a molecule or group that provides a hydrogen atom during the formation of a hydrogen bond. Usually, it is a hydrogen atom covalently bonded to a strongly electronegative atom (such as oxygen, nitrogen, fluorine). For example, among the above 32 natural plant-derived components, acids, alcohols, phenols, and sugars containing carboxyl and hydroxyl functional groups are hydrogen bond donors; the hydrogen bond acceptor is an atom or molecule that can form a hydrogen bond with a hydrogen bond donor in other molecules or groups by providing a lone pair of electrons. Usually, natural plant-derived components containing functional groups such as carboxyl, hydroxyl, and carbonyl can be used as hydrogen bond acceptors. That is, the above 32 natural plant-derived components can all be used as hydrogen bond acceptors, and other natural plant-derived components except vanillin, carvone, d-camphor, citral, and coumarin can be used as both hydrogen bond donors and hydrogen bond acceptors. In the subsequent formed deep eutectic solvent, the hydrogen bond donor and the hydrogen bond acceptor also participate in the action of these two hydrogen bond networks simultaneously. As an embodiment of the present invention, the candidate hydrogen bond donor can be menthol, and the corresponding candidate hydrogen bond acceptor can be thymol, geraniol, lauric acid, linalool, pyruvic acid, carvone, or lactic acid; the candidate hydrogen bond donor can also be thymol, and the corresponding hydrogen bond acceptor can be geraniol, linalool, carvone, or lactic acid.

[0045] After obtaining the candidate hydrogen bond donors and candidate hydrogen bond acceptors, the present invention prepares a candidate deep eutectic solvent using the candidate hydrogen bond donors and candidate hydrogen bond acceptors. As an embodiment of the present invention, the preparation method of the candidate deep eutectic solvent may include the following steps: mixing the candidate hydrogen bond donors and candidate hydrogen bond acceptors, performing a heat treatment, and then cooling to room temperature to obtain the candidate deep eutectic solvent; the temperature of the heat treatment may be 70-90 °C, specifically 70 °C, 80 °C or 90 °C, and the time may be 20-60 min, specifically 20 min, 30 min, 40 min, 50 min or 60 min. As an embodiment of the present invention, the candidate deep eutectic solvent may include a composition of menthol and thymol, a composition of menthol and geraniol, a composition of menthol and lauric acid, a composition of menthol and linalool, a composition of menthol and pyruvic acid, a composition of menthol and carvone, a composition of menthol and lactic acid, a composition of thymol and geraniol, a composition of thymol and linalool, a composition of thymol and carvone, and a composition of thymol and lactic acid; in each composition, the former is the candidate hydrogen bond donor and the latter is the candidate hydrogen bond acceptor. As an embodiment of the present invention, the molar ratio of the candidate hydrogen bond donor to the candidate hydrogen bond acceptor in the candidate deep eutectic solvent may be 1-2:1; specifically, if the molar ratio of the candidate hydrogen bond donor to the candidate hydrogen bond acceptor is limited to 1:1, a stable candidate deep eutectic solvent cannot be prepared under the above conditions, so the molar ratio of the candidate hydrogen bond donor to the candidate hydrogen bond acceptor can be limited to 2:1, such as the molar ratio of the candidate hydrogen bond donor to the candidate hydrogen bond acceptor in the composition of menthol and lauric acid, the composition of menthol and lactic acid, and the composition of thymol and lactic acid can be 2:1; in addition, in the present invention, based on the traditional organic solvent ethanol, the calculated R a value of ethanol and corosolic acid is 6.84, so for the candidate deep eutectic solvent calculated according to the molar ratio of the candidate hydrogen bond donor to the candidate hydrogen bond acceptor of 1:1, when the R a value is greater than 6, the molar ratio of the candidate hydrogen bond donor to the candidate hydrogen bond acceptor is limited to 2:1 to make the R a value of the obtained candidate deep eutectic solvent and corosolic acid as small as possible, such as the molar ratio of the candidate hydrogen bond donor to the candidate hydrogen bond acceptor in the composition of menthol and linalool, the composition of thymol and linalool, and the composition of thymol and carvone can be 2:1; for other compositions, such as the composition of menthol and thymol, the composition of menthol and geraniol, the composition of menthol and pyruvic acid, the composition of menthol and carvone, and the composition of thymol and geraniol, the molar ratio of the candidate hydrogen bond donor to the candidate hydrogen bond acceptor can be 1:1.

[0046] After obtaining the candidate deep eutectic solvents, the present invention uses the candidate deep eutectic solvents to extract the sample to be extracted, and based on the extraction amount of the target extract in the sample to be extracted, the target deep eutectic solvent is screened out. As an embodiment of the present invention, specifically, the candidate deep eutectic solvent can be mixed with the sample to be extracted for extraction; the sample to be extracted can include plant samples. Taking corosolic acid as the target extract, the sample to be extracted can specifically be the outer seed coat of hickory; the material-liquid ratio of the sample to be extracted to the candidate deep eutectic solvent can be 1 g: 15 - 40 mL, specifically 1 g: 18 mL, 1 g: 20 mL or 1 g: 25 mL. As an embodiment of the present invention, the extraction conditions can include: the temperature can be 40 - 60 °C, specifically 45 °C, 50 °C or 55 °C; the ultrasonic power can be 200 - 400 W, specifically 200 W, 250 W or 300 W; the time can be 30 - 90 min, specifically 50 min, 60 min or 70 min. The present invention screens out the candidate deep eutectic solvent corresponding to the highest extraction amount of the target extract in the sample to be extracted as the target deep eutectic solvent. In the embodiment of the present invention, taking the outer seed coat of hickory as the sample to be extracted, under the conditions of a material-liquid ratio of 1 g: 20 mL, a temperature of 50 °C, an ultrasonic power of 200 W and a time of 60 min, the optimal deep eutectic solvent is screened out with the extraction amount of corosolic acid as the target. Finally, it is determined that the deep eutectic solvent prepared from thymol and linalool (the molar ratio of thymol to linalool is 2:1) has the highest extraction rate of corosolic acid, reaching 9.21 mg / g DW. Therefore, the deep eutectic solvent prepared from thymol and linalool in a molar ratio of 2:1 is selected as the target deep eutectic solvent.

[0047] The present invention provides a method for extracting triterpenic acids from plants, comprising the following steps:

[0048] Screen the target deep eutectic solvent according to the method described in the above technical solution;

[0049] Introduce nanobubbles into the target deep eutectic solvent to obtain a deep eutectic solvent containing nanobubbles;

[0050] Mix the plant sample to be extracted with the deep eutectic solvent containing nanobubbles, and perform extraction under ultrasonic conditions to obtain the triterpenic acid.

[0051] Compared with traditional solvents, deep eutectic solvents have high designability and can optimize their dissolution performance through combinations of different components and ratios to meet the extraction requirements of specific target compounds. However, deep eutectic solvents also have certain limitations. Their high viscosity can lead to a decrease in mass transfer efficiency, thereby affecting the extraction rate and extraction amount of target components. In the present invention, deep eutectic solvents are combined with the nanobubble technology and applied to the extraction of triterpenic acids from plants, enabling the efficient extraction of triterpenic acids. Specifically, the nanobubbles have a diameter less than 200 nm, with an extremely high specific surface area and good stability. Their unique physicochemical properties can significantly improve the mass transfer behavior of the liquid. For example, nanobubbles can effectively reduce the mass transfer barrier caused by the high viscosity of deep eutectic solvents by enhancing the fluidity of the liquid and increasing the surface contact efficiency of active ingredients. In addition, microjets and ultrasonic effects are generated during the rupture of nanobubbles, which can further promote the dissolution and diffusion of solute molecules, thus improving the extraction efficiency. Therefore, based on the Hansen solubility parameter, deep eutectic solvents (specifically natural deep eutectic solvents) are screened in the present invention. On this basis, the mass transfer barrier caused by their high viscosity is overcome by combining with the nanobubble technology, and at the same time, synergistic with the ultrasonic effect, the green and efficient extraction of triterpenic acids can be achieved. This can not only provide technical support for the industrial extraction of triterpenic acids, but also open up a new research direction for the synergistic application of natural deep eutectic solvents and nanobubble technology, promoting their practical application in a wider range of fields. The method of the present invention will be described in detail below.

[0052] After screening and obtaining the target deep eutectic solvent according to the above technical solution, nano-bubbles are introduced into the target deep eutectic solvent to obtain a deep eutectic solvent containing nano-bubbles. As an embodiment of the present invention, the method of introducing nano-bubbles into the target deep eutectic solvent may include: the method of introducing nano-bubbles into the target deep eutectic solvent includes: nitrogen is introduced into the target deep eutectic solvent for pressurization, and then decompression is carried out. The pressurization and decompression steps are repeated to form nano-bubbles, and the deep eutectic solvent containing nano-bubbles is obtained. As an embodiment of the present invention, the pressure of the pressurization may be 0.3 to 0.6 MPa, specifically 0.3 MPa, 0.4 MPa, 0.5 MPa or 0.6 MPa; the decompression is specifically to reduce the pressure to atmospheric pressure; the number of cycles of the pressurization and decompression may be 20 to 60 times, specifically 20 times, 30 times, 40 times, 50 times or 60 times, where one pressurization and one decompression are recorded as one cycle; the preparation process of the nano-bubbles may be carried out at a temperature of 20 to 60 °C, specifically 25 °C, 40 °C or 50 °C. In the embodiment of the present invention, specifically, the target deep eutectic solvent prepared by thymol and linalool in a molar ratio of 2:1 is placed in a headspace bottle, a syringe is inserted into the headspace bottle below the liquid level, and nitrogen is injected into the target deep eutectic solvent through the syringe to make the gas pressure reach 0.3 to 0.6 MPa, and the gas pressure is changed by continuously pushing and pulling the syringe, that is, the nano-bubbles are prepared by decompression and pressurization. As an embodiment of the present invention, the particle size of the nano-bubbles may be 10 to 1000 nm, and further may be 10 to 500 nm.

[0053] After obtaining the deep eutectic solvent containing nano-bubbles, the present invention mixes the plant sample to be extracted with the deep eutectic solvent containing nano-bubbles and performs extraction under ultrasonic conditions to obtain the triterpenic acid. As an embodiment of the present invention, taking corosolic acid as the target extract, the plant sample to be extracted can specifically be the outer seed coat of pecan; the material-liquid ratio of the plant sample to be extracted to the deep eutectic solvent containing nano-bubbles can be 1 g: 15 - 40 mL, specifically 1 g: 18 mL, 1 g: 20 mL or 1 g: 25 mL. As an embodiment of the present invention, the response surface optimization method can be used to further optimize the extraction parameters; the extraction conditions include: the temperature can be 40 - 60 °C, specifically 40 °C, 42 °C, 45 °C, 47 °C, 50 °C, 52 °C, 55 °C, 57 °C or 60 °C; the ultrasonic power can be 160 - 320 W, specifically 160 W, 200 W, 240 W, 280 W or 320 W; the time can be 30 - 70 min, specifically 30 min, 35 min, 40 min, 46 min, 50 min, 60 min or 70 min. In the examples of the present invention, taking the outer seed coat of pecan as the plant sample to be extracted, under the conditions of a material-liquid ratio of 1 g: 20 mL, a temperature of 42 °C, an ultrasonic power of 280 W and a time of 46 min, the extraction amount of corosolic acid in the outer seed coat of pecan finally reaches 14.08 mg / g DW.

[0054] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the examples in the present invention. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0055] Example 1

[0056] This example illustrates the feasibility of the method of the present invention by taking the extraction of corosolic acid from the outer seed coat of pecan as an example, as follows:

[0057] (1) Standard samples of corosolic acid (1 mg) were respectively placed in 23 organic solvents (5 mL) with obvious Hansen solubility differences, ultrasonically treated for 5 min at a temperature of 50 °C and a power of 200 W, and then cooled to room temperature. The dissolution status of corosolic acid was observed, where complete dissolution was recorded as 1 and the rest were recorded as 0 to evaluate the solubility of corosolic acid in the organic solvents.

[0058] Figure 1It is a graph showing the dissolution of corosolic acid in some organic solvents. The complete dissolution state of corosolic acid in acetic acid is recorded as 1, and the swelling state in n - hexane, the insoluble state in 1 - pentene, and the slightly soluble state in acrylate are all recorded as 0 (from left to right). The Hansen solubility parameters of the 23 organic solvents and the evaluation results of the solubility of corosolic acid in the organic solvents are shown in Table 1.

[0059] Table 1 Hansen solubility parameters of 23 organic solvents and evaluation results of the solubility of corosolic acid in them

[0060]

[0061]

[0062] (2) Input the names of the 23 organic solvents into the HSPiP software. The three Hansen solubility parameters of the organic solvents will be automatically displayed in the HSPiP software. At the same time, input the dissolution fractions of corosolic acid in the 23 organic solvents into the HSPiP software, and draw the Hansen three - dimensional solubility parameter sphere of corosolic acid (the organic solvents with a dissolution fraction recorded as 1 are inside the sphere, and the organic solvents with a dissolution fraction recorded as 0 are outside the sphere). Determine the dissolution radius and dissolution center of corosolic acid according to the Hansen three - dimensional solubility parameter sphere of corosolic acid, where Figure 2 is the result graph of the Hansen three - dimensional solubility parameter sphere of corosolic acid. The result shows that the dissolution radius (R 0 ) of corosolic acid is 11.4, and the Hansen solubility parameters corresponding to the dissolution center are δ d = 16.49, δ p = 7.37, δ h = 12.85.

[0063] (3) According to the dissolution radius and dissolution center of corosolic acid in step (2), select hydrogen - bond donors and hydrogen - bond acceptors from 32 plant - derived natural components. Specifically, calculate the Hansen solubility parameters of the 32 plant - derived natural components respectively according to the group contribution method, and calculate the distance between each plant - derived natural component and the dissolution center of corosolic acid according to formula I:

[0064] R a 2 = 4(δ d,j - δ d,i ) 2 +(δ p,j - δ p,i ) 2 +(δ h,j - δ h,i ) 2 Formula I;

[0065] In formula I, R a is the distance between the plant-derived natural component and the dissolution center of corosolic acid, δ d,j is the δ of the plant-derived natural component d value, δ d,i is the δ of corosolic acid d value, δ p,j is the δ of the plant-derived natural component p value, δ p,i is the δ of corosolic acid p value, δ h,j is the δ of the plant-derived natural component h value, δ h,i is the δ of corosolic acid h value.

[0066] The Hansen solubility parameters of 32 plant-derived natural components and the distance in three-dimensional space from the Hansen solubility parameter of corosolic acid, that is, the distance (R a ) between each plant-derived natural component and the dissolution center of corosolic acid are shown in Table 2.

[0067] Table 2 Hansen solubility parameters of 32 plant-derived natural components and the distance between the dissolution center of corosolic acid

[0068]

[0069]

[0070] (4) Based on the results in Table 2 in step (3), initially select the hydrogen bond donor and hydrogen bond acceptor with R a < R 0 = 11.4. Then, take L-menthol and thymol as hydrogen bond donors respectively, and other plant-derived natural components as hydrogen bond acceptors. Mix the hydrogen bond donor and hydrogen bond acceptor, heat to 70 °C and keep warm for 30 min, then cool to room temperature. If the resulting product is a non-transparent liquid (such as turbid or having precipitation), or there is precipitation after standing at room temperature for 48 h, discard it. Finally, 11 candidate natural eutectic solvents are screened out, as shown in Table 3 specifically.

[0071] Table 3 11 candidate natural eutectic solvents

[0072]

[0073] (5) Mix the outer seed coat of hickory with the 11 candidate natural deep eutectic solvents in Table 3 in step (4), and perform ultrasonic extraction for 60 min under the conditions of a solid-liquid ratio of 1 g:20 mL, a temperature of 50 °C, and a power of 200 W. Screen out the target natural deep eutectic solvent with the extraction amount of corosolic acid as the target. Finally, it is determined that the natural deep eutectic solvent prepared from thymol and linalool in a molar ratio of 2:1 has the highest extraction rate of corosolic acid, reaching 9.21 mg / g DW. Therefore, the natural deep eutectic solvent prepared from thymol and linalool in a molar ratio of 2:1 is selected as the target natural deep eutectic solvent.

[0074] Example 2

[0075] Place the natural deep eutectic solvent prepared from thymol and linalool in a molar ratio of 2:1 into a headspace vial. Insert a syringe into the headspace vial below the liquid level. Inject nitrogen gas into the natural deep eutectic solvent through the syringe to make the gas pressure reach the set value, and change the gas pressure by continuously pushing and pulling the syringe, that is, prepare nano-bubbles by decompression and pressurization to obtain a natural deep eutectic solvent containing nano-bubbles.

[0076] Test the effects of the compression cycle (i.e., the number of compression cycles, specifically 20 times, 30 times, 40 times, 50 times, and 60 times) and the gas pressure in the bottle (the nitrogen pressures controlled according to Boyle's law are 0.3 MPa, 0.4 MPa, 0.5 MPa, and 0.6 MPa) on the nano-bubble size.

[0077] Figure 3 It is a graph showing the influence results of the compression cycle number and nitrogen pressure on the average size and size intensity of nano-bubbles. Among them, (A) is the influence result graph of the compression cycle number on the size intensity distribution of nano-bubbles (nitrogen pressure is 0.4 MPa), (B) is the influence result graph of the compression cycle number on the average size of nano-bubbles (nitrogen pressure is 0.4 MPa), (C) is the influence result graph of nitrogen pressure on the size intensity distribution of nano-bubbles (compression cycle number is 50 times), and (D) is the influence result graph of nitrogen pressure on the average size of nano-bubbles (compression cycle number is 50 times). The results show that the nano-bubbles obtained under the conditions of a nitrogen pressure of 0.5 MPa and a compression cycle number of 50 times have the smallest size, with an average size of 237.36 nm. Therefore, finally, according to the results of the single-factor experiment, the compression cycle number is determined to be 50 times, and the nitrogen pressure in the bottle is 0.5 MPa.

[0078] Example 3

[0079] The outer seed coat of pecan was mixed with the natural deep eutectic solvent containing nanobubbles prepared in Example 2 at a solid-liquid ratio of 1 g:20 mL, and extraction was carried out under ultrasonic conditions. Taking the extraction amount of corosolic acid as the target and ultrasonic temperature, ultrasonic power, and ultrasonic time as factors, a single-factor experiment was conducted, and the central values of the three factors required for response surface optimization were selected based on the results of the single-factor experiment; among them, the ultrasonic temperature was specifically 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C, the ultrasonic power was specifically 160 W, 200 W, 240 W, 280 W, and 320 W, and the ultrasonic time was specifically 30 min, 40 min, 50 min, 60 min, and 70 min.

[0080] Figure 4 Figure 4 shows the influence of ultrasonic temperature, ultrasonic power, and ultrasonic time on the extraction amount of corosolic acid from the outer seed coat of pecan. Among them, (A) is the influence result diagram of ultrasonic temperature on the extraction amount of corosolic acid from the outer seed coat of pecan (ultrasonic power is 200 W, ultrasonic time is 50 min); (B) is the influence result diagram of ultrasonic power on the extraction amount of corosolic acid from the outer seed coat of pecan (ultrasonic temperature is 50 °C, ultrasonic time is 50 min); (C) is the influence result diagram of ultrasonic time on the extraction amount of corosolic acid from the outer seed coat of pecan (ultrasonic temperature is 50 °C, ultrasonic power is 240 W). The results show that among the set levels, under the conditions of ultrasonic temperature of 50 °C, ultrasonic power of 240 W, and ultrasonic time of 40 min, the extraction amount of corosolic acid from the outer seed coat of pecan is the highest, which is 13.04 mg / g DW.

[0081] According to the above single-factor experiment results, in this example, a Box-Behnken response surface experiment with three factors and three levels was designed using Design-Expert version 12.0. The experimental parameters are shown in Table 4, where the ultrasonic time is set to 30 min, 40 min, 50 min, the ultrasonic power is set to 200 W, 240 W, 280 W, and the ultrasonic temperature is set to 40 °C, 45 °C, 50 °C; the specific experimental results are shown in Table 4.

[0082] The experimental results were input into the Design-Expert version 12.0 software to obtain the formula for the extraction amount of corosolic acid and the optimal parameters for ultrasonic extraction. The formula for the extraction amount of corosolic acid is shown in Equation II:

[0083] Y = -111.43 + 2.42A - 0.03B + 3.74C + 2.26×10 -2 AC - 1.65×10 -2 A 2 - 3.67×10 -2 B 2 Equation II;

[0084] In Formula II, Y is the extraction amount of corosolic acid, A is the ultrasonic time, B is the ultrasonic power, and C is the ultrasonic temperature.

[0085] When Y in Formula II reaches the maximum value, the corresponding A, B, and C are the optimal parameters for ultrasonic extraction (rounded to the nearest integer), specifically: the ultrasonic time is 46 min, the ultrasonic power is 280 W, and the ultrasonic temperature is 42 °C.

[0086] Table 4 Box-Behnken response surface experimental design and results

[0087]

[0088] Example 5

[0089] (1) Mix thymol and linalool in a molar ratio of 2:1, heat to 70 °C, keep warm for 30 min, and then cool to room temperature to obtain a natural eutectic solvent;

[0090] Place the natural eutectic solvent in a headspace vial, insert a syringe into the vial below the liquid level, inject nitrogen into the natural eutectic solvent through the syringe to make the gas pressure reach 0.5 MPa, and change the gas pressure by continuously pushing and pulling the syringe (the number of compression cycles is 50 times), that is, prepare nano-bubbles by decompression and pressurization to obtain a natural eutectic solvent containing nano-bubbles;

[0091] (2) Mix the outer seed coat of pecan with the natural eutectic solvent containing nano-bubbles prepared in step (1) at a solid-liquid ratio of 1 g:20 mL, and perform ultrasonic extraction for 46 min at a temperature of 42 °C and a power of 280 W. Finally, the extraction amount of corosolic acid in the outer seed coat of pecan reaches 14.08 mg / gDW.

[0092] Comparative Example 1

[0093] Mix the outer seed coat of pecan with ethanol at a solid-liquid ratio of 1 g:20 mL, and perform ultrasonic extraction for 60 min at a temperature of 50 °C and a power of 200 W. Finally, the extraction amount of corosolic acid in the outer seed coat of pecan is only 5.06 mg / g DW.

[0094] From the above results, it can be seen that compared with the traditional ultrasonic-assisted ethanol extraction, after screening the natural eutectic solvent, introducing nano-bubbles and optimizing the ultrasonic parameters on this basis, the extraction amount of corosolic acid in the outer seed coat of pecan has increased by more than twice.

[0095] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for screening a deep eutectic solvent based on Hansen solubility parameters, comprising the following steps: placing the target extract standard in multiple groups of organic solvents with different Hansen solubilities, respectively, to obtain solubility data of the target extract standard in the organic solvents; Obtaining a Hansen three-dimensional solubility parameter sphere according to the solubility data, and obtaining a solubility radius and a solubility center of the target extract standard according to the Hansen three-dimensional solubility parameter sphere; Screening and obtaining candidate hydrogen bond donors and candidate hydrogen bond acceptors according to the solubility radius and the solubility center; Using the candidate hydrogen bond donor and the candidate hydrogen bond acceptor to prepare a candidate deep eutectic solvent; The candidate low eutectic solvent is used to extract the sample to be extracted, and the target low eutectic solvent is screened based on the extraction amount of the target extract in the sample to be extracted.

2. The method according to claim 1, characterized in that The difference in Hansen solubility between any two groups of organic solvents in the plurality of groups of organic solvents satisfies at least one of the following conditions: the dispersion force parameter δ d Phase difference ≥ 2, polarity parameter δ p Phase difference ≥ 4, hydrogen bond parameter δ h Difference ≥ 4; The solubility data include the dispersion force parameter δ d , polarity parameter δ p , hydrogen bond parameter δ h The solubility fraction of the target extract standard in the organic solvent, the solubility fraction is obtained in the following manner: when the target extract standard is completely dissolved in the organic solvent under predetermined conditions, the solubility fraction is recorded as 1, otherwise the solubility fraction is recorded as 0; the preset conditions include: the dosage ratio of the target extract standard to the organic solvent is 1 mg: 3-10 mL; the temperature is 30-60° C., the ultrasonic power is 200-400 W, and the time is 5-20 min.

3. The method according to claim 2, characterized in that The method for obtaining the Hansen three-dimensional solubility parameter sphere includes: inputting the solubility data into HSPiP software to obtain the Hansen three-dimensional solubility parameter sphere.

4. The method according to any one of claims 1 to 3, characterized in that: The sample to be extracted includes a plant sample; the target extract includes triterpenic acid; and the candidate hydrogen bond donor and the candidate hydrogen bond acceptor are from natural components of plant origin.

5. The method according to claim 4, characterized in that The triterpene acids include pentacyclic triterpene acids; the plant-derived natural ingredients include one or more of the following: acid plant-derived natural ingredients, alcohol plant-derived natural ingredients, phenol plant-derived natural ingredients, sugar plant-derived natural ingredients, aldehyde plant-derived natural ingredients and ketone plant-derived natural ingredients.

6. The method according to claim 5, characterized in that The pentacyclic triterpene acid includes corosolic acid, hawthorn acid or ursolic acid; each of the multiple groups of organic solvents is acetic acid, acetone, acetonitrile, 1,4-butanediol, 1-n-butanol, n-butyl acetate, chloroform, cyclohexane, cyclohexanol, diacetone alcohol, diethyl ether, N,N-dimethylformamide, 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, hexane, methanol, 1-pentene, 2-propanol, acrylate, tetrachloroethylene, Ethylene, tetrahydrofuran; the plant-derived natural ingredients include acetic acid, isoleucine, leucine, proline, methionine, sorbic acid, menthol, phenylalanine, glycine, salicylic acid, pyruvic acid, glutamic acid, lauric acid, nerol, geraniol, thymol, vanillin, linalool, fructose, lactic acid, d-camphor, citral, malonic acid, carvone, citric acid, coumarin, malic acid, glycerol, glycolic acid, oxalic acid, xylitol and tartaric acid.

7. The method according to claim 6, characterized in that The dissolution radius R0 is 10.5-11.8, and the dispersion force parameter δ corresponding to the dissolution center d =15.51~16.51, polarity parameter δ p =6.51~7.92, hydrogen bond parameter δ h =12.53~14.26; The candidate low eutectic solvents include menthol and thymol composition, menthol and geraniol composition, menthol and lauric acid composition, menthol and linalool composition, menthol and pyruvic acid composition, menthol and carvone composition, menthol and lactic acid composition, thymol and geraniol composition, thymol and linalool composition, thymol and carvone composition, and thymol and lactic acid composition.

8. The method according to claim 7, characterized in that The target low eutectic solvent is a composition of thymol and linalool, and the molar ratio of thymol to linalool in the target low eutectic solvent is 1 to 2:

1.

9. A method for extracting triterpene acids from plants, comprising the following steps: Screening and obtaining a target deep eutectic solvent according to the method according to any one of claims 1 to 8; Introducing nanobubbles into the target deep eutectic solvent to obtain a deep eutectic solvent containing nanobubbles; The plant sample to be extracted is mixed with the low eutectic solvent containing nanobubbles, and extraction is performed under ultrasonic conditions to obtain the triterpene acid.

10. The method according to claim 9, characterized in that The method of introducing nanobubbles into the target deep eutectic solvent comprises: introducing nitrogen into the target deep eutectic solvent for pressurization, then reducing pressure, repeating the pressurization and decompression steps to form nanobubbles, and obtaining the deep eutectic solvent containing nanobubbles; the pressurization pressure is 0.3-0.6 MPa, and the number of pressurization and decompression cycles is 20-60 times; The extraction conditions include: temperature of 40-60° C., time of 30-70 min, and ultrasonic power of 160-320 W.

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