An ionic liquid 7-galloyltetrahydrochrysene molecularly imprinted polymer and application thereof

Abstract

The application discloses an ionic liquid 7-galloyl-tetrol flavan molecular imprinting polymer, which is prepared by using 7-galloyl-tetrol flavan as a template molecule, using an ionic liquid as a functional monomer, using an acetonitrile:1-ethyl-3-methyl imidazole tetrafluoroborate mixed solution as a porogen, and through a bulk polymerization method, under the joint action of a crosslinking agent, an initiator and the porogen, the functional monomer is polymerized with the template molecule, so that the ionic liquid 7-galloyl-tetrol flavan molecular imprinting polymer is prepared. The 7-galloyl-tetrol flavan is used as the template molecule for the first time, and the green and environment-friendly imprinting polymer material is prepared in combination with the ionic liquid, the imprinting polymer material has obvious specific adsorption, has a wide application range, can still exhibit strong specific selectivity to the template molecule in an aqueous phase, and can efficiently separate and enrich effective components.

Description

Technical Field

[0001] This invention relates to the fields of new material preparation and natural product separation and purification, specifically to an ionic liquid 7-galloyltrisulfonane molecularly imprinted polymer and its applications. Background Technology

[0002] Pithecellobium clypearia Benth., a plant belonging to the genus Pithecellobium in the legume family, is also known as the "monkey earring tree," "dragon tree," and "hair-washing tree," and is mainly distributed in Fujian, Guangdong, Guangxi, and Hainan provinces of my country. It is commonly used in traditional medicine to treat burns, snake and insect bites, and to reduce inflammation and pain. 7-Galloylterlyflavonoid (7-GTDF) is a unique component of Pithecellobium plants. Studies have found that it primarily has antiviral effects, and also exhibits certain antibacterial, hypoglycemic, antioxidant, and anti-inflammatory activities. Currently, 7-GTDF is mainly obtained from the alcohol extract of Pithecellobium clypearia through column separation (e.g., Chinese patent CN1765894A). This method is cumbersome, time-consuming, and labor-intensive, and the purity of the obtained 7-GTDF is not high, making it an undesirable method for separating and purifying 7-GTDF.

[0003] Molecular imprinting technology refers to the process of using a specific target molecule as a template, adding functional monomer molecules that can undergo a polymerization reaction with it, and then, under the action of a cross-linking agent and an initiator, cross-linking polymerization occurs between the functional monomer and the template molecule. Afterward, the template molecule is eluted and removed, thus preparing a polymer with specific selectivity only for that molecule. Even after the template molecule is removed, this molecularly imprinted polymer retains the specific structure corresponding to the template molecule, accurately identifying the template molecule used in its formation. Therefore, it can be used for target substance detection, removal of harmful substances, identification of targeted molecules, chromatographic separation, and more.

[0004] Ionic liquids, liquids composed entirely of ions, are often used as "green solvents" in organic synthesis reactions. They possess excellent physicochemical properties, such as thermal stability, strong solubility, and designability, and are low in toxicity and environmentally friendly, making them a novel type of functional monomer. In the field of molecularly imprinted polymers, ionic liquids are widely recognized for their superior physical properties, such as excellent chemical stability, non-volatility, and excellent ionic conductivity. Therefore, they are frequently used as functional monomers in the design of molecularly imprinted polymers (e.g., Chinese patents CN107383277A and CN104910339A).

[0005] The ethanol extract of monkey ear loops contains a large number of structurally similar flavonoids and flavanes. Traditional molecular imprinting techniques remain very difficult to separate 7-GTDF in the presence of other structurally similar compounds, while the aqueous extract of monkey ear loops shows less interference. However, traditional molecularly imprinted polymers typically exhibit excellent molecular recognition performance in organic solvent systems, while their molecular recognition performance in aqueous solutions is poor, limiting their application. Therefore, developing a molecularly imprinted polymer suitable for template molecule separation and purification in aqueous solutions, adhering to green production principles, using the aqueous extract as the extraction target, and reducing the use of organic solvents in the 7-GTDF extraction process, is a pressing problem in current 7-GTDF extraction technology and has significant practical value. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an ionic liquid 7-galloyltertrin molecularly imprinted polymer for separating and enriching 7-galloyltertrin compounds and its preparation method.

[0007] Another object of the present invention is to provide the application of the molecularly imprinted polymer in the separation and enrichment of 7-galloyltrisulfonane.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention first provides a molecularly imprinted polymer of the ionic liquid 7-galloylterithromane. It is prepared by bulk polymerization, in which a template molecule and a functional monomer are crosslinked and polymerized under the action of a crosslinking agent, an initiator and a porogen, and the template molecule is then eluted. Specifically, the template molecule is 7-galloylterithromane, the functional monomer is an ionic liquid, and the porogen is a mixed solution of acetonitrile and 1-ethyl-3-methylimidazolium tetrafluoroborate.

[0010] Furthermore, the pore-forming agent is wherein the molar ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate is (2-5):1.

[0011] Preferably, the molar ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate is (2-4):1.

[0012] More preferably, the molar ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate is (2-3):1.

[0013] Most preferably, the molar ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate in the porogen is 2:1.

[0014] Furthermore, the ionic liquid is 1-vinyl-3-ethylimidazolium tetrafluoroborate.

[0015] Furthermore, the crosslinking agent is ethylene glycol dimethacrylate.

[0016] Furthermore, the initiator is 2,2-azobisisobutyronitrile.

[0017] This invention also provides a method for preparing the molecularly imprinted polymer, which involves mixing template molecules and functional monomers, adding a porogen for prepolymerization, then adding a crosslinking agent and an initiator for polymerization to obtain the polymer; after removing impurities from the polymer, the template molecules are eluted and dried to obtain the molecularly imprinted polymer. The specific steps are as follows:

[0018] S1. Add the template molecule and functional monomer to a conical flask, then add the porogen, sonicate for 3-5 min, and prepolymerize at 3-5℃ for 6-8 h to obtain the prepolymer solution.

[0019] S2. Add crosslinking agent and initiator to the prepolymer liquid, stir magnetically and remove oxygen with nitrogen for 5-10 minutes, then polymerize at high temperature in the dark to obtain the polymer.

[0020] S3. The polymer is washed with methanol, dried, ground and sieved, and fine particles are removed by acetone precipitation.

[0021] S4. The polymer is washed with a methanol-glacial acetic acid mixture using a Soxhlet extraction method. After a certain period of time, the detergent is replaced to remove the template molecules. The polymer is then dried to obtain the molecularly imprinted polymer.

[0022] Furthermore, the molar ratio of the template molecule to the functional monomer is 1:(3-5).

[0023] Preferably, the molar ratio of the template molecule to the functional monomer is 1:4.

[0024] Furthermore, the molar ratio of the crosslinking agent to the template molecule is (16-24):1.

[0025] Preferably, the molar ratio of the crosslinking agent to the template molecule is 20:1.

[0026] Furthermore, the amount of the initiator is 2% to 4% of the total molar amount of the template molecule, functional monomer, and crosslinking agent.

[0027] Preferably, the amount of the initiator is 1% of the total molar amount of the template molecule, functional monomer, and crosslinking agent.

[0028] Furthermore, in step S2, the temperature for light-protected polymerization is 70–75°C, and the polymerization time is 24 hours.

[0029] Preferably, in step S2, the temperature for light-protected polymerization is 70°C.

[0030] Furthermore, in step S3, the methanol washing is performed 3 times.

[0031] Furthermore, in steps S3 and S4, the drying temperature is 60°C.

[0032] Furthermore, in the methanol-glacial acetic acid mixture, the volume ratio of methanol to glacial acetic acid is 9:1.

[0033] Furthermore, the methanol-glacial acetic acid elution rate in step S4 is 0.3–0.5 mL / min.

[0034] Furthermore, in step S4, the Soxhlet extraction washing time is 48 hours, and the detergent is replaced every 12 hours to wash away the template molecules.

[0035] Furthermore, after elution with methanol-glacial acetic acid in step S4, formic acid needs to be removed by further extraction and elution with pure methanol for 8 hours.

[0036] The molecularly imprinted polymer provided by this invention uses ionic liquids as functional monomers, enabling it to maintain structural and property stability in aqueous solutions, thus facilitating the separation and purification of template molecules in aqueous solutions. Experiments have shown that the molecularly imprinted polymer exhibits significant specific adsorption of 7-galloylterithrin in the aqueous extract of *Heliotropium indicum*, achieving a recovery rate of up to 60.01%, demonstrating its potential for specific adsorption of template molecules in aqueous phases. When applied to the separation and enrichment of the active substance 7-galloylterithrin in the aqueous extract of the traditional Chinese medicine *Heliotropium indicum*, the purity of the obtained 7-galloylterithrin can be increased from the original 30% to over 75% after adsorption and desorption by the prepared molecularly imprinted polymer.

[0037] Therefore, the present invention also provides the following applications of the ionic liquid molecularly imprinted polymer:

[0038] The application of any of the molecularly imprinted polymers described above in the separation, enrichment, and / or detection of 7-galloyltrisulfonane.

[0039] This invention also provides a method for separating and enriching 7-galloylterlioflavone using any of the molecularly imprinted polymers described above. The method involves taking the solution to be enriched, adding the molecularly imprinted polymer, shaking to adsorb, centrifuging to obtain a precipitate; after removing impurities from the precipitate, eluting the target molecule, centrifuging to obtain the supernatant, which yields the enriched 7-galloylterlioflavone solution. The specific steps are as follows:

[0040] S1. Preparation of sample solution: Take the water extract to be enriched, add water-acetonitrile mixed solution and sonicate to dissolve.

[0041] S2. Adsorption: Take the ionic liquid 7-galloyltrichloroflavon molecularly imprinted polymer, add it to the sample solution (loading solution), shake at constant temperature for 10-30 min, centrifuge and discard the supernatant.

[0042] S3. Washing: Add the remaining precipitate to an ethanol-water solution and continue shaking for 10-30 minutes. Centrifuge and discard the supernatant.

[0043] S4. Elution: Add ethanol-acetic acid mixture to the precipitate, shake for 10-30 min to elute the target molecules, centrifuge and take the supernatant to obtain the enriched 7-galloyltrisulfonane solution.

[0044] Furthermore, the ratio of water to acetonitrile in the water-acetonitrile mixed solution is 90:10 to 95:5.

[0045] Furthermore, the concentration of the sample solution is 2–4 mg / mL.

[0046] Furthermore, the concentration of the ethanol-water solution is 20%.

[0047] Furthermore, in the ethanol-acetic acid mixture, the ratio of ethanol to acetic acid is 9:1.

[0048] Further, in step S2, 30-50 ml of sample solution is added for every gram of molecularly imprinted polymer.

[0049] Furthermore, in step S2, the isothermal oscillation frequency is 80–120 r / min, and the temperature is 20–30 °C.

[0050] Furthermore, the centrifugation rate in step S2 is 12000 r / min.

[0051] Furthermore, in step S3, the amount of ethanol-water solution added is 1 to 2 times the volume of the sample solution.

[0052] Furthermore, in step S4, the amount of ethanol-acetic acid mixture added is 1 to 2 times the volume of the sample solution.

[0053] Finally, this invention provides a method for using any of the above-described molecularly imprinted polymers to detect 7-galloylterliflavones. After separating and enriching 7-galloylterliflavones with any of the above-described molecularly imprinted polymers, purity analysis is performed, and the recovery rate and purity are calculated.

[0054] Furthermore, the purity analysis was performed using HPLC.

[0055] Furthermore, the specific chromatographic conditions in the HPLC method are as follows:

[0056] Chromatographic column: ECOSIL C18 (250×4.6mm, 5μm); mobile phase A: methanol; mobile phase B: 0.2% phosphoric acid. Flow rate: 1mL / min; column temperature: 30℃; injection volume: 10.0μL; chromatographic conditions: 30–30% B, 0–5min; 30%–45% B, 5–15min; 45%–50% B, 15–20min; 50%–55% B, 20–30min; 55%–30% B, 30–32min.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] This invention utilizes 7-galloylterithromane as a template molecule, an ionic liquid as a functional monomer, and a mixed solution of acetonitrile and 1-ethyl-3-methylimidazolium tetrafluoroborate as a porogen. Through bulk polymerization, under the combined action of a crosslinking agent, initiator, and porogen, the functional monomer and template molecule are polymerized, thereby preparing an ionic liquid-based 7-galloylterithromane molecularly imprinted polymer. This invention is the first to use 7-galloylterithromane as a template molecule, combined with an ionic liquid, to prepare a green and environmentally friendly imprinted polymer material. It exhibits significant specific adsorption properties, has a wide range of applications, and maintains strong specific selectivity for the template molecule even in aqueous phases. It can efficiently separate and enrich effective components, with a convenient and rapid separation and enrichment process. The purity of the separated precipitate can reach over 70%, and the recovery rate can reach 60%. It is an ideal molecularly imprinted polymer for separating and enriching template molecules in aqueous phases, providing a reference for the further development and utilization of monkey ear fungus medicinal materials and the separation and purification of effective components. Attached Figure Description

[0059] Figure 1 Flowchart for the preparation of molecularly imprinted polymers of the ionic liquid 7-galloyltrichloroflavon.

[0060] Figure 2 Scanning electron microscopy image of the molecularly imprinted polymer of 7-galloylterithromane, in which Figure 2 a is a molecularly imprinted polymer of the ionic liquid 7-galloylterithromane. Figure 2 b represents the blank imprint polymer.

[0061] Figure 3 The isothermal static adsorption diagram is shown for the molecularly imprinted polymer of the ionic liquid 7-galloyltrisulfonane.

[0062] Figure 4 The isothermal dynamic adsorption diagram of the molecularly imprinted polymer of the ionic liquid 7-galloyltrisulfonane is shown.

[0063] Figure 5 A diagram showing four components in the aqueous extract of monkey ear rings that have structures similar to 7-galloylterithrin.

[0064] Figure 6 The chromatogram for the separation of 7-galloylterylsulforhanane from the aqueous extract of monkey ear rings. Detailed Implementation

[0065] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0066] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0067] In this embodiment of the invention, the instruments, equipment, materials, and reagents used are as follows:

[0068] 1. Instruments

[0069] CP225D electronic analytical balance (Sartorius GmbH, Germany), SK7200LHC ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), ACQUITY high performance liquid chromatograph (Waters Inc., USA), MXW-200+ mixer (Hangzhou Qiwei Instrument Co., Ltd.), KS 3000I control constant temperature shaker (IKA GmbH, Germany).

[0070] 2. Materials and Reagents

[0071] Monkey earrings were purchased from Guangdong Taihetang Industrial Co., Ltd.; 1-vinyl-3-ethylimidazolium tetrafluoroborate and 1-ethyl-3-methylimidazolium tetrafluoroborate were purchased from Shanghai Chengjie Ionic Liquid Co., Ltd.; ethylene glycol dimethacrylate (EGDMA), 2,2-azobisisobutyronitrile (AIBN), 4-vinylpyridine (4-VP), methacrylic acid (MAA), acrylic acid (AA), and acrylamide (AM) were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; acetonitrile and acetic acid were purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; methanol was of chromatographic grade, and water was purified water.

[0072] Example 1: Screening of pore-forming agents

[0073] Preparation method of ionic liquid 7-galloyltrisulfonane molecularly imprinted polymer: 0.2 mmol of 1-vinyl-3-ethylimidazolium tetrafluoroborate was added to an Erlenmeyer flask, followed by 3 mL of porogen, and then 0.05 mmol of 7-GTDF. After ultrasonic mixing for 5 min, prepolymerization was carried out at 4 °C for 6 h. 1 mmol of EGDMA and 0.025 mmol of AIBN were added, and after magnetic stirring and nitrogen deoxygenation for 5 min, polymerization was carried out at 70 °C for 24 h. After polymerization, the polymer was washed three times with methanol, dried at 60 °C, ground and sieved, and fine particles were removed by acetone precipitation. Finally, it was washed with a methanol-glacial acetic acid mixture (methanol to glacial acetic acid volume ratio of 9:1) at a rate of 0.3 mL / min for 48 h, with the washing agent replaced every 12 h to remove template molecules. The polymer was dried at 60 °C to obtain the ionic liquid 7-galloyltrisulfonane molecularly imprinted polymer.

[0074] (1) Screening of porogen types

[0075] Based on the above preparation method, different types of porogens (ionic liquid mixed solvents) were investigated. Three molecularly imprinted polymers of the ionic liquid 7-galloyltrisulfonane were prepared using different porogens. The adsorption capacity of the molecularly imprinted polymers prepared with different porogens in a water-acetonitrile mixed solution (acetonitrile to water ratio of 1:3) was investigated to screen the types of porogens. The adsorption capacity and imprinting factor were calculated according to the following formulas:

[0076] Q = v(c1 - c2) / m

[0077] IF=Q MIP / Q NIP

[0078] Where v is the volume of the adsorption solution, c1 and c2 are the concentrations before and after adsorption, and m is the sample mass; Q MIP Q represents the adsorption capacity of the molecularly imprinted polymer (mg / g); NIP This represents the amount of polymer adsorbed in the blank imprint.

[0079] Specifically, the mixed solvents used are acetonitrile: 1-butyl-3-methylimidazolium tetrafluoroborate mixed solution (molar ratio 2:1), acetonitrile: 1-hexyl-3-methylimidazolium tetrafluoroborate mixed solution (molar ratio 2:1), and acetonitrile: 1-octyl-3-methylimidazolium tetrafluoroborate mixed solution (molar ratio 2:1).

[0080] The results are shown in Table 1. The results indicate that different solvents significantly affect the adsorption performance and selectivity of the final imprinted material. With increasing pore size and alkyl chain length of the porogen, the pore size increases, the specific surface area decreases, and the adsorption capacity decreases. As the alkyl chain length of the ionic liquid solvent gradually decreases, the imprinted material can produce smaller pore sizes and larger specific surface areas, while simultaneously improving the specific adsorption of the target substance (increasing the imprinting factor). Therefore, when preparing materials with specific adsorption and separation of flavanes, ionic liquids with smaller alkyl chains should be used as the reaction polymerization solvent to improve the corresponding specific adsorption. Therefore, the preferred type of porogen is acetonitrile:1-ethyl-3-methylimidazolium tetrafluoroborate.

[0081] Table 1. Adsorption capacity and imprinting factor of ionic liquid 7-GTDF MIP and blank imprinting material NIP

[0082]

[0083] (2) Screening of the ratio of acetonitrile: 1-ethyl-3-methylimidazolium tetrafluoroborate mixed solution

[0084] Based on the above preparation method, the ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate in the acetonitrile:1-ethyl-3-methylimidazolium tetrafluoroborate mixed solution was further screened. Three molecularly imprinted polymers of the ionic liquid 7-galloylterithrolane with different molar ratios of acetonitrile:1-ethyl-3-methylimidazolium tetrafluoroborate were prepared. The adsorption capacity of the molecularly imprinted polymers prepared with different porogen ratios in a water-acetonitrile mixed solution (acetonitrile to water ratio of 1:3) was investigated to screen the porogen ratio. Thus, the optimal ratio of 1-ethyl-3-methylimidazolium tetrafluoroborate to acetonitrile was determined.

[0085] The results are shown in Table 1. With increasing acetonitrile ratio, while the adsorption capacity of the molecularly imprinted polymer remained unchanged compared to the blank adsorption capacity, the imprinting factor gradually decreased. Since 7-GTDF is a neutrally polar molecule, acetonitrile, as an aprotic solvent, can reduce the influence of the solvent on the recognition between functional monomers and template molecules during material preparation, and can effectively assist in the dissolution of 7-GTDF, making it a relatively ideal pore-forming agent. However, as shown in Table 1, although a high proportion of acetonitrile did not significantly affect the adsorption capacity of the 7-GTDF ionic liquid material, it weakened the material's specific adsorption of 7-GTDF. Ionic liquids can limit non-specific binding, promote the interaction between 7-GTDF and functional monomers, and improve the material's specific adsorption performance (increased imprinting factor). Therefore, the optimal molar ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate is 2:1.

[0086] Therefore, considering the solvent's solubility for 7-GTDF and the specificity of the prepared material, the porogen used was selected as acetonitrile:1-ethyl-3-methylimidazolium tetrafluoroborate (molar ratio 2:1).

[0087] Example 2: Preparation of the molecularly imprinted polymer of the ionic liquid 7-galloyltrisulfonane

[0088] The preparation flow chart of the molecularly imprinted polymer of the ionic liquid 7-galloyltertraceran (7-GTDF) is shown below. Figure 1 As shown, the specific steps are as follows:

[0089] 2 mmol of 1-vinyl-3-ethylimidazolium tetrafluoroborate was added to an Erlenmeyer flask, followed by 30 mL of a mixed solution of acetonitrile and 1-ethyl-3-methylimidazolium tetrafluoroborate (molar ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate: 2:1). Then, 0.5 mmol of 7-GTDF was added, and the mixture was ultrasonically mixed for 5 min, followed by prepolymerization at 4 °C for 6 h. 10 mmol of EGDMA and 0.25 mmol of AIBN were added, and the mixture was magnetically stirred under nitrogen for 5 min to remove oxygen, followed by polymerization at 70 °C for 24 h. After polymerization, the polymer was washed three times with methanol, dried at 60 °C, ground, and sieved. Fine particles were removed by acetone precipitation. Finally, the polymer was washed for 48 h with a methanol-glacial acetic acid mixture (volume ratio of methanol to glacial acetic acid: 9:1) at a rate of 0.3 mL / min, with the washing agent changed every 12 h to remove template molecules. The polymer was then dried at 60 °C to obtain the ionic liquid 7-galloyltrisulfonane molecularly imprinted polymer.

[0090] A blank polymer material (without the template molecule 7-GTDF added during the preparation process) was prepared using the same method as a control to compare and analyze the characteristics of the molecularly imprinted polymer.

[0091] Example 3: Structural characterization of the molecularly imprinted polymer of ionic liquid 7-galloyltrisulfonane.

[0092] Small amounts of the ionic liquid 7-galloyltrisulfonane molecularly imprinted polymer and the blank ionic liquid 7-galloyltrisulfonane polymer from Example 2 were added to anhydrous ethanol. After the anhydrous ethanol evaporated, scanning electron microscopy was performed to observe the pore distribution and particle size of the powder. The scanning electron microscopy results are as follows: Figure 2 As shown.

[0093] right Figure 2 Analysis of the scanning electron microscope structure in the image shows that the imprinted polymer obtained by grinding the bulk polymer ( Figure 2 a) and blank imprint polymer ( Figure 2b) The size is comparable, and compared to the blank imprinted polymer, the imprinted polymer has more pores, providing conditions for the bonding between the template molecules and the material. Comparative Example 1: Comparison of the adsorption capacity in aqueous phase between the ionic liquid 7-galloyltrisulfonane molecularly imprinted polymer and the molecularly imprinted polymer using conventional functional monomers.

[0094] In contrast, 7-GTDF molecularly imprinted polymers were prepared using the traditional functional monomer 4-vinylpyridine (4-VP), with the following specific steps:

[0095] 2 mmol of 4-vinylpyridine was added to an Erlenmeyer flask, followed by 30 mL of acetonitrile and 0.5 mmol of 7-GTDF. After ultrasonic mixing for 5 min, the mixture was prepolymerized at 4 °C for 6 h. Then, 10 mmol of EGDMA and 0.25 mmol of AIBN were added, and the mixture was magnetically stirred under nitrogen for 5 min to remove oxygen. After polymerization, the polymer was washed three times with methanol, dried at 60 °C, ground, and sieved. Fine particles were removed by acetone precipitation. Finally, the polymer was washed for 48 h with a methanol-glacial acetic acid mixture (methanol to glacial acetic acid volume ratio of 9:1) at a rate of 0.3 mL / min, with the washing agent replaced every 12 h to remove template molecules. The polymer was then dried at 60 °C to obtain the 4-VP 7-galloyltrisulfonane molecularly imprinted polymer using traditional functional monomers.

[0096] A blank polymer material (without the addition of the template molecule 7-GTDF during the preparation process) was prepared using the same method as a control.

[0097] Example 4. Comparison of the adsorption capacity of molecularly imprinted polymers in aqueous phase.

[0098] The adsorption capacities of the ionic liquid 7-GTDF molecularly imprinted polymer prepared in Example 2 and the 4-VP 7-GTDF molecularly imprinted polymer prepared in Comparative Example 1 in the aqueous phase were compared. The formulas for calculating the adsorption amount and imprinting factor are as follows:

[0099] Q = v(c1 - c2) / m

[0100] IF=Q MIP / Q NIP

[0101] Where v is the volume of the adsorption solution, c1 and c2 are the concentrations before and after adsorption, and m is the sample mass; Q MIP Q represents the adsorption capacity of the molecularly imprinted polymer (mg / g); NIP This represents the amount of polymer adsorbed in the blank imprint.

[0102] Table 2 shows the adsorption results of ionic liquid materials and traditional 4-VP materials for 1 mM 7-GTDF in a water-acetonitrile mixed solution (acetonitrile to water ratio of 1:3) at 25 °C. The results indicate that the traditional 4-VP imprinted material adsorbs more 7-GTDF in a high proportion of water than the ionic liquid material, but its adsorption is mostly non-specific, with an imprinting factor less than 1, indicating no specific adsorption. In contrast, the ionic liquid material has an imprinting factor of 2.37, demonstrating significant specific adsorption of 7-GTDF.

[0103] Table 2 Adsorption capacity and imprinting factor of ionic liquids 7-GTDF MIP and 4-VP MIP

[0104]

[0105] Example 5: Isothermal and dynamic adsorption properties of the molecularly imprinted polymer of ionic liquid 7-galloyltrisulfonane.

[0106] Weigh an appropriate amount of 7-GTDF and dissolve it in acetonitrile to prepare a stock solution with a concentration of 1 mg / mL. Take a certain amount of the stock solution and dilute it with acetonitrile to obtain sample dilutions with concentrations of 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL. Take 2 mL of each concentration of 7-GTDF dilution and add it to a centrifuge tube containing 20 mg of the material. Vortex for about 5 seconds to mix, incubate at a constant temperature (25℃, 100 rpm) for 2 hours, centrifuge at 12000 rpm for 10 minutes, and inject the sample. Each concentration is measured in triplicate.

[0107] Weigh an appropriate amount of 7-GTDF and dissolve it in acetonitrile to prepare a solution with a concentration of approximately 0.4 mg / mL. Add 2 mL of this solution to 20 mg of the sample, vortex for approximately 5 seconds to mix, and then shake at a constant temperature (25℃, 100 rpm). Collect the blank and sample at 20 min, 40 min, 60 min, 80 min, and 100 min, respectively. Centrifuge at 12000 rpm for 5 min, and inject the supernatant. Perform three parallel determinations at each sampling time point.

[0108] Isothermal static adsorption analysis was performed on the ion liquid-imprinted polymers (IL-MIPs) prepared in Example 2 and the blank ion liquid-imprinted polymers (IL-NIPs), as follows: Figure 3 As shown, at room temperature (25℃), the equilibrium adsorption capacity of IL-MIPs for 7-GTDF is approximately 18.59 mg / g, while that of IL-NIPs is approximately 7.32 mg / g. This indicates that IL-MIPs have a higher affinity for 7-GTDF than IL-NIPs and exhibit good binding to the three-dimensional cavity of the template.

[0109] The dynamic adsorption capacity of molecularly imprinted polymers was evaluated at room temperature (25 °C). The adsorption kinetics curves of IL-MIPs and IL-NIPs for 7-GTDF are shown below. Figure 4 As shown in the figure, the adsorption capacity of IL-MIPs increased rapidly in the first 30 min, then the change gradually leveled off, reaching adsorption equilibrium at around 90 min. For IL-NIPs, the adsorption capacity gradually and steadily increased within 150 min, and no significant change occurred at 120 min. Furthermore, IL-MIPs exhibited a higher adsorption capacity than IL-NIPs. In summary, ionic liquid molecularly imprinted polymers possess more template cavities, thus exhibiting advantages in faster mass transfer and binding kinetics.

[0110] Example 6: Selectivity of the ionic liquid 7-galloyltrisulfonane molecularly imprinted polymer

[0111] The adsorption capacity of the molecularly imprinted polymer 7-galloylterithromane prepared in Example 2 for structurally similar compounds epigallocatechin gallate (ECCG), epigallocatechin (ECG), myricetin, and quercetin was investigated. These four substances are known components of monkey earrings and their structures are similar to 7-GTDF, with the molecular formula shown below. Figure 5 As shown, this can easily interfere with the separation and purification of 7-GTDF. Two mL of the same 1 mM molar concentration of the above-mentioned structural analog was placed in separate centrifuge tubes, and 10 mg of the polymer was added. The tubes were shaken at 25°C for 2 h, centrifuged at 12000 r / min for 5 min, and the supernatant was injected into the HPLC system. The measurements were performed in triplicate to estimate the adsorption capacity of the material for different substances at the same concentration. The selectivity factor α was used to evaluate the MIP. S The adsorption capacity and selectivity for similar substances are calculated using the following formulas.

[0112] α = IF1 / IF2

[0113] Wherein IF1 is the imprinting factor of the imprinted polymer on the target molecule, and IF2 is the imprinting factor of the imprinted polymer on the structural analogue.

[0114] The adsorption capacity and selectivity of the molecularly imprinted polymer for epigallocatechin gallate (ECGC), epigallocatechin (ECG), myricetin, and quercetin were investigated in a water-acetonitrile mixed solution (acetonitrile to water ratio of 1:3). The results are shown in Table 3. It can be seen that the adsorption capacity of IL-MIPs for 7-GTDF is significantly better than that for the other four structural analogs. The adsorption performance of this material for different compounds is in the following order: 7-GTDF > quercetin > ECGC > ECG > myricetin. Furthermore, IL-MIPs exhibits high selectivity factors for the other four structural analogs, with the highest selectivity factors for myricetin and ECG, at 1.53 and 2.74, respectively, fully demonstrating the potential of ionic liquid imprinting for the enrichment and separation of 7-GTDF in a high-proportion aqueous phase.

[0115] Table 3. Selectivity of ionic liquid-imprinted materials for different substances in high-proportion aqueous environments.

[0116]

[0117] Example 7: Isolation and Extraction of 7-Galloylterylsulforaphane from Aqueous Extracts of Monkey Earrings

[0118] Chromatographic conditions: Column: ECOSIL C18 (250×4.6mm, 5μm), mobile phase A: methanol, mobile phase B: 0.2% phosphoric acid. Flow rate: 1mL / min, column temperature: 30℃, injection volume: 10.0μL; chromatographic conditions: 30–30% B, 0–5min; 30%–45% B, 5–15min; 45%–50% B, 15–20min; 50%–55% B, 20–30min; 55%–30% B, 30–32min.

[0119] Preparation of sample solution: Take 50 mg of monkey ear ring water extract, add 20 mL of water-acetonitrile mixed solution (the ratio of water to acetonitrile is 9:1) and sonicate to dissolve, thus obtaining the sample solution.

[0120] Take 200 mg of the ionic liquid 7-GTDF molecularly imprinted polymer prepared in Example 2, add 8 mL of the aqueous extract sample solution, and shake at a constant temperature (100 r / min, 25℃) for 20 min. Centrifuge and collect the supernatant. Add 8 mL of 20% ethanol aqueous solution to the remaining precipitate and continue shaking for 20 min. Centrifuge and collect the supernatant (elution buffer). Add 8 mL of an ethanol-acetic acid mixture (ethanol to acetic acid ratio of 9:1) to the precipitate and shake for 20 min to elute the target molecule. Centrifuge and collect the supernatant (elution buffer). The extraction and separation ability of the molecularly imprinted polymer for 7-GTDF in the aqueous extract of monkey ear rings was investigated.

[0121] Figure 6Curves a, b, and c in the figure represent the loading solution, rinsing solution, and elution solution of the monkey earring, respectively. It can be seen that the content of some glycosides, such as quercetin and myricetin, is relatively increased in the rinsing solution, indicating that the rinsing process can effectively remove interfering components. After the elution process, the purity of 7-GTDF is 76.26%, and the recovery rate reaches 60.01%, demonstrating that the molecularly imprinted polymer has the potential to specifically adsorb 7-GTDF in the aqueous phase.

Claims

1. An ionic liquid 7-galloyl-tectochrysin molecularly imprinted polymer, which is prepared by cross-linking and polymerization of a template molecule and a functional monomer under the action of a cross-linking agent, an initiator and a porogen, and then eluting the template molecule, characterized in that, The template molecule is 7-galloyl tellimaggranin, the functional monomer is an ionic liquid, and the porogen is an acetonitrile: 1-ethyl-3-methylimidazolium tetrafluoroborate mixed solution; in the porogen, the molar ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate is (2-5):1; and the ionic liquid is 1-vinyl-3-ethylimidazolium tetrafluoroborate.

2. The molecularly imprinted polymer according to claim 1, wherein In the porogen, the molar ratio of acetonitrile to 1-ethyl-3-methylimidazolium tetrafluoroborate is 2:

1.

3. The molecularly imprinted polymer of claim 1, wherein, The crosslinking agent is ethylene glycol dimethacrylate.

4. The molecularly imprinted polymer of claim 1, wherein, The initiator is 2,2-azobis isobutyronitrile.

5. A method for preparing the molecularly imprinted polymer according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: mixing the template molecule and the functional monomer, adding the porogen for pre-polymerization, and then adding the crosslinking agent and the initiator for polymerization to obtain the polymer; after the polymer is purified, the template molecule is eluted and dried to obtain the molecularly imprinted polymer.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the template molecule to the functional monomer is 1:(3-5), and the molar volume ratio of the functional monomer to the porogen is 1 mmol:(50-70) mL.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the crosslinking agent to the template molecule is (16-24):1, and the amount of the initiator is 2%-4% of the total molar amount of the template molecule, the functional monomer and the crosslinking agent.

8. Use of the molecularly imprinted polymer according to any one of claims 1-4 in separating, enriching and / or detecting 7-galloyl tellimaggranin.

9. A method for separating and enriching 7-galloylterchebin flavan by using the molecularly imprinted polymer according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: adding the molecularly imprinted polymer to a solution to be enriched, shaking and adsorbing, centrifuging to obtain a precipitate, purifying the precipitate, eluting the target molecule, centrifuging to obtain the enriched 7-galloyl tellimaggranin solution.

10. A method for detecting 7-galloylpterocaryflavan by using the molecularly imprinted polymer according to any one of claims 1 to 4, characterized in that, 7-galloyl tellimaggranin is separated and enriched by using the molecularly imprinted polymer according to any one of claims 1-4.

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