Trans-ferulic acid molecularly imprinted polymer microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of analytical chemistry, and mainly relates to trans-ferulic acid molecularly imprinted polymer microspheres as well as a preparation method and application thereof. The preparation method of the trans-ferulic acid molecularly imprinted polymer microspheres comprises the following steps: dissolving trans-ferulic acid in a solvent, adding methacrylic acid, and mixing; adding a cross-linking agent and azodiisobutyronitrile, introducing nitrogen, and sealing to obtain a molecularly imprinted polymer mixed solution; dropwise adding the molecularly imprinted polymer mixed solution into the surfactant aqueous solution through a constant-pressure dropping funnel, stirring and reacting at the reaction temperature, and separating to obtain polymer microspheres; and washing off the trans-ferulic acid from the polymer microspheres, washing the polymer microspheres to be neutral, and drying to obtain the trans-ferulic acid molecularly imprinted polymer microspheres. According to the preparation method, the trans-ferulic acid molecularly imprinted polymer microspheres with relatively good specificity and performance of selectively adsorbing a target object trans-ferulic acid can be prepared, and large-scale separation and purification of the trans-ferulic acid can be realized.

Description

Technical Field

[0001] The present application relates to the technical field of analytical chemistry, and mainly relates to a trans-ferulic acid molecularly imprinted polymer microsphere and a preparation method and application thereof. Background Art

[0002] Ferulic acid, chemically known as 4-hydroxy-3-methoxycinnamic acid, is a derivative of cinnamic acid (also known as cinnamic acid, 3-phenyl-2-propenoic acid, molecular structure). Ferulic acid (sodium ferulate) has antiplatelet aggregation, inhibits platelet serotonin release, inhibits platelet thromboxane A2 (TXA2) production, enhances prostaglandin activity, provides analgesia, and relieves vasospasm. It is a key raw material for the production of medications used to treat cardiovascular and cerebrovascular diseases and leukopenia, such as Xinxuekang, Limai Capsules, and Taitai Oral Liquid. It also has health-enhancing and skin-protecting effects in the human body. However, the large-scale extraction and preparation of ferulic acid currently faces certain limitations, making it difficult to meet the growing market demand.

[0003] In the current purification field, silica gel chromatography columns are generally used for separation. Ferulic acid can also be purified using similar applications. For example, in the prior art, different functional groups are bonded to the surface of silica spheres to meet the needs of separating different substances, such as long-chain silyldimethylsilane-bonded silica gels (such as C18 columns) used as reversed-phase columns. Furthermore, to further expand the application range and acid resistance of chromatographic packings, a combination of multiple chromatographic packings is often used. For example, Chinese invention patent CN101721980A discloses a mixed liquid chromatography column and chromatographic column using a mixture of long-chain alkyldimethylsilane-bonded silica gel and amidesilane-bonded silica gel, which can separate compounds containing both highly hydrophilic and highly lipophilic compounds. Another example is a liquid chromatography column composite packing and its application disclosed in Chinese invention patent CN113063884A, which uses a mixture of octadecylsilane-bonded silica gel and naloxone-bonded silica gel to simultaneously separate polar and non-polar components while meeting the pH range of 1 to 10. Chinese invention patent CN115364829A discloses an acid-resistant silica gel chromatographic column filler, a preparation method, and an application. The silica spheres are subjected to high-temperature treatment, dispersed with toluene, and then heated to 50-60°C. Boron trifluoride etherate and hydrophobic hydrocarbon glycidyl ether are added. After reacting for 4-6 hours, the toluene is removed to obtain an acid-resistant silica gel chromatographic column filler. It still has stable sample separation and measurement capabilities under the condition of a minimum pH of 0.5. Although the existing mixing process can expand the scope of application of chromatographic fillers and improve acid resistance, there are generally problems such as complex processes and high pressure on material quality control. It can be seen that it is necessary to provide a preparation method using molecularly imprinted polymer microspheres with good adsorption properties, and effectively apply them to the preparation of chromatographic composite fillers. Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of this application is to provide trans-ferulic acid molecularly imprinted polymer microspheres and their preparation method and application, aiming to solve the problems of complex process and high material quality control pressure in existing chromatographic fillers.

[0005] The technical solution of this application is as follows: In a first aspect, the present application provides a method for preparing trans-ferulic acid molecularly imprinted polymer microspheres, which comprises the following steps: Dissolve trans-ferulic acid in a solvent, add methacrylic acid and mix; Then, a cross-linking agent and azobisisobutyronitrile were added, nitrogen was introduced, and the mixture was sealed to obtain a molecularly imprinted polymer mixed solution; adding the molecularly imprinted polymer mixed solution dropwise into the surfactant aqueous solution through a constant pressure dropping funnel, stirring the mixture at the reaction temperature, and separating to obtain polymer microspheres; The trans-ferulic acid is washed away from the polymer microspheres, and then the polymer microspheres are washed until neutral, and dried to obtain the trans-ferulic acid molecularly imprinted polymer microspheres.

[0006] Furthermore, the surfactant aqueous solution includes span20 and tween20 accounting for 10% of the total volume, and the volume ratio of span20 to tween20 is 1:1; The dropping speed is 20 drops / min.

[0007] Furthermore, the reaction temperature is 60-70°C, and the stirring reaction time is 20-25h; After the stirring reaction, the mixture was allowed to stand at 85-95° C. for 2 h.

[0008] Furthermore, every 100-800 mg of trans-ferulic acid is dissolved in 10-50 mL of the solvent, wherein the solvent is one or more of anhydrous ethanol, acetonitrile and methanol; The cross-linking agent is ethylene glycol dimethacrylate; In the molecularly imprinted polymer mixed solution, 0.1-0.5 mL of methacrylic acid, 0.6-1.0 mL of ethylene glycol dimethacrylate and 50-150 mg of azobisisobutyronitrile are used for every 100-800 mg of trans-ferulic acid.

[0009] In a second aspect, the present application further provides a trans-ferulic acid molecularly imprinted polymer microsphere, wherein the microsphere is prepared by the preparation method of the trans-ferulic acid molecularly imprinted polymer microsphere as described in the first aspect.

[0010] In a third aspect, the present application further provides an application of the trans-ferulic acid molecularly imprinted polymer microspheres as described in the second aspect, wherein the trans-ferulic acid molecularly imprinted polymer microspheres are applied to separate and purify trans-ferulic acid by preparative chromatography, comprising the following steps: loading the molecularly imprinted polymer microspheres into a preparative chromatography column; Prepare a crude trans-ferulic acid solution as the loading solution; The sample solution is loaded onto the preparative chromatographic column, and eluted with a mixture of methanol-formic acid aqueous solution with gradient concentrations in sequence. The eluate is collected and concentrated under reduced pressure to obtain trans-ferulic acid.

[0011] Furthermore, the preparative chromatographic column is also filled with modified silica gel chromatographic column filler, and the mass ratio of the modified silica gel chromatographic column filler to the molecularly imprinted polymer microspheres is 1:9-9:1; The particle size of the trans-ferulic acid molecularly imprinted polymer microspheres is 100 mesh, and the particle size of the modified silica gel chromatographic column filler is 100 μm; The preparation method of the modified silica gel chromatographic column filler comprises the following steps: Treat the silicon spheres at 500-600°C for 2-4 hours and then disperse them in toluene; Raising the temperature to 50-60° C., adding a silane coupling agent, reacting for 4-6 hours, and removing the toluene to obtain the modified silica gel chromatographic column filler; The ratio of the silicon balls to the toluene is 1 g: (10-20) ml, and the mass ratio of the silicon balls to the silane coupling agent is 100:5-15.

[0012] Furthermore, the gradient concentration of methanol-formic acid aqueous solution includes methanol-formic acid aqueous solution with a volume proportion of methanol of 28-32%, 38-42%, 60-65%, 75-80%, and 100% respectively; In the formic acid aqueous solution, the volume proportion of formic acid is one thousandth.

[0013] Furthermore, the solid-liquid ratio of the trans-ferulic acid molecularly imprinted polymer microspheres to the trans-ferulic acid crude product solution is 1:100-500 (g / ml).

[0014] Furthermore, the concentration of trans-ferulic acid in the crude trans-ferulic acid solution is 1000-2000 mg / L.

[0015] Beneficial effects: The trans-ferulic acid molecularly imprinted polymer microspheres provided in the present application have good specific and selective adsorption performance of the target trans-ferulic acid, and can achieve efficient and highly selective separation and purification of trans-ferulic acid through preparative chromatography, and can effectively separate and obtain trans-ferulic acid with high purity and recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is a schematic diagram of the process for preparing chromatographic separation and purification of trans-ferulic acid in this application.

[0017] Figure 2 This is an electron microscope image of the trans-ferulic acid molecularly imprinted polymer microspheres prepared in Example 1 of the present application.

[0018] Figure 3 This is the rapid preparation chromatogram of Example 1 of the present application.

[0019] Figure 4 This is the rapid preparation chromatogram of Example 2 of the present application.

[0020] Figure 5 This is the rapid preparation chromatogram of Example 3 of the present application.

[0021] Figure 6 This is the rapid preparation chromatogram of Example 4 of the present application.

[0022] Figure 7 This is the ultra-high performance liquid chromatogram of trans-ferulic acid.

[0023] Figure 8 This is the ultra-high performance liquid chromatogram of trans-ferulic acid collected in Example 4 of the present application.

[0024] Figure 9 This is the NMR carbon spectrum of trans-ferulic acid collected in Example 4 of the present application. DETAILED DESCRIPTION

[0025] The present application provides trans-ferulic acid molecularly imprinted polymer microspheres and their preparation methods and applications. To make the purpose, technical solutions, and effects of the present application clearer and more specific, the present application is further described below. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present application and are not intended to limit the present application.

[0026] The present application provides a method for preparing trans-ferulic acid molecularly imprinted polymer microspheres, which comprises the following steps: S1. dissolving trans-ferulic acid in a solvent, adding methacrylic acid and mixing; S2, adding a cross-linking agent and azobisisobutyronitrile, passing nitrogen, and sealing to obtain a molecularly imprinted polymer mixed solution; S3, adding the molecularly imprinted polymer mixed solution dropwise to the surfactant aqueous solution through a constant pressure dropping funnel, stirring the reaction at the reaction temperature, and separating to obtain polymer microspheres; S4. Wash the polymer microspheres to remove trans-ferulic acid, then wash the polymer microspheres until they are neutral, and dry them to obtain trans-ferulic acid molecularly imprinted polymer microspheres.

[0027] The preparation method provided in the present application can produce trans-ferulic acid molecularly imprinted polymer microspheres with good specific and selective adsorption properties, can specifically adsorb the target trans-ferulic acid, and can achieve efficient and highly selective separation and purification of trans-ferulic acid.

[0028] Furthermore, the cross-linking agent is ethylene glycol dimethacrylate, and the solvent is one or more of anhydrous ethanol, acetonitrile and methanol.

[0029] This application uses trans-ferulic acid as a template molecule, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linking agent, and adopts emulsion polymerization technology to prepare trans-ferulic acid molecularly imprinted polymer microspheres. Among them, molecular imprinting technology is to generate an imprinted polymer with a specific spatial structure and functional groups of different sizes through a specific polymerization reaction involving a template molecule, a functional monomer and a cross-linking agent. Then, by separating the template molecule from the imprinted polymer, adsorption sites that match the template molecule are formed in the imprinted polymer. These sites constitute "memory" holes that are consistent with the spatial structure of the template molecule and have high selectivity, giving the polymer unique selectivity and recognition ability for the target molecule. A new method is provided for the preparation of chromatographic fillers, and the MIPs chromatographic fillers prepared by this method have broad application potential and development prospects in the field of separation science.

[0030] Specifically, in step S1, trans-ferulic acid is dissolved in a solvent by dissolving 100-800 mg of trans-ferulic acid in 10-50 ml of solvent, and magnetic stirring can be performed for 1 hour to ensure complete dissolution.

[0031] In step S1, methacrylic acid is added and mixed by magnetic stirring at room temperature for 2-6 hours. In this application, magnetic stirring is used to achieve good dissolution and dispersion.

[0032] Furthermore, the surfactant aqueous solution includes span20 and tween20 accounting for 10% of the total volume, and the volume ratio of span20 to tween20 is 1:1; the dripping rate is 20 drops / min. In the present application, the added span20 and tween20 as surfactants will affect the size of the polymer microspheres formed by dripping. The present application controls the appropriate amount and ratio of span20 and tween20, and at the same time controls the dripping rate, which is conducive to the formation of polymer microspheres with good surface properties and the formation of cavities that can adsorb trans-ferulic acid, which is beneficial to the separation and purification of trans-ferulic acid.

[0033] Specifically, in step S2, the nitrogen is introduced by introducing nitrogen for 5 minutes to exclude other gases and then sealing the chamber to carry out the reaction under a nitrogen atmosphere.

[0034] Furthermore, in step S3, the stirring reaction is carried out by magnetic stirring at a speed of 500-800 rpm / min for 20-25 hours at a reaction temperature of 60-70°C. After the reaction is completed, the solid is separated to obtain polymer microspheres. In the present application, the magnetic stirring reaction is used to ensure that the droplets are dispersed evenly in a timely manner. By controlling the reaction temperature at a suitable level, the droplets react to form polymer microspheres.

[0035] Furthermore, after the stirring reaction in step S3 is completed, the temperature is raised to 85-95° C. and then allowed to stand for 2 hours.

[0036] In the present application, by increasing the temperature and standing for a period of time after the reaction is complete, the formed polymer microspheres are hardened, which is beneficial to stabilizing the specific adsorption structure therein. At the same time, when a specific mesh size is required in the subsequent use, even after the grinding operation, it can avoid major damage to the specific structure. When compounded with modified silica gel filler to fill the column, it is also beneficial to improve the separation and purification effect.

[0037] Furthermore, in step S4, the step of washing the polymer microspheres to remove trans-ferulic acid includes: washing the polymer microspheres with ethanol by Soxhlet extraction 8-10 times, then vacuum drying at 50°C for 16-36 hours, and then repeatedly eluting the polymer microspheres with anhydrous ethanol-organic acid aqueous solution with a volume ratio of 8-10:1-2 to remove the template molecule trans-ferulic acid remaining in the polymer microspheres, until no trans-ferulic acid is detected in the eluate. Then, the polymer microspheres are washed with distilled water until the washing solution is neutral; and then dried by vacuum heating at a heating temperature of 50°C.

[0038] The preparation method provided in the present application provides trans-ferulic acid molecularly imprinted polymer microspheres having a high adsorption capacity, and can selectively adsorb trans-ferulic acid without adsorbing other water-soluble impurities, thereby efficiently and selectively separating and purifying trans-ferulic acid, which is beneficial to improving the recovery rate and isolating high-purity trans-ferulic acid.

[0039] The present application also provides trans-ferulic acid molecularly imprinted polymer microspheres, which are prepared by the above-mentioned preparation method of trans-ferulic acid molecularly imprinted polymer microspheres. The provided trans-ferulic acid molecularly imprinted polymer microspheres have good adsorption performance, high efficiency and good stability.

[0040] Reference Figure 1 The present application also provides an application of the trans-ferulic acid molecularly imprinted polymer microspheres as described above, wherein the trans-ferulic acid molecularly imprinted polymer microspheres are applied to separate and purify trans-ferulic acid by preparative chromatography, comprising the following steps: (I) loading the molecularly imprinted polymer microspheres onto a preparative chromatography column; (II) preparing a crude trans-ferulic acid solution as a loading solution; (III) The sample solution is loaded onto a preparative chromatography column, the eluate is collected after elution, and the eluate is concentrated under reduced pressure to collect trans-ferulic acid.

[0041] The present application separates and purifies trans-ferulic acid by preparative chromatography, specifically by rapid preparative chromatography. Using molecularly imprinted polymer microspheres as preparative chromatography column fillers, the target product, trans-ferulic acid, can be separated and purified by preparative chromatography, achieving the purpose of efficiently separating and purifying trans-ferulic acid from crude product solutions such as extracts or fermentation broths.

[0042] Furthermore, in step (I), the prepared chromatographic column is also filled with modified silica gel chromatographic column filler, and the mass ratio of molecularly imprinted polymer microspheres to modified silica gel chromatographic column filler is 1:9-9:1. The preparation method of the modified silica gel chromatographic column filler comprises the following steps: Treat the silicon spheres at 500-600°C for 2-4 hours and then disperse them in toluene; The temperature was raised to 50-60° C., a silane coupling agent was added, the reaction was carried out for 4-6 hours, and the toluene was removed to obtain a modified silica gel chromatographic column filler.

[0043] The ratio of silicon balls to toluene is 1g: (10-20) ml, and the mass ratio of silicon balls to silane coupling agent is 100: 5-15; The silane coupling agent used is KH570 silane coupling agent.

[0044] In the present application, impurities on the surface of the silicon spheres are removed by high-temperature treatment at 500-600° C. for 2-4 hours, so as to ensure the subsequent modification effect of the silane coupling agent.

[0045] In the present application, by modifying the silica spheres, the modified silica gel chromatographic filler obtained can adapt to the preparative chromatography elution system with a high water content, changing the organic solvent-resistant system to a water-resistant system, so that the service life of the prepared chromatographic column is longer and it is more suitable for the preparative separation of biosynthetic trans-ferulic acid fermentation broth. More specifically, the present application uses trans-ferulic acid molecular polymer microspheres and modified silica gel chromatographic fillers as composite fillers to achieve a good extraction effect on trans-ferulic acid. After mixing them in proportion, they can be loaded into a preparative chromatography column, and then 0.2-20 mL of a 1 g / L trans-ferulic acid solution (the solvent is methanol) is prepared. After ultrasonic degassing, the sample is loaded onto the preparative chromatography column, and trans-ferulic acid is separated by preparative chromatography to verify the separation performance.

[0046] Specifically, the molecularly imprinted polymer microspheres prepared in this application have a strong selective effect on trans-ferulic acid. When trans-ferulic acid molecularly imprinted polymer microspheres are used alone as a preparative chromatographic column, elution is normal when using a trans-ferulic acid standard solution. However, when using a trans-ferulic acid fermentation broth, a sudden increase in column pressure occurs when only the molecularly imprinted polymer microspheres are used, and severe blockage may occur. By adding modified silica gel chromatographic fillers and using them in an appropriate ratio, the preparative chromatographic column pressure is stable under the same elution gradient. When a single ferulic acid molecularly imprinted microsphere is used to prepare a chromatographic column, the column pressure will be high after repeated injection of trans-ferulic acid fermentation broth for 3-4 times. However, after using the composite filler, the trans-ferulic acid fermentation broth can be injected more than 20-50 times, and the chromatographic peak shape remains good.

[0047] Furthermore, the particle size of the trans-ferulic acid molecularly imprinted polymer microspheres is 100 mesh, and the particle size of the modified silica gel chromatographic column filler is 100 μm. In the present application, for the trans-ferulic acid fermentation broth and the chromatographic column size, the particle size of the molecularly imprinted polymer microspheres is controlled to 100 mesh, and a modified silica gel chromatographic column filler of a specific particle size is used to facilitate the smooth loading and elution, and the peak elution time and peak symmetry during the preparative chromatography are better. Specifically, the size of the preparative chromatographic column used can be 330 g (539 mL), I.D60.6 × H187, 40 g (70 mL), I.D26.8 × H125, 12 g (27 mL), I.D21.8 × H76.

[0048] Furthermore, in step (II), the solid-to-liquid ratio of the trans-ferulic acid molecularly imprinted polymer to the crude trans-ferulic acid solution is 1:100-500 (g / ml). In the present application, controlling the solid-to-liquid ratio is beneficial for maintaining the adsorption performance of the trans-ferulic acid molecularly imprinted polymer microspheres, thereby improving the separation and purification effect.

[0049] Furthermore, in step (II), the crude trans-ferulic acid solution can be an extract of a natural trans-ferulic acid product, for example. Preferably, it is a trans-ferulic acid fermentation broth, which can be produced by fermentation using a trans-ferulic acid-producing bacterium. Although trans-ferulic acid fermentation broth can easily cause column pressure and clogging issues, it can provide a high yield when used in large-scale production, facilitating large-scale separation and purification of trans-ferulic acid.

[0050] Specifically, the preparation of trans-ferulic acid by microbial fermentation currently has the advantages of high efficiency, economy and environmental protection, but the fermentation broth is generally complex in composition, which will affect the extraction effect of the target substance, and the composite filler provided by the present application can be used in the fermentation broth with complex composition. By using modified silica gel filler to remove some impurities in the fermentation broth, it is avoided to affect the specific adsorption performance of the molecularly imprinted polymer microspheres. Moreover, the molecularly imprinted polymer microspheres using a single emulsion polymerization have strong adsorption capacity and small particle size. After filling the chromatographic column, it is easy to have high pressure and high column pressure. By combining molecularly imprinted polymer microspheres and modified silica gel chromatographic column fillers for use, in addition to improving purity and recovery, the present application can also improve the problem of high quality control pressure, and can be adsorbed in a lower pressure range, which is conducive to large-scale separation and purification.

[0051] Furthermore, the concentration of trans-ferulic acid in the crude trans-ferulic acid solution is 1000-5000 mg / L, more preferably 1000-2000 mg / L.

[0052] Furthermore, in step (III), elution is performed using, in sequence, methanol-formic acid aqueous solutions containing 28-32%, 38-42%, 60-65%, 75-80%, and 100% methanol by volume, respectively; the formic acid in the formic acid aqueous solution accounts for 1 / 1000 by volume. Specifically, after gradient elution, eluates of corresponding concentrations can be collected, and trans-ferulic acid can be collected by concentrating the eluate. Preferably, collecting an eluate containing 60-65% methanol by volume in a methanol-formic acid aqueous solution is more efficient and produces higher purity, with the chromatographic purity of trans-ferulic acid reaching over 98%.

[0053] Furthermore, in step (III), the step of collecting and obtaining trans-ferulic acid comprises: The concentrate was concentrated under reduced pressure to obtain a concentrate, which was evaporated to dryness to obtain a solid. The solid was dissolved in ultrapure water to a final concentration of trans-ferulic acid of 9 g / L, and hydrochloric acid was added to adjust the pH to 3. Anhydrous ethanol was added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1, and the mixture was stirred at 20°C for 5 hours and then allowed to stand for 24 hours to separate the trans-ferulic acid.

[0054] The application method of the trans-ferulic acid molecularly imprinted polymer microspheres provided in the present application has simple process operation, low cost, is convenient for large-scale industrial production, and is of great significance for promoting the industrial development of trans-ferulic acid.

[0055] The following is further described with reference to specific examples.

[0056] The trans-ferulic acid-producing bacteria used in this example were obtained from the research group of Xiaolin Shen at Beijing University of Chemical Technology. For details, please refer to the literature (Targeting cofactors regeneration in methylation and hydroxylation for high-level production of ferulic acid, Xiaolin Shen, Beijing University of Chemical Technology, etc.).

[0057] The fermentation medium and enrichment medium used in the examples of the present application were both M9 medium, including: 2 mL of 1 M MgSO4, 0.1 mL of 1 M CaCl2, 200 mL of 5×M9 salt solution (Na2PO4·7H2O: 12.8 g, KH2PO4: 3.0 g, NaCl: 0.5 g, NH4Cl: 1.0 g, dissolved in 200 mL of double distilled water, sterilized at 121°C for 15 min.); 20 mL of 20% glucose solution, and 1000 mL of sterilized double distilled water.

[0058] Example 1 The steps of separating and purifying trans-ferulic acid by the preparative chromatography method of Example 1 include: (1) Preparation of trans-ferulic acid fermentation broth: Trans-ferulic acid fermentation broth was prepared using trans-ferulic acid-producing bacteria. The specific method was as follows: Escherichia coli was inoculated into a fermentation medium at a rate of 5%, and cultured at 37°C for 3 days with shaking to obtain a seed liquid; then, the seed liquid was inoculated into an enrichment medium at a rate of 10%, and the precursors required for fermentation were added, and cultured at 37°C for 5 days with shaking. The culture liquid was centrifuged at 5°C and 8000 r / min for 15 minutes, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 1000 mg / L.

[0059] (2) Preparative chromatographic separation: Preparation of trans-ferulic acid molecularly imprinted polymer microspheres, the preparation method comprising the following steps: 100 mg of trans-ferulic acid and 0.1 mL of methacrylic acid were added to 50 mL of anhydrous ethanol and magnetically stirred at room temperature for 6 h. 0.6 mL of ethylene glycol dimethacrylate and 50 mg of azobisisobutyronitrile were then added, nitrogen was introduced for 5 min, and the mixture was sealed to obtain a molecularly imprinted polymer mixed solution. The molecularly imprinted polymer mixed solution was added to a constant pressure dropping funnel and added dropwise to a mixed aqueous solution of span20 and tween20 at a rate of 20 drops / min (the total volume proportion of span20 and tween20 was 10%, and the volume ratio of span20 to tween20 was 1:1). , magnetic stirring is controlled at 600 rpm / min, the temperature is controlled at a reaction temperature of 65°C, the reaction is carried out with magnetic stirring for 20 hours, and the mixture is allowed to stand at 90°C for 2 hours. The solid is separated to obtain polymer microspheres; the polymer microspheres are washed with ethanol 8 times with Soxhlet extraction, and vacuum dried at 50°C for 36 hours. The dried polymer microspheres are repeatedly eluted with anhydrous ethanol-formic acid aqueous solution with a volume ratio of 9:1 (the volume proportion of formic acid is one thousandth) to remove the template molecule trans-ferulic acid in the polymer microspheres until no trans-ferulic acid is detected in the eluate. Finally, the eluted polymer microspheres are washed with distilled water until neutral, and dried at 50°C under vacuum for 12 hours to obtain the molecularly imprinted polymer microspheres of Example 1, which are ground to 100 mesh for use. The molecularly imprinted polymer prepared in Example 1 is shown in the scanning electron microscope image as shown below. Figure 2 As shown, the diameter is between 10-30nm.

[0060] 12 g of the trans-ferulic acid molecularly imprinted polymer microspheres prepared above were used as filler and loaded into a preparative chromatography column. The preparative chromatography column had a size of 12 g (27 mL) and an I.D. of 21.8 × H. The preparative chromatography column was installed on a preparative chromatograph. The trans-ferulic acid fermentation broth was separated and purified by preparative chromatography. The trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 1000 mg / L was loaded into the preparative chromatography column. The solid-to-liquid ratio of the trans-ferulic acid molecularly imprinted polymer microspheres to the trans-ferulic acid fermentation broth in the preparative chromatography column was 1:100 (g / ml). The broth was then eluted with a mixture of methanol and formic acid aqueous solution in a gradient of concentrations of 10 column volumes. The gradient elution conditions for the preparative chromatography of Example 1 are shown in Table 1: Table 1

[0061] The mobile phase in Table 1 is a mixture of methanol and formic acid aqueous solution (formic acid accounts for 1 / 1000 of the volume of formic acid aqueous solution). Figure 3 The eluate containing 62% methanol by volume was collected and concentrated under reduced pressure (the vacuum degree of the reduced pressure concentration was -0.9 MPa and the rotation speed was 170 rpm) to obtain a trans-ferulic acid sample with a chromatographic purity of more than 99.8%.

[0062] In Example 1, after sample loading and subsequent separation and purification, the column pressure easily increased, reaching 245 Pa and triggering an overpressure alarm. After adjusting the flow rate to 3 ml / min, the elution response in the chromatogram was too low. Reducing the packing weight resulted in unsatisfactory separation results, with R resolution far less than 1.

[0063] Example 2 The steps of separating and purifying trans-ferulic acid by the preparative chromatography method of Example 2 include: (1) Preparation of trans-ferulic acid fermentation broth: Trans-ferulic acid fermentation broth was prepared using trans-ferulic acid-producing bacteria. The specific method was as follows: Escherichia coli was inoculated into a fermentation medium at a rate of 5%, and cultured at 37°C for 3 days with shaking to obtain a seed liquid; then, the seed liquid was inoculated into an enrichment medium at a rate of 10%, and the precursors required for fermentation were added, and cultured at 37°C for 5 days with shaking. The culture liquid was centrifuged at 5°C and 8000 r / min for 15 minutes, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 1000 mg / L.

[0064] (2) Preparative chromatographic separation: Preparation of trans-ferulic acid molecularly imprinted polymer microspheres, the preparation method comprising the following steps: 200 mg of trans-ferulic acid and 0.3 mL of methacrylic acid were added to 50 mL of methanol and magnetically stirred at room temperature for 6 h. Then, 0.8 mL of ethylene glycol dimethacrylate and 80 mg of azobisisobutyronitrile were added, nitrogen was introduced for 5 min, and the mixture was sealed to obtain a molecularly imprinted polymer mixed solution. The molecularly imprinted polymer mixed solution was added to a constant pressure dropping funnel and added dropwise to a mixed aqueous solution of span20 and tween20 (the total volume proportion of span20 and tween20 was 10%, and the volume proportion of span20 and tween20 was 1:1) at a rate of 20 drops / min. The magnetic stirring was controlled at 600 rpm / min. n, the reaction temperature was controlled at 65° C., the reaction was carried out under magnetic stirring for 25 h, and the reaction was allowed to stand at 90° C. for 2 h, and the solid was separated to obtain polymer microspheres; the polymer microspheres were washed with ethanol using Soxhlet extraction 8 times, and vacuum-dried at 50° C. for 36 h. The dried polymer microspheres were repeatedly eluted with anhydrous ethanol-formic acid aqueous solution with a volume ratio of 8:2 (the volume proportion of formic acid was 1 / 1000) to remove the template molecule trans-ferulic acid in the polymer microspheres until no trans-ferulic acid was detected in the eluate. Finally, the eluted polymer microspheres were washed with distilled water until neutral, and dried under vacuum at 50° C. for 12 h to obtain the trans-ferulic acid molecularly imprinted polymer microspheres of Example 2, which were ground to 100 mesh for later use.

[0065] A modified silica gel chromatographic filler was prepared. The preparation method included the following steps: treating silica spheres with a particle size of 100 μm at 500° C. for 3 h; dispersing 10 g of the high-temperature treated silica spheres in 120 ml of toluene, heating the mixture to 55° C., adding 1 g of a KH570 silane coupling agent, reacting for 5 h, and removing the toluene to obtain a modified silica gel chromatographic column filler.

[0066] The trans-ferulic acid molecularly imprinted polymer microspheres prepared above and modified silica gel chromatographic filler were loaded as composite fillers into a preparative chromatographic column. The size of the preparative chromatographic column was 12 g (27 mL), I.D21.8×H76, the loading amount was 12 g, and the mass ratio of trans-ferulic acid molecularly imprinted polymer microspheres to modified silica gel chromatographic filler was 1:9. The trans-ferulic acid fermentation broth was separated and purified by preparative chromatography. The trans-ferulic acid concentration of the trans-ferulic acid fermentation broth was 1000 mg / L and loaded into the preparative chromatographic column. The solid-liquid ratio of trans-ferulic acid molecularly imprinted polymer microspheres to trans-ferulic acid fermentation broth in the preparative chromatographic column was 1:100 (g / ml). The column was then eluted with a mixture of methanol-formic acid aqueous solution in a gradient concentration of 10 column volumes. The gradient elution conditions for preparative chromatography in Example 2 are shown in Table 2: Table 2

[0067] The mobile phase in Table 2 is a mixture of methanol and formic acid aqueous solution (the volume proportion of formic acid in the formic acid aqueous solution is 1 / 1000). The column pressure during elution in Example 2 is below 80 Pa, and the preparative chromatographic results are as follows: Figure 4 The eluate containing 62% methanol by volume was collected and concentrated under reduced pressure (the vacuum degree of the reduced pressure concentration was -0.9 MPa and the rotation speed was 170 rpm) to obtain a trans-ferulic acid sample with a chromatographic purity of more than 99.8%.

[0068] Example 3 The steps of separating and purifying trans-ferulic acid by preparative chromatography in Example 3 include: (1) Preparation of trans-ferulic acid fermentation broth: Trans-ferulic acid fermentation broth was prepared using trans-ferulic acid-producing bacteria. The specific method was as follows: Escherichia coli was inoculated into a fermentation medium at a rate of 5%, and cultured at 37°C for 3 days with shaking to obtain a seed solution; then, the seed solution was inoculated into an enrichment medium at a rate of 10%, and the precursors required for fermentation were added, and cultured at 37°C for 5 days with shaking. The culture solution was centrifuged at 5°C and 8000 r / min for 15 minutes, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 2000 mg / L.

[0069] (2) Preparative chromatographic separation: Preparation of trans-ferulic acid molecularly imprinted polymer microspheres, the preparation method comprising the following steps: 200 mg of trans-ferulic acid and 0.3 mL of methacrylic acid were added to 50 mL of methanol and magnetically stirred at room temperature for 6 h. Then, 0.8 mL of ethylene glycol dimethacrylate and 80 mg of azobisisobutyronitrile were added, nitrogen was introduced for 5 min, and the mixture was sealed to obtain a molecularly imprinted polymer mixed solution. The molecularly imprinted polymer mixed solution was added to a constant pressure dropping funnel and added dropwise to a mixed aqueous solution of span20 and tween20 (the total volume proportion of span20 and tween20 was 10%, and the volume proportion of span20 and tween20 was 1:1) at a rate of 20 drops / min. The magnetic stirring was controlled at 600 rpm / min. n, the reaction temperature was controlled at 65° C., the reaction was carried out under magnetic stirring for 25 h, and the mixture was allowed to stand at 90° C. for 2 h, and the solid was separated to obtain polymer microspheres; the polymer microspheres were washed with ethanol using Soxhlet extraction 8 times, and vacuum-dried at 50° C. for 36 h. The dried polymer microspheres were repeatedly eluted with anhydrous ethanol-formic acid aqueous solution with a volume ratio of 8:2 (the volume proportion of formic acid was 1 / 1000) to remove the template molecule trans-ferulic acid in the polymer microspheres until no trans-ferulic acid was detected in the eluate. Finally, the eluted polymer microspheres were washed with distilled water until neutral, and dried under vacuum at 50° C. for 12 h to obtain the trans-ferulic acid molecularly imprinted polymer microspheres of Example 3, which were ground to 100 mesh for later use.

[0070] A modified silica gel chromatographic filler was prepared. The preparation method included the following steps: treating silicon spheres with a particle size of 100 μm at 500° C. for 3 h; dispersing 10 g of the high-temperature treated silicon spheres in 150 ml of toluene, heating the mixture to 55° C., adding 1 g of a KH570 silane coupling agent, reacting for 5 h, and removing the toluene to obtain a modified silica gel chromatographic column filler.

[0071] The prepared trans-ferulic acid molecularly imprinted polymer microspheres and modified silica gel chromatographic filler were loaded as a composite filler into a preparative chromatography column. The preparative chromatography column had a size of 12 g (27 mL), an I.D. of 21.8 × H76, a loading capacity of 6 g, and a mass ratio of trans-ferulic acid molecularly imprinted polymer microspheres to modified silica gel chromatographic filler of 1:1. The trans-ferulic acid fermentation broth was separated and purified by preparative chromatography. The trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 2000 mg / L was loaded onto the preparative chromatography column. The solid-to-liquid ratio of trans-ferulic acid molecularly imprinted polymer microspheres to trans-ferulic acid fermentation broth in the preparative chromatography column was 1:100 (g / ml). The broth was then eluted sequentially with 10 column volumes of a methanol-formic acid aqueous solution mixture (the volume ratio of methanol to formic acid aqueous solution in the methanol-formic acid aqueous solution mixture was 38:62, and the volume ratio of formic acid in the formic acid aqueous solution was 1 / 1000).

[0072] The column pressure during elution in Example 3 was below 80 Pa, and the preparative chromatographic results were as follows: Figure 5 The eluate was collected and concentrated under reduced pressure (the vacuum degree of the reduced pressure concentration was -0.9 MPa and the rotation speed was 170 rpm) to obtain a trans-ferulic acid sample with a chromatographic purity of more than 99.8%.

[0073] Example 4 The steps of separating and purifying trans-ferulic acid by preparative chromatography in Example 4 include: (1) Preparation of trans-ferulic acid fermentation broth: Trans-ferulic acid fermentation broth was prepared using trans-ferulic acid-producing bacteria. The specific method was as follows: Escherichia coli was inoculated into a fermentation medium at a rate of 5%, and cultured at 37°C for 3 days with shaking to obtain a seed solution; then, the seed solution was inoculated into an enrichment medium at a rate of 10%, and the precursors required for fermentation were added, and cultured at 37°C for 5 days with shaking. The culture solution was centrifuged at 5°C and 8000 r / min for 15 minutes, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 2000 mg / L.

[0074] (2) Preparative chromatographic separation: Preparation of trans-ferulic acid molecularly imprinted polymer microspheres, the preparation method comprising the following steps: 200 mg of trans-ferulic acid and 0.3 mL of methacrylic acid were added to 50 mL of methanol and magnetically stirred at room temperature for 6 h. Then, 0.8 mL of ethylene glycol dimethacrylate and 80 mg of azobisisobutyronitrile were added, nitrogen was introduced for 5 min, and the mixture was sealed to obtain a molecularly imprinted polymer mixed solution. The molecularly imprinted polymer mixed solution was added to a constant pressure dropping funnel and added dropwise to a mixed aqueous solution of span20 and tween20 (the total volume proportion of span20 and tween20 was 10%, and the volume proportion of span20 and tween20 was 1:1) at a rate of 20 drops / min. The magnetic stirring was controlled at 600 rpm / min. , the reaction temperature was controlled at 65°C, the reaction was carried out with magnetic stirring for 25 hours, and the mixture was allowed to stand at 90°C for 2 hours, and the solid was separated to obtain polymer microspheres; the polymer microspheres were washed with ethanol 8 times with Soxhlet extraction, and vacuum-dried at 50°C for 36 hours. The dried polymer microspheres were repeatedly eluted with anhydrous ethanol-formic acid aqueous solution with a volume ratio of 10:1 (the volume proportion of formic acid was 1 / 1000) to remove the template molecule trans-ferulic acid in the polymer microspheres until no trans-ferulic acid was detected in the eluate. Finally, the eluted polymer microspheres were washed with distilled water until neutral, and dried under vacuum at 50°C for 12 hours to obtain the trans-ferulic acid molecularly imprinted polymer microspheres of Example 4, which were ground to 100 mesh for later use.

[0075] A modified silica gel chromatographic filler was prepared. The preparation method included the following steps: treating silica spheres with a particle size of 100 μm at 500° C. for 3 h; dispersing 10 g of the high-temperature treated silica spheres in 160 ml of toluene, heating the mixture to 55° C., adding 1 g of a KH570 silane coupling agent, reacting for 5 h, and removing the toluene to obtain a modified silica gel chromatographic column filler.

[0076] The prepared trans-ferulic acid molecularly imprinted polymer microspheres and modified silica gel chromatographic filler were loaded as a composite filler into a preparative chromatography column. The preparative chromatography column had dimensions of 12 g (27 mL), an I.D. of 21.8 × H76, a loading capacity of 12 g, and a mass ratio of trans-ferulic acid molecularly imprinted polymer microspheres to modified silica gel chromatographic filler of 8:2. The trans-ferulic acid fermentation broth was separated and purified by preparative chromatography. The trans-ferulic acid fermentation broth, with a trans-ferulic acid concentration of 2000 mg / L, was loaded onto the preparative chromatography column. The solid-to-liquid ratio of trans-ferulic acid molecularly imprinted polymer microspheres to trans-ferulic acid fermentation broth in the preparative chromatography column was 1:100 (g / ml). The broth was then eluted sequentially with 10 column volumes of a methanol-formic acid aqueous solution mixture (the methanol-formic acid aqueous solution mixture had a volume ratio of methanol to formic acid aqueous solution of 38:62, with formic acid accounting for 1 / 1000 by volume in the formic acid aqueous solution).

[0077] The column pressure during elution in Example 4 was below 80 Pa, and the preparative chromatographic results were as follows: Figure 6The eluate was collected and concentrated under reduced pressure (the vacuum degree of the reduced pressure concentration was -0.9 MPa and the rotation speed was 170 rpm) to obtain a trans-ferulic acid sample with a chromatographic purity of more than 99.8%. After concentration and recovery, the recovery rate could reach more than 90%.

[0078] The purity of trans-ferulic acid collected in Example 4 was tested using an ultra-high performance liquid chromatography (UPLC) instrument (Waters, 1.7µm, 2.1*100mm column, 2µL injection volume, 320nm wavelength, and a flow rate of 0.3ml / min. The ultra-high performance liquid chromatography (UPLC) of the trans-ferulic acid standard is shown in Figure 1. Figure 7 As shown, the ultra-high performance liquid chromatography chromatogram of trans-ferulic acid obtained by preparative chromatography separation and purification in Example 4 is as follows Figure 8 As shown, the NMR carbon spectrum is as follows Figure 9 shown.

[0079] It should be understood that the application of this application is not limited to the above examples. For ordinary technicians in this field, they can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of this application.

Claims

1. A method for preparing trans-ferulic acid molecularly imprinted polymer microspheres, characterized in that: The following steps are involved: Dissolve trans-ferulic acid in a solvent, add methacrylic acid and mix; Then, a cross-linking agent and azobisisobutyronitrile were added, nitrogen was introduced, and the mixture was sealed to obtain a molecularly imprinted polymer mixed solution; adding the molecularly imprinted polymer mixed solution dropwise into the surfactant aqueous solution through a constant pressure dropping funnel, stirring the mixture at the reaction temperature, and separating to obtain polymer microspheres; The trans-ferulic acid is washed away from the polymer microspheres, and then the polymer microspheres are washed until neutral, and dried to obtain the trans-ferulic acid molecularly imprinted polymer microspheres.

2. The method for preparing trans-ferulic acid molecularly imprinted polymer microspheres according to claim 1, wherein: The surfactant aqueous solution includes span20 and tween20 accounting for 10% of the total volume, and the volume ratio of span20 to tween20 is 1:1; The dropping speed is 20 drops / min.

3. The method for preparing trans-ferulic acid molecularly imprinted polymer microspheres according to claim 2, characterized in that: The reaction temperature is 60-70°C, and the stirring reaction time is 20-25h; After the stirring reaction, the mixture was allowed to stand at 85-95° C. for 2 h.

4. The method for preparing trans-ferulic acid molecularly imprinted polymer microspheres according to claim 1, wherein Every 100-800 mg of trans-ferulic acid is dissolved in 10-50 mL of the solvent, wherein the solvent is one or more of anhydrous ethanol, acetonitrile and methanol; The cross-linking agent is ethylene glycol dimethacrylate; In the molecularly imprinted polymer mixed solution, 0.1-0.5 mL of methacrylic acid, 0.6-1.0 mL of ethylene glycol dimethacrylate and 50-150 mg of azobisisobutyronitrile are used for every 100-800 mg of trans-ferulic acid.

5. A trans-ferulic acid molecularly imprinted polymer microsphere, characterized in that: The trans-ferulic acid molecularly imprinted polymer microspheres are prepared by the preparation method of any one of claims 1 to 4.

6. A use of the trans-ferulic acid molecularly imprinted polymer microspheres according to claim 5, characterized in that: The trans-ferulic acid molecularly imprinted polymer microspheres are applied to separate and purify trans-ferulic acid by preparative chromatography, comprising the following steps: loading the molecularly imprinted polymer microspheres into a preparative chromatography column; Prepare a crude trans-ferulic acid solution as the loading solution; The sample solution is loaded onto the preparative chromatographic column, and eluted with a mixture of methanol-formic acid aqueous solution with gradient concentrations in sequence. The eluate is collected and concentrated under reduced pressure to obtain trans-ferulic acid.

7. The use of trans-ferulic acid molecularly imprinted polymer microspheres according to claim 6, characterized in that: The preparative chromatographic column is further filled with modified silica gel chromatographic column filler, and the mass ratio of the modified silica gel chromatographic column filler to the molecularly imprinted polymer microspheres is 1:9-9:1; The particle size of the trans-ferulic acid molecularly imprinted polymer microspheres is 100 mesh, and the particle size of the modified silica gel chromatographic column filler is 100 μm; The preparation method of the modified silica gel chromatographic column filler comprises the following steps: Treat the silicon spheres at 500-600°C for 2-4 hours and then disperse them in toluene; Raising the temperature to 50-60° C., adding a silane coupling agent, reacting for 4-6 hours, and removing the toluene to obtain the modified silica gel chromatographic column filler; The ratio of the silicon balls to the toluene is 1 g: (10-20) ml, and the mass ratio of the silicon balls to the silane coupling agent is 100:5-15.

8. The use of trans-ferulic acid molecularly imprinted polymer microspheres according to claim 7, characterized in that: The gradient concentration of methanol-formic acid aqueous solution includes methanol-formic acid aqueous solution with a volume proportion of methanol of 28-32%, 38-42%, 60-65%, 75-80%, and 100% respectively; In the formic acid aqueous solution, the volume proportion of formic acid is one thousandth.

9. The use of trans-ferulic acid molecularly imprinted polymer microspheres according to claim 6, characterized in that: The solid-liquid ratio of the trans-ferulic acid molecularly imprinted polymer microspheres to the trans-ferulic acid crude product solution is 1:100-500 (g / ml).

10. The use of trans-ferulic acid molecularly imprinted polymer microspheres according to claim 6, characterized in that: The concentration of trans-ferulic acid in the crude trans-ferulic acid solution is 1000-2000 mg / L.

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