Silybin oriented thin-layer molecular imprinting silica spheres for anti-aging and protective cosmetics, and preparation method and application thereof

Abstract

The application provides a silibinin directional thin-layer molecular imprinting silica ball and a preparation method and application thereof, and belongs to the technical field of natural product separation and purification. The silibinin directional thin-layer molecular imprinting silica ball is prepared by constructing a directional recognition thin layer on the surface of silica microspheres through four steps of silanization, amidation, esterification and thin-layer polymerization. The silibinin directional thin-layer molecular imprinting silica ball can specifically recognize the meta-diol structure of silibinin by virtue of the electron-deficient boron atom of phenylboronic acid, and can form a stable covalent borate ester bond under weak alkaline conditions, and the bond can be reversibly broken under acidic conditions, so that the directional capture and mild elution of silibinin are realized. In combination with the precise site replication of the thin-layer imprinting, silibinin and other flavone impurities can be efficiently separated, the separation efficiency is improved by nearly 90% compared with traditional macroporous adsorption resin, and the purity of silibinin after separation can reach more than 95%.

Description

Technical Field

[0001] This invention relates to the field of natural product separation and purification technology, and in particular to a silymarin-oriented thin-layer molecularly imprinted silicon sphere, its preparation method, and its application. Background Technology

[0002] Silymarin is a flavonoid compound extracted from the fruit of the milk thistle (Silybum marianum), a plant in the Asteraceae family. It possesses excellent antioxidant and anti-inflammatory activities and can be used in the preparation of anti-aging cosmetics, demonstrating good application development potential. Currently, the main methods for preparing silymarin include solvent extraction, column chromatography, and membrane separation, but these methods generally suffer from poor selectivity, low product purity, and low separation efficiency.

[0003] Molecularly imprinted polymers (MIPs) are widely used in the separation and purification of natural products due to their highly specific recognition of template molecules. Existing silymarin molecular imprinting techniques mostly employ bulk polymerization and embedding polymerization, which suffer from drawbacks such as random imprinting sites, low mass transfer efficiency, and severe template molecule leakage. Furthermore, existing technologies rarely utilize directional recognition mechanisms to enhance imprinting selectivity, making it difficult to achieve efficient directional separation of silymarin. Summary of the Invention

[0004] The purpose of this invention is to provide a silymarin-oriented thin-layer molecularly imprinted silicon sphere, its preparation method, and its application, so as to solve the problems of poor selectivity, template leakage, and low separation efficiency in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing silymarin-oriented thin-layer molecularly imprinted silicon spheres, comprising the following steps: Silica microspheres were added to an ethanol solution and dispersed evenly. An amination reagent was added to the resulting silica dispersion to carry out a silanization reaction, thereby obtaining amination-modified silica microspheres. The aminated silica microspheres were dispersed in a first buffer solution, and a phenylboronic acid derivative and a catalyst were added to carry out an amidation reaction to obtain phenylboronic acid modified silica microspheres. The phenylboronic acid-modified silica microspheres were dispersed in a second buffer solution, and silymarin solution was added to carry out an esterification reaction to obtain silica microspheres bound with silymarin. The silica microspheres containing silymarin were dispersed in a methanol aqueous solution, and functional monomers, crosslinking agents and initiators were added to carry out thin-layer polymerization. Subsequently, the microspheres were separated by centrifugation, eluted, washed and dried to obtain silymarin-oriented thin-layer molecularly imprinted silica spheres. The second buffer solution comprises Tris-HCl buffer solution, the pH of which is 7.5–8.5 and the concentration is 0.05–0.08 mol / L; The eluent used for elution includes a methanol-acetic acid mixture with a volume ratio of 9:1.

[0006] Preferably, the concentration of the ethanol solution is 90-100%; The amination reagent is 3-aminopropyltriethoxysilane, and the mass ratio of the amination reagent to the silica microspheres is 1:5 to 1:7.5. The silanization reaction is carried out at a temperature of 60-80°C for 8-12 hours.

[0007] Preferably, the first buffer solution comprises MES buffer, the pH of the first buffer solution is 5.0~6.0, and the concentration is 0.03~0.05 mol / L; The phenylboronic acid derivative is 3-carboxyphenylboronic acid, and the mass ratio of the phenylboronic acid derivative to the aminated silica microspheres is 1:8 to 1:11. The catalyst comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1 to 1:1.2, and the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to phenylboronic acid derivative is 1.2:1 to 1.5:1.

[0008] Preferably, the amidation reaction is carried out at a temperature of 25-30°C for 8-12 hours.

[0009] Preferably, the mass ratio of silybin to the phenylboronic acid-modified silica microspheres in the silybin solution is 1:2.0 to 1:4.2.

[0010] Preferably, the esterification reaction is carried out at a temperature of 60-70°C, under oscillation conditions, with an oscillation rate of 150-200 r / min and a duration of 4-6 h.

[0011] Preferably, the volume ratio of methanol to water in the methanol-water solution is 2:1 to 3:1; The functional monomer is methacrylic acid, and the molar ratio of the functional monomer to silybin in the silybin solution is 5:1 to 8:1. The crosslinking agent includes ethylene glycol dimethacrylate, diethylene glycol dimethacrylate or triethylene glycol dimethacrylate, and the molar ratio of the crosslinking agent to the functional monomer is 3:1 to 5:1; The initiator includes azobisisobutyronitrile, azobisisoheptanenitrile, or dimethyl azobismethylvalerate, and the mass of the initiator is 0.5 to 1% of the total mass of the functional monomer and the crosslinking agent.

[0012] Preferably, the thin-layer polymerization is carried out under anaerobic conditions, and the temperature of the thin-layer polymerization reaction is 60~70℃, and the time is 8~14 h.

[0013] This invention provides silymarin-oriented thin-layer molecularly imprinted silicon spheres prepared by the above-described method.

[0014] This invention provides the application of the above-mentioned silymarin-oriented thin-layer molecularly imprinted silicon spheres in the field of silymarin enrichment and purification.

[0015] The beneficial effects of this invention are: This invention combines the directional recognition mechanism of phenylboronic acid with thin-layer molecular imprinting technology. Through a four-step reaction of silanization, amidation, esterification and thin-layer polymerization, silybin-oriented thin-layer molecularly imprinted silicon spheres are prepared. This solves the pain points of insufficient selectivity, low efficiency and many impurities in the existing silybin separation technology. It also overcomes the defects of traditional molecular imprinting technology, such as random site, poor mass transfer and serious template leakage.

[0016] Compared with existing technologies, this invention utilizes the electron-deficient boron atoms of phenylboronic acid to specifically recognize the meta-diol structure of silybin, forming a stable covalent borate ester bond under weakly alkaline conditions. This bond reversibly breaks under acidic conditions, achieving directional capture and gentle elution of silybin. Combined with precise imprinting using thin-layer MIP, this significantly improves the selectivity of silybin recognition, achieving a product purity of over 95% after separation. Furthermore, this invention uses aminated SiO2 silica spheres as a carrier, which exhibits good dispersibility and high specific surface area, ensuring stable covalent grafting of phenylboronic acid. Additionally, the thin-layer MIP polymerization process... The MIP shell formed on the surface of silica microspheres in this invention is thin and uniform, which can reduce mass transfer resistance. The thin-layer MIP has a fast mass transfer rate, avoiding the problems of excessively deep embedding of template molecules and difficulty in elution in traditional bulk polymerization. The template leakage rate is as low as less than 1%, which effectively improves product quality. The silymarin-oriented thin-layer molecularly imprinted silica spheres prepared by this invention can achieve rapid enrichment and separation of silymarin, with a separation efficiency that is more than 80% higher than that of traditional methods. After elution, they can be reused and still maintain good recognition performance and separation efficiency after more than 6 cycles, which can further reduce production costs.

[0017] The overall preparation process of silymarin-oriented thin-layer molecularly imprinted silicon spheres of this invention is clear, the reaction conditions are mild, the reagents are readily available and highly reproducible, and it is easy to scale up for industrial production. Detailed Implementation

[0018] This invention provides a method for preparing silymarin-oriented thin-layer molecularly imprinted silicon spheres, comprising the following steps: Silica microspheres were added to an ethanol solution and dispersed evenly. An amination reagent was added to the resulting silica dispersion to carry out a silanization reaction, thereby obtaining amination-modified silica microspheres. The aminated silica microspheres were dispersed in a first buffer solution, and a phenylboronic acid derivative and a catalyst were added to carry out an amidation reaction to obtain phenylboronic acid modified silica microspheres. The phenylboronic acid-modified silica microspheres were dispersed in a second buffer solution, and silymarin solution was added to carry out an esterification reaction to obtain silica microspheres bound with silymarin. The silica microspheres containing silymarin were dispersed in a methanol aqueous solution, and functional monomers, crosslinking agents and initiators were added to carry out thin-layer polymerization. Subsequently, the microspheres were separated by centrifugation, eluted and dried to obtain silymarin-oriented thin-layer molecularly imprinted silica spheres. The second buffer solution comprises Tris-HCl buffer solution, the pH of which is 7.5–8.5 and the concentration is 0.05–0.08 mol / L; The eluent used for elution includes a methanol-acetic acid mixture with a volume ratio of 9:1.

[0019] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.

[0020] In this invention, silica microspheres are preferably added to an ethanol solution and ultrasonically dispersed to obtain a SiO2 dispersion. An amination reagent is added to the SiO2 dispersion to carry out a silanization reaction, thereby obtaining amination silica microspheres (SiO2-(CH2)3-NH2). This invention utilizes the silanization reaction between the silanol groups on the surface of SiO2 and the amination reagent to introduce amino groups onto the surface of SiO2.

[0021] In this invention, the concentration of the ethanol solution is preferably 90-100%, and more preferably 100%.

[0022] In this invention, the amination reagent is preferably 3-aminopropyltriethoxysilane (APTES); the mass ratio of the amination reagent to the silica microspheres is preferably 1:5 to 1:7.5, and more preferably 1:5.

[0023] In this invention, the temperature of the silanization reaction is preferably 60~80℃, more preferably 80℃, and the time is preferably 8~12 h, more preferably 10 h.

[0024] After the silanization reaction is completed, the present invention preferably performs a first centrifugation to separate the solid, washes it 3 to 5 times with anhydrous ethanol, and obtains aminated silica microspheres after a first vacuum drying.

[0025] In this invention, the rotational speed of the first centrifugation is preferably 4000~6000 r / min, more preferably 5000 r / min, and the time is preferably 5~15 min, more preferably 10 min.

[0026] In this invention, the temperature of the first vacuum drying is preferably 60~80℃, more preferably 80℃, and the time is preferably 2~4 h, more preferably 3 h.

[0027] In this invention, the above-mentioned aminated silica microspheres are preferably dispersed in a first buffer solution and ultrasonically dispersed; a phenylboronic acid derivative and a catalyst are added to carry out an amidation reaction to obtain phenylboronic acid-modified silica microspheres; under the catalytic action of the catalyst, the amino groups on the surface of the aminated silica microspheres undergo an amidation reaction with the carboxyl groups in the phenylboronic acid derivative to achieve covalent grafting of phenylboronic acid.

[0028] In this invention, the first buffer solution preferably includes MES buffer solution, the pH of the first buffer solution is preferably 5.0~6.0, more preferably 5.5, and the concentration is preferably 0.03~0.05 mol / L, more preferably 0.04 mol / L.

[0029] In this invention, the phenylboronic acid derivative is preferably 3-carboxyphenylboronic acid, and the mass ratio of the phenylboronic acid derivative to the aminated silica microspheres is preferably 1:8 to 1:11, and more preferably 1:10.

[0030] In this invention, the catalyst preferably comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is preferably 1:1 to 1:1.2, more preferably 1:1, and the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the phenylboronic acid derivative is preferably 1.2:1 to 1.5:1, more preferably 1.2:1. This invention achieves covalent grafting of phenylboronic acid by reacting the amino groups on the surface of aminated silica microspheres with the carboxyl groups in the phenylboronic acid derivative under the catalysis of EDC and NHS.

[0031] In this invention, the temperature of the amidation reaction is preferably 25~30°C, and the time is preferably 8~12 h, more preferably 8 h.

[0032] After the amidation reaction is completed, the present invention preferably performs a second centrifugation to separate the solid, washes it 3-4 times with anhydrous ethanol, and obtains phenylboronic acid modified silica microspheres after a second vacuum drying.

[0033] In this invention, the temperature of the second vacuum drying is preferably 60~80℃, more preferably 70℃, and the time is preferably 2~4 h, more preferably 3 h.

[0034] Preferably, the phenylboronic acid-modified silica microspheres are dispersed in a second buffer solution, and a silymarin solution is added to perform an esterification reaction to obtain silica microspheres bound with silymarin; the phenylboronic acid molecules are in the form of sp... 2 The hybrid electron-deficient boron center possesses Lewis acid properties, specifically recognizing the resorcinol-type 1,3-diol structure at the C5 / C7 position of silymarin. Through dehydration condensation, it forms a stable six-membered cyclic boron ester, with the boron atom completely converted to sp. 3 Tetrahedral hybrid configuration; when the pH of the system drops to acidic, the hybridization state of the boron atom reverses, directly triggering the directional hydrolytic breakage of the boron ester bond at this site, realizing the complete reversible cycle of this dynamic covalent bond, and completing the mild elution of silybin.

[0035] In this invention, the second buffer solution preferably comprises Tris-HCl buffer solution, the pH of the second buffer solution is preferably 7.5 to 8.5, more preferably 8.0, and the concentration is preferably 0.05 to 0.08 mol / L, more preferably 0.06 mol / L.

[0036] In this invention, the mass ratio of silybin to the phenylboronic acid-modified silica microspheres in the silybin solution is preferably 1:2.0 to 1:4.2, and more preferably 1:3 to 1:4.0.

[0037] In this invention, the solvent of the silymarin solution is preferably anhydrous ethanol, and the mass ratio of silymarin to the volume of the solvent is preferably 1:40 to 1:100, more preferably 1:100.

[0038] In this invention, the temperature of the esterification reaction is preferably 60~70℃, the esterification reaction is preferably carried out under oscillation conditions, the oscillation rate is preferably 150~200 r / min, more preferably 200 r / min, and the time is preferably 4~6h, more preferably 5h.

[0039] After the esterification reaction is completed, the present invention preferably performs centrifugation to remove the supernatant and obtain silica microspheres bound with silymarin.

[0040] In this invention, the silica microspheres containing silymarin are preferably dispersed in a methanol aqueous solution and ultrasonically dispersed until uniform. Then, functional monomers, crosslinking agents and initiators are added, nitrogen gas is introduced for 10-15 min to remove oxygen, and after sealing, thin-layer polymerization is carried out to form a thin-layer MIP shell.

[0041] In this invention, the volume ratio of methanol to water in the methanol-water solution is preferably 2:1 to 3:1, and more preferably 2:1.

[0042] In this invention, the functional monomer is preferably methacrylic acid (MAA); the molar ratio of the functional monomer to silybin in the silybin solution is preferably 5:1 to 8:1, and more preferably 5:1.

[0043] In this invention, the crosslinking agent preferably includes ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA), or triethylene glycol dimethacrylate (TEGDMA), and more preferably EGDMA; the molar ratio of the crosslinking agent to the functional monomer is preferably 3:1 to 5:1, and more preferably 3:1; this invention utilizes a small amount of functional monomer and crosslinking agent for thin-layer polymerization, forming a thin and uniform MIP shell, which can reduce mass transfer resistance, reduce template molecule leakage, and accurately replicate the spatial structure and recognition sites of silymarin, thereby improving recognition selectivity.

[0044] In this invention, the initiator preferably includes azobisisobutyronitrile (AIBN), azobisisoheptanenitrile (ABVN / V-65), or dimethyl azobismethylvalerate (AIBME / V-601), more preferably AIBN, and the mass of the initiator is preferably 0.5-1% of the total mass of the functional monomer and the crosslinking agent, more preferably 1%.

[0045] In this invention, the thin-layer polymerization is preferably carried out under anaerobic conditions, the temperature of the thin-layer polymerization reaction is preferably 60~70℃, more preferably 65℃, and the time is preferably 8~14 h, more preferably 10 h.

[0046] After the thin-layer polymerization is completed, the present invention preferably performs centrifugation and elutes the template molecule silybin with a methanol-acetic acid mixture with a volume ratio of 9:1 until the high performance liquid chromatography detection results show no obvious silybin characteristic peak in the eluent. Finally, it is washed with water until neutral and dried under a third vacuum to obtain silybin-oriented thin-layer molecularly imprinted silicon spheres.

[0047] In this invention, the temperature of the third vacuum drying is preferably 60~80℃, more preferably 60℃, and the time is preferably 4~8 h, more preferably 6 h.

[0048] This invention provides the application of the above-mentioned silybin-oriented thin-layer molecularly imprinted silica spheres in the field of silybin enrichment and purification: using the silica spheres as highly selective solid-phase extraction adsorbents, silybin is selectively adsorbed from the crude extract of milk thistle fruit. After rinsing to remove impurities, efficient elution is achieved under acidic conditions through reversible cleavage of borate ester bonds. Combined with the fast mass transfer characteristics of the thin-layer structure, rapid enrichment of silybin is achieved.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Weigh 10 g of silica microspheres, add 100 mL of anhydrous ethanol, and sonicate for 30 min; add 2 g of APTES, and stir at 60 °C for 10 h; centrifuge at 5000 r / min for 10 min, wash 4 times with anhydrous ethanol, and vacuum dry at 80 °C for 3 h to obtain aminated silica microspheres; weigh 10 g of aminated silica microspheres, disperse in 100 mL of MES buffer (pH=5.5, concentration 0.04 mol / L), and sonicate for 20 min; add 1 g of 3-carboxyphenylboronic acid, then add 10.85 mmol EDC and 10.85 mmol NHS, and stir at room temperature for 8 h; centrifuge, wash 3 times with anhydrous ethanol, and vacuum dry at 70 °C for 3 h to obtain phenylboronic acid modified silica microspheres; Weigh 10 g of phenylboronic acid-modified silica microspheres and disperse them in 25 mL of Tris-HCl buffer solution with pH=8.0 and concentration of 0.06 mol / L. Add 100 mL of alcohol solution containing 2.5 g of silymarin in anhydrous ethanol. Shake at 200 r / min at 60℃ for 5 h to bind. Centrifuge and remove the supernatant to obtain silica microspheres bound with silymarin. Silybin-bound silica spheres were dispersed in 90 mL of a methanol-water mixture with a volume ratio of 2:1 and ultrasonically dispersed for 20 min. 24.35 mmol MAA, 73.05 mmol EGDMA, and 165.70 mg AIBN were added, and nitrogen gas was purged for 12 min to remove oxygen. The mixture was then sealed and stirred at 65 °C for 10 h to polymerize. The mixture was centrifuged and eluted with a methanol-acetic acid mixture with a volume ratio of 9:1 until no obvious silybin peak was detected in the eluent by HPLC. The eluent was washed with water until neutral and then vacuum dried at 60 °C for 6 h to obtain silybin-oriented thin-layer molecularly imprinted silica spheres.

[0052] Comparative Example 1

[0053] Using AB-8 type macroporous adsorption resin (conventional industrial grade) as a comparative example, 1 molecule of imprinted silicon sphere was used.

[0054] Performance testing

[0055] 1. The silymarin-oriented thin-layer molecularly imprinted silicon spheres prepared in Example 1 were subjected to adsorption capacity testing, template leakage testing, and reusability testing according to the following methods: 1) Adsorption capacity test method: Six 8.0 mg silybin-oriented thin-layer molecularly imprinted silica spheres prepared in Example 1 were accurately weighed and placed in 10 mL centrifuge tubes. 5 mL of silybin ethanol solution (anhydrous ethanol:water = 4:1) with initial concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.10 mg / mL, and 0.12 mg / mL were added sequentially. The centrifuge tubes were placed in a 25°C constant-temperature shaker and shaken at 200 r / min in the dark for 12 h for adsorption. After adsorption, the mixture was centrifuged at 8000 r / min for 5 min. The supernatant was filtered through a 0.45 μm organic filter membrane, and the remaining concentration of silybin in the supernatant was determined by HPLC. Adsorption capacity Q = (c0 - c) t )×V / m, where c0 is the initial concentration, c t V represents the concentration of the supernatant after adsorption, V represents the solution volume, and m represents the mass of the imprinted silicon spheres.

[0056] 2) Template leakage test method: Accurately weigh 100 mg of each of the silymarin-oriented thin-layer molecularly imprinted silica spheres prepared in Example 1, place them in 100 mL stoppered conical flasks, add 50 mL of ethanol-acetic acid eluent (9:1 v / v), seal and place in a constant temperature shaker at 25 °C for 24 h at a rate of 200 r / min; take 1 mL of the sample, filter it through a 0.22 μm organic filter membrane, and determine the silymarin content in the soaking solution by HPLC; Template leakage rate (%) = (Template mass detected in immersion solution ÷ Theoretical total template bonding mass in imprint material) × 100%.

[0057] 3) Method for testing the effectiveness of repeated use: Five 20 mg portions of silymarin-oriented thin-layer molecularly imprinted silicon spheres prepared in Example 1 were accurately weighed and subjected to continuous adsorption-elution cycle experiments. Single adsorption: 10 mL of 0.08 mg / mL silymarin solution was added, and the mixture was shaken at 25 °C for 2 h for adsorption. The concentration of the supernatant was measured by centrifugation, and the adsorption capacity was calculated. Single elution: 10 mL of methanol-acetic acid (9:1) mixture was shaken and eluted for 30 min. The solid was collected by centrifugation and washed with deionized water until neutral to complete regeneration. The above cycles were performed 10 times consecutively, and the adsorption capacity was measured simultaneously after each cycle. The results are shown in Table 1.

[0058] Table 1. Test results of the reusability of silymarin-oriented thin-layer molecularly imprinted silicon spheres.

[0059] The silybin-oriented thin-layer molecularly imprinted silica spheres prepared in Example 1 were tested and found to have an adsorption capacity of 35.36 mg / g for silybin, with a template leakage rate of 0.87%. As shown in Table 1, after 6 cycles, the adsorption capacity retention rate of the silybin-oriented thin-layer molecularly imprinted silica spheres prepared in Example 1 was ≥85%, indicating that the oriented thin-layer molecularly imprinted silica spheres can be reused after elution and still maintain good recognition performance and separation efficiency after more than 6 cycles, which can further reduce production costs.

[0060] 2. The separation efficiency of the silymarin-oriented thin-layer molecularly imprinted silicon spheres prepared in Example 1 and the molecularly imprinted silicon spheres of Comparative Example 1 was tested, and the results are shown in Table 2: Weigh 2 g of silymarin-oriented thin-layer molecularly imprinted silica spheres prepared in Example 1 and molecularly imprinted silica spheres prepared in Comparative Example 1, and then wet pack them into two chromatography columns using 80% ethanol-pH=8.0 Tris-HCl buffer. The calibrated crude silymarin loading solution (silymarin concentration of 7.0 mg / mL, initial powder content of 18.2%, solvent system of anhydrous ethanol-pH=8.0 Tris-HCl buffer (ethanol content of 80%), filtered through a 0.45 μm organic filter membrane without precipitation) was simultaneously introduced into the two support columns at a flow rate of 1 BV / h. The eluent was monitored in real time. When the characteristic absorption of silymarin reached 1% of the initial concentration, it was determined as the breakthrough point, and the loading was stopped immediately. The total loading volume and loading time of the two groups were recorded respectively. Simultaneously purge 80% ethanol-pH=8.0 Tris-HCl buffer into both support columns at a flow rate of 1 BV / h until the UV signal of the effluent completely returns to baseline, and record the rinsing time. Methanol-acetic acid eluent (methanol:acetic acid = 97:3) was simultaneously introduced into both columns at a flow rate of 1 BV / h. Elution was continued until the UV signal returned to the baseline. Then, 0.5 column volumes of elution were flushed, elution was stopped, all eluent was collected, and the total elution time was recorded. Record the total operation time for a single batch from the start of sample loading to complete elution; collect all eluent, dry it, record the total product mass, and determine the silymarin content in the product; substitute the results into the formula to calculate: E=(m 产物 ×P) / (m 材料 ×t 总 ) Where E is the separation efficiency, in mg / (g·h); m 产物 The total mass of the product after rotary evaporation is expressed in mg; P represents the content of silymarin in the total product; m 材料 The mass of dry material loaded is expressed in grams (g); tons (t) 总 The total time for the entire process is expressed in hours (h).

[0061] Table 2. Separation efficiency test results for Example 1 and Comparative Example 1

[0062] As shown in Table 2, under the same filler mass and operating flow rate conditions, the loading volume of the silymarin-oriented thin-layer molecularly imprinted silica spheres prepared in this invention can reach 9.2 mL, which is 2.01 times that of the macroporous adsorption resin in Comparative Example 1; the silymarin elution recovery rate reaches 99.13%, an improvement of 21.13 percentage points compared to Comparative Example 1; the purity of silymarin in the separated product is as high as 98.20%, far exceeding the 62.70% of Comparative Example 1; the separation efficiency reaches 16.61 mg / (g·h), an improvement of 89.98% compared to Comparative Example 1. This indicates that the oriented thin-layer molecularly imprinted silica spheres exhibit significantly better adsorption selectivity, enrichment efficiency, and separation purity for silymarin than traditional macroporous adsorption resins, which can significantly shorten the production cycle, improve product quality, and have good prospects for industrial application.

[0063] In summary, this invention combines the directional recognition mechanism of phenylboronic acid with thin-layer molecular imprinting technology, and prepares silymarin-oriented thin-layer molecularly imprinted silicon spheres through a four-step reaction involving silanization, amidation, esterification, and thin-layer polymerization. Its core mechanism lies in the fact that the phenylboronic acid molecule exhibits sp... 2 The hybrid electron-deficient boron center possesses Lewis acid properties, specifically recognizing the resorcinol-type 1,3-diol structure at the C5 / C7 position of silymarin. Under weakly basic conditions, it undergoes dehydration condensation to form a stable six-membered cyclic boron ester, with the boron atom completely converted to sp. 3The tetrahedral hybrid configuration enables the directional immobilization of silybin. When the system pH drops to acidic, the hybridization state of the boron atoms reverses, directly triggering the directional hydrolytic breakage of the boron ester bond at this site, achieving a completely reversible cycle of this dynamic covalent bond and completing the gentle elution of silybin. Performance test results show that the imprinted silica spheres prepared in this invention have an adsorption capacity of up to 35.36 mg / g for silybin, a template leakage rate as low as 0.87%, and an adsorption capacity retention rate of ≥85% after 6 cycles. When used for silybin separation and purification, the purity of silybin in the separated product is as high as 98.20%, and the separation efficiency is 89.98% higher than that of traditional resins. This effectively solves the pain points of poor selectivity, low purity, and insufficient efficiency of existing silybin separation technologies, and has good prospects for industrial application.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing silymarin-oriented thin-layer molecularly imprinted silicon spheres, characterized in that, Includes the following steps: Silica microspheres were added to an ethanol solution and dispersed evenly. An amination reagent was added to the resulting silica dispersion to carry out a silanization reaction, thereby obtaining amination-modified silica microspheres. The aminated silica microspheres were dispersed in a first buffer solution, and a phenylboronic acid derivative and a catalyst were added to carry out an amidation reaction to obtain phenylboronic acid modified silica microspheres. The phenylboronic acid-modified silica microspheres were dispersed in a second buffer solution, and silymarin solution was added to carry out an esterification reaction to obtain silica microspheres bound with silymarin. The silica microspheres containing silymarin were dispersed in a methanol aqueous solution, and functional monomers, crosslinking agents and initiators were added to carry out thin-layer polymerization. Subsequently, the microspheres were separated by centrifugation, eluted, washed and dried to obtain silymarin-oriented thin-layer molecularly imprinted silica spheres. The second buffer solution comprises Tris-HCl buffer solution, the pH of which is 7.5–8.5 and the concentration is 0.05–0.08 mol / L; The eluent used for elution includes a methanol-acetic acid mixture with a volume ratio of 9:

1.

2. The preparation method according to claim 1, characterized in that, The concentration of the ethanol solution is 90-100%; The amination reagent is 3-aminopropyltriethoxysilane, and the mass ratio of the amination reagent to the silica microspheres is 1:5 to 1:7.

5. The silanization reaction is carried out at a temperature of 60-80°C for 8-12 hours.

3. The preparation method according to claim 1, characterized in that, The first buffer solution includes MES buffer, the pH of which is 5.0-6.0 and the concentration is 0.03-0.05 mol / L; The phenylboronic acid derivative is 3-carboxyphenylboronic acid, and the mass ratio of the phenylboronic acid derivative to the aminated silica microspheres is 1:8 to 1:

11. The catalyst comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1 to 1:1.2, and the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to phenylboronic acid derivative is 1.2:1 to 1.5:

1.

4. The preparation method according to claim 1 or 3, characterized in that, The amidation reaction is carried out at a temperature of 25-30°C for 8-12 hours.

5. The preparation method according to claim 1, characterized in that, The mass ratio of silybin to the phenylboronic acid-modified silica microspheres in the silybin solution is 1:2.0 to 1:4.

2.

6. The preparation method according to claim 1 or 5, characterized in that, The esterification reaction is carried out at a temperature of 60-70°C under oscillation conditions, with an oscillation rate of 150-200 r / min and a duration of 4-6 h.

7. The preparation method according to claim 1, characterized in that, The volume ratio of methanol to water in the methanol-water solution is 2:1 to 3:

1. The functional monomer is methacrylic acid, and the molar ratio of the functional monomer to silybin in the silybin solution is 5:1 to 8:

1. The crosslinking agent includes ethylene glycol dimethacrylate, diethylene glycol dimethacrylate or triethylene glycol dimethacrylate, and the molar ratio of the crosslinking agent to the functional monomer is 3:1 to 5:1; The initiator includes azobisisobutyronitrile, azobisisoheptanenitrile, or dimethyl azobismethylvalerate, and the mass of the initiator is 0.5 to 1% of the total mass of the functional monomer and the crosslinking agent.

8. The preparation method according to claim 1 or 7, characterized in that, The thin-layer polymerization is carried out under anaerobic conditions, and the temperature of the thin-layer polymerization reaction is 60~70℃, and the time is 8~14 h.

9. Silymarin-oriented thin-layer molecularly imprinted silicon spheres prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the silymarin-oriented thin-layer molecularly imprinted silicon spheres according to claim 9 in the field of silymarin enrichment and purification.

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