Boron affinity-molecular imprinting magnetic nanoparticles for directionally extracting and purifying dogwood polysaccharide as well as preparation method and application of boron affinity-molecular imprinting magnetic nanoparticles

Abstract

The invention belongs to the technical field of separation and purification of natural polysaccharides, and particularly relates to boron affinity-molecular imprinting magnetic nanoparticles for directionally extracting and purifying dogwood polysaccharides as well as a preparation method and application of the boron affinity-molecular imprinting magnetic nanoparticles. According to the invention, a dogwood polysaccharide characteristic fragment is used as a single template molecule, a three-in-one design of a Fe3O4 (at) SiO2 (at) PGMA core-shell structure magnetic carrier, a composite boron affinity ligand and a molecular imprinting layer is adopted, and a nano adsorption material with high specific recognition, high adsorption capacity and rapid magnetic separation performance is constructed by regulating and controlling the proportion of functional monomers and a polymerization process. According to the method, directional capture and efficient purification of the dogwood polysaccharide are realized through a boron affinity interaction and molecular imprinting dual recognition mechanism, and the technical problems that polysaccharide and impurities are not thoroughly separated and the loss rate of target components is high in a traditional method are solved. After optimization, the polysaccharide adsorption capacity reaches 85 mg / g or above, the purity can reach 92%, the imprinting factor (IF) is larger than or equal to 6.8, the adsorption capacity retention rate still exceeds 90% after regeneration and recycling are conducted five times, and the method is suitable for industrial precise separation and purification of the dogwood polysaccharide and has wide application prospects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural polysaccharide separation and purification, and particularly relates to a boron affinity-molecular imprinting magnetic nanoparticle for directional extraction and purification of cornus officinalis polysaccharide, and a preparation method and application thereof. BACKGROUND

[0002] The cornus officinalis polysaccharide has biological activities such as immunoregulation and antioxidant, but the existing extraction and purification technology has significant defects: (1) lack of directionality: traditional water extraction and alcohol precipitation, ultrasonic assisted methods rely on non-specific separation, and polysaccharide is co-extracted with flavonoids, tannins and other impurities, and the purity is less than 92%; (2) low separation efficiency: the steps of centrifugation, dialysis and the like take several days, and the polysaccharide recovery rate is less than 85%; (3) poor material reusability: the existing magnetic adsorption material has no specific recognition site, and the adsorption capacity is low (≤10 mg / g), and the reuse is less than 5 times. SUMMARY

[0003] In view of the problems and deficiencies in the prior art, the purpose of the present application is to provide a boron affinity-molecular imprinting magnetic nanoparticle for directional extraction and purification of cornus officinalis polysaccharide, and a preparation method and application thereof.

[0004] In order to achieve the above purpose, the present application adopts the following technical scheme: The present application provides a preparation method of a boron affinity-molecular imprinting magnetic nanoparticle for directional extraction and purification of cornus officinalis polysaccharide, comprising the following steps: (1) SiO2-coated modification is performed on Fe3O4 nanoparticles to obtain Fe3O4@SiO2 particles, and polyglycidyl methacrylate is grafted on the Fe3O4@SiO2 particles to obtain a Fe3O4@SiO2@PGMA core-shell structure magnetic carrier; (2) The Fe3O4@SiO2@PGMA core-shell structure magnetic carrier obtained in step (1) is modified with a team boron affinity molecule to obtain a boron affinity functionalized magnetic carrier; (3) The template molecule, auxiliary functional monomer, crosslinking agent and initiator are dissolved in a solvent, and the boron affinity functionalized magnetic carrier obtained in step (2) is added to the solvent, and under an inert gas atmosphere, pre-polymerization is first carried out at 45-50℃ for 2-3h, and then polymerization is carried out at 60-65℃ for 8-10h, after the reaction is completed, the template molecule is eluted and removed to obtain a boron affinity-molecular imprinting magnetic nanoparticle; wherein the template molecule is a cornus officinalis polysaccharide characteristic fragment with a molecular weight of 5-30kDa.

[0005] Preferably, in step (3), the auxiliary functional monomer is 4-vinylphenylboronic acid (VPBA).

[0006] Preferably, in step (3), the molar ratio of the template molecule, the auxiliary functional monomer, the crosslinking agent and the initiator is 1:(8-10):(30-35):(0.8-1.2).

[0007] Preferably, in step (3), the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

[0008] Preferably, in step (2), the specific method of modification is as follows: 3-aminobenzoic acid and 1,6-hexanediamine are dispersed in ethanol, and then stirred to react; after the reaction is completed, the Fe3O4@SiO2@PGMA core-shell structure magnetic carrier obtained in step (1) is added into the reaction solution to reflux, and then the solid is collected and washed by magnetic separation to obtain a boron affinity functionalized magnetic carrier.

[0009] Preferably, the mass ratio of 3-aminobenzoic acid to 1,6-hexanediamine is (1.2-1.5):1, the stirring reaction condition is stirring at 60-70℃ for 360-420min, and the reflux reaction condition is refluxing at 90-100℃ for 10-14h.

[0010] Preferably, the method for obtaining the characteristic fragment of the cornus officinalis polysaccharide is as follows: cornus officinalis powder is taken, hot water extraction is used to reflux to obtain a cornus officinalis polysaccharide extraction solution, the polysaccharide extraction solution is sequentially subjected to alcohol precipitation, Sevag method deproteinization and ultrafiltration treatment, and then the polysaccharide with a molecular weight of 5-30kDa is collected, so that the characteristic fragment of the cornus officinalis polysaccharide is obtained.

[0011] More preferably, the specific operation of the ultrafiltration treatment is as follows: first, the polysulfone (PES) ultrafiltration membrane with a molecular weight cut-off of 30kDa is used to perform ultrafiltration at an operating pressure of 0.2MPa and 25℃, and the permeate (containing polysaccharide with a molecular weight of less than 30kDa) is collected; the above permeate is subjected to ultrafiltration again through the PES ultrafiltration membrane with a molecular weight cut-off of 5kDa at the same operating pressure and temperature, and the retentate (containing polysaccharide with a molecular weight of 5-30kDa) is collected.

[0012] The second aspect of the present application provides a boron affinity-molecularly imprinted magnetic nanoparticle for directional extraction and purification of cornus officinalis polysaccharide, which is prepared by the method of the first aspect.

[0013] The third aspect of the present application provides the application of the boron affinity-molecularly imprinted magnetic nanoparticle of the second aspect in directional extraction and purification of cornus officinalis polysaccharide.

[0014] The fourth aspect of the present application provides a method for directional extraction and purification of Cornus officinalis polysaccharide, comprising the following steps: adjusting the pH value of Cornus officinalis crude extract to 7.2-7.8, adding the boron affinity-molecularly imprinted magnetic nanoparticles of claim 7, oscillating and adsorbing at 25-30 DEG C, separating and collecting the boron affinity-molecularly imprinted magnetic nanoparticles by external magnetic field after adsorption is completed, then using acetic acid-ethanol mixture to elute the collected boron affinity-molecularly imprinted magnetic nanoparticles, collecting the eluate and freeze-drying after reducing pressure concentration, to obtain Cornus officinalis polysaccharide.

[0015] Preferably, the pH value of the acetic acid-ethanol mixture is 4.5-5.0; during the oscillation and adsorption process, the dosage of the boron affinity-molecularly imprinted magnetic nanoparticles is 1 g per 30-50 mL of Cornus officinalis crude extract.

[0016] Compared with the prior art, the present application has the following beneficial effects: (1) The present application uses the characteristic fragment of Cornus officinalis polysaccharide as a single template molecule, adopts the trinity design of "Fe3O4@SiO2@PGMA core-shell structure magnetic carrier-composite boron affinity ligand-molecularly imprinted layer", and constructs a nano adsorbent material with high specific recognition, high adsorption capacity and rapid magnetic separation performance by regulating the ratio of functional monomers and the polymerization process. The method realizes the directional capture and efficient purification of Cornus officinalis polysaccharide through the dual recognition mechanism of boron affinity and molecular imprinting, solves the technical problems of incomplete separation of polysaccharide and impurities and high loss rate of target components in traditional methods. After optimization, the polysaccharide adsorption capacity is more than 85 mg / g, the purity is more than 92%, the imprinting factor (IF) is greater than or equal to 6.8, and the adsorption capacity retention rate is still more than 90% after 5 times of regeneration and recycling, which is suitable for industrialized precise separation and purification of Cornus officinalis polysaccharide and has a wide application prospect.

[0017] (2) Unlike the existing double-template or general template strategy, the present application clearly uses the characteristic fragment (5-30 kDa) of Cornus officinalis polysaccharide as a single template, which is rich in cis-dihydroxy structure and is the core active unit of Cornus officinalis polysaccharide, ensuring the precise matching of imprinting sites and target components and reducing non-specific adsorption.

[0018] (3) The present application uses the "3-aminophenylboronic acid-1,6-hexanediamine" team boron affinity molecule and 4-vinylphenylboronic acid (VPBA) composite ligand system, which not only retains the high recognition activity of the team boron affinity under neutral conditions, but also enhances the covalent binding capacity with the cis-dihydroxy group of polysaccharide through VPBA, solving the problem of insufficient adsorption capacity of single ligand, and the adsorption capacity is increased by more than 30% compared with single ligand.

[0019] (4) The Fe3O4@SiO2@PGMA three-layer core-shell structure is adopted in the application, the SiO2 shell layer improves the dispersion and chemical stability of the carrier, and the PGMA grafted layer provides abundant active sites, so that the grafting rate of boron affinity ligand is improved to 25%-30%, the thickness of the imprinted layer is uniform and controllable (10-15 nm), and the defects of easy aggregation and imprinted site embedding of the existing carrier are avoided.

[0020] (5) The adsorption process is controlled in a neutral environment with pH 7.2-7.8, so as to avoid the damage of strong acid and strong base to the structure of polysaccharide; the elution adopts a weak acid mixed solvent, so as to realize mild desorption, improve the polysaccharide recovery rate by 15%-20% compared with the traditional method, shorten the magnetic separation time to 5%-8 min, and significantly improve the separation efficiency.

[0021] (6) The application realizes the directional capture and deep purification of polysaccharide in Fructus Corni, the purity of the product reaches more than 92%, solves the bottleneck of “heavy separation and light purification” in the traditional method, simplifies the operation process of the magnetic separation process, reduces the industrialization cost, has excellent regeneration performance, meets the green production demand, and the established “composite boron affinity-molecular imprinting-magnetic carrier” technical system can be popularized to the purification of other polysaccharide substances rich in cis-dihydroxyl structure, and has universality. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the application clearer, the application is further described in detail through the following examples. It should be understood that the specific examples described herein are only used to explain the application, and are not used to limit the application.

[0023] Example 1: A preparation method of a boron affinity-molecular imprinting magnetic nanoparticle, and the specific steps are as follows: (1) 10 mmol of FeCl3·6H2O is dissolved in 50 mL of ethylene glycol, 2.0 g of sodium acetate is added, stirring is conducted for 30 min, then the mixture is transferred to a high-pressure reaction kettle, reaction is conducted at 200 ℃ for 8 h, and after cooling, Fe3O4 nanoparticles (particle size is about 100 nm) are collected by magnetic separation. The Fe3O4 is dispersed in 100 mL of an ethanol-water mixture (volume ratio is 3:1), 5 mL of tetraethyl orthosilicate (TEOS) and 2 mL of ammonia water are added, stirring is conducted at 30 ℃ for 7 h, Fe3O4@SiO2 (the thickness of the SiO2 shell layer is 18 nm) is obtained. Then, 0.5 g of CuBr, 1.0 g of pentamethyldiethylenetriamine (PMDETA) and 5 mL of glycidyl methacrylate (GMA) are added, reaction is conducted at 60 ℃ for 6 h under nitrogen protection, PGMA is grafted through atom transfer radical polymerization (ATRP), and the Fe3O4@SiO2@PGMA core-shell structure magnetic carrier with a particle size of 80-120 nm is obtained.

[0024] (2) Take 1.35 g 3-amino phenylboronic acid and 1.0 g 1,6-hexanediamine, disperse in 50 mL ethanol, stir at 65 ℃ for 390 min, then add the Fe3O4@SiO2@PGMA core-shell structure magnetic carrier prepared in step (1), reflux at 95 ℃ for 12 h, magnetically separate the solid, wash with ethanol for 3 times, to obtain the boron affinity functionalized magnetic carrier.

[0025] (3) Take 0.1 mmol of the characteristic fragment of Cornus officinalis polysaccharide (molecular weight 15 kDa, separated by gel filtration chromatography) as a template molecule, dissolve in 20 mL acetonitrile-water mixed solvent (volume ratio 6:4), sequentially add 0.9 mmol 4-vinylphenylboronic acid (VPBA), 3.2 mmol ethylene glycol dimethyl acrylate (EGDMA) and 0.1 mmol azobisisobutyronitrile (AIBN), stir and dissolve, then add the boron affinity functionalized magnetic carrier of step (2), under nitrogen protection, first pre-polymerize at 48 ℃ for 2.5 h, then heat to 62 ℃ for polymerization for 9 h. After the reaction is completed, elute 3 times (2 h each time) with a mixture of methanol-acetic acid (volume ratio 8:2), and the removal rate of the template molecule is 98.5% detected by high performance liquid chromatography, to obtain boron affinity-molecularly imprinted magnetic nanoparticles.

[0026] A method for directional extraction and purification of Cornus officinalis polysaccharide, the specific steps are as follows: Take the Cornus officinalis crude extract (polysaccharide content about 15%) and adjust the pH to 7.5, add the boron affinity-molecularly imprinted magnetic nanoparticles prepared above according to the solid-liquid ratio of 1:50 (g:mL), constant temperature oscillation adsorption at 28 ℃ for 75 min, external magnetic field separation for 5 min, separate the boron affinity-molecularly imprinted magnetic nanoparticles, elute 2 times with a mixture of acetic acid-ethanol (volume ratio 1:9) at pH 4.8, combine the eluate, concentrate under reduced pressure, then freeze-dry to obtain Cornus officinalis polysaccharide.

[0027] Example 2: A method for preparing boron affinity-molecularly imprinted magnetic nanoparticles, the specific steps are as follows: (1) 10 mmol FeCl3·6H2O was dissolved in 50 mL ethylene glycol, 2.0 g sodium acetate was added, and after stirring for 30 min, it was transferred to a high-pressure reaction kettle and reacted at 200 ℃ for 8 h. After cooling, Fe3O4 nanoparticles (particle size about 80 nm) were collected by magnetic separation. The Fe3O4 was dispersed in 100 mL of an ethanol-water mixture (volume ratio 3:1), 5 mL of tetraethyl orthosilicate (TEOS) and 2 mL of ammonia were added, and stirring was carried out at 30 ℃ for 6 h to obtain Fe3O4@SiO2 (SiO2 shell thickness 15 nm). Then 0.5 g of CuBr, 1.0 g of pentamethyldiethylenetriamine (PMDETA) and 5 mL of glycidyl methacrylate (GMA) were added, and the reaction was carried out at 60 ℃ for 6 h under nitrogen protection to graft PGMA by atom transfer radical polymerization (ATRP), thereby obtaining Fe3O4@SiO2@PGMA core-shell structure magnetic carrier with a particle size of 80-120 nm.

[0028] (2) 1.2 g of 3-aminophenylboronic acid and 1.0 g of 1,6-hexanediamine were weighed and dispersed in 50 mL of ethanol, and stirring was carried out at 65 ℃ for 390 min. Then the Fe3O4@SiO2@PGMA core-shell structure magnetic carrier prepared in step (1) was added, and reflux reaction was carried out at 95 ℃ for 12 h. The solid was collected by magnetic separation and washed with ethanol for 3 times to obtain a boron affinity functionalized magnetic carrier.

[0029] (3) 0.1 mmol of a characteristic fragment of cornus officinalis polysaccharide (molecular weight 5 kDa, separated by gel filtration chromatography) was taken as a template molecule, dissolved in 20 mL of an acetonitrile-water mixed solvent (volume ratio 6:4), and 0.8 mmol of 4-vinylphenylboronic acid (VPBA), 3.0 mmol of ethylene glycol dimethacrylate (EGDMA) and 0.08 mmol of azobisisobutyronitrile (AIBN) were added in sequence. After stirring and dissolving, the boron affinity functionalized magnetic carrier of step (2) was added, and under nitrogen protection, pre-polymerization was carried out at 45 ℃ for 2 h, and then the temperature was increased to 60 ℃ for polymerization for 8 h. After the reaction was completed, methanol-acetic acid mixture (volume ratio 8:2) was used for oscillation elution for 4 times (2 h each time), and the removal rate of the template molecule was 99.1% by high performance liquid chromatography detection, thereby obtaining boron affinity-molecularly imprinted magnetic nanoparticles.

[0030] A method for directional extraction and purification of cornus officinalis polysaccharide, and the specific steps are as follows: The crude extract of Cornus officinalis (polysaccharide content is about 15%) was adjusted to pH 7.2, and the boron affinity-molecularly imprinted magnetic nanoparticles prepared above were added at a solid-liquid ratio of 1:50 (g:mL), and adsorbed at 25°C for 60 min. The boron affinity-molecularly imprinted magnetic nanoparticles were separated under an external magnetic field for 5 min, eluted twice with acetic acid-ethanol mixture (volume ratio 1:9) at pH 4.5, combined the eluate, concentrated under reduced pressure, and freeze-dried to obtain Cornus officinalis polysaccharide.

[0031] Example 3: A preparation method of boron affinity-molecularly imprinted magnetic nanoparticles, the specific steps are as follows: (1) 10 mmol FeCl3·6H2O was dissolved in 50 mL of ethylene glycol, 2.0 g of sodium acetate was added, stirred for 30 min, then transferred to a high-pressure reaction kettle, reacted at 200°C for 8 h, and then the Fe3O4 nanoparticles (particle size about 120 nm) were collected by magnetic separation after cooling. The Fe3O4 was dispersed in 100 mL of ethanol-water mixture (volume ratio 3:1), 5 mL of tetraethyl orthosilicate (TEOS) and 2 mL of ammonia were added, stirred at 30°C for 8 h to obtain Fe3O4@SiO2 (SiO2 shell thickness 20 nm). Then 0.5 g of CuBr, 1.0 g of pentamethyl diethylene triamine (PMDETA) and 5 mL of glycidyl methacrylate (GMA) were added, and the reaction was carried out at 60°C under nitrogen protection for 6 h. The PGMA was grafted by atom transfer radical polymerization (ATRP) to obtain Fe3O4@SiO2@PGMA core-shell structure magnetic carrier with a particle size of 80-120 nm.

[0032] (2) 1.5 g of 3-aminobenzoic acid and 1.0 g of 1,6-hexanediamine were weighed and dispersed in 50 mL of ethanol, stirred at 70°C for 420 min, then the Fe3O4@SiO2@PGMA core-shell structure magnetic carrier prepared in step (1) was added, and the reaction was carried out at 100°C for 14 h. The solid was collected by magnetic separation and washed with ethanol for 3 times to obtain a boron affinity functionalized magnetic carrier.

[0033] (3) Take 0.1 mmol of the characteristic fragment of Cornus officinalis polysaccharide (molecular weight 5 kDa, separated by gel filtration chromatography) as a template molecule, dissolved in 20 mL of acetonitrile-water mixed solvent (volume ratio 6:4), and then 1.0 mmol of 4-vinylphenylboronic acid (VPBA), 3.5 mmol of ethylene glycol dimethyl acrylate (EGDMA) and 0.12 mmol of azobisisobutyronitrile (AIBN) were added, and then the boron affinity functionalized magnetic carrier of step (2) was added after stirring and dissolving. Under nitrogen protection, first pre-polymerize at 50 DEG C for 3 h, and then polymerize at 60 DEG C for 10 h. After the reaction is completed, wash and elute 4 times (2 h each time) with a mixture of methanol-acetic acid (volume ratio 8:2), and the removal rate of the template molecule is 99.3% by high performance liquid chromatography detection, to obtain boron affinity-molecularly imprinted magnetic nanoparticles.

[0034] A method for directional extraction and purification of Cornus officinalis polysaccharide, the specific steps are as follows: Take the Cornus officinalis crude extract (polysaccharide content about 15%) and adjust the pH to 7.8, then add the boron affinity-molecularly imprinted magnetic nanoparticles prepared above according to the solid-liquid ratio of 1:50 (g:mL), and adsorb at 30 DEG C for 90 min under constant temperature oscillation, and separate under the external magnetic field for 5 min. Separate the boron affinity-molecularly imprinted magnetic nanoparticles, elute 2 times with a mixture of acetic acid-ethanol (volume ratio 1:9) with a pH of 5, combine the eluate, concentrate under reduced pressure, and freeze-dry to obtain Cornus officinalis polysaccharide.

[0035] Performance analysis of the boron affinity-molecularly imprinted magnetic nanoparticles prepared in the application: 1. Adsorption capacity, imprinting factor and polysaccharide purity effect analysis of Cornus officinalis polysaccharide extraction and purification of the boron affinity-molecularly imprinted magnetic nanoparticles: The adsorption capacity, imprinting factor and polysaccharide purity effect analysis of Cornus officinalis polysaccharide extraction and purification of the boron affinity-molecularly imprinted magnetic nanoparticles prepared in the application examples 1-3 are analyzed, and comparative example 1 is designed for comparison. The specific content of comparative example 1 is as follows: Comparative example 1: A method for preparing boron affinity-molecularly imprinted magnetic nanoparticles, the specific steps are as follows: (1) Take 10 mmol FeCl3 6H2O and dissolve it in 50 mL ethylene glycol, add 2.0 g sodium acetate, stir for 30 min, then transfer to a high-pressure reaction kettle, react at 200 ℃ for 8 h, and collect Fe3O4 nanoparticles (particle size about 100 nm) after magnetic separation. Disperse Fe3O4 in 100 mL ethanol-water mixture (volume ratio 3:1), add 5 mL tetraethyl orthosilicate (TEOS) and 2 mL ammonia, stir at 30 ℃ for 7 h, and obtain Fe3O4@SiO2 (SiO2 shell thickness 18 nm). Then add 0.5 g CuBr, 1.0 g pentamethyldiethylene triamine (PMDETA) and 5 mL glycidyl methacrylate (GMA), and react at 60 ℃ for 6 h under nitrogen protection to graft PGMA by atom transfer radical polymerization (ATRP), and obtain Fe3O4@SiO2@PGMA core-shell structure magnetic carrier with a particle size of 80-120 nm.

[0036] (2) Take 0.1 mmol of the characteristic fragment of Cornus officinalis polysaccharide (molecular weight 15 kDa, separated by gel filtration chromatography) as a template molecule, dissolve it in 20 mL acetonitrile-water mixed solvent (volume ratio 6:4), and then add 0.9 mmol 4-vinylphenylboronic acid (VPBA), 3.2 mmol ethylene glycol dimethacrylate (EGDMA) and 0.1 mmol azobisisobutyronitrile (AIBN) in sequence, stir and dissolve, and then add the Fe3O4@SiO2@PGMA core-shell structure magnetic carrier of step (1), and first pre-polymerize at 48 ℃ for 2.5 h, and then polymerize at 62 ℃ for 9 h under nitrogen protection. After the reaction is completed, wash and elute 3 times (2 h each time) with a methanol-acetic acid mixture (volume ratio 8:2), and detect the removal rate of the template molecule by high performance liquid chromatography, which is 98.5%, and obtain boron affinity-molecularly imprinted magnetic nanoparticles.

[0037] A method for directional extraction and purification of Cornus officinalis polysaccharide, which has substantially the same content and embodiments as Example 1, except that the boron affinity-molecularly imprinted magnetic nanoparticles prepared in Comparative Example 1 are used.

[0038] The adsorption capacity, imprint factor and polysaccharide purity in the adsorption process of Example 1-Example 3 and Comparative Example 1 of the application are determined. The test method is as follows: (1) Adsorption capacity determination: The polysaccharide content was quantified using a static adsorption method combined with a phenol-sulfuric acid colorimetric method. The steps are as follows: A series of standard solutions of Cornus officinalis polysaccharides of known concentrations were precisely prepared, and the absorbance at 490 nm was measured to plot a standard curve. A certain amount of the boron affinity-molecularly imprinted magnetic nanoparticles (m, g) of this invention were added to a Cornus officinalis polysaccharide solution of known concentration (V0, mL, concentration C0, mg / mL). After adsorption reached equilibrium by shaking at 25℃, the supernatant was magnetically separated, and the absorbance was measured to calculate the equilibrium concentration C0. e (mg / mL). The adsorption capacity Q (mg / g) is calculated using the formula: (2) Measurement of imprinting factor (IF): Non-imprinted polymers (NIPs) were prepared (except for the absence of template molecules, the preparation conditions were exactly the same as those for imprinted polymers (MIPs), and the adsorption capacities (Q) of MIPs and NIPs were determined respectively. m and Q n The imprint factor is calculated using the following formula: (3) Polysaccharide purity determination: The "total sugar content - impurity content" difference method was used, combined with multiple detection methods: ① Total sugar content: phenol-sulfuric acid colorimetric method (same as adsorption capacity determination). ② Protein impurities: Coomassie Brilliant Blue G-250 method, measuring absorbance at 595 nm to calculate protein content. ③ Monosaccharide impurities: high performance liquid chromatography (HPLC), equipped with an amino column and a differential refractive index detector, with acetonitrile-water (75:25, v / v) as the mobile phase, column temperature 30℃, flow rate 1.0 mL / min, qualitative and quantitative analysis using standard monosaccharides (glucose, galactose, etc.). Purity calculation: Purity = Total sugar content - Protein content - Total monosaccharide content.

[0039] The test results of adsorption capacity, imprinting factor and polysaccharide purity of Examples 1-3 and Comparative Example 1 of this invention are shown in Table 1.

[0040] Table 1 Test results of Examples 1-3 and Comparative Example 1 As shown in Table 1, the adsorption capacity of Example 1 of the present invention (89.6 mg / g) is 71.3% higher than that of the single boron affinity particle of Comparative Example 1 (52.3 mg / g), and the imprinting factor (IF=7.2) is significantly higher than that of the latter (IF=3.5).

[0041] 2. Analysis of the selective adsorption effect of boron affinity-molecularly imprinted magnetic nanoparticles: The selective adsorption effect of the boron affinity-molecularly imprinted magnetic nanoparticles prepared in Example 1 of this invention was analyzed. For comparison, a boric acid composite dual-template magnetic molecularly imprinted polymer (as a control group) was prepared according to the method described in Example 1 of Chinese Invention Patent Publication No. CN114957782A. The boric acid composite dual-template magnetic molecularly imprinted polymer was used to treat the crude extract of Cornus officinalis according to the method for directional extraction and purification of Cornus officinalis polysaccharides described in Example 1 of this invention to extract Cornus officinalis polysaccharides. Then, the selective adsorption capacity and impurity removal rate of Example 1 and the control group were tested. The test methods are as follows: (1) Determination of selective adsorption capacity: Standard curve for the target polysaccharide: Accurately weigh 0.1000 g of the characteristic fragment standard of Cornus officinalis polysaccharide, dissolve it in deionized water and dilute to 100 mL to prepare a 1.0 mg / mL stock solution. Take 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the stock solution respectively, and dilute to 10 mL with deionized water to obtain a series of standard solutions of 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL. Use the phenol-sulfuric acid method to measure the absorbance at a wavelength of 490 nm. Plot a standard curve with concentration (c) on the x-axis and absorbance (A) on the y-axis.

[0042] Interference substance standard curves: The concentrations of dextran T-40, wolfberry polysaccharide, glucose, and fructose were determined by HPLC (mobile phase: 0.1 mol / L NaNO3 solution, flow rate: 0.8 mL / min, column temperature: 30℃); the concentration of gallic acid was determined by ultraviolet spectrophotometry at a wavelength of 273 nm. Standard curves were plotted and regression equations were obtained.

[0043] Single-substance adsorption: Prepare 50 mL solutions each of Cornus officinalis polysaccharide characteristic fragment, dextran T-40, Lycium barbarum polysaccharide, glucose, fructose, and gallic acid at a concentration of 0.1 mg / mL, and adjust the pH to 7.8. Take 30 mL of each solution and add 0.6 g of boron affinity-molecularly imprinted magnetic nanoparticles (solid-liquid ratio 1:50, g:mL). Incubate at 30℃ with shaking for 90 min to allow adsorption. After adsorption, separate the nanoparticles using an external magnetic field and collect the supernatant.

[0044] Adsorption of mixed system: Prepare 50 mL of mixed solution (containing 0.1 mg / mL of Cornus officinalis polysaccharide characteristic fragment, 0.05 mg / mL each of dextran T-40, Lycium barbarum polysaccharide, glucose, fructose and gallic acid), adjust pH to 7.8, and conduct adsorption experiments under the above conditions.

[0045] Formula for calculating selective adsorption capacity (Q, mg / g): In the formula: c0 is the initial concentration of the substance before adsorption (mg / mL); c1 is the remaining concentration of the substance in the supernatant after adsorption (mg / mL); V is the solution volume (mL); m is the mass of the boron affinity-molecular imprinted magnetic nanoparticles (g).

[0046] (2) Determination of impurity removal rate: Take the crude extract of Cornus officinalis (polysaccharide content approximately 15%), adjust the pH to 7.8, and use it as the "sample before purification". Determine the content of each impurity (W0). Perform extraction and purification according to the method described (30 mL crude extract + 0.6 g boron affinity-molecularly imprinted magnetic nanoparticles, adsorption at 30℃ for 90 min, elution with acetic acid-ethanol mixture, concentration and drying). Redissolve the dried Cornus officinalis polysaccharide in 30 mL of deionized water, and use this as the "sample after purification". Determine the content of each impurity (W1).

[0047] Protein removal rate: The Coomassie Brilliant Blue G-250 method was used. A standard curve was plotted at 595 nm using BSA as a standard, and the protein content of the samples before and after purification was determined.

[0048] Pigment removal rate: Ultraviolet spectrophotometry was used. The absorbance (characteristic absorption peak of pigment) of the samples before and after purification was measured at a wavelength of 420 nm. The absorbance indirectly reflects the pigment content (absorbance is positively correlated with pigment concentration).

[0049] Small molecule sugar removal rate: The phenol-sulfuric acid method combined with ethanol precipitation method was used. Take 5 mL of sample solution, add 15 mL of anhydrous ethanol (75% ethanol volume fraction), let stand at 4℃ for 2 h, centrifuge at 8000 r / min for 20 min, take the supernatant (containing small molecule sugars), and determine the total sugar content (i.e., small molecule sugar content) in the supernatant using the phenol-sulfuric acid method.

[0050] The test results of selective adsorption capacity and impurity removal rate of Example 1 and the control group of the present invention are shown in Table 2.

[0051] Table 2 Test results of Example 1 and control group As shown in Table 2, the selective adsorption capacity of Cornus officinalis polysaccharides by this invention is significantly improved compared to the control group in both single and mixed systems, and the removal rate of impurities (proteins, pigments, and small molecule sugars) is increased by more than 40%. The control group uses a dual template of "protein + general polysaccharide", and the imprint cavity needs to accommodate both types of substances, resulting in insufficient specificity for Cornus officinalis polysaccharides. In contrast, this invention uses "characteristic fragments of Cornus officinalis polysaccharides (5-30 kDa)" as a single template, and combines its cis-dihydroxy distribution and spatial structure design to create a recognition system, achieving precise capture of the target polysaccharide and significantly improving selectivity and purity.

[0052] 3. Analysis of the regeneration performance of boron affinity-molecularly imprinted magnetic nanoparticles: Boron affinity-molecularly imprinted magnetic nanoparticles from Example 1 of this invention were used to extract Cornus officinalis polysaccharides according to the method described in Example 1: The pH of the crude extract of Cornus officinalis (polysaccharide content approximately 15%) was adjusted to 7.5. The boron affinity-molecularly imprinted magnetic nanoparticles prepared in Example 1 were added at a solid-liquid ratio of 1:50 (g:mL). The mixture was subjected to constant temperature shaking at 28℃ for 75 min, followed by separation under an external magnetic field for 5 min. The boron affinity-molecularly imprinted magnetic nanoparticles were then separated and eluted twice with a pH 4.8 acetic acid-ethanol mixture (volume ratio 1:9). The eluents were combined, concentrated under reduced pressure, and then freeze-dried to obtain Cornus officinalis polysaccharides. After the first extraction, the eluted boron affinity-molecularly imprinted magnetic nanoparticles were collected by magnetic separation, washed with deionized water until neutral, and vacuum-dried for use in the next extraction. This process was repeated 5 times. The adsorption capacities (Q1 and Q5) of the first and fifth extractions were measured, and the retention rate was calculated. The formula for calculating the retention rate is shown below: Calculations show that the boron affinity-molecularly imprinted magnetic nanoparticles of Example 1 of this invention have a retention rate of 91.5% after 5 adsorption-desorption cycles, which is significantly better than existing magnetic imprinted materials (usually ≤80%). This is due to the stability of the Fe3O4@SiO2@PGMA core-shell structure (the SiO2 shell isolates Fe3O4 from external reactions, and the PGMA layer provides stable active sites), as well as the mild process of "neutral adsorption-weak acid elution" (avoiding the damage of strong acids and alkalis to the particle structure). This solves the problem of "easy degradation of structure and rapid decay of adsorption performance during recycling" of traditional magnetic materials, and meets the needs of continuous industrial production.

[0053] 4. Synergistic effect analysis of the composite ligand system in boron affinity-molecularly imprinted magnetic nanoparticles. Comparative Example 2 and Comparative Example 3 were designed for comparison. The specific contents of Comparative Example 2 and Comparative Example 3 are as follows: Comparative Example 2: Single Team Boron Affinity Molecule (APBA-HDA) System A method for preparing boron affinity-molecularly imprinted magnetic nanoparticles, the specific steps of which are as follows: (1) Dissolve 10 mmol FeCl3·6H2O in 50 mL ethylene glycol, add 2.0 g sodium acetate, stir for 30 min and transfer to a high-pressure reactor, react at 200 ℃ for 8 h, and collect Fe3O4 nanoparticles (particle size about 80 nm) by magnetic separation after cooling. Disperse Fe3O4 in 100 mL ethanol-water mixture (volume ratio 3:1), add 5 mL tetraethyl orthosilicate (TEOS) and 2 mL ammonia, stir at 30 ℃ for 6 h to obtain Fe3O4@SiO2 (SiO2 shell thickness 15 nm). Then add 0.5 g CuBr, 1.0 g pentamethyldiethylenetriamine (PMDETA) and 5 mL glycidyl methacrylate (GMA), react at 60 ℃ for 6 h under nitrogen protection, and graft PGMA by atom transfer radical polymerization (ATRP) to obtain Fe3O4@SiO2@PGMA core-shell magnetic carrier with a particle size of 80-120 nm.

[0054] (2) Weigh 1.2 g of 3-aminophenylboronic acid and 1.0 g of 1,6-hexanediamine, disperse them in 50 mL of ethanol, stir at 65 °C for 390 min, then add the Fe3O4@SiO2@PGMA core-shell magnetic support prepared in step (1), reflux at 95 °C for 12 h, collect the solid by magnetic separation, wash with ethanol 3 times to obtain the boron affinity functionalized magnetic support.

[0055] (3) Take 0.1 mmol of Cornus officinalis polysaccharide characteristic fragment (molecular weight 5 kDa, separated by gel filtration chromatography) as template molecule, dissolve it in 20 mL of acetonitrile-water mixed solvent (volume ratio 6:4), add 3.0 mmol of ethylene glycol dimethacrylate (EGDMA) and 0.08 mmol of azobisisobutyronitrile (AIBN) in sequence, stir to dissolve, and then add the boron affinity functionalized magnetic support from step (2). Under nitrogen protection, prepolymerize at 45 ℃ for 2 h, and then heat to 60 ℃ for 8 h. After the reaction is completed, elute with methanol-acetic acid mixture (volume ratio 8:2) 4 times (2 h each time). The template molecule removal rate was detected by high performance liquid chromatography as 99.1%, and boron affinity-molecularly imprinted magnetic nanoparticles were obtained.

[0056] A method for targeted extraction and purification of Cornus officinalis polysaccharides is basically the same as that in Example 2, except that the method uses boron affinity-molecularly imprinted magnetic nanoparticles prepared in Comparative Example 2.

[0057] Comparative Example 3: Single VPBA System A method for preparing boron affinity-molecularly imprinted magnetic nanoparticles, the specific steps of which are as follows: (1) Dissolve 10 mmol FeCl3·6H2O in 50 mL ethylene glycol, add 2.0 g sodium acetate, stir for 30 min and transfer to a high-pressure reactor, react at 200 ℃ for 8 h, and collect Fe3O4 nanoparticles (particle size about 80 nm) by magnetic separation after cooling. Disperse Fe3O4 in 100 mL ethanol-water mixture (volume ratio 3:1), add 5 mL tetraethyl orthosilicate (TEOS) and 2 mL ammonia, stir at 30 ℃ for 6 h to obtain Fe3O4@SiO2 (SiO2 shell thickness 15 nm). Then add 0.5 g CuBr, 1.0 g pentamethyldiethylenetriamine (PMDETA) and 5 mL glycidyl methacrylate (GMA), react at 60 ℃ for 6 h under nitrogen protection, and graft PGMA by atom transfer radical polymerization (ATRP) to obtain Fe3O4@SiO2@PGMA core-shell magnetic carrier with a particle size of 80-120 nm.

[0058] (2) 0.1 mmol of Cornus officinalis polysaccharide characteristic fragment (molecular weight 5 kDa, separated by gel filtration chromatography) was used as a template molecule and dissolved in 20 mL of acetonitrile-water mixed solvent (volume ratio 6:4). 0.8 mmol of 4-vinylphenylboronic acid (VPBA), 3.0 mmol of ethylene glycol dimethacrylate (EGDMA), and 0.08 mmol of azobisisobutyronitrile (AIBN) were added sequentially. After stirring and dissolving, the Fe3O4@SiO2@PGMA core-shell structured magnetic support from step (1) was added. Under nitrogen protection, the mixture was prepolymerized at 45 °C for 2 h, and then heated to 60 °C for 8 h of polymerization. After the reaction, the mixture was eluted 4 times (2 h each time) with a methanol-acetic acid mixture (volume ratio 8:2). The template molecule removal rate was 99.1% as determined by high performance liquid chromatography, yielding boron affinity-molecularly imprinted magnetic nanoparticles.

[0059] A method for targeted extraction and purification of Cornus officinalis polysaccharides is basically the same as that in Example 2, except that the method uses boron affinity-molecularly imprinted magnetic nanoparticles prepared in Comparative Example 3.

[0060] The selective adsorption capacity and reusable retention rate of Example 2, Comparative Example 2 and Comparative Example 3 were determined using the test method described above in this invention. The test results are shown in Table 3.

[0061] Table 3 Test results of Example 2, Comparative Example 2 and Comparative Example 3 As shown in Table 3, the selective adsorption capacity and reusability retention rate of Example 2 are much higher than those of Comparative Example 2 [single team boron affinity molecule (APBA-HDA) system (without VPBA)] and Comparative Example 3 [single VPBA system (without APBA-HDA)], indicating that the performance improvement of the two groups of single ligands and complex ligands is clearly due to "synergistic effect" rather than the individual effect of single APBA-HDA (disclosed) or single VPBA (known ligand).

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention based on the concept of the present invention, without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A preparation method of boron affinity-molecularly imprinted magnetic nanoparticles for directional extraction of purified polysaccharides from Cornus officinalis, characterized in that, It comprises the following steps: (1) Fe3O4 nanoparticles are coated with SiO2 to obtain Fe3O4@SiO2 particles, and the Fe3O4@SiO2 particles are grafted with polyglycidyl methacrylate to obtain Fe3O4@SiO2@PGMA core-shell structure magnetic carriers; (2) The Fe3O4@SiO2@PGMA core-shell structure magnetic carriers obtained in step (1) are modified with a boron affinity molecule to obtain boron affinity functionalized magnetic carriers; (3) The template molecule, auxiliary functional monomer, crosslinking agent and initiator are dissolved in a solvent, and the boron affinity functionalized magnetic carriers obtained in step (2) are added to the solvent, and under an inert gas atmosphere, pre-polymerization is first carried out at 45-50℃ for 2-3h, and then polymerization is carried out at 60-65℃ for 8-10h, after the reaction is completed, the template molecule is removed by elution to obtain boron affinity-molecularly imprinted magnetic nanoparticles; wherein the template molecule is a characteristic fragment of cornus officinalis polysaccharide with a molecular weight of 5-30kDa.

2. The preparation method of boron affinity-molecularly imprinted magnetic nanoparticles for directional extraction and purification of polysaccharides in Corni fructus according to claim 1, characterized in that, In step (3), the auxiliary functional monomer is 4-vinylphenylboronic acid. 3.The method for preparing boron affinity-molecularly imprinted magnetic nanoparticles for directional extraction of purified polysaccharides from Corni fructus according to claim 1, characterized in that, In step (3), the molar ratio of the template molecule, auxiliary functional monomer, crosslinking agent and initiator is 1:(8-10):(30-35):(0.8-1.2). 4.The method for preparing boron affinity-molecularly imprinted magnetic nanoparticles for directional extraction of purified polysaccharides from Corni fructus according to claim 1, characterized in that, In step (3), the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile. 5.The method for preparing boron affinity-molecularly imprinted magnetic nanoparticles for directional extraction of purified Cornus officinalis polysaccharides according to claim 1, characterized in that, In step (2), the specific method of modification is: 3-aminobenzeneboronic acid and 1,6-hexanediamine are dispersed in ethanol to obtain a dispersion, and the Fe3O4@SiO2@PGMA core-shell structure magnetic carriers obtained in step (1) are added to the dispersion for reflux reaction, the solid is collected by magnetic separation after the reaction is completed, and the solid is washed to obtain boron affinity functionalized magnetic carriers. 6.The method for preparing boron affinity-molecularly imprinted magnetic nanoparticles for directional extraction of purified polysaccharides of Corni Fructus according to claim 5, characterized in that, The mass ratio of 3-aminobenzeneboronic acid to 1,6-hexanediamine is (1.2-1.5):1, the stirring condition is stirring at 60-70℃ for 360-420min, and the reflux reaction condition is refluxing at 90-100℃ for 10-14h.

7. A boron affinity-molecularly imprinted magnetic nanoparticle for directional extraction and purification of cornus officinalis polysaccharide prepared by the method of any one of claims 1-6.

8. The use of the boron affinity-molecularly imprinted magnetic nanoparticle of claim 7 in directional extraction and purification of cornus officinalis polysaccharide.

9. A method for directional extraction of purified polysaccharides from Cornus officinalis, characterized in that, It comprises the following steps: The pH value of the crude extract of cornus officinalis is adjusted to 7.2-7.8, the boron affinity-molecularly imprinted magnetic nanoparticle of claim 7 is added, and the adsorption is carried out by oscillation at 25-30℃, then the boron affinity-molecularly imprinted magnetic nanoparticle is separated and collected by an external magnetic field, and then the collected boron affinity-molecularly imprinted magnetic nanoparticle is eluted with acetic acid-ethanol mixed solution, the eluate is collected and concentrated under reduced pressure, and freeze-dried to obtain cornus officinalis polysaccharide.

10. The method for directional extraction and purification of polysaccharides from Cornus officinalis according to claim 9, characterized in that, The pH of the acetic acid-ethanol mixed solution is 4.5-5.0; during the oscillation adsorption process, the amount of boron affinity-molecularly imprinted magnetic nanoparticle used is 1g per 30-50mL of cornus officinalis crude extract.

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