Template molecule, molecularly imprinted polymer and application of molecularly imprinted polymer in separation of naringin

Abstract

The invention relates to a template molecule, a molecularly imprinted polymer and application of the molecularly imprinted polymer in separation of naringin, and belongs to the technical field of plant component refining. According to the molecularly imprinted polymer disclosed by the invention, (S)-2-(4-hydroxymethyl phenyl)-7-(((2S, 3R, 4S, 5S, 6R)-3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzodihydropyran-4-one is used as a template molecule, and the template molecule and a target product naringin can form multiple acting sites through non-covalent bonds such as hydrogen bonds and hydrophobic interaction, so that the target product naringin can be used for preparing the target product naringin. Therefore, specific binding is carried out, namely, high selectivity to naringin is achieved. The virtual template molecule is low in cost and can be used for large-scale preparation of molecularly imprinted polymers, and the extraction cost of naringin is remarkably reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of plant component refining, and specifically relates to a template molecule, a molecular imprinting polymer and applications thereof in separating naringin. Background Art

[0002] Molecularly imprinted polymers are a new type of enrichment material, which are widely used in biochemical separation analysis due to their structural stability and specificity of target recognition. Naringin is a flavonoid derived from the Chinese medicinal herbs tangerine peel, green peel, immature bitter orange, bitter orange, bone drynaria, tangerine peel and citron, or the fruits or peels of Rutaceae plants such as pomelo, grapefruit, citron, tangerine and orange, and has multiple effects such as anti-inflammatory, antioxidant, anti-inflammatory ultraviolet damage, and oil control. Molecularly imprinted polymers prepared using naringin template molecules can be used to extract naringin from plants, but the cost of naringin template molecules is high. Summary of the invention

[0003] The first object of the present invention is to provide a template molecule to reduce costs by designing a virtual template molecule.

[0004] The second object of the present invention is to provide a molecularly imprinted polymer prepared by the above template molecule.

[0005] The third object of the present invention is to provide the application of the molecularly imprinted polymer.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A template molecule with the following structural formula:

[0008]

[0009] The molecularly imprinted polymer microspheres prepared by the above template molecules are characterized in that the preparation method thereof comprises the following operations:

[0010] Prepare dispersed phase: mix hydrophilic functional monomer, crosslinking agent, water and template molecule to form solution A;

[0011] Prepare the continuous phase: mix the emulsifier with n-hexane under an inert gas atmosphere to form a continuous phase as solution B;

[0012] Preparation of inverse dispersion system: Mix solution A and solution B under an inert gas atmosphere to obtain a white emulsion;

[0013] Polymerization reaction: Mix the white latex with the initiator to obtain a polymer microsphere mixture;

[0014] Post-treatment: Mix the polymer microsphere mixture with methanol, filter out the precipitate, wash the precipitate to neutrality after eluting the template molecules, and then dry it to constant weight to obtain molecularly imprinted polymer microspheres.

[0015] As a preferred embodiment of the present invention, in preparing the dispersed phase, the hydrophilic functional monomer is one of acrylamide, acrylic acid and allylamine.

[0016] As a preferred embodiment of the present invention, in preparing the dispersed phase, the crosslinking agent is at least one of N,N'-methylenebisacrylamide, polyethylene glycol dimethacrylate, and trimethoxypropyl trimethacrylate.

[0017] As a preferred embodiment of the present invention, in preparing the dispersed phase, the molar ratio of the hydrophilic functional monomer to the template molecule is 1:4.5-7.5.

[0018] As a preferred embodiment of the present invention, in the preparation of the continuous phase, the emulsifier is at least one of docusate sodium, polyoxyethylene lauryl ether, and Span 80.

[0019] As a preferred embodiment of the present invention, in the polymerization reaction, the initiator is one of potassium persulfate, benzoyl peroxide and ammonium persulfate.

[0020] As a preferred embodiment of the present invention, in the polymerization reaction, the polymerization reaction temperature is 30-75°C.

[0021] As a preferred embodiment of the present invention, in the polymerization reaction, the polymerization reaction time is 1 to 4 hours, and stirring is continued during the polymerization reaction.

[0022] Application of the molecular imprinting polymer in the separation of naringin.

[0023] Beneficial effects of the present invention:

[0024] The molecular imprinted polymer of the present invention uses (S)-2-(4-hydroxymethylphenyl)-7-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)chroman-4-one as a template molecule. The template molecule contains a hydrogen bond donor and a strong electronic effect, and can form multiple action sites with the target product naringin through non-covalent bonds such as hydrogen bonds and hydrophobic interactions, thereby specifically binding, that is, having a high selectivity for naringin. The virtual template molecule has a low cost and can be used for large-scale preparation of molecular imprinted polymers, significantly reducing the extraction cost of naringin.

[0025] The naringin molecularly imprinted polymer microspheres prepared by the preparation method of the present invention can increase the adsorption amount of the target molecule and improve the purity of naringin in application, and can be used for the separation, enrichment and purification of naringin compounds in natural product extracts, with high enrichment, high accuracy and wide application range. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with specific implementation methods.

[0027] Example 1

[0028] A template molecule (S)-2-(4-hydroxymethylphenyl)-7-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)chroman-4-one was prepared, and its structural formula is:

[0029]

[0030] The specific experimental operations are as follows:

[0031] (1) Add 4.0 mmol of compound A1-[2-hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)phenyl]-1-ethanone, 4.8 mmol of compound B benzaldehyde and 8 mL of methanol to a round-bottom flask. Then, a methanol solution of KOH (2M, 20 mL) was added dropwise to the solution. The mixture was stirred at room temperature for 48 hours. The crude product was diluted with 40 mL of water, and the aqueous layer was then extracted with ethyl acetate (30 mL×3). The organic layers were combined and washed with 40 mL of saturated sodium chloride aqueous solution. The organic layer was dried over anhydrous sodium sulfate, and the organic solvent was then removed by rotary evaporator. Compound C was obtained after purification by column chromatography (n-hexane / ethyl acetate volume ratio of 2:1) with a yield of 85%.

[0032] (2) Add 3.4 mmol of compound C, 6 mL of ethanol and 6 mL of 4 M hydrochloric acid to a round-bottom flask. Stir the reaction at room temperature for 4 hours. Then dilute with 20 mL of water, extract the aqueous layer with ethyl acetate (20 mL×3), and wash the organic layer with 30 mL of saturated sodium chloride aqueous solution. Dry the organic layer with anhydrous sodium sulfate, and then remove the organic solvent by rotary evaporator. After purification by column chromatography (n-hexane / ethyl acetate volume ratio of 3:1), compound D was obtained with a yield of 80%.

[0033] (3) Add 10.0 mmol of D-glucose and 10 mL of acetic anhydride to a round-bottom flask to obtain a mixture, and cool the mixture to 0°C. Then, add 0.1 m of perchloric acid (70 wt%) dropwise to the mixture while stirring. After the addition is completed, continue stirring at 0°C for 5 h to obtain a reaction solution. Dilute the reaction solution with dichloromethane (40 mL). Wash the diluted reaction solution with water (20 mL), saturated NaHCO3 solution (20 mL) and brine (20 mL), respectively. Dry the organic layer with anhydrous sodium sulfate, and then remove the organic solvent in the organic layer by a rotary evaporator to obtain acetyl-protected glucose with a yield of 94%.

[0034] (4) Add 2.0 mmol of acetyl-protected glucose and 20 mL of dichloromethane to a round-bottom flask, and add 0.83 mL of 48 wt% boron trifluoride ether solution (Article No.: Titan Technology 01375737) to the mixed solution at 0°C. Continue to stir the mixture at 0°C for 10 minutes, and then add 2.6 mmol of compound D to the mixture. Then warm to room temperature and continue stirring for 24 hours. After the reaction is completed, cool to 0°C, add 10 mL of saturated NaHCO3 aqueous solution to the mixture at 0°C, and wash the organic layer with water (10 mL×3). After separation, further extract the aqueous layer with 20 mL of dichloromethane. The combined dichloromethane organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure. Compound E is obtained after column chromatography purification with a yield of 90%.

[0035] (5) 1.5 mmol of compound E was dissolved in 10 mL of methanol and 1.5 mL of 25 wt% aqueous ammonia solution, stirred at room temperature for 24 hours, and then the solvent was removed under reduced pressure. The template molecule was obtained after column chromatography purification with a yield of 91%.

[0036] The template molecule map is as follows:

[0037] 1 H NMR(400MHz,DMS0-d6)δ7.72-7.69(d,J=8.68Hz,1H),7.34-7.25(m,J=8.44Hz,2H),6.80-6.69(m,J=8.31Hz,2H),6.68 -6.77(m,2H),5.50-5.39(m,1H),5.36-5.32(d,J=4.28Hz,1H),5.10(d,J=3.67Hz,1H),5.09-5.01(d,J=5.14Hz,1H),4.99-4.93(t,J =7.46Hz,1H),4.55(m,1H),3.81(s,3H),3.66(m,1H),3.44(d,J=11.52,5.79Hz,1H),3.38(d,J=9.66Hz,1H),3.12-3.28(m,4H),2.61 -2.76(m,1H). 13 C NMR(101MHz,DMSO-d6)δ197.1,165.4,162.9,159.6,130.4,128.4(2C),113.9(2C),103. 3,99.7,96.7,95.5,78.5,77.1,76.4,73.1,69.6,60.7,55.8,55.2,42.1.HRMS(ESI)m / z calcd for C 22 H 24 O9[M+H] +:433.1568,found:433.1570.

[0038] Example 2 Preparation and performance testing of molecularly imprinted polymers

[0039] 1. Prepare molecularly imprinted polymers, the steps are as follows:

[0040] 1.1 Preparation of dispersed phase: 6 mmol acrylic acid and 30 mmol polyethylene glycol dimethacrylate (EGDMA) were dissolved in 30 mL water, and 1 mmol of the template molecule (S)-2-(4-hydroxymethylphenyl)-7-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)chroman-4-one prepared in Example 1 was added, uniformly dispersed, and magnetically stirred at room temperature for 0.5 h to form a prepolymer to obtain solution A;

[0041] 1.2 Preparation of continuous phase: 12 g of sodium docusate (AOT) and 23 g of L4 polyoxyethylene lauryl ether (BRIJ 30) were dissolved in 450 mL of n-hexane under nitrogen atmosphere to form a continuous phase as solution B;

[0042] 1.3 Preparation of reverse phase dispersion system: slowly transfer solution A to solution B while stirring, and continue stirring at room temperature for 60 min under nitrogen atmosphere to obtain a white emulsion;

[0043] 1.4 Polymerization reaction: 0.75 mL of 10% (w / v) ammonium persulfate (APS) was added to the white emulsion to initiate the polymerization reaction, and the reaction was continued at 45°C and 380 r / min for 3 h to obtain a polymer microsphere mixture;

[0044] 1.5 Post-treatment: Methanol is added to the polymer microsphere mixture to aggregate and precipitate the polymer, and the polymer is obtained by filtration. Transfer to a 200-mesh sieve, wash with water to remove fine powder, and then dry. The dried polymer is placed in a Soxhlet extractor, and the template molecules in the polymer are extracted with methanol. The polymer is vacuum dried at 50°C to constant weight. After drying, the imprinted material is classified with sieves of different mesh sizes to obtain molecular imprinted polymers of different sizes.

[0045] 2. Performance Testing

[0046] 2.1 Study on the adsorption performance of naringin:

[0047] i) Analytical method: Refer to the Chinese Pharmacopoeia 2015 edition P246 Detection method of naringin in Citrus aurantium (HPLC)

[0048] ii) Determination of adsorption amount: Accurately weigh 0.2g of molecularly imprinted nanospheres and place them in a ground-mouth conical flask, add 5.0mL of naringin methanol solution with a concentration of 3mg / mL, and adsorb at a constant temperature for 5h after oscillation. Centrifuge and dilute the supernatant with methanol to 50-300μg / mL, then detect and calculate the naringin concentration according to method i. The adsorption amount of the imprinted polymer (58.77mg / g) was calculated based on the change in naringin concentration in the solution before and after adsorption. Calculation formula:

[0049] Q=(C0-C)*V / W

[0050] Where: Q: adsorption amount per gram of polymer (mg / g);

[0051] C0: original mass concentration of adsorbed compound in equilibrium adsorption experiment (mg / L);

[0052] C: mass concentration of adsorbed compound in the supernatant after reaching adsorption equilibrium (mg / L);

[0053] V: volume of adsorption solution (L);

[0054] W: mass of imprinted polymer (g).

[0055] Experimental Example 1: Investigating the effect of different emulsifiers on polymer shape during the preparation process

[0056] Based on the preparation method in Example 2, different emulsifiers were used as dispersions (see Table 1) to obtain molecularly imprinted polymers. The adsorption of naringin by the molecularly imprinted polymers was measured according to 2.1. The results are shown in Table 1:

[0057] Table 1

[0058] Emulsifier Polymer adsorption Docusate Sodium(AOT) 57.04mg / g L4 Polyoxyethylene Laureth Ether (BRIJ 30) 52.90mg / g Span 80 39.56mg / g

[0059] As shown in Table 1, the molecular imprinted polymers prepared by using docusate sodium (AOT) and L4 polyoxyethylene lauryl ether (BR I J30) as emulsifiers have stronger adsorption performance for naringin; the adsorption performance of the molecular imprinted polymer prepared by using Span 80 as an emulsifier is relatively lower than that of the other two.

[0060] Experimental Example 2 Investigating the effects of different functional monomer types and proportions used in the dispersed phase preparation step on the properties of molecularly imprinted polymers

[0061] Based on the preparation method in Example 2, different functional monomer types and ratios (see Table 2) were used to obtain different molecularly imprinted polymers, and their adsorption amounts for naringin were measured. The results are shown in Table 2:

[0062] Table 2

[0063] Serial number Functional monomer and dosage (molar ratio) Polymer adsorption 1 Template molecule / acrylamide (1:6) 42.63mg / g 2 Template molecule / acrylic acid (1:6) 58.02mg / g 3 Template molecule / allylamine (1:6) 50.89mg / g 4 Template molecule / acrylic acid (1:3) 17.93mg / g 5 Template molecule / acrylic acid (1:4.5) 47.09mg / g 6 Template molecule / acrylic acid (1:7.5) 45.37mg / g

[0064] As shown in Table 2, when acrylamide, acrylic acid, and allylamine are selected as hydrophilic functional monomers and the template molecule is used in combination with them at a molar ratio of 1:6, the prepared molecular imprinted polymer has a good adsorption capacity for naringin. The adsorption capacity is the largest when the template molecule is used in combination with acrylic acid at a molar ratio of 1:6, which is 58.02 mg / g. When the template molecule and acrylic acid are used in combination at different molar ratios, the adsorption capacity of naringin by the prepared molecular imprinted polymer changes greatly. When the molar ratio of the template molecule to acrylic acid is 1:3, the adsorption capacity is significantly reduced to 17.93 mg / g. When the molar ratio of the template molecule to acrylic acid is 1:4.5 and 1:7.5, the adsorption capacity is slightly lower than that when the molar ratio is 1:6.

[0065] Experimental Example 3 Investigating the Effect of Using Different Crosslinking Agents in the Preparation of Dispersed Phase on Molecularly Imprinted Polymers

[0066] Based on the preparation method in Example 2, different cross-linking agents (see Table 3) were used to obtain different molecularly imprinted polymers, and their adsorption amounts for naringin were measured. The results are shown in Table 3:

[0067] Table 3

[0068] Crosslinking agent Polymer adsorption N,N'-Methylenebisacrylamide (MBA) 45.72mg / g Polyethylene glycol dimethacrylate (EGDMA) 57.83mg / g Trimethoxypropyl trimethacrylate (TRIM) 31.33mg / g

[0069] As shown in Table 3, different molecular imprinting polymers prepared by using N, N'-methylenebisacrylamide and polyethylene glycol dimethacrylate as cross-linking agents have better adsorption effects on naringin, and the effect of trimethoxypropyl trimethacrylate is slightly lower than the first two.

[0070] Experimental Example 4 Investigating the Effect of Using Different Initiators in the Polymerization Step on the Properties of Molecularly Imprinted Polymers

[0071] Based on the preparation method in Example 2, different initiators (see Table 4) were used to observe the reaction performance of different molecularly imprinted polymers. The results are shown in Table 4:

[0072] Table 4

[0073] Initiator Polymerization Potassium Persulfate Simple operation, quick response, stable process Benzoyl peroxide Simple operation, quick response, slightly poor process stability Ammonium Persulfate Simple operation, quick response, stable process

[0074] As shown in Table 4, different molecular imprinted polymers were prepared by using potassium persulfate, benzoyl peroxide and ammonium persulfate as initiators, all of which were simple to operate and had rapid reactions. The difference was that the molecular imprinted polymers prepared by potassium persulfate and ammonium persulfate had stable reaction processes, while the molecular imprinted polymers prepared by benzoyl peroxide had slightly less stable reaction processes.

[0075] Experimental Example 5 Investigating the Effect of Different Reaction Conditions in the Polymerization Step on the Properties of Molecularly Imprinted Polymers in the Preparation Method

[0076] Based on the preparation method in Example 2, different molecular imprinted polymers were prepared by changing the reaction conditions of the polymerization reaction, including reaction temperature, reaction time, and mechanical stirring speed (see Table 5), and their adsorption amounts for naringin were measured respectively. The results are shown in Table 5:

[0077] Table 5

[0078]

[0079] It can be seen from Table 5 that different molecularly imprinted polymers prepared under the conditions of polymerization reaction temperature of 30-75°C and reaction time of 1-4h can form microspheres; the optimal reaction conditions are reaction temperature of 45°C and reaction time of 3h, and the molecularly imprinted polymer microspheres have a strong adsorption capacity for naringin.

[0080] Example 3 Application of the molecularly imprinted polymer obtained in Example 2

[0081] In order to verify the separation performance and application potential of the molecularly imprinted polymer microspheres, the molecularly imprinted polymer microspheres were used as an adsorbent material for internal phase extraction to enrich and separate naringin from grapefruit extract.

[0082] The specific experiments are as follows:

[0083] (1) Preparation of the extract of pomelo of Rutaceae: Weigh 5 g of fresh pomelo, add 80 mL of 60% ethanol solution, heat and reflux in a water bath for 5 h, separate the supernatant after centrifugation, repeat the operation to wash the residue 3 times, combine the supernatants, and concentrate them by rotary evaporator to obtain pomelo extract (medicinal material: extract = 10:1, W / W), seal and keep at low temperature for later use.

[0084] (2) Adsorption experiment: First, 200 mg of molecularly imprinted polymer was loaded into a solid phase extraction column and activated with 5 mL of methanol. Then, 10 mg / mL grapefruit extract loading solution was prepared with methanol, and 3 mL of loading solution was loaded; 5 mL of methanol was used for elution; and ethanol-acetic acid (90:10, v / v) was used for elution.

[0085] (3) Result analysis: The collected solution was tested by high performance liquid chromatography according to the detection method of naringin in Fructus Aurantii Immaturus in the Chinese Pharmacopoeia 2015 edition P246. The concentration of naringin in the solution before and after adsorption was calculated based on the standard curve of naringin molecularly imprinted polymer microspheres, and the recovery rate was 86%.

[0086] The above is only an implementation method of the invention, and does not limit the scope of patent protection. Those skilled in the art can make non-substantial changes or substitutions based on the invention, which still fall within the scope of patent protection.

Claims

1. A template molecule, characterized in that The structural formula is as follows:

2. A molecularly imprinted polymer prepared from the template molecule according to claim 1, characterized in that: Preparation method thereof The following operations are included: Prepare dispersed phase: mix hydrophilic functional monomer, crosslinking agent, water and template molecule to form solution A; Prepare the continuous phase: mix the emulsifier with n-hexane under an inert gas atmosphere to form a continuous phase as solution B; Preparation of inverse dispersion system: Mix solution A and solution B under an inert gas atmosphere to obtain a white emulsion; Polymerization reaction: Mix the white latex with the initiator to obtain a polymer microsphere mixture; Post-treatment: Mix the polymer microsphere mixture with methanol, filter out the precipitate, wash the precipitate to neutrality after eluting the template molecules, and then dry it to constant weight to obtain molecularly imprinted polymer microspheres.

3. The molecularly imprinted polymer according to claim 2, characterized in that In preparing the dispersed phase, the hydrophilic functional monomer is one of acrylamide, acrylic acid and allylamine.

4. The molecularly imprinted polymer according to claim 2, characterized in that In preparing the dispersed phase, the crosslinking agent is at least one of N,N'-methylenebisacrylamide, polyethylene glycol dimethacrylate, and trimethoxypropyl trimethacrylate.

5. The molecularly imprinted polymer according to claim 2, characterized in that In preparing the dispersed phase, the molar ratio of the hydrophilic functional monomer to the template molecule is 1:4.5-7.

5.

6. The molecularly imprinted polymer according to claim 2, characterized in that In the preparation of the continuous phase, the emulsifier is at least one of docusate sodium, polyoxyethylene lauryl ether, and Span 80.

7. The molecularly imprinted polymer according to claim 2, characterized in that In the polymerization reaction, the initiator is one of potassium persulfate, benzoyl peroxide and ammonium persulfate.

8. The molecularly imprinted polymer according to claim 2, characterized in that During the polymerization reaction, the polymerization temperature is 30 to 75°C.

9. The molecularly imprinted polymer according to claim 2, characterized in that During the polymerization reaction, the polymerization reaction time is 1 to 4 hours, and stirring is continued during the polymerization reaction.

10. Use of the molecularly imprinted polymer according to any one of claims 2 to 9 in separating naringin.

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