Template molecule, molecularly imprinted polymer and application of molecularly imprinted polymer in separation of natural flavonoid compounds

Abstract

The invention relates to a template molecule, a molecularly imprinted polymer and application of the molecularly imprinted polymer in separation of natural flavonoid compounds, and belongs to the technical field of plant component refining. According to the molecularly imprinted polymer disclosed by the invention, 5, 7-dimethoxy-2-(4-methoxyphenyl)-3-(((2S, 3R, 4S, 5S, 6R)-3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy)-4H-benzo-4-ketone is used as a template molecule. The method can be used for separating, enriching and purifying the natural flavonoid compounds in a natural product extracting solution, and is high in enrichment degree, high in accuracy and wide in application range; according to the present invention, the natural flavonoid compound molecules such as quercitrin, kaempferol, kaempferol-3-O-rutinoside, rutin, puerarin, kaempferol-3-O-glucoside, myricetin, and myricetin can be well adsorbed.

Description

Technical Field

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

[0002] Molecularly imprinted polymers are a new type of enrichment material, which are widely used in biochemical separation analysis due to their stable structure and specific target recognition. Flavonoids are widely present in various plants, most of which contain sugar structures, that is, they have strong hydrogen bond receptors and can form very stable intermolecular hydrogen bonds with strong hydrogen bond donors, which is conducive to the construction of molecular imprinting. Molecularly imprinted polymers are prepared using molecular imprinting technology to extract flavonoids from plants. The cost of the template molecules used for extraction is high, and the high cost of the template molecules accounts for a considerable proportion of the extraction cost. Summary of the invention

[0003] The first object of the present invention is to provide a template molecule to reduce the extraction cost 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 through the following technical solutions:

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

[0008]

[0009] The molecularly imprinted polymer prepared by the above template molecule comprises the following steps:

[0010] Prepare dispersed phase: mix hydrophilic functional monomer, crosslinking agent, water and template molecule to prepare 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, elute the template molecules, wash until neutral, and then dry 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 acrylic acid, allylamine and methacrylic acid.

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

[0017] As a preferred embodiment of the present invention, in preparing the dispersed phase, the molar ratio of the template molecule to the hydrophilic functional monomer is 1:5-9.

[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, octylphenol polyoxyethylene ether, and Triton X-100.

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

[0020] As a preferred embodiment of the present invention, in the polymerization reaction, the polymerization reaction temperature is 30-60°C.

[0021] As a preferred embodiment of the present invention, in the polymerization reaction, the polymerization reaction time is 1.5 to 3.5 hours, and stirring is continued during the polymerization reaction.

[0022] The molecular imprinting polymer is used in separating natural flavonoid compounds, wherein the natural flavonoid compound is at least one of quercetin, kaempferol, kaempferol-3-O-rutinoside, rutin, puerarin, kaempferol-3-O-glucoside, myricetin and myricetin.

[0023] Beneficial effects of the present invention:

[0024] The molecular imprinted polymer of the present invention uses 5,7-dimethoxy-2-(4-methoxyphenyl)-3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-benzo-4-one as a template molecule. The template molecule contains a hydrogen bond donor (hydroxyl group), has strong hydrophobic interaction and π-π stacking (aromatic ring), and when natural flavonoid compounds are separated from natural products, the molecular imprinted polymer prepared by using the virtual template molecule can form a strong intermolecular force and a spatial pocket with higher specificity with the flavonoid compounds, that is, it has a higher selectivity for natural flavonoid compounds. The virtual template molecule has low cost and can be used for large-scale preparation of molecular imprinted polymers, which significantly reduces the extraction cost of natural flavonoid compounds.

[0025] The natural flavonoid compound molecular imprinting polymer prepared by the preparation method of the present invention can increase the adsorption amount of the target molecule during application, improve the purity of the natural flavonoid compound, can be used for the separation, enrichment and purification of the natural flavonoid compound in the natural product extract, has high enrichment, strong accuracy and wide application range, has good adsorption performance for the natural flavonoid compound molecules of quercetin, kaempferol, kaempferol-3-O-rutinoside, rutin, puerarin, kaempferol-3-O-glucoside, myricetin and myricetin, and has broad application prospects in the preparation and detection of natural flavonoid compounds. 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 is prepared: 5,7-dimethoxy-2-(4-methoxyphenyl)-3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-benzo-4-one, whose structural formula is:

[0029]

[0030] The specific experimental operations are as follows:

[0031] (1) Add 4.0mmol of compound A 2-hydroxy-4,6-dimethoxyacetophenone, 4.2mmol of compound B p-methoxybenzaldehyde and 8mL of water into a round-bottom flask. Then, add 40mmol of tetrahydropyrrole dropwise into the reaction system to obtain a mixture. Stir the resulting mixture at 50°C for 24 hours and then cool it to room temperature. Pour the mixture into 20mL of ice water and acidify it to pH=4 with aqueous hydrochloric acid solution (30%, v / v). Filter the resulting precipitate and wash it twice with water and ethanol respectively. After purification by column chromatography, compound C is obtained with a yield of 90%.

[0032] (2) 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 93%.

[0033] (3) 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% mass fraction boron trifluoride ether solution (Article No.: Titan Technology 01375737) to the mixed solution at 0°C to obtain a mixture. After continuing to stir the mixture at 0°C for 10 min, add 2.6 mmol of compound C to the mixture, and then continue to stir at room temperature for 24 h. 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. Combine the dichloromethane organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. After column chromatography purification, compound D is obtained with a yield of 92%.

[0034] (4) Compound D was dissolved in a mixture of 10 mL of methanol and 2.0 mL of a 25% aqueous ammonia solution, stirred at room temperature for 24 h, and then the solvent was removed under reduced pressure. The template molecule was purified by column chromatography with a yield of 95%.

[0035] The spectral data of the template molecule are as follows: 1H NMR (400MHz, DMSO-d6) δ8.13-7.99 (m, 2H), 6.95-6.86 (m, 2H), 6.43 (d, J = 2.0 Hz, 1H), 6.21 (d, J = 2.0 Hz, 1H), 5.46 (d, J = 7.3 Hz, 1H), 5.34 (d, J = 4.5 Hz, 1H), 5.05 (d, J = 4.6 Hz, 1H), 4.99 -4.91(m,1H),4.26(t,J=5.6Hz,1H),3.90(s,3H),3.81(s,6H),3.57(dd,J=11.5,5.5Hz,1 H), 3.32 (d, J = 10.3Hz, 2H), 3.19 (dtd, J = 13.5, 8.6, 4.7Hz, 1H), 3.08 (q, J = 4.2, 3.4Hz, 2H). 13 C NMR(101MHz,DMSO-d6)δ177.9,164.6,161.7,160.4,156.8,156.7,133.6,131.3(2C),121.3, 115.5(2C),104.4,101.2,99.1,94.1,77.9,76.8,74.6,70.3,61.3,55.8(3C).HRMS(ESI)m / z calcd for C 24 H 26 O 16 [M+H] + :491.2058,found:491.2055.

[0036] Example 2 Preparation and performance testing of molecularly imprinted polymers

[0037] 1. Prepare molecularly imprinted polymers, the steps are as follows:

[0038] 1.1 Preparation of dispersed phase: 5 mmol acrylic acid and 25 mmol N, N'-methylenebisacrylamide (MBA) were dissolved in 25 mL water, and 1 mmol of the template molecule 5,7-dimethoxy-2-(4-methoxyphenyl)-3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-benzo-4-one prepared in Example 1 was added, uniformly dispersed, and magnetically stirred for 0.5 h at room temperature to form a prepolymer to obtain solution A;

[0039] 1.2 Preparation of continuous phase: Dissolve 7.5 g of octylphenol polyoxyethylene ether (OP-10) in 400 mL of n-hexane under nitrogen atmosphere to form a continuous phase as solution B;

[0040] 1.3 Preparation of reverse phase dispersion system: slowly transfer solution A to solution B while stirring, and then stir at room temperature for 60 min under nitrogen atmosphere to obtain a white emulsion;

[0041] 1.4 Polymerization reaction: 0.6 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 50° C. and 400 r / min for 2.5 h to obtain a polymer microsphere mixture;

[0042] 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 230-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 45°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.

[0043] 2. Performance Testing

[0044] 2.1 Study on the adsorption performance of kaempferol:

[0045] i) Brief description of the detection method: Prepare a 4 μg / mL kaempferol standard solution with methanol as solvent, take 0, 1.0, 2.0, 3.0, 4.0, 5.0 mL of kaempferol standard solution into a volumetric flask, add methanol to a total of 5 mL, mix well, detect the absorbance at a wavelength of 365 nm with a UV-visible spectrophotometer, and draw a standard curve with absorbance (A) as the ordinate and concentration as the abscissa. Dilute the sample to be tested with methanol to a concentration of 0.8-4 μg / mL for detection, and calculate the content using the standard curve.

[0046] 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 3mg / mL kaempferol methanol solution, and adsorb at constant temperature and oscillate for 5h, then centrifuge and measure the absorbance of the supernatant three times in parallel using a UV-visible spectrophotometer according to method i, take the arithmetic mean, and calculate the kaempferol concentration using the standard curve. The adsorption amount of the imprinted polymer (52.91mg / g) was calculated based on the change in kaempferol concentration in the solution before and after adsorption. Calculation formula:

[0047] Q=(C0-C)*V / W

[0048] Where: Q: adsorption amount per gram of polymer (mg / g);

[0049] C0: original mass concentration of adsorbed compound in equilibrium adsorption experiment (mg / L);

[0050] C: mass concentration of adsorbed compound in the supernatant after reaching adsorption equilibrium (mg / L);

[0051] V: volume of adsorption solution (L);

[0052] W: mass of imprinted polymer (g).

[0053] Experimental Example 1: Investigating the effect of different emulsifiers on polymer shape during the preparation process

[0054] Based on the preparation method in Example 2, different emulsifiers were used as dispersions (see Table 1) to obtain molecularly imprinted polymers. The adsorption amount of kaempferol was measured according to 2.1, and the results are shown in Table 1:

[0055] Table 1

[0056] Emulsifier Polymer adsorption Docusate Sodium(AOT) 44.67mg / g Octylphenol polyoxyethylene ether (OP-10) 50.23mg / g Triton X-100 35.72mg / g

[0057] As shown in Table 1, the molecular imprinted polymers prepared by using docusate sodium (AOT) and octylphenol polyoxyethylene ether (OP-10) as emulsifiers have a higher adsorption capacity for kaempferol. The molecular imprinted polymers prepared by using Triton X-100 as an emulsifier have a slightly lower adsorption capacity for kaempferol than the other two.

[0058] 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

[0059] 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 the flavonoid compound kaempferol were measured. The results are shown in Table 2:

[0060] Table 2

[0061]

[0062]

[0063] As shown in Table 2, when acrylic acid, allylamine and methacrylic acid are selected as hydrophilic functional monomers and the template molecule is used in combination with them at a molar ratio of 1:5, the prepared molecular imprinted polymer has a good adsorption capacity for kaempferol. The adsorption capacity is the largest when the template molecule is used in combination with acrylic acid at a molar ratio of 1:5, which is 53.1 mg / g. When the template molecule and acrylic acid are used in combination at different molar ratios, the adsorption capacity of the prepared molecular imprinted polymer for kaempferol changes greatly. When the molar ratio of the template molecule to acrylic acid is 1:3, the adsorption capacity is significantly reduced to 13.07 mg / g. When the molar ratio of the template molecule to acrylamide is 1:7 and 1:9, the adsorption capacity is slightly lower than that when the molar ratio is 1:5.

[0064] Experimental Example 3 Investigating the Effect of Using Different Crosslinking Agents in the Preparation of Dispersed Phase on Molecularly Imprinted Polymers

[0065] Based on the preparation method in Example 2, different cross-linking agents (see Table 3) were used to obtain different molecularly imprinted polymers. The adsorption amount of kaempferol was measured according to 2.1. The results are shown in Table 3:

[0066] Table 3

[0067] Crosslinking agent Polymer adsorption N,N'-Methylenebisacrylamide (MBA) 51.15mg / g Polyethylene glycol dimethacrylate (EGDMA) 48.43mg / g Trimethoxypropyl trimethacrylate (TRIM) 36.89mg / g Divinylbenzene (DVB) 32.44mg / g

[0068] As shown in Table 3, different molecular imprinting polymers prepared by using N, N'-methylenebisacrylamide and polyethylene glycol dimethacrylate as cross-linking agents all have good adsorption capacity for kaempferol; while the adsorption performance of different molecular imprinting polymers prepared by using trimethoxypropyl trimethacrylate and divinylbenzene as cross-linking agents is slightly lower than that of the other two.

[0069] Experimental Example 4 Investigating the Effect of Using Different Initiators in the Polymerization Step on the Properties of Molecularly Imprinted Polymers

[0070] 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:

[0071] Table 4

[0072] Initiator Polymerization Ammonium Persulfate Simple operation, quick response, stable process Benzoyl peroxide Simple operation, quick response, slightly poor process stability

[0073] As shown in Table 4, different molecular imprinting polymers prepared by using ammonium persulfate as an initiator are simple to operate and react rapidly, while the molecular imprinting polymer prepared by using benzoyl peroxide has a slightly less stable reaction process.

[0074] 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

[0075] Based on the preparation method in Example 2, different molecularly 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). The adsorption amount of kaempferol was measured according to 2.1 to verify the performance of the molecularly imprinted microspheres. The results are shown in Table 5:

[0076] Table 5

[0077]

[0078] Different molecular imprinted polymers were prepared under the conditions of polymerization reaction temperature of 30-60℃ and reaction time of 1.5-3.5h, and all of them could form microspheres. It can be seen from Table 5 that the best reaction conditions are reaction temperature of 50℃ and reaction time of 2.5h, and the molecular imprinted polymers prepared under this condition have better adsorption performance.

[0079] Example 3 Testing the adsorption performance of the molecularly imprinted polymer obtained in Example 2 on various natural flavonoids:

[0080] The adsorption performance test of kaempferol was carried out according to the method described in 2.1, except that the test molecule was replaced by an alternative molecule (natural flavonoid molecule) instead of kaempferol and the detection wavelength was adjusted. The obtained data are shown in Table 6.

[0081] Table 6

[0082]

[0083] As shown in Table 6, the molecular imprinted polymer prepared in Example 2 has good adsorption performance for natural flavonoids molecules such as quercetin, kaempferol-3-O-rutinoside, rutin, puerarin, kaempferol-3-O-glucoside, myricetin and myricetin.

[0084] Example 4 Application of the molecularly imprinted polymer obtained in Test Example 2

[0085] In order to verify the separation performance and application potential of the molecularly imprinted polymer obtained in Example 2, the template molecularly imprinted polymer microspheres were used as an adsorbent material for internal phase extraction to enrich and separate quercetin and myricetin from the Platycladus orientalis leaf extract.

[0086] The specific experiments are as follows:

[0087] (1) Preparation of Platycladus orientalis leaf extract: Weigh 0.5 g Platycladus orientalis leaf, add 80 mL 60% ethanol solution, extract under ultrasonic reflux for 0.5 h, separate the supernatant after centrifugation, repeat the extraction residue 3 times, combine the supernatants, and concentrate by rotary evaporator to obtain Platycladus orientalis leaf extract (medicinal material: extract = 5:1, W / W), seal and keep at low temperature for later use.

[0088] (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, 20 mg / mL of Platycladus orientalis leaf extract was prepared with methanol, and 3 mL of the sample solution was loaded; then, it was rinsed with 5 mL of methanol; and then eluted with ethanol-acetic acid (90:10, v / v).

[0089] (3) Result analysis: The collected solution was tested by HPLC according to the detection method of quercetin in Platycladus orientalis leaves in the Chinese Pharmacopoeia 2015 edition P123. The contents of quercetin and myricetin were detected at the same time. The concentrations of quercetin and myricetin in the solution before and after adsorption were calculated. The recoveries were 89% (quercetin) and 85% (myricetin), respectively.

[0090] 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 prepare 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, elute the template molecules, wash until neutral, and then dry 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 acrylic acid, allylamine and methacrylic acid.

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, trimethoxypropyl trimethacrylate and divinylbenzene.

5. The molecularly imprinted polymer according to claim 2, characterized in that In preparing the dispersed phase, the molar ratio of the template molecule to the hydrophilic functional monomer is 1:5-9.

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, octylphenol polyoxyethylene ether, and Triton X-100.

7. The molecularly imprinted polymer according to claim 2, characterized in that In the polymerization reaction, the initiator is one of 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 60°C.

9. The molecularly imprinted polymer according to claim 2, characterized in that During the polymerization reaction, the polymerization reaction time is 1.5 to 3.5 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 natural flavonoid compounds, characterized in that: The natural flavonoid compound is at least one of quercetin, kaempferol, kaempferol-3-O-rutinoside, rutin, puerarin, kaempferol-3-O-glucoside, myricetin and myricetin.

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