Preparation method of mixed flavone molecularly imprinted polymer
By preparing mixed flavonoid molecular imprinted polymers, the problems of poor selectivity and complex operation of flavonoid glycoside compounds in the prior art are solved, and the selective adsorption and efficient separation of high-purity flavonoid glycoside compounds are achieved, which is suitable for large-scale production.
Patent Information
- Application Number
- CN202510584936.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-08-15
AI Technical Summary
The prior art When separating and enriching flavonoid glycoside compounds from ginkgo leaf extract, the selectivity is poor, the cost is high, and the operation is complicated, making it difficult to achieve high purity and efficient separation and purification.
The preparation method of mixed flavonoid molecular imprinted polymers is adopted, and molecular imprinted polymers are prepared through self-assembly reactions and polymerization reactions, combining specific eluents and adsorption conditions to achieve selective adsorption of flavonoid glycoside compounds.
The extraction purity of flavonoid glycoside compounds is improved, from 20% to more than 90%, simplifies the operation process, reduces costs, and maintains good reusability and thermal stability.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant processing, and more particularly to a method for preparing a mixed template molecule flavonoid molecular imprinted polymer and a method for selectively enriching high-purity flavonoid glycoside compounds from a ginkgo extract. Background Art
[0002] Ginkgo biloba extract, derived from ginkgo leaves using appropriate solvents to extract the active ingredients, is widely used in health foods, pharmaceuticals, cosmetics, and other fields. Its main active ingredients are flavonoids and terpene lactones. Flavonoid glycosides, a type of flavonoid compound composed of flavonols and sugars linked by glycosidic bonds, have been shown to lower blood pressure, relieve spasms, provide anti-inflammatory and analgesic effects, and enhance immunity. They are used to treat conditions such as diabetes, hyperlipidemia, anxiety, and acute pancreatitis. Research is focused on selectively isolating and enriching flavonoid glycosides with high content and excellent pharmacological activity.
[0003] Numerous methods for extracting and separating Ginkgo biloba flavonoids have been reported, most often employing solvent extraction, ultrasonic extraction, resin adsorption, and supercritical fluid extraction. These methods are costly, complex, and exhibit poor selectivity for structurally similar flavonoids. Molecular imprinting technology, a separation technique that specifically adsorbs specific substances, offers advantages such as high selectivity, ease of operation, acid and alkali resistance, and excellent stability, and holds great promise for the separation and purification of flavonoids. Therefore, providing a molecularly imprinted polymer with a simple preparation method and strong selective adsorption capacity would be of great value in the separation and enrichment of high-purity flavonoid glycosides. Summary of the Invention
[0004] In light of this, the present invention aims to provide a method for preparing a molecularly imprinted flavonoid polymer and its application in selectively adsorbing high-purity flavonoid glycosides from Ginkgo biloba extracts. The molecularly imprinted polymer preparation method of the present invention is simple and can specifically separate, enrich, and purify flavonoid glycosides from complex natural extracts, significantly enhancing the bioactivity and economic value of the extract product.
[0005] To achieve the above object, the present invention adopts the following technical solutions:
[0006] 1. A method for preparing a mixed flavonoid molecularly imprinted polymer, characterized by comprising the following raw materials:
[0007] 1 part of template molecule, 1-10 parts of functional monomer, 10-50 parts of cross-linking agent, 1-3 parts of initiator; preferably, 4-8 parts of functional monomer and 20-40 parts of cross-linking agent;
[0008] The mixed flavonoids are template molecules, namely at least two of rutin, isoquercetin, apigenin-7-O-glucoside, 3-O-rutinoside, and quercetin;
[0009] The functional monomer is one of 4-vinylpyridine (4-VP), acrylamide (AM) or α-methylacrylic acid (MAA).
[0010] 2. The method for preparing the mixed flavonoid molecularly imprinted polymer according to claim 1, wherein the preparation steps are as follows:
[0011] The template molecule and the functional monomer are fully dissolved in an alcohol solution by ultrasonication, and the self-assembly reaction is carried out for 6 to 12 hours, preferably 10 to 12 hours to allow the self-assembly reaction to be more complete, to obtain a prepolymer solution; the prepolymer solution, the cross-linking agent and the initiator are mixed, and then ultrasonically deoxygenated to make the reactants more uniform; after the polymerization reaction is carried out under a nitrogen atmosphere for 12 to 24 hours, an eluent is used to remove the template molecule to obtain a molecularly imprinted polymer.
[0012] 3. The method according to claim 2, wherein the eluent is a solution of methanol and acetic acid mixed in a volume ratio of 7:3 to 9:1; the cross-linking agent is ethylene glycol dimethacrylate or butyl acrylate, preferably, the cross-linking agent is ethylene glycol dimethacrylate; the initiator is azobisisobutyronitrile or benzoyl peroxide, preferably, the initiator is azobisisobutyronitrile; and the solvent is ethanol or methanol.
[0013] 4. The method according to claim 2, wherein the flavonoid glycosides are enriched and prepared by dissolving a crude ginkgo extract having a total flavonoid content of 20-24% in alcohol, and filtering the solution through a microporous filter membrane. The filtrate is mixed with a molecularly imprinted polymer for adsorption, and the molecularly imprinted polymer adsorbed with the flavonoid glycosides is eluted to obtain four flavonoid glycoside compounds; the flavonoid glycoside compounds include rutin, isoquercetin, 3-O-rutinoside, and apigenin-7-O-glucoside.
[0014] 5. The method according to claim 4, characterized in that the alcohol dissolution comprises dissolving the crude ginkgo extract in methanol and ultrasonically dissolving it for 30 minutes to fully assist dissolution; the crude ginkgo extract is added to methanol at a concentration of 0.2-0.5 g / L; the adsorption temperature is 30-60°C, and the adsorption time on a closed oscillating constant temperature shaker is 6-12 hours; and the mass ratio of the crude ginkgo extract to the molecularly imprinted polymer during the adsorption is 1:0.1-3.
[0015] 6. A method for preparing a mixed flavonoid molecular imprinted polymer, characterized in that the ratio of mixed flavonoid template molecules is designed, the template molecules are rutin, isoquercetin, and apigenin-7-O-glucoside, and the molar ratio of rutin: isoquercetin: apigenin-7-O-glucoside is 3:2:1.
[0016] The present invention has the following beneficial effects:
[0017] 1. The mixed flavonoid molecularly imprinted polymer of the present invention forms a specific spatial structure with high strength and stable structure. It can selectively extract template molecules or compounds with very similar structures, providing a new approach for the high-purity preparation of rutin, isoquercetin, apigenin-7-O-glucoside, and 3-O-rutinoside.
[0018] 2. The mixed flavonoid molecularly imprinted polymer of the present invention has good reusability and thermal stability, conforms to the development concept of green chemistry and chemical industry, is simple to synthesize, has high yield, and is suitable for large-scale production and application.
[0019] 3. The mixed flavonoid molecularly imprinted polymer of the present invention selectively adsorbs flavonoid glycosides (molecular weight greater than 400), rutin, isoquercetin, apigenin-7-O-glucoside, and 3-O-rutinoside, but does not adsorb flavonols, thereby increasing the total flavonoid content to more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 HPLC spectra of Ginkgo biloba extract before and after MIP adsorption;
[0021] Figure 2 It is the reuse performance of MIP. DETAILED DESCRIPTION
[0022] The present invention is further described below with reference to the following embodiments. The following embodiments are illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.
[0023] Example 1
[0024] Screening and optimization of functional monomers
[0025] A 10 mL test solution was prepared by mixing a 0.25 mmol / L methanol solution of the template molecule and various concentrations of the functional monomer (template molecule to functional monomer molar ratios of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8). After mixing, the solution was allowed to rest for 1 hour, and UV absorption spectra were measured over the full wavelength range of 200 nm to 800 nm. The absorbance between the template molecule (T) and the solvent (SO), and between the template, solvent, and functional monomer (F) was measured under UV light. The absorbance change before and after the interaction of the functional monomer with the template molecule was calculated using the formula ΔAbs(T*) = Abs(F+T+SO) - Abs(F+SO). This absorbance change was used to measure the interaction between the template molecule and the functional monomer, allowing the optimal functional monomer and ratio to be screened. The results are shown in Table 1.
[0026] Table 1 Comparison of absorbance changes of different functional monomers interacting with template molecules at wavelength λ = 360nm
[0027]
[0028] Example 2
[0029] Preparation of Mixed Flavonoid Molecularly Imprinted Polymers (MIPs)
[0030] 0.3 mmol of rutin, 0.2 mmol of isoquercetin, and 0.1 mmol of apigenin-7-O-glucoside were dissolved in methanol, followed by the addition of 2.5 mmol of 4-VP and mechanical stirring at room temperature for 4 hours. Then, 12.5 mmol of ethylene glycol dimethacrylate and 40 mg of azobisisobutyronitrile were added. The mixture was thoroughly mixed and ultrasonicated for 20 minutes to remove dissolved oxygen. Nitrogen was then purged for 30 minutes, sealed, and thermally polymerized at 60°C for 24 hours. The product was eluted by Soxhlet extraction using a mixture of methanol and acetic acid (9:1 by volume) until the UV absorption peak of rutin could no longer be detected in the eluate, indicating that the template molecule in the molecularly imprinted polymer had been removed. Excess acetic acid was then washed away with methanol, and the product was freeze-dried to obtain a rutin molecularly imprinted polymer (MIP).
[0031] Example 3
[0032] Weigh 35 mg of ginkgo extract with a total flavonoid content of 24% into a 100 mL volumetric flask and dilute to the mark with methanol. Weigh 20 mg of MIP into a 50 mL conical flask and add 20 mL of the ginkgo extract solution. Shake the mixture in a closed, thermostatic shaker at 40°C for 6 and 12 hours, then filter through a 0.45 μm microporous membrane. Liquid chromatography analyzes the filtrate to examine the adsorption performance of MIP on ginkgo extract.
[0033] Chromatographic conditions: BDS Hypersil™ C18 column (250 mm × 4.6 mm, 5 μm); acetonitrile as mobile phase A, 0.1% trifluoroacetic acid aqueous solution as mobile phase B for gradient elution, gradient elution conditions refer to the Chinese Pharmacopoeia 2020 edition, flow rate 1.0 mL / min, detection wavelength 360 nm, injection volume 10 μL, column temperature 45°C.
[0034] See the results Figure 1 The MIP had no adsorption for flavonols, but it did adsorb four flavonoid glycosides: rutin, isoquercetin, apigenin-7-O-glucoside, and 3-O-rutinoside. This is due to the high structural similarity between 3-O-rutinoside and the template molecule, providing a new method for the enrichment of flavonoid glycosides.
[0035] Example 4
[0036] MIP reuse experiment
[0037] The initial concentration of the template molecule solution was 0.1 mg / mL, and adsorption was performed under oscillation for 60 minutes. The adsorbed MIPs were placed in a methanol-acetic acid mixture (volume ratio 9:1) and eluted using Soxhlet extraction until no UV absorption peak could be detected in the eluate, indicating that the template molecule had been removed from the molecularly imprinted polymer. The eluted MIPs and NIPs were then dried to obtain the eluted MIPs. The eluted MIPs were subjected to repeated adsorption experiments, and the amount of flavonoid glycoside adsorbed by the MIPs after each elution was recorded to investigate their reusability.
[0038] The results are as follows Figure 2 As shown, the amount of flavonoid glycosides adsorbed by the MIP gradually decreased. After five adsorption cycles, the MIP lost approximately 17% of its initial adsorption capacity. This is because the increasing number of adsorption and desorption cycles destroyed some of the pores on the MIP surface. Although the adsorption capacity of the imprinted polymer decreased slightly after five repeated adsorption experiments, it remained greater than 80% of the initial adsorption capacity, demonstrating the MIP's high reusability.
[0039] In summary, the mixed flavonoid molecularly imprinted polymer (MIP) of the present invention can provide a simple and rapid method for enriching high-purity flavonoid glycosides from natural extracts with complex components.
[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. A method for preparing a mixed flavonoid molecularly imprinted polymer, characterized in that: The preparation includes the following raw materials: 1 part of template molecule, 1-10 parts of functional monomer, 10-50 parts of cross-linking agent, 1-3 parts of initiator; The mixed flavonoids are template molecules, namely at least two of rutin, isoquercetin, apigenin-7-O-glucoside, 3-O-rutinoside, and quercetin; The functional monomer is one of 4-vinylpyridine (4-VP), acrylamide (AM) or α-methylacrylic acid (MAA).
2. The method for preparing the mixed flavonoid molecularly imprinted polymer according to claim 1, characterized in that: The preparation steps are as follows: The template molecule and the functional monomer are ultrasonically dissolved in an alcohol solution, and the self-assembly reaction is carried out for 6 to 12 hours to obtain a prepolymer solution; the prepolymer solution, the crosslinking agent and the initiator are mixed and then ultrasonically deoxygenated, and the polymerization reaction is carried out under a nitrogen atmosphere for 12 to 24 hours, preferably 18 to 24 hours to allow the polymerization reaction to be more complete, and the template molecule is removed by an eluent to obtain a molecularly imprinted polymer.
3. The method according to claim 2, wherein the eluent is a solution of methanol and acetic acid mixed in a volume ratio of 7:3 to 9:1; the crosslinking agent is ethylene glycol dimethacrylate or butyl acrylate; the initiator is azobisisobutyronitrile or benzoyl peroxide; and the solvent is ethanol or methanol.
4. The method according to claim 2, wherein the flavonoid glycosides are enriched and prepared by dissolving a crude ginkgo extract having a total flavonoid content of 20-24% in alcohol, and filtering the solution through a microporous filter membrane. The filtrate is mixed with a molecularly imprinted polymer for adsorption, and the molecularly imprinted polymer adsorbed with the flavonoid glycosides is eluted to obtain four flavonoid glycoside compounds; the flavonoid glycoside compounds include rutin, isoquercetin, 3-O-rutinoside, and apigenin-7-O-glucoside.
5. The method according to claim 4, characterized in that The alcohol dissolution comprises dissolving the crude ginkgo extract in methanol and performing ultrasonic treatment for 30 minutes to fully assist dissolution; the crude ginkgo extract is added to methanol at a concentration of 0.2 to 0.5 g / L; the adsorption temperature is 30 to 60° C., and the adsorption time is 6 to 12 hours on a closed oscillating constant temperature shaker; and the mass ratio of the crude ginkgo extract to the molecularly imprinted polymer during the adsorption is 1:0.1 to 3.
6. A method for preparing a mixed flavonoid molecular imprinted polymer, characterized in that The ratio of mixed flavonoid template molecules was designed, and the template molecules were rutin, isoquercetin, and apigenin-7-O-glucoside, and the molar ratio was rutin: isoquercetin: apigenin-7-O-glucoside = 3:2:1.