Preparation and application of kaempferol temperature-responsive imprinted material

Abstract

The application discloses a preparation method of a kaempferol temperature-responsive imprinting material and application thereof, and the preparation method comprises the steps of pre-assembly, polymerization reaction and elution of a template molecule. The application is specially designed for a high-viscosity vegetable oil matrix, breaks through the bottleneck that traditional imprinting materials are prone to collapse and block in a pure lipid matrix, adopts trimethylolpropane trimethacrylate, 1,6-hexanediol dimethacrylate and azobisisobutyronitrile to form an organic ternary copolymer network, and mixes a crosslinking agent to construct a pure organic skeleton which has rigid compression channels and micro-flexible space, and the synergistic effect of a functional monomer p-vinylphenol and a temperature-sensitive monomer N-isopropyl acrylamide is combined to realize high-selectivity recognition of kaempferol, oil resistance to collapse and temperature-controllable adsorption and release. The kaempferol temperature-responsive imprinting material of the application eliminates the complex and time-consuming pretreatment process before vegetable oil extraction, and realizes efficient extraction of kaempferol in vegetable oil.

Description

Technical Field

[0001] This invention relates to the field of molecular trace materials technology, and in particular to a method for preparing a kaempferol temperature-responsive imprinted material and its application. Background Technology

[0002] Kaempferol is a flavonoid compound with significant antioxidant, anti-inflammatory, and cardiovascular protective bioactivities, and it has important research and application value in the fields of functional foods and pharmaceutical preparations. Natural kaempferol exists mostly in the form of glycosides and is water-soluble, but it is easily degraded into non-glycoside forms during processing and extraction. These non-glycoside forms are basically insoluble in water but soluble in vegetable oils. Non-glycoside forms of kaempferol cannot be obtained by conventional aqueous extraction. Kaempferol in vegetable oils is more easily absorbed by the human body, so achieving efficient separation and enrichment of kaempferol in oil matrices is crucial for product development. Currently used solvent extraction, macroporous resins, and chromatographic separation techniques generally suffer from insufficient selectivity, complex operation, and difficulty in reusing in high-viscosity matrices (Li Xia, He Jia, Wang Jiandong, et al. Determination of quercetin and kaempferol content in Anoectochilus roxburghii by reversed-phase high-performance liquid chromatography [J]. Zhejiang Agricultural Sciences, 2023, 64(2): 446-449).

[0003] While molecular imprinting technology, introduced in recent years, possesses specific recognition capabilities, traditional imprinted materials face significant application bottlenecks in complex vegetable oil systems. Chinese patent CN110669172A discloses a method for preparing a molecularly imprinted material and its application. The kaempferol molecularly imprinted material described is a hydrophilic material, suitable only for the separation of kaempferol in aqueous systems. Due to the high viscosity of the triglyceride environment in vegetable oils creating strong mass transfer resistance, traditional imprinted materials are prone to conformational shrinkage and pore blockage in nonpolar high-lipid systems. Simultaneously, the non-specific adsorption of large lipid molecules easily triggers the fouling effect, causing the material to directly lose its recognition and mass transfer capabilities. To avoid such matrix interference, existing extraction processes have to rely on cumbersome pretreatment steps such as high-temperature saponification or large-volume solvent liquid-liquid extraction. This not only increases the difficulty and cost of operation, but also easily leads to the oxidative degradation of active ingredients (Li Wenting, Liu Ling, Xia Jiaen, et al. Optimization of molecularly imprinted solid-phase extraction-high performance liquid chromatography and its application in the determination of benzo(a)pyrene content in vegetable oils [J]. Grain and Oil Food Science and Technology, 2023, 31(1): 150-157.). Therefore, developing a composite imprinting material that has high selectivity, can resist oil phase compression and collapse, and has temperature response regulation properties to achieve direct targeted enrichment and temperature-controlled release of kaempferol in vegetable oils without pretreatment has important application value. Summary of the Invention

[0004] To address the challenges of conformational shrinkage and pore closure of traditional imprinted materials in nonpolar high-lipid systems, where the non-specific adsorption of macromolecular lipids easily triggers fouling, causing the material to lose its recognition and mass transfer capabilities, existing extraction processes rely on cumbersome pretreatment steps such as high-temperature saponification or large-volume solvent liquid-liquid extraction to avoid matrix interference. This not only increases operational difficulty and cost but also easily leads to oxidative degradation of active ingredients. This invention provides a method for preparing a kaempferol temperature-responsive imprinted material. Unlike traditional imprinted materials that rely on rigid short-chain crosslinking agents, this invention is specifically designed for high-viscosity vegetable oil matrices. It employs an organic ternary copolymer network and a mixed crosslinking strategy, combining the synergistic effect of the functional monomer p-vinylphenol and the temperature-sensitive monomer N-isopropylacrylamide to achieve highly selective recognition of kaempferol, resistance to oil collapse, and temperature-controlled adsorption and release. This kaempferol temperature-responsive imprinted material eliminates the cumbersome pretreatment steps before vegetable oil extraction, exhibits excellent oil-phase mass transfer kinetics and reversible temperature response characteristics, and can be used for the efficient separation of kaempferol from vegetable oils.

[0005] To achieve the above objectives, the present invention provides a method for preparing a kaempferol temperature-responsive imprinted material, characterized by comprising the following steps: S1. Pre-assembly: Kaempferol, p-vinylphenol and N-isopropylacrylamide are dissolved in a mixture of methanol and vegetable oil. The mixture is ultrasonically mixed for 10-20 min at an ultrasonic power of 200-300 W, and then reacted at room temperature for 2-6 h to obtain a pre-assembled complex solution, denoted as product A. S2. Polymerization reaction: Add a mixed crosslinking agent and initiator azobisisobutyronitrile to product A obtained in step S1. The mixed crosslinking agent is composed of trimethylolpropane trimethacrylate and 1,6-hexanediol dimethacrylate. Under nitrogen protection, react at 50-70 °C for 12-24 h, and cool to room temperature to obtain a white powder material, denoted as product B. S3. Elution of template molecules: The product B obtained in step S2 is continuously washed with a mixture of methanol and acetic acid for 6-24 hours to remove unreacted components and template molecules. The resulting solid is then washed with methanol 2-3 times. Finally, the washed solid is vacuum dried at 40-60 °C for 12-24 hours to obtain kaempferol temperature-responsive imprinted material.

[0006] Preferably, the vegetable oil in step S1 is one or a combination of several of the following: tea oil, soybean oil, rapeseed oil, corn germ oil, and sunflower seed oil.

[0007] Preferably, in step S1, the mass ratio of kaempferol, p-vinylphenol, and N-isopropylacrylamide is 1:2-4:2-4; the volume of the mixture of methanol and vegetable oil is 10-20 times the total mass of the reactants (volume unit is ml, mass unit is g); the reactants are kaempferol, p-vinylphenol, and N-isopropylacrylamide; and the volume ratio of methanol to vegetable oil is 1:5-20.

[0008] Preferably, in step S2, the mass ratio of the total mass of the mixed crosslinking agent to the total mass of the monomers is 10-20:1, the total monomers are p-vinylphenol and N-isopropylacrylamide, the mass of azobisisobutyronitrile added is 1-5% of the total polymerizable component mass, and the total polymerizable component is product A and the mixed crosslinking agent.

[0009] Preferably, in step S2, the mass ratio of trimethylolpropane trimethacrylate to 1,6-hexanediol dimethacrylate is 1:1 to 2:1.

[0010] Preferably, in step S3, the volume ratio of methanol to acetic acid in the eluent is 8-10:1, the volume of the methanol-acetic acid mixture is 10-20 times the mass of product B, and the volume of the methanol washing solution is 10-20 times the mass of product B. The volume unit is ml, and the mass unit is g.

[0011] The kaempferol temperature-responsive imprinted material prepared by the above method can reversibly regulate the adsorption and desorption behavior of kaempferol molecules under different temperature conditions, achieving highly selective enrichment and green release of target components in complex plant oil matrices.

[0012] This invention also provides the application of the kaempferol temperature-responsive imprinted material prepared by the above preparation method, characterized in that the kaempferol temperature-responsive imprinted material is suitable for the separation of kaempferol from high-viscosity vegetable oils, specifically including the following steps: 1) The kaempferol temperature-responsive imprint material is directly added to the unsaponified vegetable oil stock solution, the vegetable oil being the same as the vegetable oil in step S1 above. The material is shaken and adsorbed at 20-30℃ for 0.5-1 h, and then the saturated material C is separated by filtration. 2) Use n-hexane to quickly rinse the surface of material C obtained in step 1) to remove residual grease. Then place material C in an aqueous ethanol eluent and elute at a constant temperature of 50-60 ℃ for 0.5-1 h. After filtration and separation, heat the filtrate to evaporate and remove the eluent to obtain the product kaempferol. The filter cake is a kaempferol temperature-responsive imprinted material that can be reused.

[0013] Preferably, in step 1), the vegetable oil concentrate is diluted with n-hexane beforehand, and the volume ratio of vegetable oil concentrate to n-hexane is 1:1-3.

[0014] Preferably, in step 1), the ratio of the mass of the kaempferol temperature-responsive imprint material to the volume of the vegetable oil stock solution is 1:300-500, where the mass unit is g and the volume unit is ml.

[0015] Preferably, in step 1), the ratio of the mass of the kaempferol temperature-responsive imprint material to the volume of the vegetable oil concentrate diluted with n-hexane is 1:300-500, where the mass unit is g and the volume unit is ml.

[0016] Preferably, in step 2), the volume of hexane used for rinsing is 10-20 times the mass of the kaempferol temperature-responsive imprint material, with volume in ml and mass in g; the aqueous ethanol eluent is an aqueous ethanol solution with a volume fraction of 50-80%, and the volume of the aqueous ethanol eluent is 50-100 times the mass of the kaempferol temperature-responsive imprint material, with volume in ml and mass in g.

[0017] The working principle of this invention is as follows: A ternary copolymer organic network is constructed using a mixed crosslinking agent composed of trimethylolpropane trimethacrylate and 1,6-hexanediol dimethacrylate, and an initiator, azobisisobutyronitrile, to overcome the problems of high mass transfer resistance and pore collapse in the pure oil phase. During the pre-assembly and polymerization stages, vinylphenol and kaempferol construct highly matched imprinted cavities through intermolecular hydrogen bonds and π-π interactions. In practical applications, the nonpolar oil phase eliminates hydrogen bond competition from the polar solvent, significantly enhancing the specific targeting recognition ability of the cavity. In the design of the support system, this invention utilizes trimethylolpropane trimethacrylate to construct a rigid mesoporous framework, which resists triglyceride extrusion with excellent mechanical strength, preventing pore blockage and ensuring oil phase penetration. Simultaneously, 1,6-hexanediol dimethacrylate, containing long carbon chains, is used as a flexible lipophilic crosslinking agent, improving interfacial compatibility and providing sufficient three-dimensional conformational freedom for the internal temperature-sensitive monomer (N-isopropylacrylamide). During low-temperature adsorption, the temperature-sensitive network fully expands in the oil phase, effectively exposing specific cavities to capture the target analyte. During high-temperature desorption, the chain segments undergo a strong hydrophobic contraction phase transition, utilizing microscopic extrusion force to efficiently pump kaempferol into the polar eluent. This achieves highly efficient, highly selective, and temperature-sensitive on / off separation of kaempferol from pretreatment-free vegetable oil systems.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention breaks through the bottleneck of traditional imprinting materials being prone to collapse and closure in pure lipid matrices. By using a mixed crosslinking agent to construct a pure organic framework that combines rigid and pressure-resistant channels with microscopic flexible space, the complex and time-consuming pretreatment process is eliminated, and efficient extraction of kaempferol from plant oils is achieved.

[0019] 2. This invention achieves highly selective recognition and temperature-controlled regulation in complex oil-phase matrices. By utilizing the advantage of eliminating solvent competition in a non-polar environment, it strengthens the specific binding between functional monomers and kaempferol, exhibiting rapid and stable temperature-controlled adsorption and desorption behavior, thereby improving the separation and enrichment efficiency of trace active ingredient kaempferol.

[0020] 3. The kaempferol temperature-responsive imprint material preparation system of this invention is carried out under normal pressure and mild conditions. The overall synthesis process is simple. The elution solvent used in the application of kaempferol temperature-responsive imprint material prepared from vegetable oil is easy to recycle and reuse, which has outstanding green and environmental protection advantages and industrial application potential. Attached Figure Description

[0021] Figure 1 Infrared spectra of the kaempferol temperature-responsive imprinted material in Example 1 and the imprinted material in Comparative Example 1.

[0022] Figure 2 Example 1: Scanning electron microscope image of kaempferol temperature-responsive imprinted material.

[0023] Figure 3 : Scanning electron microscope image of the white powder material in Comparative Example 1.

[0024] Figure 4 Example 1: Comparison of the adsorption of kaempferol in tea oil by temperature-responsive imprinted material and Comparative Example 3: non-temperature-responsive imprinted material under different temperature conditions. Detailed Implementation

[0025] The following specific embodiments, in conjunction with the accompanying drawings, further illustrate the preparation method and application of a kaempferol temperature-responsive imprint material according to the present invention. However, the scope of protection of the present invention is not limited to the scope described in the embodiments.

[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques described in the literature or reference books in this field or in accordance with the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products.

[0027] Example 1 describes the preparation of a kaempferol temperature-responsive imprinted material, comprising the following steps: S1. Weigh 1.0g kaempferol, 3.0g p-vinylphenol, and 2.0g N-isopropylacrylamide, add them to 80 mL of methanol / tea oil mixture (methanol and tea oil volume ratio 1:5), sonicate for 15 min under ultrasonic power of 200 W, and then react at room temperature for 4 h to obtain a pre-assembled complex solution, denoted as product A. S2. Add 25.0 g of trimethylolpropane trimethacrylate, 25.0 g of 1,6-hexanediol dimethacrylate and 0.15 g of initiator azobisisobutyronitrile to product A. After stirring evenly, purge with nitrogen gas and place in an oil bath at 60 ℃ for polymerization reaction for 18 h. After the reaction is completed, cool to room temperature to obtain 12 g of white powder material, which is denoted as product B. S3. Product B was washed with a mixture of 150 mL methanol and acetic acid in a volume ratio of 9:1 for 12 h to remove unreacted components and template molecules. The resulting solid was then washed three times with 150 mL methanol and dried under vacuum at 60 °C to obtain 8.2 g of kaempferol temperature-responsive imprinted material.

[0028] The following describes the application of the kaempferol temperature-responsive imprinted material prepared in Example 1 for separating kaempferol from tea oil, including the following steps: 1) Take 400 mL of tea oil stock solution with an initial kaempferol concentration of 8.0 mg / L, add 1.00 g of the kaempferol temperature-responsive imprinted material prepared in Example 1, and allow it to adsorb under shaking conditions at 30℃ for 0.5 h. Separate the saturated material C by filtration. The remaining concentration of kaempferol in the filtrate was measured to be 1.31 mg / L. The kaempferol adsorption rate was calculated to be 83.6%, corresponding to a unit adsorption capacity of 2.68 mg / g.

[0029] 2) The surface of material C was rapidly washed with 15 mL of n-hexane to remove residual grease. Then, material C was placed in 50 mL of 80% (v / v) ethanol aqueous solution and eluted at 50 °C for 0.5 h. After filtration, the filtrate was heated and evaporated to remove the eluent, yielding 4.8 mg of product. The product contained 53.5% kaempferol, with a desorption rate of 96.0%. The filter cake was a temperature-responsive imprinted material of kaempferol and could be reused.

[0030] Example 2 describes the preparation of a kaempferol temperature-responsive imprinted material, comprising the following steps: S1. Weigh 1.0g kaempferol, 4.0g p-vinylphenol, and 2.0g N-isopropylacrylamide, add them to 100mL of methanol / soybean oil mixture (volume ratio 1:10), and sonicate for 20min under ultrasonic power of 200W. Then react at room temperature for 6h to obtain a pre-assembled complex solution, which is denoted as product A.

[0031] S2. Add 40.0 g of trimethylolpropane trimethacrylate, 20.0 g of 1,6-hexanediol dimethacrylate, and 0.15 g of initiator azobisisobutyronitrile to product A. After stirring evenly, purge with nitrogen and place in an oil bath at 65°C for polymerization reaction for 24 h. After the reaction is complete, cool to room temperature to obtain 15 g of white powder material, denoted as product B.

[0032] S3. Product B was eluted with a mixture of 150 mL methanol and acetic acid in a volume ratio of 8:1 for 24 h to remove unreacted components and template molecules. The resulting solid was then washed three times with 150 mL methanol and dried under vacuum at 55 °C to obtain 9.7 g of kaempferol temperature-responsive imprinted material.

[0033] The following describes the application of the kaempferol temperature-responsive imprinted material prepared in Example 2 for the separation of kaempferol from soybean oil, including the following steps: 1) Mix 200 mL of soybean oil concentrate with 200 mL of n-hexane to prepare a diluted oil phase system with a total volume of 400 mL and an initial kaempferol concentration of 6.0 mg / L. Add 1.00 g of kaempferol temperature-responsive imprinted material to this diluted oil phase system, and allow it to adsorb under shaking conditions at 20℃ for 1.0 h. Filter to separate the saturated material C. The residual concentration of kaempferol in the filtrate was measured to be 0.97 mg / L, and the adsorption rate of kaempferol was calculated to be 83.8%, corresponding to an adsorption capacity of 2.01 mg / g.

[0034] 2) The residual grease on the surface of material C was rapidly washed with 20 mL of n-hexane. Material C was then placed in 100 mL of 50% (v / v) ethanol aqueous solution and eluted at a constant temperature of 60 °C for 1.0 h. After filtration, the filtrate was heated and evaporated to remove the eluent, yielding 8 mg of product. The product contained 51.5% kaempferol, with a desorption rate of 96.5%. The filter cake was a temperature-responsive imprinted material of kaempferol and could be reused.

[0035] Example 3 describes the preparation of a kaempferol temperature-responsive imprinted material, comprising the following steps: S1. Weigh 1.0g kaempferol, 3.0g p-vinylphenol, and 3.0g N-isopropylacrylamide, add them to 80mL of methanol / rapeseed oil mixture (volume ratio 1:20), and sonicate for 10min under ultrasonic power of 300W. Then react at room temperature for 2h to obtain a pre-assembled complex solution, which is denoted as product A.

[0036] S2. Add 36.0 g of trimethylolpropane trimethacrylate, 24.0 g of 1,6-hexanediol dimethacrylate, and 0.18 g of azobisisobutyronitrile (AIB) initiator to product A. After stirring until homogeneous, purge with nitrogen and polymerize in an oil bath at 60°C for 12 h. After the reaction is complete, cool to room temperature to obtain 11.0 g of white powder, denoted as product B.

[0037] S3. Product B was eluted with a mixture of 220 mL methanol and acetic acid in a volume ratio of 10:1 for 12 h to remove unreacted components and template molecules. The resulting solid was then washed three times with 150 mL methanol and dried under vacuum at 50 °C to obtain 7.7 g of kaempferol temperature-responsive imprinted material.

[0038] The following describes the application of the kaempferol temperature-responsive imprinted material prepared in Example 3 for the separation of kaempferol from soybean oil, including the following steps: 1) Take 400 mL of rapeseed oil stock solution with an initial kaempferol concentration of 5.0 mg / L, add 1.00 g of kaempferol temperature-responsive imprinted material, shake and adsorb at 25℃ for 0.5 h, and filter to separate the material C that is saturated with adsorption; The residual concentration of kaempferol in the filtrate was measured to be 1.32 mg / L, and the adsorption rate of kaempferol was calculated to be 73.6%, corresponding to an adsorption capacity of 1.47 mg / g.

[0039] 2) Use 10 mL of n-hexane to quickly wash the residual grease on the surface of material C. Place material C in 70 mL of 60% ethanol aqueous solution and elute at 55 ℃ for 45 min. Then filter and separate. Heat the filtrate to evaporate and remove the eluent to obtain 2.4 mg of product with kaempferol content of 58.4% and desorption rate of 95.0%.

[0040] Comparative Example 1 The reaction was essentially the same as in Example 1, except that kaempferol (template molecule) was not added in step S1. After the reaction, 7.4 g of white powdery material was obtained, with a yield of 71.2%. This white powdery material was directly used for a comparative enrichment test of kaempferol in tea oil concentrate, and its adsorption rate was measured to be 14.0%, the unit adsorption capacity was 0.45 mg / g, and the desorption rate was 42.0%.

[0041] Comparative Example 2 The reaction was essentially the same as in Example 1, except that the ratio of kaempferol, p-vinylphenol, and N-isopropylacrylamide in step S1 was 1:1:1. All other steps and components remained unchanged. After the reaction, 5.2 g of a white powder was obtained, with a yield of 50.1%. This white powder was used for a comparative enrichment test of kaempferol in tea oil, and its adsorption rate was measured to be 36.5%, with a unit adsorption capacity of 1.17 mg / g and a desorption rate of 43.6%.

[0042] Comparative Example 3 The reaction was essentially the same as in Example 1, except that N-isopropylacrylamide (a temperature-sensitive monomer) was not added in step S1. The proportions and operations of other components, solvents, and mixed crosslinking agents remained unchanged. After the reaction, 4.6 g of a white powdery non-temperature-responsive molecularly imprinted material was finally obtained. This white powdery non-temperature-responsive molecularly imprinted material was used for a comparative enrichment test of kaempferol in tea oil, and its adsorption rate was measured to be 40.0%, with a unit adsorption capacity of 1.28 mg / g. The desorption rate at 50 °C was 31.5%.

[0043] Comparative Example 4 The reaction was essentially the same as in Example 1, except that in step S2, the crosslinking agent used was conventional ethylene glycol dimethacrylate, with an amount of 50.0 g. Other steps and components remained unchanged, and 10.8 g of a white powder was obtained after the reaction. This white powder was used for a comparative enrichment test of kaempferol in tea oil, and its adsorption rate was measured to be 18.5%, with a unit adsorption capacity of 0.59 mg / g and a desorption rate of 28.0%.

[0044] Performance testing: (a) The kaempferol temperature-responsive imprint material prepared in Example 1 and the white powder material prepared in Comparative Example 1 were subjected to Fourier transform infrared spectroscopy (FT-IR) testing.

[0045] Test results are as follows Figure 1 As shown, the material obtained in Example 1 was measured at 1257 cm. -1 The materials prepared in Comparative Example 1 were placed at 1255 cm. -1 The absorption peak at 1246 cm⁻¹ is attributed to the C=O vibration of the amide bond. -1 The C=O absorption peak at this point corresponds to the carbonyl group of N-isopropylacrylamide. Furthermore, it also shows an absorption peak at 989 cm⁻¹. -1 and 915 cm -1 Although the C=C vibrational absorption peak still exists at this point, its intensity is weak, indicating that N-isopropylacrylamide has undergone polymerization. The material obtained in Example 1 was measured at 1455 cm⁻¹. -1 The materials prepared in Comparative Example 1 were at 1458 cm. -1 The absorption peak at this location corresponds to the CH stretching vibration on the pyridine ring backbone of p-vinylphenol, indicating that p-vinylphenol exists in both material structures. These characteristics demonstrate the successful preparation of the separated material.

[0046] (II) Morphological observation of the kaempferol temperature-responsive imprint material prepared in Example 1 and the white powder material prepared in Comparative Example 1.

[0047] Methods: The surface morphology of the kaempferol temperature-responsive imprint material prepared in Example 1 and the white powder material prepared in Comparative Example 1 were observed using scanning electron microscopy (SEM). After freeze-drying, a small amount of powder was placed on conductive adhesive and fixed. After gold sputtering, the samples were scanned under an accelerating voltage of 5-10 kV to compare the differences in microstructure.

[0048] Test results are as follows Figure 2 , Figure 3As shown, the material particles prepared in Comparative Example 1 are nearly spherical with a smooth and even surface, and the particles are relatively densely and uniformly distributed. In contrast, the material prepared in Example 1 exhibits an irregular particle morphology, with relatively loose particle distribution, a rougher surface, and obvious wrinkles and textures in some areas. Compared with the material prepared in Comparative Example 1, the difference in surface morphology of the material prepared in Example 1 is due to the presence of the template molecule kaempferol and the introduction of the thermosensitive monomer N-isopropylacrylamide during the polymerization process, which makes the polymer network structure more porous and loose. These surface features help to form more adsorbable recognition sites and mass transfer channels, thereby providing a larger specific surface area and a better molecular recognition environment for the subsequent adsorption process.

[0049] (III) Comparative test of the adsorption performance of kaempferol temperature-responsive imprinted materials prepared in Examples 1-3 and white powder materials prepared in Comparative Examples 1-4 on kaempferol in vegetable oil.

[0050] Methods: To further verify the selective adsorption performance, oil-phase mass transfer capacity, and temperature response performance of the material of the present invention for kaempferol in a vegetable oil matrix, the adsorption performance of the materials in Examples 1-3 and Comparative Examples 1-4 was compared, with commercially available macroporous adsorption resin as a control. 1.00 g of each of the materials from Examples 1-3, Comparative Examples 1-4, and the control were weighed. Except for Examples 2 and 3, which were added to 400 mL of soybean oil and rapeseed oil with a kaempferol concentration of 8.0 mg / L respectively, the others were added to 400 mL of tea oil with a kaempferol concentration of 8.0 mg / L. Adsorption was carried out at 30 ℃ with shaking for 0.5 h. The saturated material was separated by filtration. The remaining concentration of kaempferol in the filtrate was determined by liquid chromatography, and the adsorption capacity and adsorption rate were calculated. The separated material was rapidly eluted with 15 mL of n-hexane to remove residual oil from the surface. Then, the material was placed in 50 mL of 80% (v / v) ethanol aqueous solution and eluted at 50 ℃ for 0.5 h. The eluent was evaporated to obtain the product, the kaempferol content in the product was determined, and the desorption rate was calculated.

[0051] Chromatographic conditions: Welch Ultimate XB-C18 reversed-phase column (250 mm × 4.6 mm, 3 μm), mobile phase methanol-formic acid solution (70:30, v / v), flow rate 0.8 mL / min, column temperature 30 °C, injection volume 10 μL, detection wavelength 365 nm.

[0052] The test results are shown in Table 1.

[0053] Table 1 Comparison of the adsorption and desorption properties of various materials for kaempferol in tea oil samples

[0054] Table 1 shows that the kaempferol temperature-responsive imprinted materials prepared in Examples 1-3 all exhibited significantly better adsorption and desorption rates than the comparative white powder material and commercially available macroporous adsorption resin under the same conditions, with the optimal ratio in Example 1 showing the best performance. Comparative Example 1, lacking the specific cavity formed by template molecules, only exhibited weak non-specific physical adsorption. Comparative Example 3, lacking the temperature-sensitive responsive segment of the temperature-sensitive monomer N-isopropylacrylamide, could not generate microscopic shrinkage extrusion force during elution at 50 °C, resulting in a low desorption rate. Comparative Example 4 used the traditional single rigid crosslinking agent ethylene glycol dimethacrylate, whose crosslinking network was locked in the non-polar oil phase and could not provide sufficient phase transition relaxation space, leading to hindered mass transfer in the oil phase and a significant decrease in both adsorption and desorption rates. These comparative data verify the selective and temperature-responsive separation characteristics of the materials of this invention for kaempferol in vegetable oils.

[0055] (iv) Comparative test of the selective adsorption performance of different flavonoid components on the kaempferol temperature-responsive imprinted materials of Examples 1-3 and the white powder materials prepared in Comparative Examples 1-4.

[0056] Methods: To verify the specific molecular recognition ability and anti-interference performance of the material of the present invention for the target compound kaempferol in a complex untreated vegetable oil matrix, the materials prepared in Examples 1-3 and Comparative Examples 1-4, as well as a commercially available macroporous adsorption resin, were selected as controls. Their selective adsorption behavior for three structurally similar flavonoids—kaempferol, quercetin, and myricetin—was investigated. The three flavonoids were added to vegetable oil matrices containing none of the three flavonoids to prepare simulated oil phases with an initial concentration of 8.0 mg / L for each flavonoid. 1.00 g of each material was weighed and added to 400 mL of each simulated oil phase system. Adsorption was carried out at 25 ℃ under constant temperature and shaking for 1.0 h in the dark. After adsorption, the solution was filtered through a 0.45 μm filter membrane, and the concentration of each flavonoid in the filtrate was measured. The unit adsorption capacity was then calculated. Subsequently, using the adsorption capacity of commercially available macroporous adsorption resins for each corresponding flavonoid component as a benchmark, the selectivity coefficient of each material (i.e., the ratio of the material's adsorption capacity for a certain flavonoid to the macroporous resin's adsorption capacity for that flavonoid) was calculated to evaluate the material's specific enrichment capacity. The chromatographic conditions and procedures were the same as those in test (III) above.

[0057] The test results are shown in Table 2.

[0058] Table 2 Comparison of adsorption properties of various materials for kaempferol, quercetin, and myricetin

[0059] As shown in Table 2, the kaempferol temperature-responsive imprinted materials prepared in Examples 1-3 exhibited significantly higher adsorption capacity for the target kaempferol than structurally similar quercetin and myricetin in the comparative tests of the three flavonoid components, demonstrating a significant selectivity advantage. Compared with the comparative examples and macroporous resin control materials, the materials in the examples showed higher selectivity coefficients for kaempferol, while the adsorption capacity for the other two similar compounds was significantly lower. This indicates that the efficient adsorption of kaempferol by the temperature-responsive imprinted materials of this invention is not due to simple physical partitioning, but rather relies on highly matched specific binding sites within the molecular imprint cavity. These test results verify the specific recognition effect of the material of this invention on the three-dimensional space and chemical forces of the target molecule.

[0060] (V) Comparative test of the adsorption performance of the kaempferol temperature-responsive imprinted material prepared in Example 1 and the white powder non-temperature-responsive molecular imprinted material prepared in Comparative Example 3 under different temperature conditions.

[0061] Methods: To investigate the temperature-responsive adsorption behavior of the materials, 1.00 g each of the kaempferol temperature-responsive imprinted material prepared in Example 1 and the white powder non-temperature-responsive molecularly imprinted material prepared in Comparative Example 3 were added to 400 mL of kaempferol standard solution with an initial concentration of 8.0 mg / L, and the solutions were shaken and adsorbed for 1.0 h at 20 ℃, 30 ℃, 40 ℃, and 50 ℃ in a constant temperature shaker. After adsorption, the filtrate was filtered, the equilibrium concentration was determined, and the unit adsorption capacity at different temperatures was calculated.

[0062] Test results are as follows Figure 4 As shown, the adsorption performance of the kaempferol temperature-responsive imprinted material in Example 1 is highly sensitive to temperature changes, while the adsorption capacity of the non-temperature-responsive material in Comparative Example 2 is low and shows no significant fluctuation. At 20 °C, the temperature-sensitive segments inside the kaempferol temperature-responsive imprinted material of Example 1 are in a highly swollen hydrophilic state, and the imprinted cavity expands, resulting in a decrease in the spatial matching degree between the specific recognition site and the target molecule, and a lower apparent adsorption capacity. When the system is heated to 30 °C, close to the low critical dissolution temperature of the temperature-sensitive monomer, the temperature-sensitive segments undergo moderate micro-contraction, and the three-dimensional conformation of the imprinted cavity reaches the optimal geometric spatial matching state with the kaempferol molecule, resulting in the highest adsorption capacity. When the temperature rises to 40 °C and 50 °C, a violent hydrophobic phase transition occurs inside the material, the three-dimensional cross-linked network excessively contracts and causes pore blockage, generating micro-compression forces that repel the target molecule, leading to a decrease in adsorption performance. In contrast, the non-temperature-responsive material in Comparative Example 3, lacking a temperature-sensitive responsive structure, exhibits adsorption behavior that is not driven by a phase transition and changes gradually with temperature. These results demonstrate that temperature-responsive molecularly imprinted materials possess temperature-responsive characteristics and can achieve efficient adsorption and controlled desorption of target molecules through reversible contraction and relaxation regulation of physical conformation.

[0063] (vi) Comparative test of the recovery rate of kaempferol in vegetable oil by the temperature-responsive imprinted materials of kaempferol prepared in Examples 1-3 and the white powder materials prepared in Comparative Examples 1-4.

[0064] Methods: To evaluate the anti-interference ability and extraction reliability of each material in a real, complex, high-viscosity oil matrix, the kaempferol temperature-responsive imprinted materials of Examples 1-3, the white powder materials of Comparative Examples 1-4, and a commercially available macroporous adsorption resin were selected for spiking recovery tests. A suitable amount of kaempferol standard was added to vegetable oil to prepare a spiking stock solution with a kaempferol concentration of 5.0 mg / L. Then, 1.00 g each of the kaempferol temperature-responsive imprinted materials of Examples 1-3 and the white powder materials of Comparative Examples 1-4 were weighed and added to 400 mL of the spiking sample solution. Adsorption was carried out at a constant temperature of 30 ℃ with shaking in the dark for 0.5 h. After filtering to separate the saturated adsorption material, the surface of the material was rapidly washed with 15 mL of n-hexane to remove residual oil, and then placed in 50 mL of 80% (v / v) ethanol aqueous solution for temperature-controlled desorption (elution) at 50 ℃ for 0.5 h. After the desorption solution was filtered through a 0.45 μm filter membrane, the concentration of kaempferol in the filtrate (recovery solution) was measured. The equivalent concentration of the recovery solution was obtained by volume conversion, and the actual recovery rate was calculated.

[0065] The test results are shown in Table 3.

[0066] Table 3 Comparison of actual recovery capacity of kaempferol in vegetable oil spiked samples using different materials

[0067] As shown in Table 3, the kaempferol temperature-responsive composite imprinted materials prepared in Examples 1-3 all exhibited significantly higher recovery rates than the comparative materials, maintaining a stable recovery rate of over 93% in complex pure oil matrices. This demonstrates that the kaempferol temperature-responsive composite imprinted material of this invention possesses excellent resistance to macromolecular lipid extrusion and high matrix selectivity. Comparative Example 1 lacked imprinted cavities due to the absence of template molecules, resulting in only weak physical adsorption; Comparative Example 2 had poor monomer ratios and insufficient effective site density; Comparative Example 3 lacked temperature-sensitive segments, leading to loss of high-temperature desorption kinetics and retention of a large amount of target material in the pores; Comparative Example 4 used a traditional single rigid crosslinking agent, which caused conformational shrinkage and pore closure in the non-polar oil phase, hindering oil mass transfer and reducing its recovery rate. Furthermore, macroporous adsorption resins lack specificity and are highly susceptible to interference from impurities. These results further demonstrate that the kaempferol temperature-responsive composite imprinted material prepared in this invention can achieve efficient enrichment and recovery of the target component kaempferol in vegetable oil samples.

[0068] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and substitutions without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a kaempferol temperature-responsive imprinted material, characterized in that, Includes the following steps: S1. Pre-assembly: Kaempferol, p-vinylphenol and N-isopropylacrylamide are dissolved in a mixture of methanol and vegetable oil. The mixture is ultrasonically mixed for 10-20 min at an ultrasonic power of 200-300 W, and then reacted at room temperature for 2-6 h to obtain a pre-assembled complex solution, denoted as product A. S2. Polymerization reaction: Add a mixed crosslinking agent and initiator azobisisobutyronitrile to product A obtained in step S1. The mixed crosslinking agent is composed of trimethylolpropane trimethacrylate and 1,6-hexanediol dimethacrylate. Under nitrogen protection, react at 50-70 °C for 12-24 h, and cool to room temperature to obtain a white powder material, denoted as product B. S3. Elution of template molecules: The product B obtained in step S2 is continuously washed with a mixture of methanol and acetic acid for 6-24 hours to remove unreacted components and template molecules. The resulting solid is then washed with methanol 2-3 times. Finally, the washed solid is vacuum dried at 40-60 °C for 12-24 hours to obtain kaempferol temperature-responsive imprinted material.

2. The method for preparing a kaempferol temperature-responsive imprinted material according to claim 1, characterized in that: In step S1, the vegetable oil is one or a combination of several of the following: tea oil, soybean oil, rapeseed oil, corn germ oil, and sunflower seed oil.

3. The method for preparing a kaempferol temperature-responsive imprinted material according to claim 1, characterized in that: In step S1, the mass ratio of kaempferol, p-vinylphenol, and N-isopropylacrylamide is 1:2-4:2-4; the volume of the mixture of methanol and vegetable oil is 10-20 times the total mass of the reactants (volume unit is ml, mass unit is g); the reactants are kaempferol, p-vinylphenol, and N-isopropylacrylamide; and the volume ratio of methanol to vegetable oil is 1:5-20.

4. The method for preparing a kaempferol temperature-responsive imprinted material according to claim 1, characterized in that: In step S2, the mass ratio of the total mass of the mixed crosslinking agent to the total mass of the monomers is 10-20:

1. The total monomers are p-vinylphenol and N-isopropylacrylamide. The mass of azobisisobutyronitrile added is 1-5% of the total polymerizable component mass. The total polymerizable component is product A and the mixed crosslinking agent.

5. The method for preparing a kaempferol temperature-responsive imprinted material according to claim 1, characterized in that: In step S2, the mass ratio of trimethylolpropane trimethacrylate to 1,6-hexanediol dimethacrylate is 1:1 to 2:

1.

6. The method for preparing a kaempferol temperature-responsive imprinted material according to claim 1, characterized in that: In step S3, the volume ratio of methanol to acetic acid in the eluent is 8-10:1, the volume of the methanol-acetic acid mixture is 10-20 times the mass of product B, and the volume of the methanol washing solution is 10-20 times the mass of product B. The volume unit is ml, and the mass unit is g.

7. The application of a kaempferol temperature-responsive imprinted material prepared by the preparation method according to any one of claims 1 to 6, characterized in that, The aforementioned kaempferol temperature-responsive imprint material is suitable for the separation of kaempferol from high-viscosity vegetable oils, and specifically includes the following steps: 1) The kaempferol temperature-responsive imprint material is directly added to the unsaponified vegetable oil stock solution, wherein the vegetable oil is the same as the vegetable oil in step S1 of claim 1, and the adsorption is shaken at 20-30℃ for 0.5-1 h, and then the adsorbed saturated material C is separated by filtration; 2) Use n-hexane to quickly rinse the surface of material C obtained in step 1) to remove residual grease. Then place material C in an aqueous ethanol eluent and elute at a constant temperature of 50-60 ℃ for 0.5-1 h. After filtration and separation, heat the filtrate to evaporate and remove the eluent to obtain the product kaempferol. The filter cake is a kaempferol temperature-responsive imprinted material that can be reused.

8. The application according to claim 7, characterized in that: In step 1), the ratio of the mass of the kaempferol temperature-responsive imprint material to the volume of the vegetable oil stock solution is 1:300-500, with the mass unit being g and the volume unit being ml.

9. The application according to claim 7, characterized in that: In step 1), the vegetable oil stock solution is diluted with n-hexane beforehand. The volume ratio of the vegetable oil stock solution to n-hexane is 1:1-3. The ratio of the mass of the kaempferol temperature-responsive imprint material to the volume of the vegetable oil stock solution diluted with n-hexane is 1:300-500. The mass unit is g, and the volume unit is ml.

10. The application according to claim 7, characterized in that: In step 2), the volume of hexane used for rinsing is 10-20 times the mass of the kaempferol temperature-responsive imprint material, with volume in ml and mass in g; the aqueous ethanol eluent is an aqueous ethanol solution with a volume fraction of 50-80%, and the volume of the aqueous ethanol eluent is 50-100 times the mass of the kaempferol temperature-responsive imprint material, with volume in ml and mass in g.

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