A template molecule, molecular imprinting material and its application in separation of iridoid glycoside compounds

Abstract

The present invention relates to a template molecule, a molecular imprinting material and an application thereof in separating iridoid glycoside compounds, and belongs to the technical field of plant component refining. The molecular imprinting material of the iridoid glycoside compounds of the present invention uses 2-(hydroxymethyl)-6-((4-ethoxynaphthalene-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol as a template molecule, can specifically bind to a target product, and has high selectivity. It can be used for the separation, enrichment and purification of iridoid glycoside compound molecules of gentiana scabra glycoside methyl ester, geniposide, swertiamarin, swertiarin, morroniside, loganin, gentiopicroside, leonurin A, oleuropein, scutellarin, aucubin and deacetylscutellarin methyl ester. It has high enrichment, high accuracy and wide application range.

Description

Technical Field

[0001] The invention belongs to the technical field of plant component purification, and in particular relates to a template molecule, a molecular imprinting material and application thereof in separating iridoid glycoside compounds. Background Art

[0002] Molecular imprinting technology (MIT) simulates the specific binding of substrates and enzymes to achieve rapid extraction and separation of a specific target substance. Its greatest advantage lies in personalized customization and specific binding, which is highly consistent with the separation process of natural products. Iridoids are a type of monoterpene compound with structures such as binary rings, olefin bonds, and ether bonds, which are helpful in constructing molecular imprinting. However, since the separation and synthesis of a single variety of iridium terpene compounds are very difficult, the price of pure compounds is very expensive, that is, the high cost of the template molecule accounts for a considerable proportion of the extraction cost. Summary of the invention

[0003] The object of the present invention is to provide a template molecule, which can reduce the extraction cost by using a virtual template molecule.

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

[0005] The third object of the present invention is to provide the application of the molecular imprinting material.

[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 molecular imprinted material 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 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 ethanol, filter out the precipitate, elute the template molecules, wash until neutral, and then dry to constant weight to obtain the molecular imprinting material.

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

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

[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 1:4-8.

[0018] As a preferred embodiment of the present invention, in the preparation of the continuous phase, the emulsifier is at least one of polyoxyethylene lauryl ether, Triton X-100 and Span 80.

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

[0020] As a preferred embodiment of the present invention, in the polymerization reaction, the polymerization reaction temperature is 25-50°C.

[0021] As a preferred embodiment of the present invention, in the polymerization reaction, the polymerization reaction time is 2 to 4 hours, and stirring is continued during the polymerization reaction.

[0022] The molecular imprinting material is used for separating iridoid glycoside compounds, wherein the iridoid glycoside compound is one of gentiana glycoside methyl ester, geniposide, scutellaria glycoside, scutellaria glycoside, morroniside, loganin, gentiopicroside, leonurin A, oleuropein, scutellaria glycoside, aucubin, and deacetylscutellaria glycoside methyl ester.

[0023] Beneficial effects of the present invention:

[0024] The molecular imprinting material of iridoid glycoside compounds of the present invention uses 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol as a template molecule, the template molecule contains a hydrogen bond donor (glycoside fragment) and a strong electronic effect (naphthalene ring fragment), has a high-specificity space pocket, can form multiple action sites with the target product through non-covalent bonds such as hydrogen bonds and hydrophobic interactions, thereby specifically binding, so it has high selectivity. The virtual template molecule is low in price and can be used for large-scale preparation of molecular imprinting materials. The use of the virtual template molecule to prepare molecular imprinting materials significantly reduces the extraction cost of iridoid glycoside compounds.

[0025] The preparation method of the present invention selects the inverse emulsion polymerization method to prepare the molecular imprinting material of iridoid glycosides, thereby increasing the adsorption amount of the target molecules, and the target molecules are the molecules of iridoid glycosides for gentiana glycoside methyl ester, geniposide, swertiamarin, swertiain, morroniside, loganin, gentiopicroside, leonurin A, oleuropein, scutellarin, aucubin, and deacetylated scutellarin methyl ester. The molecular imprinting material improves the purity of the iridoid glycosides, can be used for the separation, enrichment, and purification of the iridoid glycosides in the natural product extract, has high enrichment, high accuracy, and wide application range, and has broad application prospects in the preparation and detection of iridoid glycosides. 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: 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol, whose structural formula is:

[0029]

[0030] The specific experimental operations are as follows:

[0031] (1) 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 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). Then add water (20 mL), saturated NaHCO 3 The diluted reaction solution was washed with 1% paraformaldehyde (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate, and then the organic solvent in the organic layer was removed by a rotary evaporator to obtain acetyl-protected glucose with a yield of 95%.

[0032] (2) Add 2.0 mmol of acetyl-protected glucose and 20 mL of dichloromethane to a round-bottom flask to obtain a mixed solution, 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. Continue to stir the mixed solution at 0°C for 10 minutes, then add 2.6 mmol of 1-ethoxy-4-hydroxynaphthalene to the mixed solution, then heat it to room temperature and continue stirring for 24 hours. After the reaction is completed, cool it to 0°C, and add 10 mL of saturated NaHCO 3The aqueous solution was washed with water (10 mL x 3). After separation, the aqueous layer was further extracted with 20 mL of dichloromethane. The dichloromethane organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. After column chromatography purification, the acetyl-protected template molecule was obtained with a yield of 88%.

[0033] (3) 1.5 mmol of the acetyl-protected template molecule was dissolved in a mixture of 10 mL of methanol and 1.5 mL of 25% by mass aqueous ammonia solution, stirred at room temperature for 24 hours, and then the solvent was removed under reduced pressure. The template molecule was purified by column chromatography with a yield of 89%.

[0034] The template molecular map data is as follows: 1 H NMR (500MHz, CD 3 OD)δ(ppm)8.38-8.36(m,1H),7.79-7.77(m,1H),7.50-7.44(m,3H),7.40-7.37(m,1H),7.24-7.22(m,1H),5.10(d,J=7.5 Hz,1H),4.54(m,2H),4.39(m,1H),4.21(dd,J1=11.0Hz,J2=5.5Hz,1H),3.77-3.67(m,2H),3.59-3.50(m,2H),161(m,3H). 13 C NMR (125MHz, CD 3 OD)δ(ppm)154.4,135.8,128.4,127.3,127.1,127.0,126.3,123.2,123.0,110.6,102.6,77.7,75.9,75.0,71.2,68.1,60.9,15.8.HRMS(ESI)m / z calcd.for C 16 H 18 O 6 [M+H] + 350.1366, found 350.1354.

[0035] Example 2 Preparation and performance testing of molecularly imprinted polymers of iridoid glycosides

[0036] 1. Preparation of iridoid glycoside molecular imprinted polymers, the steps are as follows:

[0037] 1.1 Preparation of dispersed phase: 4 mmol acrylamide and 20 mmol N, N'-methylenebisacrylamide were dissolved in 20 mL water, and 1 mmol template molecule 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol was added, uniformly dispersed, and magnetically stirred for 0.5 h at room temperature to form a prepolymer to obtain solution A;

[0038] 1.2 Preparation of continuous phase: Dissolve 24 g of L4 polyoxyethylene lauryl ether (BRIJ 30) in 300 mL of n-hexane under nitrogen atmosphere to form a continuous phase as solution B;

[0039] 1.3 Preparation of inverse dispersion system: Under magnetic stirring, slowly transfer solution A to solution B and stir at room temperature for 60 min under nitrogen atmosphere to obtain a white emulsion;

[0040] 1.4 Polymerization reaction: 0.5 mL of 10% (w / v) potassium persulfate was added to the white emulsion to initiate the polymerization reaction, and the reaction was continued at 50° C. and 300 r / min for 2 h to obtain a polymer microsphere mixture;

[0041] 1.5 Post-treatment: Add ethanol to the polymer microsphere mixture to aggregate and precipitate the polymer, and filter to obtain the polymer. Transfer to a 240-mesh sieve, wash with water to remove fine powder, and then dry. Put the dried polymer into a Soxhlet extractor, extract the template molecules in the polymer with ethanol, and vacuum dry the polymer at 50-55°C to constant weight. After drying, classify the imprinted material with sieves of different mesh sizes to obtain molecular imprinted polymers of different sizes.

[0042] 2. Performance Testing

[0043] 2.1 Test on the adsorption performance of gentiopicroside:

[0044] i) Analysis conditions: Methanol was used as blank control and the absorbance was measured by UV-visible spectrophotometer;

[0045] ii) Drawing of standard curve: ① Accurately weigh the gentiopicroside standard and dilute it with methanol to 10, 20, 30, 45, and 60 ppm; ② Use methanol as blank and measure the absorbance at a wavelength of 254 nm with a UV-visible spectrophotometer; ③ Use absorbance (A) as the ordinate and concentration as the abscissa to draw the standard curve. Obtain the regression equation;

[0046] iii) Determination of adsorption amount: Accurately weigh 0.2g of molecular imprinted polymer of cyclopentadiene ether terpenoid glycosides and place it in a ground-mouth conical flask, add 5.0mL of gentiopicroside methanol solution with a concentration of 3mg / mL, adsorb at constant temperature and oscillate for 5h, centrifuge, dilute the supernatant with methanol to a concentration of 10-60ppm, then measure the absorbance at a wavelength of 254nm in parallel 3 times, take the arithmetic mean, and calculate the gentiopicroside concentration using the standard curve. Based on the change in the concentration of gentiopicroside in the solution before and after adsorption, the adsorption amount of the imprinted polymer was calculated to be 54.4mg / g. Calculation formula:

[0047] Q=(C 0 -C)*V / W

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

[0049] C 0 : the original mass concentration of the adsorbed compound in the 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 Test on the adsorption performance of iridoid glycosides molecularly imprinted polymers on various iridoid glycosides

[0054] The adsorption test of gentiopicroside was carried out according to the method for testing the adsorption performance of gentiopicroside in 2.1, except that the test molecule was replaced by a substitute molecule (iridoid glycoside compound molecule) instead of gentiopicroside, and the detection wavelength was adjusted synchronously. The obtained data are shown in Table 1.

[0055] Table 1

[0056]

[0057]

[0058] It can be seen from Table 1 that the molecular imprinting polymer of iridoid glycosides prepared in Example 1 has good adsorption performance for iridoid glycoside molecules of gentiana glycoside methyl ester, geniposide, scutellaria glycoside, scutellaria glycoside, morroniside, loganin, leonurin A, oleuropein, scutellaria glycoside, aucubin and deacetylscutellaria glycoside methyl ester.

[0059] Experimental Example 2 Investigating the Effect of Different Emulsifiers on the Shape of Molecularly Imprinted Polymers of Iridoid Glycosides in the Preparation Method

[0060] Based on the preparation method in Example 2, different emulsifiers were used as dispersants (see Table 2) to obtain iridoid glycoside molecularly imprinted polymers. The adsorption amounts of gentiopicroside were measured respectively, and the results are shown in Table 2:

[0061] Table 2

[0062] Emulsifier Polymer adsorption L4 Polyoxyethylene Laureth Ether (BRIJ 30) 55.02mg / g Triton X-100 47.09mg / g Span 80 36.71mg / g

[0063] It can be seen from Table 2 that the iridoid glycoside molecularly imprinted polymers prepared with emulsifiers L4 polyoxyethylene lauryl ether (BRIJ 30) and Triton X-100 have good adsorption amounts for gentiopicroside. Span 80 can also achieve adsorption, but the effect is slightly worse than the previous two.

[0064] In Test Example 3, the effects of different types and ratios of functional monomers in the step of preparing the dispersion phase on the performance of iridoid glycoside molecularly imprinted polymers were investigated.

[0065] Based on the preparation method in Example 2, different types and ratios of functional monomers were used (see Table 3) to obtain different iridoid glycoside molecularly imprinted polymers. The adsorption amounts of gentiopicroside were measured respectively, and the results are shown in Table 3:

[0066] Table 3

[0067]

[0068] It can be seen from Table 3 that when acrylamide and methacrylic acid are selected as hydrophilic functional monomers and the template molecule and the hydrophilic functional monomer are used in a molar ratio of 1:4, the iridoid glycoside molecularly imprinted polymers prepared have good adsorption amounts for gentiopicroside. The adsorption amount is the largest when the template molecule and acrylamide are used in a molar ratio of 1:4, which is 56.20 mg / g. When the template molecule and acrylamide are used in different molar ratios, the adsorption amounts of the iridoid glycoside molecularly imprinted polymers for gentiopicroside change greatly. When the molar ratio of the template molecule to acrylamide is 1:2, the adsorption amount decreases significantly, which is 8.54 mg / g. When the molar ratios of the template molecule to acrylamide are 1:6 and 1:8, the adsorption amounts are slightly lower than that when the molar ratio is 1:4.

[0069] In Test Example 4, the effects of different crosslinking agents in the step of preparing the dispersion phase on iridoid glycoside molecularly imprinted polymers were investigated.

[0070] Based on the preparation method in Example 2, different crosslinking agents were used (see Table 4) to obtain different iridoid glycoside molecularly imprinted polymers. The adsorption amounts of gentiopicroside were measured respectively, and the results are shown in Table 4:

[0071] Table 4

[0072] Crosslinking agent Polymer adsorption N,N'-Methylenebisacrylamide (MBA) 52.70mg / g Polyethylene glycol dimethacrylate (EGDMA) 49.24mg / g

[0073] As shown in Table 4, the molecular imprinting polymers of different iridoid glycosides prepared by using N,N'-methylenebisacrylamide and polyethylene glycol dimethacrylate as cross-linking agents all have good adsorption capacity for gentiopicroside.

[0074] Experimental Example 5 Investigating the effects of different initiators used in the polymerization step on the properties of molecularly imprinted polymers of iridoid ether terpenoid glycosides in the preparation method

[0075] Based on the preparation method in Example 2, different initiators (see Table 5) were used to observe the reaction performance of molecularly imprinted polymers of different iridoid glycosides. The results are shown in Table 5:

[0076] Table 5

[0077] Initiator Polymerization Potassium Persulfate Simple operation, quick response, stable process Benzoyl peroxide Simple operation, quick response, slightly poor process stability

[0078] As shown in Table 5, the molecular imprinting polymers of different iridoid glycosides were prepared by using potassium persulfate and benzoyl peroxide as initiators, which were simple to operate and had rapid reactions. The difference was that the molecular imprinting polymer prepared by potassium persulfate had a stable reaction process, while the molecular imprinting polymer prepared by benzoyl peroxide had a slightly less stable reaction process.

[0079] Experimental Example 6 Investigating the Effect of Different Reaction Conditions in the Polymerization Step on the Properties of Iridoid Glycoside Molecular Imprinting Polymers in the Preparation Method

[0080] Based on the preparation method in Example 2, the reaction conditions of the polymerization reaction were changed to prepare different iridoid glycosides molecularly imprinted polymers, including reaction temperature, reaction time, and mechanical stirring speed (see Table 6), and the adsorption amount of gentiopicroside by the obtained imprinted microspheres was measured respectively. The results are shown in Table 6:

[0081] Table 6

[0082]

[0083] It can be seen from Table 6 that different iridoid glycosides molecular imprinted polymers prepared under the conditions of polymerization reaction temperature of 25-80℃ and reaction time of 1-4h can form microspheres; the iridoid glycosides molecular imprinted polymers prepared under the conditions of reaction temperature of 40℃, reaction time of 4h and reaction temperature of 50℃, reaction time of 2h have better adsorption performance. However, the iridoid glycosides molecular imprinted polymer microspheres prepared under the conditions of reaction temperature of 25℃, reaction time of 4h and reaction temperature of 80℃, reaction time of 1h have poor adsorption performance.

[0084] 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 molecular imprinting material 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 ethanol, filter out the precipitate, elute the template molecules, wash until neutral, and then dry to constant weight to obtain the molecular imprinting material.

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

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

5. The molecularly imprinted material 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 1:4-8.

6. The molecularly imprinted material according to claim 2, characterized in that In preparing the continuous phase, the emulsifier is at least one of polyoxyethylene lauryl ether, Triton X-100, and Span 80.

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

8. The molecularly imprinted material according to claim 2, characterized in that During the polymerization reaction, the polymerization temperature is 25 to 50°C.

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

10. Use of the molecularly imprinted material according to any one of claims 2 to 9 in separating iridoid glycosides, characterized in that: The iridoid ether terpenoid glycoside compound is one of methyl ester of gentiana glycoside, geniposide, scutellaria glycoside, scutellaria glycoside, morroniside, loganin, leonurin A, oleuropein, scutellaria glycoside, aucubin, gentiopicroside and methyl deacetylscutellaria glycoside.

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