A method for extracting and purifying phloretin

Abstract

The present invention is applicable to the technical field of phloretin purification, and provides a phloretin extraction and purification method, comprising the following steps: synthesizing magnetic Fe3O4 nanoparticles by a solvent thermal method; preparing a molecularly imprinted polymer; preparing a crude phloretin product; adding a molecularly imprinted polymer to the crude product, obtaining a phloretin eluate through adsorption and elution processes, evaporating the solvent to obtain a secondary crude phloretin product; dissolving the secondary crude phloretin product in ethyl acetate, and adding n-hexane or cyclohexane dropwise to obtain pure phloretin. The present invention is simple and easy to operate, has high yield, low cost, low pollution and energy consumption, and obtains phloretin after two purifications. Using a new purification process, the purity is greater than 99.0%.

Description

Technical Field

[0001] The invention belongs to the technical field of phloretin purification, and in particular relates to a phloretin extraction and purification method. Background Art

[0002] Phloretin has extremely strong antioxidant, anti-inflammatory, antibacterial, and freckle-removing and whitening activities. It also has the functions of delaying aging, immunosuppression, sun protection, promoting transdermal absorption, moisturizing, preventing hair loss, improving endothelial dysfunction and disorders, promoting membrane absorption, and regulating glucose transport. It is an important raw material ingredient.

[0003] Commercially available phloretin mainly comes from two sources: chemical synthesis and extraction and separation. The plants that can be used for extraction and separation are mainly apple trees, apple peels, and Lithospermum multiflorum. Lithospermum multiflorum leaves are Chinese medicinal materials, and the pharmacopoeia stipulates that the phlorizin content must not be less than 2%. Therefore, the usual method of obtaining phloretin is to first extract phloretin, and then obtain phloretin through hydrolysis, separation and purification. The extraction process is cumbersome and requires repeated purification processes such as column chromatography and recrystallization. A large amount of organic solvents are used, which causes greater pollution. Impurities are easily generated during the extraction, hydrolysis and purification process, and the type and amount of impurities are difficult to control. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide a method for extracting and purifying phloretin, aiming to solve the problems existing in the above-mentioned background technology.

[0005] The embodiment of the present invention is achieved by a method for extracting and purifying phloretin, comprising the following steps:

[0006] (1) FeCl3•6H2O was dissolved in alcohol and magnetically stirred at room temperature to form a transparent solution. Anhydrous sodium acetate and sodium citrate were then added and stirred to mix the solution evenly. The mixed solution was then transferred to a stainless steel autoclave, which was placed in an oven for reaction to obtain a black material. The black material was washed with anhydrous ethanol and distilled water in sequence, and the excess raw materials were removed and dried under vacuum to obtain magnetic nanoparticles.

[0007] (2) The crude phloretin was dissolved in methanol, and then magnetic nanoparticles and zein were added, stirred evenly at room temperature, and pure water was added and stirred to carry out self-polymerization reaction. After the reaction was completed, the product was collected with a magnet, washed several times with a mixed solution of ethanol and water to remove the remaining raw materials, and then washed several times with a mixed solution of methanol and organic acid to remove the template molecules in the polymer body, thereby obtaining a molecularly imprinted polymer;

[0008] (3) crushing the leaves of the multi-spike lycopodium, adding the crushed multi-spike lycopodium leaves to a reactor, adding water and concentrated hydrochloric acid, fully mixing, soaking and dissociating, heating and stirring, reflux extraction, filtering the extract, and concentrating the filtrate under reduced pressure to obtain a concentrate, adding an equal amount of petroleum ether to the concentrate, and degreasing the petroleum ether to obtain a crude product;

[0009] (4) adding molecularly imprinted polymer to the primary crude product, obtaining phloretin eluate through adsorption and elution processes, and evaporating the solvent to obtain secondary crude phloretin;

[0010] (5) Dissolve the secondary crude phloretin in ethyl acetate and add n-hexane or cyclohexane dropwise to obtain pure phloretin.

[0011] Preferably, in step (1), the alcohol is an alcohol having a boiling point higher than 150°C.

[0012] Preferably, in step (1), the concentration of FeCl3•6H2O dissolved in alcohol is 220-320 g / L.

[0013] Preferably, in step (1), the autoclave is placed in an oven and the reaction temperature is 150-250°C.

[0014] Preferably, in step (2), the organic acid is formic acid or acetic acid.

[0015] Preferably, in step (2), the self-polymerization reaction time is 10-15 hours.

[0016] Preferably, in step (3), the dissociation adopts one or more of C1-C4 alcohol, ethyl acetate, ethyl formate, tetrahydrofuran, acetone, butanone, and 1,4-dioxane.

[0017] The embodiment of the present invention provides a method for extracting and purifying phloretin, which is simple and easy to operate, has high yield, low cost, low pollution and energy consumption, can obtain phloretin in one step, and adopts a new purification process, with a purity greater than 99.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 a is the SEM image of MBMIPs (magnetic molecular imprinted materials) provided in Example 2 of the present invention, Figure 1 b is the SEM image of MBNIPs (magnetic reference material), Figure 1 c is the SEM spectrum of Fe3O4, Figure 1 d is the TEM spectrum of MBMIPs, Figure 1 e is the TEM spectrum of MBNIPs, Figure 1 f is the TEM spectrum of Fe3O4;

[0019] Figure 2This is a liquid chromatogram of a crude product provided in Example 2 of the present invention;

[0020] Figure 3 This is a liquid chromatogram of the secondary crude product provided in Example 2 of the present invention;

[0021] Figure 4 This is a liquid chromatogram of the pure product provided in Example 2 of the present invention. Implementation Method

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] A method for extracting and purifying phloretin comprises the following steps:

[0024] (1) FeCl3•6H2O was dissolved in ethylene glycol (concentration of 220-320 g / L) and magnetically stirred at room temperature to form a transparent solution. Then, anhydrous sodium acetate was added at a ratio of 62 g / L and sodium citrate was added at a ratio of 8 g / L. The mixture was stirred at 150°C to mix the solution evenly. The mixed black solution was then transferred to a Teflon-lined stainless steel autoclave. The autoclave was placed in an oven at 200°C for 8 h to react. The obtained black material was then washed with anhydrous ethanol and distilled water in sequence to remove excess raw materials. After washing, the mixture was dried under vacuum at 50°C to obtain the prepared magnetic nanoparticles.

[0025] (2) The crude phloretin was dissolved in 2 L of 75% methanol at a concentration of 50 g / L. Then, 500 g of magnetic nanoparticles and five times the amount of zein were mixed and added to the reaction system. The mixed solution was stirred evenly at room temperature. 5 L of pure water was added and self-polymerized under stirring at room temperature for more than 10 h. After the reaction, the product was collected with a magnet and washed three times with 75% ethanol / water (v / v) solution to remove the remaining raw materials. Then, it was washed three times with methanol and organic acid solution (v / v 9:1) to remove the template molecules in the polymer body and obtain molecularly imprinted polymer.

[0026] (3) Crush 5000 g of Lithospermum multiflorum leaves, add the crushed Lithospermum multiflorum leaves into a reactor, add 15 L of water and 1 L of concentrated hydrochloric acid, mix thoroughly and soak for 12 h, then add 5 L of ethanol, heat to 80 °C, stir, reflux and extract for 4 h, filter the extract, and concentrate the filtrate under reduced pressure to obtain a 1.1% concentrate. Add an equal amount of petroleum ether to the concentrate, and defat the petroleum ether to obtain a crude product;

[0027] (4) adding molecularly imprinted polymer to the primary crude product, obtaining phloretin through adsorption and elution, testing and drying, and obtaining a secondary crude phloretin;

[0028] (5) Dissolve the crude product in ethyl acetate and add n-hexane or cyclohexane dropwise to obtain pure phloretin.

[0029] The specific implementation of the present invention is described in detail below with reference to specific embodiments. Example 1

[0030] A method for extracting and purifying phloretin comprises the following steps:

[0031] (1) Synthesis of magnetic Fe3O4 nanoparticles using a solvothermal method: 2700 g of FeCl3•6H2O was dissolved in 10 L of ethylene glycol and stirred magnetically at room temperature until a transparent solution was formed. Then, 620 g of anhydrous sodium acetate and 80 g of sodium citrate were added and stirred at 150°C for about 30 min to mix the solution evenly. The mixed black solution was then transferred to a Teflon-lined stainless steel autoclave and placed in an oven at 200°C for 8 h. Finally, the obtained black material was washed with anhydrous ethanol and distilled water once each to clean the material with poor magnetic properties or unsuccessful synthesis. The material was then dried under vacuum at 50°C for 8 h to obtain the prepared Fe3O4.

[0032] (2) 100 g of crude phloretin was dissolved in 2 L of 75% ethanol, and then 500 g of synthesized magnetic nanoparticles and 2500 g of zein were added to the mixed solution. The mixed solution was stirred at room temperature for 2 h, and 5 L of pure water was added to self-polymerize at room temperature under stirring for about 10 h. After the reaction was completed, the product was collected with a magnet and washed three times with 75% ethanol / water (v / v) solution to wash away other materials that failed to synthesize molecular imprints. Then, methanol and acetic acid solution (v / v 9:1) were repeated three times to wash away the template molecules in the polymer shell to obtain molecularly imprinted polymers.

[0033] (3) Crush 5000 g of Lithospermum multiflorum leaves, add the crushed Lithospermum multiflorum leaves into a reactor, add 15 L of water and 1 L of concentrated hydrochloric acid, mix thoroughly and soak for 12 h, then add 5 L of ethanol, heat to 80 °C, stir, reflux and extract for 4 h, filter the extract, and concentrate the filtrate under reduced pressure to obtain a 1.1% concentrate. Add an equal amount of petroleum ether to the concentrate, and defat the petroleum ether to obtain a crude product;

[0034] (4) adding molecularly imprinted polymer to the primary crude product, obtaining phloretin through adsorption and elution, testing and drying, and obtaining a secondary crude phloretin;

[0035] (5) Dissolve the secondary crude phloretin in ethyl acetate and add n-hexane dropwise to obtain pure phloretin. Example 2

[0036] A method for extracting and purifying phloretin comprises the following steps:

[0037] (1) Synthesis of magnetic Fe3O4 nanoparticles using the solvent thermal method: 2700 g of FeCl3•6H2O was dissolved in 10 L of propylene glycol and stirred magnetically at room temperature until a transparent solution was formed. Then, 620 g of anhydrous sodium acetate and 80 g of sodium citrate were added and stirred at 150°C for about 30 min to mix the solution evenly. The mixed black solution was then transferred to a Teflon-lined stainless steel autoclave and placed in an oven at 200°C for 8 h. Finally, the obtained black material was washed three times with anhydrous ethanol and distilled water in turn to clean the material with poor magnetic properties or that was not successfully synthesized. Then, the prepared Fe3O4 was dried at 50°C in vacuum for 8 h. The scanning electron microscopy and projection electron microscopy results are shown in Figure 2. Figure 1 As shown;

[0038] (2) 100 g of crude phloretin was dissolved in 2 L of 75% methanol, and then 500 g of synthesized magnetic nanoparticles and 2500 g of zein were added to the mixed solution. The mixed solution was stirred at room temperature for 2 h, and 5 L of pure water was added to self-polymerize at room temperature under stirring for about 10 h. After the reaction was completed, the product was collected with a magnet and washed three times with 75% ethanol / water (v / v) solution to wash away other materials that failed to synthesize molecular imprints. Then, the template molecules in the polymer shell were washed three times with methanol and acetic acid solution (v / v 9:1) to obtain molecularly imprinted polymers. The scanning electron microscopy and transmission electron microscopy results are shown in Figure 2. Figure 1 As shown;

[0039] (3) Crush 5000 g of Lithops multiflora leaves, add the crushed Lithops multiflora leaves into a reactor, add 5 L of methanol, add 300 mL of concentrated sulfuric acid, heat to 80 °C and stir to extract for 4 h; filter the extract, evaporate the filtrate to dryness under reduced pressure, add 2 L of water to the dried product, extract with methyl tert-butyl ether 4 times, combine the organic phases and evaporate to dryness under reduced pressure to obtain a crude product of phloretin, the liquid chromatogram of which is shown in the figure below. Figure 2 As shown;

[0040] (4) Molecularly imprinted polymer was added to the primary crude product for adsorption and desorption to obtain phloretin. After testing, the product was dried by spin drying to obtain the secondary crude phloretin. The liquid chromatogram of the product is shown in FIG. Figure 3 As shown;

[0041] (5) The secondary crude phloretin was dissolved in ethyl acetate and cyclohexane was added dropwise to obtain pure phloretin. Its liquid chromatogram is shown in the following figure: Figure 4 shown.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting and purifying phloretin, characterized in that: The following steps are involved: (1) FeCl3•6H2O was dissolved in alcohol and magnetically stirred at room temperature to form a transparent solution. Anhydrous sodium acetate and sodium citrate were then added and stirred to mix the solution evenly. The mixed solution was then transferred to a stainless steel autoclave, which was placed in an oven for reaction to obtain a black material. The black material was washed with anhydrous ethanol and distilled water in sequence, and the excess raw materials were removed and dried under vacuum to obtain magnetic nanoparticles. (2) The crude phloretin was dissolved in methanol, and then magnetic nanoparticles and zein were added, stirred evenly at room temperature, and pure water was added and stirred to carry out self-polymerization reaction. After the reaction was completed, the product was collected with a magnet, washed several times with a mixed solution of ethanol and water to remove the remaining raw materials, and then washed several times with a mixed solution of methanol and organic acid to remove the template molecules in the polymer body, thereby obtaining a molecularly imprinted polymer; (3) crushing the leaves of the multi-spike schizonepeta, adding the crushed multi-spike schizonepeta leaves into a reactor, adding water and concentrated hydrochloric acid, fully mixing, soaking and dissociating, heating and stirring, reflux extraction, filtering the extract, and concentrating the filtrate under reduced pressure to obtain a concentrate, adding an equal amount of petroleum ether to the concentrate, and defatting the petroleum ether to obtain a crude product, wherein the dissociation adopts one or more of C1-C4 alcohol, ethyl acetate, ethyl formate, tetrahydrofuran, acetone, butanone, and 1,4-dioxane; (4) adding molecularly imprinted polymer to the primary crude product, obtaining phloretin eluate through adsorption and elution processes, and evaporating the solvent to obtain secondary crude phloretin; (5) Dissolve the secondary crude phloretin in ethyl acetate and add n-hexane or cyclohexane dropwise to obtain pure phloretin.

2. The phloretin extraction and purification method according to claim 1, wherein In step (1), the alcohol is an alcohol having a boiling point higher than 150°C.

3. The phloretin extraction and purification method according to claim 2, wherein In step (1), the concentration of FeCl3•6H2O dissolved in alcohol is 220-320 g / L.

4. The method for extracting and purifying phloretin according to claim 3, wherein In step (1), the autoclave is placed in an oven and the reaction temperature is 150-250°C.

5. The method for extracting and purifying phloretin according to claim 1, wherein In step (2), the organic acid is formic acid or acetic acid.

6. The method for extracting and purifying phloretin according to claim 5, wherein In step (2), the self-polymerization reaction time is 10-15 hours.

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