Method for preparing extract for preventing and treating non-alcoholic steatohepatitis from olive leaves

Abstract

The invention discloses a method for preparing an extract for preventing and treating non-alcoholic steatohepatitis from olive leaves, which comprises the following steps of: extracting and concentrating the olive leaves to obtain a sample solution, adsorbing the sample solution by using a synthetic separation material, and respectively eluting by using water and an ethanol water solution, so that the content of oleuropein is greater than 50%, and the yield is greater than 60%. The extract for preventing and treating the non-alcoholic steatohepatitis can be prepared by utilizing a static adsorption / desorption method, the process route is simple and environment-friendly, and animal experiments prove that the obtained extract has a good drug effect on the non-alcoholic steatohepatitis and can be used as a raw material for developing new drugs.

Description

Technical Field

[0001] The present invention relates to a method for preparing an extract for preventing and treating nonalcoholic fatty hepatitis and application thereof, and in particular to a method for preparing an extract for preventing and treating nonalcoholic fatty hepatitis from olive leaves and application thereof, belonging to the technical field of separation and purification of natural medicines. Background Art

[0002] Nonalcoholic Steatohepatitis (NASH) is a critical stage in the progression of nonalcoholic fatty liver disease (NAFLD). It is also called metabolic steatohepatitis. It is a clinical syndrome with pathological changes similar to alcoholic hepatitis but without a history of excessive drinking. It is more common in middle-aged people, especially overweight and obese individuals.

[0003] Oleuropein has antioxidant, anti-cancer, hypoglycemic and lipid-lowering effects. At present, the literature has reported the use of oleuropein with a content of about 80% to prevent and treat non-alcoholic fatty liver disease (Experimental & Molecular Medicine (2014) 46, e92). Oleuropein is present in large quantities in olive (Oleaeuropaea L.) leaves, and olive leaves, as a by-product of the olive planting industry, are discarded every year when pruning and harvesting olive fruits, causing a serious waste of resources. Therefore, separating and purifying high-purity oleuropein from olive leaves and using it to prevent and treat non-alcoholic fatty liver disease is of great significance to the comprehensive utilization and development of olive leaves and the high-value utilization of olive by-products. Summary of the invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies in the prior art and to provide a method and application of preparing an extract for preventing and treating non-alcoholic fatty liver disease from olive leaves. The extract obtained by the present invention has good pharmacological effects on non-alcoholic fatty liver disease and can be used as a raw material for new drug development.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention first provides a method for preparing an extract for preventing and treating non-alcoholic fatty hepatitis from olive leaves, comprising the following steps:

[0007] (1) Olive Leaf Extract:

[0008] Olive leaves are used as raw materials, and the extraction solvent is used for reflux extraction twice. The extracts are combined and filtered and concentrated in sequence, and the concentrated solution is used as the sample solution.

[0009] (2) Synthesis of separation materials:

[0010] The swellable polymer seed microspheres are placed in deionized water containing a surfactant for dispersion to obtain a solution A, a swelling agent is taken and emulsified in the deionized water containing a surfactant to obtain a solution B, and the solution A and the solution B are combined in a counter container, and the two are swollen under certain conditions; a polymer monomer, a crosslinking agent, a nitrogen-containing polymer monomer, a porogen and an initiator are mixed and dissolved, and then emulsified in deionized water containing a surfactant to obtain a solution C, and the solution C is added to a reactor to continue swelling; then a deionized water solution containing a stabilizer is added to the reactor, and a reaction is carried out under certain conditions, and after the reaction is completed, the reaction solution is filtered and washed to obtain a separation material;

[0011] (3) Separating and purifying the sample solution using separation materials:

[0012] An appropriate amount of the separation material obtained in step (2) is added to a certain amount of the sample solution obtained in step (1) for stirring and adsorption. After the adsorption is completed, the solution is filtered, and then a desorbent is added to the filtrate for stirring and desorption. The desorbed solution is collected, filtered, concentrated, and dried in sequence to obtain an olive leaf extract for preventing and treating non-alcoholic fatty liver disease.

[0013] In the above technical scheme, in step (1), the extraction solvent is an ethanol aqueous solution with a mass fraction of 10% to 90%, and the solid-liquid ratio of olive leaves to the extractant is 1:3 to 1:30, wherein the mass of the olive leaves is measured in kg, the volume of the extractant is measured in L, and the reflux extraction time for each time is 1 to 5 hours.

[0014] In the above technical scheme, in step (1), the filtration is carried out using a 10-100 mesh filter; the concentration is carried out using a rotary evaporator at a temperature of 20-80° C. and a pressure of 5-30 kPa, and the concentration is carried out to a concentration of oleuropein of 0.1-15 mg / mL.

[0015] In the above technical solution, in step (2), the surfactant is sodium dodecyl sulfate; and the mass concentration of the solute in the deionized water containing the surfactant is 0.1% to 5%.

[0016] In the above technical solution, in step (2), the swellable polymer seed microspheres are polystyrene microspheres with a particle size of 1 to 50 μm; and the total volume of the deionized water containing the surfactant is 50 to 1000 times the mass of the swellable polymer seed microspheres.

[0017] In the above technical scheme, in step (2), the swelling agent is any one of dibutyl phthalate, 1-chlorododecane, etc., and the mass ratio of the swelling agent to the swellable polymer seed microspheres is 0.1 to 10:1; the volume ratio of the swelling agent to the deionized water containing the surfactant is 1:50 to 1000, and the emulsification method when the two are emulsified is ultrasonic emulsification, the emulsification time is 10 to 60 minutes, and the power is 100 to 750W.

[0018] In the above technical solution, in step (2), when solution A and solution B are swelled, the swelling conditions are: temperature 10-50° C., stirring speed 50-300 rpm, and time 6-72 h.

[0019] In the above technical scheme, in step (2), the polymerizable monomer is styrene; the cross-linking agent is any one of divinylbenzene, ethylene glycol dimethacrylate, etc.; the nitrogen-containing polymerizable monomer is any one of acrylamide, vinylaniline, vinylimidazole, vinylpyrrolidone, etc.; the porogen is any one of toluene, xylene, n-heptane, etc.; the initiator is any one of azobisisobutyronitrile, benzoyl peroxide, etc., wherein the polymerizable monomer: cross-linking agent: nitrogen-containing polymerizable monomer = 1: (0.1~10): (0.1~10); the mass of the porogen is 0.1~10 times the total mass of the polymerizable monomer, the cross-linking agent and the nitrogen-containing polymerizable monomer, and the mass of the initiator is 0.1%~5% of the total mass of the polymerizable monomer, the cross-linking agent and the nitrogen-containing polymerizable monomer.

[0020] In the above technical scheme, in step (2), the polymerizable monomer, the cross-linking agent, the nitrogen-containing polymerizable monomer, the porogen and the initiator are mixed and dissolved and then emulsified in deionized water containing a surfactant, the mass of the deionized water containing the surfactant is 1 to 20 times the total mass of the polymerizable monomer, the cross-linking agent and the nitrogen-containing polymerizable monomer, the emulsification method is ultrasonic emulsification, the emulsification time is 10 to 60 minutes, and the power is 100 to 750W.

[0021] In the above technical scheme, in step (2), solution C is added to the reactor to continue swelling, and the mass ratio of the total mass of the polymerizable monomer, the crosslinking agent, and the nitrogen-containing polymerizable monomer in solution C to the swellable polymerizable seed microspheres is (10-50):1, and the swelling conditions are: temperature 10-50°C, stirring speed 50-300rpm, and time 6-72h.

[0022] In the above technical scheme, in step (2), the stabilizer is any one of polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, etc., the mass concentration of the solute in the deionized water solution containing the stabilizer is 0.1% to 5%; the mass of the deionized water solution containing the stabilizer is 0.1 to 20 times the total mass of the polymerized monomer, the cross-linking agent and the nitrogen-containing polymerized monomer; the reaction temperature is 50 to 100° C., the stirring speed is 100 to 1000 rpm, and the reaction time is 6 to 72 hours;

[0023] In the above technical solution, in step (2), the filtration is performed using an 800-mesh filter; the washing is performed by first washing with ethanol for 1 to 5 times and then washing with deionized water for 1 to 5 times to obtain a separated material.

[0024] In the above technical scheme, in step (3), the ratio of the mass of the separation material to the volume of the sample solution is 1: (1-100), wherein the separation material is measured in grams (wet weight) and the sample solution is measured in milliliters; the stirring adsorption is mechanical stirring, the stirring speed is 10-500 rpm, the adsorption temperature is 5-50°C, the adsorption time is 5-500 min, and the filtration after the adsorption is completed is carried out using an 800-mesh filter.

[0025] In the above technical scheme, in step (3), the desorbent is an ethanol solution with a mass fraction of 10% to 90%, and the ratio of the volume of the desorbent (in milliliters) to the mass of the adsorbent (in grams) is (5 to 200):1; the stirring desorption is mechanical stirring, the stirring speed is 10 to 500 rpm, the desorption temperature is 5 to 50°C, and the desorption time is 5 to 500 min; the filtration after desorption is carried out by 800 mesh filter, and the desorption liquid is concentrated by a rotary evaporator at a concentration temperature of 20 to 80°C and a pressure of 5 to 30 kPa, and concentrated to a paste; the drying is carried out in a vacuum drying oven at a drying temperature of 20 to 80°C and a drying pressure of 2 to 40 kPa, and dried to a water content of less than 5%.

[0026] The second object of the present invention is to provide an extract for preventing and treating non-alcoholic fatty hepatitis prepared by the above method, wherein the content of oleuropein is 60% to 98%.

[0027] The second purpose of the present invention is to provide an application of the extract for preventing and treating non-alcoholic fatty hepatitis in the preparation of a drug for preventing and treating non-alcoholic fatty hepatitis.

[0028] The extract for preventing and treating non-alcoholic fatty hepatitis obtained by the present invention is used in an animal experiment with non-alcoholic fatty hepatitis, wherein the animal experiment is conducted on mice with non-alcoholic fatty hepatitis induced by MCD feed. From the animal experiment, it can be seen that the extract obtained by the present invention has good efficacy in preventing and treating non-alcoholic fatty hepatitis. The process method of the present invention is simple to operate and is green and environmentally friendly, and can provide guidance for the comprehensive utilization of olive leaves and the development of health foods, functional foods and medicines, thereby improving the comprehensive utilization rate of olive leaves. DETAILED DESCRIPTION

[0029] The specific implementation of the technical solution of the present invention is described in detail below, but the present invention is not limited to the following description:

[0030] Embodiment 1:

[0031] An extract for preventing and treating nonalcoholic fatty hepatitis is prepared from olive leaves by the following method:

[0032] (1) Olive Leaf Extract:

[0033] Weigh 200g of olive leaves, place in a 5L round-bottom flask, add 3L of 50% ethanol solution, heat in an electric heating mantle and reflux at normal pressure for 2h, pour out the extract. Add 2.4L of 50% ethanol solution to the residue, reflux at normal pressure for 2h, pour out the extract, and discard the residue. Combine the two extracts, filter with a 40-mesh filter, and use a rotary evaporator at a pressure of 15kPa and 50°C to concentrate to a concentration of oleuropein of 2mg / mL to obtain a sample solution;

[0034] (2) Synthesis of separation materials:

[0035] 1 g of 10 μm polystyrene seed microspheres was dispersed in 70 mL of 0.25% sodium dodecyl sulfate aqueous solution to obtain solution A, which was placed in a 500 mL three-necked round-bottom flask; 1 g of dibutyl phthalate was emulsified in 80 mL of 0.25% sodium dodecyl sulfate aqueous solution in an ultrasonic device with a power of 500 W for 30 min to obtain an emulsion, which was solution B. Solution A and solution B were swollen in a reactor (three-necked round-bottom flask) at 40° C. and 150 rpm for 24 h; 10 g of styrene, 10 g of divinylbenzene, 5 g of vinylpyrrolidone, 20 g of xylene and 0.5 g of azobisisobutyronitrile were mixed and dissolved, and then added to 100 mL of 0.25% sodium dodecyl sulfate aqueous solution, and emulsified in an ultrasonic device with a power of 500 W for 30 min to obtain an emulsion, which was placed in a reactor and continued to swell at 40° C. and 150 rpm for 24 h; 50 mL 1% polyvinyl alcohol solution was added to the reactor, the temperature was raised to 75°C, and the reaction was carried out at 300rpm for 24h. After the reaction was completed, it was filtered with an 800-mesh filter, washed twice with anhydrous ethanol, and then washed twice with deionized water to finally obtain a separated material.

[0036] (3) Separating and purifying the sample solution using separation materials:

[0037] 10 g of separation material (wet weight) was added to 100 mL of sample solution, adsorbed at 30° C. and 100 rpm for 1 h, filtered with an 800-mesh filter after adsorption, and the adsorbed residual liquid was discarded. 100 mL of 60% ethanol solution was added to the adsorbed material, and desorbed at 30° C. and 100 rpm for 1 h. After desorption, the desorbed liquid was filtered with an 800-mesh filter and the desorbed liquid was collected. The solvent was recovered to an extract on a rotary evaporator at a temperature of 40° C. and a pressure of 20 kPa, and finally dried in an oven at a temperature of 50° C. and a pressure of 10 kPa to a water content of less than 5%, thereby obtaining an olive leaf extract, wherein the content of oleuropein was 67.34%, and the yield was 94.47%.

[0038] Embodiment 2:

[0039] An extract for preventing and treating nonalcoholic fatty hepatitis is prepared from olive leaves by the following method:

[0040] (1) Olive Leaf Extract:

[0041] Weigh 200g of olive leaves, place in a 5L round-bottom flask, add 2L of 50% ethanol solution, heat under normal pressure and reflux for 3h in an electric heating mantle, pour out the extract. Add 2L of 50% ethanol solution to the residue, reflux under normal pressure for 3h, pour out the extract, and discard the residue. Combine the two extracts, filter with a 40-mesh filter, and use a rotary evaporator at a pressure of 15kPa and 50°C to concentrate to a concentration of oleuropein of 4mg / mL to obtain a sample solution;

[0042] (2) Synthesis of separation materials:

[0043] 1 g of 5 μm polystyrene seed microspheres was dispersed in 70 mL of 0.35% sodium dodecyl sulfate aqueous solution to obtain solution A, which was placed in a 500 mL three-necked round-bottom flask; 1.5 g of dibutyl phthalate was dissolved in 80 mL of 0.35% sodium dodecyl sulfate aqueous solution and emulsified in an ultrasonic device with a power of 500 W for 30 min to obtain an emulsion, which was solution B. Solution A and solution B were swollen in a reactor (three-necked round-bottom flask) at 40° C. and 150 rpm for 24 h; 5 g of styrene, 15 g of divinylbenzene, 5 g of vinylaniline, 20 g of xylene and 0.5 g of azobisisobutyronitrile were mixed and dissolved, and then added to 100 mL of 0.25% sodium dodecyl sulfate aqueous solution, and emulsified in an ultrasonic device with a power of 500 W for 30 min to obtain an emulsion, which was placed in a reactor and continued to swell at 40° C. and 150 rpm for 12 h; 50 mL 1% polyvinyl pyrrolidone solution was added to the reactor, the temperature was raised to 80°C, and the reaction was carried out at 200rpm for 20h. After the reaction was completed, it was filtered through an 800-mesh filter, washed 3 times with anhydrous ethanol, and then washed 3 times with deionized water to finally obtain the separated material.

[0044] (3) Separating and purifying the sample solution using separation materials:

[0045] 10 g of separation material (wet weight) was added to 150 mL of sample solution, adsorbed at 30° C. and 100 rpm for 2 h, filtered with an 800-mesh filter after adsorption, and the adsorbed residual liquid was discarded. 100 mL of 60% ethanol solution was added to the adsorbed material, and desorbed at 30° C. and 100 rpm for 1 h. After desorption, the desorbed liquid was filtered with an 800-mesh filter and the desorbed liquid was collected. The solvent was recovered to an extract on a rotary evaporator at a temperature of 40° C. and a pressure of 20 kPa, and finally dried in an oven at a temperature of 50° C. and a pressure of 10 kPa to a water content of less than 5%, thereby obtaining an olive leaf extract, wherein the content of oleuropein was 65.32%, and the yield was 92.58%.

[0046] Embodiment 3:

[0047] An extract for preventing and treating nonalcoholic fatty hepatitis is prepared from olive leaves by the following method:

[0048] (1) Olive Leaf Extract:

[0049] Weigh 200g of olive leaves, place in a 5L round-bottom flask, add 3L of 50% ethanol solution, heat in an electric heating mantle and reflux at normal pressure for 2h, pour out the extract. Add 3L of 50% ethanol solution to the residue, reflux at normal pressure for 2h, pour out the extract, and discard the residue. Combine the two extracts, filter with an 80-mesh filter, and use a rotary evaporator at a pressure of 15kPa and 50°C to concentrate to a concentration of oleuropein of 2mg / mL to obtain a sample solution;

[0050] (2) Synthesis of separation materials:

[0051] 1 g of 5 μm polystyrene seed microspheres was dispersed in 70 mL of 0.25% sodium dodecyl sulfate aqueous solution to obtain solution A, which was placed in a 500 mL three-necked round-bottom flask; 2 g of dibutyl phthalate was dissolved in 80 mL of 0.25% sodium dodecyl sulfate aqueous solution and emulsified in an ultrasonic device with a power of 500 W for 40 min to obtain an emulsion, which was solution B. Solution A and solution B were swollen in a reactor (three-necked round-bottom flask) at 35° C. and 100 rpm for 24 h; 10 g of styrene, 10 g of divinylbenzene, 5 g of vinylpyrrolidone, 25 g of toluene and 0.5 g of azobisisobutyronitrile were mixed and dissolved, and then added to 100 mL of 0.25% sodium dodecyl sulfate aqueous solution, and emulsified in an ultrasonic device with a power of 500 W for 30 min to obtain an emulsion, which was placed in a reactor and continued to swell at 35° C. and 100 rpm for 24 h; 50 mL 1% polyvinyl alcohol solution was added to the reactor, the temperature was raised to 75°C, and the reaction was carried out at 250rpm for 24 hours. After the reaction was completed, it was filtered through an 800-mesh filter, washed twice with anhydrous ethanol, and then washed twice with deionized water to finally obtain a separated material.

[0052] (3) Separating and purifying the sample solution using separation materials:

[0053] 10 g of separation material (wet weight) was added to 200 mL of sample solution, adsorbed at 30° C. and 150 rpm for 1 h, filtered with an 800-mesh filter after adsorption, and the adsorbed residual liquid was discarded. 150 mL of 70% ethanol solution was added to the adsorbed material, and desorbed at 30° C. and 150 rpm for 2 h. After desorption, the desorbed liquid was filtered with an 800-mesh filter and the desorbed liquid was collected. The solvent was recovered to an extract on a rotary evaporator at a temperature of 40° C. and a pressure of 20 kPa, and finally dried in an oven at a temperature of 60° C. and a pressure of 10 kPa to a water content of less than 5%, thereby obtaining an olive leaf extract, wherein the content of oleuropein was 68.53%, and the yield was 96.13%.

[0054] Application examples:

[0055] Animal experiment was carried out with the olive leaf extract obtained in Example 1 as raw material. Male Kunming mice weighing 18-22g were taken and randomly divided into a control group, a model group, a low-dose group (5.0 original drug g / kg) and a high-dose group (20.0 original drug g / kg) by body weight after adaptive feeding. Except that the control group was given ordinary feed, the model group and all the other groups were given MCD feed for 1 month. Meanwhile, the drug administration group was gavaged once a day, and the administration volume was 10mL / kg. After continuous administration for 1 month, the control group and the model group were gavaged with equal volume of water. After the last administration, mice were fasted and water was not allowed to be forbidden. After 14 hours, serum was taken to detect AST, ALT, TC, TG, HDL, LDL, and the liver was taken to weigh the wet weight and calculate the liver index.

[0056] Table 1 Effects of olive leaf extract on body weight and liver index in mice

[0057]

[0058] Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0059] As shown in Table 1, compared with the control group, the body weight of mice in the model group was significantly reduced and the liver index was significantly increased. Compared with the model group, the body weight of mice in the high-dose group was significantly reduced.

[0060] Table 2 Effects of olive leaf extract on serum liver function in mice

[0061]

[0062] Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0063] As shown in Table 2, compared with the control group, the ALT, AST, and TBA in the serum of the mice in the model group were significantly increased, and the liver function was significantly decreased. Compared with the model group, the low-dose group and the high-dose group could significantly reduce the ALT content in the serum of the mice; each administration group could significantly reduce the AST content in the serum of the mice; the high-dose group could significantly reduce the TBA content in the serum of the mice.

[0064] Table 3 Effects of olive leaf extract on blood lipids in mice

[0065]

[0066] Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0067] As shown in Table 3, compared with the control group, the TG, TC, HDL and LDL in the serum of the mice in the model group were significantly reduced. Compared with the model group, the high-dose group could significantly reduce the TG content in the serum of the mice, and each drug-treated group could significantly increase the HDL content in the serum of the mice.

[0068] Table 4 Effects of olive leaf extract on liver lipids in mice

[0069]

[0070] Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0071] As shown in Table 4, compared with the control group, the TG content in the liver of mice in the model group was significantly increased. Compared with the model group, the high-dose group could significantly reduce the TG content in the liver of mice.

[0072] The above examples are only for illustrating the technical concept and technical features of the present invention, and cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an extract for preventing and treating nonalcoholic fatty hepatitis from olive leaves, characterized in that: The following steps are involved: (1) Olive leaf extract: Olive leaves are used as raw materials, and the extraction solvent is used for reflux extraction twice. The extracts are combined and filtered and concentrated in sequence, and the concentrated solution is used as the sample solution. (2) Synthesis of separation materials: The swellable polymer seed microspheres are placed in deionized water containing a surfactant for dispersion to obtain a solution A, a swelling agent is taken and emulsified in the deionized water containing a surfactant to obtain a solution B, and the solution A and the solution B are combined in a counter container, and the two are swollen under certain conditions; a polymer monomer, a crosslinking agent, a nitrogen-containing polymer monomer, a porogen and an initiator are mixed and dissolved, and then emulsified in deionized water containing a surfactant to obtain a solution C, and the solution C is added to a reactor to continue swelling; then a deionized water solution containing a stabilizer is added to the reactor, and a reaction is carried out under certain conditions, and after the reaction is completed, the reaction solution is filtered and washed to obtain a separation material; (3) Separate and purify the sample solution using separation materials: An appropriate amount of the separation material obtained in step (2) is added to a certain amount of the sample solution obtained in step (1) for stirring and adsorption. After the adsorption is completed, the solution is filtered. Then, a desorbent is added to the filtrate for stirring and desorption. The desorbed solution is collected, filtered, concentrated, and dried in sequence to obtain an olive leaf extract for preventing and treating non-alcoholic fatty liver disease.

2. The method according to claim 1, characterized in that: In step (1), the extraction solvent is an ethanol aqueous solution with a mass fraction of 10% to 90%, the solid-liquid ratio of olive leaves to the extractant is 1:3 to 1:30, and the reflux extraction time is 1 to 5 hours each time; the filtration is performed using a 10 to 100 mesh filter; the concentration is performed using a rotary evaporator at a temperature of 20 to 80° C. and a pressure of 5 to 30 kPa, and the concentration is concentrated to a concentration of oleuropein of 0.1 to 15 mg / mL.

3. The method according to claim 1, characterized in that In step (2), the surfactant is sodium dodecyl sulfate, the mass concentration of the solute in the deionized water containing the surfactant is 0.1% to 5%; the swellable polymer seed microspheres are polystyrene microspheres with a particle size of 1 to 50 μm; and the total volume of the deionized water containing the surfactant is 50 to 1000 times the mass of the swellable polymer seed microspheres.

4. The method according to claim 1, characterized in that: In step (2), the swelling agent is any one of dibutyl phthalate, 1-chlorododecane, etc., and the mass ratio of the swelling agent to the swellable polymer seed microspheres is 0.1~10:1; the volume ratio of the swelling agent to the deionized water containing the surfactant is 1:50~1000, and the emulsification method when the two are emulsified is ultrasonic emulsification, the emulsification time is 10~60min, and the power is 100~750W; when solution A and solution B are swelled, the swelling conditions are: temperature 10~50℃, stirring speed 50~300rpm, and time 6~72h.

5. The method according to claim 1, characterized in that: In step (2), the polymerizable monomer is styrene; the cross-linking agent is any one of divinylbenzene, ethylene glycol dimethacrylate, etc.; the nitrogen-containing polymerizable monomer is any one of acrylamide, vinylaniline, vinylimidazole, vinylpyrrolidone, etc.; the porogen is any one of toluene, xylene, n-heptane, etc.; the initiator is any one of azobisisobutyronitrile, benzoyl peroxide, etc., wherein the polymerizable monomer: cross-linking agent: nitrogen-containing polymerizable monomer = 1: (0.1~10): (0.1~10); the mass of the porogen is 0.1~10 times the total mass of the polymerizable monomer, the cross-linking agent and the nitrogen-containing polymerizable monomer, and the mass of the initiator is 0.1%~5% of the total mass of the polymerizable monomer, the cross-linking agent and the nitrogen-containing polymerizable monomer.

6. The method according to claim 1, characterized in that In step (2), the polymer monomer, cross-linking agent, nitrogen-containing polymer monomer, porogen and initiator are mixed and dissolved, and then emulsified in deionized water containing a surfactant, wherein the mass of the deionized water containing a surfactant is 1 to 20 times the total mass of the polymer monomer, the cross-linking agent and the nitrogen-containing polymer monomer, and the emulsification method is ultrasonic emulsification, the emulsification time is 10 to 60 minutes, and the power is 100 to 750W; solution C is added to the reactor to continue swelling, and the mass ratio of the total mass of the polymer monomer, the cross-linking agent and the nitrogen-containing polymer monomer in solution C to the swellable polymer seed microspheres is (10 to 50): 1, and the swelling conditions are: temperature 10 to 50°C, stirring speed 50 to 300rpm, and time 6 to 72h.

7. The method according to claim 1, characterized in that In step (2), the stabilizer is any one of polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, etc., and the mass concentration of the solute in the deionized water solution containing the stabilizer is 0.1%~5%; the mass of the deionized water solution containing the stabilizer is 0.1~20 times the total mass of the polymer monomer, the cross-linking agent and the nitrogen-containing polymer monomer; the reaction temperature is 50~100°C, the stirring speed is 100~1000rpm, and the reaction time is 6~72h; the filtration is performed using an 800-mesh filter; the washing is first washed with ethanol for 1~5 times, and then washed with deionized water for 1~5 times to obtain a separated material.

8. The method according to claim 1, characterized in that In step (3), the ratio of the mass of the separation material to the volume of the sample solution is 1: (1-100), wherein the separation material is measured in grams (wet weight) and the sample solution is measured in milliliters. The stirring adsorption is mechanical stirring, the stirring speed is 10-500 rpm, the adsorption temperature is 5-50°C, the adsorption time is 5-500 min, and the filtration after the adsorption is completed is carried out using an 800-mesh filter. The desorbent is an ethanol solution with a mass fraction of 10%-90%, the stirring desorption is mechanical stirring, the stirring speed is 10-500 rpm, the desorption temperature is 5-50°C, and the desorption time is 5-500 min. The filtration after desorption is carried out using an 800-mesh filter, and the desorption liquid is concentrated by a rotary evaporator at a concentration temperature of 20-80°C and a pressure of 5-30 kPa to a paste. The drying is carried out in a vacuum drying oven at a drying temperature of 20-80°C and a drying pressure of 2-40 kPa to a water content of less than 5%.

9. An extract for preventing and treating nonalcoholic fatty liver disease prepared by the method according to any one of claims 1 to 8, characterized in that: The content of oleuropein is 60%~98%.

10. Use of the extract for preventing and treating non-alcoholic fatty hepatitis according to claim 9 in the preparation of a drug for preventing and treating non-alcoholic fatty hepatitis.

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