Mulberry leaf polyphenol-silk fibroin microcapsule as well as preparation method and application thereof

Abstract

The invention provides a mulberry leaf polyphenol-silk fibroin microcapsule as well as a preparation method and application thereof, and belongs to the technical field of silk fibroin microcapsules. After a silk fibroin solution and template particles are mixed and incubated, methanol is used for treatment, conversion of silk fibroin conformation to beta-folding can be promoted to achieve a more stable structure, layer-by-layer wrapping is achieved through multiple times of repetition, and therefore a large number of stable and shaped silk fibroin microcapsules can be obtained; the purity of the mulberry leaf polyphenol reaches 70% or above through the extraction and purification mode, and the prepared mulberry leaf polyphenol-silk fibroin micro-capsule can effectively treat hepatic fibrosis of mice.

Description

Technical Field

[0001] The invention relates to the technical field of silk fibroin microcapsules, and in particular to a mulberry leaf polyphenol-silk fibroin microcapsule and a preparation method and application thereof. Background Art

[0002] Liver fibrosis is an abnormal proliferation of connective tissue in the liver common to liver diseases caused by multiple etiologies and chronic stimulation. It is reversible. Early diagnosis and anti-fibrosis treatment may reverse the pathological process. Otherwise, it may develop into cirrhosis or hepatocellular carcinoma, turning into an irreversible process. Mulberry leaves are a dual-purpose resource for medicine and food, and have the effect of clearing the liver and improving eyesight. Polyphenol compounds are present in high content and rich variety in mulberry leaves. They are the main active ingredients of mulberry leaves and are an ideal natural antioxidant and liver protection raw material. However, due to their poor water solubility, short circulation time in the body, low absorption rate, lack of targeting and other problems, their application is greatly limited. At present, domestic research on technologies for improving the bioavailability of mulberry leaf polyphenols is very limited. In addition, the mulberry leaf polyphenol extracts obtained by the prior art have the problem of low purity.

[0003] New drug carriers based on nanomaterials can improve the blood solubility and stability of drugs, enhance therapeutic targeting and bioavailability. As a natural protein, silk fibroin has good biocompatibility and low immunogenicity, and is an excellent drug carrier material. Silk hollow microspheres or hollow microcapsules have attracted widespread attention because their internal hollow structure can provide a huge drug-loading space and the outer structure can more effectively protect the drug. At present, the method for preparing silk fibroin microcapsules is still mainly based on the template method combined with layer-by-layer self-assembly technology, using organic or inorganic substances as soft / hard templates, and through weak interactions (such as electrostatic attraction, hydrogen bonds, coordination bonds, etc.), silk fibroin is alternately deposited layer by layer, and molecular aggregates are formed after repeated repetitions. Finally, the template is removed to obtain silk fibroin hollow microcapsules.

[0004] The invention patent with application number "201910558742.9" discloses a silk drug-loaded nano-microcapsule, which uses silk protein and 3-aminopropyltriethoxysilane to alternately coat functionally modified polystyrene microspheres layer by layer, and finally obtains a multilayer structure of coated polystyrene complexes with negative or positive potentials, and finally uses a template removal agent to remove the functionally modified polystyrene template to obtain a silk drug-loaded nano-microcapsule. The silk protein nano-microcapsule coating prepared by the microcapsule preparation method described in the patent is thin and not obvious, and it is difficult to obtain a large number of microcapsules after removing the template. Summary of the invention

[0005] The purpose of the present invention is to provide a mulberry leaf polyphenol-silk fibroin microcapsule and a preparation method and application thereof, so as to solve the defects of the prior art that the silk fibroin microcapsule coating is relatively thin and it is difficult to obtain a large number of microcapsules after removing the template, and at the same time solve the problem of low purity of mulberry leaf polyphenol extract.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a silk fibroin drug-loaded microcapsule, comprising the following steps:

[0008] S1. mixing the silk fibroin solution with the template microparticles, incubating at 3-5° C. for 30-40 min, washing after incubation, and treating with a methanol solution to obtain first microparticles;

[0009] S2. mixing the silk fibroin solution with the first microparticles, incubating at 3-5° C. for 15-20 min, washing after incubation, and treating with a methanol solution to obtain second microparticles;

[0010] S3. Repeat step (2) 5 to 6 times;

[0011] S4. The microparticles obtained in step (3) are mixed with a template removal reagent, incubated for 24 to 28 hours, and then centrifuged to obtain a precipitate, which is a silk fibroin microcapsule;

[0012] S5. Mix the silk fibroin microcapsule solution with the mulberry leaf polyphenol extract solution, incubate with shaking at 35-40°C for 22-26 hours, collect the supernatant after centrifugation; mix the supernatant with the mulberry leaf polyphenol extract solution again, incubate with shaking, repeat 3-4 times, and the resulting precipitate is the silk fibroin drug-loaded microcapsule.

[0013] Preferably, the template microparticles include polystyrene microparticles, and the de-template agent includes N,N-dimethylformamide.

[0014] Preferably, the concentration of the silk fibroin solution is 5-10 mg / mL; the volume mass ratio of the silk fibroin solution to the template particles is 1-3 mL:1 g.

[0015] Preferably, the methanol solution treatment time is 10 to 15 minutes.

[0016] Preferably, the extraction of the mulberry leaf polyphenol extract comprises the following steps:

[0017] (1) mixing mulberry leaf powder with an ethanol solution, and performing ultrasonic extraction at 50 to 60° C. for 30 to 45 minutes to obtain a mulberry leaf polyphenol extract;

[0018] (2) subjecting the mulberry leaf polyphenol extract to rotary evaporation concentration to obtain a mulberry leaf polyphenol concentrate;

[0019] (3) diluting the mulberry leaf polyphenol concentrate by 3 to 4 times for chromatography elution, and collecting the eluate;

[0020] (4) The eluate is concentrated by rotary evaporation and then freeze-dried in vacuum to obtain a mulberry leaf polyphenol extract.

[0021] Preferably, the mass volume ratio of the mulberry leaf powder to the ethanol solution is 50-100 g / L, and the concentration of the ethanol solution is 65%-75%.

[0022] Preferably, during chromatography elution, the concentration of the mulberry leaf polyphenol loading solution is 1 to 1.5 mg / mL, the loading flow rate is 2 to 3 mL / min, and the loading amount is 8 to 10 times the column bed volume.

[0023] Preferably, the concentrations of the silk fibroin microcapsule solution and the mulberry leaf polyphenol extract solution in S5 are independently 0.4-0.6 mg / mL, and the volume ratio of the silk fibroin microcapsule solution to the mulberry leaf polyphenol extract solution is 1:1-3.

[0024] The invention also provides mulberry leaf polyphenol-silk fibroin microcapsules prepared by the method.

[0025] The invention provides application of the mulberry leaf polyphenol-silk fibroin microcapsule in preparing medicine for preventing and treating liver fibrosis.

[0026] By adopting the above technical scheme, the present invention has the following beneficial effects: after the silk fibroin solution is mixed and incubated with the template particles, the silk fibroin is treated with methanol, which can promote the transformation of the silk fibroin conformation to β-folding to achieve a more stable structure, and layer-by-layer wrapping is achieved through repeated multiple times, so that a large number of stable and well-shaped silk fibroin microcapsules can be obtained, and the extraction and purification method of the present invention can make the purity of mulberry leaf polyphenol reach more than 70%, and the mulberry leaf polyphenol-silk fibroin microcapsules prepared by the present invention can effectively treat mouse liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The scanning electron microscope morphology images of polystyrene microspheres, silk-polystyrene microspheres and silk microcapsules, among which A is polystyrene microspheres; B is silk-polystyrene microspheres; and C is silk microcapsules.

[0028] Figure 2 This is a picture of mulberry leaf polyphenol microcapsule powder.

[0029] Figure 3 This is a scanning electron microscope image of mulberry leaf polyphenol-silk fibroin microcapsule; A is a scale of 200 μm; B is a scale of 100 μm.

[0030] Figure 4 This is a comparison of Masson staining results of liver tissue.

[0031] Figure 5This is a comparison chart of the expression results of α-SMA protein in liver tissue. DETAILED DESCRIPTION

[0032] The present invention provides a method for preparing a silk fibroin drug-loaded microcapsule, comprising the following steps:

[0033] S1. mixing the silk fibroin solution with the template microparticles, incubating at 3-5° C. for 30-40 min, washing and treating with a methanol solution to obtain first microparticles;

[0034] S2. mixing the silk fibroin solution with the first microparticles, incubating at 3-5° C. for 15-20 min, washing and treating with a methanol solution to obtain second microparticles;

[0035] S3. Repeat step (2) 5 to 6 times;

[0036] S4. The microparticles obtained in step (3) are mixed with a template removal reagent, incubated for 24 to 28 hours, and then centrifuged to obtain a precipitate, which is a silk fibroin microcapsule;

[0037] S5. Mix the silk fibroin microcapsule solution with the mulberry leaf polyphenol extract solution, incubate with shaking at 35-40°C for 22-26 hours, collect the supernatant after centrifugation; mix the supernatant with the mulberry leaf polyphenol extract solution again, incubate with shaking, repeat 3-4 times, and the resulting precipitate is the silk fibroin drug-loaded microcapsule.

[0038] The present invention dissolves silk fibroin in water to prepare a silk fibroin solution, then mixes the silk fibroin solution with template microparticles and places them for incubation, then washes, and treats them with a methanol solution to obtain first microparticles. The concentration of the silk fibroin solution is 5 to 10 mg / mL, more preferably 6 to 9 mg / mL, and more preferably 8 mg / mL; the volume mass ratio of the silk fibroin solution to the template microparticles is 1 to 3 mL:1 g, more preferably 1.5 to 2.5 mL:1 g, and more preferably 2 mL:1 g; the washing is preferably performed using distilled water, and the template microparticles include polystyrene microparticles. The incubation temperature is 3-5°C, more preferably 3.5-4.5°C, and more preferably 4°C; the incubation time is 30-40min, more preferably 33-38min, and more preferably 35min; after the incubation, the template particles are treated with methanol, and the volume concentration of the methanol solution is 80-90%, more preferably 82-88%, and more preferably 85%; the treatment time is 10-15min, more preferably 12-14min, and more preferably 13min; after treatment with the methanol solution, a layer of silk fibroin-coated polystyrene silk fibroin micron particles are obtained, which are the first particles.

[0039] The present invention mixes a silk fibroin solution with first microparticles for incubation, the ratio of the silk fibroin solution to the first microparticles being the same as the ratio of the silk fibroin solution to the template microparticles, and the microparticles are washed and treated with a methanol solution to obtain second microparticles; the mixing method and incubation temperature are the same as those in the previous step; the incubation time is 15 to 20 minutes, more preferably 16 to 19 minutes, and more preferably 18 minutes; the methanol solution treatment is the same as in the previous step; and this step is repeated 5 to 6 times.

[0040] The microparticles obtained in the above steps are mixed with a de-template reagent, centrifuged after incubation, and the precipitate is obtained to obtain a silk fibroin microcapsule; the de-template reagent includes N,N-dimethylformamide. The incubation is preferably a shaking incubation, and the incubation time is 24 to 28 hours, more preferably 25 to 27 hours, and more preferably 26 hours. Centrifuge after incubation, the centrifuge speed is 11000 to 13000 rpm, more preferably 11500 to 12500 rpm, and more preferably 12000 rpm; the centrifuge time is 10 to 15 minutes, more preferably 12 to 14 minutes, and more preferably 13 minutes; after centrifugation, the supernatant is removed, the precipitate is taken, and washed 2 to 3 times to obtain a silk fibroin microcapsule; the washing is preferably done with deionized water.

[0041] The extraction of the mulberry leaf polyphenol extract of the present invention comprises the following steps:

[0042] (1) mixing mulberry leaf powder with an ethanol solution, and performing ultrasonic extraction at 50 to 60° C. for 30 to 45 minutes to obtain a mulberry leaf polyphenol extract;

[0043] (2) subjecting the mulberry leaf polyphenol extract to rotary evaporation concentration to obtain a mulberry leaf polyphenol concentrate;

[0044] (3) diluting the mulberry leaf polyphenol concentrate by 3 to 4 times for chromatography elution, and collecting the eluate;

[0045] (4) The eluate is concentrated by rotary evaporation and then freeze-dried in vacuum to obtain a mulberry leaf polyphenol extract.

[0046] The present invention first mixes mulberry leaf powder with an ethanol solution, performs ultrasonic extraction, and obtains a mulberry leaf polyphenol extract; the volume concentration of the ethanol solution is 65% to 75%, more preferably 68 to 72%, and more preferably 70%; the mass volume ratio of the mulberry leaf powder to the ethanol solution is 50 to 100 g / L, more preferably 60 to 90 g / L, and more preferably 80 g / L; the extraction temperature is 50 to 60° C., more preferably 53 to 57° C., and more preferably 55° C.; the extraction time is 30 to 45 min, more preferably 35 to 45 min, and more preferably 40 min.

[0047] The present invention performs rotary evaporation concentration on the mulberry leaf polyphenol extract to remove ethanol and obtain a mulberry leaf polyphenol concentrated solution. The rotary evaporation concentration temperature is 40-45°C, the rotary evaporation concentration speed is 70-80rpm, the rotary evaporation concentration vacuum is -0.095--0.075MPa, and the rotary evaporation to the mulberry leaf polyphenol concentrated solution has a volume of 1 / 4-1 / 5 of the original extract volume.

[0048] The invention dilutes the mulberry leaf polyphenol concentrated solution and then performs chromatography, collects the eluate, and then places the eluate in a rotary evaporator to concentrate and remove ethanol, and performs vacuum freeze drying to obtain the mulberry leaf polyphenol extract. The concentration of the diluted leaf polyphenol concentrate (loading solution) is 1-1.5 mg / mL, more preferably 1.2-1.4 mg / mL, and more preferably 1.3 mg / mL; the loading flow rate is 2-4 mL / min, more preferably 2.5-3.5 mL / min, and more preferably 3 mL / min; the loading amount is 8-10 times the column bed volume, and more preferably 9 times; after the adsorption is completed, first use 1-1.5 times the column volume of distilled water to elute to remove some impurities, and then use ethanol solution for elution, the volume concentration of the ethanol solution is 60-70%, more preferably 63-67%, and more preferably 65%; the elution flow rate of the ethanol solution is 2.5-3.5 mL / min, more preferably 2.7-3.2 mL / min, and more preferably 3.0 mL / min, and the eluent volume is 5-8 times the column bed volume, and more preferably 6 times. The eluate is collected, and then the eluate is placed in a rotary evaporator and concentrated until there is no alcohol taste, and then vacuum freeze-dried to obtain the mulberry leaf polyphenol extract. The temperature of the rotary evaporation concentration is 40 to 45°C, the rotation speed of the rotary evaporation concentration is 70 to 80 rpm, and the vacuum degree of the rotary evaporation concentration is -0.095 to -0.075 MPa; the present invention has no special restrictions on the parameters of vacuum freeze drying, and can obtain a dry mulberry leaf polyphenol extract.

[0049] The method comprises preparing a solution of prepared silk fibroin microcapsules and mulberry leaf polyphenol extract, mixing the solution, incubating the solution together, and then centrifuging the solution. The supernatant is collected and then added into the mulberry leaf polyphenol extract solution for incubation. The process is repeated 3 to 4 times. The obtained precipitate is vacuum freeze-dried to obtain mulberry leaf polyphenol-silk fibroin microcapsules. In the present invention, the concentrations of the silk fibroin microcapsule solution and the mulberry leaf polyphenol extract solution are independently 0.4-0.6 mg / mL, more preferably 0.45-0.55 mg / mL, and more preferably 0.5 mg / mL; the volume of the mulberry leaf polyphenol extract solution added each time is 1-1.5 times, more preferably 1-1.2 times, and more preferably 1 times of the silk fibroin microcapsule solution; the incubation temperature is 35-40°C, more preferably 36-38°C, and more preferably 37°C; the incubation is preferably carried out in a constant temperature oscillator, the oscillation speed is 100-150 rpm, and the oscillation incubation time is 22-26 h, more preferably 23-25 ​​h, and more preferably 24 h; the centrifugal speed is 14000-16000 rpm, more preferably 14500-15500 rpm, and more preferably 15000 rpm, and the centrifugal time is 10-15 min, more preferably 12-14 min, and more preferably 13 min.

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1

[0052] (1) Preparation of silk fibroin microcapsules

[0053] Silk fibroin was dissolved in water to prepare a 5 mg / mL silk fibroin solution. 50 mL of the silk fibroin solution was mixed with 25 mg of polystyrene micron particles, incubated at 4°C for 30 min, washed with distilled water after incubation, and then treated with 80% by volume methanol for 10 min. After washing with distilled water, the first microparticles were obtained.

[0054] 50 mL of silk fibroin solution was mixed with the first microparticles, incubated at 4°C for 15 min, washed with distilled water and then treated with 80% methanol solution by volume for 10 min, and this step was repeated 6 times. The obtained microparticles were resuspended with N,N-dimethylformamide, incubated with shaking for 24 h, centrifuged at 12000 rpm for 10 min, the supernatant was removed, and the precipitate was obtained, which was then washed twice with deionized water to obtain silk fibroin microcapsules.

[0055] (2) Extraction and purification of mulberry leaf polyphenols

[0056] Weigh 50g of mulberry leaf powder, add 1L of 70% ethanol solution, extract by ultrasonic at 60℃ for 40min, collect the supernatant by centrifugation, and obtain mulberry leaf polyphenol extract. Place the mulberry leaf polyphenol extract in a rotary evaporator to concentrate and remove ethanol to obtain mulberry leaf polyphenol concentrate. Dilute the mulberry leaf polyphenol concentrate with distilled water to a loading solution with a concentration of 1mg / mL, load the pretreated D101 macroporous adsorption resin into a glass chromatography column with a diameter of 1.6cm and a height of 50cm, load mulberry leaf polyphenol into the glass chromatography column at a flow rate of 2mL / min, and the loading amount is 800mL; after the adsorption is completed, first use 100mL of distilled water to elute to remove some impurities, then use 600mL of 60% ethanol solution with a flow rate of 3mL / min to elute, and collect the eluate. Place the eluate in a rotary evaporator to concentrate and remove ethanol, and then vacuum freeze-dry to obtain the mulberry leaf polyphenol extract. The temperature of the above two rotary evaporation concentrations was 40° C., the rotation speed was 70 rpm, the vacuum degree was -0.095 MPa, and the purity of mulberry leaf polyphenols was 80.07% as determined by HPLC.

[0057] (3) Preparation of mulberry leaf polyphenol-silk fibroin microcapsules

[0058] The silk fibroin microcapsules obtained in step (1) and the mulberry leaf polyphenols obtained in step (2) are respectively prepared into solutions with a concentration of 0.5 mg / mL, 100 mL of the silk fibroin microcapsule solution is mixed with 100 mL of the mulberry leaf polyphenols solution, and the mixture is placed in a constant temperature oscillator for co-incubation, and continuously oscillated at 35° C. and 120 rpm for 24 hours, and then centrifuged at 15000 rpm for 10 minutes, the supernatant is collected and then added with 100 mL of the mulberry leaf polyphenols extract solution for co-incubation, and the mixture is repeated 4 times. The obtained precipitate is washed and freeze-dried to obtain mulberry leaf polyphenol-silk fibroin microcapsule powder.

[0059] Example 2

[0060] (1) Preparation of silk fibroin microcapsules

[0061] Silk fibroin was dissolved in water to prepare a silk fibroin solution with a concentration of 8 mg / mL. 50 mL of the silk fibroin solution was mixed with 25 mg of polystyrene micron particles, incubated at 4°C for 35 min, washed with distilled water after incubation, and then treated with methanol with a volume concentration of 85% for 13 min. After washing with distilled water, the first microparticles were obtained.

[0062] The silk fibroin solution was mixed with the first microparticles, incubated at 4°C for 18 minutes, washed with distilled water and then treated with a methanol solution with a volume concentration of 85% for 13 minutes, and this step was repeated 6 times. The obtained microparticles were resuspended with N,N-dimethylformamide, incubated with shaking for 26 hours, centrifuged at 12000 rpm for 12 minutes, the supernatant was removed, and the precipitate was obtained, which was then washed twice with deionized water to obtain silk fibroin microcapsules.

[0063] (2) Extraction and purification of mulberry leaf polyphenols

[0064] Weigh 80g of mulberry leaf powder, add 1L of 70% ethanol solution, extract by ultrasonic at 55℃ for 40min, collect the supernatant by centrifugation, and obtain mulberry leaf polyphenol extract. Place the mulberry leaf polyphenol extract in a rotary evaporator to concentrate and remove ethanol to obtain mulberry leaf polyphenol concentrate. Dilute the mulberry leaf polyphenol concentrate with distilled water to a loading solution with a concentration of 1.3mg / mL, load the pretreated D101 macroporous adsorption resin into a glass chromatography column with a diameter of 1.6cm and a height of 50cm, load mulberry leaf polyphenol into the glass chromatography column at a flow rate of 3mL / min, and the loading amount is 800mL; after the adsorption is completed, first use 100mL of distilled water to elute to remove some impurities, then use 600mL of 65% ethanol solution to elute at a flow rate of 3mL / min, and collect the eluate. Place the eluate in a rotary evaporator to concentrate and remove ethanol, and then vacuum freeze-dry to obtain the mulberry leaf polyphenol extract. The temperature of the above two rotary evaporation concentrations was 42° C., the rotation speed was 75 rpm, the vacuum degree was -0.08 MPa, and the purity of mulberry leaf polyphenols was 81.15% as determined by HPLC.

[0065] (3) Preparation of mulberry leaf polyphenol-silk fibroin microcapsules

[0066] The silk fibroin microcapsules obtained in step (1) and the mulberry leaf polyphenols obtained in step (2) are prepared into solutions with a concentration of 0.5 mg / mL, 100 mL of the silk fibroin microcapsule solution is mixed with 120 mL of the mulberry leaf polyphenols solution, and the mixture is placed in a constant temperature oscillator for co-incubation, and continuously oscillated at 37° C. and 120 rpm for 24 hours, and then centrifuged at 15000 rpm for 15 minutes, the supernatant is collected and then added with 120 mL of the mulberry leaf polyphenols extract solution for co-incubation, and the mixture is repeated 4 times. The obtained precipitate is washed and freeze-dried to obtain mulberry leaf polyphenol-silk fibroin microcapsule powder.

[0067] Example 3

[0068] (1) Preparation of silk fibroin microcapsules

[0069] Silk fibroin was dissolved in water to prepare a 10 mg / mL silk fibroin solution. 50 mL of the silk fibroin solution was mixed with 25 mg of polystyrene micron particles, incubated at 4°C for 40 min, washed with distilled water after incubation, and then treated with 90% by volume methanol for 15 min. After washing with distilled water, the first microparticles were obtained.

[0070] The silk fibroin solution was mixed with the first microparticles, incubated at 4°C for 20 minutes, washed with distilled water and then treated with a 90% volume concentration methanol solution for 15 minutes, and this step was repeated 6 times. The obtained microparticles were resuspended with N,N-dimethylformamide, incubated with shaking for 28 hours, centrifuged at 12000 rpm for 15 minutes, the supernatant was removed, and the precipitate was obtained, which was then washed twice with deionized water to obtain silk fibroin microcapsules.

[0071] (2) Extraction and purification of mulberry leaf polyphenols

[0072] Weigh 100g of mulberry leaf powder, add 1L of 75% ethanol solution, extract by ultrasonic at 60℃ for 45min, collect the supernatant by centrifugation, and obtain mulberry leaf polyphenol extract. Place the mulberry leaf polyphenol extract in a rotary evaporator to concentrate and remove ethanol to obtain mulberry leaf polyphenol concentrate. Dilute the mulberry leaf polyphenol concentrate with distilled water to a loading solution with a concentration of 1.5mg / mL, load the pretreated D101 macroporous adsorption resin into a glass chromatography column with a diameter of 1.6cm and a height of 50cm, load mulberry leaf polyphenol into the glass chromatography column at a flow rate of 3mL / min, and the loading amount is 800mL; after the adsorption is completed, first use 100mL of distilled water to elute to remove some impurities, then use 600mL of 70% ethanol solution with a flow rate of 3.5mL / min to elute, and collect the eluate. Place the eluate in a rotary evaporator to concentrate and remove ethanol, and then vacuum freeze-dry to obtain the mulberry leaf polyphenol extract. The temperature of the above two rotary evaporation concentrations was 45° C., the rotation speed was 80 rpm, the vacuum degree was -0.075 MPa, and the purity of mulberry leaf polyphenols was 72.17% as determined by HPLC.

[0073] (3) Preparation of mulberry leaf polyphenol-silk fibroin microcapsules

[0074] The silk fibroin microcapsules obtained in step (1) and the mulberry leaf polyphenols obtained in step (2) were prepared into solutions with a concentration of 0.5 mg / mL, 100 mL of the silk fibroin microcapsule solution was mixed with 150 mL of the mulberry leaf polyphenols solution, and the mixture was placed in a constant temperature oscillator for co-incubation, and continuously oscillated at 35° C. and 120 rpm for 24 hours, and then centrifuged at 15000 rpm for 10 minutes, the supernatant was collected and then added with 150 mL of the mulberry leaf polyphenols extract solution for co-incubation, and the mixture was repeated 4 times. The obtained precipitate was washed and freeze-dried to obtain mulberry leaf polyphenol-silk fibroin microcapsule powder.

[0075] Experimental Example 1

[0076] The polystyrene microparticles, polystyrene microspheres wrapped with silk fibroin, silk fibroin microcapsules and mulberry leaf polyphenol-silk fibroin microcapsules prepared in Example 2 were observed under a scanning electron microscope. Figure 1 , Figure 3 As shown. Figure 1It can be seen that the silk coating on the polystyrene microspheres is thicker before removing the template ( Figure 1 B), the morphology of the silk microcapsules obtained after removing the template is better ( Figure 1 C in Figure 3 It can be seen that the mulberry leaf polyphenol-silk fibroin micronized microcapsules prepared by the present invention are uniform and regular.

[0077] Experimental Example 2 Study on the anti-liver fibrosis effect of mulberry leaf polyphenol-silk fibroin microcapsules

[0078] 54 SPF male Kunming mice weighing 30-40g (purchased from Sibeifu Biotechnology Co., Ltd.) were randomly divided into 9 groups, namely normal group, model group, colchicine group, mulberry leaf polyphenol-silk protein microcapsule low-dose group (prepared in Example 2), mulberry leaf polyphenol-silk protein microcapsule medium-dose group, mulberry leaf polyphenol-silk protein microcapsule high-dose group (mulberry leaf polyphenol-silk protein microcapsule was prepared according to the method in Example 2), 6 mice in each group. Mouse liver fibrosis model was prepared by intraperitoneal injection of carbon tetrachloride. Except for the normal group mice intraperitoneally injected with olive oil, the mice in the other groups were intraperitoneally injected with 10% CCl4 olive oil solution (5mL / kg), twice a week for 8 consecutive weeks, to establish the mouse liver fibrosis model. From the third week onwards, the low-dose, medium-dose and high-dose groups of mulberry leaf polyphenol-silk protein microcapsules were gavaged at doses of 75 mg / kg, 150 mg / kg and 300 mg / kg, respectively. The mice in the normal group and the model group were gavaged with 150 mg / kg of distilled water once a day for 6 consecutive weeks. 10 hours after the last administration, the eyeballs of all mice were removed to collect blood, and the serum was separated. The ALT, AST and HA levels in their serum were detected using a kit. After the blood collection was completed, the mice were killed by cervical dislocation, and the liver was quickly dissected and separated to remove the surrounding fat. The liver was rinsed with saline until it changed color to remove residual red blood cells as much as possible. Tissue blocks of appropriate size were cut and fixed with 40 g / L paraformaldehyde for immunohistochemical staining to detect the expression of α-SAM in liver tissue and Masson staining to observe liver fibrosis. The results are shown in Table 1. Figure 4 and Figure 5 shown.

[0079] Table 1 Serum ALT, AST and HA levels of mice in each group

[0080]

[0081] Note: *, significant difference compared with the normal group (P<0.05); #, significant difference compared with the model group (P<0.05); the same below.

[0082] As shown in Table 1, the levels of ALT, AST and HA in the model group were significantly increased (P < 0.05); compared with the liver fibrosis group, the serum ALT, AST and HA levels in the mulberry leaf polyphenol-silk fibroin microcapsule group were significantly decreased (P < 0.05), indicating that mulberry leaf polyphenol-silk fibroin microcapsule can reduce the degree of liver damage in mice.

[0083] Figure 4 The results showed that the liver structure of the normal group was normal, and no obvious fibrous tissue hyperplasia and hepatic lobule formation were observed; a large number of proliferative collagen fibers were observed in the model group, and the hepatic lobule structure was severely damaged. Compared with the normal group, the percentage of Masson positive staining area of ​​the liver tissue in the model group was significantly increased (P < 0.05); the deposition of collagen fibers and fibrous hyperplasia in the liver of the microcapsule group was reduced compared with the model group, and the fibrous interval was smaller than that of the liver fibrosis group.

[0084] Figure 5 The results showed that compared with the normal group, the percentage of α-SAM positive staining area in liver tissue of the liver fibrosis group was increased (P<0.05); compared with the liver fibrosis group, the percentage of α-SAM positive staining area in liver tissue of the microcapsule group was decreased (P<0.05).

[0085] It can be seen from the above embodiments that the present invention provides a mulberry leaf polyphenol-silk fibroin microcapsule and a preparation method and application thereof. The mulberry leaf polyphenol-silk fibroin microcapsule of the present invention can effectively prevent and treat liver fibrosis in mice.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing mulberry leaf polyphenol-silk fibroin microcapsules, characterized in that: The following steps are involved: S1. mixing the silk fibroin solution with the template microparticles, incubating at 3-5° C. for 30-40 min, washing after incubation, and treating with a methanol solution to obtain first microparticles; S2. mixing the silk fibroin solution with the first microparticles, incubating at 3-5° C. for 15-20 min, washing after incubation, and treating with a methanol solution to obtain second microparticles; S3. Repeat step (2) 5 to 6 times; S4. The microparticles obtained in step (3) are mixed with a template removal reagent, incubated for 24 to 28 hours, and then centrifuged to obtain a precipitate, which is a silk fibroin microcapsule; S5. Mix the silk fibroin microcapsule solution with the mulberry leaf polyphenol extract solution, incubate with shaking at 35-40°C for 22-26 hours, collect the supernatant after centrifugation; mix the supernatant with the mulberry leaf polyphenol extract solution again, incubate with shaking, repeat 3-4 times, and the resulting precipitate is the silk fibroin drug-loaded microcapsule.

2. The preparation method according to claim 1, characterized in that: The template particles include polystyrene microparticles, and the de-template agent includes N,N-dimethylformamide.

3. The preparation method according to claim 2, characterized in that: The concentration of the silk fibroin solution is 5-10 mg / mL; the volume mass ratio of the silk fibroin solution to the template particles is 1-3 mL:1 g.

4. The preparation method according to claim 1, characterized in that: The methanol solution treatment time is 10 to 15 minutes.

5. The preparation method according to claim 1, characterized in that: The extraction of the mulberry leaf polyphenol extract comprises the following steps: (1) mixing mulberry leaf powder with an ethanol solution, and performing ultrasonic extraction at 50 to 60° C. for 30 to 45 minutes to obtain a mulberry leaf polyphenol extract; (2) subjecting the mulberry leaf polyphenol extract to rotary evaporation concentration to obtain a mulberry leaf polyphenol concentrate; (3) diluting the mulberry leaf polyphenol concentrate by 3 to 4 times for chromatography elution, and collecting the eluate; (4) The eluate is concentrated by rotary evaporation and then freeze-dried in vacuum to obtain a mulberry leaf polyphenol extract.

6. The preparation method according to claim 5, characterized in that: During extraction, the mass volume ratio of the mulberry leaf powder to the ethanol solution is 50-100 g / L, and the concentration of the ethanol solution is 65%-75%.

7. The preparation method according to claim 5, characterized in that: During chromatography elution, the concentration of the mulberry leaf polyphenol loading solution is 1-1.5 mg / mL, the loading flow rate is 2-3 mL / min, and the loading amount is 8-10 times the column bed volume; the eluent is 60-70% ethanol solution, and the eluent volume is 6-8 times the column bed volume.

8. The preparation method according to claim 1, characterized in that: The concentrations of the silk fibroin microcapsule solution and the mulberry leaf polyphenol extract solution in S5 are independently 0.4-0.6 mg / mL, and the volume of the mulberry leaf polyphenol extract solution is 1-1.5 times that of the silk fibroin microcapsule solution.

9. Mulberry leaf polyphenol-silk fibroin microcapsules prepared by any one of the methods of claims 1 to 8.

10. Use of the mulberry leaf polyphenol-silk fibroin microcapsule according to claim 9 in the preparation of a drug for preventing and treating liver fibrosis.

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