Epimedium extract liposome prepared by using soluble solid phase particles, preparation method and application thereof

Abstract

The application provides a liposome of an extract of Epimedium herb prepared by using soluble solid-phase particles, a preparation method and application thereof, and belongs to the field of traditional Chinese medicine preparations. First, a wrapping material is added into an anhydrous ethanol solution to prepare a wrapping material solution; then, the soluble solid-phase particles are immersed in the wrapping material solution, and the anhydrous ethanol is removed, so that the soluble solid-phase particles are combined with the wrapping material to obtain empty liposomes. The empty liposomes are mixed with an extract of Epimedium herb solution to obtain an extract of Epimedium herb liposome emulsion. The liposomes prepared by the method have high particle embedding rate, good particle uniformity and high stability. The method provides a reference for the innovation of liposome preparation technology and the preparation of more ideal liposomes, and lays a foundation for the clinical application of icariin and the further application research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine preparations, in particular to a liposome of icariin extract prepared by using soluble solid particles, a preparation method and application thereof. BACKGROUND

[0002] Icariin is a small molecule compound extracted from traditional Chinese medicine Epimedium, which has various pharmacological effects, including reproductive function, neuroprotection, bone protection, cardiovascular protection, anti-tumor, anti-inflammatory, and immune enhancement. In the field of cosmetic applications, icariin also has the effects of skin ultraviolet protection, anti-ultraviolet damage, and anti-aging. Liu Wenjie et al. proved through experiments that icariin can improve the survival ability of fibroblasts, reduce the proportion of senescence-associated β-galactosidase staining positive cells, and up-regulate the synthesis of extracellular matrix and down-regulate the expression of senescence-associated cyclin-dependent kinase inhibitors, thereby reducing anti-aging and resisting ultraviolet damage by reducing related cell apoptosis caused by ultraviolet rays. Icariin has a wide range of applications in the pharmaceutical and cosmetic industries. However, icariin has low oral bioavailability and low solubility in water, which to some extent affects the clinical application of icariin.

[0003] Nanometer liposomes can successfully encapsulate icariin, improving its stability and bioavailability. The hydrophilic environment inside the liposome can load hydrophilic drugs, while the phospholipid molecular layer constituting the liposome can load lipophilic drugs, which can isolate the drug from external air and other influences, improve its stability, and prolong its storage time. Moreover, the small particle size of the liposome is similar to the structure of the cell membrane, which can improve the bioavailability of the drug. Currently, liposomes are mainly prepared by organic solvent injection method, thin film dispersion method, reverse evaporation method, etc. Zhang Xinyue et al. used the thin film dispersion method and ammonium sulfate hydration method to prepare icariin liposomes modified by Angiopep-2, and the results showed that the icariin encapsulation rate was about 92%, the drug encapsulation rate was high, and the liposomes had good active targeting property (Modern Chinese Medicine Research and Practice, 2022, No. 2, pp. 57-62). Lu Ruitao et al. prepared icariin-loaded liposomes and optimized the process, observed the appearance and shape, and investigated the stability. The results showed that the icariin-loaded liposomes had ideal microstructure and suitable particle size distribution, and excellent stability. Sun Xiaoduan prepared targeted liposome BBL by thin film dispersion method, and the particle size of the targeted liposome BBL was less than 200 nm and the polydispersity coefficient was about 0.19, indicating that the prepared liposome had uniform particle size. The encapsulation rate was about 78.6% measured by liquid chromatography (Southwest Medical University, 2018, Master's Thesis). However, the current liposome preparation generally has complex preparation steps and uses a large amount of organic solvent, which seriously affects the clinical application and development of icariin. SUMMARY

[0004] The present application provides a liposome of icariin extract prepared by using soluble solid particles, a preparation method thereof and application thereof.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0006] A blank liposome, which comprises soluble solid particles and a wrapping material, the soluble solid particles are selected from one of sucrose or sodium chloride, and the wrapping material is lecithin or a mixture of lecithin and cholesterol; the wrapping material is wrapped on the surface of the solid particles in the form of a membrane; and the thickness of the membrane is 2.5-16.7 μm.

[0007] The particle size of the soluble solid particles is 150-1250 μm, preferably 500-800 μm, and further preferably 600-650 μm. By controlling the particle size of the solid particles, the thickness of the membrane can be controlled, thereby controlling the drug loading capacity.

[0008] The blank liposome provided by the present application has the following advantages compared with the prior art:

[0009] 1) The precursor liposome is prepared by mixing the drug and the membrane material into solid particles, and the preparation process of the present application is simpler, and the drug does not need to be dissolved in an organic solvent.

[0010] 2) The present application does not affect the stability of the drug. The precursor liposome is prepared by dissolving the film-forming material and the drug into an organic solvent solution, and then evaporating the organic solvent to coat the film-forming material and the drug on the solid particles. This process may affect the stability of the drug components due to the heat and long exposure time, and may cause the residue of toxic and harmful organic solvents. The present application is to dissolve the film-forming material into an organic solvent (using anhydrous ethanol) solution, and then coat it on the solid particles, which will not cause the residue of toxic and harmful organic solvents, and will not affect the stability of the drug.

[0011] 3) The liposome can be prepared on demand, and the drug-loaded liposome can be obtained by mixing the blank liposome and the aqueous drug solution. The application is more widely used. The precursor liposome is packaged in the form of a solid preparation after preparation, and the storage and transportation need to consider the properties of the drug components. The blank liposome of the present application is packaged in the form of a solid raw material after preparation, and can be used for on-demand preparation of various drug components due to the absence of drug components.

[0012] The present application also provides a preparation method of the above-mentioned blank liposome,

[0013] (1) adding the wrapping material into an anhydrous ethanol solution to prepare a wrapping material solution;

[0014] (2) immerse the soluble solid phase particles in the solution of the coating material, remove the anhydrous ethanol, and combine the soluble solid phase particles with the coating material to obtain the empty liposome.

[0015] Preferably, in step (1), the coating material is one of lecithin, a mixture of lecithin and cholesterol; the mass ratio of the mixture of lecithin and cholesterol is 5-7:1.

[0016] Preferably, the mass concentration of the coating material in the coating solution is 0.0189-0.2 g / mL; preferably 0.15-0.2 g / mL. The coating material is calculated based on lecithin.

[0017] Preferably, in step (2), the mass ratio of the soluble solid phase particles to the coating material is (2.08-26.45):1.

[0018] The soluble solid phase particles are NaCl, the particle size is 600-700 μm, preferably the average particle size is 650 μm, and the particle size is between 25-30 mesh. The mass ratio of the NaCl to the coating material is (2.08-4.17):1.

[0019] The soluble solid phase particles are sucrose, the particle size is 550-650 μm, preferably the average particle size is 600 μm, and the particle size is between 25-35 mesh. The mass ratio of the sucrose to the coating material is (2.08-26.45):1; preferably (2.08-4.17):1.

[0020] Preferably, in step (2), the method for removing the anhydrous ethanol includes one of water bath evaporation, blowing dry, and standing at room temperature. The water bath evaporation includes normal pressure water bath evaporation and reduced pressure water bath evaporation.

[0021] Preferably, in step (b), the coating material solution is added dropwise while stirring, so that the coating material is uniformly adhered to the surface of the solid phase particles.

[0022] An object of the present application is to provide a liposome of Epimedium extract, which comprises 0.0149-0.22% of Epimedium extract, 0.05-2.2% of coating material, 0.47-7.31% of soluble solid phase particles, and water.

[0023] The main component of the Epimedium extract is icariin, and the mass percentage of icariin in the extract is more than 30.45%.

[0024] Preferably, the encapsulation efficiency of the Epimedium liposome is 100%. When the solid phase particles are sucrose, the encapsulation efficiency is 84-100%, preferably 100%.

[0025] Preferably, the transdermal diffusion experiment of the said Epimedium liposome, when the solid phase particle is NaCl, the 48h permeation rate is not less than 50%; when the solid phase particle is sucrose, the 48h permeation rate is not less than 23.6%.

[0026] Preferably, the solid phase particle is NaCl, the particle size of the said Epimedium liposome is 117.8-172.5nm, the polydispersity index (PDI) is 0.307-0.482, and the average potential is -28.9--44.5mV. The solid phase particle is sucrose, the particle size of the said Epimedium liposome is 116.83-197.9nm, the polydispersity index (PDI) is 0.248-0.368, and the average potential is -41.7--54.4mV.

[0027] The present application also provides a method for preparing Epimedium extract liposome by using soluble solid phase particle, mixing empty liposome with Epimedium extract solution to obtain Epimedium extract liposome emulsion.

[0028] Preferably, the mixing method of the said empty liposome and Epimedium extract solution comprises vortexing, shaking, and ultrasonic.

[0029] Preferably, the mass ratio of Epimedium extract to wrapping material is (7.4-27.8):100; the solvent of the said Epimedium extract solution is water, and the concentration of the Epimedium extract solution is 0.15-2.4mg / mL.

[0030] When the solid phase particle is NaCl, the mass ratio of Epimedium extract to wrapping material is (7.4-12.4):100; when the solid phase particle is sucrose, the mass ratio of Epimedium extract to wrapping material is (7.4-27.8):100.

[0031] Preferably, the above preparation method further comprises a particle size control method, i.e. a whole particle method, which refers to the method of shearing, ultrasonic, homogenizing, and extruding the obtained Epimedium extract liposome at high speed, preferably extruding, and more preferably microfiltration extruding.

[0032] The present application also provides the application of the said Epimedium extract liposome and the Epimedium extract liposome prepared by the above method in the preparation of anti-osteoporosis drugs.

[0033] When the soluble solid phase particle is sucrose, the said Epimedium extract liposome can be prepared into oral liquid dosage form.

[0034] When the soluble solid phase particle is sodium chloride, the said Epimedium extract liposome can be prepared into injection dosage form.

[0035] The application also provides application of the above-mentioned liposome of the Epimedium extract and the liposome of the Epimedium extract prepared by the above-mentioned method in preparing cosmetics. Preferably, the liposome is used in preparing anti-ultraviolet cosmetics. The type of the cosmetic can be liquid or paste.

[0036] Advantages of the application:

[0037] 1. The application utilizes the large specific surface area of the solid phase particles, and forms a thin film of the wrapping material with large specific surface area by volatilizing the solution of the wrapping material on the surface of the soluble solid phase particles, so as to obtain the empty liposome. The empty liposome provided by the application is convenient for transportation and storage, and is convenient for use. The water solution of the loaded drug is mixed with the empty liposome uniformly, so as to obtain the drug-loaded liposome.

[0038] 2. The application utilizes the spherical structure of the soluble solid phase particles and the easy water-solubility, and for the first time, a new liposome preparation method is adopted to embed the Epimedium into the liposome. Since the large specific surface area of the solid phase particles is utilized, the thickness of the obtained thin film of the wrapping material is small, so that the required wrapping material is low, and the amount of the used anhydrous ethanol is greatly reduced. Compared with the prior art, the amount of the used ethanol is reduced by about 90%. The application constructs a new liposome preparation process which is simple in process, low in organic solvent consumption, simple in preparation method and green and pollution-free, and solves the problems of the prior art, such as complex preparation process, large amount of organic solvent and difficult scale production.

[0039] 3. The liposome particles prepared by the method of the application have high embedding rate, good particle uniformity and high stability. The application provides a reference for the innovation of the liposome preparation technology and the preparation of more ideal liposomes, and lays a foundation for the clinical application of icariin and the further application research.

[0040] 4. The prior art loads the drug and the lipids on the soluble solid phase particles by using an organic solvent, and then obtains the liposome suspension by dissolving with water. The method of the application overcomes the following deficiencies of the prior art: in order to remove the solvent, heat treatment is inevitable, which will affect the stability of the drug, especially for the heat-sensitive drug, and increases the time and difficulty of the heat treatment. In addition, the performance of the drug is inevitably affected during the transportation and storage of the precursor liposome loaded with the drug, and the cost of the transportation and storage is increased. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 HPLC spectra of the permeation liquid and the concentrated liquid of the Epimedium samples of Example 1, Example 2 and Comparative Example 2 of the application;

[0042] Figure 2HPLC chromatograms of the permeate and concentrate of the Epimedium sagittatum sample of Example 3, Example 4, Comparative Example 3 of the present application;

[0043] Figure 3 HPLC chromatograms of the permeate and concentrate of the Epimedium sagittatum sample of Example 6-8 of the present application;

[0044] Figure 4 HPLC chromatograms of the permeate and concentrate of the Epimedium sagittatum sample of Example 9-11 of the present application;

[0045] Figure 5 TEM electron microscope image of the Epimedium sagittatum liposome of Example 2 of the present application. DETAILED DESCRIPTION

[0046] The content of the present application will be further described in detail through the embodiments below, but should not be understood as limiting the present application to the following embodiments, and any technology realized based on the content of the present application above falls within the scope of the present application.

[0047] The lecithin mentioned in the examples was purchased from Beijing Aoboxing Biological Co., Ltd., with the product code 01-02; anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; salt was of a regular size, a commercially available product; cholesterol was purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd., with the batch number 20191008. Sucrose was of a regular size, a commercially available product. Artificial skin was Strat-MTM Membrane (25mm Discs) purchased from Merck Millipore Ltd.

[0048] Preparation method of Epimedium sagittatum extract: 5000g of Epimedium sagittatum decoction pieces were added with water for decoction, filtered, and the filtrate was concentrated into a clear paste, dried under reduced pressure to obtain dry extract 71.6g, packaged, and used, and the content of icariin in the Epimedium sagittatum extract was measured by HPLC to be 30.45%.

[0049] Example 1

[0050] Preparation of Epimedium sagittatum extract liposome with NaCl as solid phase particles:

[0051] Lecithin was weighed into an anhydrous ethanol solution, ultrasonicated for 10 minutes to obtain a 0.2g / mL lecithin solution, which was stored in the dark. NaCl particles (average particle size 650μm) were placed in an evaporating dish, heated in a water bath at 50℃, and then solid particles were added to the lecithin solution at a mass ratio of salt to lecithin 2.08:1, while stirring, and the ethanol was evaporated in a water bath to obtain empty liposomes. The empty liposomes were taken, and Epimedium sagittatum extract solution was added at a mass ratio of Epimedium sagittatum extract to lecithin 7.4:100, with the concentration of the Epimedium sagittatum extract solution being 0.6mg / mL, and the mixture was shaken for 10min to obtain an Epimedium sagittatum extract liposome emulsion. The encapsulation efficiency of the liposome was tested to be 100%.

[0052] Example 2

[0053] Preparation of liposome of Epimedium extract with NaCI as solid phase particle:

[0054] Take lecithin and add to anhydrous ethanol solution, ultrasonic for 10 minutes, get 0.2 g / mL lecithin solution, keep in dark condition. Put salt (average particle size is 650 μm) in evaporating dish, heat in water bath at 50 ℃, then add solid particle to lecithin solution according to mass ratio of salt solid phase particle to lecithin 3.125:1, stir while adding, dry in water bath, get empty liposome. Take empty liposome, add Epimedium extract solution with concentration of 0.15 mg / mL according to mass ratio of Epimedium extract to lecithin 9.9:100, shake for 10 minutes, get Epimedium extract liposome emulsion. Calculate encapsulation efficiency as 100%.

[0055] Example 3

[0056] Preparation of liposome of Epimedium extract with NaCI as solid phase particle:

[0057] Take lecithin and add to anhydrous ethanol solution, ultrasonic for 10 minutes, get 0.2 g / mL lecithin solution, keep in dark condition. Put salt (average particle size is 650 μm) in evaporating dish, heat in water bath at 50 ℃, then add solid particle to lecithin solution according to mass ratio of salt solid phase particle to lecithin 3.125:1, stir while adding, dry in water bath, get empty liposome. Take empty liposome, add Epimedium extract solution with concentration of 0.15 mg / mL according to mass ratio of Epimedium extract to lecithin 9.9:100, shake for 10 minutes, get Epimedium extract liposome emulsion. Calculate encapsulation efficiency as 100%.

[0058] Example 4

[0059] Preparation of liposome of Epimedium extract with NaCI as solid phase particle:

[0060] Take lecithin and add to anhydrous ethanol solution, ultrasonic for 10 minutes, get 0.2 g / mL lecithin solution, keep in dark condition. Put salt (average particle size is 650 μm) in evaporating dish, heat in water bath at 50 ℃, then add solid particle to lecithin solution according to mass ratio of salt solid phase particle to lecithin 3.125:1, stir while adding, dry in water bath, get empty liposome. Take empty liposome, add Epimedium extract solution with concentration of 0.15 mg / mL according to mass ratio of Epimedium extract to lecithin 9.9:100, shake for 10 minutes, get Epimedium extract liposome emulsion. Calculate encapsulation efficiency as 100%.

[0061] Example 5

[0062] Preparation of Epimedium extract liposome with NaCl as solid phase particle:

[0063] The preparation conditions of this example are the same as those of Example 1, except that cholesterol is added to the coating material, and the mass ratio of lecithin-cholesterol is 6:1.

[0064] Comparative Example 1

[0065] Preparation of Epimedium extract liposome with NaCl as solid phase particle:

[0066] This comparative example has the same preparation conditions as Example 1, except that no salt solid phase particle is added.

[0067] Lecithin is weighed and added to anhydrous ethanol solution, and a solution with a lecithin concentration of 0.2 g / mL is prepared, ultrasonicated for 10 minutes, and stored in the dark. Lecithin solution is added dropwise to an evaporating dish, and lecithin film is obtained by heating in a water bath at 50°C until dry. Epimedium solution is taken, and 0.6 mg / mL of Epimedium extract solution is added according to the mass ratio of Epimedium extract to lecithin of 32.4:100, ultrasonicated for 10 min, and Epimedium liposome emulsion is obtained.

[0068] Comparative Example 2

[0069] Preparation of Epimedium extract liposome with NaCl as solid phase particle:

[0070] Lecithin is weighed and added to anhydrous ethanol solution, and a solution with a lecithin concentration of 0.2 g / mL is prepared, ultrasonicated for 10 minutes, and stored in the dark. Lecithin solution is added dropwise to an evaporating dish, and lecithin film is obtained by heating in a water bath at 50°C until dry. Epimedium solution is taken, and 0.6 mg / mL of Epimedium extract solution is added according to the mass ratio of Epimedium extract to lecithin of 32.4:100, ultrasonicated for 10 min, and Epimedium liposome emulsion is obtained.

[0071] Comparative Example 3

[0072] Preparation of Epimedium extract liposome with NaCl as solid phase particle:

[0073] The lecithin was weighed into anhydrous ethanol solution, and ultrasonic treatment was performed for 10 minutes to obtain a 0.0189 g / mL lecithin solution, which was stored in the dark. The salt (average particle size 650 μm) was placed in an evaporating dish, heated in a water bath at 50°C, and then the solid particles were added to the lecithin solution at a mass ratio of salt solid phase particles to lecithin of 26.455:1, while stirring, and the water bath was evaporated to obtain empty liposomes. The empty liposomes were taken, and the Epimedium extract was added at a mass ratio of Epimedium extract to lecithin of 65.89:100, and a 0.6 mg / mL Epimedium extract solution was obtained. The mixture was shaken for 10 minutes to obtain an Epimedium extract liposome emulsion.

[0074] Comparative Example 4

[0075] The preparation conditions and raw material amounts of the present comparative example were the same as those of Example 1, except that the Epimedium extract was first added to the lecithin ethanol solution, and then the Epimedium extract and lecithin were loaded onto the NaCl particles, and finally water was added to obtain the product. The specific method is as follows:

[0076] The lecithin and Epimedium extract were weighed into anhydrous ethanol, ultrasonic treatment was performed for 10 minutes, and centrifugation was performed to obtain an organic solution containing lecithin and Epimedium extract (in which the mass ratio of Epimedium extract to lecithin was 7.4:100, and the mass concentration of lecithin in the solution was 0.2 g / mL), which was stored in the dark. The NaCl particles (particle size 650 μm) were placed in an evaporating dish, heated in a water bath at 50°C, and then the organic solution containing lecithin and Epimedium extract was added at a mass ratio of NaCl solid phase particles to coating material of 2.08:1, while stirring, and the water bath was evaporated. Subsequently, deionized water was added to obtain an Epimedium extract liposome emulsion (in which the mass ratio of deionized water to Epimedium extract was 1000:0.6).

[0077] Comparative Example 5

[0078] The raw material amounts and preparation conditions of the present comparative example were the same as those of Example 1, except that the combination method of the coating material and the solid phase particles was different.

[0079] The lecithin was weighed into anhydrous ethanol solution, and a 0.2 g / mL lecithin solution was prepared, ultrasonic treatment was performed for 10 minutes, and the solution was stored in the dark. The NaCl particles were placed in a rotary evaporating flask, and then the lecithin solution was sprayed into the rotary evaporating flask at a mass ratio of NaCl to coating material of 2.08:1, and the rotary evaporation temperature was 50°C to obtain empty liposomes. The empty liposomes were taken, and a 0.6 mg / mL Epimedium extract aqueous solution was added at a mass ratio of Epimedium extract to lecithin of 7.4:100, and the mixture was shaken for 10 minutes to obtain an Epimedium extract liposome suspension.

[0080] Example 6

[0081] Preparation of Epimedium liposome with sucrose as solid particles:

[0082] The lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution, which was ultrasonically treated for 10 minutes and stored in the dark. The sucrose (average particle size 600 μm) was placed in an evaporating dish and heated in a water bath at 50°C. Then, solid particles were added to the lecithin solution at a mass ratio of sucrose to lecithin of 2.08:1 while stirring, and the water bath was evaporated to dryness to obtain empty liposomes. The empty liposomes were taken and 0.6 mg / mL Epimedium solution was added at a mass ratio of Epimedium extract to lecithin of 7.4:100, and the mixture was shaken for 10 minutes to obtain an Epimedium liposome emulsion.

[0083] Example 7

[0084] Preparation of Epimedium liposome with sucrose as solid particles:

[0085] The lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.0189 g / mL lecithin solution, which was ultrasonically treated for 10 minutes and stored in the dark. The sucrose (average particle size 600 μm) was placed in an evaporating dish and heated in a water bath at 50°C. Then, solid particles were added to the lecithin solution at a mass ratio of sucrose to lecithin of 26.45:1 while stirring, and the water bath was evaporated to dryness to obtain empty liposomes. The empty liposomes were taken and 0.6 mg / mL Epimedium solution was added at a mass ratio of Epimedium extract to lecithin of 14.7:100, and the mixture was shaken for 10 minutes to obtain an Epimedium liposome emulsion.

[0086] Example 8

[0087] Preparation of Epimedium liposome with sucrose as solid particles:

[0088] The lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution, which was ultrasonically treated for 10 minutes and stored in the dark. The sucrose (average particle size 600 μm) was placed in an evaporating dish and heated in a water bath at 50°C. Then, solid particles were added to the lecithin solution at a mass ratio of sucrose to lecithin of 2.08:1 while stirring, and the water bath was evaporated to dryness to obtain empty liposomes. The empty liposomes were taken and 0.6 mg / mL Epimedium solution was added at a mass ratio of Epimedium extract to lecithin of 7.4:100, and the mixture was shaken for 10 minutes to obtain an Epimedium liposome emulsion.

[0089] Example 9

[0090] Preparation of Epimedium liposome with sucrose as solid particles:

[0091] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sucrose (average particle size 600 μm) was placed in an evaporating dish and heated in a water bath at 50°C. Then, solid particles were added to the lecithin solution at a mass ratio of 4.17:1 (sucrose solid particles to lecithin), stirring constantly. The mixture was evaporated to dryness in a water bath to obtain empty liposomes. The empty liposomes were then taken, and 2.4 mg / mL of epimedium solution was added, adjusting the mass ratio of empty liposomes to epimedium solution solid content to lecithin to 12.4:100. The mixture was shaken for 10 minutes to obtain an epimedium liposome emulsion.

[0092] Example 10

[0093] Preparation of Epimedium liposomes using sucrose as solid particles:

[0094] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sucrose (average particle size 600 μm) was placed in an evaporating dish and heated in a water bath at 50°C. Then, solid particles were added to the lecithin solution at a mass ratio of 2.5:1 (sucrose to lecithin), stirring constantly. The mixture was evaporated to dryness in a water bath to obtain empty liposomes. The empty liposomes were then mixed with 0.6 mg / mL epimedium solution, adjusted to a mass ratio of 8.4:100 (ephemeral epimedium solution to lecithin), and shaken for 10 minutes to obtain an epimedium liposome emulsion.

[0095] Example 11

[0096] Epimedium liposomes were prepared using sucrose as solid particles: Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.02363 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sucrose (average particle size 600 μm) was placed in an evaporating dish and heated in a water bath at 50°C. Then, solid particles were added to the lecithin solution at a mass ratio of 21.16:1 (sucrose solid particles to lecithin), stirring continuously. The mixture was evaporated to dryness in a water bath to obtain empty liposomes. The empty liposomes were then converted to an epimedium solution solid content to lecithin mass ratio of 27.8:100 (empty liposome to epimedium solution solid content to lecithin mass ratio). 0.6 mg / mL epimedium solution was added, and the mixture was shaken for 10 minutes to obtain an epimedium liposome emulsion.

[0097] Example 12

[0098] Epimedium liposomes prepared using sucrose as solid particles: The preparation conditions in this embodiment are the same as in Example 6, except that cholesterol is added to the encapsulation material, and the mass ratio of lecithin to cholesterol is 6:1.

[0099] Performance test:

[0100] 1. Test of the Epimedium liposome encapsulation rate:

[0101] Test method of the encapsulation rate:

[0102] Take 0.1 mL of the Epimedium liposome emulsion in a common centrifuge tube, centrifuge at 8000 rpm for 10 min, take out 0.05 mL of the supernatant and add to an ultrafiltration centrifuge tube, then add 0.35 mL of purified water, ultrafiltrate for 10 min (in which, the ultrafiltration membrane pore size is 30 KD, and the centrifuge speed is 6000 rpm), take the supernatant (permeate) of the first ultrafiltration centrifugation for liquid chromatography analysis. Take the centrifuged sediment of the common centrifugation and add 0.75 mL of anhydrous ethanol, ultrasonically dissolve, take out 0.2 mL for the second ultrafiltration centrifugation for 10 min (in which, the ultrafiltration membrane pore size is 30 KD, and the centrifuge speed is 6000 rpm), take the supernatant (concentrate) of the second ultrafiltration centrifugation for liquid chromatography analysis. According to the HPLC method, detect the Epimedium glycoside chromatographic peak area of the permeate and the concentrate, and calculate the encapsulation rate (%) = concentrate peak area / (permeate peak area + concentrate peak area) x 100%.

[0103] Test conditions of high performance liquid chromatography: bonded silica gel C 18 (4.6 mm x 250 mm, 5 μm), with acetonitrile (A) - water solution (B) as the mobile phase, gradient elution (0-15 min, B 10%-25%; 15-38 min, B 25%; 38-61 min, B 25%-71%; 61-75 min, B 71%-100%), flow rate 1.0 ml / min, column temperature 30°C, detection wavelength 270 nm, sample amount: 10 μL.

[0104] The properties of the Epimedium liposomes obtained from each example and the comparative example are summarized in Table 1 and Table 2.

[0105] Table 1: Encapsulation rate (%) of the Epimedium liposomes obtained when the solid phase particles are NaCl (the encapsulated material is calculated based on lecithin)

[0106]

[0107] Table 1 shows the encapsulation efficiency of the Epimedium liposomes prepared in the examples and comparative examples. It can be seen that in Examples 1-5, the encapsulation efficiency reached 100% when the ratio of Epimedium extract to encapsulating material was in the range of 7.4:100 to 12.4:100, indicating that the encapsulating material effectively encapsulated the Epimedium extract. In Comparative Example 1, without using solid particles as a carrier, the encapsulation efficiency decreased significantly, indicating that this method can more effectively encapsulate the active ingredients of Epimedium extract than the traditional rotary evaporation film-forming method. In Comparative Examples 2 and 3, increasing the ratio of Epimedium extract to encapsulating material to 104:100 and 65.89:100 respectively resulted in a significant decrease in encapsulation efficiency, indicating that the drug loading capacity of the encapsulating material was exceeded. Therefore, the ratio of Epimedium extract to encapsulating material is a key factor affecting the solute encapsulation effect. In Comparative Example 4, the epimedium extract was placed in the encapsulation material solution. After film formation and hydration, the encapsulation efficiency decreased. This may be because the active ingredients in the epimedium extract participated in the film-forming process of the encapsulation material, becoming doped into the film on the surface of the carrier particles, affecting the film's uniformity and further impacting the encapsulation effect of the epimedium extract. Comparative Example 4 had a poor encapsulation efficiency because, during the spraying process, the system was under vacuum, making it difficult for the solution to effectively spray onto the surface of the solid particles to form a film. Instead, more tiny spots and some film particles formed on the solid particle surface. Therefore, for the same amount of solution, the film-forming area in Comparative Example 4 was much smaller than that in Examples 1-5, affecting the encapsulation efficiency.

[0108] Compared to Examples 1-5, the encapsulation efficiency without solid particles is significantly lower than that using solid particles. Although existing methods can achieve encapsulation efficiencies of 85-100% with optimized experimental parameters, their complex preparation processes, increased organic solvent usage, and long preparation times hinder large-scale production.

[0109] like Figure 1 , 2 The figures show the HPLC spectra of the epimedium liposome permeate and concentrate prepared in Examples 1-4 and Comparative Examples 2 and 3, respectively. It can be seen from the figures that both the permeate and the concentrate have peaks at the icariin chromatographic peak (the retention time of the icariin chromatographic peak is about 37 min). When the peak of the permeate is small and almost linear, while the concentrate has a strong peak, it indicates that the drug encapsulation effect is ideal.

[0110] Table 2 Encapsulation efficiency (%) of Epimedium liposomes prepared using sucrose as the solid phase particle (encapsulation material is based on lecithin)

[0111]

[0112] The encapsulation efficiency data of the Epimedium liposome prepared by using sucrose as the solid phase particle is shown in Table 2. As shown in Table 2, lecithin is used as the raw material for forming the liposome shell. When the lecithin amount is within a certain range and other conditions remain unchanged, the amount of the formed liposome is small when the lecithin amount is small, and the embedding rate is poor. When the lecithin amount is too large, the lecithin adheres to each other and cannot form a well-shaped spherical shape, the Epimedium containing amount is small or the thickness of the liposome shell is large, and a small amount of drug is difficult to enter, and the embedding rate is small. The encapsulation efficiency of the liposome obtained by the present application is generally 84-100%, which has a significant advantage compared with the prior art. A high embedding rate can be obtained within a wide parameter range, and the preparation process is simple, the amount of organic solvent is small, the preparation time is short, and large-scale preparation can be achieved.

[0113] As shown in Figure 4 , 5 The HPLC chromatograms of the permeation liquid and the concentrated liquid of the Epimedium liposome prepared in Examples 6-11 are shown in the figures. As shown in the figures, the permeation liquid and the concentrated liquid both have peaks at the icariin chromatographic peak (the icariin chromatographic peak has a retention time of about 37 min). The smaller the peak of the permeation liquid and the stronger the peak of the concentrated liquid, the better the drug wrapping effect.

[0114] 2. Particle size determination of the Epimedium liposome:

[0115] Test method: The prepared Epimedium liposome emulsion is filtered through a 0.45 μm microfiltration membrane, 0.1 mL of the filtrate is taken, and diluted with water to 1 mL, and then detected three times by a laser particle size analyzer Zetasizer Nano ZS90. The particle size test results of the liposomes obtained in each example and the comparative example are summarized in Table 3.

[0116] 3. Transdermal diffusion experiment:

[0117] Strat-MTM Membrane (Merck Millipore Ltd) and a transdermal diffusion test device are used. A 40% PEG400 aqueous solution is added to the diffusion cell, and 0.2 mL of the Epimedium liposome emulsion and 0.2 mL of the Epimedium extract aqueous solution (0.6 mg / mL) obtained in the example or the comparative example are uniformly dispersed on the smooth surface of the artificial skin. The transdermal diffusion is carried out at room temperature, and 0.2 mL of the permeation solution is taken out from the diffusion cell after 12 h, and then subjected to liquid chromatography. At the same time, the volume is supplemented with a 40% PEG400 aqueous solution, and the permeation rate is calculated by high performance liquid chromatography. The permeation rate is shown in Table 3.

[0118] Table 3 Particle size of the Epimedium liposome obtained in each example and the comparative example

[0119]

[0120]

[0121] It can be seen from the analysis that the particle size of the liposome after microfiltration is small, about 220 nm, and the dispersion coefficient is small, stable and uniformly dispersed. The average electric potential of the liposome is negative, indicating that the surface of the liposome particles has a negative charge and good stability. The average particle size is reduced, the particle size is more uniform, and the aggregation index PDI is reduced.

[0122] The TEM electron microscope images of the Epimedium liposome emulsion prepared in Example 2 after copper mesh filtration and drying are shown in FIGS. 1 and 2, respectively. Figure 5 As can be seen from the figures, the Epimedium liposome is similar to a spherical shape, and the particle size is significantly smaller than that of the particles in the Epimedium liposome emulsion, which may be caused by the drying and water loss of the particles in the emulsion during the sample preparation process; and the Epimedium liposome particles are uniformly distributed.

[0123] 4. DPPH free radical scavenging activity determination:

[0124] (1) Solution preparation:

[0125] DPPH-ethanol solution: weigh 1 mg of DPPH, add 22.2 ml of anhydrous ethanol, and ultrasonically dissolve to obtain.

[0126] A 0.03 mg / mL vitamin C sample solution was used as a positive control to verify the accuracy of the test results.

[0127] (2) Experimental steps:

[0128] The experiment set a determination group, a control group, and a blank group

[0129] Determination group: take 80 μl of the Epimedium liposome transdermal diffusion test 48 h transudate to the 96 well plate, then add 120 ul of DPPH-ethanol solution, shake well, and let stand at room temperature (25℃) for 10 min, measure the absorbance, and record it as A1;

[0130] Control group: according to the method of the determination group, replace the DPPH-ethanol solution with an equal volume of 60 wt% ethanol solution, and measure the absorbance, record it as A2;

[0131] Blank group: take 80 μL of 60% ethanol to the 96 well plate, add 120 μL of DPPH-ethanol solution, shake well, and let stand at room temperature for 10 min, measure the absorbance, and record it as A0.

[0132] The positive control group was tested according to the determination group.

[0133] The absorbance of each sample was measured at 517 nm in the enzyme marker, and the 48 h free radical scavenging rate (i.e. inhibition rate) was calculated according to the following formula:

[0134]

[0135] Table 4 Antioxidant activity of liposome of Epimedium extract prepared according to the present application

[0136]

[0137]

[0138] As shown in Table 4, artificial skin membrane permeation concentration was detected in Examples 1-12 and Comparative Examples 1-5, and antioxidant activity was detected in Examples 1-12 and Comparative Examples 1-5 except for Example 8 and Comparative Example 2. The Epimedium aqueous solution permeate did not detect antioxidant activity, and the measured permeation rate data was 0, indicating that the uncoated Epimedium extract aqueous solution cannot permeate the artificial skin membrane, and the permeation capacity can be greatly improved by coating with lecithin to form liposomes.

Claims

1. A method for preparing liposomes of Epimedium extract using soluble solid phase particles, characterized in that, The method comprises the following steps: (1) adding a wrapping material into an anhydrous ethanol solution to prepare a wrapping material solution; (2) immersing soluble solid-phase particles into the wrapping material solution, removing the anhydrous ethanol, and combining the soluble solid-phase particles with the wrapping material to obtain empty liposomes; (3) mixing the empty liposomes with an extract solution of Epimedium to obtain an extract liposome emulsion of Epimedium; The mass ratio of the extract of Epimedium to the wrapping material is (7.4-27.8):100, The soluble solid-phase particles are NaCl or sucrose, When the soluble solid-phase particles are NaCl, the mass ratio of NaCl to the wrapping material is (2.08-4.17):1, and when the soluble solid-phase particles are sucrose, the mass ratio of sucrose to the wrapping material is (2.08-26.45):

1.

2. The production method according to claim 1, characterized by, In step (3), the solvent of the extract solution of Epimedium is water, and the concentration of the extract solution of Epimedium is 0.15-2.4 mg / mL.

3. The preparation method according to claim 1, characterized in that, When the solid-phase particles are NaCl, the mass ratio of the extract of Epimedium to the wrapping material is (7.4-12.4):100; and when the solid-phase particles are sucrose, the mass ratio of the extract of Epimedium to the wrapping material is (7.4-27.8):

100.

4. The production method according to claim 1, characterized by, When the solid-phase particles are sucrose, the mass ratio of sucrose to the wrapping material is (2.08-4.17):

1.

5. The method of claim 1, wherein, In step (2), the empty liposomes are: the wrapping material is wrapped around the surface of the solid-phase particles in the form of a membrane, and the thickness of the membrane is 2.5-16.7 μm.

6. The method of claim 1, wherein, In step (2), the particle size of the soluble solid-phase particles is 150-1250 μm.

7. The preparation method according to claim 1, characterized in that, In step (2), the particle size of the soluble solid-phase particles is 500-800 μm.

8. The method of claim 1, wherein, In step (2), the particle size of the soluble solid-phase particles is 600-650 μm.

9. The method of claim 1, wherein, In step (1), the wrapping material is one of lecithin, a mixture of lecithin and cholesterol, or the like; the mass ratio of the mixture of lecithin and cholesterol is 5-7:1; and the mass concentration of the wrapping material in the wrapping solution is 0.0189-0.25 g / mL.

10. The method of claim 1, wherein, In step (1), the mass concentration of the wrapping material in the wrapping solution is 0.15-0.2 g / mL.

11. The method of claim 1, wherein, The method comprises a whole-grain method, and the obtained extract liposomes of Epimedium are subjected to one of high-speed shearing, ultrasonic, homogenization, and extrusion.

12. The method of claim 11, wherein, The whole-grain method is extrusion.

13. The method of claim 12, wherein, The extrusion is microfiltration extrusion.

14. The liposome of Epimedium extract prepared by the method of any one of claims 1-13, characterized in that, The method comprises 0.0149-0.22% of the extract of Epimedium, 0.05-2.2% of the wrapping material, 0.47-7.31% of the soluble solid-phase particles, and the balance of water.

15. The Epimedium extract liposome according to claim 14, characterized in that, The main component of the extract of Epimedium is icariin, and the mass percentage of icariin in the extract is more than 30.45%.

16. The Epimedium extract liposome according to claim 14, characterized in that, When the solid-phase particles are NaCl, the particle size of the extract liposomes of Epimedium is 117.8-172.5 nm, the aggregation index PDI is 0.307-0.482, and the average electric potential is -28.9--44.5 mV. The solid phase particle is sucrose, the particle size of the Epimedium extract liposome is 116.83-197.9 nm, the aggregation index PDI is 0.248-0.368, and the average potential is-41.7--54.4 mV.

17. The use of the Epimedium extract liposome prepared by the method of any one of claims 1-13 in the preparation of a drug for treating and preventing osteoporosis.

18. The use according to claim 17, characterized in that, The Epimedium extract liposome is in the form of an oral liquid or an injection.

19. The use of the Epimedium extract liposome prepared by the method of any one of claims 1-13 in the preparation of a cosmetic.

20. The use of the Epimedium extract liposome prepared by the method of any one of claims 1-13 in the preparation of an anti-ultraviolet cosmetic.

21. The use according to claim 20, characterized in that, The cosmetic is in the form of a liquid or a paste.

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