Epimedium extract liposome prepared from soluble solid-phase particles as well as preparation method and application of epimedium extract liposome

Abstract

The invention provides a herba epimedii extract liposome prepared from soluble solid-phase particles as well as a preparation method and application thereof, and belongs to the field of traditional Chinese medicine preparations. The preparation method comprises the following steps: firstly, adding a wrapping material into an absolute ethyl alcohol solution to prepare a wrapping material solution; then, the soluble solid-phase particles are soaked in a wrapping material solution, absolute ethyl alcohol is removed, and the soluble solid-phase particles and the wrapping material are combined to obtain the no-load liposome. And mixing the no-load liposome with the herba epimedii extract solution to obtain the herba epimedii extract liposome emulsion. The liposome particles prepared by the method disclosed by the invention are high in embedding rate, good in particle uniformity and high in stability. Reference is provided for innovation of a liposome preparation technology and more ideal liposome preparation, convenience is provided for clinical application of icariin, and a foundation is laid for deeper application research.

Description

Technical Field

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

[0002] Icariin is a small molecule compound extracted from the traditional Chinese medicine Epimedium, which has a variety of pharmacological effects, including reproductive function, neuroprotection, bone protection, cardiovascular protection, anti-tumor, anti-inflammatory, and immunity enhancement. In the field of cosmetic applications, icariin also has the effects of skin UV protection, anti-UV damage and aging. Liu Wenjie et al. proved through experiments that icariin can improve the survival ability of fibroblasts, reduce the proportion of senescence-related β-galactosidase staining positive cells, upregulate the synthesis of extracellular matrix, and downregulate the expression of senescence-related cyclin-dependent kinase inhibitors, so as to reduce anti-aging, and resist UV damage by reducing UV-related cell apoptosis. Icariin is widely used 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] Nanoliposomes can successfully encapsulate icariin and improve its stability and bioavailability. The hydrophilic environment inside the liposome can load hydrophilic drugs, while the phospholipid molecular layers that constitute the liposome can load lipophilic drugs, so that the drugs are isolated from the influence of external air, etc., improving their stability and extending the storage time. The liposome particle size is small and similar to the cell membrane structure, which can improve the bioavailability of the drug. At present, liposome preparation mostly uses organic solvent injection method, thin film dispersion method, reverse evaporation method, etc. For example, Zhang Xinyue et al. used thin film dispersion method and ammonium sulfate hydration method to modify the salidroside / icariin liposome modified by Angiopep-2. The results show that the icariin encapsulation rate is about 92%, the drug encapsulation rate is high, and it has good active targeting ("Modern Chinese Medicine Research and Practice", 2022 Issue 2 |, pp. 57-62). Lu Cuitao and others prepared liposomes encapsulating icariin, and conducted process optimization, appearance shape and stability investigation. The results showed that the liposomes encapsulating icariin had an ideal microscopic morphology and suitable particle size distribution, and excellent stability. Sun Xiaoduan used a thin film dispersion method to prepare targeted liposomes BBL. The particle size of the targeted liposomes BBL was less than 200nm and the polydispersity coefficient was about 0.19 measured by Malvern laser particle size analyzer, indicating that the particle size of the prepared liposomes was uniform, and 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 problems such as complex preparation steps and the use of a large amount of organic solvents, which seriously affects the clinical application and development of icariin. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an epimedium extract liposome prepared by using soluble solid phase particles, a preparation method and application thereof.

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

[0006] An empty liposome, the liposome comprises soluble solid phase particles and a wrapping material, the soluble solid phase particles are selected from one of sucrose and sodium chloride, the wrapping material is lecithin, or a mixture of lecithin and cholesterol; the wrapping material is wrapped on the surface of the solid phase particles in the form of a film, and the thickness of the film is 2.5 to 16.7 μm.

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

[0008] Compared with the prior art, the empty liposomes provided by the present invention have the following advantages:

[0009] 1) Proliposomes are solid particles made of drugs and membrane materials. The method of the present invention is to make the membrane material into solid particles. The preparation process is simpler and there is no need to dissolve the drug in an organic solvent.

[0010] 2) The present invention will not affect the stability of the drug. To prepare proliposomes, the film-forming material and the drug need to be made into a solution of an organic solvent, and then the organic solvent is evaporated to remove it, and the film-forming material and the drug are coated on solid particles. In this process, the drug components are heated and exposed for a long time, which may affect their stability and may cause toxic organic solvent residues. This method is to make the film-forming material into a solution of an organic solvent (using anhydrous ethanol), and then coat it on solid particles. It will not cause toxic organic solvent residues and will not affect the stability of the drug.

[0011] 3) Liposomes can be prepared on the spot. Simply mix empty liposomes with drug aqueous solution to obtain drug-loaded liposomes. The application is more extensive. After the preparation of the proliposomes, they are packaged in the form of solid preparations. Storage and transportation need to consider the properties of the drug components. After the preparation of the empty liposomes of the present invention, they are packaged in solid raw materials. Since they do not contain drug components, they can be used for the preparation of multiple drug components on the spot.

[0012] The present invention also provides a method for preparing the above-mentioned empty liposome.

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

[0014] (2) Immersing the soluble solid phase particles in the encapsulating material solution, removing the anhydrous ethanol, and combining the soluble solid phase particles with the encapsulating material to obtain empty liposomes.

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

[0016] Preferably, the mass concentration of the encapsulating material in the encapsulating solution is 0.0189 to 0.2 g / mL, preferably 0.15 to 0.2 g / mL. The encapsulating material is calculated as lecithin.

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

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

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

[0020] Preferably, in step (2), the method of removing anhydrous ethanol includes one of water bath evaporation, air drying, 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 particles.

[0022] One object of the present invention is to provide an epimedium extract liposome, comprising 0.0149-0.22% by mass of epimedium extract, 0.05-2.2% by mass of encapsulating material, 0.47-7.31% by mass of soluble solid phase particles, and water.

[0023] The main component of the epimedium extract of the invention 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, in the transdermal diffusion experiment of the epimedium liposome, when the solid phase particles are NaCl, the 48h permeability is not less than 50%, and when the solid phase particles are sucrose, the 48h permeability is not less than 23.6%.

[0026] Preferably, the solid phase particles are NaCl, the particle size of the epimedium liposome is 117.8-172.5 nm, the aggregation index (PDI) is 0.307-0.482, and the average potential is -28.9--44.5 mV. The solid phase particles are sucrose, the particle size of the epimedium 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.

[0027] The present invention also provides a method for preparing epimedium extract liposomes by using soluble solid phase particles, wherein empty liposomes are mixed with epimedium extract solution to obtain epimedium extract liposome emulsion.

[0028] Preferably, the mixing method of the empty liposomes and the epimedium extract solution includes: vortex oscillation, shaking, and ultrasound.

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

[0030] When the solid phase particles are NaCl, the mass ratio of the epimedium extract to the encapsulating material is (7.4-12.4):100, and when the solid phase particles are sucrose, the mass ratio of the epimedium extract to the encapsulating material is (7.4-27.8):100.

[0031] Preferably, the above preparation method also includes a granulation method, which refers to a particle size control method, wherein the obtained epimedium extract liposomes are subjected to one of four methods: high-speed shearing, ultrasound, homogenization, and extrusion, preferably extrusion, and more preferably microfiltration extrusion.

[0032] The present invention also provides the epimedium extract liposome and the use of the epimedium extract liposome prepared by the above method in preparing a drug for treating osteoporosis.

[0033] When the soluble solid phase particles are sucrose, the epimedium extract liposome can be prepared into an oral liquid dosage form.

[0034] When the soluble solid phase particles are sodium chloride, the epimedium extract liposomes can be prepared into an injection form.

[0035] The present invention also provides the above-mentioned epimedium extract liposome and the use of the epimedium extract liposome prepared by the above-mentioned method in the preparation of cosmetics. Preferably, it is used in the preparation of anti-ultraviolet cosmetics. The type of cosmetics can be liquid or paste.

[0036] Beneficial effects of the present invention:

[0037] 1. The present invention utilizes the large specific surface area of ​​solid phase particles, and forms a wrapping material film with a large specific surface area by volatilizing the wrapping material solution on the surface of the soluble solid phase particles to obtain empty liposomes. The empty liposomes provided by the present invention are convenient for transportation, storage, and use. The drug-loaded liposomes can be obtained by uniformly mixing the aqueous solution of the drug carried with the empty liposomes.

[0038] 2. The present invention utilizes the spherical structure of soluble solid phase particles and the characteristics of being easily soluble in water, and adopts a new liposome preparation method for the first time to embed Epimedium in liposomes. Since the characteristics of large specific surface area of ​​solid phase particles are utilized, the thickness of the obtained wrapping material film is small. Therefore, during the preparation process, the required wrapping material is low, which greatly reduces the amount of anhydrous ethanol used. Compared with the prior art, the amount of ethanol used is reduced by about 90%. The present invention constructs a new liposome preparation process with simple process, small amount of organic solvent, simple preparation method, and green and pollution-free, which solves the problems of complex existing liposome preparation process, large amount of organic solvent, and difficulty in large-scale preparation.

[0039] 3. The liposome particles prepared by the method of the present invention have high embedding rate, good particle uniformity and high stability. It provides a reference for the innovation of liposome preparation technology and more ideal liposome preparation, and at the same time provides convenience for the clinical application of icariin and lays a foundation for more in-depth application research.

[0040] 4. The existing liposomes load drugs and lipids on soluble solid phase particles through organic solvents, and then use water to dissolve to obtain liposome suspensions. The present invention overcomes the following shortcomings of this method: heat treatment is inevitably required to remove the solvent, which will have a certain impact on the stability of the drug, especially for thermosensitive drugs, increasing the time and difficulty of heat treatment. In addition, during the transportation and storage process of the proliposome loaded with drugs, the performance of each material component must be taken into account, which will inevitably have a certain impact on the performance of the drug and increase the cost of transportation and storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 HPLC spectra of the permeate and concentrate of the Epimedium samples of Example 1, Example 2 and Comparative Example 2 of the present invention;

[0042] Figure 2HPLC spectra of the permeate and concentrate of the Epimedium samples of Example 3, Example 4 and Comparative Example 3 of the present invention;

[0043] Figure 3 HPLC spectra of the permeate and concentrate of the Epimedium samples of Examples 6-8 of the present invention;

[0044] Figure 4 HPLC spectra of the permeate and concentrate of the Epimedium samples of Examples 9-11 of the present invention;

[0045] Figure 5 This is a TEM image of the Epimedium liposome of Example 2 of the present invention. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below through specific implementation examples, but it should not be understood that the present invention is limited to the following examples, and all technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0047] The lecithin described in the examples was purchased from Beijing Aoboxing Biological Co., Ltd., item number 01-02; anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; salt was purchased from Sinopharm Chemical Reagent Co., Ltd., batch number: 20191008. Sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd., batch number: 20191008. The artificial skin was Strat-MTM Membrane (25 mm Discs) purchased from Merck Millipore Ltd.

[0048] Preparation method of epimedium extract: take 5000g of epimedium slices, add water to boil, filter, concentrate the filtrate into a clear paste, dry under reduced pressure to obtain 71.6g of dry extract, package and set aside, HPLC measured the icariin content in the epimedium extract to be 30.45%.

[0049] Example 1

[0050] Preparation of epimedium extract liposomes using NaCl as solid phase particles:

[0051] Weigh lecithin and add anhydrous ethanol solution, ultrasonicate for 10 minutes to obtain 0.2g / mL lecithin solution, and store it under light-proof conditions. Place NaCl particles (average particle size is 650μm) in an evaporating dish, heat in a water bath at 50°C, then add solid particles to the lecithin solution at a mass ratio of 2.08:1 of salt to lecithin, stir while adding, evaporate ethanol in a water bath to obtain empty liposomes. Take empty liposomes, add epimedium extract solution according to the mass ratio of epimedium extract to lecithin of 7.4:100, the concentration of epimedium extract solution is 0.6mg / mL, shake for 10min, and obtain epimedium extract liposome emulsion. The encapsulation efficiency of the liposomes was tested to be 100%.

[0052] Example 2

[0053] Preparation of epimedium extract liposomes using NaCl as solid phase particles:

[0054] Weigh lecithin and add anhydrous ethanol solution, ultrasonicate for 10 minutes to obtain 0.2g / mL lecithin solution, and store it under light-proof conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat it in a water bath at 50°C, then add solid particles to the lecithin solution according to the mass ratio of salt solid phase particles to lecithin of 3.125:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.15mg / mL epimedium extract solution according to the mass ratio of epimedium extract to lecithin of 9.9:100, shake for 10min, and obtain epimedium liposome emulsion. The encapsulation efficiency is calculated to be 100%.

[0055] Example 3

[0056] Preparation of epimedium extract liposomes using NaCl as solid phase particles:

[0057] Weigh lecithin and add anhydrous ethanol solution, ultrasonicate for 10 minutes to obtain 0.2g / mL lecithin solution, and store it under light-proof conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat it in a water bath at 50°C, then add solid particles to the lecithin solution according to the mass ratio of salt solid phase particles to lecithin of 3.125:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 2.4mg / mL epimedium extract solution according to the mass ratio of epimedium extract to lecithin of 9.9:100, shake for 10min, and obtain epimedium liposome emulsion. The encapsulation efficiency is calculated to be 100%.

[0058] Example 4

[0059] Preparation of epimedium extract liposomes using NaCl as solid phase particles:

[0060] Weigh lecithin and add anhydrous ethanol solution, ultrasonicate for 10 minutes to obtain 0.2g / mL lecithin solution, and store it in dark conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat it in a water bath at 50°C, then add solid particles to the lecithin solution according to the mass ratio of salt solid phase particles to lecithin 4.17:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL epimedium extract solution according to the mass ratio of epimedium extract to lecithin 12.4:100, shake for 10min, and obtain epimedium liposome emulsion. The encapsulation efficiency is calculated to be 100%.

[0061] Example 5

[0062] Preparation of epimedium extract liposomes using NaCl as solid phase particles:

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

[0064] Comparative Example 1

[0065] Preparation of epimedium extract liposomes using NaCl as solid phase particles:

[0066] The preparation conditions of this comparative example are the same as those of Example 1, except that no salt solid phase particles are added.

[0067] Weigh lecithin and add anhydrous ethanol solution to prepare a solution with a lecithin concentration of 0.2 g / mL, ultrasonicate for 10 minutes, and store in dark conditions. Add lecithin solution dropwise to an evaporating dish, heat in a water bath at 50°C to evaporate, and obtain a lecithin film. Take the epimedium solution, add 0.6 mg / mL of epimedium extract solution according to the mass ratio of epimedium extract to lecithin of 32.4:100, and ultrasonicate for 10 minutes to obtain an epimedium liposome emulsion.

[0068] Comparative Example 2

[0069] Preparation of epimedium extract liposomes using NaCl as solid phase particles:

[0070] Weigh lecithin and add anhydrous ethanol solution, ultrasonicate for 10 minutes to obtain 0.01181g / mL lecithin solution, and store it in dark conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat it in a water bath at 50°C, then add solid particles to the lecithin solution at a mass ratio of 42.34:1 of salt solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL of epimedium extract solution according to a mass ratio of 104:100 of epimedium extract to lecithin, shake for 10min, and obtain epimedium extract liposome emulsion.

[0071] Comparative Example 3

[0072] Preparation of epimedium extract liposomes using NaCl as solid phase particles:

[0073] Weigh lecithin and add anhydrous ethanol solution, ultrasonicate for 10 minutes to obtain 0.0189g / mL lecithin solution, and store it in dark conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat it in a water bath at 50°C, then add solid particles to the lecithin solution at a mass ratio of 26.455:1 of salt solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL of epimedium extract solution according to a mass ratio of 65.89:100 of epimedium extract to lecithin, shake for 10min, and obtain epimedium liposome emulsion.

[0074] Comparative Example 4

[0075] The preparation conditions and raw material dosage of this comparative example are the same as those of Example 1, except that the epimedium extract is first added to the lecithin ethanol solution, then the epimedium extract and lecithin are loaded on NaCl particles, and finally water is added to obtain the product. The specific method is as follows:

[0076] Weigh lecithin and epimedium extract, add anhydrous ethanol, ultrasonically dissolve, centrifuge, obtain an organic solution containing lecithin and epimedium extract (wherein, the mass ratio of epimedium extract to lecithin is 7.4:100, and the mass concentration of lecithin in the solution is 0.2g / mL), ultrasonicate for 10 minutes, and store in the dark. Take NaCl particles (particle size 650μm) and place them in an evaporating dish. Under the condition of heating in a water bath at 50°C, add the organic solution containing lecithin and epimedium extract at a mass ratio of 2.08:1 of NaCl solid phase particles to encapsulating material, stir while adding, evaporate in a water bath, and then add deionized water to obtain epimedium extract liposome emulsion (wherein, the mass ratio of deionized water to epimedium extract is 1000:0.6).

[0077] Comparative Example 5

[0078] The raw material dosage and preparation conditions of this comparative example are the same as those of Example 1, except that the combination method of the encapsulating material and the solid phase particles is different.

[0079] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark. Take NaCl particles and place them in a rotary evaporator, then spray lecithin solution into the rotary evaporator at a mass ratio of NaCl to packaging material of 2.08:1, and the rotary evaporation temperature is 50°C to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL epimedium extract aqueous solution according to the mass ratio of epimedium extract to lecithin of 7.4:100, shake for 10 minutes, and obtain epimedium extract liposome suspension.

[0080] Example 6

[0081] Preparation of Epimedium liposomes using sucrose as solid particles:

[0082] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store under light-proof conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat in a water bath at 50°C, then add solid particles to the lecithin solution at a mass ratio of sucrose to lecithin of 2.08:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL epimedium solution according to a mass ratio of epimedium extract to lecithin of 7.4:100, shake for 10min, and obtain epimedium liposome emulsion.

[0083] Example 7

[0084] Preparation of Epimedium liposomes using sucrose as solid particles:

[0085] Weigh lecithin and add anhydrous ethanol solution to prepare 0.0189g / mL lecithin solution, ultrasonicate for 10 minutes, and store under light-proof conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat in a water bath at 50°C, then add solid particles to the lecithin solution at a mass ratio of sucrose to lecithin of 26.45:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL epimedium solution according to a mass ratio of epimedium extract to lecithin of 14.7:100, shake for 10min, and obtain epimedium liposome emulsion.

[0086] Example 8

[0087] Preparation of Epimedium liposomes using sucrose as solid particles:

[0088] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store under light-proof conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat in a water bath at 50°C, then add solid particles to the lecithin solution at a mass ratio of 4.17:1 of sucrose solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.15mg / mL of epimedium solution according to the mass ratio of the solid content of the empty liposomes to the epimedium solution, and shake for 10min to obtain an epimedium liposome emulsion.

[0089] Example 9

[0090] Preparation of Epimedium liposomes using sucrose as solid particles:

[0091] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store under light-proof conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat in a water bath at 50°C, then add solid particles to the lecithin solution at a mass ratio of 4.17:1 of sucrose solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 2.4mg / mL of epimedium solution according to the mass ratio of the solid content of the empty liposomes to the epimedium solution, and shake for 10min to obtain an epimedium liposome emulsion.

[0092] Example 10

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

[0094] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store under light-proof conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat in a water bath at 50°C, then add solid particles to the lecithin solution at a mass ratio of sucrose to lecithin of 2.5:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL of epimedium solution according to the mass ratio of the solid content of the empty liposomes to the epimedium solution, and shake for 10min to obtain an epimedium liposome emulsion.

[0095] Embodiment 11

[0096] Preparation of Epimedium liposomes with sucrose as solid particles: weigh lecithin and add anhydrous ethanol solution to prepare 0.02363g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat it in a water bath at 50°C, then add the solid particles to the lecithin solution at a mass ratio of sucrose solid phase particles to lecithin of 21.16:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL epimedium solution according to the mass ratio of the solid content of the empty liposomes to the epimedium solution, which is converted to a mass ratio of the solid content of the epimedium solution to the mass ratio of lecithin of 27.8:100, shake for 10 minutes, and obtain an epimedium liposome emulsion.

[0097] Example 12

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

[0099] Performance Test:

[0100] 1. Epimedium liposome encapsulation efficiency test:

[0101] Encapsulation efficiency test method:

[0102] Take 0.1mL of epimedium liposome emulsion in a common centrifuge tube, centrifuge at 8000rpm for 10min, take out 0.05mL of supernatant and add it to an ultrafiltration centrifuge tube, then add 0.35mL of purified water, ultrafiltration centrifuge for 10min (wherein, the ultrafiltration membrane pore size is 30KD, and the centrifugal speed is 6000rpm), take the supernatant (permeate) of the first ultrafiltration centrifugation for liquid chromatography analysis. Take the centrifugal sediment of common centrifugation and add 0.75mL of anhydrous ethanol, ultrasonically dissolve, take out 0.2mL and perform the second ultrafiltration centrifugation for 10min (wherein, the ultrafiltration membrane pore size is 30KD, and the centrifugal speed is 6000rpm), take the supernatant (concentrate) of the second ultrafiltration centrifugation for liquid chromatography analysis. According to the HPLC method, the peak area of ​​the icariin chromatographic peak of the permeate and the concentrate is detected, and the encapsulation rate (%) is calculated as the peak area of ​​the concentrate / (peak area of ​​the permeate+peak area of ​​the concentrate)×100%.

[0103] HPLC test conditions: bonded silica gel C 18 (4.6mm×250mm, 5μm), with acetonitrile (A)-aqueous solution (B) as the mobile phase, gradient elution (0-15min, B 10%-25%; 15-38min, B 25%; 38-61min, B 25%-71%; 61-75min, B 71%-100%), flow rate 1.0ml / min, column temperature 30℃, detection wavelength 270nm, injection volume: 10μL.

[0104] The properties of the epimedium liposomes obtained in the examples and comparative examples are summarized in Tables 1 and 2.

[0105] Table 1 Encapsulation efficiency of epimedium liposomes obtained when the solid phase particles are NaCl (%, encapsulation material is calculated based on lecithin)

[0106]

[0107] As shown in Table 1, the encapsulation rate of the epimedium liposomes prepared in the examples and comparative examples, it can be seen that: in Examples 1 to 5, the epimedium extract: encapsulation material dosage ratio is in the range of 7.4:100 to 12.4:100, and the encapsulation rate is 100%, and the encapsulation material can effectively encapsulate the epimedium extract; Comparative Example 1 does not use solid phase particles as carriers, and the encapsulation rate is greatly reduced, indicating that this method can more effectively encapsulate the active ingredients of the epimedium extract than the traditional rotary evaporation film forming method. Comparative Examples 2 and 3 increase the epimedium extract: encapsulation material dosage ratio to 104:100 and 65.89:100, and the encapsulation rate is greatly reduced, indicating that the drug loading capacity of the encapsulation material is exceeded, so the epimedium extract: encapsulation material dosage ratio is a key factor affecting the solute encapsulation effect. In Comparative Example 4, the epimedium extract was placed in the encapsulating material solution, and the film was formed and hydrated, and the encapsulation rate was reduced. It may be that the active ingredients in the epimedium extract participated in the film-forming process of the encapsulating material, and were doped in the film of the encapsulating material on the surface of the carrier particles, affecting the uniformity of the film, and further affecting the encapsulation effect of the epimedium extract. The encapsulation rate of Comparative Example 4 is poor, because during the spraying process of the film material solution, since the system is in a vacuum state, the film material solution is difficult to be effectively sprayed onto the surface of the solid phase particles to form a film, and more tiny spots are formed on the surface of the solid phase particles, and some of them form film material particles. Therefore, for the same amount of film material solution, the film-forming area of ​​Comparative Example 4 is much smaller than the film-forming area of ​​Examples 1 to 5, which affects the encapsulation efficiency.

[0108] Compared with Examples 1 to 5, without using solid phase particles, the encapsulation efficiency is much lower than that of using solid phase particles. Although the encapsulation efficiency of the existing method can reach 85-100% under the optimized experimental parameters, the preparation process is complicated, the amount of organic solvent is increased, and the preparation time is long, which is not conducive to large-scale preparation.

[0109] like Figure 1 , 2 As shown, they are HPLC spectra of the epimedium liposome permeate and concentrate prepared in Examples 1 to 4 and Comparative Examples 2 and 3, respectively. It can be seen from the figure that the permeate and the concentrate both 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 close to a straight line, the concentrate has a strong peak, indicating that the drug encapsulation effect is ideal.

[0110] Table 2 Encapsulation efficiency (%) of epimedium liposomes prepared with sucrose as solid phase particles (encapsulation material is calculated as lecithin)

[0111]

[0112] As shown in Table 2, the encapsulation rate data of the epimedium liposome prepared with sucrose as solid phase particles, as can be seen from Table 2: lecithin is the raw material for forming the liposome shell, and within a certain dosage range of lecithin and other conditions remain unchanged, the amount of lecithin is less, the number of liposomes formed is small, and then the embedding rate is poor; the amount of lecithin is too much, causing the lecithin to adhere to each other, unable to form a spherical shape with good morphology, small capacity for epimedium or large thickness of the liposome shell formed, causing a small amount of drugs to be difficult to enter, and the embedding rate is small. The encapsulation rate of the liposome obtained by the present invention is generally 84-100%, which has significant advantages over the prior art, can obtain a higher embedding rate within a wider parameter range, and the preparation process is simple, the amount of organic solvent is small, the preparation time is short, and it can be prepared on a large scale.

[0113] like Figure 4 , 5 As shown in the figure, they are the HPLC spectra of the epimedium liposome permeate and the concentrated solution prepared in Examples 6 to 11, respectively. It can be seen from the figure that the permeate and the concentrated solution have peaks at the icariin chromatographic peak (the retention time of the icariin chromatographic peak is about 37 min). The smaller the peak of the permeate and the stronger the peak of the concentrated solution, the better the drug encapsulation effect.

[0114] 2. Particle size determination of Epimedium liposomes:

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

[0116] 3. Transdermal diffusion experiment:

[0117] Using Strat-MTM Membrane (Merck Millipore Ltd) and a transdermal diffusion test apparatus, a PEG400 aqueous solution with a mass concentration of 40% was added to the diffusion cell, and 0.2 mL of the epimedium liposome emulsion and the epimedium extract aqueous solution (0.6 mg / mL) obtained in the examples or comparative examples were uniformly dispersed on the smooth surface of the artificial skin, and permeated at room temperature. After an interval of 12 hours, 0.2 mL of the permeated solution was taken out from the diffusion cell for liquid chromatography determination, and the volume was supplemented with a PEG400 aqueous solution with a mass concentration of 40%. The permeability was calculated by high performance liquid chromatography. The permeability is shown in Table 3.

[0118] Table 3 Particle size of Epimedium liposomes obtained in various examples and comparative examples

[0119]

[0120]

[0121] Analysis shows that the particle size of the liposomes after microfiltration is small, about 220nm, and the dispersion coefficient is small, relatively stable, and evenly dispersed. The average potential of the liposomes is measured to be negative, indicating that the surface of the liposome particles is negatively charged and has good stability. The average particle size is reduced, the particle size is more uniform, and the aggregation index PDI is reduced.

[0122] The TEM images of the epimedium liposome emulsion prepared in Example 2 after being filtered through a copper mesh and dried are as follows: Figure 5 As shown in the figure, it can be seen that the epimedium liposome is similar to a sphere, and its particle size is significantly smaller than the particle size in the epimedium liposome emulsion, which may be caused by the drying and water loss of the particles in the emulsion during the electron microscopy sample preparation process; and the epimedium liposome particles are more evenly distributed.

[0123] 4. Determination of DPPH free radical scavenging activity:

[0124] (1) Solution preparation:

[0125] DPPH-ethanol solution: weigh 1 mg DPPH, add 22.2 ml anhydrous ethanol, and dissolve by ultrasonication.

[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 up a test group, a control group, and a blank group.

[0129] Determination group: Take 80 μl of the permeate of the epimedium liposome transdermal diffusion test for 48 hours into a 96-well plate, add 120 μl of DPPH-ethanol solution, shake well, and stand at room temperature (25°C) in the dark for 10 minutes, and measure its absorbance, which is recorded as A1;

[0130] Control group: According to the method of the above-mentioned test group, the DPPH-ethanol solution was replaced by an equal volume of 60wt% ethanol solution, and its absorbance was measured, which was recorded as A2;

[0131] Blank group: Pipette 80 μL of 60% ethanol into a 96-well plate, add 120 μL of DPPH-ethanol solution, shake well, and stand at room temperature away from light for 10 min. Measure the absorbance and record it as A0.

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

[0133] The absorbance of the above samples was measured at 517 nm in an ELISA instrument, and the 48-hour free radical scavenging rate (i.e., inhibition rate) was calculated according to the following formula:

[0134]

[0135] Table 4 Antioxidant activity of epimedium extract liposomes prepared by the present invention

[0136]

[0137]

[0138] As shown in Table 4, certain artificial skin membrane permeation concentrations were detected in Examples 1-12 and Comparative Examples 1-5, and certain antioxidant activities were detected in the remaining Examples and Comparative Examples except Example 8 and Comparative Example 2. No antioxidant activity was detected in the permeate of the epimedium aqueous solution, and the measured permeability data was also 0, indicating that the unencapsulated epimedium extract aqueous solution could not permeate the artificial skin membrane, and its permeability could be greatly improved by encapsulating with lecithin to form liposomes.

Claims

1. An empty liposome, characterized in that: The liposome comprises soluble solid phase particles and a wrapping material, wherein the soluble solid phase particles are selected from 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 phase particles in the form of a membrane.

2. The empty liposome according to claim 1, characterized in that The thickness of the film is 2.5 to 16.7 μm; the particle size of the soluble solid phase particles is 150 to 1250 μm, preferably 500 to 800 μm, and more preferably 600 to 650 μm.

3. The method for preparing empty liposomes according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) adding a wrapping material into an anhydrous ethanol solution to prepare a wrapping material solution; (2) Immersing the soluble solid phase particles in the encapsulating material solution, removing the anhydrous ethanol, and combining the soluble solid phase particles with the encapsulating material to obtain empty liposomes.

4. The preparation method according to claim 3, characterized in that: In step (1), the encapsulating material is one of lecithin and a mixture of lecithin and cholesterol; the mass ratio of the lecithin and cholesterol mixture is 5 to 7:1; the mass concentration of the encapsulating material in the encapsulating solution is 0.0189 to 0.25 g / mL; preferably 0.15 to 0.2 g / mL. In step (2), the mass ratio of the soluble solid phase particles to the encapsulating material is (2.08-26.45):

1.

5. The preparation method according to claim 3, characterized in that: The soluble solid phase particles are NaCl, with a particle size of 600-700 μm, preferably 650 μm. The mass ratio of NaCl to the packaging material is (2.08-4.17):

1. The soluble solid phase particles are sucrose with a particle size of 550-650 μm, preferably 600 μm. The mass ratio of sucrose to the packaging material is (2.08-26.45):1, preferably (2.08-4.17):

1.

6. An epimedium extract liposome, characterized in that: The invention comprises 0.0149-0.22% of epimedium extract by weight, 0.05-2.2% of packaging material by weight, 0.47-7.31% of soluble solid phase particles by weight, and the balance of water.

7. The epimedium extract liposome according to claim 6, characterized in that The main component of the epimedium extract is icariin, and the mass percentage of icariin in the extract is more than 30.45%.

8. The epimedium extract liposome according to claim 6, characterized in that The solid phase particles are preferably NaCl, the particle size of the epimedium extract liposome is 117.8 to 172.5 nm, the aggregation index (PDI) is 0.307 to 0.482, and the average potential is -28.9 to -44.5 mV. The solid phase particles are preferably sucrose, and the particle size of the epimedium extract liposome is 116.83 to 197.9 nm, the aggregation index (PDI) is 0.248 to 0.368, and the average potential is -41.7 to -54.4 mV.

9. A method for preparing epimedium extract liposomes using soluble solid phase particles, characterized in that: The empty liposomes described in any one of claims 1 to 2 or the empty liposomes prepared by the method described in any one of claims 3 to 5 are mixed with an epimedium extract solution to obtain an epimedium extract liposome emulsion.

10. The preparation method according to claim 9, characterized in that: The mass ratio of the epimedium extract to the packaging material is (7.4-27.8):100; the solvent of the epimedium extract solution is water, and the concentration of the epimedium extract solution is 0.15-2.4 mg / mL.

11. The preparation method according to claim 9, characterized in that: When the solid phase particles are NaCl, the mass ratio of the epimedium extract to the encapsulating material is (7.4-12.4):100; when the solid phase particles are sucrose, the mass ratio of the epimedium extract to the encapsulating material is (7.4-27.8):

100.

12. The preparation method according to claim 9, characterized in that: The method comprises a granulation method, wherein the obtained epimedium extract liposome is subjected to one of the following methods: high-speed shearing, ultrasound, homogenization, and extrusion, preferably extrusion, and more preferably microfiltration extrusion.

13. Use of the epimedium extract liposome according to any one of claims 6 to 8 or the epimedium extract liposome prepared by the method according to any one of claims 9 to 12 in the preparation of a drug for treating osteoporosis.

14. The use according to claim 13, characterized in that The epimedium extract liposome is in the form of oral liquid or injection.

15. Use of the epimedium extract liposome according to any one of claims 6 to 8 or the epimedium extract liposome prepared by the method according to any one of claims 9 to 12 in the preparation of cosmetics, preferably in the preparation of anti-ultraviolet cosmetics, the cosmetics can be in liquid or paste form.

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