Nuciferine liposome emulsion as well as preparation method and application thereof

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to a nuciferine liposome emulsion, a preparation method and application thereof, sucrose / sodium chloride solid-phase particles and a wrapping material are combined through a dipping film forming method to prepare a no-load liposome, and the no-load liposome is coated with nuciferine to prepare the nuciferine liposome emulsion. The preparation method provided by the invention is simple in process; the raw materials are wide in source, green and environment-friendly, and do not affect the biological activity of active ingredients; the preparation process is mild in condition, does not need special equipment, does not change the pH value of the solution, and is suitable for transdermal oral administration; the dosage of an organic solvent is small, the encapsulation efficiency is high, and large-scale preparation is easy.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to a nuciferine liposome emulsion, a preparation method and application thereof. Background Art

[0002] Nuciferine is an aporphine alkaloid found in lotus leaves, which has multiple pharmacological activities such as lowering blood lipids, anti-inflammatory, anti-tumor, regulating sugar metabolism, antipsychotic, and weight loss. In addition, studies have shown that nuciferine also has anti-acetylcholinesterase activity, which can not only be developed as an anti-AD drug, but also can be used as an inhibitor to regulate AChE activity to treat skin pigmentation. Nuciferine has the characteristics of low solubility, fast elimination rate, and low bioavailability in the body, which to a certain extent limits the development and utilization of lotus leaf alkaloids. Liposomes are closed vesicles composed of lipid materials such as phospholipids and cholesterol. They have a phospholipid bilayer structure similar to that of cell membranes and the potential to enhance drug skin permeability. Currently, the commonly used methods for preparing liposomes include ethanol injection method, thin film dispersion method, high pressure homogenization method, freeze drying method, etc., which have the disadvantages of too complicated preparation process, high production cost, large amount of organic solvent, and difficulty in large-scale preparation. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention uses sucrose / sodium chloride solid phase particles as carriers for the first time, combines sucrose / sodium chloride solid phase particles with encapsulation materials by an impregnation film-forming method to prepare empty liposomes, and encapsulates the drug nuciferine to prepare nuciferine liposome emulsion. The preparation method provided by the present invention has a simple process; the raw material source is wide, green and environmentally friendly, and does not affect the biological activity of the active ingredient; the preparation process conditions are mild, no special equipment is required, the pH value of the solution is not changed, and it is suitable for transdermal and oral administration; the amount of organic solvent used is small, the encapsulation rate is high, and it is easy to prepare on a large scale.

[0004] To achieve the above object, the present invention provides a method for preparing a nuciferine liposome emulsion, comprising:

[0005] (a) adding a wrapping material into anhydrous ethanol to prepare a wrapping material solution;

[0006] (b) immersing the solid phase particles in a coating material solution, removing the anhydrous ethanol, and combining the solid phase particles with the coating material to obtain empty liposomes;

[0007] (c) mixing the empty liposomes with the nuciferine solution to obtain the nuciferine liposome emulsion.

[0008] The solid phase particles are one of sucrose and sodium chloride.

[0009] Wherein, the packaging material is one of lecithin and lecithin-cholesterol.

[0010] The solid phase particles utilize their huge specific surface area to allow the coating material solution to volatilize on the surface of the solid phase particles to form a film.

[0011] The particle size of the solid phase particles is 150 to 1250 μm. When the particle size is too large, the specific surface area is reduced, the film thickness is too large, and the encapsulation rate is too low; when the particle size is too small, the film thickness is too small and it is easy to break.

[0012] Preferably, the particle size of the sodium chloride solid phase particles is 600-700 μm;

[0013] Preferably, the particle size of the sucrose solid phase particles is 300-900 μm.

[0014] In step (b), the method for removing the anhydrous ethanol comprises one of evaporating in a water bath, blowing dry, and standing at room temperature;

[0015] The water bath drying includes normal pressure water bath drying and reduced pressure water bath drying.

[0016] In step (c), the empty liposomes and the nuciferine solution are mixed in a manner including: vortexing, shaking, and ultrasound;

[0017] The concentration of the encapsulating material in the encapsulating material solution is 0.1 to 0.2 g / mL; preferably 0.2 g / mL;

[0018] The mass ratio of the solid phase particles to the encapsulating material is (1.3-2.6):1; preferably (2.08-2.6):1.

[0019] The solvent of the nuciferine solution is water, and the concentration is 0.1-2 mg / mL;

[0020] More preferably, when the solid phase particles are sucrose, the concentration of the nuciferine solution is 0.3-0.6 mg / mL;

[0021] When the solid phase particles are sodium chloride, the concentration of the nuciferine solution is 0.6-2 mg / mL.

[0022] The mass ratio of the nuciferine drug to lecithin is (5.5-8.6):100.

[0023] In the prior art, thin film dispersion method is commonly used to prepare liposomes. During the preparation process, the solvent needs to be removed by rotary evaporation to form a thin film on the wall of the rotary evaporation bottle, thereby obtaining liposomes. However, the specific surface area of ​​the rotary evaporation bottle is small, and the concentration of the solution is too high in the later stage of solvent evaporation, resulting in a large difference in the thickness of the film on the inner surface of the rotary evaporation bottle, thereby resulting in a wide distribution of the particle size of the liposome particles formed later. In the present invention, the solid phase particles have a large specific surface area, and the film formation time and film formation rate of the encapsulating material on the surface of the solid phase particles are similar. Therefore, the thickness of the film on the surface of the solid phase particles is uniform and extremely thin, which can greatly increase the drug loading while rapidly encapsulating the drug.

[0024] In the traditional rotary evaporation spray method, the sprayed droplets form uneven and discontinuous spots on the surface of the solid phase particles, and the microspheres formed by the encapsulating material itself affect the drug loading rate, and the liposome particles formed are large and dispersed, usually more than microns. In the present invention, the solid phase particle impregnation film-forming method is used, and a high concentration of encapsulating material solution is used, so that the encapsulating material solution on the surface of the solid phase particles can not only be quickly evaporated to form a film, but also greatly reduce the amount of encapsulating material solution and solvent, improve the drug loading rate, and the liposome particles formed are smaller and more uniform in size, usually about 10 times smaller than the particle size prepared by the traditional rotary evaporation spray method.

[0025] The invention does not need to dissolve the nuciferine drug with anhydrous ethanol, but removes the anhydrous ethanol to obtain empty liposomes, which are then mixed with the nuciferine solution. On the one hand, the influence of the anhydrous ethanol on the nuciferine drug is reduced, and on the other hand, the heating time caused by removing the anhydrous ethanol is reduced, thereby avoiding the influence of heat on the stability of the nuciferine drug to a great extent.

[0026] In addition, the present invention first prepares empty liposomes and then mixes them with the nuciferol drug solution to obtain the nuciferol liposome emulsion. During the application process, empty liposomes can be prepared first, and the nuciferol liposome emulsion can be prepared immediately without considering the impact of storage and transportation conditions on the nuciferol drug.

[0027] The present invention also provides a nuciferine liposome emulsion prepared by the above preparation method, wherein the nuciferine liposome emulsion has a 48h transmittance of not less than 38%;

[0028] The encapsulation rate of the nuciferine liposome emulsion is 58.9% to 100%;

[0029] Furthermore, the encapsulation efficiency of the nuciferine liposome emulsion is measured by HPLC-ultrafiltration centrifugation method.

[0030] The ultrafiltration centrifugal speed is 6000-12000 rpm, and the centrifugal time is 10-20 minutes.

[0031] After granulation, the particle size of the nuciferine liposome is 90-230.1 nm, and the aggregation index (PDI) is 0.176-0.329.

[0032] The granulation method used for the nuciferine liposome is one of the following: ultrasound, shearing, homogenization, and extrusion, preferably extrusion, and more preferably microfiltration extrusion.

[0033] The present invention also provides an application of the nuciferine liposome emulsion prepared by the above preparation method in the preparation of anti-AD and weight loss drugs.

[0034] When the solid phase particles are sucrose, the nuciferine liposome emulsion can be prepared into an oral emulsion.

[0035] When the solid phase particles are sodium chloride, the nuciferine liposome emulsion can be prepared into an injection.

[0036] The nuciferine liposome emulsion does not contain an organic solvent and does not need to be dried, thereby reducing the effect of drying on the stability of the liposome.

[0037] The present invention also provides an application of the nuciferine liposome emulsion prepared by the above preparation method in the preparation of freckle-removing and whitening cosmetics.

[0038] Beneficial effects:

[0039] 1. The present invention adopts the immersion film-forming method for the first time, using sucrose / sodium chloride solid phase particles as carriers, combined with packaging materials, to prepare empty liposomes, and then prepares nuciferine liposomes by coating nuciferine. The preparation process is simple, the amount of organic solvent used is small, and it is easy to prepare on a large scale. The amount of anhydrous ethanol used in the present invention is about 10% of the prior art.

[0040] 2. The present invention uses solid phase particles as carriers, which are not only widely available and environmentally friendly, but also do not affect the biological activity of the active ingredients.

[0041] 3. The nuciferine liposomes provided by the present invention are spherical in shape, have a narrow particle size distribution range and uniform particle size, and have good stability and encapsulation efficiency.

[0042] 4. The present invention adopts solid phase particles as carriers to prepare nuciferine liposomes, which does not change the pH value of the emulsion and can be administered percutaneously or orally, thereby improving the bioavailability and stability of nuciferine in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 HPLC spectra of the permeate and concentrate of the nuciferine samples of Examples 1 and 2;

[0044] Figure 2 HPLC spectra of the permeate and concentrate of the nuciferine samples of Examples 6 and 7;

[0045] Figure 3 HPLC spectra of the permeate and concentrate of the nuciferine samples of Examples 8 and 9;

[0046] Figure 4 The HPLC spectra of the permeate and concentrate of the nuciferine samples of Comparative Examples 1 and 4;

[0047] Figure 5 This is the particle size distribution diagram of the nuciferine liposomes prepared in Example 2 after microfiltration and extrusion;

[0048] Figure 6 This is the particle size distribution diagram of the nuciferine liposomes prepared in Example 7 after microfiltration and extrusion;

[0049] Figure 7 This is a TEM image of the nuciferine liposomes prepared in Example 2;

[0050] Figure 8 This is the TEM image of the nuciferine liposomes prepared in Example 7. DETAILED DESCRIPTION

[0051] 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.

[0052] The lecithin described in the examples was purchased from Beijing Aoboxing Biological Co., Ltd., item number 01-021; anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; cholesterol was purchased from Sinopharm Chemical Reagent Co., Ltd.; anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; montmorillonite was provided by Dezhou Bocheng Pharmaceutical Co., Ltd.; the preparation method of TiO2 was referred to "Li Shitong, et al. Co. 2+ -Preparation and photocatalytic performance of TiO2 nanoparticles[J]. Applied Chemical Industry, 2005, 34(12):737-740”; artificial skin is Strat-MTM Membrane purchased from Merck Millipore Ltd; sucrose and sodium chloride are conventional commercial products.

[0053] The preparation method of the lotus leaf alkaloid is as follows: weigh the lotus leaf extract residue, soak it in acidic ethanol for 24 hours, spin dry, collect the leachate, adjust the pH value to neutral, refrigerate for 48 hours, centrifuge at 8000rpm, remove the ethanol by rotary evaporation of the supernatant, add the concentrated solution to a crystallizer, stir, and crystallize at 45°C for 24 hours; cool it step by step, cool it to 25°C for crystallization for 24 hours; cool it again to 15°C, maintain the temperature for crystallization for 3 days; centrifuge it at 3000r / min, wash the centrifugal sediment twice with distilled water, and vacuum dry it at 60°C for 24 hours to obtain gray-white granular crystals, which are the lotus leaf alkaloid sample.

[0054] Embodiment 1:

[0055] Preparation of nuciferine liposome emulsion using sucrose as solid phase particles:

[0056] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in the dark. Take sucrose (average particle size of 850μm) and add it to the lecithin solution at a mass ratio of 2.5:1 of sucrose solid phase particles and encapsulating material, stir while adding, and evaporate in a 50℃ normal pressure water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL of nelumboide solution at a mass ratio of 8.4:100 of nelumboide to lecithin, shake for 10min, and obtain nelumboide liposome emulsion.

[0057] Example 2

[0058] Preparation of nuciferine liposome emulsion using sucrose as solid phase particles:

[0059] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in the dark. Take sucrose (average particle size of 850μm) and add it to the lecithin solution at a mass ratio of 1.4:1 of sucrose solid phase particles and encapsulating material, stir while adding, evaporate in a 50℃ water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL of nelumboide solution at a mass ratio of 5.7:100 of nelumboide to lecithin, shake for 10min, and obtain nelumboide liposome emulsion.

[0060] Example 3

[0061] Preparation of nuciferine liposome emulsion using sucrose as solid phase particles:

[0062] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, store in dark, take sucrose (average particle size of 850μm) and add it to the lecithin solution according to the mass ratio of sucrose solid phase particles to encapsulating material of 1.4:1, stir while adding, evaporate in a 50℃ water bath to obtain empty liposomes. According to the mass ratio of nuciferine to lecithin of 5.7:100, add 0.1mg / mL nuciferine solution, shake for 10min, and obtain nuciferine liposome emulsion.

[0063] Example 4

[0064] Preparation of nuciferine liposome emulsion using sucrose as solid phase particles:

[0065] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, store in dark, take sucrose (average particle size of 850μm) and add it to the lecithin solution according to the mass ratio of sucrose solid phase particles to encapsulating material of 1.4:1, stir while adding, evaporate in a 50℃ water bath to obtain empty liposomes. According to the mass ratio of nuciferine to lecithin of 5.7:100, add 2mg / mL nuciferine solution, shake for 10min, and obtain nuciferine liposome emulsion.

[0066] Example 5

[0067] Preparation of nuciferine liposome emulsion using sucrose as solid phase particles:

[0068] The preparation conditions of this example are the same as those of Example 2, except that lecithin is replaced by lecithin-cholesterol.

[0069] Among them, the mass ratio of lecithin to cholesterol is 6:1.

[0070] Example 6

[0071] Preparation of nuciferine liposome emulsion using sodium chloride as solid phase particles:

[0072] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Add sodium chloride (average particle size of 650μm) to the lecithin solution at a mass ratio of 2.6:1 of sodium chloride solid phase particles to lecithin, stir while adding, evaporate in a 50℃ water bath to obtain empty liposomes. Take the empty liposomes, add 0.6mg / mL of lecithin solution at a mass ratio of 8.6:100 of lecithin solution solid content to lecithin, shake for 10min, and obtain lecithin liposome emulsion.

[0073] Example 7

[0074] Preparation of nuciferine liposome emulsion using sodium chloride as solid phase particles:

[0075] Weigh lecithin and add anhydrous ethanol solution to prepare a 0.2g / mL solution, ultrasonicate for 10 minutes, and store in dark conditions. Add sodium chloride (average particle size of 650μm) to the lecithin solution at a mass ratio of 2.08:1 of sodium chloride to packaging material, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Take the empty liposomes, add 0.6mg / mL of lecithin solution at a mass ratio of 7.4:100 of the solid content of the lecithin solution to lecithin, shake for 10 minutes, and obtain the lecithin liposome emulsion.

[0076] Embodiment 8:

[0077] Preparation of nuciferine liposome emulsion using sodium chloride as solid phase particles:

[0078] Weigh lecithin and add it to anhydrous ethanol solution to prepare a 0.2g / ml solution, ultrasonicate for 10 minutes, and store it in dark conditions. Weigh sodium chloride (average particle size of 650μm) and add it to the lecithin solution at a mass ratio of 1.3:1 between sodium chloride and packaging material, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Take the empty liposomes, add 0.6mg / ml of lecithin solution at a mass ratio of 5.5:100 between the solid content of the lecithin solution and lecithin, and shake for 10 minutes to obtain a lecithin liposome emulsion.

[0079] Embodiment 9:

[0080] Preparation of nuciferine liposome emulsion using sodium chloride as solid phase particles:

[0081] Weigh lecithin and add it to anhydrous ethanol solution to prepare a 0.2g / mL solution, ultrasonicate for 10 minutes, and store it in dark conditions. Weigh sodium chloride (average particle size of 650μm) and add it to the lecithin solution at a mass ratio of 1.3:1 between sodium chloride and packaging material, stir while adding, and evaporate in a 50°C water bath to obtain empty liposomes. Take the empty liposomes, add 0.1mg / mL of nuciferine solution at a mass ratio of 7.4:100 between the solid content of nuciferine solution and lecithin, and shake for 10 minutes to obtain nuciferine liposome emulsion.

[0082] Embodiment 10:

[0083] Preparation of nuciferine liposome emulsion using sodium chloride as solid phase particles:

[0084] Weigh lecithin and add anhydrous ethanol solution to prepare a 0.2g / mL solution, ultrasonicate for 10 minutes, and store in dark conditions. Add sodium chloride (average particle size of 650μm) to the lecithin solution at a mass ratio of 2.08:1 between solid phase particles and encapsulating materials, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Take the empty liposomes, add 2mg / mL of homemade lecithin solution at a mass ratio of 7.4:100 between the solid content of the lecithin solution and the mass ratio of lecithin, and shake for 10 minutes to obtain a lecithin liposome emulsion.

[0085] Comparative Example 1

[0086] The preparation conditions of this comparative example are the same as those of Example 2, except that no sucrose solid phase particles are added.

[0087] The specific preparation method is as follows: weigh lecithin and add anhydrous ethanol solution to prepare a 0.2g / ml lecithin solution, ultrasonicate for 10 minutes, and store in a dark environment. Put the lecithin solution in a rotary evaporator, evaporate it in a 50°C water bath, add 0.6mg / mL of lecithin aqueous solution according to the mass ratio of solid content of lecithin solution to 5.7:100, and ultrasonicate for 10 minutes to obtain lecithin liposome emulsion.

[0088] Comparative Example 2:

[0089] The preparation conditions of this comparative example are the same as those of Example 2, except that the sucrose solid phase particles are replaced by montmorillonite of the same particle size.

[0090] Comparative Example 3:

[0091] The preparation conditions of this comparative example are the same as those of Example 2, except that the sucrose solid phase particles are replaced with TiO2 of the same particle size.

[0092] Comparative Example 4:

[0093] Weigh lecithin and nuciferine and add them to anhydrous ethanol solution to obtain an organic solution of lecithin and nuciferine (wherein, the mass ratio of nuciferine to lecithin is 5.7:100, and the mass concentration of lecithin is 0.2g / mL), ultrasonicate for 10 minutes, and store in the dark. Take sucrose and place it in a rotary evaporator. Under the condition of heating at 50°C, add the organic solution of lecithin and nuciferine at a mass ratio of 1.4:1 of sucrose solid phase particles to encapsulating material, stir while adding, evaporate in a water bath, and then add deionized water to obtain a nuciferine liposome emulsion (wherein, the mass ratio of deionized water to nuciferine is 1000:0.6).

[0094] Comparative Example 5:

[0095] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark. Take sucrose and place it in a rotary evaporator, then spray lecithin solution into the rotary evaporator according to the mass ratio of sucrose solid phase particles to encapsulating material 1.4:1, the rotary evaporation temperature is 50°C, the vacuum degree is -0.098MPa, and empty liposomes are obtained. Take empty liposomes, add 0.6mg / mL of nuciferine solution according to the mass ratio of nuciferine to lecithin of 5.7:100, shake for 10min, and obtain nuciferine liposome emulsion.

[0096] Test example 1: Encapsulation efficiency test

[0097] The method for detecting the encapsulation rate of liposomes of nuciferine is as follows: 0.1 mL of liposomes of nuciferine is placed in a centrifuge tube (CF), centrifuged at 8000 rpm for 10 min, the supernatant is taken out and added to an ultrafiltration centrifuge tube (UF-CF, the pore size of the ultrafiltration membrane is 30KD), and then 0.3 mL of pure water is added, and the centrifugation time is 8000 rpm for 10 min, and the permeated liquid is taken as the test solution for free nuciferine detection (defined as the permeated liquid). 0.4 mL of anhydrous ethanol is added to the above CF, weighed, ultrasonically dissolved, added to the above UF-CF, ultrafiltration centrifuged at 8000 rpm for 10 min, and the permeated liquid is taken to make up the lost weight with anhydrous ethanol as the test solution for the detection of encapsulated nuciferine (defined as the concentrated solution).

[0098] HPLC conditions: bonded silica gel C 18 (4.6mm*250mm, 5μm), with acetonitrile (A)-0.1% triethylamine aqueous solution (B) as the mobile phase, gradient elution (0-20min, A% (45%~60%); 21-30min, A% 100%), flow rate 1.0mL·min, column temperature 30℃, detection wavelength: 270nm, injection volume: 10μL.

[0099] Wherein, encapsulation rate (%) = peak area of ​​concentrated solution / (peak area of ​​permeate+peak area of ​​concentrated solution)×100%.

[0100] like Figure 1 As shown, they are the HPLC spectra of the nuciferine liposome permeate and the concentrated solution of Examples 1-2, respectively. It can be seen from the figure that the nuciferine liposome permeate prepared in Examples 1-2 has basically no peak at the nuciferine chromatographic peak (about 20min), and the concentrated solution has a corresponding strong peak at the nuciferine chromatographic peak, indicating that the nuciferine is completely encapsulated.

[0101] like Figure 3 As shown, they are the HPLC spectra of the permeate and concentrate of the nuciferine liposome prepared in Examples 8 to 9, respectively. It can be seen from the figure that both the permeate and the concentrate have peaks at the nuciferine chromatographic peak (the retention time of the nuciferine chromatographic peak is about 20 min), but the peak of the permeate is smaller and close to a straight line, while the concentrate has a strong peak, indicating that the encapsulated drug leaks little and has strong encapsulation.

[0102] Table 1 Liposome encapsulation efficiency of nuciferine in various embodiments and comparative examples (%)

[0103]

[0104]

[0105] Table 1 shows the encapsulation efficiency of the nuciferine liposomes prepared in various examples and comparative examples.

[0106] Compared with Example 2, the amount of encapsulating material used in Example 1 is small, the number of liposomes formed is small, and the encapsulation efficiency is poor. Compared with Example 7, the amount of encapsulating material used in Example 8 is too much, the encapsulating materials adhere to each other, and cannot form a spherical shape with good morphology. The liposome encapsulation amount is small or the thickness of the liposome shell is large, making it difficult for a small amount of drug to enter.

[0107] The concentration of nuciferine has a great influence on the encapsulation rate. As shown in Examples 2, 3, and 4, compared with Example 2, the concentration of nuciferine in Example 3 is lower, and the volume of nuciferine solution is larger. Under the same dosage of empty liposomes, the concentration of liposomes in the solution is small and it is difficult to capture and embed all the nuciferine; compared with Example 2, the concentration of nuciferine in Example 4 is higher, the volume of nuciferine is smaller, and the concentration of the formed liposomes is higher, which will cause the encapsulation materials to adhere to each other and cannot form a spherical shape with a good morphology. The liposome embedding amount is small or the thickness of the formed liposome shell is large, which makes it difficult for a small amount of drugs to enter.

[0108] Comparative Example 1 uses the same amount of packaging material, but the encapsulation efficiency is poor. This is because solid phase particles are not used as carriers and the film-forming area is far less than that of Examples 1-10.

[0109] In Comparative Examples 2 and 3, due to the rough surface of montmorillonite and its strong adsorption capacity, the encapsulating material agglomerates on the surface of montmorillonite and cannot form a well-shaped sphere; since the surface of TiO2 is too smooth, the surface of the carrier is difficult to wet, resulting in poor adsorption of the TiO2 carrier on the encapsulating material, making it difficult to form a film on its surface.

[0110] Comparative Examples 4 and 5 have poor encapsulation efficiency compared to Examples 1 to 10. This is because in the process of preparing nuciferin liposomes by thin film dispersion method in Comparative Example 4, the film formed by the wrapping material on the wall of the rotary evaporation bottle is generally thick and uneven, resulting in a low encapsulation efficiency of nuciferin liposomes. The encapsulation efficiency of Comparative Example 5 is poor because during the spraying process of the membrane material solution, since the system is in a vacuum state, it is difficult for the membrane material solution to be effectively sprayed onto the surface of the solid phase particles to form a film. More tiny spots are formed on the surface of the solid phase particles, and some membrane material particles are formed. Therefore, for the same amount of membrane material solution, the film-forming area of ​​Comparative Example 6 is much smaller than that of Examples 1 to 10.

[0111] Test Example 2: Determination of Particle Size

[0112] 0.1 mL of the nuciferine liposome solution prepared in each of Examples 2 and 7 was taken and diluted to 1 mL with water. The particle size of the nuciferine liposome before microfiltration and extrusion was measured by a laser particle size analyzer ZetasizerNano ZS90.

[0113] The nuciferine liposome emulsion prepared in Example 2 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 the particle size of the nuciferine liposome after microfiltration extrusion was measured by a laser particle size analyzer Zetasizer Nano ZS90.

[0114] The nuciferin liposomes prepared in Example 2 had an average particle size of 170 nm and a PDI of 0.227 before microfiltration extrusion; after granulation, the average particle size and PDI were reduced to 112.7 nm and 0.223, respectively. The particle size and polydispersity coefficient of the liposomes after microfiltration extrusion in Example 7 were 90 nm and 0.176, respectively, indicating that the microfiltration extrusion effect was obvious. The particle size distribution diagram of the nuciferin liposomes prepared in Examples 2 and 7 after microfiltration extrusion is shown in Figure 2. Figure 5 , Figure 6 The average potentials of the liposomes prepared in Examples 2 and 7 after microfiltration extrusion were measured to be -65.1mv and -41.0mv, respectively, indicating that the surface of the liposome particles was negatively charged, and the absolute value of the potential was between 30-80, which was relatively stable. The particle size detection method of the nuciferine liposomes after microfiltration extrusion in the remaining Examples and Comparative Examples was the same as that in Examples 2 and 7, and the specific results are shown in Table 2.

[0115] The TEM images of the nuciferine liposome emulsions prepared in Example 2 and Example 7 after being filtered through a copper mesh and dried are as follows: Figure 7 , 8 As shown in the figure, it can be seen that the nuciferine liposomes prepared in Examples 2 and 7 are similar to spheres, and their particle size is significantly smaller than the particle size of the particles in the nuciferine 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 nuciferine liposome particles are distributed more evenly.

[0116] Test Example 3: Transdermal Diffusion Experiment

[0117] Using a transdermal diffusion test device, a 40wt% PEG400 aqueous solution was added to the diffusion cell, and 0.3mL of each of the nuciferine liposome emulsion and nuciferine aqueous solution prepared in each embodiment and comparative example was evenly applied on the smooth surface of the artificial skin, and permeated at room temperature to obtain a nuciferine liposome permeation solution and a nuciferine aqueous solution permeation solution, respectively. At intervals of 6 and 48 hours, 0.2mL of the sample solution was taken out from the diffusion cell each time for liquid chromatography determination, and the volume was supplemented with a 40wt% PEG400 aqueous solution, and the high performance liquid chromatography was used to determine the permeability, and the permeability was calculated.

[0118] Table 2 Transmission rate, average particle size and potential of the nuciferine liposomes prepared in each embodiment and comparative example at different time

[0119]

[0120]

[0121] As shown in Table 2, the transmittance, average particle size and potential of the nuciferine liposomes prepared in each embodiment and comparative example at different times are shown, wherein "ND" means not detected and "-" means not detected. It can be seen from the table that the average particle size of Examples 1 to 10 is between 90 and 123.1 nm, indicating that liposomes with smaller particle sizes can be obtained using sucrose and sodium chloride as solid phase particle carriers, and the wide PDI range may be related to the pore size distribution of the microfiltration membrane.

[0122] Test example 4: ACE enzyme activity test:

[0123] 1. Solution preparation

[0124] Phosphate buffered saline (PBS): Take 1.36 g of potassium dihydrogen phosphate, add 29.1 mL of 0.1 mol / L sodium hydroxide solution, and dilute with water to 100 mL.

[0125] DTNB solution (0.01 M): Dissolve 3.9 mg DTNB in ​​1 mL DMSO and store at 4°C in the dark.

[0126] ATCI solution: Dissolve 108.35 mg of acetylthiocholine iodide (ATCI) in 4 mL of water and store at 4°C.

[0127] AChE solution: Dissolve 3 mg of acetylcholinesterase (AChE) in 3.5 mL of PBS and store at 4°C.

[0128] Huperzine A solution: Dissolve 2 mg of Huperzine A in 1 mL of water and dilute to 0.01 mg / mL.

[0129] 2. Experimental steps

[0130] The experiment set up a blank reference group, a blank control group, a sample reference group, and a sample group.

[0131] Sample group: 5 μL sample solution, 170 μL phosphate buffer (0.1 mol / L, pH 7.0), 15 μL AChE solution and 10 μL DTNB solution were added to the wells in sequence, shaken thoroughly, and placed at 4°C for 20 min. After 20 min, 10 μL ATCI solution was added dropwise, and the mixture was reacted in a water bath at 37°C for 20 min. The absorbance S was measured at 405 nm in an ELISA reader.

[0132] The sample solutions are respectively: the permeation solution of Examples 1-10 obtained in Detection Example 3, a nuciferine aqueous solution, and a huperzine A solution (10 μg / mL).

[0133] Sample reference group: 15 μL of PBS solution replaced 15 μL of AChE solution, and the other steps were the same as the sample group, and the absorbance SR was measured.

[0134] Blank reference group: 175 μL of PBS solution replaced 5 μL of sample solution and 170 μL of phosphate buffer, and the other steps were the same as the sample group, and the absorbance BR was measured.

[0135] Blank control group: 20 μL of dimethyl sulfoxide (DMSO) and 140 μL of PBS solution replaced 5 μL of sample solution and 170 μL of phosphate buffer. Other steps were the same as the sample group, and the absorbance BC was measured.

[0136] The inhibition rate was calculated as follows:

[0137]

[0138] Table 3 Acetylcholinesterase inhibition rate

[0139]

[0140]

[0141] As shown in Table 3, the inhibition rates of acetylcholinesterase by the nuciferine sample, the nuciferine liposome permeate, the nuciferine aqueous solution and the huperzine A solution are shown respectively. The concentrations are the permeate concentrations of each embodiment, the permeate concentrations of the nuciferine aqueous solution and the huperzine A reference substance concentration, which are obtained by high performance liquid chromatography. According to the inhibition rate of the huperzine A reference substance, it can be seen that the experimental results are reliable. Both Example 1-0 and the nuciferine aqueous solution have a certain inhibitory effect on the activity of acetylcholinesterase, and since the transdermal effect of nuciferine after encapsulation is greatly improved, the inhibition rate of nuciferine on acetylcholinesterase after transdermalization is improved.

Claims

1. A method for preparing a nuciferine liposome emulsion, characterized in that: The preparation method comprises: (a) adding a wrapping material into anhydrous ethanol to prepare a wrapping material solution; (b) immersing the solid phase particles in a coating material solution, removing the anhydrous ethanol, and combining the solid phase particles with the coating material to obtain empty liposomes; (c) mixing the empty liposomes with the nuciferine solution to obtain a nuciferine liposome emulsion; The solid phase particles are one of sucrose and sodium chloride.

2. The method for preparing the nuciferine liposome emulsion according to claim 1, characterized in that: The particle size of the solid phase particles is 150 to 1250 μm; Preferably, the particle size of the sodium chloride solid phase particles is 600-700 μm; Preferably, the particle size of the sucrose solid phase particles is 300-900 μm.

3. The method for preparing the nuciferine liposome emulsion according to claim 1, characterized in that: In step (b), the removal method of the anhydrous ethanol includes evaporation in a water bath, air drying, and standing at room temperature.

4. The method for preparing the nuciferine liposome emulsion according to claim 1, characterized in that: The packaging material is one of lecithin and lecithin-cholesterol.

5. The method for preparing the nuciferine liposome emulsion according to claim 4, characterized in that: The concentration of the encapsulating material in the encapsulating material solution is 0.1 to 0.2 g / mL; The mass ratio of the solid phase particles to the encapsulating material is (1.3-2.6):1; preferably (2.08-2.6):1; The mass ratio of the nuciferine drug to lecithin is (5.5-8.6):

100.

6. The method for preparing the nuciferine liposome emulsion according to claim 1, characterized in that: The solvent of the nuciferine solution is water; The concentration of the nuciferine solution is 0.1-2 mg / mL. Preferably, when the solid phase particles are sucrose, the concentration of the nuciferine solution is 0.3-0.6 mg / mL; Preferably, when the solid phase particles are sodium chloride, the concentration of the nuciferine solution is 0.6-2 mg / mL.

7. A nuciferine liposome emulsion prepared by the preparation method according to claim 1, characterized in that: The encapsulation rate of the nuciferine liposome emulsion is 58.9% to 100%.

8. The nuciferine liposome emulsion according to claim 7, characterized in that: The 48h permeability of the nuciferine liposome emulsion is not less than 38%; The particle size of the nuciferine liposome is 90-230.1 nm, and the PDI is 0.176-0.

329.

9. Use of the nuciferine liposome emulsion prepared by the preparation method according to any one of claims 1 to 6 or the nuciferine liposome emulsion according to any one of claims 7 to 8 in the preparation of anti-AD and weight loss drugs, characterized in that: When the solid phase particles are sucrose, the nuciferine liposome emulsion can be made into an oral emulsion; When the solid phase particles are sodium chloride, the nuciferine liposome emulsion can be prepared into an injection.

10. Use of the nuciferine liposome emulsion prepared by the preparation method according to any one of claims 1 to 6 or the nuciferine liposome emulsion according to any one of claims 7 to 8 in the preparation of freckle-removing and whitening cosmetics.

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