Nuciferine liposome prepared by using silica gel as carrier as well as preparation method and application of nuciferine liposome

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to nuciferine lipidosome prepared by using silica gel as a carrier as well as a preparation method and application of the nuciferine lipidosome. Silica gel solid-phase particles and a coating material are combined together by adopting a dipping film forming method to prepare a no-load liposome, and nuciferine is coated to prepare the nuciferine liposome. The preparation method provided by the invention is mild in wrapping process conditions, does not need special equipment, does not change the pH value of the solution, and is suitable for transdermal oral administration; in addition, the preparation method provided by the invention is simple in process, low in organic solvent consumption, high in encapsulation efficiency and easy for large-scale preparation.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to a nuciferine liposome prepared by using silica gel as a carrier, a preparation method and an 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-free radicals, anti-tumor, inhibiting hypercholesterolemia, anti-fatty liver, and weight loss. Nuciferine has the characteristics of short half-life, fast elimination rate, high excretion rate and low bioavailability in the body, which is not conducive to the development and utilization of lotus leaf alkaloids. How to select suitable drug carriers to improve the bioavailability of drugs and increase the stability of drug release is of great research significance.

[0003] As an important drug delivery system, liposomes have attracted widespread attention in drug delivery and therapy. Liposomes are tiny droplets surrounded by one or more lipid bilayers, which can be used to encapsulate and deliver various drugs, and also have good biocompatibility and stability. The outer surface of liposomes can be modified to have specific targeting, thereby achieving targeted drug delivery. Compared with traditional drug delivery forms, liposomes can improve the bioavailability of drugs, reduce adverse reactions, and improve therapeutic effects. Common preparation methods for liposomes include ethanol injection method, thin film dispersion method, ultrasonic dispersion method, melting method, reverse evaporation method, etc. The existing liposome preparation process is too cumbersome, the production cost is too high, a large amount of organic solvents are used, and it is difficult to achieve large-scale preparation. Researchers are still exploring new liposome preparation methods to improve their performance and stability. Summary of the invention

[0004] The existing liposome preparation process is complicated, the production cost is too high, the amount of organic solvent used is large, and it is difficult to prepare on a large scale. The present invention discloses a nuciferine liposome prepared by using silica gel as a carrier, a preparation method and its application. The silica gel solid phase particles are combined with the encapsulation material by an impregnation film-forming method to prepare empty liposomes, and nuciferine is encapsulated to prepare nuciferine liposomes. Silica gel is widely available, green and environmentally friendly, and does not affect the biological activity of the active ingredients. The encapsulation process conditions are mild, no special equipment is required, and the pH value of the solution is not changed, which is suitable for transdermal and oral administration. In addition, the preparation method provided by the present invention is simple in process, uses less organic solvent, has a high encapsulation rate, and is easy to prepare on a large scale.

[0005] To achieve the above object, the present invention provides a method for preparing nuciferine liposomes using silica gel as a carrier, comprising:

[0006] (a) adding a wrapping material into an anhydrous ethanol solution to prepare a wrapping material solution;

[0007] (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;

[0008] (c) mixing the empty liposomes with the nuciferine solution to obtain a nuciferine liposome suspension.

[0009] The encapsulating material is one of lecithin and lecithin-cholesterol;

[0010] The solid phase particles are silica gel solid phase particles; the huge specific surface area of ​​the silica gel solid phase particles is utilized to make the wrapping material solution volatilize on the surface of the solid phase particles to form a wrapping material film with a huge specific surface area, thereby quickly dispersing in the nuciferine aqueous solution to achieve the purpose of wrapping.

[0011] The particle size of the silica gel is 48 to 150 μm, preferably 48 to 74 μm.

[0012] The method for removing the anhydrous ethanol is one of heating in a water bath to evaporate, freeze drying, and standing at room temperature.

[0013] Preferably, in step (c), the empty liposomes and the nuciferine solution are mixed in a manner comprising: vortexing, shaking, and ultrasound.

[0014] The mass concentration of the coating material in the coating solution is 0.01 to 0.2 g / mL, preferably 0.1 to 0.2 g / mL.

[0015] The mass ratio of the solid phase particles to the encapsulating material is (2-3):1;

[0016] The mass ratio of the solid content of the nuciferine solution to lecithin is (7.2-9.6):100;

[0017] The solvent of the nuciferine solution is water, and the concentration of the nuciferine is 0.1-2 mg / mL, preferably the concentration of the nuciferine is 0.6-2 mg / mL.

[0018] Preferably, in step (c), the nuciferine solution contains hydrolyzed sodium hyaluronate.

[0019] More preferably, the mass of the hydrolyzed sodium hyaluronate accounts for no more than 2% of the total mass of the nuciferine liposome suspension.

[0020] In the traditional rotary evaporation method, the coating material cannot be evenly spread in the rotary evaporation bottle, but the present invention uses silica gel solid phase particles as carriers, and immerses them in a high concentration of coating material solution, so that the coating material can still be spread on the surface of the silica gel solid phase particles. Therefore, the present invention can not only make the coating material solution on the surface of the silica gel solid phase particles evaporate quickly to form a film, reduce the amount of coating material and anhydrous ethanol, and increase the drug loading, but also prepare nuciferine liposomes with small and uniform particle size.

[0021] In addition, if the immersion film-forming method is replaced by the spray method, the small droplets sprayed by the spray method will form uneven and discontinuous spots on the surface of the silica gel solid phase particles, and the nuciferine liposomes formed will have larger and more dispersed particles.

[0022] The present invention also provides a nuciferol liposome prepared by the above preparation method, wherein the nuciferol liposome has a 48h permeability of not less than 30%;

[0023] The encapsulation rate of the nuciferine liposome is 83.9% to 98.6%.

[0024] Furthermore, the encapsulation efficiency of the nuciferine liposomes was measured by HPLC-centrifugation method;

[0025] The centrifugation is performed at 6000-12000 rpm for 10-20 min, preferably at 8000 rpm for 10 min.

[0026] The present invention also provides an application of the nuciferine liposome suspension prepared by the above preparation method in the preparation of anti-AD and weight loss drugs.

[0027] Preferably, the dosage form of the nuciferine liposome is a tablet.

[0028] The present invention also provides an application of the nuciferine liposome suspension prepared by the above preparation method in the preparation of whitening and spot-lightening skin care products.

[0029] Preferably, the dosage form of the nuciferine liposome is an ointment.

[0030] Beneficial Effects

[0031] 1. In view of the problem that the thickness of the film in the rotary evaporator is different in the traditional thin film dispersion method for preparing liposomes, which leads to a wide distribution of liposome particle sizes, the present invention uses silica gel solid phase particles as carriers for the first time, combines them with encapsulating materials to prepare empty liposomes, and then adds nuciferine solution to prepare nuciferine liposome suspension. Since the silica gel solid phase particles have a large specific surface area, the film formed by the encapsulating material on the surface of the silica gel solid phase particles is uniform and extremely thin, so that the prepared nuciferine liposomes are spherical and have a narrow particle size distribution range.

[0032] 2. In view of the problem of instability of liposomes in the prior art due to long drying time during the drying process of liposomes, the present invention adopts silica gel solid phase particles as carriers and utilizes the high density of silica gel to completely separate liposomes from the dispersed system through only a short-time, low-speed centrifugation operation, thereby greatly reducing the risk of instability of liposomes during the drying process.

[0033] 3. The present invention does not need to dissolve the nuciferine drug with anhydrous ethanol, but removes the anhydrous ethanol to obtain empty liposomes, and then mixes with the nuciferine drug solution, which reduces the influence of anhydrous ethanol on the nuciferine drug on the one hand, and reduces the heating time caused by removing anhydrous ethanol on the other hand, and greatly avoids the influence of heat on the stability of the nuciferine drug. In addition, due to the huge specific surface area of ​​the silica gel solid phase particles, the utilization rate of the packaging material is improved, so a large amount of packaging material is not required, and a large amount of anhydrous ethanol is not required for dissolution, thereby reducing costs and reducing preparation time.

[0034] 4. The present invention first prepares empty liposomes and then mixes them with the nuciferol drug solution to obtain the nuciferol liposome suspension. During the application process, empty liposomes can be prepared first, and the nuciferol liposome suspension can be prepared immediately without considering the impact of storage and transportation conditions on the nuciferol drug.

[0035] 5. The present invention forms a uniform and continuous film on the surface of the silica gel solid phase particles by immersing the silica gel solid phase particles in the coating material solution, rather than the traditional rotary evaporation spray method. This not only avoids the formation of uneven and discontinuous spots on the surface of the silica gel solid phase particles by droplets, but also avoids the formation of microspheres by the coating material itself that affects the drug loading. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 HPLC spectra of the permeate and concentrate of the nuciferine samples of Examples 1 and 2;

[0037] Figure 2 HPLC spectra of the permeate and concentrate of the nuciferine samples of Examples 3-5 and Comparative Example 1;

[0038] Figure 3 This is a microscope image (40X) of the silica gel aqueous dispersion in Example 1;

[0039] Figure 4 This is a microscopic image (40X) of the silica gel empty liposome aqueous dispersion in Example 1;

[0040] Figure 5 This is a microscope image (40X) of the silica gel nuciferine liposome suspension in Example 1. DETAILED DESCRIPTION

[0041] 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.

[0042] Lecithin was purchased from Beijing Aoboxing Biological Co., Ltd.; anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; silica gel was manufactured by Haihua Desiccant Factory, Shibei District, Qingdao City, model HG / T2354-92; hydrolyzed sodium hyaluronate was purchased from Shandong Baifu Freda Pharmaceutical Co., Ltd., with a molecular weight of 400 Da; artificial skin was Strat-MTM Membrane purchased from Merck Millipore Ltd; the remaining raw materials were all commercially available products unless otherwise specified.

[0043] 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 the crystallizer, adjust the high stirring speed, 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 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 for 24 hours to obtain gray-white granular crystals, which are the lotus leaf alkaloid sample (detected by HPLC area normalization method, the purity of the lotus leaf alkaloid sample is 72.9%).

[0044] Embodiment 1:

[0045] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, store under light-proof conditions, weigh silica gel solid phase particles (particle size 200-300 mesh) and place in an evaporating dish, heat in a water bath at 50°C, then add lecithin solution according to a mass ratio of 3:1 of silica gel solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add a 0.6mg / mL nuciferine aqueous solution according to a mass ratio of 9.6:100 of nuciferine to lecithin, add hydrolyzed sodium hyaluronate powder, the hydrolyzed sodium hyaluronate powder accounts for 2% of the total mass of the nuciferine liposome suspension, shake for 10 minutes, and obtain a nuciferine liposome suspension.

[0046] Microscope images (40X) of silica gel aqueous dispersion, silica gel empty liposome aqueous dispersion and silica gel nuciferine liposome suspension are shown in Figure 3 to Figure 5 .

[0047] Embodiment 2:

[0048] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, store in dark conditions, weigh silica gel solid phase particles (particle size 200-300 mesh) and place in an evaporating dish, heat in a water bath at 50°C, then add lecithin solution at a mass ratio of 2:1 of silica gel solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take the empty liposomes, add 0.6mg / mL of lecithin aqueous solution at a mass ratio of lecithin to lecithin of 7.2:100, shake for 10 minutes, and obtain a lecithin liposome suspension.

[0049] Embodiment 3:

[0050] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, store in dark conditions, weigh silica gel (particle size 200-300 mesh) and place in an evaporating dish, heat in a water bath at 50°C, then add lecithin solution at a mass ratio of 2:1 between solid phase particles and encapsulating material, stir while adding, evaporate in a water bath to obtain empty liposomes. Take the empty liposomes, add 0.1mg / mL of nelumboide aqueous solution at a mass ratio of nelumboide to lecithin of 7.2:100, shake for 10 minutes, and obtain nelumboide liposome suspension.

[0051] Embodiment 4:

[0052] Weigh lecithin and add it to anhydrous ethanol solution to prepare a 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store it in a dark environment. Weigh silica gel solid phase particles (particle size 200-300 mesh) and place them in an evaporating dish, heat them in a water bath at 50°C, then add them to the lecithin solution at a mass ratio of 2:1 of silica gel solid phase particles to encapsulating material, stir while adding, evaporate in a water bath to obtain empty liposomes. Take the empty liposomes, add 2mg / mL of lecithin aqueous solution at a mass ratio of lecithin to lecithin of 7.2:100, shake for 10 minutes, and obtain a lecithin liposome suspension.

[0053] Embodiment 5:

[0054] The preparation conditions of this example are the same as those of Example 2, except that lecithin is replaced by lecithin-cholesterol.

[0055] Among them, the mass ratio of lecithin to cholesterol is 6:1.

[0056] Comparative Example 1:

[0057] The preparation conditions of this comparative example are the same as those of Example 2, except that no silica gel solid phase particles are added, and the mass ratio of nuciferine to lecithin is 5.7:100.

[0058] Comparative Example 2:

[0059] 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 7.2:100, and the mass concentration of lecithin is 0.2 g / mL), ultrasonicate for 10 minutes, and store in the dark. Take silica gel solid phase particles and place them in a rotary evaporator. Under the condition of heating at 50°C, add the organic solution of lecithin and nuciferine in a mass ratio of 2:1 of silica gel solid phase particles to encapsulating material, evaporate to dryness, and then add deionized water to obtain a nuciferine suspension (wherein, the mass ratio of deionized water to nuciferine is 1000:0.6).

[0060] Comparative Example 3:

[0061] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Weigh silica gel and place it in a rotary evaporator, then spray the lecithin solution into the rotary evaporator at a mass ratio of 2:1 between solid phase particles and encapsulating material, heat the rotary evaporator at 50°C, and the vacuum degree is -0.098MPa to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL of nuciferine solution according to the mass ratio of nuciferine to lecithin 7.2:100, shake for 10min, and obtain nuciferine liposome suspension.

[0062] Comparative Example 4:

[0063] The preparation conditions of this comparative example are the same as those of Example 2, except that the silica gel solid phase particles are replaced by montmorillonite of the same particle size.

[0064] Comparative Example 5:

[0065] The preparation conditions of this comparative example are the same as those of Example 2, except that the silica gel solid phase particles are replaced with TiO2 of the same particle size.

[0066] Test example 1:

[0067] The method for detecting the encapsulation rate of nuciferine liposome is as follows: after centrifuging 0.2 mL of nuciferine liposome suspension at 8000 rpm for 10 min, the supernatant (permeate) is taken out, and the supernatant is filtered through a 0.45 micron filter membrane and then analyzed by liquid chromatography. After adding 0.15 mL of 60% ethanol by volume to the centrifugal sediment, ultrasonically dissolve it, centrifuge it for 10 min, take out the supernatant (concentrate), and filter it through a 0.45 micron filter membrane and then analyze it by liquid chromatography.

[0068] Since silica gel solid phase particles are not used as carriers in Comparative Example 1, effective data cannot be obtained by the above encapsulation rate detection method. Therefore, the encapsulation rate detection method of the nuciferine liposome prepared in Comparative Example 1 is as follows: 0.1 mL of nuciferine liposome suspension is placed in a centrifuge tube, and after ordinary centrifugation at 8000 rpm for 10 min, the supernatant is taken out and added to an ultrafiltration centrifuge tube, and 0.3 mL of pure water is added. After the first ultrafiltration centrifugation for 10 min (wherein, the ultrafiltration membrane specification is 30KD, and the centrifugal speed is 8000 rpm), the permeated liquid of the first ultrafiltration centrifugation is taken as the test solution for free nuciferine detection (defined as the permeated liquid). Take the centrifugal sediment of ordinary centrifugation, add 0.4 mL of anhydrous ethanol, ultrasonically dissolve, add to the ultrafiltration centrifuge tube of the first ultrafiltration centrifugation, ultrafiltration centrifugation at 8000 rpm for 10 min, take the permeated liquid and supplement the weight lost with anhydrous ethanol, as the test solution for the detection of nuciferine (defined as the concentrated solution).

[0069] Wherein, encapsulation rate (100%) = amount of encapsulated nuciferine / (amount of free nuciferine+amount of encapsulated nuciferine)×100%.

[0070] Liquid chromatography 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.

[0071] Table 1 Nuciferine liposome encapsulation efficiency (%)

[0072]

[0073]

[0074] As shown in Table 1, the encapsulation rates of the nuciferine liposomes obtained by testing the various examples and comparative examples are shown. It can be seen from Table 1 that the encapsulation rates of Examples 1-5 are between 83.9% and 98.6%, and the encapsulation effect is ideal. It can be seen from Examples 2, 3, and 4 that when the concentration of the nuciferine solution is too low, the encapsulation rate is poor. Compared with Example 2, the encapsulation rate of Example 5 is reduced, which is because cholesterol is added to the encapsulation material, resulting in a slightly lower density of the lecithin film.

[0075] In Comparative Example 1, since silica gel solid phase particles were not used as carriers, the thickness of the formed lecithin film was relatively large and uneven, resulting in a significant decrease in the encapsulation efficiency.

[0076] Comparative Examples 2 and 3 have poorer encapsulation rates than Examples 1 to 5. This is because in the process of preparing nuciferin liposomes by thin film dispersion method in Comparative Example 2, 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 rate of nuciferin liposomes. The encapsulation rate of Comparative Example 3 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 3 is much smaller than that of Examples 1 to 5.

[0077] In Comparative Examples 4 and 5, 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.

[0078] like Figure 1 , 2 As shown, the HPLC spectra of the nuciferine liposome permeate and concentrate prepared in Examples 1 and 2 and the HPLC spectra of the nuciferine liposome permeate and concentrate prepared in Examples 3-5 and Comparative Example 1, respectively, can be seen from the figure that the nuciferine liposome permeate and concentrate prepared in Examples 1-5 have peaks at the nuciferine chromatographic peak, but the corresponding chromatographic peak of the permeate is extremely small, while the concentrate has a strong peak, indicating that the nuciferine liposome prepared in Examples 1-5 has an ideal encapsulation effect. However, the nuciferine liposome permeate prepared in Comparative Example 1 has a strong nuciferine chromatographic peak, and the encapsulation effect is not good.

[0079] Test Example 2: 48h Transdermal Diffusion Experiment

[0080] Using a transdermal diffusion test device, a PEG400 solution with a mass concentration of 40% was added to a diffusion cell, and 0.3 mL of a nuciferine liposome solution and a nuciferine aqueous solution with a mass concentration of 0.6 mg / mL were 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. The preparation method of the nuciferine liposome solution is as follows: the nuciferine liposome suspension prepared in Example 2 was centrifuged (8000 rpm, 5 min), the supernatant was discarded, and 5% Tween 20 was added to obtain 0.3 mL of a nuciferine liposome solution. 0.2 mL of the sample solution was taken out from the diffusion cell at intervals of 48 h for liquid chromatography determination, and the volume was supplemented with a PEG400 solution with a mass concentration of 40%, and the high performance liquid chromatography analysis was performed to calculate the permeability.

[0081] Test Example 3: 12h Transdermal Diffusion Experiment

[0082] Using a self-made transdermal diffusion test device, a PEG400 solution with a mass concentration of 40% was added to the diffusion cell, and 0.3 mL of the nuciferine liposome solution was evenly applied on the skin of mice in vitro, and the solution was permeated at room temperature to obtain a nuciferine liposome permeation solution, wherein the preparation method of the nuciferine liposome solution is as follows: the nuciferine liposome suspension prepared in Example 2 was centrifuged (8000rmp, 5min), the supernatant was discarded, and 0.3 mL of the nuciferine liposome solution was obtained. 0.2 mL of the sample solution was taken out from the diffusion cell at intervals of 12h for liquid chromatography determination, and the volume was supplemented with a PEG400 solution with a mass concentration of 40%, and the permeability was calculated by high performance liquid chromatography.

[0083] Test Example 4: Acetylcholinesterase Inhibitory Activity

[0084] 1. Solution preparation

[0085] 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.

[0086] Nuciferine solution: Take an appropriate amount of nuciferine, weigh accurately, and dissolve it in water to prepare a 0.072 mg / mL nuciferine solution.

[0087] DTNB solution (0.01 M): Dissolve 3.9 mg DTNB in ​​1 mL DMSO and store at 4°C in the dark.

[0088] ATCI solution: Dissolve 108.35 mg of ATCI in 4 mL of water and store at 4°C.

[0089] AChE solution: Dissolve 3 mg of AChE in 3.5 mL of PBS and store at 4°C.

[0090] Huperzine A solution: Take an appropriate amount of Huperzine A reference substance, weigh it accurately, and dissolve it in water to prepare a 0.01 mg / mL Huperzine A solution.

[0091] 2. Experimental steps

[0092] The experiment set up a blank reference group (BR), a blank control group (BC), a sample reference group (SR), a sample group (S), and a positive control group (C).

[0093] Group S: 5 μL of sample solution, 170 μL of phosphate buffer (0.1 mol / L, pH 7.0), 15 μL of AChE solution and 10 μL of DTNB solution were added to the wells in sequence, shaken thoroughly, and placed at 4°C for 20 min. Then 10 μL of ATCI solution was added dropwise and allowed to react in a 37°C water bath for 20 min. After the reaction, the absorbance A was measured in an ELISA reader. S .

[0094] The sample solutions are respectively a nuciferine solution, a nuciferine liposome permeation solution prepared in Detection Example 2, a nuciferine aqueous solution permeation solution prepared in Detection Example 2, a nuciferine liposome permeation solution prepared in Detection Example 3, and a huperzine A solution.

[0095] SR group: 15 μL of PBS solution was used to replace 15 μL of AChE solution. Other steps remained unchanged. The absorbance A was measured. SR .

[0096] BR group: 175 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. The absorbance A was measured. BR .

[0097] BC 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. The absorbance A was measured. BC .

[0098] The inhibition rate of nuciferine samples was calculated using the formula:

[0099] Table 2 Nuciferine acetylcholinesterase inhibition rate

[0100]

[0101]

[0102] As shown in the results of Table 2, both the nuciferine solution and the huperzine A reference solution have significant acetylcholinesterase inhibitory activity. The results of the nuciferine liposome permeate in Test Example 2 and the nuciferine aqueous solution permeate in Test Example 2 show that there is a significant difference in the artificial skin permeability before and after nuciferine encapsulation, and nuciferine encapsulation can significantly improve the artificial skin permeability of nuciferine. The nuciferine liposome permeate in Test Example 3 was used to conduct a permeability experiment on mouse skin. The results showed that after nuciferine was applied to mouse skin, a small amount of nuciferine permeation could be detected after 12 hours. Due to the low concentration of nuciferine in the permeate, its anti-acetylcholinesterase inhibitory activity was not detected.

[0103] Application example 1: Nuciferine liposome tablets

[0104] 62.5 g of pharmaceutical grade silica powder (Shaanxi Zhengyi Pharmaceutical Excipients Co., Ltd.) was taken to prepare empty liposomes according to Example 1. 2.0 g of nuciferine was weighed and dissolved in an appropriate amount of water, and 88 g of blank liposomes were added. The mixture was ultrasonically treated for 5 min (40 kHz, 700 W) and centrifuged at 8000 rpm for 5 min. The precipitate was freeze-dried to obtain nuciferine liposome fine powder for later use.

[0105] Take 30g of the above-mentioned nuciferine liposome fine powder, mix it evenly with 90g of sorbitol, then add 50g of disintegrant (the ratio of cross-linked carboxymethyl cellulose sodium and cross-linked polyvinylpyrrolidone is 2:1), mix and place in a fluidized bed for granulation; dry, mix with 10g of magnesium stearate and then press into tablets to obtain the core of nuciferine liposome tablets. The core is film-coated to obtain 1000 nuciferine liposome tablets.

[0106] Application Example 2: Nuciferine Liposome Ointment

[0107] 0.6g sodium carboxymethylcellulose and 1.0g glycerol were ground in a mortar, 5mg ethylparaben alcohol and 8.4mL distilled water were added and ground, and 1.0g nuciferol liposome fine powder was added in portions and ground to obtain nuciferol liposome ointment. The prepared ointment was applied to the skin, which was easy to disperse, smooth and non-greasy, and easy to wash off.

Claims

1. A method for preparing nuciferine liposomes using silica gel as a carrier, characterized in that: The preparation method comprises: (a) adding a wrapping material into an anhydrous ethanol solution 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 suspension; The solid phase particles are silica gel solid phase particles.

2. The method for preparing nuciferine liposomes using silica gel as a carrier according to claim 1, characterized in that: The packaging material is one of lecithin and lecithin-cholesterol.

3. The method for preparing nuciferine liposomes using silica gel as a carrier according to claim 1, characterized in that: The particle size of the silica gel is 48 to 150 μm, preferably 48 to 74 μm.

4. The method for preparing nuciferine liposomes using silica gel as a carrier according to claim 2, characterized in that: The mass concentration of the coating material in the coating solution is 0.01 to 0.2 g / mL, preferably 0.1 to 0.2 g / mL.

5. The method for preparing nuciferine liposomes using silica gel as a carrier according to claim 1, characterized in that: The mass ratio of the solid phase particles to the encapsulating material is (2-3):1; The solvent of the nuciferine solution is water. Preferably, the concentration of nuciferine is 0.1-2 mg / mL. More preferably, the concentration of nuciferine is 0.6-2 mg / mL.

6. The method for preparing nuciferine liposomes using silica gel as a carrier according to claim 2, characterized in that: The mass ratio of the solid content of the nuciferine solution to lecithin is (7.2-9.6):

100.

7. The method for preparing nuciferine liposomes using silica gel as a carrier according to claim 1, characterized in that: In step (c), the nuciferine solution contains hydrolyzed sodium hyaluronate. Preferably, the mass of the hydrolyzed sodium hyaluronate accounts for no more than 2% of the total mass of the nuciferine liposome suspension.

8. A nuciferine liposome prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The 48h permeability of the nuciferine liposome is not less than 30%; The encapsulation rate of the nuciferine liposome is 83.9% to 98.6%.

9. Use of the nuciferine liposome prepared by the preparation method of any one of claims 1 to 7 or the nuciferine liposome according to claim 8 in the preparation of anti-AD and weight loss drugs, characterized in that: The dosage form of the nuciferine liposome is a solid preparation, preferably a tablet.

10. Use of the nuciferine liposome prepared by the preparation method of any one of claims 1 to 7 or the nuciferine liposome according to claim 8 in the preparation of whitening and spot-lightening skin care products, characterized in that: The dosage form of the nuciferine liposome is an ointment.

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