Nuciferine liposome emulsion, preparation method and application thereof

Abstract

The present application relates to the technical field of pharmaceutical preparation, in particular to a lotus leaf alkaloid liposome emulsion, a preparation method and application thereof, wherein sucrose / sodium chloride solid particles are combined with a wrapping material by a dipping film formation method to prepare empty liposomes, and a drug lotus leaf alkaloid is coated to prepare a lotus leaf alkaloid liposome emulsion.The preparation method provided by the present application is simple in process; raw materials are widely sourced and green and environmentally friendly, without affecting the biological activity of active ingredients; the preparation process is mild in conditions, without the need for special equipment, without changing the pH value of the solution, suitable for transdermal and oral administration; the amount of organic solvent is small, the encapsulation rate is high, and the preparation is easy to scale up.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a lotus leaf alkaloid liposome emulsion, its preparation method, and its application. Background Technology

[0002] Lotus leaf alkaloids are apophene-like alkaloids found in lotus leaves, possessing various pharmacological activities such as lowering blood lipids, anti-inflammation, anti-tumor, regulating glucose metabolism, antipsychotic, and weight loss. Furthermore, studies have shown that lotus leaf alkaloids also exhibit anti-acetylcholinesterase activity, making them potential candidates not only for anti-AD drugs but also for inhibiting AChE activity to treat skin pigmentation. However, lotus leaf alkaloids suffer from low solubility, rapid elimination rate, and low bioavailability in vivo, which limits their development and utilization to some extent. Liposomes are closed vesicles composed of lipid materials such as phospholipids and cholesterol, possessing a phospholipid bilayer structure similar to cell membranes and the potential to enhance drug permeability through the skin. Currently, commonly used liposome preparation methods include ethanol injection, thin-film dispersion, high-pressure homogenization, and freeze-drying, but these methods suffer from drawbacks such as overly complex preparation processes, high production costs, large amounts of organic solvents, and difficulty in large-scale production. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention, for the first time, uses sucrose / sodium chloride solid particles as a carrier. Through an impregnation film-forming method, sucrose / sodium chloride solid particles are combined with an encapsulation material to prepare empty liposomes, which are then encapsulated with lotus leaf alkaloids to prepare a lotus leaf alkaloid liposome emulsion. The preparation method provided by this invention is simple; the raw materials are widely available, environmentally friendly, and do not affect the bioactivity of the active ingredients; the preparation process is mild, requires no special equipment, does not change the pH of the solution, and is suitable for transdermal and oral administration; it uses a small amount of organic solvent, has a high encapsulation efficiency, and is easy to scale up.

[0004] To achieve the above objectives, the present invention provides a method for preparing lotus leaf alkaloid liposome emulsion, comprising:

[0005] (a) Prepare a coating material solution by adding the coating material to anhydrous ethanol;

[0006] (b) The solid particles were immersed in the encapsulation material solution, and the anhydrous ethanol was removed. The solid particles and the encapsulation material were combined to obtain empty liposomes.

[0007] (c) Mix empty liposomes with lotus leaf alkali solution to obtain lotus leaf alkali liposome emulsion.

[0008] The solid particles are either sucrose or sodium chloride.

[0009] The encapsulating material is either lecithin or lecithin-cholesterol.

[0010] The solid particles utilize their large specific surface area to cause the coating material solution to evaporate on the surface of the solid particles, forming a thin film.

[0011] The particle size of the solid particles is 150–1250 μm. When the particle size is too large, the specific surface area decreases, the film thickness becomes too large, and the encapsulation efficiency becomes too low; when the particle size is too small, the film thickness becomes too small, and it is prone to breakage.

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

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

[0014] In step (b), the removal of anhydrous ethanol includes one of the following methods: water bath evaporation, air drying, or standing at room temperature.

[0015] The water bath drying includes atmospheric pressure water bath drying and depressurized water bath drying.

[0016] In step (c), the mixing methods of the empty liposomes and lotus leaf alkaline solution include: vortexing, shaking, and sonication;

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

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

[0019] The solvent for the lotus leaf alkali 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 lotus leaf alkali solution is 0.3 to 0.6 mg / mL;

[0021] When the solid phase particles are sodium chloride, the concentration of the lotus leaf alkaline solution is 0.6–2 mg / mL.

[0022] The mass ratio of lotus leaf alkaloid to lecithin is (5.5-8.6):100.

[0023] In existing technologies, thin-film dispersion is commonly used to prepare liposomes. This process requires rotary evaporation to remove the solvent, forming a thin film on the walls of the evaporation flask to obtain liposomes. However, the small surface area of ​​the evaporation flask and the excessively high solution concentration in the later stages of solvent evaporation lead to significant differences in the film thickness on the inner surface of the flask, resulting in a wide particle size distribution of the final liposomes. In contrast, the solid-phase particles in this invention have a large surface area, and the film-forming time and rate of the encapsulating material on the solid-phase particle surface are similar. Therefore, the film thickness on the solid-phase particle surface is uniform and extremely thin, which can significantly increase drug loading while rapidly encapsulating the drug.

[0024] In traditional rotary evaporation spraying, the formation of unevenly thick and discontinuous spots on the surface of solid particles by the sprayed droplets, along with the formation of microspheres by the encapsulating material itself, affects the drug loading rate. Furthermore, the resulting liposome particles are large and dispersed, typically exceeding micrometer size. In contrast, this invention utilizes a solid-phase particle impregnation film-forming method with a high-concentration encapsulating material solution. This allows the encapsulating material solution on the surface of the solid particles to not only evaporate rapidly to form a film but also significantly reduces the amount of encapsulating material solution and solvent used, thereby improving the drug loading rate. The resulting liposome particles are smaller and more uniform in size, typically about 10 times smaller than those prepared by traditional rotary evaporation spraying.

[0025] This invention eliminates the need to dissolve lotus leaf alkaloids in anhydrous ethanol. Instead, it removes the anhydrous ethanol to obtain empty liposomes, which are then mixed with the lotus leaf alkaloid solution. This reduces the impact of anhydrous ethanol on the lotus leaf alkaloids and also reduces the heating time required to remove the anhydrous ethanol, thus greatly avoiding the impact of heating on the stability of the lotus leaf alkaloids.

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

[0027] The present invention also provides a lotus leaf alkaloid liposome emulsion prepared by the above preparation method, wherein the lotus leaf alkaloid liposome emulsion has a permeability of not less than 38% after 48 hours;

[0028] The encapsulation efficiency of the lotus leaf alkaloid liposome emulsion is 58.9%–100%;

[0029] Furthermore, the encapsulation efficiency of the lotus leaf alkaloid liposome emulsion was measured by HPLC-ultrafiltration centrifugation.

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

[0031] After granulation, the lotus leaf alkaloid liposomes have a particle size of 90–230.1 nm and an aggregation index (PDI) of 0.176–0.329.

[0032] The granulation method used for the lotus leaf alkaloid liposomes is one of four: ultrasonication, shearing, homogenization, and extrusion, with extrusion being preferred, and microfiltration extrusion being more preferred.

[0033] This invention also provides an application of the lotus leaf alkaloid 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 lotus leaf alkaloid liposome emulsion can be formulated into an oral emulsion.

[0035] When the solid phase particles are sodium chloride, the lotus leaf alkaloid liposome emulsion can be formulated into an injection.

[0036] The lotus leaf alkaloid liposome emulsion does not contain organic solvents and does not require drying, thus reducing the impact of drying on the stability of liposomes.

[0037] This invention also provides an application of the lotus leaf alkaloid liposome emulsion prepared by the above preparation method in the preparation of skin-whitening and freckle-removing cosmetics.

[0038] Beneficial effects:

[0039] 1. This invention is the first to use an impregnation film-forming method, using sucrose / sodium chloride solid particles as carriers, combined with encapsulation materials to prepare empty liposomes, and then preparing lotus leaf alkaloid liposomes by encapsulating lotus leaf alkaloids. 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 this invention is about 10% of that in the prior art.

[0040] 2. This invention uses solid particles as a carrier, which are not only widely available and environmentally friendly, but also do not affect the bioactivity of the active ingredients.

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

[0042] 4. This invention uses solid-phase particles as a carrier to prepare lotus leaf alkaloid liposomes without changing the pH value of the emulsion. It can be administered transdermally and orally, which improves the bioavailability and stability of lotus leaf alkaloid in the human body. Attached Figure Description

[0043] Figure 1 The HPLC chromatograms are of the permeate and concentrate of the lotus leaf alkaloid samples from Examples 1 and 2.

[0044] Figure 2 The HPLC chromatograms are of the permeate and concentrate of the lotus leaf alkaloid samples from Examples 6 and 7.

[0045] Figure 3 The HPLC chromatograms are of the permeate and concentrate of the lotus leaf alkaloid samples from Examples 8 and 9.

[0046] Figure 4 The HPLC chromatograms are of the permeate and concentrate of the lotus leaf alkaloid samples from Comparative Examples 1 and 4.

[0047] Figure 5 This is a particle size distribution diagram of the lotus leaf alkaloid liposomes after microfiltration extrusion prepared in Example 2;

[0048] Figure 6 This is a particle size distribution diagram of the lotus leaf alkaloid liposomes after microfiltration extrusion prepared in Example 7;

[0049] Figure 7 TEM image of lotus leaf alkaloid liposomes prepared in Example 2;

[0050] Figure 8 The image shown is a TEM image of the lotus leaf alkaloid liposomes prepared in Example 7. Detailed Implementation

[0051] The present invention will be further described in detail below through specific embodiments, but it should not be construed as the present invention being limited to the following embodiments. All technologies implemented based on the above content of the present invention are within the scope of the present invention.

[0052] The lecithin mentioned in the examples was purchased from Beijing Aoboxing Biotechnology Co., Ltd., catalog 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”; the artificial skin was Strat-MTM Membrane purchased from Merck Millipore Ltd; sucrose and sodium chloride were commercially available products.

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

[0054] Example 1:

[0055] Preparation of lotus leaf alkaloid liposome emulsion using sucrose as the solid phase particle:

[0056] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sucrose (average particle size 850 μm) was added to the lecithin solution at a mass ratio of 2.5:1 (sucrose solid particles to encapsulating material), stirred continuously, and evaporated to dryness in a 50°C, atmospheric pressure water bath to obtain empty liposomes. The empty liposomes were then mixed with a 0.6 mg / mL lotus leaf alkaloid solution at a mass ratio of 8.4:100 (lotus leaf alkaloid to lecithin), and shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0057] Example 2

[0058] Preparation of lotus leaf alkaloid liposome emulsion using sucrose as the solid phase particle:

[0059] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sucrose (average particle size 850 μm) was added to the lecithin solution at a mass ratio of 1.4:1 (sucrose solid particles to encapsulating material) while stirring. The solution was then evaporated to dryness in a 50°C water bath to obtain empty liposomes. The empty liposomes were then mixed with a 0.6 mg / mL lotus leaf alkaloid solution at a mass ratio of 5.7:100 (lotus leaf alkaloid to lecithin). The mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0060] Example 3

[0061] Preparation of lotus leaf alkaloid liposome emulsion using sucrose as the solid phase particle:

[0062] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sucrose (average particle size 850 μm) was added to the lecithin solution at a mass ratio of 1.4:1 (sucrose solid particles to encapsulating material) while stirring. The solution was then evaporated to dryness in a 50°C water bath to obtain empty liposomes. Lotus leaf alkaloid solution (0.1 mg / mL) was added at a mass ratio of 5.7:100 (lotus leaf alkaloid to lecithin), and the mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0063] Example 4

[0064] Preparation of lotus leaf alkaloid liposome emulsion using sucrose as the solid phase particle:

[0065] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sucrose (average particle size 850 μm) was added to the lecithin solution at a mass ratio of 1.4:1 (sucrose solid particles to encapsulating material), stirred continuously, and evaporated to dryness in a 50°C water bath to obtain empty liposomes. Lotus leaf alkaloid solution (2 mg / mL) was added at a mass ratio of 5.7:100 (lotus leaf alkaloid to lecithin), and the mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0066] Example 5

[0067] Preparation of lotus leaf alkaloid liposome emulsion using sucrose as the solid phase particle:

[0068] The preparation conditions in this embodiment are the same as those in Example 2, except that lecithin is replaced with lecithin-cholesterol.

[0069] The mass ratio of lecithin to cholesterol is 6:1.

[0070] Example 6

[0071] Preparation of lotus leaf alkaloid liposome emulsion using sodium chloride as the solid phase particle:

[0072] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sodium chloride (average particle size 650 μm) was added to the lecithin solution at a mass ratio of sodium chloride solid particles to lecithin of 2.6:1, while stirring continuously. The solution was then evaporated to dryness in a 50°C water bath to obtain empty liposomes. The empty liposomes were then taken, and 0.6 mg / mL of lotus leaf alkaloid solution was added at a mass ratio of lotus leaf alkaloid solution solid content to lecithin of 8.6:100. The mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0073] Example 7

[0074] Preparation of lotus leaf alkaloid liposome emulsion using sodium chloride as the solid phase particle:

[0075] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL solution. The solution was sonicated for 10 minutes and stored in the dark. Sodium chloride (average particle size 650 μm) was added to the lecithin solution at a mass ratio of sodium chloride to encapsulation material of 2.08:1, while stirring continuously. The solution was then evaporated to dryness in a 50°C water bath to obtain empty liposomes. The empty liposomes were then mixed with 0.6 mg / mL of lotus leaf alkaloid solution at a mass ratio of 7.4:100 (solid content of lotus leaf alkaloid solution to lecithin). The mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0076] Example 8:

[0077] Preparation of lotus leaf alkaloid liposome emulsion using sodium chloride as the solid phase particle:

[0078] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / ml solution. The solution was sonicated for 10 minutes and stored in the dark. Sodium chloride (average particle size 650 μm) was weighed and added to the lecithin solution at a mass ratio of sodium chloride to encapsulation material of 1.3:1, while stirring continuously. The solution was then evaporated to dryness in a 50°C water bath to obtain empty liposomes. The empty liposomes were then mixed with 0.6 mg / ml of lotus leaf alkaloid solution at a mass ratio of 5.5:100 (solid content of lotus leaf alkaloid solution to lecithin). The mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0079] Example 9:

[0080] Preparation of lotus leaf alkaloid liposome emulsion using sodium chloride as the solid phase particle:

[0081] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL solution. The solution was sonicated for 10 minutes and stored in the dark. Sodium chloride (average particle size 650 μm) was weighed and added to the lecithin solution at a mass ratio of 1.3:1 (sodium chloride to encapsulation material), stirring constantly. The solution was then evaporated to dryness in a 50°C water bath to obtain empty liposomes. The empty liposomes were then taken, and 0.1 mg / mL of lotus leaf alkaloid solution was added at a mass ratio of 7.4:100 (lotus leaf alkaloid solution solid content to lecithin). The mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0082] Example 10:

[0083] Preparation of lotus leaf alkaloid liposome emulsion using sodium chloride as the solid phase particle:

[0084] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL solution. The solution was sonicated for 10 minutes and stored in the dark. Sodium chloride (average particle size 650 μm) was added to the lecithin solution at a solid-phase particle to encapsulation material mass ratio of 2.08:1, while stirring continuously. The solution was then evaporated to dryness in a 50°C water bath to obtain empty liposomes. The empty liposomes were then mixed with 2 mg / mL of a self-made lotus leaf alkaloid solution at a solid content to lecithin mass ratio of 7.4:100. The mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0085] Comparative Example 1

[0086] The comparative example was prepared under the same conditions as Example 2, except that no sucrose solid particles were added.

[0087] The specific preparation method is as follows: Weigh lecithin and add it to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. Sonicate for 10 minutes and store under light-protected conditions. Place the lecithin solution in a rotary evaporator flask and evaporate to dryness in a 50°C water bath. Add 0.6 mg / mL of lotus leaf alkaloid aqueous solution according to a mass ratio of 5.7:100 between the solid content of the lotus leaf alkaloid solution and lecithin. Sonicate for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0088] Comparative Example 2:

[0089] The comparative example was prepared under the same conditions as Example 2, except that the sucrose solid particles were replaced with montmorillonite of the same particle size.

[0090] Comparative Example 3:

[0091] The comparative example was prepared under the same conditions as Example 2, except that the sucrose solid particles were replaced with TiO2 of the same particle size.

[0092] Comparative Example 4:

[0093] Lecithin and lotus leaf alkaloids were weighed and added to anhydrous ethanol solution to obtain an organic solution of lecithin and lotus leaf alkaloids (wherein the mass ratio of lotus leaf alkaloids to lecithin was 5.7:100, and the mass concentration of lecithin was 0.2 g / mL). The solution was sonicated for 10 minutes and stored in the dark. Sucrose was placed in a rotary evaporator flask and heated at 50°C. The organic solution of lecithin and lotus leaf alkaloids was added at a mass ratio of sucrose solid particles to encapsulating material of 1.4:1 while stirring. The solution was evaporated to dryness in a water bath. Subsequently, deionized water was added to obtain a lotus leaf alkaloid liposome emulsion (wherein the mass ratio of deionized water to lotus leaf alkaloids was 1000:0.6).

[0094] Comparative Example 5:

[0095] Lecithin was weighed and added to anhydrous ethanol solution to prepare a 0.2 g / mL lecithin solution. The solution was sonicated for 10 minutes and stored in the dark. Sucrose was placed in a rotary evaporator flask, and the lecithin solution was sprayed into the flask at a mass ratio of 1.4:1 (sucrose solid particles to encapsulating material). The evaporation temperature was 50℃, and the vacuum degree was -0.098 MPa to obtain empty liposomes. The empty liposomes were then mixed with 0.6 mg / mL lotus leaf alkaloid solution at a mass ratio of 5.7:100 (lotus leaf alkaloid to lecithin). The mixture was shaken for 10 minutes to obtain a lotus leaf alkaloid liposome emulsion.

[0096] Example 1: Encapsulation efficiency testing

[0097] The method for detecting the encapsulation efficiency of lotus leaf alkaloid liposomes is as follows: Take 0.1 mL of lotus leaf alkaloid liposome emulsion into a centrifuge tube (CF), centrifuge at 8000 rpm for 10 min, collect the supernatant and add it to an ultrafiltration centrifuge tube (UF-CF, ultrafiltration membrane pore size 30 KD), then add 0.3 mL of purified water, centrifuge at 8000 rpm for 10 min, and take the permeate as the test solution for detecting free lotus leaf alkaloids (defined as permeate). Continue to add 0.4 mL of anhydrous ethanol to the above CF, weigh, sonicate to dissolve, add to the above UF-CF, ultrafilter and centrifuge at 8000 rpm for 10 min, take the permeate and replenish the lost weight with anhydrous ethanol, and use this as the test solution for detecting encapsulated lotus leaf alkaloids (defined as concentrate).

[0098] High performance liquid chromatography conditions: bonded silica gel C 18 (4.6mm*250mm, 5μm), using 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 efficiency (%) = peak area of ​​concentrate / (peak area of ​​permeate + peak area of ​​concentrate) × 100%.

[0100] like Figure 1 The figures show the HPLC chromatograms of the lotus leaf alkaloid liposome permeate and concentrate of Examples 1 and 2, respectively. As can be seen from the figures, the lotus leaf alkaloid liposome permeate prepared in Examples 1 and 2 has almost no peak at the lotus leaf alkaloid chromatographic peak (around 20 min), while the concentrate has corresponding strong peaks at the lotus leaf alkaloid chromatographic peak, indicating that the lotus leaf alkaloid is completely encapsulated.

[0101] like Figure 3 The figures show the HPLC spectra of the permeate and concentrate of the lotus leaf alkaloid liposomes prepared in Examples 8-9, respectively. It can be seen from the figures that both the permeate and the concentrate have peaks at the lotus leaf alkaloid chromatographic peak (the retention time of the lotus leaf alkaloid chromatographic peak is about 20 min), but the peak of the permeate is smaller and almost a straight line, while the concentrate has a strong peak, indicating that the encapsulated drug leakage is very small and the encapsulation is strong.

[0102] Table 1. Encapsulation efficiency (%) of lotus leaf alkaloid liposomes in each example and comparative example.

[0103]

[0104]

[0105] Table 1 shows the encapsulation efficiency of lotus leaf alkaloid liposomes prepared in each example and comparative example.

[0106] Compared to Example 2, Example 1 used less encapsulating material, resulting in fewer liposomes and a poorer encapsulation rate. Compared to Example 7, Example 8 used too much encapsulating material, causing the encapsulating materials to stick together and fail to form well-shaped spheres. This resulted in a small amount of liposomes encapsulated or a thick outer shell, making it difficult for a small amount of drug to enter.

[0107] The concentration of lotus leaf alkaloids has a significant impact on the encapsulation efficiency. As shown in Examples 2, 3, and 4, compared to Example 2, the concentration of lotus leaf alkaloids in Example 3 is lower, and the volume of the lotus leaf alkaloid solution is larger. Under the same amount of empty liposomes, the concentration of liposomes in the solution is low, making it difficult to capture and encapsulate all the lotus leaf alkaloids. Compared to Example 2, the concentration of lotus leaf alkaloids in Example 4 is higher, and the volume of lotus leaf alkaloids is smaller. The resulting liposomes have a higher concentration, which can cause the encapsulation materials to stick together and not form well-shaped spheres. The amount of liposomes encapsulated is small, or the thickness of the liposome shell is large, making it difficult for a small amount of drug to enter.

[0108] Comparative Example 1 used the same amount of encapsulation material, but the encapsulation rate was poor. This was because solid particles were not used as a carrier, and the film area was much smaller than that of Examples 1-10.

[0109] In Comparative Examples 2 and 3, the rough surface of montmorillonite and its strong adsorption capacity caused the coating material to clump together on the surface of montmorillonite and fail to form a well-shaped sphere. The smooth surface of TiO2 made it difficult to wet the carrier surface, resulting in poor adsorption of the coating material by the TiO2 carrier and making it difficult to form a film on its surface.

[0110] Compared to Examples 1-10, Comparative Examples 4 and 5 exhibited poorer encapsulation rates. This is because in Comparative Example 4, during the preparation of lotus leaf alkaloid liposomes using the thin-film dispersion method, the film formed by the encapsulating material on the rotary evaporator flask wall was generally thick and uneven, resulting in a low encapsulation rate of the lotus leaf alkaloid liposomes. Comparative Example 5 also had a poor encapsulation rate because, during the spraying process of the membrane solution, due to the vacuum state in the system, the membrane solution was difficult to effectively spray onto the surface of the solid particles to form a film. Instead, more tiny spots were formed on the surface of the solid particles, and some membrane particles were formed. Therefore, for the same amount of membrane solution, the film-forming area of ​​Comparative Example 6 was much smaller than that of Examples 1-10.

[0111] Example 2: Particle size determination

[0112] Take 0.1 mL of the lotus leaf alkaloid liposome solution prepared in Examples 2 and 7, and dilute it with water to 1 mL. Measure the particle size of the lotus leaf alkaloid liposomes before microfiltration extrusion using a Zetasizer Nano ZS90 laser particle size analyzer.

[0113] The lotus leaf alkaloid 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 with water to 1 mL. The particle size of the lotus leaf alkaloid liposomes after microfiltration extrusion was measured by a Zetasizer Nano ZS90 laser particle size analyzer.

[0114] The lotus leaf alkaloid liposomes prepared in Example 2 had an average particle size of 170 nm and a polydispersity index (PDI) of 0.227 before microfiltration extrusion. After granulation, the average particle size and PDI decreased to 112.7 nm and 0.223, respectively. The liposomes in Example 7, after microfiltration extrusion, had a particle size and polydispersity index of 90 nm and 0.176, respectively, indicating a significant microfiltration extrusion effect. The particle size distribution of the lotus leaf alkaloid liposomes prepared in Examples 2 and 7 after microfiltration extrusion is shown in the figure below. Figure 5 , Figure 6 As shown in Table 2, the average potentials of the liposomes prepared in Examples 2 and 7 after microfiltration extrusion were -65.1 mV and -41.0 mV, respectively, indicating that the liposome particles have a negatively charged surface with an absolute potential value between 30 and 80, which is relatively stable. The particle size detection method for the lotus leaf alkaloid liposomes after microfiltration extrusion in the other examples and comparative examples was the same as in Examples 2 and 7, and the specific results are shown in Table 2.

[0115] The lotus leaf alkaloid liposome emulsions prepared in Examples 2 and 7, after being filtered through a copper mesh and dried, are shown in the TEM images below. Figure 7 , 8 As shown in the figure, the lotus leaf alkaloid liposomes prepared in Examples 2 and 7 are similar to spheres, and their particle size is significantly smaller than that of the particles in the lotus leaf alkaloid liposome emulsion. This may be due to the drying and water loss of the particles in the emulsion during the electron microscopy sample preparation process. In addition, the lotus leaf alkaloid liposome particles are relatively evenly distributed.

[0116] Example 3: Transdermal diffusion test

[0117] Using a transdermal diffusion assay apparatus, 40 wt% PEG400 aqueous solution was added to the diffusion cell. 0.3 mL each of the lotus leaf alkaloid liposome emulsion and lotus leaf alkaloid aqueous solution prepared in each example and comparative example were uniformly applied to the smooth surface of artificial skin. The solution was allowed to permeate at room temperature, yielding lotus leaf alkaloid liposome permeation solution and lotus leaf alkaloid aqueous solution permeation solution, respectively. At 6 and 48 h intervals, 0.2 mL of sample solution was taken from the diffusion cell for liquid chromatography analysis. Simultaneously, the volume was replenished with 40 wt% PEG400 aqueous solution, and the permeability was calculated using high-performance liquid chromatography.

[0118] Table 2 shows the permeability, average particle size, and potential of lotus leaf alkaloid liposomes prepared in each example and comparative example at different times.

[0119]

[0120]

[0121] Table 2 shows the permeability, average particle size, and potential of lotus leaf alkaloid liposomes prepared in each example and comparative example at different times. "ND" indicates not detected, and "-" indicates not detected. As can be seen from the table, the average particle size of Examples 1 to 10 is between 90 and 123.1 nm, indicating that liposomes with small particle size can be obtained by using sucrose and sodium chloride as solid phase particle carriers. The wide PDI range may be related to the pore size distribution of the microfiltration membrane.

[0122] Example 4: ACE enzyme activity assay:

[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 (0.01M): Dissolve 3.9 mg DTNB in ​​1 mL DMSO and store at 4°C protected from light.

[0126] ATCI solution: Dissolve 108.35 mg of thioacetylcholine 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 Procedure

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

[0131] Sample group: Add 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 to the wells in sequence, shake well, and incubate at 4°C for 20 min. After 20 min, add 10 μL of ATCI solution and incubate in a water bath at 37°C for 20 min. Measure the absorbance (S) at 405 nm using a microplate reader.

[0132] The sample solutions were: the permeate solutions of Examples 1-10 obtained in Detection Example 3, lotus leaf alkaloid aqueous solution, and huperzine A solution (10 μg / mL).

[0133] Sample reference group: 15 μL of PBS solution was used instead of 15 μL of AChE solution. 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. 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 were used to replace 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 is calculated using the following formula:

[0137]

[0138] Table 3 Acetylcholinesterase inhibition rate

[0139]

[0140]

[0141] Table 3 shows the inhibition rates of acetylcholinesterase by lotus leaf alkaloid samples, lotus leaf alkaloid liposome permeation solution, lotus leaf alkaloid aqueous solution, and huperzine A solution, respectively. The concentrations mentioned are the concentrations of the permeation solution, lotus leaf alkaloid aqueous solution, and huperzine A reference standard in each example, obtained by high-performance liquid chromatography (HPLC). Based on the inhibition rate of huperzine A reference standard, the experimental results are reliable. Examples 1-0 and lotus leaf alkaloid aqueous solution both showed a certain inhibitory effect on acetylcholinesterase activity. Furthermore, due to the significantly improved transdermal effect of the encapsulated lotus leaf alkaloid, the inhibition rate of acetylcholinesterase by the transdermal lotus leaf alkaloid was increased.

Claims

1. A method for preparing a lotus leaf alkaloid liposome emulsion, characterized in that, The preparation method includes: (a) Prepare a coating material solution by adding the coating material to anhydrous ethanol; (b) The solid particles were immersed in the encapsulation material solution, and the anhydrous ethanol was removed. The solid particles and the encapsulation material were combined to obtain empty liposomes. (c) Mix empty liposomes with lotus leaf alkaloid solution to obtain lotus leaf alkaloid liposome emulsion; The solid particles are either sucrose or sodium chloride. The encapsulating material is one of lecithin and lecithin-cholesterol; The concentration of the encapsulating material in the encapsulating material solution is 0.1~0.2 g / mL; The mass ratio of lotus leaf alkaloid to lecithin is (5.5~8.6):100; When the solid particles are sucrose, the mass ratio of the solid particles to the encapsulating material is (1.4~2.5):1; When the solid particles are sodium chloride, the mass ratio of the solid particles to the encapsulating material is (2.08~2.6):

1.

2. The method for preparing lotus leaf alkaloid liposome emulsion according to claim 1, characterized in that, The particle size of the solid particles is 150~1250μm.

3. The method for preparing lotus leaf alkaloid liposome emulsion according to claim 2, characterized in that, When the solid particles are sodium chloride, the particle size of the solid particles is 600~700μm.

4. The method for preparing lotus leaf alkaloid liposome emulsion according to claim 2, characterized in that, When the solid particles are sucrose, the particle size of the solid particles is 300~900μm.

5. The method for preparing lotus leaf alkaloid liposome emulsion according to claim 1, characterized in that, In step (b), the removal of anhydrous ethanol includes evaporation in a water bath, drying by blowing, and standing at room temperature.

6. The method for preparing lotus leaf alkaloid liposome emulsion according to claim 1, characterized in that, The solvent for the lotus leaf alkaline solution is water; The concentration of the lotus leaf alkali solution is 0.1~2 mg / mL.

7. The method for preparing lotus leaf alkaloid liposome emulsion according to claim 6, characterized in that, When the solid phase particles are sucrose, the concentration of the lotus leaf alkali solution is 0.3~0.6 mg / mL.

8. The method for preparing the lotus leaf alkaloid liposome emulsion according to claim 6, characterized in that, When the solid phase particles are sodium chloride, the concentration of the lotus leaf alkali solution is 0.6~2 mg / mL.

9. A lotus leaf alkaloid liposome emulsion prepared by the preparation method according to claim 1, characterized in that, The encapsulation efficiency of the lotus leaf alkaloid liposome emulsion is 58.9%~100%.

10. The lotus leaf alkaloid liposome emulsion according to claim 9, characterized in that, The lotus leaf alkaloid liposome emulsion has a permeability of no less than 38% after 48 hours. The lotus leaf alkaloid liposomes have a particle size of 90~230.1 nm and a PDI of 0.176~0.329.

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