Lotus leaf flavone 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 lotus leaf flavone liposome emulsion, a preparation method and application thereof, and the lotus leaf flavone liposome emulsion is prepared by coating the surfaces of sucrose / sodium chloride solid-phase particles with a layer of coating material film containing lotus leaf flavone through a dipping film-forming method to obtain a dry matter, coating the dry matter with a layer of coating material film, and hydrating to obtain the lotus leaf flavone liposome emulsion. And preparing to obtain the lotus leaf flavone 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 lotus leaf flavonoid liposome emulsion, a preparation method and application thereof. Background Art

[0002] Flavonoids are an important class of active ingredients in natural medicines, with biological activities such as antioxidant, antimutagenic, anti-inflammatory, antibacterial, antiviral, insecticidal, liver protection, and protection of the cardiovascular and cerebrovascular systems. Flavonoids can also be used in the field of cosmetics, such as licorice flavonoids, etc., which have significant antioxidant, whitening, and after-sun repair effects, achieving the effect of slowing down skin aging. Studies have shown that flavonoids in lotus leaves can significantly inhibit the oxidation process of linoleic acid and have a good effect of scavenging DPPH free radicals; lotus leaf flavonoids have significant antioxidant activity on hyperlipidemia rats, can significantly increase the activity of glutathione peroxidase and serum superoxide dismutase, and reduce the content of malondialdehyde. Flavonoids in lotus leaves have important medicinal development value.

[0003] Due to the presence of phenolic hydroxyl groups in the molecular structure of flavonoid compounds, they are easily oxidized and deteriorated during application. Liposome encapsulation can effectively improve their stability. Liposomes are a closed vesicle 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 liposome preparation methods include ethanol injection, thin film dispersion, high-pressure homogenization, freeze drying, etc., which have the disadvantages of too complicated preparation process, high production cost, large amount of organic solvent, and difficulty in large-scale preparation. Existing encapsulation technology is applied to the encapsulation of lotus leaf flavonoids, but there is still the problem of unsatisfactory encapsulation rate. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a lotus leaf flavonoid liposome emulsion, a preparation method and an application thereof.

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

[0006] A method for preparing a lotus leaf flavonoid liposome emulsion comprises:

[0007] (a) dissolving lotus leaf flavonoids in anhydrous ethanol, adding a wrapping material, and preparing a membrane material solution (I);

[0008] (b) immersing the solid phase particles in a film-forming material solution (I), removing the anhydrous ethanol, performing a first film-forming step, and combining the solid phase particles with the drug-containing encapsulation material to obtain an encapsulation (I);

[0009] (c) immersing the inclusion (I) in a film-forming material solution (II), removing the anhydrous ethanol, performing secondary film formation to obtain the inclusion (II), and hydrating to obtain a lotus leaf flavonoid liposome emulsion; the solid phase particles are one of sucrose and sodium chloride.

[0010] Wherein, the packaging material is one of lecithin and lecithin-cholesterol.

[0011] The present invention utilizes the huge specific surface area of ​​solid phase particles to make the coating material solution volatilize on the surface of the solid phase particles to form a film.

[0012] 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.

[0013] Preferably, the particle size of the sodium chloride solid phase particles is 600-700 μm;

[0014] Preferably, the particle size of the sucrose solid phase particles is 300-900 μm, preferably 600-700 μm.

[0015] In step (b), the removal method of the anhydrous ethanol includes one of water bath evaporation, air drying, and standing at room temperature; the water bath evaporation includes normal pressure water bath evaporation and reduced pressure water bath evaporation.

[0016] In step (c), the hydration method of the dried product includes: vortex oscillation, shaking, and ultrasound.

[0017] In the film-forming material solution (I), the concentration of lotus leaf flavonoids is 0.1-2 mg / mL; the concentration of the encapsulating material lecithin is 0.1-0.2 g / mL, preferably 0.2 g / mL.

[0018] In the inclusion (I), the mass ratio of solid phase particles to lecithin is (1.25-6.25):1; preferably (1.43-2.5):1.

[0019] In the package (I), the mass ratio of lotus leaf flavonoids to lecithin is (0.03-1.875):100; preferably (0.03-1.5):100; and more preferably (0.06-0.5):100.

[0020] In the secondary film-forming, the concentration of the encapsulating material in the film-forming material solution (II) is 0.2 g / mL.

[0021] The mass ratio of the solid phase particles to the lecithin in the membrane-forming material solution (II) is 10:3.

[0022] 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. The traditional method has a low encapsulation efficiency for lotus leaf flavonoid components. In the present invention, lotus leaf flavonoids are uniformly dispersed in the encapsulation material solution, and the encapsulation material carries lotus leaf flavonoids to form a film quickly on the surface of the solid phase particles through the solid phase particle impregnation film forming method, and a secondary film is formed on the surface of the formed precursor liposome, which greatly improves the encapsulation efficiency of lotus leaf flavonoid components.

[0023] Preferably, the present invention further comprises step (d) granulation process.

[0024] The present invention also provides a lotus leaf flavonoid liposome emulsion prepared by the above-mentioned preparation method, wherein the lotus leaf flavonoid liposome emulsion has a 12h permeability (skin permeability) of not less than 36.8%;

[0025] The encapsulation rate of the lotus leaf flavonoids liposome emulsion is 55.5% to 94.8%, and preferably the encapsulation rate is not less than 90%. The encapsulation rate of the lotus leaf flavonoids liposome emulsion is measured by HPLC-ultrafiltration centrifugation. The ultrafiltration centrifugation speed is 8000 rpm, and the centrifugation time is 10 minutes.

[0026] After granulation, the particle size of the lotus leaf flavonoid liposome is 106.7-186.1 nm, and the aggregation index (PDI) is 0.144-0.214. The granulation method of the lotus leaf flavonoid liposome is one of ultrasound, shearing, homogenization, and extrusion, preferably extrusion, more preferably microfiltration extrusion.

[0027] The present invention also provides an application of the lotus leaf flavonoid liposome emulsion prepared by the above preparation method in the preparation of antioxidant drugs.

[0028] When the solid phase particles are sucrose, the lotus leaf flavonoids liposome emulsion can be prepared into an oral preparation.

[0029] When the solid phase particles are sodium chloride, the lotus leaf flavonoids liposome emulsion can be prepared into an injection.

[0030] The lotus leaf flavonoids liposome emulsion solves the problem that the organic solvent is difficult to completely remove in the liposome emulsion in the traditional preparation method.

[0031] The present invention also provides an application of the lotus leaf flavonoid liposome emulsion prepared by the above-mentioned preparation method in the preparation of anti-aging and whitening cosmetics.

[0032] Beneficial effects:

[0033] 1. The present invention adopts the secondary impregnation film-forming method for the first time, using sucrose / sodium chloride solid phase particles as carriers to prepare lotus leaf flavonoid liposomes. 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.

[0034] 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. The preparation process is mild, does not require special equipment, does not change the pH value of the solution, and 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.

[0035] 3. The lotus leaf flavonoid 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.

[0036] 4. The present invention adopts solid phase particles as carriers to encapsulate lotus leaf flavonoid components, which can be prepared immediately and can be administered percutaneously or orally, thereby improving the stability of lotus leaf flavonoids and their bioavailability in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The HPLC spectrum of the free lotus leaf flavonoids and the encapsulated lotus leaf flavonoids test solution in the lotus leaf flavonoids liposome emulsion of Examples 1-3;

[0038] Figure 2 The HPLC spectrum of the free lotus leaf flavonoids and the encapsulated lotus leaf flavonoids test solution in the lotus leaf flavonoids liposome emulsion of Examples 4-6;

[0039] Figure 3 HPLC spectra of free lotus leaf flavonoids and encapsulated lotus leaf flavonoids test solution in the lotus leaf flavonoids liposome emulsions of Examples 7-8 and Comparative Example 1;

[0040] Figure 4 This is the particle size distribution diagram of the lotus leaf flavonoid liposomes prepared in Example 1 after microfiltration and extrusion;

[0041] Figure 5 This is the TEM image of the lotus leaf flavonoid liposomes prepared in Example 1. DETAILED DESCRIPTION

[0042] 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.

[0043] The lecithin described in the embodiments was purchased from Beijing Aoboxing Biological Co., Ltd.; the preparation method of lotus leaf flavonoids refers to the authorized patent (a method for simultaneously separating and purifying lotus leaf flavonoids and polysaccharides, ZL2015101508152); 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.; artificial skin was Strat-MTM Membrane (25mm Discs) purchased from Merck Millipore Ltd; sucrose and sodium chloride are conventional commercially available products.

[0044] Embodiment 1:

[0045] Prepare 0.12mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.2g / mL solution, ultrasonic for 10 minutes, prepare film-forming material solution (I), keep away from light for later use. Take 0.2g sodium chloride solid phase particles (average particle size is 650μm), add to the film-forming material solution (I) at a solid phase particle to lecithin mass ratio of 2.5:1, stir while adding, evaporate in a 50℃ water bath to obtain inclusion (I), continue to add 0.3ml film-forming material solution (II) (lecithin ethanol solution with a concentration of 0.2g / mL), stir while adding, evaporate ethanol in a 50℃ water bath to obtain inclusion (II). Take 0.05g of inclusion (II), add 1ml water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion with an encapsulation rate of 94.84%.

[0046] Example 2

[0047] Prepare 0.12mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.2g / mL solution, ultrasonicate for 10 minutes, prepare membrane material solution (I), keep away from light for later use. Take 0.2g sodium chloride solid phase particles (average particle size is 650μm), add to the membrane material solution (I) at a mass ratio of 2.5:1 of solid phase particles to lecithin, stir while adding, evaporate in a 50℃ water bath to obtain inclusion (I). Take 0.05g of inclusion (I), add to 1ml water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion, with an encapsulation rate of 55.5%.

[0048] Example 3

[0049] Prepare 0.12mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.2g / mL solution, ultrasonicate for 10 minutes, prepare film-forming material solution (I), keep away from light for later use. Take 0.2g sucrose solid phase particles (average particle size is 650μm), add to the film-forming material solution (I) at a solid phase particle to lecithin mass ratio of 2.5:1, stir while adding, evaporate in a 50℃ water bath to obtain inclusion (I). Continue to add 0.3ml of film-forming material solution (II) (ethanol solution of lecithin with a concentration of 0.2g / mL), stir while adding, evaporate ethanol in a 50℃ water bath to obtain inclusion (II). Take 0.05g of inclusion (II), add 1ml of water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion. Encapsulation efficiency is 80.84%.

[0050] Example 4

[0051] Prepare 0.12mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.2g / mL solution, ultrasonic for 10 minutes, prepare film-forming material solution (I), keep away from light, and set aside. Take 0.2g sodium chloride solid phase particles (average particle size is 650μm), add to the film-forming material solution (I) at a solid phase particle to lecithin mass ratio of 1.43:1, stir while adding, evaporate in a 50℃ water bath, and obtain inclusion (I), continue to add 0.3ml film-forming material solution (II) (lecithin ethanol solution with a concentration of 0.2g / mL), stir while adding, evaporate ethanol in a 50℃ water bath, and obtain inclusion (II). Take 0.05g of inclusion (II), add 1ml water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion, with an encapsulation rate of 94.12%.

[0052] Example 5

[0053] Prepare 0.12mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.2g / mL solution, ultrasonicate for 10 minutes, prepare film-forming material solution (I), keep away from light for later use. Take 0.2g sodium chloride solid phase particles (average particle size is 650μm), add to the film-forming material solution (I) at a solid phase particle to lecithin mass ratio of 1.25:1, stir while adding, evaporate in a 50℃ water bath to obtain inclusion (I). Continue to add 0.3ml of film-forming material solution (II) (ethanol solution of lecithin with a concentration of 0.2g / mL), stir while adding, evaporate ethanol in a 50℃ water bath to obtain inclusion (II). Take 0.05g of inclusion (II), add 1ml water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion with an encapsulation rate of 56.29%.

[0054] Example 6

[0055] Prepare 1.0 mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.2 g / mL solution, ultrasonicate for 10 minutes, prepare film-forming material solution (I), keep away from light, and set aside. Take 0.2g sodium chloride solid phase particles (average particle size is 650μm), add to the film-forming material solution (I) at a solid phase particle to lecithin mass ratio of 2.5:1, stir while adding, evaporate in a 50℃ water bath, and obtain inclusion (I), continue to add 0.3ml film-forming material solution (II) (lecithin ethanol solution with a concentration of 0.2g / mL), stir while adding, evaporate ethanol in a 50℃ water bath, and obtain inclusion (II). Take 0.05g of inclusion (II), add 1ml water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion, with an encapsulation rate of 92.52%.

[0056] Example 7

[0057] Prepare 1.0 mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.16 g / mL solution, ultrasonicate for 10 minutes, prepare film-forming material solution (I), keep away from light for later use. Take 0.2g sodium chloride solid phase particles (average particle size is 650μm), add to the film-forming material solution (I) at a solid phase particle to lecithin mass ratio of 1.56:1, stir while adding, evaporate in a 50℃ water bath to obtain inclusion (I). Continue to add 0.3ml of film-forming material solution (II) (ethanol solution of lecithin with a concentration of 0.2g / mL), stir while adding, evaporate ethanol in a 50℃ water bath to obtain inclusion (II). Take 0.05g of inclusion (II), add 1ml of water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion with an encapsulation rate of 78.21%.

[0058] Example 8

[0059] Prepare 1.0 mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.2 g / mL solution, ultrasonicate for 10 minutes, prepare film-forming material solution (I), keep away from light, and set aside. Take 0.2g sucrose solid phase particles (average particle size is 650μm), add to the film-forming material solution (I) at a solid phase particle to lecithin mass ratio of 2.5:1, stir while adding, evaporate in a 50℃ water bath, and obtain inclusion (I), continue to add 0.3ml film-forming material solution (II) (lecithin ethanol solution with a concentration of 0.2g / mL), stir while adding, evaporate ethanol in a 50℃ water bath, and obtain inclusion (II). Take 0.05g of inclusion (II), add 1ml water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion, with an encapsulation rate of 62.78%.

[0060] Example 9

[0061] Prepare 0.625mg / mL lotus leaf flavonoids ethanol solution, add lecithin to dissolve, make the lecithin concentration reach 0.2g / mL solution, ultrasonic for 10 minutes, prepare film-forming material solution (I), keep away from light for later use. Take 0.2g sodium chloride solid phase particles (average particle size is 650μm), add to the film-forming material solution (I) at a solid phase particle to lecithin mass ratio of 2.5:1, stir while adding, evaporate in a 50℃ water bath to obtain inclusion (I), continue to add 0.3ml film-forming material solution (II) (lecithin ethanol solution with a concentration of 0.2g / mL), stir while adding, evaporate ethanol in a 50℃ water bath to obtain inclusion (II). Take 0.05g of inclusion (II), add 1ml water, shake for 10min, and obtain lotus leaf flavonoids liposome emulsion with an encapsulation rate of 78.83%.

[0062] Comparative Example 1

[0063] The preparation conditions are the same as those in Example 2, except that the film-forming material solution does not contain lotus leaf flavonoids.

[0064] 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 dark conditions. Sodium chloride (average particle size of 650μm) is added to the lecithin solution at a mass ratio of 2.5:1 between solid phase particles and encapsulating materials, stirring while adding, and evaporating in a 50°C water bath to obtain empty liposomes. Take the empty liposomes, add 0.12mg / mL of lotus leaf flavonoids aqueous solution according to the mass ratio of the solid content of the lotus leaf flavonoids solution to the mass ratio of lecithin of 0.06:100, shake for 10 minutes, and obtain the lotus leaf flavonoids liposome emulsion. The encapsulation rate is 41.25%.

[0065] Test example 1: Encapsulation efficiency test

[0066] The method for detecting the encapsulation rate of lotus leaf flavonoid liposome is as follows: take 0.1mL lotus leaf flavonoid liposome emulsion in a centrifuge tube, centrifuge at 8000rpm for 10min, take out the supernatant and add it to an ultrafiltration centrifuge tube, then add 0.3mL pure water, centrifuge for the first time for 10min (wherein, the ultrafiltration membrane specification is 30KD, and the centrifuge speed is 8000rpm), take the permeate as the free lotus leaf flavonoid test solution, and perform liquid chromatography analysis. Add 0.4mL of anhydrous ethanol to the above centrifuge tube, ultrasonically dissolve, perform a second ultrafiltration centrifugation for 10min (wherein, the ultrafiltration membrane specification is 30KD, and the centrifuge speed is 8000rpm), take the permeate to make up the lost weight with anhydrous ethanol, and use it as the encapsulated lotus leaf flavonoid test solution for liquid chromatography detection.

[0067] HPLC conditions: bonded silica gel C 18(4.6mm*250mm, 5μm), with methanol (A) and 0.2% (v / v) phosphoric acid water (B) as the mobile phase, gradient elution (0-8min, A% (10%-40%); 8-23min, A% (40%-70%); 23-30min, A% (70%-85%), flow rate 1.0mL·min -1 , column temperature 30℃, detection wavelength: 360nm, injection volume: 20μL.

[0068] Here, encapsulation rate (%) = amount of encapsulated components / (amount of free components + amount of encapsulated components)×100%.

[0069] like Figures 1 to 3 As shown, the HPLC spectra of free and encapsulated lotus leaf flavonoids of liposomes of Examples 1 to 3, Examples 4 to 6, Examples 7 to 9 and Comparative Example 1 are respectively shown. It can be seen from the figure that the lotus leaf flavonoids liposome free lotus leaf flavonoids test solution prepared in Example 1 has basically no peak at the lotus leaf flavonoids chromatographic peak (about 17.5min), and the encapsulated lotus leaf flavonoids test solution has corresponding strong peaks at the lotus leaf flavonoids chromatographic peak, indicating that the lotus leaf flavonoids are completely encapsulated.

[0070] Table 1 Encapsulation efficiency of lotus leaf flavonoids liposomes in various examples and comparative examples (%)

[0071]

[0072] Table 1 shows the encapsulation efficiency of lotus leaf flavonoids liposomes prepared in various examples and comparative examples.

[0073] Compared with Example 3 using sucrose as the solid phase particles, the solid phase particles in Example 1 are sodium chloride, and the coating material solution containing lotus leaf flavonoids is easy to spread on its surface and form a film on its surface, with an ideal coating effect.

[0074] Compared with Example 2 which adopts a one-time film-forming method, Example 1 further adds a wrapping material solution (II) to perform a second film-forming after the first film-forming, which significantly improves the wrapping effect.

[0075] In Examples 1, 4 and 6, the encapsulation rate can reach more than 92.5%, indicating that the mass ratio of lotus leaf flavonoids to lecithin in the range of 0.06:100-0.5:100 has little effect on the encapsulation rate and the encapsulation effect is ideal. When the mass ratio of lotus leaf flavonoids to lecithin in the encapsulation (I) is 0.625:100, as in Example 7, the encapsulation rate is as low as 78.21%.

[0076] Using sodium chloride as the solid phase particles, the results of Example 4 and Example 6 also show that the encapsulation rate effect is ideal when the mass ratio of the solid phase particles to the encapsulating material is in the range of 1.43:1-2.5:1.

[0077] The concentration of lotus leaf flavonoids is in the range of 0.12-1.0 mg / ml, and the encapsulation effect is ideal, with no significant effect on the encapsulation rate.

[0078] Comparative Example 1 uses the same amount of solid particles and encapsulating materials as Example 1 to first prepare empty liposomes, but the encapsulation efficiency is poor. This is because the lotus leaf flavonoids component exists in the aqueous solution and it is difficult to effectively encapsulate it during the hydration process of the empty liposomes.

[0079] Test Example 2: Determination of Particle Size

[0080] Take 0.1 mL of the lotus leaf flavonoids liposome solution prepared in Examples 1 to 9, and dilute it to 1 mL with water. The particle size of the lotus leaf flavonoids liposome after microfiltration and extrusion is measured by a laser particle size analyzer Zetasizer Nano ZS90.

[0081] The lotus leaf flavonoid liposomes prepared in Example 1 had an average particle size of 188.3 nm and a PDI of 0.338 before microfiltration extrusion; after granulation, the average particle size and PDI were reduced to 162.8 nm and 0.160, respectively. This shows that the microfiltration extrusion effect is obvious. The particle size distribution of the lotus leaf flavonoid liposomes prepared in Example 1 after microfiltration extrusion is shown in Figure 2. Figure 4 As shown. The average potentials of the liposomes prepared in Example 1 before and after microfiltration extrusion were measured to be -23.9mv and -22.8mv, respectively, indicating that the surface of the liposome particles is negatively charged and relatively stable. The detection methods of the remaining examples and comparative examples are the same as those of Example 1, and the specific results are shown in Table 2. As can be seen from Table 2, the average particle sizes of Examples 1 to 9 are between 106.7 and 186.1nm, indicating that liposomes with smaller particle sizes can be obtained using sodium chloride and sucrose as solid phase particle carriers.

[0082] The lotus leaf flavonoids liposome emulsion prepared in Example 1 was filtered through a copper mesh and dried. The TEM electron microscope image is as follows: Figure 5 As shown in the figure, it can be seen that the lotus leaf flavonoid liposomes prepared in Example 1 are similar to spheres with regular shapes. Their particle size is significantly smaller than that of particles in the lotus leaf flavonoid liposome emulsion, which may be caused by the drying and water loss of particles in the emulsion during the electron microscopy sample preparation process; and the lotus leaf flavonoid liposome particles are relatively evenly distributed.

[0083] Table 2 Average particle size and potential of lotus leaf flavonoid liposomes prepared in Examples 1 to 9 and Comparative Example 1

[0084]

[0085]

[0086] Test Example 3: Transdermal Diffusion Experiment

[0087] Using Strat-MTM Membrane (Merck Millipore Ltd) and a transdermal diffusion test device, a 40 wt% PEG400 aqueous solution was added to the diffusion cell, and 0.3 mL of the lotus leaf flavonoid liposome emulsion prepared in Example 6 and a lotus leaf flavonoid aqueous solution (0.071 mg / ml) of a concentration corresponding to Example 6 were evenly applied on the smooth surface of the artificial skin, and permeated at room temperature. At intervals of 12 hours, 0.2 mL of the sample solution was taken out from the diffusion cell each time, and the volume was supplemented with a 40 wt% PEG400 aqueous solution to obtain a lotus leaf flavonoid liposome skin permeation solution and a lotus leaf flavonoid aqueous solution skin permeation solution, respectively. 0.3 mL of a lotus leaf flavonoid aqueous solution (0.071 mg / ml) of a concentration corresponding to Example 6 was taken and diluted in accordance with the lotus leaf flavonoid liposome skin permeation solution to obtain a lotus leaf flavonoid control solution of a concentration corresponding to Example 6. The lotus leaf flavonoid liposome skin permeation solution of Example 7, a lotus leaf flavonoid aqueous solution skin permeation solution of a concentration corresponding to Example 7, and a lotus leaf flavonoid control solution were prepared in the same manner. Liquid chromatography was performed to determine the amount of lotus leaf flavonoids, using the peak area of ​​hyperoside, the main component of lotus leaf flavonoids, as the amount of lotus leaf flavonoids to calculate the skin permeability. The results are shown in Table 3.

[0088] Skin penetration rate % = (amount of drug passing through the skin / total amount of drug) × 100%. In the present invention, skin penetration rate % = peak area of ​​hyperoside in skin penetration solution / (peak area of ​​hyperoside in lotus leaf flavonoid control solution) * 100%.

[0089] Test Example 4: Antioxidant Activity

[0090] 1. Solution preparation

[0091] Solution preparation

[0092] DPPH-ethanol solution: Weigh 9 mg of DPPH and dissolve it in 200 mL of ethanol. Dissolve it by ultrasonication to prepare a 0.045 mg / mL DPPH-ethanol solution.

[0093] Lotus leaf flavonoids solution: Take an appropriate amount of lotus leaf flavonoids, dissolve it in 60% ethanol and make up to volume to prepare a 0.0045 mg / mL solution.

[0094] Vitamin C solution: Dissolve in 60% ethanol and make up to volume to prepare a solution with a concentration of 0.005 mg / mL.

[0095] 2. Sample group: Take 80 μL of sample solution into a 96-well plate, add 120 μL of DPPH free radical solution, shake well, and stand at room temperature (25°C) away from light for 10 min. Measure its absorbance, which is recorded as A1;

[0096] The sample solutions are: the skin permeation solution of detection example 3, the skin permeation solution of lotus leaf flavonoids aqueous solution corresponding to the embodiment, and the vitamin C solution.

[0097] Control group: According to the above method, the DPPH free radical solution was replaced by an equal volume of 60% ethanol, and its absorbance was measured and recorded as A2;

[0098] Blank control group: Pipette 80 μL of 60% ethanol into a 96-well plate, add 120 μL of DPPH free radical solution, shake well, and stand at room temperature in the dark for 10 min. Measure the absorbance, which is recorded as A0;

[0099] The scavenging rate of DPPH free radicals was calculated according to the following formula. The results are shown in Table 3.

[0100]

[0101] Table 3 Scavenging rate of DPPH free radicals by skin permeation liquid of lotus leaf flavonoid liposome emulsion

[0102]

[0103] As shown in Table 3, the skin permeability and scavenging rate of DPPH free radicals of lotus leaf flavonoids aqueous solution, lotus leaf flavonoids liposome emulsion 12h skin permeation liquid, lotus leaf flavonoids aqueous solution 12h skin permeation liquid and vitamin C solution are respectively shown, where "ND" means not detected. Vitamin C solution and lotus leaf flavonoids aqueous solution were used as controls, and the DPPH free radical scavenging rate results were normal, indicating that the results of the method of the present invention are accurate. The lotus leaf flavonoids aqueous solution 12h skin permeation liquid cannot penetrate the artificial skin, and the lotus leaf flavonoids liposome emulsion 12h skin permeation performance is ideal, and its skin permeation liquid has a certain scavenging effect of DPPH free radicals, which confirms that liposome encapsulation greatly improves the transdermal effect of lotus leaf flavonoids.

Claims

1. A method for preparing lotus leaf flavonoid liposome emulsion, characterized in that: The following steps are involved: (a) dissolving lotus leaf flavonoids in anhydrous ethanol, and then adding encapsulating materials to prepare a membrane material solution (I); (b) immersing the solid phase particles in a film-forming material solution (I), removing the anhydrous ethanol, performing a first film-forming step, and combining the solid phase particles with the drug-containing encapsulation material to obtain an encapsulation (I); (c) immersing the inclusion (I) in a film-forming material solution (II), removing the anhydrous ethanol, performing secondary film formation to obtain the inclusion (II), and hydrating to obtain a lotus leaf flavonoid liposome emulsion; the solid phase particles are one of sucrose and sodium chloride.

2. The method for preparing the lotus leaf flavonoids liposome emulsion according to claim 1, characterized in that: The particle size of the solid phase particles is 150-1250 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 lotus leaf flavonoids liposome emulsion according to claim 1, characterized in that: The packaging material is one of lecithin and lecithin-cholesterol.

4. The method for preparing the lotus leaf flavonoids liposome emulsion according to claim 1, characterized in that: In the film-forming material solution (I), the concentration of lotus leaf flavonoids is 0.1-2 mg / mL, and the concentration of the encapsulating material lecithin is 0.1-0.2 g / mL, preferably 0.2 g / mL. In the film-forming material solution (II), the concentration of the encapsulating material was 0.2 g / mL.

5. The method for preparing the lotus leaf flavonoid liposome emulsion according to claim 4, characterized in that: In the inclusion (I), the mass ratio of solid phase particles to lecithin is (1.25-6.25):1; preferably (1.43-2.5):1; In the package (I), the mass ratio of lotus leaf flavonoids to lecithin is (0.03-1.875):100; preferably (0.03-1.5):100; and more preferably (0.06-0.5):

100.

6. A lotus leaf flavonoids liposome emulsion prepared by the preparation method according to any one of claims 1 to 5.

7. The lotus leaf flavonoids liposome emulsion according to claim 6, characterized in that: The encapsulation rate of the lotus leaf flavonoids liposome emulsion is 55.5% to 94.8%.

8. The lotus leaf flavonoids liposome emulsion according to claim 6, characterized in that: The particle size of the lotus leaf flavonoid liposome is 106.7-186.1 nm, and the aggregation index (PDI) is 0.144-0.

214.

9. Use of the lotus leaf flavonoid liposome emulsion prepared by the preparation method according to any one of claims 1 to 6 or the lotus leaf flavonoid liposome emulsion according to any one of claims 7 to 8 in the preparation of antioxidant drugs.

10. Use of the lotus leaf flavonoid liposome emulsion prepared by the preparation method according to any one of claims 1 to 6 or the lotus leaf flavonoid liposome emulsion according to any one of claims 7 to 8 in the preparation of anti-aging and whitening cosmetics.

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