Hydroxytyrosol berberine hydrochloride composite liposome emulsion, preparation method and application thereof

Abstract

The present invention relates to the technical field of pharmaceutical preparations, and in particular to a hydroxytyrosol berberine hydrochloride composite liposome emulsion, a preparation method and an application thereof. After adjusting the hydrophilicity and hydrophobicity of hydroxytyrosol by berberine hydrochloride, sucrose or sodium chloride solid phase particles are used as carriers, and empty liposomes are formed by combining with a coating material solution, and then hydroxytyrosol double salt is coated to prepare the hydroxytyrosol berberine hydrochloride composite liposome emulsion. The present invention utilizes the excellent specific surface area of ​​the solid phase particles to uniformly coat the coating material on the surface of the solid phase particles to form a thin film, thereby greatly increasing the drug loading while quickly encapsulating the hydroxytyrosol double salt.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to a hydroxytyrosol-berberine hydrochloride composite liposome emulsion, a preparation method and an application thereof. Background Art

[0002] Hydroxytyrosol is a phenolic compound extracted from olive leaves and olive oil, which has anti-inflammatory, anti-tumor, antiviral, antibacterial and antifungal biological activities. Hydroxytyrosol has significant antioxidant activity. It can directly remove oxidative substances and indirectly inhibit oxidative stress by regulating the phase II enzyme antioxidant system with nuclear transcription factor E2-related factor 2 as the core. It has great potential in the prevention / treatment of various oxidative damage-related diseases. Due to its significant biological activity and good safety, hydroxytyrosol has important application value in food, medicine and cosmetics.

[0003] As a polyphenol compound, hydroxytyrosol has poor stability during storage and use. Encapsulation by nanotechnology can greatly improve its stability and bioavailability. Liposomes are aqueous core microvesicles formed by phospholipid bilayers, which can encapsulate drugs, reduce toxic side effects, and improve their bioavailability, thereby solving the problems of short half-life in the body, fast elimination rate, low efficacy, and low bioavailability. Li Wenjun et al. used hydrogenated lecithin and cholesterol as emulsifiers and prepared HT flexible nanoliposomes by thin film dispersion method and studied their storage stability. The encapsulation efficiency, particle size, polydispersity index (PDI) and Zeta potential of the prepared HT flexible nanoliposomes were 46.78%, 104.7 nm, 0.231 and -42.5 mV, respectively; after being stored at 4, 25 and 60 °C for 28 days, the encapsulation efficiency of HT nanoliposomes at 4 °C was 37.56%, the particle size was 152.7 nm, the PDI and Zeta potential did not change much, while the above indicators of HT nanoliposomes at 25 and 60 °C changed greatly. Therefore, 4 °C was more conducive to its stable storage (Li Wenjun, Wang Chengzhang, Lei Jiandu et al., Preparation of HT flexible nanoliposomes and study on their storage stability, Biomass Chemical Engineering, 2023, 57(5): 25-34). In order to enable HT nanoliposomes to be stored for a long time at low temperature, Li Wenjun et al. quickly froze them at -50°C for 8 h and freeze-dried them for 72 h. Among them, when the freeze-drying protective agent was a D-trehalose / sucrose / mannitol solution with a mass ratio of 4:2:1 and the mass fraction of the solution was 10%, the average particle size of the HT nanoliposomes obtained after reconstitution was 125.24 nm, and the encapsulation rate was 69.74% (Li Wenjun et al., Preparation and performance evaluation of hydroxytyrosol nanoliposome freeze-dried powder, Chemistry and Industry of Forest Products, 2023, 43(5):125). However, the hydroxytyrosol liposomes prepared by them still have the disadvantages of low encapsulation rate, unsatisfactory stability, and overly complicated preparation process, which limits the research and development and application of hydroxytyrosol liposome series products. Summary of the invention

[0004] The present invention aims at the problems of low encapsulation rate, unsatisfactory stability and overly complicated preparation process of existing hydroxytyrosol liposomes. Solid phase particles are used as carriers in combination with encapsulation materials to prepare empty liposomes, and then hydroxytyrosol is converted into a double salt, and the empty liposomes are used to coat the hydroxytyrosol double salt to prepare a hydroxytyrosol hydrochloride berberine composite liposome emulsion. Among them, the preparation process of the empty liposomes is mild, does not require special equipment, does not affect the biological activity of the active ingredient, does not change the pH value of the solution, has no solvent residue, and is suitable for oral, transdermal and injection administration. As solid phase particle carriers, sucrose and sodium chloride are green, safe, and low in cost, and do not affect the application of liposomes in subsequent medical cosmetics. The present invention constructs a new preparation process for hydroxytyrosol double salt liposomes with simple process, low organic solvent consumption, high encapsulation rate, high stability and high drug loading, which provides a new idea for the large-scale preparation of hydroxytyrosol liposomes and has significant application value.

[0005] To achieve the above object, the present invention provides a method for preparing a hydroxytyrosol-berberine hydrochloride composite liposome emulsion, 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 solution of an encapsulating material, removing the anhydrous ethanol, and combining the solid phase particles with the encapsulating material to obtain empty liposomes;

[0008] (c) adding the empty liposomes to the mixed solution of hydroxytyrosol and berberine hydrochloride, mixing, and obtaining a hydroxytyrosol-berberine hydrochloride composite liposome emulsion.

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

[0010] In the lecithin-cholesterol, the mass ratio of lecithin to cholesterol is greater than 3, preferably 4-8.

[0011] The solid phase particles are sucrose or sodium chloride. By utilizing the excellent specific surface area of ​​the solid phase particles, the wrapping material solution is volatilized on the surface of the solid phase particles to form a wrapping material film.

[0012] The quaternary ammonium group of the berberine hydrochloride reacts with hydroxytyrosol to generate a hydroxytyrosol complex, which adjusts the hydrophilicity and hydrophobicity of hydroxytyrosol, thereby improving the encapsulation rate of hydroxytyrosol.

[0013] The particle size of the solid phase particles is 150-1250 μm, preferably 300-900 μm.

[0014] Preferably, in step (b), the method for removing the anhydrous ethanol comprises: evaporation in a high-temperature water bath, freeze-drying, and standing at room temperature.

[0015] Preferably, in step (c), the empty liposomes and the mixed solution of hydroxytyrosol and berberine hydrochloride are mixed by vortexing, shaking, and ultrasound.

[0016] The mass concentration of the coating material in the coating solution is 0.01-0.2 g / mL, preferably 0.1-0.2 g / mL.

[0017] The mass ratio of the solid phase particles to lecithin is (4.5-6.5):1.

[0018] Preferably, when the solid phase particles are sucrose, the mass ratio of the solid phase particles to lecithin is (4.5-5):1.

[0019] Preferably, when the solid phase particles are sodium chloride, the mass ratio of the solid phase particles to lecithin is (4.68-6.25):1.

[0020] The mass ratio of hydroxytyrosol to lecithin is (6.09-15.53):100;

[0021] Preferably, when the solid phase particles are sucrose, the mass ratio of hydroxytyrosol to lecithin is (6.09-12.17):100.

[0022] The solvent of the hydroxytyrosol and berberine hydrochloride mixed solution is water, and the mass ratio of hydroxytyrosol to berberine hydrochloride is (0.5~2):1; when the mass ratio is greater than 2, the amount of berberine hydrochloride is too small, and part of the hydroxytyrosol forms a complex and part exists in a free state, so that the hydroxytyrosol complex cannot be completely encapsulated.

[0023] In the mixed solution of hydroxytyrosol and berberine hydrochloride, the concentration of hydroxytyrosol is 0.3-1.2 mg / mL.

[0024] The present invention also provides a hydroxytyrosol berberine hydrochloride composite liposome emulsion prepared by the above-mentioned preparation method. When the solid phase particles are sucrose, the 24-hour transmittance of the hydroxytyrosol berberine hydrochloride composite liposome emulsion is not less than 50%, and the encapsulation efficiency is 38% to 90%; when the solid phase particles are sodium chloride, the 24-hour transmittance of the hydroxytyrosol berberine hydrochloride composite liposome emulsion is not less than 16%, and the encapsulation efficiency is 56% to 75%.

[0025] Furthermore, the encapsulation efficiency of the hydroxytyrosol berberine hydrochloride composite liposomes was measured by HPLC-ultrafiltration centrifugation method;

[0026] The ultrafiltration centrifugal speed is 6000-12000 rpm, and the centrifugal time is 10-20 min.

[0027] After granulation, the particle size of the hydroxytyrosol berberine hydrochloride composite liposome is 129.2-223.8 nm, and the polydispersity index (PDI) is 0.220-0.342.

[0028] The granulation method is one of the following: ultrasound, shearing, homogenization, and extrusion, preferably extrusion, and more preferably microfiltration extrusion.

[0029] The present invention also provides an application of the hydroxytyrosol-berberine hydrochloride composite liposome emulsion prepared by the above-mentioned preparation method in the preparation of antioxidant drugs.

[0030] When the solid phase particles are sucrose, the dosage form of the hydroxytyrosol berberine hydrochloride complex liposome emulsion is an oral emulsion.

[0031] When the solid phase particles are sodium chloride, the dosage form of the hydroxytyrosol berberine hydrochloride complex liposome emulsion is an injection.

[0032] The present invention also provides an application of the hydroxytyrosol-berberine hydrochloride composite liposome emulsion prepared by the above-mentioned preparation method in the preparation of antioxidant cosmetics.

[0033] Beneficial Effects

[0034] 1. The present invention is the first to utilize the quaternary ammonium group of berberine hydrochloride to react with hydroxytyrosol to generate a hydroxytyrosol complex, thereby adjusting the hydrophilicity and hydrophobicity of hydroxytyrosol and thus improving the encapsulation rate of hydroxytyrosol.

[0035] 2. The present invention uses sucrose or sodium chloride solid phase particles as carriers for the first time, combines with the encapsulating material solution to form empty liposomes, and encapsulates hydroxytyrosol double salt to prepare hydroxytyrosol berberine hydrochloride composite liposome emulsion. Due to the excellent specific surface area of ​​the solid phase particles, the film thickness formed by the encapsulating material on the surface of the solid phase particles is thin and uniform, which greatly increases the drug loading while quickly encapsulating the drug.

[0036] 3. Compared with the spraying method of spraying the coating material solution onto the surface of the solid phase particles to form a spot film with uneven thickness and discontinuousness, the present invention adopts the immersion film-forming method to immerse the solid phase particles in a high concentration of the coating material solution. The obtained film thickness is uniform, resulting in smaller and more uniform liposome particle size, thereby increasing the drug loading capacity.

[0037] 4. The present invention prepares the empty liposomes and then mixes them with the hydroxytyrosol and berberine hydrochloride mixture, which not only avoids the influence of anhydrous ethanol on hydroxytyrosol, but also reduces the heating time of hydroxytyrosol produced by removing ethanol.

[0038] 5. In actual application, empty liposomes can be prepared first, and the hydroxytyrosol-berberine hydrochloride composite liposomes can be prepared immediately without considering the effects of storage and transportation conditions on the hydroxytyrosol drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The UV absorption spectra of hydroxytyrosol and berberine hydrochloride before and after compounding;

[0040] Figure 2 HPLC spectrum of the test solution for free hydroxytyrosol and encapsulated hydroxytyrosol detected in the hydroxytyrosol-berberine hydrochloride composite liposomes of Example 1-4;

[0041] Figure 3 HPLC spectra of the test solution for free hydroxytyrosol and encapsulated hydroxytyrosol detected in hydroxytyrosol-berberine hydrochloride composite liposomes of Examples 8-10 and Comparative Examples 1-3;

[0042] Figure 4 This is the particle size distribution diagram of the hydroxytyrosol berberine hydrochloride composite liposomes prepared in Example 1. DETAILED DESCRIPTION

[0043] 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.

[0044] In the following examples, the lecithin was purchased from Beijing Aoboxing Biological Co., Ltd.; hydroxytyrosol, berberine hydrochloride, and cholesterol were purchased from Sinopharm Chemical Reagent Co., Ltd.; anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; the artificial skin was Strat-MTM Membrane purchased from Merck Millipore Ltd; and sucrose and sodium chloride were conventional commercial products.

[0045] Example 1

[0046] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sucrose (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of 4.68:1 of sucrose solid phase particles to lecithin, stir while adding, and evaporate in a 50°C water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed in a mass ratio of 1:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 0.3mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 12.17:100, shake for 10 minutes, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0047] like Figure 1 The UV absorption spectra of hydroxytyrosol and berberine hydrochloride before and after the complexation are shown. The detection solutions are 0.6 mg / mL hydroxytyrosol ethanol solution, 0.6 mg / mL berberine hydrochloride ethanol solution, and 0.6 mg / mL hydroxytyrosol-berberine hydrochloride ethanol solution, respectively. The mass ratio of hydroxytyrosol to berberine hydrochloride in the hydroxytyrosol-berberine hydrochloride ethanol solution is 1:1. As can be seen from the figure, hydroxytyrosol has an absorption peak near 293nm, berberine hydrochloride has an absorption peak near 351nm, and the hydroxytyrosol-berberine hydrochloride complex has an absorption peak near 354nm. After the complexation, the shape of the absorption curve has changed, the absorption peak of hydroxytyrosol near 293nm disappears, and the absorption peak of berberine hydrochloride near 351nm red-shifts, all of which prove the formation of the hydroxytyrosol-berberine hydrochloride complex.

[0048] Example 2

[0049] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sucrose (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of sucrose solid phase particles to lecithin of 6.25:1, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed in a mass ratio of 1:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 0.3mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 15.53:100, shake for 10min, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0050] Example 3

[0051] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sucrose (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of 4.68:1 of sucrose solid phase particles to lecithin, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed in a mass ratio of 1:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 1.2mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 12.17:100, shake for 10min, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0052] Example 4

[0053] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sucrose (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of 4.68:1 of sucrose solid phase particles to lecithin, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed in a mass ratio of 2:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 0.3mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 12.17:100, shake for 10min, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0054] Example 5

[0055] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sucrose (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of 4.68:1 of sucrose solid phase particles to lecithin, stir while adding, and evaporate in a 50°C water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed at a mass ratio of 0.5:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 0.3mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 6.09:100, shake for 10 minutes, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0056] Example 6

[0057] The preparation conditions of this example are the same as those of Example 1, except that the mass ratio of lecithin to cholesterol is replaced with 3:1.

[0058] Example 7

[0059] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sodium chloride (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of sodium chloride solid phase particles to lecithin of 4.68:1, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed at a mass ratio of 0.5:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 0.3mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 6.09:100, shake for 10min, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0060] Example 8

[0061] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sodium chloride (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of sodium chloride solid phase particles to lecithin of 6.25:1, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed in a mass ratio of 1:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 0.3mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 15.53:100, shake for 10min, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0062] Example 9

[0063] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sodium chloride (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of sodium chloride solid phase particles to lecithin of 4.68:1, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed in a mass ratio of 1:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 1.2mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 15.53:100, shake for 10min, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0064] Example 10

[0065] Weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in dark conditions. Add sodium chloride (average particle size of 650μm) to the lecithin-cholesterol solution at a mass ratio of sodium chloride solid phase particles to lecithin of 4.68:1, stir while adding, and evaporate in a 50℃ water bath to obtain empty liposomes. Hydroxytyrosol and berberine hydrochloride are mixed in a mass ratio of 2:1 to obtain a mixed solution of hydroxytyrosol and berberine hydrochloride. Take the empty liposomes, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 0.3mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin of 12.17:100, shake for 10min, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0066] Embodiment 11

[0067] The preparation conditions of this example are the same as those of Example 7, except that the lecithin-cholesterol in Example 7 is replaced by lecithin.

[0068] Comparative Example 1

[0069] The preparation conditions of this comparative example are the same as those of Example 1, except that no sucrose solid phase particles are added.

[0070] Weigh lecithin and cholesterol according to the mass ratio of lecithin to cholesterol of 6:1, add anhydrous ethanol solution, prepare a solution with a lecithin-cholesterol concentration of 0.2g / mL, ultrasonicate for 10 minutes, and store under light-proof conditions. Add lecithin-cholesterol solution dropwise to the evaporating dish, heat in a water bath at 50°C to evaporate, and obtain a lecithin-cholesterol membrane. A mixed solution of hydroxytyrosol and berberine hydrochloride was prepared according to the mass ratio of hydroxytyrosol to berberine hydrochloride of 1:1. Take a mixed solution of hydroxytyrosol and berberine hydrochloride, add a mixed solution of hydroxytyrosol and berberine hydrochloride with a hydroxytyrosol concentration of 0.3mg / mL according to the mass ratio of hydroxytyrosol solid content to lecithin-cholesterol of 12.17:100, and ultrasonicate for 10min to obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0071] Comparative Example 2

[0072] The preparation conditions of this comparative example are the same as those of Example 1, except that the mixed solution of hydroxytyrosol and berberine hydrochloride is replaced by a hydroxytyrosol solution with the same solid content.

[0073] Comparative Example 3

[0074] The preparation conditions of this comparative example are the same as those of Example 1, except that berberine hydrochloride is replaced by nuciferine.

[0075] Comparative Example 4

[0076] The preparation conditions of this comparative example are the same as those of Example 1, except that the mass ratio of hydroxytyrosol to berberine hydrochloride is 3:1.

[0077] Comparative Example 5

[0078] The experimental conditions of this comparative example are the same as those of Example 1, wherein hydroxytyrosol and berberine hydrochloride are added to an anhydrous ethanol solution of lecithin-cholesterol (the concentration of lecithin-cholesterol in the anhydrous ethanol solution of lecithin-cholesterol is 0.2 g / mL), the mass ratio of hydroxytyrosol to berberine hydrochloride is 1:1, and the mass ratio of hydroxytyrosol to lecithin is 12.17:100.

[0079] Sucrose was placed in a rotary evaporator, and added to a lecithin-cholesterol solution containing hydroxytyrosol at a mass ratio of sucrose solid phase particles to lecithin of 4.68:1 under heating at 50°C. The solvent was evaporated and deionized water was added to obtain a hydroxytyrosol-berberine hydrochloride complex liposome emulsion (wherein the mass ratio of deionized water to hydroxytyrosol was 1000:0.3).

[0080] Comparative Example 6

[0081] The experimental conditions of this comparative example refer to Example 1, weigh lecithin and cholesterol (the mass ratio of lecithin to cholesterol is 6:1), add anhydrous ethanol solution, prepare a lecithin-cholesterol solution containing 0.2g / mL lecithin, ultrasonicate for 10 minutes, and store in the dark. Take sucrose and place it in a rotary evaporator, then spray the lecithin-cholesterol solution into the rotary evaporator at a mass ratio of 4.68:1 of sucrose solid phase particles and lecithin, the rotary evaporation temperature is 50°C, the vacuum degree is -0.098MPa, and empty liposomes are obtained. Take empty liposomes, according to the mass ratio of hydroxytyrosol to lecithin of 12.17:100, add hydroxytyrosol and berberine hydrochloride solution with a hydroxytyrosol concentration of 0.3mg / mL (wherein, the mass ratio of hydroxytyrosol to berberine hydrochloride is 1:1), shake for 10min, and obtain a hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0082] Test example 1: Encapsulation efficiency test

[0083] The encapsulation efficiency of the hydroxytyrosol hydrochloride berberine composite liposome emulsion was calculated by high performance liquid chromatography: 0.1 mL of the hydroxytyrosol hydrochloride berberine composite liposome emulsion was placed in a centrifuge tube (CF), centrifuged at 8000 rpm for 10 min, the supernatant was taken out and added to an ultrafiltration centrifuge tube (UF-CF, ultrafiltration membrane pore size is 30KD), and then 0.3 mL of pure water was added, and the centrifugation time at 8000 rpm was 10 min. The permeated liquid was taken as the test solution for free hydroxytyrosol detection. 0.4 mL of anhydrous ethanol was added to the above CF, weighed, ultrasonically dissolved, added to the above UF-CF, ultrafiltration centrifuged at 8000 rpm for 10 min, and the permeated liquid was taken to make up the lost weight with anhydrous ethanol as the test solution for the detection of encapsulated hydroxytyrosol.

[0084] Chromatographic conditions: bonded silica gel C18 (4.6 mm *250 mm, 5 μm), acetonitrile-water (10:90) as mobile phase, flow rate 1.0 mL·min, column temperature 30°C, detection wavelength: 280 nm, injection volume: 10 μL.

[0085] Among them, encapsulation efficiency (100%) = encapsulated hydroxytyrosol peak area / (free hydroxytyrosol peak area + encapsulated hydroxytyrosol peak area).

[0086] Table 1 Encapsulation efficiency of hydroxytyrosol-berberine hydrochloride composite liposome emulsion

[0087]

[0088] Hydroxytyrosol is highly water-soluble, and the traditional method of using lecithin to encapsulate the hydroxytyrosol has a very poor effect. The present invention proposes for the first time to use a hydroxytyrosol-berberine hydrochloride complex to solve the problem of poor lecithin encapsulation effect of hydroxytyrosol.

[0089] As shown in Table 1, the encapsulation efficiency of the hydroxytyrosol-berberine hydrochloride composite liposome emulsion prepared in each example and comparative example is shown. Each example examines the effects of the amount of lecithin, the concentration of hydroxytyrosol and the concentration of berberine hydrochloride on the encapsulation efficiency.

[0090] Compared with Example 1, the amount of wrapping material used in Example 2 is less, resulting in a low encapsulation rate. This is because the amount of wrapping material used as the raw material for forming the liposome shell is too small, resulting in a thin film thickness on the surface of the solid particles, which in turn leads to a poor encapsulation rate. If the amount of wrapping material is too much, the wrapping materials will adhere to each other, the surface area of ​​the film formed will be reduced, the liposome embedding amount will be small or the thickness of the liposome shell will be large, making it difficult for a small amount of drug to enter.

[0091] Compared with Example 1, the hydroxytyrosol concentration in Example 3 is too high, resulting in a poor encapsulation rate. This is because the concentrations of hydroxytyrosol and berberine hydrochloride have a greater influence on the encapsulation rate. When other conditions remain unchanged, when the hydroxytyrosol concentration is high, the solution volume is small, and the concentration of the formed liposomes is high, which will also cause the encapsulation materials to adhere to each other and fail to form a spherical shape with a good morphology. The liposome encapsulation amount is small or the thickness of the liposome shell is large, making it difficult for a small amount of drugs to enter. When the hydroxytyrosol concentration is low, the volume of the hydroxytyrosol solution is large. Under the same amount of empty liposomes, the liposome concentration in the solution is small and it is difficult to capture and embed all the hydroxytyrosol.

[0092] Compared with Example 1, the mass ratio of hydroxytyrosol to berberine hydrochloride in Example 4 is larger, and part of hydroxytyrosol forms a complex and part exists in a free state, resulting in a poor encapsulation efficiency.

[0093] In Example 1, hydroxytyrosol and berberine hydrochloride form a complex salt in a mass ratio of 1:1, and the encapsulation rate can reach more than 87%. In addition, the preparation process is simple, the amount of organic solvent used is small, and large-scale preparation is possible. The present application has significant advantages over the prior art.

[0094] like Figure 2 , 3 As shown, the HPLC spectra of free and encapsulated hydroxytyrosol test solutions in the hydroxytyrosol hydrochloride berberine composite liposome emulsions prepared in Examples 1-4, Examples 8-10, and Comparative Examples 1-3 are shown. Figure 2 It can be seen that both the free hydroxytyrosol test solution and the encapsulated hydroxytyrosol test solution have peaks at the hydroxytyrosol chromatographic peak (the retention time of the hydroxytyrosol chromatographic peak is about 5 minutes). Figure 3 It can also be seen that each test solution has a peak at the hydroxytyrosol chromatographic peak (the retention time of the hydroxytyrosol chromatographic peak is about 5 minutes); however, the peak of the free hydroxytyrosol test solution is smaller and close to a straight line, while the encapsulated hydroxytyrosol test solution has a strong peak, indicating that the encapsulated drug leaks very little and the encapsulation effect is ideal.

[0095] Test Example 2: Particle Size Determination of Hydroxytyrosol-Berberine Hydrochloride Complex Liposomes

[0096] The hydroxytyrosol berberine hydrochloride composite liposome emulsion prepared in Example 1 was filtered through a 0.45 μm microfiltration membrane, 0.1 mL of the filtrate was taken and diluted to 1 mL with water, and the average particle size of the hydroxytyrosol berberine hydrochloride composite liposome after microfiltration was detected three times by a laser particle size analyzer Zetasizer Nano ZS90. The average particle size, average PDI, and average potential of the hydroxytyrosol berberine hydrochloride composite liposome were 213.1 nm, 0.283, and -17.6 mv. Figure 4 The results showed that the particle size of hydroxytyrosol-berberine hydrochloride composite liposomes after microfiltration extrusion was smaller, the dispersion coefficient was smaller, the particles were more stable, and the dispersion was uniform.

[0097] The particle size detection methods of the remaining embodiments and comparative examples are the same as those of embodiment 1, and the detection data are shown in Table 2.

[0098] Test Example 3: Transdermal Diffusion Experiment

[0099] Using a transdermal diffusion test apparatus, a PEG400 solution with a mass concentration of 40% was added to the diffusion cell, and 200 µL of each hydroxytyrosol berberine hydrochloride composite liposome emulsion prepared in each embodiment and comparative example was taken, diluted to 0.3 mL with water, and evenly dispersed on the smooth surface of the artificial skin. It was permeated at room temperature. After intervals of 12 h and 24 h, 0.2 mL of the permeated solution was taken out from the diffusion cell each time for liquid chromatography determination. At the same time, the volume was supplemented with a PEG400 solution with a mass concentration of 40%, and the permeability was determined and calculated by high performance liquid chromatography.

[0100] Table 2 Average particle size, potential and permeability of hydroxytyrosol-berberine hydrochloride composite liposomes

[0101]

[0102] As can be seen from Table 2, in Comparative Example 2, berberine hydrochloride is replaced by hydroxytyrosol, and in Comparative Example 3, berberine hydrochloride is replaced by nuciferine, and the permeability is 0 at intervals of 12h and 24h, while the permeability can be detected in other embodiments and comparative examples, indicating that the use of berberine hydrochloride to form a hydroxytyrosol berberine hydrochloride composite liposome emulsion can effectively promote transdermal absorption, because the quaternary ammonium group of berberine hydrochloride reacts with hydroxytyrosol to form a hydroxytyrosol complex, which adjusts the hydrophilicity and hydrophobicity of hydroxytyrosol, thereby increasing the encapsulation rate of hydroxytyrosol, and then promoting its transdermal absorption. In addition, the mass ratio of hydroxytyrosol to berberine hydrochloride is increased in Comparative Example 4, resulting in a significant decrease in its permeability, indicating that the mass ratio of hydroxytyrosol to berberine hydrochloride has a great influence on the hydroxytyrosol berberine hydrochloride composite liposome emulsion.

[0103] The results show that in Comparative Example 1, solid phase particles were not added as carriers, and its transmittance decreased significantly. This is because there are no solid phase particles with large specific surface area as carriers, resulting in a smaller film-forming area of ​​the wrapping material, which affects its encapsulation rate and further affects its transmittance. In Comparative Example 5, since the organic solution of lecithin and drugs is used to form a film on the surface of the solid phase particles, the density of the film is far less than that of the wrapping material on the surface of the solid phase particles in Example 1, which affects its encapsulation rate and further affects its transmittance. In Comparative Example 6, during the spraying process of the lecithin-cholesterol solution, since the rotary evaporator is in a vacuum state, it is difficult to effectively spray the film on the surface of the solid phase particles. More tiny spots are formed on the surface of the solid phase particles, and some particles are formed. Therefore, for the same amount of wrapping material solution, the film-forming area of ​​Comparative Example 6 is much smaller than the film-forming area of ​​Examples 1 to 10, thereby affecting its transmittance.

[0104] The results also showed that the permeability of hydroxytyrosol-berberine hydrochloride composite liposomes prepared with sucrose particles as carrier was higher than that prepared with sodium chloride as carrier.

[0105] Test Example 4: Antioxidant Activity

[0106] The results of the DPPH method for determining the antioxidant activity of hydroxytyrosol are as follows:

[0107] 1. Solution preparation

[0108] DPPH-ethanol solution: Weigh 9 mg of DPPH and dissolve it in 200 mL of anhydrous ethanol. Dissolve it by ultrasonication to prepare a 0.045 mg / mL DPPH-ethanol solution.

[0109] Vitamin C solution: Dissolve and dilute with anhydrous ethanol to prepare a solution with a concentration of 0.0056 mg / mL.

[0110] 2. Detection

[0111] 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;

[0112] The sample solutions are respectively: the permeation solutions of the embodiments and comparative examples obtained in Test Example 3, and the vitamin C solution.

[0113] Control group: According to the above method, the DPPH free radical solution was replaced by an equal volume of 60% (volume fraction) ethanol, and its absorbance was measured and recorded as A2;

[0114] Blank control group: Pipette 80 μL of 60% (volume fraction) 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;

[0115] The scavenging rate of DPPH free radical was calculated as follows. The results are shown in Table 3.

[0116] .

[0117] Table 3 Scavenging rate of DPPH free radicals

[0118]

[0119] Vitamin C was used as a positive control, indicating that the experimental results of the DPPH clearance rate of the hydroxytyrosol berberine hydrochloride composite liposome emulsions prepared in the examples and comparative examples were reliable.

[0120] The results in Table 3 show that the clearance rates of Examples 1-11 are higher than those of Comparative Examples 1-6, which is related to the better transdermal performance of the prepared hydroxytyrosol berberine hydrochloride composite liposome emulsion. The penetration concentrations in the Examples and Comparative Examples are between 0.0026 and 0.00695 mg / mL, and the clearance rate and the penetration concentration show a certain dose-effect relationship; between 0.00695 and 0.0504 mg / mL, the clearance rate no longer increases with the increase of the penetration concentration, which is related to the excessive penetration concentration.

Claims

1. A method for preparing a hydroxytyrosol-berberine hydrochloride composite liposome emulsion, characterized in that: include: (a) adding a wrapping material into an anhydrous ethanol solution to prepare a wrapping material solution; (b) immersing the solid phase particles in a solution of an encapsulating material, removing the anhydrous ethanol, and combining the solid phase particles with the encapsulating material to obtain empty liposomes; (c) adding the empty liposomes to the mixed solution of hydroxytyrosol and berberine hydrochloride, mixing, and obtaining a hydroxytyrosol-berberine hydrochloride composite liposome emulsion; The solid phase particles are sucrose or sodium chloride; The mass ratio of hydroxytyrosol to berberine hydrochloride is (0.5-2):1; In the mixed solution of hydroxytyrosol and berberine hydrochloride, the concentration of hydroxytyrosol is 0.3-1.2 mg / mL; The encapsulating material is one of lecithin and lecithin-cholesterol; When the solid phase particles are sucrose, the mass ratio of the solid phase particles to lecithin is (4.5-5):1; When the solid phase particles are sodium chloride, the mass ratio of the solid phase particles to lecithin is (4.68-6.25):

1.

2. The method for preparing the hydroxytyrosol berberine hydrochloride composite liposome emulsion according to claim 1, characterized in that: The solvent of the hydroxytyrosol and berberine hydrochloride mixed solution is water.

3. The method for preparing the hydroxytyrosol berberine hydrochloride composite liposome emulsion according to claim 1, characterized in that: The mass concentration of the encapsulation material in the encapsulation solution is 0.01-0.2 mg / mL; In the lecithin-cholesterol, the mass ratio of lecithin to cholesterol is greater than 3.

4. The method for preparing the hydroxytyrosol berberine hydrochloride composite liposome emulsion according to claim 1, characterized in that: The mass ratio of hydroxytyrosol to lecithin is (6.09-15.53):

100.

5. The method for preparing the hydroxytyrosol berberine hydrochloride composite liposome emulsion according to claim 4, characterized in that: When the solid phase particles are sucrose, the mass ratio of hydroxytyrosol to lecithin is (6.09-12.17):

100.

6. The method for preparing the hydroxytyrosol berberine hydrochloride composite liposome emulsion according to claim 1, characterized in that: The particle size of the solid phase particles is 150-1250 μm.

7. A hydroxytyrosol-berberine hydrochloride composite liposome emulsion prepared by the preparation method as claimed in claim 1, characterized in that: When the solid phase particles are sucrose, the 24h permeability of the hydroxytyrosol berberine hydrochloride composite liposome emulsion is not less than 50%, and the encapsulation efficiency is 38% to 90%; When the solid phase particles are sodium chloride, the 24h permeability of the hydroxytyrosol berberine hydrochloride composite liposome emulsion is not less than 16%, and the encapsulation efficiency is 56%-75%.

8. The hydroxytyrosol-berberine hydrochloride composite liposome emulsion according to claim 7, characterized in that: After granulation, the particle size of the hydroxytyrosol berberine hydrochloride composite liposome is 129.2-223.8 nm, and the polydispersity coefficient is 0.220-0.

342.

9. Use of the hydroxytyrosol berberine hydrochloride complex liposome emulsion prepared by the preparation method according to any one of claims 1 to 6 or the hydroxytyrosol berberine hydrochloride complex liposome emulsion according to any one of claims 7 and 8 in the preparation of antioxidant cosmetics.

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