Liposome with inner cavity solubilizing effect as well as preparation method and application thereof

Abstract

The invention discloses a lipidosome with an inner cavity solubilizing effect and a preparation method and application thereof. The lipidosome comprises the following components: phospholipid, cholesterol, a stabilizer, a cosolvent and a pentacyclic triterpenoid. The stabilizer comprises sodium hyaluronate and betaine. According to the lipidosome prepared by the scheme, glycyrrhizic acid and derivatives thereof are solubilized to a certain extent through the aid (betaine) with a specific chemical structure, the problem of extrusion caused by coating in a bimolecular layer is avoided, the lipidosome is good in solubility, and the problem that traditional glycyrrhizic acid is difficult to dissolve and apply is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and in particular relates to a liposome with an inner cavity solubilizing effect, a preparation method and an application thereof. Background Art

[0002] Pentacyclic triterpenoid compounds are widely distributed in plants. As important active ingredients of natural plants, their structural diversity and wide range of biological activities have attracted much attention from all walks of life. However, due to the particularity of the core structure, this type of compound has poor water solubility, is not conducive to absorption, lacks storage stability, and has low bioavailability. These side effects have greatly limited its application in the field of cosmetics. Representative and common natural compounds of this structure include oleanolic acid, betulin, hawthorn acid, ivy saponin, ursolic acid, betulinic acid, boswellic acid, glycyrrhetinic acid, farnesol, etc. Existing liposomes generally have low drug loading, and require a large amount of surfactants and organic solvents to assist in dissolution and stability. Therefore, it is urgent to provide a new liposome. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a liposome with lumen solubilization effect.

[0004] The invention also provides a preparation method of the liposome.

[0005] The present invention also provides the application of the liposome.

[0006] The invention also provides a product.

[0007] According to one aspect of the present invention, a liposome with lumen solubilization is provided, wherein the liposome contains the following components: phospholipids, cholesterol, a stabilizer, a cosolvent and a pentacyclic triterpenoid compound;

[0008] The stabilizer comprises sodium hyaluronate and betaine.

[0009] In some embodiments of the present invention, the stabilizer is sodium hyaluronate and betaine. Hyaluronic acid participates in the skeleton structure of the liposome composition, and its molecular fat chain is embedded with the phospholipid double chain to form a spatial balloon skeleton structure with certain flexibility and deformation ability. Betaine improves the stability and solubility of pentacyclic triterpenoid compounds (glycyrrhetinic acid or its derivatives) in the inner cavity aqueous phase through ionic bond forces, and it has a strong hydration effect between itself and water molecules, thereby playing a role in anchoring the pentacyclic triterpenoid compounds (glycyrrhetinic acid or its derivatives) in the inner cavity of the vesicle.

[0010] In some embodiments of the present invention, the phospholipid includes at least one of lecithin, soybean lecithin, phosphatidylcholine, hydrogenated lecithin, hydrogenated phosphatidylcholine and hydrogenated soybean lecithin.

[0011] In some embodiments of the present invention, the co-solvent includes at least one of ethanol, propylene glycol, dipropylene glycol, butylene glycol, glycerol, hexylene glycol, pentylene glycol and ethoxydiglycol.

[0012] In some embodiments of the present invention, the pentacyclic triterpenoid compound includes oleanolic acid, betulin, maslinic acid, hedera saponin, ursolic acid, betulinic acid, boswellic acid, farnesol, glycyrrhetinic acid or a derivative thereof.

[0013] In some embodiments of the present invention, the derivative of glycyrrhetinic acid includes at least one of fatty alcohol glycyrrhetinic acid ester and glycyrrhetinic acid salt.

[0014] In some embodiments of the present invention, the fatty alcohol glycyrrhetinic acid ester includes at least one of glycyrrhetinic acid stearate and stearyl glycyrrhetinic acid ester.

[0015] In some embodiments of the present invention, the glycyrrhetinic acid salt includes at least one of disodium succinylglycyrrhetinate, trisodium glycyrrhizate, ammonium glycyrrhizate and zinc glycyrrhetinate.

[0016] In some embodiments of the present invention, the pentacyclic triterpenoid compound is glycyrrhetinic acid or a derivative thereof.

[0017] In some embodiments of the present invention, the mass ratio of phospholipid to cholesterol is (45-55):(1-10).

[0018] In some embodiments of the present invention, the mass ratio of phospholipid to cholesterol is (45-55):(1-3).

[0019] In some embodiments of the present invention, the mass ratio of phospholipid to cholesterol is 50:2.

[0020] In some embodiments of the present invention, the molar ratio of sodium hyaluronate to betaine is (0.05-2):100.

[0021] In some embodiments of the present invention, the molar ratio of sodium hyaluronate to betaine is (0.1-1):100.

[0022] In some embodiments of the present invention, the molar ratio of the betaine to the pentacyclic triterpenoid compound is (0.2-2):1.

[0023] In some embodiments of the present invention, the molar ratio of betaine to pentacyclic triterpenoid compound is (0.4-1.2):1.

[0024] In some embodiments of the present invention, the molar ratio of betaine to pentacyclic triterpenoid compound is (0.5-1):1.

[0025] In some embodiments of the present invention, the stabilizer may further include at least one of dextran, chitosan, mannan, arginine, aspartic acid, polylysine and hydroxyethylpiperazineethane sulfonic acid.

[0026] In some embodiments of the present invention, the liposome further comprises a solvent, and the solvent comprises water.

[0027] According to a second aspect of the present invention, a method for preparing the above liposome is provided, the method comprising the following steps:

[0028] (1) mixing phospholipids, cholesterol and a cosolvent to obtain an oil phase;

[0029] (2) mixing sodium hyaluronate, betaine, pentacyclic triterpenoid compounds and a solvent to obtain an aqueous phase; or,

[0030] Sodium hyaluronate and betaine are prepared to obtain a combination, and a pentacyclic triterpenoid compound and a cosolvent are added to obtain an aqueous phase;

[0031] (3) The aqueous phase prepared in (2) is added to the oil phase to prepare liposomes.

[0032] In some embodiments of the present invention, the mass ratio of phospholipids, cholesterol and co-solvent in the oil phase is (45-55): (20-30): (1-10).

[0033] In some embodiments of the present invention, the mass ratio of phospholipids, cholesterol and co-solvent in the oil phase is (45-55): (22-27): (1-3).

[0034] In some embodiments of the present invention, the mass ratio of phospholipids, cholesterol and co-solvent in the oil phase is 50:25:2.

[0035] In some embodiments of the present invention, the molar ratio of sodium hyaluronate to betaine is (0.05-2):100.

[0036] In some embodiments of the present invention, the molar ratio of sodium hyaluronate to betaine is (0.1-1):100.

[0037] In some embodiments of the present invention, the molar ratio of the betaine to the pentacyclic triterpenoid compound is (0.2-2):1.

[0038] In some embodiments of the present invention, the molar ratio of betaine to pentacyclic triterpenoid compound is (0.4-1.2):1.

[0039] In some embodiments of the present invention, the molar ratio of betaine to pentacyclic triterpenoid compound is (0.5-1):1.

[0040] In some embodiments of the present invention, the mass ratio of the cosolvent to betaine in the aqueous phase is (8-12):1.

[0041] In some embodiments of the present invention, the specific step of mixing in step (1) is to heat at 55-65°C to dissolve and mix uniformly.

[0042] In some embodiments of the present invention, the heating method is water bath heating.

[0043] In some embodiments of the present invention, the specific steps of mixing sodium hyaluronate, betaine, pentacyclic triterpenoid compounds and cosolvents to obtain an aqueous phase are: heating and dissolving sodium hyaluronate at 35-45°C to obtain a sodium hyaluronate solution; heating and dissolving betaine and cosolvents at 80-88°C to obtain a mixed solution; and mixing the sodium hyaluronate solution, the mixed solution and the pentacyclic triterpenoid compounds to obtain an aqueous phase.

[0044] In some embodiments of the present invention, the heating method is water bath heating.

[0045] In some embodiments of the present invention, the preparation of the combination comprises the following steps:

[0046] 1) subjecting betaine, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and ethylenediamine to a first reaction to obtain an intermediate NMF-N;

[0047] 2) subjecting sodium hyaluronate and the intermediate NMF-N to a second reaction to obtain a reaction product;

[0048] 3) Adding 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to the reaction product for a third reaction to obtain a combination.

[0049] In some embodiments of the present invention, the solvent for dissolving betaine is an organic solvent.

[0050] In some embodiments of the present invention, the organic solvent comprises methanol.

[0051] In some embodiments of the present invention, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride added is 0.005-0.05 g / mL.

[0052] In some embodiments of the present invention, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride added is 0.005-0.02 g / mL.

[0053] In some embodiments of the present invention, the added volume of ethylenediamine is 25-35 mL.

[0054] In some embodiments of the present invention, the specific steps of the first reaction are: after activating betaine and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride at 20-28° C. for 0.5-2 h, adding ethylenediamine and reacting at 30-38° C. for 5-10 h.

[0055] In some embodiments of the present invention, the first reaction further comprises a stirring step.

[0056] In some embodiments of the present invention, the specific steps of the second reaction are: adding sodium hyaluronate to the NMF-N, and adding ethanol at the same time, and reacting for 2-4 hours.

[0057] In some embodiments of the present invention, the second reaction further comprises a stirring step.

[0058] In some embodiments of the present invention, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride added to the reaction product is 0.005-0.05 g / mL.

[0059] In some embodiments of the present invention, the reaction temperature of the third reaction is 20-28°C.

[0060] In some embodiments of the present invention, the third reaction further comprises a stirring step.

[0061] In some embodiments of the present invention, the method further comprises a step of purifying the combination.

[0062] In some embodiments of the present invention, the purification method is ultrafiltration.

[0063] In some embodiments of the present invention, the method further comprises a step of drying the assembly.

[0064] In some embodiments of the present invention, the drying method comprises freeze drying.

[0065] In some embodiments of the present invention, the water phase is added to the oil phase under stirring at a rotation speed of 600-1000 rpm.

[0066] In some embodiments of the present invention, the added volume ratio of the water phase and the oil phase is (0.4-1.5):1.

[0067] In some embodiments of the present invention, the added volume ratio of the water phase and the oil phase is (1.2-1.5):1.

[0068] In some embodiments of the present invention, the step of treating the liposomes is further included, the step comprising: adding the liposomes to a solvent, treating at 40-50° C. for 1-2 hours, homogenizing under high pressure, and performing granulation treatment at 500-800 bar.

[0069] In some embodiments of the invention, the solvent comprises water.

[0070] According to the third aspect of the present invention, the application of the above liposome is proposed, and the application is application in any of the following items:

[0071] (1) preparing products with anti-inflammatory effects;

[0072] (2) preparing products for skin repair;

[0073] (3) preparing products with soothing effects;

[0074] (4) Prepare anti-allergic products.

[0075] In some embodiments of the invention, said repairing the skin comprises repairing the skin barrier.

[0076] In some embodiments of the present invention, the skin repair includes wound repair.

[0077] In some embodiments of the present invention, the product is a cosmetic or a medicine.

[0078] According to a fourth aspect of the present invention, a product is provided, comprising the above-mentioned liposome.

[0079] In some embodiments of the present invention, the product is a cosmetic or a medicine.

[0080] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the scheme of the present invention provides a liposome with lumen solubilization, wherein betaine and hyaluronic acid are chemically synthesized to form a stable bridge, and the betaine hyaluronic acid bridge is assembled with phospholipids in a chimeric manner to form a more flexible and deformable liposome vesicle, and then glycyrrhetinic acid or its derivatives are anchored and loaded in the lumen of the liposome vesicle to prepare the liposome. The liposome prepared by the scheme of the present invention achieves a certain degree of solubilization of glycyrrhetinic acid and its derivatives by an auxiliary agent (betaine) with a specific chemical structure, avoids the problem of extrusion when coated in a bilayer, and has good solubility, which improves the dissolution and application difficulties of traditional glycyrrhetinic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0082] Figure 1 This is a graph showing the morphology characterization results of the glycyrrhetinic acid liposomes in the test example of the present invention;

[0083] Figure 2 This is a test result diagram of the soothing effect of the product in the test example of the present invention. DETAILED DESCRIPTION

[0084] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0085] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0086] Example 1

[0087] This embodiment provides a glycyrrhetinic acid liposome with lumen solubilization effect, and the specific preparation process is as follows:

[0088] (1) dissolving lecithin, hexylene glycol and cholesterol in a mass ratio of 50:25:2 in a water bath at 60° C., stirring and mixing to obtain an oil phase for standby use;

[0089] (2) weighing sodium hyaluronate and betaine at a molar ratio of 0.6:100 respectively; dissolving water and sodium hyaluronate at a mass ratio of 30:1 in a water bath at 40° C. to obtain a sodium hyaluronate solution;

[0090] (3) dissolving and mixing hexylene glycol and the betaine weighed above in a mass ratio of 10:1 in a water bath at 80° C., and then adding glycyrrhetinic acid in a molar ratio of 1:1 to betaine, stirring and dissolving completely under the same conditions to obtain a mixed solution;

[0091] (4) mixing the sodium hyaluronic acid solution obtained in step (2) and the mixed solution obtained in step (3) at a volume ratio of 2.1:1 and stirring to obtain an aqueous phase;

[0092] (5) slowly adding the aqueous phase to the oil phase while stirring at 800 rpm to obtain pre-liposomes, wherein the volume ratio of the aqueous phase to the oil phase is 1.5:1;

[0093] (6) The pre-liposomes obtained above were slowly added to 616 mL of water while stirring at 800 rpm to prepare a system with a volume of 1 L. The system was hydrated and incubated in a 45°C water bath for 1.5 h (1 h to 2 h), and then added to a high-pressure homogenizer and granulated at 700 bar to obtain glycyrrhetinic acid liposomes.

[0094] Example 2

[0095] This embodiment provides a glycyrrhetinic acid liposome with lumen solubilization effect, and the specific preparation process is as follows:

[0096] (1) dissolving lecithin, hexylene glycol and cholesterol in a mass ratio of 50:25:2 in a water bath at 60° C., stirring and mixing to obtain an oil phase for standby use;

[0097] (2) weighing sodium hyaluronate and betaine at a molar ratio of 1:100 respectively; dissolving water and sodium hyaluronate at a mass ratio of 30:1 in a water bath at 40° C. to obtain a sodium hyaluronate solution;

[0098] (3) Hexylene glycol and the betaine weighed above were dissolved and mixed in a water bath at 80° C. in a mass ratio of 10:1, and then glycyrrhetinic acid was weighed in a molar ratio of 0.5:1 to betaine, and stirred under the same conditions to completely dissolve, to obtain a mixed solution;

[0099] (4) mixing the sodium hyaluronic acid solution obtained in step (2) and the mixed solution obtained in step (3) at a volume ratio of 2.7:1 and stirring to obtain an aqueous phase;

[0100] (5) slowly adding the aqueous phase to the oil phase while stirring at 800 rpm to obtain pre-liposomes, wherein the volume ratio of the aqueous phase to the oil phase is 1.2:1;

[0101] (6) The pre-liposomes obtained above were slowly added to 670 mL of water while stirring at 800 rpm to prepare a system with a volume of 1 L. The system was hydrated and incubated in a 45°C water bath for 1.5 h (1 h to 2 h), and then added to a high-pressure homogenizer and granulated at 700 bar to obtain glycyrrhetinic acid liposomes.

[0102] Example 3

[0103] This embodiment provides a glycyrrhetinic acid liposome with lumen solubilization effect, and the specific preparation process is as follows:

[0104] (1) dissolving lecithin, hexylene glycol and cholesterol in a mass ratio of 50:25:2 in a water bath at 60° C., stirring and mixing to obtain an oil phase for standby use;

[0105] (2) Sodium hyaluronate and betaine were weighed at a molar ratio of 0.6:100, betaine was dissolved in methanol (betaine to methanol addition mass ratio was 1:15), stirred until dissolved, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride was added at a final concentration of 0.01 g / mL, stirred and activated at room temperature for 1 h, the solvent was removed by rotary evaporation, 30 mL of ethylenediamine was added, stirred at 35° C. for 8 h, and excess ethylenediamine was removed by rotary evaporation. The obtained solid was dissolved in ethanol, and the intermediate NMF-N was obtained after crystallization and purification. Add distilled water and sodium hyaluronate (the mass ratio of water to sodium hyaluronate is 30:1) to a round-bottom flask, stir at room temperature until dissolved, add the above intermediate NMF-N, and at the same time drop an appropriate amount of anhydrous ethanol, stir for 3 hours until the solution is clear, add 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride with a final concentration of 0.01g / mL, stir at room temperature until the reaction is complete. The solution is purified by ultrafiltration concentration, and then freeze-dried to obtain a white powder product, which is the hyaluronic acid-betaine combination.

[0106] The preparation flow chart of the hyaluronic acid-betaine combination is as follows:

[0107]

[0108] (3) dissolving hexylene glycol and the above combination in a mass ratio of 10:1 in a water bath at 80° C., and then adding glycyrrhetinic acid in a molar ratio of 1:1 to betaine in a water bath to dissolve and mix them in the same water bath to obtain a mixture;

[0109] (4) mixing water and the mixture obtained in step (3) in a mass ratio of 30:1 in a 40° C. water bath to form an aqueous phase;

[0110] (5) slowly adding the aqueous phase to the oil phase while stirring at 800 rpm to obtain pre-liposomes, wherein the volume ratio of the aqueous phase to the oil phase is 1.5:1;

[0111] (6) The pre-liposomes obtained above were slowly added to 670 mL of water while stirring at 800 rpm to prepare a system with a volume of 1 L. The system was hydrated and incubated in a 45°C water bath for 1.5 h (1 h to 2 h), and then added to a high-pressure homogenizer and granulated at 700 bar to obtain glycyrrhetinic acid liposomes.

[0112] Example 4

[0113] This embodiment provides a glycyrrhetinic acid liposome with lumen solubilization effect, and the specific preparation process is as follows:

[0114] (1) dissolving lecithin, hexylene glycol and cholesterol in a mass ratio of 50:25:2 in a water bath at 60° C., stirring and mixing to obtain an oil phase for standby use;

[0115] (2) weighing sodium hyaluronate and betaine at a molar ratio of 0.1:100 respectively; dissolving water and sodium hyaluronate in a mass ratio of 30:1 in a water bath at 40° C. to obtain a sodium hyaluronate solution;

[0116] (3) dissolving and mixing hexylene glycol and the betaine weighed above in a mass ratio of 10:1 in a water bath at 80° C., and then adding glycyrrhetinic acid in a molar ratio of 0.5:1 to betaine, stirring and dissolving completely under the same conditions to obtain a mixed solution;

[0117] (4) mixing the sodium hyaluronate solution obtained in step (2) and the mixed solution obtained in step (3) at a volume ratio of 0.3:1 and stirring to obtain an aqueous phase;

[0118] (5) slowly adding the aqueous phase to the oil phase while stirring at 800 rpm to obtain pre-liposomes, wherein the volume ratio of the aqueous phase to the oil phase is 0.4:1;

[0119] (6) The pre-liposomes obtained above were slowly added to 786 ml of water under stirring at 800 rpm to prepare a system with a volume of 1 L. The system was hydrated and incubated in a 45°C water bath for 1.5 h (1 h to 2 h), and then added to a high-pressure homogenizer and granulated at 700 bar to obtain glycyrrhetinic acid liposomes.

[0120] Example 5

[0121] This embodiment provides a glycyrrhetinic acid liposome with lumen solubilization effect, and the specific preparation process is as follows:

[0122] (1) dissolving lecithin, hexylene glycol, cholesterol and glycyrrhetinic acid in a mass ratio of 50:50:2:10 in a water bath at 60° C., stirring and mixing to obtain an oil phase for standby use;

[0123] (2) weighing sodium hyaluronate at a molar ratio of 0.6:100 to glycyrrhetinic acid, and dissolving water and sodium hyaluronate in a mass ratio of 30:1 in a water bath at 40° C. to obtain an aqueous phase;

[0124] (3) slowly adding the aqueous phase to the oil phase while stirring at 800 rpm to obtain pre-liposomes, wherein the volume ratio of the aqueous phase to the oil phase is 0.7:1;

[0125] (4) The pre-liposomes obtained above were slowly added to 620 mL of water under stirring at 800 rpm to prepare a system with a volume of 1 L. The system was hydrated and incubated in a 45° C. water bath for 1.5 h (1 h to 2 h), and then added to a high-pressure homogenizer and granulated at 700 bar to obtain glycyrrhetinic acid liposomes.

[0126] Comparative Example 1

[0127] This comparative example prepared a glycyrrhetinic acid liposome, and the specific preparation process was as follows:

[0128] (1) dissolving lecithin, hexylene glycol, cholesterol and glycyrrhetinic acid in a mass ratio of 50:50:2:10 in a water bath at 60° C., stirring and mixing to obtain an oil phase for standby use;

[0129] (2) The oil phase is slowly added to the remaining water (the volume ratio of water to oil phase is 3.5:1) while stirring at 800 rpm, and incubated in a 45°C water bath for 1.5 h (1 h to 2 h), and then added to a high-pressure homogenizer and granulated at 700 bar to obtain glycyrrhetinic acid liposomes.

[0130] Test example

[0131] 1. Morphological observation and characterization

[0132] The glycyrrhetinic acid liposomes prepared in Example 1-4 were diluted 10 times with deionized water, and an appropriate amount was added to a copper mesh. After natural drying at room temperature, 2% phosphotungstic acid was added to stain the sample for 10 minutes, and the sample was washed twice with deionized water. After natural drying, the morphology of the glycyrrhetinic acid liposomes was observed under a transmission electron microscope. A typical morphology of the glycyrrhetinic acid liposome is shown in FIG. Figure 1 shown.

[0133] 2. Particle size and potential determination

[0134] 1 g of the glycyrrhetinic acid liposomes prepared in Examples 1-5 and Comparative Example 1 was accurately weighed and diluted to 20 mL with pure water to obtain a liposome suspension. The particle size and PDI distribution of each group of liposome suspensions were measured using a nanoparticle size and Zeta potential instrument at room temperature, and the Zeta potential was measured at the same time. The experiment was repeated 3 times, and the results are shown in Table 1.

[0135] Table 1

[0136] Group Particle size Zeta Potential PDI Encapsulation efficiency Example 1 168.4nm -22 0.3 90.5％ Example 2 169.1nm -35 0.301 93.4％ Example 3 166.3nm -20 0.302 93.8％ Example 4 151.5nm -18 0.285 88.5％ Example 5 139nm -30 0.209 85.3％ Comparative Example 1 132nm -19 0.218 86.4％

[0137] 3. Determination of encapsulation efficiency

[0138] 200 μL of the liposomes prepared in Examples 1-5 and Comparative Example 1 were respectively placed in a 10kda ultrafiltration centrifuge tube, centrifuged at 4000 rpm / min for 60 min, the lower supernatant was taken and fixed to volume with methanol, and then filtered using an organic membrane, and the content of free glycyrrhetinic acid drug was determined by HPLC; 200 μL of the liposomes prepared in Examples 1-5 and Comparative Example 1 were taken in a centrifuge tube, 800 μL of methanol was added to oscillate and suspend evenly, and then filtered with a 0.45 μm filter membrane, methanol-water-glacial acetic acid (90:10:06) was used as the mobile phase, the chromatographic column was a C18 column (150 mm × 4.6 mm, 3.5 μm), the detection wavelength was 250 nm, the flow rate was 10 mL / min, the column temperature was 30 ° C, the injection volume was 20 μL, the glycyrrhetinic acid content in the sample was determined and the encapsulation efficiency was calculated.

[0139] The results are shown in Table 1. It can be seen from Table 1 that the encapsulation rate of the glycyrrhetinic acid liposomes prepared in Examples 1-4 of the present invention can reach more than 88%, which is significantly higher than that of the glycyrrhetinic acid liposomes prepared in Example 1. The results show that the inner cavity solubilization method is used to encapsulate glycyrrhetinic acid, because its shell has good deformation properties, which is more conducive to the retention and preservation of glycyrrhetinic acid in the inner cavity of the vesicle, thereby achieving an increase in drug loading and encapsulation rate.

[0140] 4. Determination of retention rate

[0141] Take 200 μL of liposomes prepared in Examples 1-5 and Comparative Example 1 (liposomes are specifically placed at 4°C, room temperature and 42°C for a period of time (1 month, 2 months and 3 months respectively)), add 800 μL of methanol to oscillate and suspend evenly in a centrifuge tube, filter with a 0.45 μm filter membrane, use methanol-water-glacial acetic acid (90:10:06) as the mobile phase, the chromatographic column is a C18 column (150 mm × 4.6 mm, 3.5 μm), the detection wavelength is 250 nm, the flow rate is 10 mL / min, the column temperature is 30°C, the injection volume is 20 μL, the glycyrrhetinic acid content in the sample is determined, and the retention rate under different storage conditions is calculated. The results are shown in Table 2.

[0142] Table 2

[0143]

[0144]

[0145] The results are shown in Table 2. It can be seen from Table 2 that the liposomes prepared in Examples 1-3 of the present invention can achieve long-term storage and stable encapsulation effects under different environments.

[0146] 5. Determination of product soothing efficacy

[0147] Artificially induced skin erythema reaction: under closed conditions, fresh yam was peeled and thin slices were cut and applied to the skin for 2 hours or 3% sodium dodecyl sulfate (SLS) aqueous solution was applied to the skin for 24 hours and then the patch was removed.

[0148] The soothing effect of the product was evaluated by measuring the skin characteristic states (skin erythema state retention, subjective sensitivity and irritation rating) at different times (1 h and 2 h, respectively) after induction and after using the product (liposomes prepared in Examples 1 and 3, respectively). The application amount of liposomes was 0.2 mL / cm 2 The positive control group was treated with an equal amount of 2% dipotassium glycyrrhizinate solution, while the blank group was not treated. The evaluation indicators are shown in Table 3.

[0149] Table 3

[0150]

[0151]

[0152] The results are as follows Figure 2 As shown, from Figure 2 It can be seen that the glycyrrhizic acid liposomes prepared in the embodiment of the present invention are significantly better than the known effective substance dipotassium glycyrrhizinate in soothing effect, slightly better than dipotassium glycyrrhizinate in eliminating itching, stinging and discomfort, and significantly better than the dipotassium glycyrrhizinate test group in the effect of fading redness and repairing within the same test time.

[0153] Licorice is not only a traditional Chinese medicine, but also widely used in modern medicine, food, cosmetics and other fields. In the field of cosmetics, licorice is known as the "whitening queen". The types of compounds with chemical structures that have been discovered and determined in licorice include triterpenoid saponins (4% to 24%, mainly glycyrrhizic acid), total flavonoids of licorice (1% to 5%), alkaloids, polysaccharides, amino acids (1% to 2%), etc.

[0154] Glycyrrhizic acid and its derivatives, the active ingredient in licorice, have significant anti-allergic, anti-inflammatory, and antioxidant effects. Glycyrrhizic acid can only exert its effects after being metabolized into glycyrrhetinic acid by specific enzymes in the body. 30 H 46O4), also known as glycyrrhetinic acid, belongs to the pentacyclic triterpenoids. It also has a wide range of pharmacological activities, especially its anti-inflammatory properties, which are related to the inhibition of superoxide radical production by neutrophils, which are the main mediators of inflammation. In addition, the steroid-like structure of glycyrrhetinic acid gives it anti-inflammatory and anti-immune effects. Glycyrrhetinic acid has anti-inflammatory, repairing and anti-allergic effects and is widely used in the field of medicine and cosmetics.

[0155] Glycyrrhetinic acid and its derivatives (fatty alcohol glycyrrhetinic acid ester, glycyrrhetinic acid salt) on the market are mainly solid powder dosage forms. Since the logP (oil-water partition coefficient) of glycyrrhetinic acid is too high and it is highly hydrophobic, it is difficult to penetrate the stratum corneum of the skin, and GA has extremely poor water solubility, with a solubility in water of only 6.32 mg / L, which greatly limits the application of glycyrrhetinic acid and its derivatives. Existing glycyrrhetinic acid liposomes generally have low drug loading, require a large amount of surfactants and organic solvents to assist in dissolution and stability, and glycyrrhetinic acid is often encapsulated between phospholipid bilayers, and this encapsulation method is prone to unstable drug extrusion.

[0156] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A liposome having lumen solubilization effect, characterized in that: The liposome contains the following components: phospholipid, cholesterol, stabilizer, cosolvent and pentacyclic triterpenoid compound; The stabilizer comprises sodium hyaluronate and betaine.

2. The liposome according to claim 1, characterized in that The phospholipids include at least one of lecithin, soybean lecithin, phosphatidylcholine, hydrogenated lecithin, hydrogenated phosphatidylcholine and hydrogenated soybean lecithin; And / or, the co-solvent includes at least one of ethanol, propylene glycol, dipropylene glycol, butylene glycol, glycerol, hexylene glycol, pentylene glycol and ethoxydiglycol.

3. The liposome according to claim 1, characterized in that The pentacyclic triterpenoid compounds include oleanolic acid, betulin, hawthorn acid, hederaconin, ursolic acid, betulinic acid, boswellic acid, farnesol, glycyrrhetinic acid or their derivatives; Preferably, the derivative of glycyrrhetinic acid comprises at least one of fatty alcohol glycyrrhetinic acid ester and glycyrrhetinic acid salt; More preferably, the fatty alcohol glycyrrhetinic acid ester includes at least one of glycyrrhetinic acid stearate and stearyl glycyrrhetinic acid ester; More preferably, the glycyrrhetinic acid salt includes at least one of disodium succinylglycyrrhetinate, trisodium glycyrrhizate, ammonium glycyrrhizate and zinc glycyrrhetinate.

4. The liposome according to claim 1, characterized in that The mass ratio of phospholipid to cholesterol is (45-55):(1-10); And / or, the molar ratio of sodium hyaluronate to betaine is (0.05-2):100; And / or, the molar ratio of betaine to pentacyclic triterpenoid compound is (0.2-2):1; And / or, the stabilizer further comprises dextran, chitosan, mannan, arginine, aspartic acid, polylysine and / or hydroxyethylpiperazineethane sulfonic acid.

5. A method for preparing the liposome according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) mixing phospholipids, cholesterol and a cosolvent to obtain an oil phase; (2) mixing sodium hyaluronate, betaine, pentacyclic triterpenoid compounds and a solvent to obtain an aqueous phase; or, Sodium hyaluronate and betaine are prepared to obtain a combination, and a pentacyclic triterpenoid compound and a cosolvent are added to obtain an aqueous phase; (3) The aqueous phase prepared in (2) is added to the oil phase to prepare liposomes.

6. The preparation method according to claim 5, characterized in that: The mass ratio of phospholipid, cholesterol and cosolvent in the oil phase is (45-55): (20-30): (1-10); And / or, the mass ratio of the cosolvent to betaine in the aqueous phase is (8-12):

1.

7. The preparation method according to claim 5, characterized in that: The assembly is prepared by the following steps: 1) subjecting betaine, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and ethylenediamine to a first reaction to obtain an intermediate NMF-N; 2) subjecting sodium hyaluronate and the intermediate NMF-N to a second reaction to obtain a reaction product; 3) Adding 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to the reaction product for a third reaction to obtain a combination.

8. The preparation method according to claim 7, characterized in that: The added concentration of the 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 0.005-0.05 g / mL; And / or, the added volume of ethylenediamine is 25-35 mL.

9. Use of the liposome according to any one of claims 1 to 4 in any one of the following: (1) preparing products with anti-inflammatory effects; (2) preparing products for skin repair; (3) preparing products with soothing effects; (4) Preparation of anti-allergic products; Preferably, the product is a cosmetic or a medicine.

10. A product, characterized in that The liposome comprises the liposome according to any one of claims 1 to 4.

Images