Dihydromyricetin liposome as well as preparation method and application thereof

Abstract

The invention relates to dihydromyricetin liposome as well as a preparation method and application thereof. The dihydromyricetin liposome comprises the following components in percentage by mass: (1) an oil phase: 1%-6% of phospholipid, 0.06%-1% of cholesterol and 1%-7.5% of ethanol; (2) a water phase: 0.2%-0.5% of dihydromyricetin, 0.5%-2.5% of an emulsifier, 1%-10% of glycerol, 0.1%-2.5% of a pH regulator and 60%-96.14% of water; and the emulsifying agent is beheneth-25. The dihydromyricetin liposome has good solubility in water, is mild to a human body, has good antioxidant, whitening and anti-inflammatory effects, shows excellent stability in seven different severe environments of normal temperature, refrigeration, freezing, heating, freezing and thawing, illumination and shading, has a good slow release effect, and can be well applied to the field of cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of liposomes, and particularly relates to a dihydromyricetin liposome, a preparation method thereof and an application thereof. Background Art

[0002] Dihydromyricetin (DMY) is widely present in plants of the genus Ampelopsis. As a natural flavonoid compound, it has good anti-inflammatory, antioxidant and whitening effects. However, DMY has poor solubility in cold water and better solubility in hot water, which is the main reason for its poor membrane permeability and poor bioavailability.

[0003] To address the problem of poor solubility of dihydromyricetin, traditionally, its solubility has been improved mainly by preparing it into liposomes. However, the current dihydromyricetin liposomes have poor stability, becoming a turbid emulsion after being stored at about 25°C for a period of time at room temperature, and have even worse stability under high temperature conditions, and the encapsulation rate of DMY is not high, so improvement is urgently needed. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a dihydromyricetin liposome with good solubility, good stability, high encapsulation rate, sustained release effect and being safe and non-irritating, which can be applied to cosmetics.

[0005] The technical solution is as follows:

[0006] A dihydromyricetin liposome is mainly made of the following components by mass percentage:

[0007] (1) Oil phase:

[0008] Phospholipid 1% - 6%,

[0009] Cholesterol 0.06% - 1%,

[0010] Ethanol 1% - 7.5%;

[0011] (2) Aqueous phase:

[0012]

[0013] The emulsifier is behenyl alcohol polyether-25.

[0014] In one embodiment, the phospholipid is selected from one or a combination of several of S45 soybean lecithin, S75 soybean lecithin, 90G soybean lecithin and PC50 soybean lecithin.

[0015] In one embodiment, the mass ratio of the phospholipid to cholesterol is (6 - 90):1.

[0016] In one embodiment, the mass ratio of dihydromyricetin to the phospholipid is 1:(5 - 20).

[0017] In one embodiment, the mass ratio of dihydromyricetin to the phospholipid is 1:(12 - 20).

[0018] In one embodiment, the pH regulator is citric acid.

[0019] In one embodiment, the oil phase further includes an antioxidant.

[0020] In one embodiment, the antioxidant is selected from one or a combination of several of pentaerythritol tetra(bis - tert - butylhydroxyhydrocinnamate), vitamin E, and vitamin E acetate.

[0021] In one embodiment, the aqueous phase further includes a first preservative.

[0022] In one embodiment, the first preservative is pentylene glycol.

[0023] In one embodiment, the dihydromyricetin liposome further includes a second preservative.

[0024] In one embodiment, the second preservative is selected from one or a mixture of several of hydroxyacetophenone, pentylene glycol, and phenoxyethanol.

[0025] In one embodiment, the dihydromyricetin liposome is mainly made of the following components by mass percentage:

[0026] (1) Oil phase:

[0027]

[0028] (2) Aqueous phase:

[0029]

[0030] In one embodiment, the particle size of the dihydromyricetin liposome is 30 nm - 200 nm, the PDI is 0.1 - 0.5, and the encapsulation efficiency is ≥60%.

[0031] The present invention also provides a preparation method of the dihydromyricetin liposome as described above, including the following steps:

[0032] Mix the phospholipid and cholesterol in ethanol to prepare an oil - phase mixture;

[0033] Mix the dihydromyricetin, emulsifier, glycerol, pH regulator, and water to prepare an aqueous - phase mixture;

[0034] Mix the oil-phase mixture with the water-phase mixture, and perform atmospheric pressure homogenization treatment and high-pressure homogenization treatment.

[0035] In one embodiment, the process parameters for preparing the oil-phase mixture include: the temperature is 40°C to 60°C.

[0036] In one embodiment, the process parameters for preparing the water-phase mixture include: the pH is 5 to 5.5, and the temperature is 70°C to 75°C.

[0037] In one embodiment, the process parameters for atmospheric pressure homogenization treatment include: the mechanical stirring speed is 300 rpm to 700 rpm, the stirring time is 20 min to 40 min, and the temperature is 40°C to 60°C.

[0038] In one embodiment, the process parameters for high-pressure homogenization treatment include: the pressure is 300 bar to 900 bar, and the number of times is 1 to 9 times.

[0039] The present invention also provides a cosmetic, which includes the dihydromyricetin liposome as described above.

[0040] In one embodiment, the cosmetic is selected from at least one of facial masks, eye creams, facial creams, primer creams, essence, lotions, and skin softeners.

[0041] The present invention has at least the following beneficial effects:

[0042] The dihydromyricetin liposome provided by the present invention has an oil phase including specific mass percentages of phospholipids, cholesterol, and ethanol, and a water phase including specific mass percentages of dihydromyricetin, an emulsifier, glycerol, a pH regulator, and water, and the emulsifier is behenyl polyether-25.

[0043] While ensuring good solubility of dihydromyricetin, the present invention also significantly improves the stability of the dihydromyricetin liposome. Moreover, under the combined action of the emulsifier and other raw materials in the present invention, the encapsulation efficiency of dihydromyricetin can be significantly improved, and the particle size and PDI of the dihydromyricetin liposome can be significantly reduced, and the slow release rate of dihydromyricetin can be prolonged. In addition, the dihydromyricetin liposome provided by the present invention contains no harmful substances, is gentle to the human body, and is safe and non-irritating.

[0044] It has been confirmed that the dihydromyricetin liposome provided by the present invention has good solubility in water, is gentle to the human body, has good skin physiological effects of antioxidant, whitening, and anti-inflammatory, and exhibits excellent stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark, slowly releases dihydromyricetin, and improves its dose-effect relationship and bioavailability. In view of the above-mentioned many advantages of the dihydromyricetin liposome, it can be well used in the cosmetic field.

[0045] In addition, the preparation method of the dihydromyricetin liposome of the present invention is simple, has high repeatability, short preparation time, no residue of harmful substances, high encapsulation rate, high mechanization degree in the whole process, so that the product quality and process have good reproducibility and stability, and are easy to be industrially produced. Description of the Drawings

[0046] Figure 1 Results of the encapsulation rate of the dihydromyricetin nano-liposome prepared in Examples 3 to 8 varying with influencing factors:

[0047] Among them, Figure 1 Figure A is a trend graph of the encapsulation rate of the dihydromyricetin nano-liposome prepared in Example 3 varying with the mass ratio of soybean lecithin to cholesterol;

[0048] Figure 1 Figure B is a trend graph of the encapsulation rate of the dihydromyricetin nano-liposome prepared in Example 4 varying with the mass ratio of dihydromyricetin to soybean lecithin;

[0049] Figure 1 Figure C is a trend graph of the encapsulation rate of the dihydromyricetin nano-liposome prepared in Example 5 varying with the mass content of polyoxyethylene 25 behenate;

[0050] Figure 1 Figure D is a trend graph of the encapsulation rate of the dihydromyricetin nano-liposome prepared in Example 6 varying with the mass content of glycerol;

[0051] Figure 1 Figure E is a trend graph of the encapsulation rate of the dihydromyricetin nano-liposome prepared in Example 7 varying with the high-pressure homogenization pressure;

[0052] Figure 1 Figure F is a trend graph of the encapsulation rate of the dihydromyricetin nano-liposome prepared in Example 8 varying with the number of high-pressure homogenization times.

[0053] Figure 2 Results of the morphology and particle size of the dihydromyricetin nano-liposome prepared in Example 12:

[0054] Among them, Figure 2 Figure A is the TEM (scale 100 nm) of the dihydromyricetin nano-liposome prepared in Example 12;

[0055] Figure 2 Figure B is the TEM (scale 500 nm) of the dihydromyricetin nano-liposome prepared in Example 12;

[0056] Figure 2In figure C, it is the particle size distribution trend diagram of the dihydromyricetin nanoliposomes prepared in Example 12 after being processed by Image and Origin with TEM.

[0057] Figure 3 It is the in vitro slow release amount trend diagram of the dihydromyricetin nanoliposomes prepared in Example 12.

[0058] Figure 4 It is the results of the change trends of the particle size and PDI of the dihydromyricetin nanoliposomes prepared in Example 12 under 7 different environments:

[0059] Among them, Figure 4 In figure A, it is the change trend diagram of the particle size and PDI of the dihydromyricetin nanoliposomes prepared in Example 12 under normal temperature environment;

[0060] Figure 4 In figure B, it is the change trend diagram of the particle size and PDI of the dihydromyricetin nanoliposomes prepared in Example 12 under refrigerated environment;

[0061] Figure 4 In figure C, it is the change trend diagram of the particle size and PDI of the dihydromyricetin nanoliposomes prepared in Example 12 under frozen environment;

[0062] Figure 4 In figure D, it is the change trend diagram of the particle size and PDI of the dihydromyricetin nanoliposomes prepared in Example 12 under heating environment;

[0063] Figure 4 In figure E, it is the change trend diagram of the particle size and PDI of the dihydromyricetin nanoliposomes prepared in Example 12 under freeze-thaw environment;

[0064] Figure 4 In figure F, it is the change trend diagram of the particle size and PDI of the dihydromyricetin nanoliposomes prepared in Example 12 under light environment;

[0065] Figure 4 In figure G, it is the change trend diagram of the particle size and PDI of the dihydromyricetin nanoliposomes prepared in Example 12 under light-proof environment.

[0066] Figure 5 It is the results of the change trends of the encapsulation efficiency and pH of the dihydromyricetin nanoliposomes prepared in Example 12 under 7 different environments:

[0067] Among them, Figure 5 In figure A, it is the change trend diagram of the encapsulation efficiency and pH of the dihydromyricetin nanoliposomes prepared in Example 12 under normal temperature environment;

[0068] Figure 5In Figure B, it is the graph of the encapsulation efficiency and pH change trend of the dihydromyricetin nano-liposomes prepared in Example 12 under refrigerated conditions;

[0069] Figure 5 In Figure C, it is the graph of the encapsulation efficiency and pH change trend of the dihydromyricetin nano-liposomes prepared in Example 12 under frozen conditions;

[0070] Figure 5 In Figure D, it is the graph of the encapsulation efficiency and pH change trend of the dihydromyricetin nano-liposomes prepared in Example 12 under heating conditions;

[0071] Figure 5 In Figure E, it is the graph of the encapsulation efficiency and pH change trend of the dihydromyricetin nano-liposomes prepared in Example 12 under freeze-thaw conditions;

[0072] Figure 5 In Figure F, it is the graph of the encapsulation efficiency and pH change trend of the dihydromyricetin nano-liposomes prepared in Example 12 under light irradiation conditions;

[0073] Figure 5 In Figure G, it is the graph of the encapsulation efficiency and pH change trend of the dihydromyricetin nano-liposomes prepared in Example 12 under light-shielded conditions.

[0074] Figure 6 It is the result of the change trend of the dihydromyricetin content of the dihydromyricetin nano-liposomes and the dihydromyricetin aqueous solution prepared in Example 12 under 7 different conditions:

[0075] Among them, Figure 6 In Figure A, it is the graph of the change trend of the dihydromyricetin content of the dihydromyricetin nano-liposomes and the dihydromyricetin aqueous solution prepared in Example 12 under normal temperature conditions;

[0076] Figure 6 In Figure B, it is the graph of the change trend of the dihydromyricetin content of the dihydromyricetin nano-liposomes and the dihydromyricetin aqueous solution prepared in Example 12 under heating conditions;

[0077] Figure 6 In Figure C, it is the graph of the change trend of the dihydromyricetin content of the dihydromyricetin nano-liposomes and the dihydromyricetin aqueous solution prepared in Example 12 under sunlight irradiation conditions.

[0078] Figure 7 It is the linear regression equation of the red blood cell hemolysis rate of the dihydromyricetin nano-liposomes prepared in Example 12.

[0079] Figure 8 It is the test result of the antioxidant capacity of the dihydromyricetin nano-liposomes or the dihydromyricetin aqueous solution prepared in Example 12 at different concentrations:

[0080] Among them,Figure 8 In A, it is the change trend of the DPPH radical scavenging rate of dihydromyricetin nano-liposomes prepared in Example 12 at different concentrations;

[0081] Figure 8 In B, it is the change trend of the DPPH radical scavenging rate of aqueous solutions of dihydromyricetin prepared in Example 12 at different concentrations.

[0082] Figure 9 It is the result of the anti-irritation test of the dihydromyricetin nano-liposomes prepared in Example 12:

[0083] Among them, Figure 9 In A, it is the linear regression equation of the erythrocyte hemolysis rate at different volumes of 0.1% SDS;

[0084] Figure 9 In B, it is the change trend of the erythrocyte hemolysis inhibition rate of dihydromyricetin nano-liposomes prepared in Example 12 at different concentrations;

[0085] Figure 9 In C, it is the change trend of the erythrocyte hemolysis inhibition rate of aqueous solutions of dihydromyricetin prepared in Example 12 at different concentrations. Detailed implementation manners

[0086] The present invention will be further described in detail below with reference to specific examples. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0088] In the present invention, "above" and "below" both include the number itself. For example, below 1 means ≥1.

[0089] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to an integer, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed in the present invention should be understood to include any and all sub-ranges subsumed therein.

[0090] In the present invention, for units related to data ranges, if a unit is only attached after the right endpoint, it means that the units of the left and right endpoints are the same. For example, 800~850nm means that the units of both the left endpoint "800" and the right endpoint "850" are nm (nanometers).

[0091] In the present invention, for temperature parameters, unless otherwise specified, both constant temperature treatment and treatment within a certain temperature range are allowed. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. The room temperature in the present invention refers to 0℃~40℃, preferably 10℃~35℃, and more preferably 20℃~30℃. The normal pressure in the present invention refers to about 1atm, one atmospheric pressure.

[0092] In the present invention, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the invention, the meaning of "at least one kind" is one kind, two kinds, or more than two kinds, and the meaning of "several kinds" and "multiple kinds" is at least two kinds, such as two kinds, three kinds, etc., unless otherwise clearly and specifically defined. In the description of the present application, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0093] If there is no special instruction, all steps of the present invention can be carried out sequentially or randomly, and preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) carried out sequentially, or may also include steps (b) and (a) carried out sequentially. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may also include steps (a), (c) and (b), or may also include steps (c), (a) and (b), etc.

[0094] In the present invention, unless otherwise mentioned, terms in the singular form may include the plural form and should not be construed as having a quantity of one.

[0095] In addition, the drawings of the present invention are not drawn to a scale of 1:1, and the relative sizes of the components are only drawn by way of example in the drawings for the convenience of understanding the present invention, but are not necessarily drawn to the actual scale, and the scale in the drawings does not constitute a limitation to the present invention.

[0096] Dihydromyricetin (DMY) is widely present in plants of the genus Ampelopsis. As a natural flavonoid compound, it has good anti-inflammatory, antioxidant, and whitening effects. However, the poor solubility of DMY, which is better in hot water, is the main reason for its poor membrane permeability and low bioavailability.

[0097] To address the problem of poor solubility of dihydromyricetin, traditionally, its solubility has been improved mainly by preparing it into liposomes. However, the current dihydromyricetin liposomes have poor stability and will become a turbid emulsion after being stored at about 25°C for a period of time at room temperature, let alone the storage stability under high-temperature conditions. For example, a reported multi-vesicle type dihydromyricetin liposome uses Tween-80 and PEG-4000 as the aqueous phase, and dihydromyricetin, cholesterol, and egg yolk lecithin as the oil phase. However, the drug loading rate of this dihydromyricetin liposome is only 42.93%, and the solution becomes a white turbid emulsion after being stored at 25°C or 37°C for 30 days, showing poor stability.

[0098] In addition, problems such as irreversible oxidation of DMY in aqueous solution with the increase of time and temperature, and easy color change under heating and light conditions have emerged, which have limited its application to a certain extent. Therefore, improving the solubility, stability, color change problem of DMY, and prolonging the slow release rate of DMY are a major prerequisite for its wide application.

[0099] To address the above problems, the present invention provides a dihydromyricetin liposome with good solubility, good stability, high encapsulation rate, slow release effect, and safety and non-irritation, which can be applied to cosmetics.

[0100] The technical solution is as follows:

[0101] A dihydromyricetin liposome is mainly made of the following components in mass percentage:

[0102] (1) Oil phase:

[0103] Phospholipid 1% - 6%,

[0104] Cholesterol 0.06% - 1%,

[0105] Ethanol 1% - 7.5%;

[0106] (2) Aqueous phase:

[0107]

[0108] The emulsifier is behenyl alcohol polyether-25.

[0109] While ensuring good solubility of dihydromyricetin, the present invention also significantly improves the stability of dihydromyricetin liposomes. Moreover, under the combined action of the emulsifier and other raw materials of the present invention, the encapsulation efficiency of dihydromyricetin can be significantly improved, and the particle size and PDI of dihydromyricetin liposomes can be significantly reduced, and the slow release rate of dihydromyricetin can be prolonged. In addition, the dihydromyricetin liposomes provided by the present invention are free of harmful substances, gentle to the human body, and safe and non-irritating.

[0110] It has been confirmed that the dihydromyricetin liposomes provided by the present invention have good solubility in water (good solubility in cold water and hot water), are gentle to the human body, have good skin physiological effects of antioxidant, whitening, and anti-inflammatory, and exhibit excellent stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thawing, light, and dark, can be stably stored for 90 days, slowly release dihydromyricetin to play a role, and improve its dose-effect relationship and bioavailability.

[0111] (1) In the present invention, in the dihydromyricetin liposomes, the oil phase comprises components in the following mass percentages: phospholipid 1% - 6%, cholesterol 0.06% - 1%, and ethanol 1% - 7.5%. The phospholipid and cholesterol are set in the oil phase, and ethanol is used as a solvent to completely dissolve the liposome preparation film material in advance so as to form a closed vesicle structure when encountering water to encapsulate dihydromyricetin.

[0112] It can be understood that, by mass percentage, the oil phase of the dihydromyricetin liposomes contains 1% - 6% of phospholipid, including but not limited to 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6%. Preferably, by mass percentage, the oil phase of the dihydromyricetin liposomes contains 6% of phospholipid.

[0113] In one embodiment, the phospholipid is selected from one or a combination of several of S45 soy lecithin, S75 soy lecithin, 90G soy lecithin, and PC50 soy lecithin. Preferably, compared with the liposomes prepared using other phospholipids, the dihydromyricetin liposomes prepared using 90G soy lecithin are more clarified and transparent, and the particle size (30nm - 100nm) and PDI (0.1 - 0.4) are significantly more stable.

[0114] In one embodiment, the mass ratio of dihydromyricetin to the phospholipid is 1:(5 - 20), including but not limited to 1:5, 1:8, 1:10, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. Preferably, the mass ratio of dihydromyricetin to the phospholipid is 1:(12 - 20).

[0115] Understandably, in terms of mass percentage, the oil phase of the dihydromyricetin liposome contains 0.06% to 1% of cholesterol, including but not limited to 0.06%, 0.067%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.

[0116] In one embodiment, the mass ratio of the phospholipid to cholesterol is (6 - 90):1, which is beneficial to enhancing the stability of the vesicles and reducing the permeability of the lipid membrane to solutes.

[0117] Understandably, the mass ratio of the lecithin to cholesterol includes but not limited to 6:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1 or 90:1. Preferably, the mass ratio of the lecithin to cholesterol is (6 - 60):1. Further preferably, the mass ratio of the lecithin to cholesterol is 33:1.

[0118] Understandably, in terms of mass percentage, the oil phase of the dihydromyricetin liposome contains 1% to 7.5% of ethanol, including but not limited to 1%, 2%, 3%, 4%, 5%, 6%, 7% or 7.5%. Preferably, the mass percentage of the ethanol in the dihydromyricetin liposome is 5%.

[0119] In one embodiment, the oil phase of the dihydromyricetin liposome further contains an antioxidant. Adding the antioxidant can further enhance the antioxidant property and stability of the dihydromyricetin liposome, and improve the stability and discoloration problem of DMY.

[0120] In one embodiment, the oil phase of the dihydromyricetin liposome contains 0.03% to 0.05% of the antioxidant, including but not limited to 0.03%, 0.035%, 0.04%, 0.045% or 0.05%.

[0121] In one embodiment, the antioxidant is selected from one or a combination of several of pentaerythrityl tetra(bis - tert - butylhydroxyhydrocinnamate), vitamin E and vitamin E acetate.

[0122] (2) In the present invention, in the dihydromyricetin liposome, the aqueous phase comprises components in the following mass percentages: 0.2% - 0.5% of dihydromyricetin, 0.5% - 2.5% of emulsifier, 1% - 10% of glycerol, 0.1% - 2.5% of pH regulator, and 60% - 96.14% of water. Dissolving dihydromyricetin in the aqueous phase is beneficial for the complete dispersion of dihydromyricetin in the solution, preventing the incomplete dissolution of dihydromyricetin and reducing the encapsulation efficiency. Setting the emulsifier and glycerol in the aqueous phase can enable the emulsifier to adhere well to the surface of the vesicles, enhancing the interaction force between particles in the solution and the stability of the solution. At the same time, glycerol can improve the freeze-thaw resistance, enabling the vesicles and the solution to be placed in an environment of -15°C, which can protect the vesicles and the solution. Setting the pH regulator in the aqueous phase is beneficial for improving the stability of dihydromyricetin.

[0123] It is understandable that, by mass percentage, the aqueous phase of the dihydromyricetin liposome contains 0.2% - 0.5% of dihydromyricetin, including but not limited to 0.2%, 0.3%, 0.4%, or 0.5%. Preferably, by mass percentage, the aqueous phase of the dihydromyricetin liposome contains 0.5% of dihydromyricetin.

[0124] In the present invention, the emulsifier is behenyl polyether-25. Compared with the complex of glyceryl caprylate / caprate (GTCC), Tween 80, SS, and SSE20 with a specific HLB value, cetearyl glucoside, and poloxamer 407, which show large particle size changes, flocculent floating, and delamination phenomena of liposomes under normal temperature and centrifugation conditions, the present invention selects behenyl polyether-25 as the aqueous phase emulsifier, which exhibits excellent stability under 7 different harsh environments including normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark.

[0125] It is understandable that, by mass percentage, the aqueous phase of the dihydromyricetin liposome contains 0.5% - 2.5% of emulsifier, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5%.

[0126] It is understandable that, by mass percentage, the aqueous phase of the dihydromyricetin liposome contains 1% - 10% of glycerol, including but not limited to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. Preferably, by mass percentage, the aqueous phase of the dihydromyricetin liposome contains 5% - 10% of glycerol.

[0127] Understandably, in terms of mass percentage, the aqueous phase of the dihydromyricetin liposome further contains 0.1% - 2.5% of a pH regulator, including but not limited to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4% or 2.5%. Further, the pH of the aqueous phase is 5 - 5.5.

[0128] In one embodiment, the pH regulator is citric acid.

[0129] In one embodiment, the aqueous phase of the dihydromyricetin liposome further contains a first preservative.

[0130] In one embodiment, in terms of mass percentage, the aqueous phase of the dihydromyricetin liposome contains 0.01% - 1% of a first preservative, including but not limited to 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%. Preferably, in terms of mass percentage, the aqueous phase of the dihydromyricetin liposome contains 1% of a first preservative.

[0131] In one embodiment, the first preservative is pentylene glycol.

[0132] Understandably, in terms of mass percentage, the aqueous phase of the dihydromyricetin liposome contains 60% - 96.14% of water, including but not limited to 60%, 65%, 70%, 72%, 77%, 77.9%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 96.14%. Preferably, in terms of mass percentage, the aqueous phase of the dihydromyricetin liposome contains 60% - 96.05% of water.

[0133] In one embodiment, the dihydromyricetin liposome further includes (3) a second preservative.

[0134] Further, in terms of mass percentage, the dihydromyricetin liposome contains 0.05% to 1.05% of a second preservative, including but not limited to 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or 1.05%.

[0135] In one embodiment, the second preservative is selected from one or a mixture of several of p-hydroxyacetophenone, pentylene glycol and phenoxyethanol. Further, the second preservative is a mixture of p-hydroxyacetophenone and pentylene glycol.

[0136] In one embodiment, in terms of mass percentage, the second preservative of the dihydromyricetin liposome contains 0.01% to 1% of pentylene glycol, including but not limited to 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%. Preferably, in terms of mass percentage, the second preservative of the dihydromyricetin liposome contains 1% of pentylene glycol.

[0137] In one embodiment, in terms of mass percentage, the dihydromyricetin liposome contains 0.01% to 0.1% of p-hydroxyacetophenone, including but not limited to 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09% or 0.1%. Preferably, in terms of mass percentage, the dihydromyricetin liposome contains 0.05% of p-hydroxyacetophenone.

[0138] In one embodiment, the dihydromyricetin liposome comprises the following components in mass percentage:

[0139] (1) Oil phase:

[0140]

[0141] (2) Aqueous phase:

[0142]

[0143]

[0144] In one embodiment, the dihydromyricetin liposome comprises the following components in mass percentage: (1) Oil phase:

[0145]

[0146] (2) Aqueous phase:

[0147]

[0148] In one embodiment, the dihydromyricetin liposome comprises the following components in mass percentage: (1) Oil phase:

[0149]

[0150] (2) Aqueous phase:

[0151]

[0152] (3) Preservative:

[0153] p-Hydroxyacetophenone 0.05%,

[0154] Pentylene glycol 1%.

[0155] In one embodiment, the dihydromyricetin liposome comprises the following components in mass percentage:

[0156] (1) Oil phase:

[0157]

[0158] (2) Aqueous phase:

[0159]

[0160] (3) Preservative:

[0161] p-Hydroxyacetophenone 0.05%,

[0162] Pentylene glycol 1%.

[0163] In one embodiment, the particle size of the dihydromyricetin liposome is 30 nm to 200 nm, the PDI is 0.1 to 0.5, and the encapsulation efficiency is ≥60%. Further, the pH is 5 to 5.5.

[0164] In one embodiment, the particle size of the dihydromyricetin liposome is 30 nm to 100 nm, the PDI is 0.1 to 0.5, and the encapsulation efficiency is ≥80%. Further, the pH is 5 to 5.5.

[0165] In one embodiment, the particle size of the dihydromyricetin liposome is 100 nm to 200 nm, the PDI is 0.1 to 0.3, and the encapsulation efficiency is ≥90%. Further, the pH is 5 to 5.5.

[0166] Traditional methods for preparing dihydromyricetin liposomes include the thin-film ultrasonic method, the ethanol injection method-calcium acetate gradient method, and the ethanol injection-ammonium sulfate gradient method. Among them, although the thin-film ultrasonic method is easy to prepare, it has problems such as low method reproducibility, low encapsulation efficiency, poor stability, possible degradation of phospholipids and active ingredients, uneven particle size, and residues of toxic reagents, which make it difficult to achieve industrial production. The ethanol injection method is simple, fast, and mild, and can be further scaled up for production. Combining the calcium acetate gradient and ammonium sulfate gradient methods is for a higher ratio of active ingredient to lipid and encapsulation efficiency. However, the encapsulation rates in traditional technologies are mostly below 60% and do not reach 75%, and the preparation time is significantly increased, and the product stability is also poor.

[0167] In view of the above problems, the present invention provides a method for preparing dihydromyricetin liposomes, which has high repeatability, short preparation time, no residue of harmful substances, and high encapsulation rate. By encapsulating dihydromyricetin with this method, the release rate of dihydromyricetin can be prolonged, its solubility, encapsulation efficiency, and stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark can be improved.

[0168] The technical solution is as follows:

[0169] A method for preparing dihydromyricetin liposomes as described above, comprising the following steps:

[0170] Mix the phospholipids and cholesterol in ethanol to prepare an oil-phase mixture;

[0171] Mix the dihydromyricetin, emulsifier, glycerol, pH regulator with water to prepare an aqueous-phase mixture;

[0172] Mix the oil-phase mixture and the aqueous-phase mixture, and perform normal-pressure homogenization treatment and high-pressure homogenization treatment.

[0173] The present invention uses the method of ethanol injection combined with high-pressure homogenization to prepare dihydromyricetin liposomes. The preparation method is simple, fast, and the process is simple, suitable for large-scale industrial production.

[0174] In one embodiment, the process parameters for preparing the oil-phase mixture include: the temperature is 40°C to 60°C.

[0175] In one embodiment, the process parameters for preparing the aqueous-phase mixture include: the pH of the system is 5.0 to 5.5, and the temperature is 70°C to 75°C.

[0176] In one embodiment, mix the dihydromyricetin, emulsifier, glycerol and water to prepare an aqueous-phase mixture; it includes the following steps:

[0177] Adjust the pH to 5.0 - 5.5 with 0.1 mol / L citric acid solution in water, add an emulsifier, glycerol, and stir and heat to 70°C - 75°C, then dissolve dihydromyricetin for 10 min to obtain an aqueous phase mixture.

[0178] In one embodiment, slowly drip the oil phase mixture into the above-mentioned aqueous phase mixture and mix to obtain a primary emulsion.

[0179] In one embodiment, the process parameters of atmospheric pressure homogenization treatment include: the mechanical stirring speed is 300 rpm - 700 rpm, the stirring time is 20 min - 40 min, and the temperature is 40°C - 60°C. It can be understood that in the step of atmospheric pressure homogenization treatment, the mechanical stirring speed includes but is not limited to 300 rpm, 400 rpm, 500 rpm, 600 rpm, or 700 rpm; the time includes but is not limited to 20 min, 25 min, 30 min, 35 min, or 40 min. Preferably, in the step of atmospheric pressure homogenization treatment, the mechanical stirring speed is 500 rpm and the emulsification time is 30 min.

[0180] In one embodiment, the process parameters of high-pressure homogenization treatment include: the pressure is 300 bar - 900 bar, and the number of times is 1 - 9 times. The high-pressure homogenizer used in each embodiment of the present application is produced by GEA Niro Soavi, and the model is PANDAPLUS 2000. It can be understood that in the process of high-pressure homogenization treatment, the high-pressure homogenization pressure includes but is not limited to 300 bar, 400 bar, 500 bar, 600 bar, 700 bar, 800 bar, or 900 bar; the number of high-pressure homogenization times includes but is not limited to 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, or 9 times. Preferably, in the step of high-pressure homogenization treatment, the high-pressure homogenization pressure is 500 bar and the high-pressure homogenization number of times is 3 times.

[0181] In one embodiment, a method for preparing dihydromyricetin liposomes as described above includes the following steps:

[0182] Mix the phospholipids, cholesterol, and antioxidant in ethanol to prepare an oil phase mixture;

[0183] Mix the dihydromyricetin, emulsifier, glycerol, and water to prepare an aqueous phase mixture;

[0184] Slowly drip the oil phase mixture into the aqueous phase mixture and mechanically stir to prepare a primary emulsion;

[0185] After subjecting the primary emulsion to high-pressure homogenization treatment, add a preservative and mix well.

[0186] In one embodiment, the dihydromyricetin, emulsifier, glycerol, pentylene glycol and water are mixed to prepare an aqueous phase mixture; the method comprises the following steps:

[0187] In water, the pH is adjusted to 5.0 - 5.5 with 0.1 mol / L citric acid solution, the emulsifier, glycerol and pentylene glycol are added, and the mixture is stirred and heated to 70°C - 75°C, then dihydromyricetin is dissolved for 10 min to obtain the aqueous phase mixture.

[0188] It can be understood that for the dihydromyricetin liposome in which the aqueous phase contains a first preservative (such as pentylene glycol), in the step of preparing the aqueous phase mixture, the step of adding the first preservative and mixing it evenly with the emulsifier, glycerol and water is further included.

[0189] It can be understood that for the dihydromyricetin liposome which contains (1) an oil phase, (2) an aqueous phase and (3) a second preservative, after the step of preparing the high-pressure homogenization treatment, the step of adding the second preservative to the treated sample and mixing it evenly is further included.

[0190] The present invention also provides the application of the dihydromyricetin liposome as described above. The technical solution is as follows:

[0191] A cosmetic, characterized in that it comprises the dihydromyricetin liposome as described above.

[0192] In one embodiment, the cosmetic is selected from at least one of a facial mask, eye cream, facial cream, primer, essence, lotion and skin softener.

[0193] The following is the specific embodiment part.

[0194] Unless otherwise specified, all raw materials are commercially available products.

[0195] Example 1

[0196] Prepare the sample according to the ratio in Table 1. Dissolve lecithin, cholesterol and antioxidant in absolute ethanol to obtain the oil phase, adjust the pH of the water in the aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, add the emulsifier, glycerol and pentylene glycol, then heat to 70°C, stir and dissolve dihydromyricetin for 10 min to obtain the aqueous phase. Slowly add the oil phase to the aqueous phase, mechanically stir at 500 rpm for 30 min to form the primary emulsion, then subject the primary emulsion to high-pressure homogenization at 500 bar for 3 times (a total of 3 times of high-pressure homogenization), add the preservative and mix evenly to obtain the sample.

[0197] Table 1

[0198]

[0199] The experimental results show that after samples were prepared with S45 soy lecithin and PC50 soy lecithin and placed in an environment at 45°C for more than two months, the appearance of the samples showed demulsification, changing from a clear and transparent solution to a milky turbid solution. After samples were prepared with 90G soy lecithin and S75 soy lecithin and placed in an environment at 45°C for three months, the appearance of the samples remained a clear and transparent solution. Therefore, S75 soy lecithin and 90G soy lecithin are preferably used as the raw materials of lecithin in the liposome system of the present invention.

[0200] Example 2

[0201] Prepare samples according to the ratios in Table 2. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution. After adding emulsifier, glycerol, and pentylene glycol, heat to 70°C and stir for 10 min to dissolve dihydromyricetin to obtain the aqueous phase. Slowly add the oil phase to the aqueous phase and mechanically stir at 500 rpm for 30 min to form a primary emulsion. Then, homogenize the primary emulsion at 500 bar three times (a total of three times of high-pressure homogenization), add preservative and mix well to obtain the sample.

[0202] After centrifuging all the prepared samples at 10000 rpm for 10 min, measure the particle size and polydispersity index, and at the same time place the samples at room temperature and observe the change in the appearance of the samples.

[0203] Table 2

[0204]

[0205]

[0206] The results show that when GTCC, cetearyl glucoside, and poloxamer 407 are used as emulsifiers in the system, the samples show delamination after being placed at room temperature for 1 day, 2 days, and 14 days respectively. The polydispersity index of the sample with the HLB value of 8.55 by compounding SS and SSE20 is higher than 50%, indicating that the dispersion is not uniform enough. After centrifuging the sample with the HLB value of 14.1 by compounding polyglyceryl-10 laurate, SS, and SSE20, the changing trends of the particle size and polydispersity index of the sample become larger, indicating that the sample is not stable enough. Therefore, ceteth-25 is selected as the raw material of the emulsifier in the system.

[0207] Example 3

[0208] Preparation experiment with different mass ratios of soy lecithin to cholesterol

[0209] Prepare samples according to the ratios in Table 3. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water phase to 5 - 5.5 with 0.1 mol / L citric acid solution. After adding emulsifier, glycerol, and pentylene glycol, heat to 70 °C and stir for 10 min to dissolve dihydromyricetin to obtain the water phase. Slowly add the oil phase to the water phase and stir mechanically at 500 rpm for 30 min to form the primary emulsion. Then, subject the primary emulsion to high-pressure homogenization at 500 bar three times (a total of three times of high-pressure homogenization). Add preservative and mix well to obtain the sample.

[0210] Table 3

[0211]

[0212] Take the prepared dihydromyricetin nano-liposomes, add ethanol to demulsify and dilute 40 times. At the same time, use an ultrafiltration centrifugal tube with a molecular cut-off of 3000 for the dihydromyricetin nano-liposomes, centrifuge at a speed of 10000 rpm for 30 min. Take the filtrate, dilute it 40 times with ethanol, and measure the content of dihydromyricetin with a high-performance liquid chromatograph to calculate the encapsulation efficiency. The results are as shown in Appendix Figure 1 A.

[0213] From Figure 1 it can be seen from A that as the mass ratio of soybean lecithin to cholesterol increases, the encapsulation efficiency shows a trend of first decreasing, then increasing, and then decreasing. Among them, the encapsulation efficiency of the samples with a mass ratio of 6:1 and 60:1 is higher than 90%. In order to improve the encapsulation efficiency of dihydromyricetin, the mass ratio of soybean lecithin to cholesterol is preferably (6 - 60):1.

[0214] Example 4

[0215] Preparation experiments with different mass ratios of dihydromyricetin to soybean lecithin

[0216] Prepare samples according to the ratios in Table 4. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water phase to 5 - 5.5 with 0.1 mol / L citric acid solution. After adding emulsifier, glycerol, and pentylene glycol, heat to 70 °C and stir for 10 min to dissolve dihydromyricetin to obtain the water phase. Slowly add the oil phase to the water phase and stir mechanically at 500 rpm for 30 min to form the primary emulsion. Then, subject the primary emulsion to high-pressure homogenization at 500 bar three times (a total of three times of high-pressure homogenization). Add preservative and mix well to obtain the sample.

[0217] Table 4

[0218]

[0219] Take the prepared dihydromyricetin nano-liposomes, add ethanol to demulsify and dilute 40 times. At the same time, centrifuge the dihydromyricetin nano-liposomes with an ultrafiltration centrifugal tube with a molecular cut-off of 3000 at a speed of 10,000 rpm for 30 min. Take the filtrate, dilute it 40 times with ethanol, measure the content of dihydromyricetin with a high-performance liquid chromatograph, and calculate the encapsulation efficiency. The results are shown in Appendix Figure 1 as shown in B.

[0220] As can be seen Figure 1 from B, as the drug-lipid ratio increases, the encapsulation efficiency shows a trend of first increasing, then decreasing, and then increasing. Moreover, the encapsulation efficiencies of the drug-lipid ratios of 1:20 and 1:12 are significantly higher than 80%. In order to improve the encapsulation efficiency as much as possible, the mass ratio of dihydromyricetin to soybean lecithin is preferably 1:(12 - 20).

[0221] Example 5

[0222] Preparation experiments with different contents of polyoxyethylene 25 behenate

[0223] Prepare samples according to the ratios in Table 5. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water phase to 5 - 5.5 with 0.1 mol / L citric acid solution. After adding emulsifier, glycerol, and pentylene glycol, heat to 70 °C and stir for 10 min to dissolve dihydromyricetin to obtain the water phase. Slowly add the oil phase to the water phase and mechanically stir at 500 rpm for 30 min to form the primary emulsion. Then, homogenize the primary emulsion at 500 bar three times (a total of three times of high-pressure homogenization), add preservative and mix well to obtain the sample.

[0224] Table 5

[0225]

[0226] Take the prepared dihydromyricetin nano-liposomes, add ethanol to demulsify and dilute 40 times. At the same time, centrifuge the dihydromyricetin nano-liposomes with an ultrafiltration centrifugal tube with a molecular cut-off of 3000 at a speed of 10,000 rpm for 30 min. Take the filtrate, dilute it 40 times with ethanol, measure the content of dihydromyricetin with a high-performance liquid chromatograph, and calculate the encapsulation efficiency. The results are shown in Appendix Figure 1 as shown in C.

[0227] As can be seen Figure 1 from C, as the mass content of polyoxyethylene 25 behenate increases, the encapsulation efficiency shows a trend of first decreasing, then increasing, and then decreasing. Moreover, the encapsulation efficiencies of 0.5% and 1.5% are higher than 80%. Considering its influence on skin irritation and preparation cost, while ensuring high encapsulation efficiency and good emulsification and stability performance, the mass content of polyoxyethylene 25 behenate in the liposomes with the mass ratio of dihydromyricetin to soybean lecithin is optimized to be 0.5% - 2.5%.

[0228] Example 6

[0229] Preparation experiment with different glycerol contents

[0230] Prepare samples according to the ratios in Table 6. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution. After adding emulsifier, glycerol, and pentylene glycol, heat to 70°C and stir for 10 min to dissolve dihydromyricetin to obtain the aqueous phase. Slowly add the oil phase to the aqueous phase and mechanically stir at 500 rpm for 30 min to form the primary emulsion. Then, homogenize the primary emulsion at 500 bar under high pressure for 3 times (a total of 3 high-pressure homogenizations). Add preservative and mix well to obtain the sample.

[0231] Table 6

[0232]

[0233]

[0234] Take the prepared dihydromyricetin nanoliposomes, add ethanol to demulsify and dilute 40 times. At the same time, use an ultrafiltration centrifugal tube with a molecular cut-off of 3000 for the dihydromyricetin nanoliposomes, centrifuge at a speed of 10000 rpm for 30 min. Take the filtrate, dilute it 40 times with ethanol, and measure the content of dihydromyricetin with a high-performance liquid chromatograph to calculate the encapsulation efficiency. The results are as shown in Figure 1 Figure D in the appendix.

[0235] From Figure 1 Figure D, it can be seen that as the glycerol content increases, the encapsulation efficiency first increases and then decreases. Although the encapsulation efficiencies at glycerol contents of 0% and 10% are not very different, the addition of glycerol can significantly improve the stability of the sample under freezing conditions. Therefore, a glycerol content of 5% - 10% is preferably selected.

[0236] Example 7

[0237] Preparation experiment with different high-pressure homogenization pressures

[0238] Prepare samples according to the ratios in Table 7. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution. After adding emulsifier, glycerol, and pentylene glycol, heat to 70°C and stir for 10 min to dissolve dihydromyricetin to obtain the aqueous phase. Slowly add the oil phase to the aqueous phase and mechanically stir at 500 rpm for 30 min to form the primary emulsion. Then, homogenize at different high-pressure homogenization pressures for 3 times (a total of 3 high-pressure homogenizations). Add preservative and mix well to obtain the sample.

[0239] Table 7

[0240]

[0241] Then, take the prepared dihydromyricetin nano-liposomes, add ethanol to demulsify and dilute 40 times. At the same time, use an ultrafiltration centrifuge tube with a molecular cut-off of 3000 to centrifuge the dihydromyricetin nano-liposomes at a speed of 10,000 rpm for 30 min. Take the filtrate, dilute it 40 times with ethanol, measure the content of dihydromyricetin with a high-performance liquid chromatograph, and calculate the encapsulation efficiency. The results are as shown in Figure 1 Figure E in the appendix.

[0242] From Figure 1 Figure E in the appendix, it can be seen that as the high-pressure homogenization pressure gradually increases, the encapsulation efficiency gradually increases. When the high-pressure homogenization pressure increases to 700 bar, the encapsulation efficiency hardly changes any more, and starting from 500 bar of the high-pressure homogenization pressure, there is no significant change in the encapsulation efficiency. Therefore, the high-pressure homogenization pressure of 500 bar is preferably selected.

[0243] Example 8

[0244] Preparation experiments with different numbers of high-pressure homogenization

[0245] Prepare samples according to the ratios in Table 8. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water phase to 5 - 5.5 with 0.1 mol / L citric acid solution. After adding emulsifier, glycerol, and pentylene glycol, heat to 70 °C and stir for 10 min to dissolve dihydromyricetin to obtain the water phase. Slowly drip the oil phase into the water phase and mechanically stir at 500 rpm for 30 min to form the primary emulsion. Then homogenize at a high-pressure homogenization pressure of 500 bar for different numbers of times, add preservative and mix well to obtain the samples.

[0246] Table 8

[0247]

[0248] Take the prepared dihydromyricetin nano-liposomes, add ethanol to demulsify and dilute 40 times. At the same time, use an ultrafiltration centrifuge tube with a molecular cut-off of 3000 to centrifuge the dihydromyricetin nano-liposomes at 10,000 rpm for 30 min. Take the filtrate, dilute it 40 times with ethanol, measure the content of dihydromyricetin with a high-performance liquid chromatograph, and calculate the encapsulation efficiency. The results are as shown in Figure 1 Figure F in the appendix.

[0249] From Figure 1 Figure F in the appendix, it can be seen that as the number of high-pressure homogenization gradually increases, the encapsulation efficiency first increases and then decreases. When the number of high-pressure homogenization increases to 5 times, the encapsulation efficiency starts to decline, and starting from 3 times of the high-pressure homogenization, there is no significant change in the encapsulation efficiency. Therefore, the number of high-pressure homogenization of 3 times is preferably selected.

[0250] Example 9

[0251] Orthogonal experiment

[0252] Soybean lecithin (6%), cholesterol (0.1%, 0.1818%, 1%), pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate) 0.05%, and absolute ethanol 5% were completely dissolved by mass percentage as the oil phase; the pH of water was adjusted to 5 - 5.5 with 0.1 mol / L citric acid solution (added to 100%), and behenyl alcohol polyether-25 (0.5%, 1.5%, 2.5%), glycerol (0%, 5%, 10%), and pentylene glycol 1% were added, then heated to 70°C to dissolve dihydromyricetin (0.3%, 0.375%, 0.5%) as the water phase; the oil phase was slowly added dropwise to the water phase, and the primary emulsion was obtained after mechanical stirring at 40°C - 60°C and 500 rpm for 30 min; the primary emulsion was homogenized at 500 bar under high pressure three times, and then 1 g of pentylene glycol and 0.05 g of p-hydroxyacetophenone were added and mixed evenly. Then, the prepared dihydromyricetin nanoliposomes were taken, ethanol was added to demulsify and diluted 40 times. At the same time, the dihydromyricetin nanoliposomes were centrifuged at 10,000 rpm for 30 min using an ultrafiltration centrifugal tube with a molecular cut-off of 3000. The filtrate was taken and diluted 40 times with ethanol, and then the content of dihydromyricetin was measured using a high performance liquid chromatograph, and the encapsulation efficiency was calculated. The results are shown in Table 9 below.

[0253] Table 9 Results of process optimization data

[0254]

[0255] Note: A represents the mass percentage of lecithin and cholesterol, B represents the mass percentage of lecithin and dihydromyricetin, C represents the emulsifier content, and D represents the glycerol content.

[0256] The results showed that the primary and secondary order of the factors affecting the encapsulation efficiency of dihydromyricetin nanoliposomes was B > A > D > C, and the optimal plan was A2B3C2D1. It was found that when it was placed in normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark environments for 5 days, the appearance of the sample changed from a transparent solution to a turbid solution in the freezing and freeze-thaw environments, while there was no change under heating conditions, indicating that the sample had poor stability under freezing conditions. Glycerol, as a raw material that could adjust the freezing stability of the solution, was a secondary influencing factor in the orthogonal experiment. The glycerol content could be appropriately adjusted according to the experimental stability requirements to make the sample more stable. Therefore, by adjusting the glycerol content to 7.5% and 10% for comparison, and at the same time measuring its encapsulation efficiency and the stability of the sample under different conditions, the optimal plan was screened for subsequent experiments.

[0257] Example 10

[0258] Optimization process experiment 1

[0259] Dissolve 6% soy lecithin, 0.1818% cholesterol, 0.05% pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate), and 5% absolute ethanol by mass percentage to form the oil phase; adjust the pH of water to 5 - 5.5 with 0.1 mol / L citric acid solution (added to 100%), add 1.5% polyoxyethylene 25 behenyl ether, 5% glycerol, and 1% pentylene glycol, then heat to 70°C and dissolve 0.5% dihydromyricetin to form the water phase; slowly drip the oil phase into the water phase, mechanically stir at 40°C - 60°C and 500 rpm for 30 min to obtain the primary emulsion; subject the primary emulsion to high-pressure homogenization at 500 bar for 3 times (a total of 3 times of high-pressure homogenization), and add 1 g pentylene glycol and 0.05 g p-hydroxyacetophenone and mix well.

[0260] Example 11

[0261] Optimization process experiment 2

[0262] Dissolve 6% soy lecithin, 0.1818% cholesterol, 0.05% pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate), and 5% absolute ethanol by mass percentage to form the oil phase; adjust the pH of water to 5 - 5.5 with 0.1 mol / L citric acid solution (added to 100%), add 1.5% polyoxyethylene 25 behenyl ether, 7.5% glycerol, and 1% pentylene glycol, then heat to 70°C and dissolve 0.5% dihydromyricetin to form the water phase; slowly drip the oil phase into the water phase, mechanically stir at 40°C - 60°C and 500 rpm for 30 min to obtain the primary emulsion; subject the primary emulsion to high-pressure homogenization at 500 bar for 3 times (a total of 3 times of high-pressure homogenization), and add 1 g pentylene glycol and 0.05 g p-hydroxyacetophenone and mix well.

[0263] Example 12

[0264] Optimization process experiment 3

[0265] Dissolve 6% soy lecithin, 0.1818% cholesterol, 0.05% pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate), and 5% absolute ethanol by mass percentage to form the oil phase; adjust the pH of water to 5 - 5.5 with 0.1 mol / L citric acid solution (added to 100%), add 1.5% polyoxyethylene 25 behenyl ether, 10% glycerol, and 1% pentylene glycol, then heat to 70°C and dissolve 0.5% dihydromyricetin to form the water phase; slowly drip the oil phase into the water phase, mechanically stir at 40°C - 60°C and 500 rpm for 30 min to obtain the primary emulsion; subject the primary emulsion to high-pressure homogenization at 500 bar for 3 times (a total of 3 times of high-pressure homogenization), and add 1 g pentylene glycol and 0.05 g p-hydroxyacetophenone and mix well.

[0266] Test

[0267] (1) The encapsulation efficiency of the dihydromyricetin nano-liposomes prepared in Examples 10, 11, and 12 was tested as follows:

[0268] Precisely weigh 0.002 g of dihydromyricetin standard, dissolve it in a 10 mL volumetric flask with 75% ethanol to prepare a reference substance solution with a concentration of 200 μg / mL. Respectively take 0, 15 μL, 30 μL, 75 μL, 150 μL, 300 μL, 600 μL, 900 μL, 1200 μL, 1500 μL of the reference substance solution, make up the volume to 3 mL with 75% ethanol and shake well to prepare a series of solutions with concentrations of 0, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL. Use an Agilent 1260 liquid chromatograph, a C18 chromatographic column, with the mobile phase being methanol and 0.1% (v / v) formic acid solution, the mobile phase ratio being 35:65 (v / v), the flow rate being 1.0 ml / min, and measure the peak area of DMY at 290 nm to plot the standard curve y = 25.3445x - 25.8835, R2 = 0.9994.

[0269] The ultrafiltration centrifugation method was used to determine the encapsulation efficiency of dihydromyricetin nano-liposomes. First, take 400 μL of DMY nano-liposomes and place them in an ultrafiltration centrifugal tube with a molecular cut-off of 3000. After ultrafiltration centrifugation at 10000 rpm for 30 min, take out 125 μL of the filtrate from the outer tube and dilute it to 5 mL, and use liquid chromatography to measure the peak area as the content of free DMY. Take another 125 μL of DMY nano-liposomes, add ethanol to demulsify and dilute it to 5 mL. After centrifugation at 10000 rpm for 30 min, let it stand for a while and take the supernatant to pass through a 0.22 μm organic membrane to measure the peak area as the total DMY content. Calculate the total DMY content C1 and the free DMY content C0 according to the linear regression equation of the standard curve respectively. Finally, calculate the encapsulation efficiency according to the calculation formula: Encapsulation efficiency = [(C1 - C0) / C1] × 100%.

[0270] The results showed that the encapsulation efficiency of Example 10 was (96.05 ± 0.14)%, the encapsulation efficiency of Example 11 was (95.60 ± 0.57)%, and the encapsulation efficiency of Example 12 was (96.02 ± 0.59)%. The encapsulation efficiencies of these three examples were not significantly different. Therefore, under the condition of ensuring a very small change in the encapsulation efficiency, the most stable example was selected as the final optimized process formula, and TEM, sustained release, stability investigation, stability improvement, safety evaluation, and anti-irritation evaluation experiments were carried out.

[0271] (2) The particle size and stability of the dihydromyricetin nano-liposomes prepared in Examples 10, 11, and 12 were tested as follows:

[0272] Take 30 mL each of Examples 10, 11, and 12 and place them in sample bottles. Place them under normal temperature, refrigeration, freezing, freeze-thaw, heating, light, and dark conditions for 7 days and 30 days. After taking out the samples and diluting them 10 times, measure the particle size of the samples using a ZSU 3200 nanoparticle size analyzer, with the unit being nm.

[0273] The results are shown in Table 10. After freezing for 7 days, the particle size of Example 10 increased by 30 nm. After freeze-thaw for 7 days, the particle size increased by 20 nm. However, the change in particle size under heating conditions was less than 10 nm. Therefore, it was determined that Example 10 was unstable under freezing conditions. After 30 days of freeze-thaw, the particle size of Example 11 increased by 10 nm, while the change in particle size of Example 12 under 7 conditions for 30 days was less than 10 nm. Therefore, Example 12 was selected as the final optimized process formula for TEM, sustained release, stability investigation, stability improvement, safety evaluation, and anti-irritation evaluation experiments.

[0274] The results showed that the particle size of the sample prepared from Example 12 was (52.27 ± 0.54) nm, the PDI was (0.31 ± 0.03), and the Zeta potential was -2.5 mV.

[0275] Table 10 Results of stability investigation

[0276]

[0277] (3) Conduct TEM detection on the dihydromyricetin nano-liposomes prepared from Example 12 as follows:

[0278] Weigh the sample prepared from Example 12, dilute it by a certain multiple, and then drop it on a copper grid. After a few seconds, gently pick up the copper grid sample with forceps, and use filter paper to absorb the excess liquid along one side. After it is slightly dry, place the copper grid on a 2% phosphotungstic acid staining solution drop for floating staining for 60 s. After picking it up with forceps, also use filter paper to absorb the excess liquid along one side, and place it membrane-side up on the filter paper to dry. Observe and take pictures using a transmission electron microscope. The results are as Figure 2 shown in Figures 2A and 2B. The particles are spherical-like and are relatively evenly distributed.

[0279] (4) Conduct in vitro slow release testing on the dihydromyricetin nano-liposomes prepared from Example 12.

[0280] For the in vitro release test of dihydromyricetin nano-liposomes, the dihydromyricetin in the release medium was measured by dialysis with PBS containing 0.5% Tween. 5 mL of the sample was placed into a dialysis bag (molecular weight cut-off: 6000 - 8000), and the dialysis bag was placed into a 250 mL beaker containing 200 mL of the release medium. The beaker was incubated in a shaking water bath thermostat at 37 °C and shaken at 100 rpm by a magnetic stirrer. At predetermined time intervals (1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120 hours after the start of the incubation), all of the release medium (3 mL) was taken out and replaced with the same volume of fresh release medium (3 mL). The release amount of DMY was determined by HPLC, and the slow release curve was plotted.

[0281]

[0282] Qi: Cumulative release amount at the i-th sampling time point, %;

[0283] V: Volume of the release medium, mL;

[0284] ρ n : Concentration of dihydromyricetin in the release medium during the n-th sampling, μg / mL;

[0285] ρ i : Concentration of dihydromyricetin in the release medium during the i-th sampling, μg / mL;

[0286] Vi: Sampling volume at the i-th sampling, mL;

[0287] m: Mass of dihydromyricetin in the nano-liposome sample, μg;

[0288] The slow release results were as Figure 3 shown. The release amount of the dihydromyricetin solution reached the highest at 8 h, and no more dihydromyricetin was released after 8 h. However, the release amount of the dihydromyricetin nano-liposomes did not reach the highest at 8 h and continued to release dihydromyricetin until 48 h, indicating that dihydromyricetin was encapsulated in the liposomes, which helped with the slow release of dihydromyricetin. And Figure 3 after reaching the highest release amount, the dihydromyricetin solution in [[ ]] showed a downward trend, indicating that after dihydromyricetin was released, it might be due to being in a free state and standing for too long, resulting in the instability of dihydromyricetin and ultimately a decrease in the content of dihydromyricetin.

[0289] (5) The stability of the dihydromyricetin nano-liposomes prepared in Example 12 was investigated as follows:

[0290] The samples prepared in Example 12 were respectively filled into 7 vials and sealed, and then placed in environments of normal temperature, refrigeration (4°C), freezing (-15°C), heating (45°C), freeze-thaw (-15°C and 45°C alternating), light avoidance, and light exposure (28°C) for storage. Particle size, PDI test, encapsulation efficiency, and pH test were carried out on the 7th, 14th, 30th, 60th, and 90th days respectively. The results are as Figure 4 shown. The changes in particle size and PDI were relatively small, as Figure 5 shown. The change in encapsulation efficiency was relatively small. Among them, when stored in a hot environment ( Figure 5 D) for 90 days, the encapsulation efficiency decreased to less than 80%. After inspecting the appearance of the samples, the color of the samples gradually deepened, but no demulsification phenomenon occurred. This may be because soy lecithin accelerated its oxidation and decomposition under the heating state, resulting in the leakage of dihydromyricetin, thereby reducing the encapsulation efficiency. The pH range was still between 4 and 6. Therefore, the dihydromyricetin nanoliposomes prepared by the present invention can be stably stored for 90 days in environments of normal temperature, refrigeration, freezing, heating, freeze-thaw, light exposure, and light avoidance.

[0291] (6) The stability improvement test was carried out on the dihydromyricetin nanoliposomes prepared in Example 12, and the specific steps are as follows:

[0292] The samples were respectively filled into 3 vials and sealed, and then placed in environments of normal temperature, heating (45°C), and sunlight exposure for storage. The dihydromyricetin content was tested at the beginning, 14th, and 30th days respectively. The results are as Figure 6 shown. When the dihydromyricetin liposomes were stored in the three environments for 30 days, no crystal precipitation phenomenon occurred in the appearance of the samples. While the dihydromyricetin aqueous solution showed different degrees of crystal precipitation phenomenon after being placed in these three environments for 7 days, indicating that the encapsulation of dihydromyricetin by liposomes can improve its tendency to crystallize and precipitate. At the same time, when the dihydromyricetin liposomes were placed in normal temperature and light exposure environments for 30 days, the dihydromyricetin content decreased only by 3.82% and 29.55% respectively, while the dihydromyricetin content in the dihydromyricetin aqueous solution decreased by 52.53% and 57.77% respectively. Therefore, the dihydromyricetin nanoliposomes prepared by the present invention can not only improve the tendency of dihydromyricetin to crystallize and precipitate, but also improve the trend of the decrease in dihydromyricetin content to a certain extent.

[0293] (7) The safety test was carried out on the dihydromyricetin nanoliposomes prepared in Example 12, and the specific steps are as follows:

[0294] First, defibrinated sheep blood was centrifuged and washed 4 times at 3800 rpm using PBS, and then the blood was diluted with PBS to an absorbance of 2 - 2.3 to obtain the standby blood. Meanwhile, 0.01 g of sodium dodecylsulfate (SDS) was weighed and dissolved in 10 g of water to prepare a 0.1% SDS solution. The dihydromyricetin liposome sample obtained in Preparation Example 12 was diluted with a PBS solution with a pH of 6.8 to samples with concentrations of 0.5%, 1%, 2%, 4%, 8%, 16%, 32%, 64%, and 100%. 500 μL of the diluted sample and 500 μL of the standby blood were added, incubated in a shaker at 200 rpm for 10 min, centrifuged at 10000 rpm for 1 min, then added to a 96-well plate. The OD value was measured at 560 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and a linear regression equation of sample concentration - hemolysis rate was plotted, as shown in Figure 7 shown. The sample concentration L value at a hemolysis rate of 50% was calculated according to the linear regression equation. Meanwhile, the OD values of the 96-well plate were measured at 540 nm and 575 nm, and the protein denaturation rate DI values of samples with different concentrations were calculated.

[0295]

[0296] Negative control: Replace the sample with a PBS solution

[0297] Positive control: Replace the sample with a 0.1% SDS solution

[0298]

[0299] R1: OD when the sample is replaced with water 575nm / 540nm ;

[0300] R2: OD when the sample is replaced with a 0.1% SDS solution 575nm / 540nm ;

[0301] R i : OD of the sample 575nm / 540nm ;

[0302] The results are shown in Table 11, and the L / D values of samples with different concentrations were calculated to be > 100. Therefore, the dihydromyricetin nano-liposomes prepared in the present invention are safe and non-irritating.

[0303] Table 11 Results of safety test

[0304]

[0305] (8) The antioxidant capacity of the dihydromyricetin nano-liposomes prepared in Preparation Example 12 was tested as follows:

[0306] Since reactive oxygen species are involved in the process of inflammation, antioxidant activity is also an important pathway for soothing effects. First, the sample of Example 12 was diluted with pure water into samples with concentrations of 10%, 50%, and 100% for standby. At the same time, aqueous solutions with concentrations of 10%, 50%, and 100% of dihydromyricetin were prepared for standby. These samples were placed in a normal temperature environment for 7 days. Then, the samples in the starting state and the samples placed in a normal temperature environment for 7 days were filtered through a 0.45 μm filter membrane at the same time. 20 μL of the sample solution and 180 μL of DPPH ethanol solution were added to a 96-well plate. After mixing, the reaction was carried out in the dark at room temperature for 30 min, and the OD value was measured at 517 nm.

[0307]

[0308] A s : The OD value when the DPPH ethanol solution was replaced with absolute ethanol solution;

[0309] A b : The OD values of samples with different concentrations;

[0310] A c : The OD value when the sample solution was replaced with absolute ethanol solution;

[0311] The results are as Figure 8 shown. For samples with different concentrations of dihydromyricetin liposomes and dihydromyricetin aqueous solutions in the starting state, their DPPH radical scavenging rates were comparable, both higher than 80%. After being placed in a normal temperature environment for 7 days, the dihydromyricetin liposomes significantly protected the antioxidant ability of dihydromyricetin, and the DPPH radical scavenging efficiency was significantly higher than that of the dihydromyricetin aqueous solution. Therefore, the dihydromyricetin nano-liposomes prepared in the present invention not only have good antioxidant ability, but also have the ability to protect the degree of decline in the antioxidant ability of dihydromyricetin.

[0312] (9) The anti-irritation test was carried out on the dihydromyricetin nano-liposomes prepared in Example 12, and the specific steps are as follows:

[0313] First, defibrinated sheep blood was centrifuged and washed 4 times at 3800 rpm using PBS, and then the blood was diluted with PBS to an absorbance of 2 - 2.3 to obtain standby blood. Meanwhile, 0.01 g of SDS was dissolved in 10 g of water to prepare a 0.1% SDS solution. 1 μL, 2 μL, 4 μL, 8 μL, 16 μL, and 32 μL of the 0.1% SDS solution were added respectively, and the volume was supplemented to 500 μL with PBS solution. Then 500 μL of the standby blood was added. After incubation in a shaker at 200 rpm for 10 min, centrifugation was carried out at 10000 rpm for 1 min, and then the mixture was added to a 96 - well plate. The OD value was measured at 560 nm using an enzyme - labeled instrument, and a linear fitting curve of the volume - hemolysis rate of the 0.1% SDS solution was plotted, as shown in Figure 9 shown in A. The volume of the 0.1% SDS solution when the hemolysis rate was 50% was calculated to be 9.7 μL. The dihydromyricetin liposome sample obtained in Preparation Example 12 and an aqueous solution with the same dihydromyricetin content were diluted with PBS solution at pH 6.8 to samples with concentrations of 10%, 50%, and 100% for standby, and these samples were placed at room temperature for 7 days. Then, both the initial - state samples and the samples placed at room temperature for 7 days were filtered through a 0.45 - μm filter membrane. First, 9.7 μL of the 0.1% SDS solution was added, then 490.3 μL of the diluted sample and 500 μL of the standby blood were added. After incubation in a shaker at 200 rpm for 10 min, centrifugation was carried out at 10000 rpm for 1 min, and then the mixture was added to a 96 - well plate. The OD value was measured at 560 nm using an enzyme - labeled instrument.

[0314]

[0315]

[0316] Negative control: Replace the sample with PBS solution;

[0317] Positive control: Replace the sample with 0.1% SDS solution;

[0318] The results are shown in Figure 9 B and Figure 9As shown in C, the hemolysis inhibition rate of dihydromyricetin liposomes and aqueous solutions gradually decreases with the increase of sample concentration. This may be because in the erythrocyte hemolysis experiment, the hemolysis rate of water in erythrocytes is 100%, and the higher the sample concentration, the higher the water content, thus affecting the measurement of the erythrocyte hemolysis inhibition rate. However, after being placed at room temperature for 7 days, the hemolysis inhibition rate of dihydromyricetin liposomes is not affected, and the hemolysis inhibition rate is still higher than 50%, while the hemolysis inhibition rate of dihydromyricetin aqueous solution decreases to varying degrees. Therefore, the dihydromyricetin nano-liposomes prepared by the present invention not only have good anti-stimulus ability, but also have the ability to protect the decline of the anti-stimulus ability of dihydromyricetin.

[0319] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0320] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A dihydromyricetin liposome, characterized in that, It is mainly made of components with the following mass percentages: (1) Oil phase: Phospholipid 1% - 6%, Cholesterol 0.06% - 1%, Ethanol 1% - 7.5%; (2) Aqueous phase: The emulsifier is behenyl alcohol polyether - 25.

2. The dihydromyricetin liposome according to claim 1, wherein The phospholipid is selected from one or a combination of several of S45 soy lecithin, S75 soy lecithin, 90G soy lecithin, and PC50 soy lecithin.

3. The dihydromyricetin liposome according to claim 1, characterized in that, It satisfies at least one of the following (1) - (2): (1) The mass ratio of the phospholipid to the cholesterol is (6 - 90):1; (2) The mass ratio of dihydromyricetin to the phospholipid is 1:(5 - 20).

4. The dihydromyricetin liposome according to claim 1, wherein The pH regulator is citric acid.

5. The dihydromyricetin liposome according to any one of claims 1 to 4, characterized in that, It satisfies at least one of the following (1) - (3): (1) The oil phase further includes an antioxidant; (2) The aqueous phase further includes a first preservative; (3) The dihydromyricetin liposome further includes a second preservative.

6. The dihydromyricetin liposome according to claim 5, characterized in that, It is mainly made of components with the following mass percentages: (1) Oil phase: (2) Aqueous phase: (3) Second preservative 0.05% - 1.05%.

7. The dihydromyricetin liposome according to claim 6, characterized in that, It satisfies at least one of the following (1) - (3): (1) The antioxidant is selected from one or a combination of several of pentaerythritol tetra(bis - tert - butylhydroxyhydrocinnamate), vitamin E, and vitamin E acetate; (2) The first preservative is pentylene glycol; (3) The second preservative is selected from one or a mixture of several of hydroxyacetophenone, pentylene glycol, and phenoxyethanol.

8. The dihydromyricetin liposome according to any one of claims 1 to 4, characterized in that, The particle size of the dihydromyricetin liposome is 30nm - 200nm, the PDI is 0.1 - 0.5, and the encapsulation efficiency is ≥60%.

9. A method for preparing the dihydromyricetin liposome according to any one of claims 1 to 8, characterized in that, It includes the following steps: Mix the phospholipid and cholesterol in ethanol to prepare an oil - phase mixture; Mix dihydromyricetin, emulsifier, glycerol, pH regulator with water to prepare an aqueous - phase mixture; Mix the oil - phase mixture with the aqueous - phase mixture and perform normal - pressure homogenization treatment and high - pressure homogenization treatment.

10. The preparation method of dihydromyricetin liposome according to claim 9, characterized in that, It satisfies at least one of the following (1) - (4): (1) The process parameters for preparing the oil - phase mixture include: temperature 40°C - 60°C; (2) The process parameters for preparing the aqueous - phase mixture include: pH 5 - 5.5, temperature 70°C - 75°C; (3) The process parameters for normal - pressure homogenization treatment include: mechanical stirring speed 300rpm - 700rpm, stirring time 20min - 40min, temperature 40°C - 60°C; (4) The process parameters for high - pressure homogenization treatment include: pressure 300bar - 900bar, number of times 1 - 9 times.

11. A cosmetic, characterized in that, It includes the dihydromyricetin liposome according to any one of claims 1 to 8.

12. The cosmetic according to claim 11, characterized in that, The cosmetic is selected from at least one of facial masks, eye creams, face creams, primer creams, essence, lotions, and skin softeners.

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