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 has a W / O / W multiple emulsion structure and comprises the following components in percentage by mass: (1) an inner water phase: 0.05-0.2% of dihydromyricetin and 10-20% of water; (2) an oil phase: 3%-9% of phospholipid and 0.03%-0.1% of cholesterol; (3) an external water phase: 0.5%-1.5% of an emulsifier, 1%-10% of glycerol and 59.2%-85.42% of water; and the emulsifier is selected from one or a mixture of two of glyceryl polyether-26 and 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 specifically relates to a dihydromyricetin liposome, a preparation method thereof, and an application thereof. Background Art

[0002] Dihydromyricetin (DMY) widely exists 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 in cold water and good solubility in hot water are the main reasons for its poor membrane permeability and low bioavailability.

[0003] To address the problem of poor solubility of dihydromyricetin, it has traditionally been mainly prepared into liposomes to improve solubility. However, the current dihydromyricetin liposomes have poor stability and will become turbid emulsions after being stored at about 25°C for a period of time, and their stability is even worse under high-temperature conditions. 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, and a sustained-release effect, which can be applied to cosmetics.

[0005] The technical solution is as follows:

[0006] A dihydromyricetin liposome having a W / O / W multiple emulsion structure is mainly made of the following components by mass percentage:

[0007] (1) Inner aqueous phase:

[0008] Dihydromyricetin 0.05% - 0.2%,

[0009] Water 10% - 20%;

[0010] (2) Oil phase:

[0011] Phospholipid 3% - 9%,

[0012] Cholesterol 0.03% - 0.1%;

[0013] (3) Outer aqueous phase:

[0014] Emulsifier 0.5% - 1.5%,

[0015] Glycerol 1% - 10%,

[0016] Water 59.2% - 85.42%;

[0017] The emulsifier is selected from one or a mixture of two of glycerol polyether-26 and behenyl alcohol polyether-25.

[0018] In one embodiment, the mass ratio of the oil phase to the inner aqueous phase is (0.2 - 1):1, and the mass ratio of the outer aqueous phase to the oil phase is (5 - 20):1.

[0019] In one embodiment, the phospholipid is selected from one or a combination of several of 80H hydrogenated lecithin, 90H hydrogenated lecithin, 90G soy lecithin, and PC50 soy lecithin.

[0020] In one embodiment, the mass ratio of the phospholipid to cholesterol is (6 - 90):1.

[0021] In one embodiment, the oil phase further includes an antioxidant.

[0022] In one embodiment, the outer aqueous phase further includes a first preservative.

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

[0024] In one embodiment, the dihydromyricetin liposome is mainly made of the following components by mass percentage:

[0025] (1) Inner aqueous phase:

[0026] Dihydromyricetin 0.05% - 0.2%,

[0027] Water 10% - 20%;

[0028] (2) Oil phase:

[0029] Phospholipid 3% - 9%,

[0030] Cholesterol 0.03% - 0.1%,

[0031] Antioxidant 0.03% - 0.1%;

[0032] (3) Outer aqueous phase:

[0033]

[0034] (4) Second preservative 0.05% - 1.05%.

[0035] 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.

[0036] In one embodiment, the first preservative is pentylene glycol.

[0037] In one embodiment, the second preservative is selected from one or a mixture of two of hydroxyacetophenone and pentylene glycol.

[0038] 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 ≥60%.

[0039] The present invention also provides a method for preparing the dihydromyricetin liposome as described above, and the technical solution is as follows:

[0040] A method for preparing the dihydromyricetin liposome as described above, comprising the following steps:

[0041] Mix the phospholipid and cholesterol in an alcohol solvent to prepare an oil-phase mixture.

[0042] Mix the dihydromyricetin with water to prepare a first aqueous-phase mixture.

[0043] Mix the emulsifier and glycerol in water to prepare a second aqueous-phase mixture.

[0044] Mix the oil-phase mixture with the first aqueous-phase mixture to prepare a primary emulsion, and the primary emulsion has a W / O structure.

[0045] Mix the primary emulsion with the second aqueous-phase mixture to prepare a first multiple emulsion, and the first multiple emulsion has a W / O / W structure.

[0046] Remove the alcohol solvent in the first multiple emulsion, and add water to make up the mass of the alcohol solvent to prepare a second multiple emulsion, and the second multiple emulsion has a W / O / W structure.

[0047] In one embodiment, the process parameters for preparing the first aqueous-phase mixture include: the pH of the system is 5.0 to 5.5, and the temperature is 70 °C to 75 °C.

[0048] In one embodiment, the process parameters for preparing the primary emulsion include: the emulsification speed is 5000 rpm to 9000 rpm, and the time is 5 min to 15 min.

[0049] In one embodiment, the process parameters for preparing the first multiple emulsion include: the emulsification speed is 3000 rpm to 5000 rpm, and the time is 5 min to 15 min.

[0050] In one embodiment, the alcohol solvent is ethanol.

[0051] In one embodiment, the process parameters for preparing the second multiple emulsion include: vacuum concentration, the temperature is 40 °C to 50 °C, and the time is 15 min to 30 min.

[0052] The present invention also provides the application of the dihydromyricetin liposome as described above. The technical solution is as follows:

[0053] A cosmetic product, characterized in that it comprises the dihydromyricetin liposome as described above.

[0054] In one embodiment, the cosmetic product is selected from at least one of a facial mask, eye cream, facial cream, primer, essence, lotion and skin softener.

[0055] The present invention has at least the following beneficial effects:

[0056] The dihydromyricetin liposome provided by the present invention has a W / O / W multiple emulsion structure, wherein the internal aqueous phase comprises dihydromyricetin and water in specific mass percentages, the oil phase comprises phospholipids and cholesterol in specific mass percentages, and the external aqueous phase comprises an emulsifier, glycerol and water in specific mass percentages, and the emulsifier is selected from one or a mixture of two of polyglyceryl-26 and behenyl alcohol polyether-25.

[0057] Through the specific W / O / W multiple emulsion structure, the present invention not only ensures good solubility of dihydromyricetin, but also significantly improves the stability of the dihydromyricetin liposome. 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, 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 and is gentle to the human body.

[0058] It has been confirmed that the dihydromyricetin liposome provided by the present invention has good solubility in water (good solubility in cold water and hot water), is gentle to the human body, has good skin physiological functions 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 field of cosmetics.

[0059] In addition, the preparation method of the dihydromyricetin liposome described in the present invention is simple, has high repeatability, short preparation time, no harmful substance residues, high encapsulation efficiency, high mechanization degree in the whole process, enables the product quality and process to have good reproducibility and stability, and is easy for industrial production. Description of the Drawings

[0060] Figure 1 It is a trend graph of the particle size of the dihydromyricetin nano-liposome prepared in Example 3 and Example 4 changing with the mass ratio of soybean lecithin to cholesterol; wherein, Figure 1 A is the trend graph of the particle size of the dihydromyricetin nano-liposome prepared in Example 3 changing with the mass ratio of soybean lecithin to cholesterol; Figure 1In Figure B, it is a trend graph showing the change of the particle size of dihydromyricetin nano-liposomes prepared in Example 4 with the change of the mass ratio of dihydromyricetin to soy lecithin.

[0061] Figure 2 They are trend graphs showing the change of the particle size of dihydromyricetin nano-liposomes prepared in Example 5, Example 6, and Example 7 with the change of different factors; among them, Figure 2 In Figure C, it is a trend graph showing the change of the particle size of dihydromyricetin nano-liposomes prepared in Example 5 with the change of the mass content of glycerol polyether-26; Figure 2 In Figure D, it is a trend graph showing the change of the particle size of dihydromyricetin nano-liposomes prepared in Example 6 with the change of the mass content of glycerol; Figure 2 In Figure E, it is a trend graph showing the change of the particle size of dihydromyricetin nano-liposomes prepared in Example 7 with the change of the stirring speed of the primary emulsion.

[0062] Figure 3 They are the results of the morphology and particle size of dihydromyricetin nano-liposomes prepared in Example 9; among them, Figure 3 In Figure A, it is the TEM of dihydromyricetin nano-liposomes prepared in Example 9; Figure 3 In Figure B, it is a trend graph of the particle size distribution of the sample after the TEM of dihydromyricetin nano-liposomes prepared in Example 9 is processed by Image and Origin.

[0063] Figure 4 It is a trend graph of the in vitro slow release amount of dihydromyricetin nano-liposomes prepared in Example 9.

[0064] Figure 5 They are trend graphs of the change of the particle size and PDI of dihydromyricetin nano-liposomes prepared in Example 9 under 7 different environments; among them, Figure 5 In Figure A, it is a trend graph of the change of the particle size and PDI of dihydromyricetin nano-liposomes prepared in Example 9 under normal temperature environment; Figure 5 In Figure B, it is a trend graph of the change of the particle size and PDI of dihydromyricetin nano-liposomes prepared in Example 9 under refrigerated environment; Figure 5 In Figure C, it is a trend graph of the change of the particle size and PDI of dihydromyricetin nano-liposomes prepared in Example 9 under frozen environment; Figure 5 In Figure D, it is a trend graph of the change of the particle size and PDI of dihydromyricetin nano-liposomes prepared in Example 9 under heating environment; Figure 5 In Figure E, it is a trend graph of the change of the particle size and PDI of dihydromyricetin nano-liposomes prepared in Example 9 under freeze-thaw environment; Figure 5 In Figure F, it is a trend graph of the change of the particle size and PDI of dihydromyricetin nano-liposomes prepared in Example 9 under light environment; Figure 5In Figure G, it is the trend chart of the particle size and PDI of dihydromyricetin nanoliposomes prepared in Example 9 under a light - shielded environment. Detailed implementation manners

[0065] The present invention will be further described in detail below with reference to specific embodiments. 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 so that the disclosure of the present invention can be more thoroughly and comprehensively understood.

[0066] 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 specification of the present invention herein 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.

[0067] In the present invention, "above" and "below" both include the number itself. For example, 1 below means ≥1.

[0068] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above - mentioned 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 integers, 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.

[0069] In the present invention, for the unit of a data range, if only the unit is attached after the right - hand endpoint, it means that the units of the left - hand endpoint and the right - hand endpoint are the same. For example, 800~850nm means that the units of the left - hand endpoint "800" and the right - hand endpoint "850" are both nm (nanometers).

[0070] In the present invention, for temperature parameters, unless otherwise specifically limited, it is allowed to be a constant - temperature treatment or a treatment within a certain temperature range. The constant - temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0071] In the present invention, the terms "first" and "second" are used for descriptive purposes only and should not 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 such feature. 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 specifically defined. In the description of the present application, the meaning of "several" is at least one, such as one, two, etc., unless otherwise specifically defined.

[0072] If there is no special instruction, all steps of the present invention can be carried out sequentially or randomly, 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, it is mentioned that the method may further include step (c), which 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.

[0073] 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.

[0074] 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. The scale in the drawings does not constitute a limitation to the present invention.

[0075] Dihydromyricetin (DMY) widely exists 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, especially its poor solubility in cold water and better solubility in hot water, is the main reason for its poor membrane permeability and bioavailability.

[0076] 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 turn into a turbid emulsion after being stored at about 25°C for a period of time, 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 turns into a white turbid emulsion after being stored at 25°C or 37°C for 30 days, showing poor stability.

[0077] 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 restricted its application to a certain extent. Therefore, improving the stability and color change problem of DMY and prolonging the slow release rate of DMY are a major prerequisite for its wide application.

[0078] To address the above problems, the present invention provides a dihydromyricetin liposome with good solubility, good stability, high encapsulation rate, and a slow release effect, which can be applied to cosmetics.

[0079] The technical solution is as follows:

[0080] A dihydromyricetin liposome having a W / O / W multiple emulsion structure is mainly made of the following components by mass percentage:

[0081] (1) Inner aqueous phase:

[0082] Dihydromyricetin 0.05% - 0.2%,

[0083] Water 10% - 20%;

[0084] (2) Oil phase:

[0085] Phospholipid 3% - 9%,

[0086] Cholesterol 0.03% - 0.1%;

[0087] (3) Outer aqueous phase:

[0088] Emulsifier 0.5% - 1.5%,

[0089] Glycerol 1% - 10%,

[0090] Water 59.2% - 85.42%;

[0091] The emulsifier is selected from one or a mixture of two of glycerol polyether-26 and behenyl alcohol polyether-25.

[0092] Through a specific W / O / W multiple emulsion structure, while ensuring good solubility of dihydromyricetin, the stability of dihydromyricetin liposomes is significantly improved. Moreover, under the combined action of the emulsifier and other raw materials of the present invention, the encapsulation efficiency of dihydromyricetin can be significantly increased, and the particle size and PDI of dihydromyricetin liposomes can be significantly reduced, and the slow release rate can be prolonged. In addition, the dihydromyricetin liposomes provided by the present invention are free of harmful substances and are gentle to the human body.

[0093] It has been confirmed that the dihydromyricetin liposomes provided by the present invention have good solubility in water, are gentle to the human body, have good skin physiological functions 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 darkness, can be stably stored for 30 days, slowly release dihydromyricetin to play a role, and improve its dose-effect relationship and bioavailability.

[0094] (1) In the present invention, the dihydromyricetin liposomes have a W / O / W multiple emulsion structure, wherein the inner aqueous phase comprises components in the following mass percentages: 0.05% - 0.2% of dihydromyricetin and 10% - 20% of water. Setting dihydromyricetin in the inner aqueous phase is beneficial to the stable preservation of dihydromyricetin in liposome vesicles and avoids direct leakage caused by vesicle rupture.

[0095] It can be understood that, by mass percentage, the inner aqueous phase of the dihydromyricetin liposomes contains 0.05% - 0.2% of dihydromyricetin, including but not limited to 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2%. Preferably, by mass percentage, the inner aqueous phase of the dihydromyricetin liposomes contains 0.1% of dihydromyricetin.

[0096] It can be understood that, by mass percentage, the inner aqueous phase of the dihydromyricetin liposomes contains 10% - 20% of water, including but not limited to 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. Preferably, by mass percentage, the inner aqueous phase of the dihydromyricetin liposomes contains 15% of water.

[0097] (2) In the present invention, the dihydromyricetin liposomes have a W / O / W multiple emulsion structure, wherein the oil phase comprises components in the following mass percentages: 3% - 9% of phospholipids and 0.03% - 0.1% of cholesterol. Setting phospholipids and cholesterol in the oil phase completely dissolves the liposome preparation membrane material in advance so as to form a closed vesicle structure when encountering water to encapsulate dihydromyricetin.

[0098] Understandably, in terms of mass percentage, the oil phase of the dihydromyricetin liposome contains 3% to 9% of phospholipids, including but not limited to 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5% or 9%. Preferably, in terms of mass percentage, the oil phase of the dihydromyricetin liposome contains 3% to 6% of phospholipids. Further preferably, in terms of mass percentage, the oil phase of the dihydromyricetin liposome contains 3% of phospholipids.

[0099] In one embodiment, the phospholipid is selected from one or a combination of several of 80H hydrogenated lecithin, 90H hydrogenated lecithin, 90G soy lecithin and PC50 soy lecithin. Preferably, the phospholipid is PC50 soy lecithin, and the prepared dihydromyricetin liposome is a clear and transparent solution, and the particle size (100 nm to 200 nm) and PDI (0.1 to 0.3) are significantly smaller than those of liposomes prepared with other phospholipids.

[0100] Understandably, in terms of mass percentage, the oil phase of the dihydromyricetin liposome contains 0.03% to 0.1% of cholesterol, including but not limited to 0.03%, 0.033%, 0.04%, 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09% or 0.1%.

[0101] In one embodiment, the mass ratio of the phospholipid to cholesterol is (6 to 90):1, which is beneficial to enhancing the stability of the vesicles and reducing the permeability of the lipid membrane to solutes.

[0102] 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 (30 to 90):1. Further preferably, the mass ratio of the lecithin to cholesterol is 90:1.

[0103] 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.

[0104] In one embodiment, the oil phase of the dihydromyricetin liposome contains 0.03% to 0.1% of an antioxidant, including but not limited to 0.03%, 0.033%, 0.04%, 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09% or 0.1%.

[0105] 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.

[0106] (3) In the present invention, the dihydromyricetin liposome has a W / O / W multiple emulsion structure, wherein the outer aqueous phase comprises components in the following mass percentages: 0.5% - 1.5% of an emulsifier, 1% - 10% of glycerol, and 59.2% - 85.42% of water. The emulsifier is selected from one or a mixture of two of glycerol polyether-26 and behenyl alcohol polyether-25. Setting the emulsifier and glycerol in the outer aqueous phase can enable the emulsifier to adhere well to the surface of the vesicles, enhancing the interaction force between the 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.

[0107] In the present invention, the emulsifier is selected from one or a mixture of two of glycerol polyether-26 and behenyl alcohol polyether-25. Compared with glyceryl tributyrate (GTCC), Tween 80, pentaerythritol distearate, and glyceryl stearate, which show floc floating and delamination phenomena under freeze-thaw or heating conditions, the present invention selects glycerol polyether-26 and behenyl alcohol polyether-25 as the emulsifier, which exhibits excellent stability under 7 different harsh environments including normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark.

[0108] It can be understood that, by mass percentage, the outer aqueous phase of the dihydromyricetin liposome contains 0.5% - 1.5% of an 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%, or 1.5%.

[0109] It can be understood that, by mass percentage, the outer 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 outer aqueous phase of the dihydromyricetin liposome contains 5% - 10% of glycerol.

[0110] In one embodiment, the outer aqueous phase of the dihydromyricetin liposome further contains a first preservative. Further, the mass content of the first preservative in the dihydromyricetin liposome is 0.01% - 1%. Further, the first preservative is pentylene glycol.

[0111] In one embodiment, in terms of mass percentage, the outer aqueous phase 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 outer aqueous phase of the dihydromyricetin liposome contains 1% of pentylene glycol.

[0112] Understandably, in terms of mass percentage, the outer aqueous phase of the dihydromyricetin liposome contains 59.2% to 85.42% of water, including but not limited to 59.2%, 60%, 61%, 62%, 63%, 64%, 65%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 85% or 85.42%. Preferably, in terms of mass percentage, the outer aqueous phase of the dihydromyricetin liposome contains 59.2% to 85.33% of water.

[0113] In one embodiment, the mass ratio of the oil phase to the inner aqueous phase is (0.2 - 1):1, and the mass ratio of the outer aqueous phase to the oil phase is (5 - 20):1, maintaining the vesicle space of the inner aqueous phase and encapsulating as much dihydromyricetin as possible. Understandably, the mass ratio of the oil phase to the inner aqueous phase includes but is not limited to 0.2:1, 0.3:1, 0.4:1, 0.407:1, 0.437:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1. Preferably, the mass ratio of the oil phase to the inner aqueous phase is (0.3 - 0.8):1; the mass ratio of the outer aqueous phase to the oil phase includes but is not limited to 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 10.68:1, 11:1, 11.71:1, 12:1, 12.63:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20:1.

[0114] In one embodiment, the dihydromyricetin liposome further includes a second preservative.

[0115] Furthermore, in terms of mass percentage, the dihydromyricetin liposome contains 0.05% to 1.05% of the 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%.

[0116] In one embodiment, the second preservative is selected from one or a mixture of two of p - hydroxyacetophenone and pentylene glycol. Further, the second preservative is a mixture of p - hydroxyacetophenone and pentylene glycol.

[0117] In one embodiment, based on the mass percentage, the dihydromyricetin liposome contains 0.01% - 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, based on the mass percentage, the dihydromyricetin liposome contains 0.05% of p - hydroxyacetophenone.

[0118] In one embodiment, based on the mass percentage of the dihydromyricetin liposome, the second preservative contains 0.01% - 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, based on the mass percentage of the dihydromyricetin liposome, the second preservative contains 1% of pentylene glycol.

[0119] In one embodiment, the dihydromyricetin liposome comprises the following components in mass percentage:

[0120] (1) Inner aqueous phase:

[0121] Dihydromyricetin 0.05% - 0.2%,

[0122] Water 10% - 20%;

[0123] (2) Oil phase:

[0124] Phospholipid 3% - 9%,

[0125] Cholesterol 0.03% - 0.1%,

[0126] Antioxidant 0.03% - 0.1%;

[0127] (3) Outer aqueous phase:

[0128]

[0129] (4) Second preservative 0.05% - 1.05%.

[0130] In one embodiment, the dihydromyricetin liposome comprises the following components in mass percentage: (1) Inner aqueous phase:

[0131] Dihydromyricetin 0.05% - 0.2%,

[0132] Water 10% - 20%;

[0133] (2) Oil phase:

[0134] Phospholipid 3% - 9%,

[0135] Cholesterol 0.033% - 0.1%,

[0136] Antioxidant 0.05%;

[0137] (3) Outer aqueous phase:

[0138]

[0139] (4) Preservative:

[0140] p - Hydroxyacetophenone 0.05%,

[0141] Pentylene glycol 1%.

[0142] In one embodiment, the dihydromyricetin liposome comprises the following components in mass percentage: (1) Inner aqueous phase:

[0143] Dihydromyricetin 0.1%,

[0144] Water 15%;

[0145] (2) Oil phase:

[0146] Phospholipid 3% - 9%,

[0147] Cholesterol 0.033% - 0.1%,

[0148] Antioxidant 0.05%;

[0149] (3) Outer aqueous phase:

[0150]

[0151] (4) Preservative:

[0152] p - Hydroxyacetophenone 0.05%,

[0153] Pentylene glycol 1%.

[0154] In one embodiment, the particle size of the dihydromyricetin liposome is 100nm - 200nm, the PDI is 0.1 - 0.3, and the encapsulation efficiency is ≥60%. Further, the particle size of the dihydromyricetin liposome is 100nm - 200nm, the PDI is 0.1 - 0.3, and the encapsulation efficiency is ≥75%.

[0155] Traditional methods for preparing dihydromyricetin liposomes include the thin film ultrasonic method, the ethanol injection - 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, non - uniform particle size, and residual toxic reagents, making 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 rate in traditional technologies is mostly below 60%, and hardly reaches 75%. Moreover, the preparation time is significantly increased, and the product stability is also poor.

[0156] In view of the above problems, the present invention provides a method for preparing dihydromyricetin liposomes, which has high repeatability, short preparation time, no harmful substance residues, and high encapsulation rate. By encapsulating dihydromyricetin with this method, it can extend the release rate of dihydromyricetin, improve its solubility (especially water solubility), encapsulation efficiency, and stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze - thawing, light, and dark.

[0157] The technical solution is as follows:

[0158] A method for preparing dihydromyricetin liposomes as described above, comprising the following steps:

[0159] Mix the phospholipids and cholesterol in an alcohol solvent to prepare an oil - phase mixture.

[0160] Mix the dihydromyricetin with water to prepare a first aqueous - phase mixture.

[0161] Mix the emulsifier and glycerol in water to prepare a second aqueous - phase mixture.

[0162] Mix the oil - phase mixture with the first aqueous - phase mixture to prepare a primary emulsion, and the primary emulsion has a W / O structure.

[0163] Mix the primary emulsion with the second aqueous - phase mixture to prepare a first multiple emulsion, and the first multiple emulsion has a W / O / W structure.

[0164] Remove the alcohol solvent from the first multiple emulsion, and add water to make up the mass of the alcohol solvent to prepare a second multiple emulsion, and the second multiple emulsion has a W / O / W structure.

[0165] In one embodiment, the alcohol solvent is ethanol. The present invention uses the multiple - emulsion method to prepare dihydromyricetin liposomes. The preparation method is simple, fast, and the process is simple, suitable for large - scale industrial production.

[0166] Further, the mass percentage of ethanol in the dihydromyricetin liposome is 20% to 30%, including but not limited to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%. Preferably, the mass percentage of ethanol in the dihydromyricetin liposome is 24%.

[0167] In one embodiment, the process parameters for preparing the first aqueous phase mixture include: the pH of the system is 5.0 to 5.5, and the temperature is 70°C to 75°C.

[0168] In one embodiment, the dihydromyricetin is mixed with water to prepare a first aqueous phase mixture; the following steps are included:

[0169] Using a 0.1 mol / L citric acid solution in water to adjust the pH to 5.0 to 5.5, heating to 70°C to 75°C and dissolving dihydromyricetin for 10 min to obtain the first aqueous phase mixture.

[0170] In one embodiment, the process parameters for preparing the primary emulsion include: the emulsification speed is 5000 rpm to 9000 rpm, and the time is 5 min to 15 min. It can be understood that in the step of preparing the primary emulsion, the emulsification speed includes but is not limited to 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm or 9000 rpm; the time includes but is not limited to 5 min, 6 min, 8 min, 10 min, 12 min or 15 min. Preferably, in the step of preparing the primary emulsion, the emulsification speed is 7000 rpm and the emulsification time is 10 min.

[0171] In one embodiment, mixing the oil phase mixture with the first aqueous phase mixture to prepare a primary emulsion with a W / O structure includes the following steps:

[0172] Adding the first aqueous phase mixture dropwise to the oil phase mixture and emulsifying for 5 min to 15 min at a speed of 5000 rpm to 9000 rpm to form a W / O system and obtain the primary emulsion.

[0173] In one embodiment, the process parameters for preparing the first multiple emulsion include: the emulsification speed is 3000 rpm to 5000 rpm, and the time is 5 min to 15 min. It can be understood that in the step of preparing the first multiple emulsion, the emulsification speed includes but is not limited to 3000 rpm, 4000 rpm or 5000 rpm; the time includes but is not limited to 5 min, 6 min, 8 min, 10 min, 12 min or 15 min. Preferably, in the step of preparing the first multiple emulsion, the emulsification speed is 5000 rpm and the emulsification time is 10 min.

[0174] In one embodiment, mixing the colostrum with the second aqueous phase mixture to prepare a first multiple emulsion having a W / O / W structure includes the following steps:

[0175] Adding the colostrum dropwise to the second aqueous phase mixture and emulsifying it for 5 min to 15 min under the condition that the rotation speed is 3000 rpm to 5000 rpm to form a W / O / W system, thereby obtaining the first multiple emulsion.

[0176] In one embodiment, the process parameters for preparing the second multiple emulsion include: reduced pressure concentration, at a temperature of 40°C to 50°C, and for a time of 15 min to 30 min. It can be understood that in the step of preparing the second multiple emulsion, the temperature includes but is not limited to 40°C, 45°C or 50°C; the time includes but is not limited to 15 min, 16 min, 18 min, 20 min, 22 min, 25 min or 30 min.

[0177] In one embodiment, removing the alcohol solvent from the first multiple emulsion and adding water to make up for the mass of the alcohol solvent to prepare a second multiple emulsion having a W / O / W structure includes the following steps:

[0178] Rotating and evaporating the first multiple emulsion with a rotary evaporator at 40°C to 50°C for 15 min to 30 min to remove absolute ethanol, and making up the weight difference before and after rotary evaporation with pure water.

[0179] It can be understood that for the dihydromyricetin liposomes with a preservative (such as pentylene glycol) in the outer aqueous phase, in the step of preparing the second aqueous phase mixture, it further includes the step of adding the preservative and mixing it evenly with the emulsifier, glycerol and water.

[0180] It can be understood that for the dihydromyricetin liposomes which contain (1) inner aqueous phase, (2) oil phase, (3) outer aqueous phase and also contain (4) preservative, after the step of preparing the second multiple emulsion, it further includes the step of adding the second multiple emulsion and mixing it evenly with the preservative.

[0181] The present invention also provides the application of the dihydromyricetin liposomes as described above. The technical solution is as follows:

[0182] A cosmetic, characterized in that it includes the dihydromyricetin liposomes as described above.

[0183] 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.

[0184] The following is the specific embodiment part.

[0185] Unless otherwise specified, all raw materials are commercially available products.

[0186] In each embodiment, the antioxidant is selected from pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate).

[0187] Example 1

[0188] Prepare samples according to the ratios in Table 1. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 70 °C, stir and dissolve dihydromyricetin for 10 min to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 50 °C and dissolve glycerol, glycerol polyether-26, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40 °C - 50 °C for 15 min - 30 min to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add preservatives and mix well to obtain the samples.

[0189] Among them, when 80H hydrogenated lecithin and 90H hydrogenated lecithin are used to prepare the multiple emulsion, the samples are viscous and in the form of a creamy solid, and it is impossible to remove absolute ethanol by rotary evaporation. After rotary evaporation, 90G soybean lecithin gives a milky white solution with a particle size as high as 1665 nm and a PDI as high as 0.8796. While PC50 soybean lecithin prepares a clear and transparent solution, and the particle size and PDI are significantly smaller. Therefore, PC50 soybean lecithin is preferably used as the raw material of lecithin in the system.

[0190] Table 1

[0191]

[0192] Example 2

[0193] Prepare samples according to the ratios in Table 1. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 70 °C, stir and dissolve dihydromyricetin for 10 min to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 50 °C and dissolve glycerol, glycerol polyether-26, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40 °C - 50 °C for 15 min - 30 min to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add preservatives and mix well to obtain the samples.

[0194] All the prepared samples were placed in a heating environment (45°C) and a freeze-thaw environment (-15°C and 45°C alternating) for 7 days. The appearance of the samples was observed, and the particle size was measured simultaneously. The results showed that except for glycerol polyether-26, floccules floating and stratification occurred in other emulsifiers under the heating or freeze-thaw environment. Therefore, glycerol polyether-26 was selected as the raw material of the emulsifier in the system.

[0195] Table 2

[0196]

[0197] Example 3

[0198] Preparation experiments with different mass ratios of soybean lecithin to cholesterol

[0199] Samples were prepared according to the ratios in Table 3. Lecithin, cholesterol, and antioxidant were dissolved in absolute ethanol to obtain the oil phase. The pH of the water in the inner aqueous phase was adjusted to 5 - 5.5 with 0.1 mol / L citric acid solution, heated to 70°C, and dihydromyricetin was stirred and dissolved for 10 min to obtain the inner aqueous phase. Then, the pH of the water in the outer aqueous phase was adjusted to 5 - 5.5 with 0.1 mol / L citric acid solution, and glycerol, emulsifier, and pentylene glycol were dissolved by heating to 50°C to obtain the outer aqueous phase. The inner aqueous phase was added to the oil phase and emulsified at 7000 rpm for 10 min to form the primary emulsion, and then the primary emulsion was added to the outer aqueous phase and emulsified at 5000 rpm for 10 min to form the double emulsion. The double emulsion was rotary evaporated at 40°C - 50°C for 15 min - 30 min using a rotary evaporator to remove absolute ethanol. After making up the weight difference before and after rotary evaporation with pure water, preservatives were added and mixed evenly to obtain the samples. Then, 15 μL of the prepared dihydromyricetin nanoliposomes were taken respectively, added with pure water to 3000 μL and mixed evenly, and the particle size of the samples was measured with a ZSU 3200 nanoparticle size analyzer. The results are as shown in Figure 1 Figure A.

[0200] As can be seen from Figure 1 Figure A, as the mass ratio of soybean lecithin to cholesterol increases, the particle size continuously decreases, and the decreasing trend of the particle size gradually decreases starting from 30:1. Generally, liposomes with smaller particle sizes and PDI are more uniform and stable. The mass ratio of soybean lecithin to cholesterol is preferably (30 - 90):1.

[0201] Table 3

[0202]

[0203] Example 4

[0204] Preparation experiments with different mass ratios of dihydromyricetin to soybean lecithin

[0205] 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 in the inner aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 70 °C, and stir for 10 min to dissolve dihydromyricetin to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 50 °C, and dissolve glycerol, emulsifier, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40 - 50 °C for 15 - 30 min with a rotary evaporator to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add preservative and mix evenly to obtain the sample. Then, respectively take 15 μL of the prepared dihydromyricetin nanoliposomes, add pure water to 3000 μL and mix evenly, and measure the particle size of the sample with a ZSU 3200 nanoparticle size analyzer. The results are as shown in Figure 1 Figure B in the appendix.

[0206] As can be seen Figure 1 from Figure B in the appendix, the particle size gradually decreases with the increase of the drug-lipid ratio, and there is no significant difference between the particle sizes of the drug-lipid ratios of 1:90 and 1:60. Considering the preparation cost, the mass ratio of dihydromyricetin to soybean lecithin is preferably 1:(30 - 60).

[0207] Table 4

[0208]

[0209] Example 5

[0210] Preparation experiments with different contents of glycerol polyether - 26

[0211] 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 in the inner aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 70 °C, and stir for 10 min to dissolve dihydromyricetin to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 50 °C, and dissolve glycerol, emulsifier, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40 °C - 50 °C for 15 min - 30 min with a rotary evaporator to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add preservative and mix evenly to obtain the sample. Then, respectively take 15 μL of the prepared dihydromyricetin nanoliposomes, add pure water to 3000 μL and mix evenly, and measure the particle size of the sample with a ZSU 3200 nanoparticle size analyzer. The results are as shown inFigure 2 As shown in C.

[0212] From Figure 2 As can be seen from C, as the mass content of glycerol polyether-26 increases, the particle size continuously decreases, and the decreasing trend becomes lower and lower. In order to consider its impact on skin irritation and preparation cost, while ensuring good emulsification effect and stability performance, the optimized mass content of glycerol polyether-26 in the liposome with the mass ratio of dihydromyricetin to soybean lecithin is 0.5% - 1.5%.

[0213] Table 5

[0214]

[0215]

[0216] Example 6

[0217] Preparation experiments with different glycerol contents

[0218] 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 inner aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 70°C, and stir for 10 min to dissolve dihydromyricetin to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, and heat to 50°C to dissolve glycerol, emulsifier, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40°C - 50°C for 15 min - 30 min using a rotary evaporator to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add preservatives and mix well to obtain the samples. Then, respectively take 15 μL of the prepared dihydromyricetin nanoliposomes, add pure water to 3000 μL and mix evenly, and measure the particle size of the samples with a ZSU 3200 nanoparticle size analyzer. The results are as shown in Figure 2 As shown in D.

[0219] From Figure 2 As can be seen from D, as the glycerol content increases, the particle size gradually decreases. However, after the glycerol content is added to 5%, the change trend of the particle size is not significant. The preferred glycerol content is 5% - 10%.

[0220] Table 6

[0221]

[0222]

[0223] Example 7

[0224] Preparation experiment with different initial milk stirring speeds

[0225] Prepare samples according to the ratio in Table 7. Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 70 °C, and stir and dissolve dihydromyricetin for 10 min to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 50 °C, and dissolve glycerol, emulsifier, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify for 10 min at the stirring speed in Table 7 to form the initial milk. Then add the initial milk to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate and evaporate the multiple emulsion with a rotary evaporator at 40 °C - 50 °C for 15 min - 30 min to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add preservatives and mix evenly to obtain the samples. Then, respectively take 15 μL of the prepared dihydromyricetin nano-liposomes, add pure water to 3000 μL and mix evenly, and measure the particle size of the samples with a ZSU 3200 nanoparticle size analyzer. The results are as shown in Figure 2 Figure E in the appendix.

[0226] From Figure 2 Figure E, it can be seen that the particle size first decreases and then increases with the increase of the rotation speed. Maybe the rotation speed reaches a critical value, resulting in the instability and rupture of the vesicles. Moreover, the change trend of the particle size is not significant, indicating that this single factor has a very small impact on the particle size. The preferred initial milk stirring speed is 7000 rpm.

[0227] Table 7

[0228]

[0229]

[0230] Example 8

[0231] Orthogonal experiment

[0232] Soybean lecithin (3%, 4%, 6%), cholesterol (0.033% - 0.2%), pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate) 0.05%, and absolute ethanol 24% were completely dissolved by mass percentage to form the oil phase; the pH of 15% water was adjusted to 5 - 5.5 with 0.1 mol / L citric acid solution, and after heating to 70 °C, 0.1% dihydromyricetin was dissolved to form the inner aqueous phase; the pH of (added to 100%) water was adjusted to 5 - 5.5 with 0.1 mol / L citric acid solution, and after heating to 50 °C, glycerol polyether-26 (0.5%, 1%, 1.5%), glycerol (0%, 5%, 10%), and pentylene glycol 1% were dissolved to form the outer aqueous phase; the inner aqueous phase was added dropwise to the oil phase and homogenized at 7000 rpm for 10 min to obtain the primary emulsion; the primary emulsion was added dropwise to the outer aqueous phase and homogenized at 5000 rpm for 10 min to obtain the multiple emulsion; finally, the multiple emulsion was poured into an eggplant-shaped flask and weighed as W1, and after rotary evaporation at 50 °C for 15 - 30 min using a rotary evaporator and weighed as W2, the weight difference was supplemented with water, and then 1 g of pentylene glycol and 0.05 g of p-hydroxyacetophenone were added and mixed evenly. Then, 15 μL of the prepared dihydromyricetin nanoliposomes were taken respectively, added with pure water to 3000 μL and mixed evenly, and the particle size of the sample was measured with a ZSU 3200 nanoparticle size analyzer. The results are shown in Table 1 below.

[0233] Table 1 Results of Process Optimization Data

[0234]

[0235] 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.

[0236] Example 9

[0237] Optimization Process Experiment 1

[0238] Dissolve 6% soy lecithin, 0.067% cholesterol, 0.05% pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate), and 24% absolute ethanol by mass percentage to form the oil phase; adjust the pH of 15% water to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 70 °C, and dissolve 0.1% dihydromyricetin to form the inner aqueous phase; adjust the pH of (added to 100%) water to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 50 °C, and dissolve 0.5% glycerol polyether-26, 10% glycerol, and 1% pentylene glycol to form the outer aqueous phase; drop the inner aqueous phase into the oil phase and homogenize at 7000 rpm for 10 min to obtain the primary emulsion; drop the primary emulsion into the outer aqueous phase and homogenize at 5000 rpm for 10 min to obtain the multiple emulsion; finally, pour the multiple emulsion into an eggplant-shaped flask, weigh it as W1, rotate and evaporate it at 50 °C for 30 min with a rotary evaporator, then weigh it as W2, supplement the weight difference with water, and add 1 g pentylene glycol and 0.05 g p-hydroxyacetophenone and mix well.

[0239] Example 10

[0240] Optimization Process Experiment 2

[0241] Dissolve 3% soy lecithin, 0.033% cholesterol, 0.05% pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate), and 24% absolute ethanol by mass percentage to form the oil phase; adjust the pH of 15% water to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 70 °C, and dissolve 0.1% dihydromyricetin to form the inner aqueous phase; adjust the pH of (added to 100%) water to 5 - 5.5 with 0.1 mol / L citric acid solution, heat to 50 °C, and dissolve 1.5% glycerol polyether-26, 10% glycerol, and 1% pentylene glycol to form the outer aqueous phase; drop the inner aqueous phase into the oil phase and homogenize at 7000 rpm for 10 min to obtain the primary emulsion; drop the primary emulsion into the outer aqueous phase and homogenize at 5000 rpm for 10 min to obtain the multiple emulsion; finally, pour the multiple emulsion into an eggplant-shaped flask, weigh it as W1, rotate and evaporate it at 50 °C for 30 min with a rotary evaporator, then weigh it as W2, supplement the weight difference with water, and add 1 g pentylene glycol and 0.05 g p-hydroxyacetophenone and mix well.

[0242] Test

[0243] (1) Perform encapsulation efficiency tests on the dihydromyricetin nanoliposomes prepared in Examples 9 and 10.

[0244] Accurately weigh 0.002 g of dihydromyricetin standard product, dissolve it in a 10 mL volumetric flask with 75% ethanol, and prepare a reference substance solution with a concentration of 200 μg / mL. Respectively take 0 μL, 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, shake well, and prepare a series of solutions with concentrations of 0 μg / mL, 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, measure the peak area of DMY at 290 nm, and draw a standard curve y = 25.3445x - 25.8835, R2 = 0.9994. The encapsulation efficiency of dihydromyricetin nano-liposomes was determined by the ultrafiltration centrifugation method. 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 then 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%.

[0245] The results showed that the encapsulation efficiency of Example 9 was (79.15 ± 0.88)%, and the encapsulation efficiency of Example 10 was (74.60 ± 1.04)%. Among them, the encapsulation efficiency of Example 9 was higher. Therefore, Example 9 was selected as the final optimized process formula for TEM, transdermal absorption, and stability investigation experiments.

[0246] (2) Particle size measurement was carried out on the dihydromyricetin nano-liposomes prepared in Example 9.

[0247] Respectively take 15 μL of the dihydromyricetin nano-liposomes prepared in Example 9, add pure water to 3000 μL, mix evenly, and measure the particle size of the sample with a ZSU 3200 nano particle size analyzer. The results showed that the particle size of the sample prepared in Example 9 was 161.2 nm, the PDI was 0.1583, and the Zeta potential was -32.86 mV.

[0248] (3) TEM detection was carried out on the dihydromyricetin nano-liposomes prepared in Example 9.

[0249] Weigh the sample prepared in Example 9 and 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 dried, 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, place it on the filter paper with the film side up to dry, and observe and take pictures with a transmission electron microscope. The results are as Figure 3 shown in A in the figure. The particles are spherical-like and the distribution is relatively uniform. The particle size distribution trend diagram of the dihydromyricetin nanoliposomes prepared in Example 9 after being processed by Image and Origin is shown in Figure 3 B in the figure.

[0250] (4) Conduct an in vitro slow release test on the dihydromyricetin nanoliposomes prepared in Example 9.

[0251] For the in vitro release test of the dihydromyricetin nanoliposomes, the dihydromyricetin in the release medium is measured by dialysis with PBS containing 30% ethanol. Put 5 mL of the sample into a dialysis bag (cut-off molecular weight: 6000 - 8000), and place the dialysis bag into a 250 mL beaker containing 200 mL of the release medium. The beaker is incubated in a 37 °C shaking water bath thermostat and shaken at 100 rpm by a magnetic stirrer. At predetermined time intervals (1, 2, 4, 6, 8, 12, 24, 48, 72, 96 hours after the start of the culture), take out all the release medium (3 mL) and replace it with the same volume of fresh release medium (3 mL). Determine the release amount of DMY by HPLC and plot the slow release curve.

[0252]

[0253] Qi: Cumulative release amount at the i-th sampling time point, %;

[0254] V: Volume of the release medium, mL;

[0255] ρ n : Concentration of dihydromyricetin in the release medium during the n-th sampling process, μg / mL;

[0256] ρ i : Concentration of dihydromyricetin in the release medium during the i-th sampling process, μg / mL;

[0257] Vi: Sampling volume at the i-th time, mL;

[0258] m: Mass of dihydromyricetin in the nanoliposome sample, μg;

[0259] The slow release results are as Figure 4As shown, the release amount of dihydromyricetin solution reached the highest at 8 h, and dihydromyricetin was no longer released after 8 h. However, the release amount of dihydromyricetin nano-liposomes did not reach the highest at 8 h and continued to release dihydromyricetin until 12 h, indicating that dihydromyricetin was encapsulated in the liposomes, which helped the slow release of dihydromyricetin. After the highest release amount, the two samples in the figure showed a downward trend, indicating that after dihydromyricetin was released, it might be in a free state and the storage time was too long, resulting in the instability of dihydromyricetin and ultimately the decrease in the content of dihydromyricetin.

[0260] (5) The stability of the dihydromyricetin nano-liposomes prepared in Example 9 was investigated.

[0261] The samples prepared in Example 9 were respectively filled into 7 vials and sealed, and stored in normal temperature, refrigeration (4 °C), freezing (-15 °C), heating (45 °C), freeze-thaw (-15 °C and 45 °C alternating), dark, and light (28 °C) environments. The particle size and PDI were tested on the 7th, 14th, and 30th days, and the results are as Figure 5 shown. The changes in particle size and PDI were small. Among them, when stored in a hot environment ( Figure 5 in D) for 30 d, the particle size increased, and after examining the appearance of the sample, demulsification occurred on the 34th day, and the sample changed from a translucent state to a turbid state. This might be because soy lecithin accelerated its oxidation and decomposition under the heating state, resulting in the demulsification of the sample. Therefore, the dihydromyricetin nano-liposomes prepared in the present invention can be stably stored for 30 d in normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark environments.

[0262] 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 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 described in this specification.

[0263] The above-described embodiments only 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 still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A dihydromyricetin liposome, characterized in that, It has a W / O / W multiple emulsion structure and is mainly made of components with the following mass percentages: (1) Inner aqueous phase: Dihydromyricetin 0.05% - 0.2%, Water 10% - 20%; (2) Oil phase: Phospholipid 3% - 9%, Cholesterol 0.03% - 0.1%; (3) Outer aqueous phase: Emulsifier 0.5% - 1.5%, Glycerol 1% - 10%, Water 59.2% - 85.42%; The emulsifier is selected from one or a mixture of two of glycerol polyether - 26 and behenyl alcohol polyether - 25.

2. The dihydromyricetin liposome according to claim 1, characterized in that, The mass ratio of the oil phase to the inner aqueous phase is (0.2 - 1):1, and the mass ratio of the outer aqueous phase to the oil phase is (5 - 20):

1.

3. The dihydromyricetin liposome according to claim 1, wherein The phospholipid is selected from one or a combination of several of 80H hydrogenated lecithin, 90H hydrogenated lecithin, 90G soy lecithin, and PC50 soy lecithin.

4. The dihydromyricetin liposome according to claim 1, characterized in that, The mass ratio of the phospholipid to cholesterol is (6 - 90):

1.

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

6. The dihydromyricetin liposome according to claim 5, wherein It is mainly made of components with the following mass percentages: (1) Inner aqueous phase: Dihydromyricetin 0.05% - 0.2%, Water 10% - 20%; (2) Oil phase: Phospholipid 3% - 9%, Cholesterol 0.03% - 0.1%, Antioxidant 0.03% - 0.1%; (3) Outer aqueous phase: (4) Second preservative 0.05% - 1.05%.

7. The dihydromyricetin liposome according to claim 6, wherein Meet 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 two of p - hydroxyacetophenone and pentylene glycol.

8. The dihydromyricetin liposome according to any one of claims 1 to 4, characterized in that, The particle size of the dihydromyricetin liposome is 100nm - 200nm, the PDI is 0.1 - 0.3, and the encapsulation efficiency ≥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 an alcohol solvent to prepare an oil - phase mixture; Mix the dihydromyricetin with water to prepare a first aqueous - phase mixture; Mix the emulsifier and glycerol in water to prepare a second aqueous - phase mixture; Mix the oil - phase mixture with the first aqueous - phase mixture to prepare a primary emulsion, and the primary emulsion has a W / O structure; Mix the primary emulsion with the second aqueous - phase mixture to prepare a first multiple emulsion, and the first multiple emulsion has a W / O / W structure; Remove the alcohol solvent from the first multiple emulsion and add water to make up for the mass of the alcohol solvent to prepare a second multiple emulsion, and the second multiple emulsion has a W / O / W structure.

10. The preparation method of the dihydromyricetin liposome according to claim 9, wherein Meet at least one of the following (7) - (11): (7) The process parameters for preparing the first aqueous - phase mixture include: the pH of the system is 5.0 - 5.5, and the temperature is 70°C - 75°C; (7) The process parameters for preparing the primary emulsion include: the emulsification speed is 5000rpm - 9000rpm, and the time is 5min - 15min; (8) The process parameters for preparing the first multiple emulsion include: the emulsification rotation speed is 3000 rpm to 5000 rpm, and the time is 5 min to 15 min; (9) The alcohol solvent is ethanol; (10) The process parameters for preparing the second multiple emulsion include: vacuum concentration, the temperature is 40 °C to 50 °C, and the time is 15 min to 30 min.

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 a facial mask, eye cream, facial cream, primer, essence, lotion and skin softener.

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