Oil-in-water nanoemulsion with high drug loading and preparation method and application thereof

Abstract

The present invention relates to a water-in-oil nanoemulsion with high drug loading, and a preparation method and application thereof. The water-in-oil nanoemulsion is prepared by dropping an aqueous phase solution containing tocosolan into an oil phase solution; the aqueous phase solution is composed of tocosolan, a co-surfactant, a hydrophilic active substance, a preservative and water; the oil phase solution is composed of liquid oil and surfactant; the mass percentage of each component in the water-in-oil nanoemulsion is: liquid oil 50-64%, surfactant 10-17%, tocosolan 5-8, surfactant 5-12%, hydrophilic active substance 0.1-6%, preservative 0.2-0.6%, water 8-15%. The water-in-oil nanoemulsion has the advantages of high drug loading, good skin permeability, high retention in skin cells, high bioavailability, good stability, high safety, etc., and can be used in pure oil cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics and relates to nanoemulsion, in particular to a water-in-oil nanoemulsion with high drug loading, and a preparation method and application thereof. Background Art

[0002] For cosmetic active ingredients to have an effect on the human body, two conditions must be met at the same time: one is that they have specific efficacy and activity, and the other is that they can be enriched in the skin target cells to an effective concentration. Active ingredients enter the active epidermis through the stratum corneum and diffuse into the dermis, which is the main way for active ingredients to be absorbed through the skin. The stratum corneum is composed of inanimate keratinocytes and non-polar intercellular lipids. Active ingredients with strong water solubility usually have high polarity, and polar molecules have difficulty passing through the stratum corneum, showing poor permeability.

[0003] To solve the above problems, technicians in the cosmetics field have proposed a variety of solutions, such as preparing water-soluble functional ingredients into liposomes, bicontinuous phase nanoemulsions and other dosage forms to improve their skin permeability. However, with the concept of "using oil to nourish the skin", pure oil system products have been favored in the field of skin care. Liposomes and bicontinuous phase nanoemulsions are mainly used in O / W cosmetic products and cannot be used in pure oil systems. Therefore, these two types of dosage forms have certain limitations and cannot meet the development needs of pure oil system products.

[0004] Chinese invention patent document CN110882178A discloses a water-in-oil system whitening polypeptide composition, and Chinese invention patent document CN117224420A discloses an oil-soluble lip plumping complex peptide and its preparation method. In both patent documents, W / O compositions are prepared by non-ionic surfactants. The compositions can be used in pure oil system products and have a transparent appearance, which can meet some market needs. However, the proportion of internal water phase is relatively low, about 5%. This relatively low proportion of internal water phase is difficult to load a high content of hydrophilic active substances, resulting in a low amount of active substance added in the combination, which still has great limitations. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a water-in-oil nanoemulsion with a high drug loading, which can not only effectively improve the skin permeability of active substances, but also can be applied to pure oil system products.

[0006] In order to achieve the above-mentioned object of the invention, the present invention includes the following technical solutions.

[0007] In a first aspect, the present invention provides a water-in-oil nanoemulsion with a high drug loading, which is prepared by dropping an aqueous solution containing tocosolan into an oily solution;

[0008] The aqueous phase solution is composed of tocopherol, a co-surfactant, a hydrophilic active substance, a preservative and water; the oil phase solution is composed of liquid oil and a surfactant;

[0009] The mass percentage of each component in the water-in-oil nanoemulsion is:

[0010] Liquid oil 50~64%

[0011] Surfactant 10~17%

[0012] Tocosolan 5~8%

[0013] Co-surfactant 5~12%

[0014] Hydrophilic active matter 0.1~6%

[0015] Preservatives 0.2~0.6%

[0016] Water 8~15%;

[0017] Wherein, the hydrophilic active substance is a hydrophilic functional ingredient used in cosmetics; for example, it can be one or more of carnosine, decarboxylated carnosine, nonapeptide-1, hexapeptide-2, acetyl hexapeptide-8, arginine / lysine polypeptide, dipeptide diaminobutyryl benzylamide diacetate, acetyl tetrapeptide-5, tripeptide-1, oligopeptide-1, biotin tripeptide-1, myristoyl pentapeptide-17, hexapeptide-9, pentapeptide-3, acetyl hexapeptide-1, glutathione, hydroxypropyl tetrahydropyrantriol (bosine), tetrahydromethylpyrimidine carboxylic acid (ectoine), ergothioneine and dipotassium glycyrrhizinate;

[0018] The surfactant is selected from one or more of sorbitan monooleate, lecithin, hydrogenated lecithin and polyglycerol-3 polyricinoleate.

[0019] In a second aspect, the present invention also provides the use of the water-in-oil nanoemulsion in the preparation of cosmetics (especially pure oil cosmetics, such as essence oil, lipstick and cleansing oil, etc.).

[0020] In a third aspect, the present invention also provides a method for preparing the water-in-oil nanoemulsion, comprising the following steps:

[0021] S1. The liquid oil and surfactant are mixed, heated until completely dissolved, and cooled to obtain an oil phase;

[0022] S2. The tocosolan and co-surfactant are mixed, heated until completely dissolved, and cooled to obtain the tocosolan phase;

[0023] S3. The hydrophilic active substance and preservative are added to water, heated until completely dissolved, cooled to obtain an aqueous phase;

[0024] S4. The tocosolan phase prepared in S2 was added to the aqueous phase prepared in S3 and mixed to obtain an aqueous solution;

[0025] S5. dropping the aqueous solution prepared in S4 into the oil phase prepared in S1 and stirring for 5 min to 20 min to obtain the water-in-oil nanoemulsion; or,

[0026] The aqueous phase solution prepared in S4 is dropped into the oil phase prepared in S1, stirred for 5 min to 20 min, and then sheared and emulsified at a rotation speed of 5000 rpm to 7000 rpm for 5 min to 10 min to obtain the water-in-oil nanoemulsion.

[0027] The present invention has the following beneficial effects:

[0028] The invention modifies the aqueous phase with tocosolan, then drips the aqueous phase solution containing tocosolan into the oil phase, and combines with specific surfactants and other components to prepare an oil-in-water nanoemulsion with high drug loading, good skin permeability, good stability and high safety.

[0029] The invention can significantly increase the drug loading of the hydrophilic compound in the W / O nanoemulsion by adding tocosolan in a specific proportion, so that the hydrophilic compound can be dissolved in various vegetable oils, synthetic oils and mineral oils at a relatively high concentration, and can be directly applied to a variety of pure oil cosmetics for "skin care with oil". After being dissolved, the appearance is clear and transparent, thus solving the problem of poor oil solubility compatibility of high-concentration hydrophilic compounds.

[0030] The tocorsolan-modified water-in-oil nanoemulsion prepared by the present invention not only significantly increases the content of water-soluble active ingredients in the water-in-oil nanoemulsion, but also significantly increases the skin permeability of the water-soluble active ingredients, increases the cell uptake of the active ingredients, and can also inhibit the efflux effect of the cells in the later stage, increase the retention of hydrophilic active substances in the cells, and has a sustained release effect, thereby significantly improving the bioavailability of the hydrophilic active substances.

[0031] The tocopherol modified high drug loading water-in-oil nanoemulsion of the present invention has good stability, and after being placed for a long time under different conditions, there is no flocculation, aggregation, precipitation and stratification, and the particle size and PDI have no obvious changes.

[0032] The tocosolan modified high-drug-loaded water-in-oil nanoemulsion of the present invention has high safety, no irritating ingredients are used in the components or preparation process, and the nanoemulsion is mild and non-irritating to the skin within the use concentration. In addition, since tocosolan has an antioxidant effect and can effectively prevent oil rancidity, no additional antioxidant needs to be added to the oil phase.

[0033] The preparation method of the present invention is simple, and a stable nano system can be formed under stirring at room temperature. At the same time, the equipment requirements are low, and there is no need for high-pressure homogenizers and other equipment to provide a large amount of energy. The process is simple and controllable, and industrial production is easy to achieve. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the particle size distribution diagram of the tocosolan modified water-in-oil nanoemulsion prepared in Example 3.

[0035] Figure 2 The appearance of the tocosolan-modified water-in-oil nanoemulsion prepared in Example 3 (left) and the appearance of the essential oil obtained by dissolving the oil shown in Formula 1 in Example 7 at a concentration of 10% (right).

[0036] Figure 3 The following is a bar graph showing the skin penetration and skin retention of the water-in-oil nanoemulsion or aqueous solution prepared in Example 3 and Comparative Examples 4 and 5.

[0037] Figure 4 The bar graph is a graph showing the cellular uptake of the water-in-oil nanoemulsions or aqueous solutions prepared in Example 3 and Comparative Examples 4 and 5.

[0038] Figure 5 It is a bar graph of the cell melanin content after being acted upon by the water-in-oil nanoemulsion or aqueous solution prepared in Example 3 and Comparative Examples 4 and 5. DETAILED DESCRIPTION

[0039] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0040] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0041] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0042] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0043] Some of the embodiments involve a water-in-oil nanoemulsion prepared by dropping an aqueous solution containing tocosolan into an oily solution;

[0044] The aqueous phase solution is composed of tocopherol, a co-surfactant, a hydrophilic active substance, a preservative and water; the oil phase solution is composed of liquid oil and a surfactant;

[0045] The mass percentage of each component in the water-in-oil nanoemulsion is:

[0046] Liquid oil 50~64%

[0047] Surfactant 10~17%

[0048] Tocosolan 5~8%

[0049] Co-surfactant 5~12%

[0050] Hydrophilic active matter 0.1~6%

[0051] Preservatives 0.2~0.6%

[0052] Water 8~15%;

[0053] The hydrophilic active substance is a hydrophilic functional ingredient used in cosmetics;

[0054] The surfactant is selected from one or more of sorbitan monooleate, lecithin (such as soybean lecithin), hydrogenated lecithin and polyglycerol-3 polyricinoleate. For example, it can be a combination of sorbitan monooleate, soybean lecithin and polyglycerol-3 polyricinoleate in a mass ratio of 1:0.6-0.8:0.6-0.8; it can be a combination of sorbitan monooleate and soybean lecithin in a mass ratio of 1:1-1.5; it can be a combination of polyglycerol-3 polyricinoleate and hydrogenated lecithin in a mass ratio of 1:1-2; it can be a combination of polyglycerol-3 polyricinoleate and sorbitan monooleate in a mass ratio of 1:1-2, etc.

[0055] In some preferred embodiments, the mass percentage of each component in the water-in-oil nanoemulsion is:

[0056] Liquid oil 53~62%

[0057] Surfactant 12~15%

[0058] Tocosolan 5~7%

[0059] Co-surfactant 6~10%

[0060] Hydrophilic active ingredients 1~5%

[0061] Preservatives 0.3~0.5%

[0062] Water 9~14%.

[0063] The water-in-oil nanoemulsion of the present invention can significantly improve the water phase content and stability of the water-in-oil nanoemulsion by adding a specific proportion of tocosolan and matching a specific type of surfactant. Tocosolan is a hydrophilic compound. If a stable W / O nanoemulsion with a transparent and clear appearance is required, the addition of tocosolan will theoretically reduce the proportion of the water phase. The present invention unexpectedly found through a large number of experimental studies that by adding a specific proportion of tocosolan, the drug loading of the hydrophilic compound in the W / O nanoemulsion can be significantly increased, so that the hydrophilic compound can be dissolved in various vegetable oils, synthetic oils and mineral oils at a relatively high concentration, and can be directly applied to a variety of "oil-nourishing skin" pure oil cosmetics. After dissolution, the appearance is clear and transparent, which solves the problem of poor oil solubility of high-concentration hydrophilic compounds.

[0064] In some preferred embodiments, the hydrophilic active ingredient is selected from one or more of carnosine, decarboxylated carnosine, nonapeptide-1, hexapeptide-2, acetyl hexapeptide-8, arginine / lysine polypeptide, dipeptide diaminobutyryl benzylamide diacetate, acetyl tetrapeptide-5, tripeptide-1, oligopeptide-1, biotin tripeptide-1, myristoyl pentapeptide-17, hexapeptide-9, pentapeptide-3, acetyl hexapeptide-1, glutathione, bosine, ectoine, ergothioneine and dipotassium glycyrrhizinate.

[0065] In some preferred embodiments, the liquid oil is one or more of isohexadecane, isopropyl myristate, caprylic capric triglyceride, isopropyl palmitate, and isononyl isononanoate. For example, it can be a combination of caprylic capric triglyceride and isohexadecane in a mass ratio of 1:1-3; it can be a combination of isopropyl palmitate and isononyl isononanoate in a mass ratio of 1:1-3; it can be a combination of caprylic capric triglyceride and isononyl isononanoate in a mass ratio of 1:1-3; it can be a combination of isohexadecane, caprylic capric triglyceride and isononyl isononanoate in a mass ratio of 1:1-2:1-2; it can be a combination of isononyl isononanoate, caprylic capric triglyceride, isopropyl myristate and isohexadecane in a mass ratio of 1:1-1.5:1-3:1-2, etc.

[0066] In some preferred embodiments, the cosurfactant is one or more of 1,3-butylene glycol, 2,3-butylene glycol, dipropylene glycol, glycerol and ethoxydiglycol. For example, it can be a combination of 1,3-butylene glycol and dipropylene glycol in a mass ratio of 1:0.5-1.5; it can be a combination of glycerol and ethoxydiglycol in a mass ratio of 1:0.5-1.5, and so on.

[0067] In some preferred embodiments, the preservative is 1,2-hexanediol and / or 1,2-pentanediol.

[0068] In some embodiments, the particle size of the water-in-oil nanoemulsion of the present invention is 40nm~150nm, preferably 40~110nm, more preferably 60nm~80nm, and the PDI is 0.05~0.2. The nanoemulsion of this particle size can maintain good stability, and after being placed for a long time under different conditions, there is no flocculation, aggregation, precipitation and stratification, and the particle size and PDI do not change significantly; and the hydrophilic active substance can effectively penetrate the stratum corneum and penetrate into the living epidermis and dermis, significantly improving the skin retention of hydrophilic compounds.

[0069] Some embodiments of the present invention also relate to a method for preparing the water-in-oil nanoemulsion, comprising the following steps:

[0070] S1. The liquid oil and surfactant are mixed, heated until completely dissolved, and cooled to obtain an oil phase;

[0071] S2. The tocosolan and co-surfactant are mixed, heated until completely dissolved, and cooled to obtain the tocosolan phase;

[0072] S3. The hydrophilic active substance and preservative are added to water, heated until completely dissolved, cooled to obtain an aqueous phase;

[0073] S4. The tocosolan phase prepared in S2 was added to the aqueous phase prepared in S3 and mixed to obtain an aqueous solution;

[0074] S5. dropping the aqueous solution prepared in S4 into the oil phase prepared in S1, stirring for 5 min~20 min, and then shearing and emulsifying at a speed of 5000rpm~7000 rpm for 5min~10 min to obtain the water-in-oil nanoemulsion; or,

[0075] The aqueous phase solution prepared in S4 is dropped into the oil phase prepared in S1, stirred for 5 min to 20 min, and then sheared and emulsified at a rotation speed of 5000 rpm to 7000 rpm for 5 min to 10 min to obtain the water-in-oil nanoemulsion.

[0076] In some embodiments, the heating temperature in S1 is 60°C-70°C.

[0077] In some embodiments, the heating temperature in S2 is 45°C-55°C.

[0078] In some embodiments, the heating temperature in S3 is 35°C-40°C.

[0079] In some embodiments, the temperature of the tocosolan phase and the aqueous phase is first cooled to 30° C. to 35° C. before adding the tocosolan phase prepared in S2 to the aqueous phase prepared in S3 in S4.

[0080] In some embodiments, the mixing in S4 is stirring at a temperature of 30° C. to 35° C. for 3 min to 8 min.

[0081] In some embodiments, the oil phase is cooled to 30° C.-35° C. before the aqueous phase solution prepared in S4 is dripped into the oil phase prepared in S1 in S5.

[0082] In some embodiments, the stirring temperature in S5 is 30° C. to 35° C., and the stirring time is 8 min to 12 min.

[0083] The present invention is further described in detail below with reference to specific embodiments.

[0084] The percentages of raw materials in the following examples and comparative examples, unless otherwise specified, refer to the mass percentages of the corresponding raw material components in the water-in-oil nanoemulsion.

[0085] Example 1

[0086] S1. Preparation of oil phase: Mix 41% isohexadecane, 20% caprylic capric triglyceride, 5% sorbitan monooleate, 3.5% soy lecithin and 3.5% polyglycerol-3 polyricinoleate, heat in a water bath at 60°C until completely dissolved, cool to 30°C and set aside.

[0087] S2. Preparation of tocopherol phase: Mix 6% tocopherol, 3% 1,3-butanediol and 3% dipropylene glycol, heat in a water bath at 50°C until completely dissolved, cool to 30°C and set aside.

[0088] S3. Preparation of aqueous phase: Add 0.05% acetyl hexapeptide-8, 0.05% arginine / lysine peptide, 0.05% dipeptide diaminobutyryl benzylamide diacetate, 1.85% carnosine and 0.4% 1,2-hexanediol to 12.6% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0089] S4. Add the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, and stir in a water bath at 30°C for 5 min.

[0090] S5. The solution prepared in S4 was dripped dropwise into the oil phase prepared in S1, stirred continuously in a water bath at 30°C for 10 min, and high-speed shear emulsification was performed at 5000 rpm for 5 min to obtain a tocopherol modified water-in-oil nanoemulsion.

[0091] Example 2

[0092] S1. Preparation of oil phase: Mix 40% isononyl isononanoate, 17% isopropyl palmitate, 7% soy lecithin and 6% sorbitan monooleate, heat in a water bath at 65°C until completely dissolved, cool to 30°C and set aside.

[0093] S2. Preparation of tocopherol phase: Mix 5% tocopherol, 5% 1,3-butanediol and 5% dipropylene glycol, heat in a water bath at 55°C until completely dissolved, cool to 30°C and set aside.

[0094] S3. Preparation of aqueous phase: Add 1% ergothioneine, 1% decarboxylated carnosine, 1% glutathione, 0.2% 1,2-pentanediol and 0.2% 1,2-hexanediol to 11.6% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0095] S4. Add the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, and stir in a water bath at 30°C for 5 min.

[0096] S5. The solution prepared in S4 was dripped dropwise into the oil phase prepared in S1, stirred continuously in a water bath at 30°C for 10 min, and high-speed shear emulsification was performed at 6000 rpm for 7 min to obtain a tocopherol modified water-in-oil nanoemulsion.

[0097] Example 3

[0098] S1. Preparation of oil phase: Mix 41% isopropyl myristate, 15% caprylic capric triglyceride, 6% sorbitan monooleate and 9% soy lecithin, heat in a water bath at 65°C until completely dissolved, cool to 30°C and set aside.

[0099] S2. Preparation of tocosolan phase: Mix 6% tocosolan and 8% ethoxydiglycol, heat in a water bath at 50°C until completely dissolved, cool to 30°C and set aside.

[0100] S3. Preparation of aqueous phase: Add 1% carnosine, 0.01% hexapeptide-2, 0.01% nonapeptide-1 and 0.4% 1,2-hexanediol to 13.58% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0101] S4. Add the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, and stir in a water bath at 30°C for 5 min.

[0102] S5. The solution prepared in S4 was dripped dropwise into the oil phase prepared in S1, stirred continuously in a water bath at 30°C for 10 min, and high-speed shear emulsification was performed at 6000 rpm for 5 min to obtain a tocopherol modified water-in-oil nanoemulsion.

[0103] The particle size distribution of the tocosolone-modified water-in-oil nanoemulsion obtained in this example is as follows: Figure 1 As shown, its appearance is Figure 2 shown.

[0104] Example 4

[0105] S1. Preparation of oil phase: Mix 20% isononyl isononanoate, 20% caprylic capric triglyceride, 13% isohexadecane, 10% hydrogenated lecithin and 5% polyglycerol-3 polyricinoleate, heat in a water bath at 70°C until completely dissolved, cool to 30°C and set aside.

[0106] S2. Preparation of tocopherol phase: Mix 7% tocopherol, 5% glycerol and 5% ethoxydiglycol, heat in a water bath at 55°C until completely dissolved, cool to 30°C and set aside.

[0107] S3. Preparation of aqueous phase: Add 3% bosine, 0.01% tripeptide-1, 0.01% acetyl tetrapeptide-5, 0.98% carnosine, 0.2% 1,2-pentanediol and 0.2% 1,2-hexanediol to 10.6% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0108] S4. Add the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, and stir in a water bath at 30°C for 5 min.

[0109] S5. The solution prepared in S4 was dripped dropwise into the oil phase prepared in S1, stirred continuously in a water bath at 30°C for 10 min, and high-speed shear emulsification was performed at 7000 rpm for 10 min to obtain a tocopherol modified water-in-oil nanoemulsion.

[0110] Example 5

[0111] S1. Preparation of oil phase: Mix 10% isononyl isononanoate, 10% caprylic capric triglyceride, 20% isopropyl myristate, 16% isohexadecane, 9% sorbitan monooleate and 6% polyglycerol-3 polyricinoleate, heat in a water bath at 60°C until completely dissolved, cool to 30°C and set aside.

[0112] S2. Preparation of tocopherol phase: Mix 7% tocopherol, 4% dipropylene glycol and 3% 1,3-butylene glycol, heat in a water bath at 50°C until completely dissolved, cool to 30°C and set aside.

[0113] S3. Preparation of aqueous phase: Add 4% dipotassium glycyrrhizinate, 1% ectoine and 0.4% 1,2-hexanediol to 9.6% pure water, heat in a water bath at 40°C until completely dissolved, cool to 30°C and set aside.

[0114] S4. Add the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, and stir in a water bath at 30°C for 5 min.

[0115] S5. The solution prepared in S4 was added dropwise into the oil phase prepared in S1, and the mixture was stirred in a water bath at 30° C. for 10 min to obtain a tocopherol modified water-in-oil nanoemulsion.

[0116] Comparative Example 1

[0117] The difference between this comparative example 1 and example 1 is that the added amount of tocosolan is 10%.

[0118] S1. Preparation of oil phase: Mix 37% isohexadecane, 20% caprylic capric triglyceride, 5% sorbitan monooleate, 3.5% soy lecithin and 3.5% polyglycerol-3 polyricinoleate, heat in a water bath at 60°C until completely dissolved, cool to 30°C and set aside.

[0119] S2. Preparation of tocopherol phase: Mix 10% tocopherol, 3% 1,3-butanediol and 3% dipropylene glycol, heat in a water bath at 50°C until completely dissolved, cool to 30°C and set aside.

[0120] S3. Preparation of aqueous phase: Add 0.05% acetyl hexapeptide-8, 0.05% arginine / lysine peptide, 0.05% dipeptide diaminobutyryl benzylamide diacetate, 1.85% carnosine and 0.4% 1,2-hexanediol to 12.6% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0121] S4. Add the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, and stir in a water bath at 30°C for 5 min.

[0122] S5. The solution prepared in S4 was dripped dropwise into the oil phase prepared in S1, stirred continuously in a water bath at 30°C for 10 min, and high-speed shear emulsification was performed at 5000 rpm for 5 min to obtain a tocopherol modified water-in-oil nanoemulsion.

[0123] Comparative Example 2

[0124] The difference between this comparative example 2 and example 2 is that the added amount of tocosolan is 3%.

[0125] S1. Preparation of oil phase: Mix 40% isononyl isononanoate, 19% isopropyl palmitate, 7% soy lecithin and 6% sorbitan monooleate, heat in a water bath at 65°C until completely dissolved, cool to 30°C and set aside.

[0126] S2. Preparation of tocopherol phase: Mix 3% tocopherol, 5% 1,3-butanediol and 5% dipropylene glycol, heat in a water bath at 55°C until completely dissolved, cool to 30°C and set aside.

[0127] S3. Preparation of aqueous phase: Add 1% ergothioneine, 1% decarboxylated carnosine, 1% glutathione, 0.2% 1,2-pentanediol and 0.2% 1,2-hexanediol to 11.6% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0128] S4. Add the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, and stir in a water bath at 30°C for 5 min.

[0129] S5. The solution prepared in S4 was dripped dropwise into the oil phase prepared in S1, stirred continuously in a water bath at 30°C for 10 min, and high-speed shear emulsification was performed at 6000 rpm for 7 min to obtain a tocopherol modified water-in-oil nanoemulsion.

[0130] Comparative Example 3

[0131] The difference between this comparative example and Example 3 lies in the surfactant. The surfactant in this comparative example is polyglyceryl-4 isostearate and polyglyceryl-2 triisostearate.

[0132] S1. Preparation of oil phase: Mix 41% isopropyl myristate, 15% caprylic capric triglyceride, 6% polyglyceryl-4 isostearate and 9% polyglyceryl-2 triisostearate, heat in a water bath at 65°C until completely dissolved, cool to 30°C and set aside.

[0133] S2. Preparation of tocosolan phase: Mix 6% tocosolan and 8% ethoxydiglycol, heat in a water bath at 50°C until completely dissolved, cool to 30°C and set aside.

[0134] S3. Preparation of aqueous phase: Add 1% carnosine, 0.01% hexapeptide-2, 0.01% nonapeptide-1 and 0.4% 1,2-hexanediol to 13.58% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0135] S4. Add the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, and stir in a water bath at 30°C for 5 min.

[0136] S5. The solution prepared in S4 was dripped dropwise into the oil phase prepared in S1, stirred continuously in a water bath at 30°C for 10 min, and high-speed shear emulsification was performed at 6000 rpm for 5 min to obtain a tocopherol modified water-in-oil nanoemulsion.

[0137] Comparative Example 4

[0138] 1% carnosine, 0.01% hexapeptide-2, 0.01% nonapeptide-1, 0.4% 1,2-hexanediol, 8% ethoxydiglycol and 6% tocopherol were added to 84.58% pure water, heated in a water bath at 35°C until completely dissolved, to obtain an aqueous solution containing tocopherol and active ingredients.

[0139] The raw material percentages in this comparative example refer to the mass percentages of the corresponding raw material components in the aqueous solution.

[0140] Comparative Example 5

[0141] This comparative example is a conventional preparation method of water-in-oil nanoemulsion, in which the proportion of water phase is relatively low, otherwise a stable water-in-oil nanoemulsion cannot be prepared.

[0142] S1. Preparation of oil phase: Mix 50.1% isopropyl myristate, 15% caprylic capric triglyceride, 9% polyglyceryl-6 polyricinoleate and 6% polyglyceryl-4 laurate, heat in a water bath at 65°C until completely dissolved, cool to 30°C and set aside.

[0143] S2. Preparation of aqueous phase: Add 0.30% carnosine, 0.006% hexapeptide-2, 0.006% nonapeptide-1, 0.1% 1,2-hexanediol and 15% glycerol to 4.49% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0144] S3. The water phase prepared in S2 was added dropwise into the oil phase prepared in S1, and the mixture was stirred continuously in a water bath at 30°C for 10 min. The mixture was high-speed sheared and emulsified at 6000 rpm for 5 min to obtain a water-in-oil nanoemulsion.

[0145] Comparative Example 6

[0146] The difference between this comparative example and Example 1 is that no modification with tocosolan is used.

[0147] S1. Preparation of oil phase: Mix 49% isohexadecane, 24% caprylic capric triglyceride, 5% sorbitan monooleate, 3.5% soy lecithin and 3.5% polyglycerol-3 polyricinoleate, heat in a water bath at 60°C until completely dissolved, cool to 30°C and set aside.

[0148] S2. Preparation of aqueous phase: Add 0.05% acetyl hexapeptide-8, 0.05% arginine / lysine peptide, 0.05% dipeptide diaminobutyryl benzylamide diacetate, 1.85% carnosine and 0.4% 1,2-hexanediol to 12.6% pure water, heat in a water bath at 35°C until completely dissolved, cool to 30°C and set aside.

[0149] S3. The water phase prepared in S2 was added dropwise into the oil phase prepared in S1, and the mixture was stirred in a water bath at 30°C for 10 min. The mixture was high-speed sheared at 5000 rpm for 5 min to obtain a water-in-oil nanoemulsion.

[0150] Example 6 Stability test of tocosolan-modified water-in-oil nanoemulsion

[0151] The water-in-oil nanoemulsions prepared in Examples 1 to 5 and Comparative Examples 1 to 3 and Comparative Example 6 were placed at room temperature, 4° C. and 40° C. for 90 days, respectively, and their appearance, particle size and PDI were tested.

[0152] Table 1 Stability results of water-in-oil nanoemulsions prepared in Examples 1 to 5 and Comparative Examples 1 to 3 and 6

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] From the results in Table 1, it can be seen that the water-in-oil nanoemulsions prepared in Examples 1 to 5 of the present invention are all light yellow clear and transparent liquids, with a particle size in the range of 60 to 110 nm, and a PDI of less than 0.20, which meets the requirements of practical applications; and after being placed under different conditions for 90 days, there is no flocculation, agglomeration, precipitation and stratification, and the particle size and PDI have no obvious changes, which still meets the requirements of practical applications, indicating that the tocosolone-modified water-in-oil nanoemulsion prepared by the present invention has excellent stability.

[0160] The stability results of the water-in-oil nanoemulsion prepared in Comparative Examples 1-2 show that the amount of tocosolan added has a very large effect on the stability of the obtained water-in-oil nanoemulsion. When the amount of tocosolan added is 3% or 10% and 15% of the aqueous phase is added, a stable water-in-oil nanoemulsion cannot be formed. The obtained water-in-oil nanoemulsion will show stratification and precipitation as the storage time increases.

[0161] The stability results of the water-in-oil nanoemulsion prepared in Comparative Example 3 show that the choice of surfactant also has a great influence on the stability of the obtained water-in-oil nanoemulsion. When polyglycerol-4 isostearate and polyglycerol-2 triisostearate are used as surfactants, their stability is significantly reduced compared with the water-in-oil nanoemulsion prepared in Example.

[0162] The water-in-oil nanoemulsion prepared in Comparative Example 6 had the same internal water phase ratio as that of Example 1, but no tocosolone was added. The resulting water-in-oil nanoemulsion system was not stable and stratified within 15 days.

[0163] In summary, only a specific amount of tocosolan combined with a specific surfactant can produce an oil-in-water nanoemulsion with high drug loading and good stability.

[0164] Example 7 Oil Solubility Test of Tocosolan-Modified Water-in-Oil Nanoemulsion

[0165] The tocosolan-modified water-in-oil nanoemulsion prepared in Examples 1 to 5 and the water-in-oil nanoemulsion prepared in Comparative Example 5 were respectively added to commonly used cosmetic oils (olive oil, squalane, silicone oil, jojoba seed oil, isohexadecane, isopropyl myristate and a combination thereof) to prepare the corresponding pure oil cosmetics, i.e., essence oil, with the water-in-oil nanoemulsion added in an amount of 10% (mass ratio), and its dissolution in different pure oil systems was observed.

[0166] Table 2 The miscibility of water-in-oil nanoemulsions with commonly used cosmetic oils

[0167]

[0168] From the results in Table 2, it can be seen that the water-in-oil nanoemulsion sample prepared by the present invention can be well miscible with the commonly used oils in cosmetics. Although it is not miscible with single oil olive oil and silicone oil, it is soluble in the compounded completely miscible base oil. After miscibility, the appearance is clear and transparent without precipitation. The content of the efficacy of the essence oil obtained after the nanoemulsion is dissolved at 10% can reach its effective concentration on the cells, meeting the requirements of practical application.

[0169] Example 8 Testing the Skin Penetration Ability of Tocosolan-Modified Water-in-Oil Nanoemulsion

[0170] The skin permeability of the water-in-oil nanoemulsion prepared in Example 3 and Comparative Example 5 and the solution prepared in Comparative Example 4 was tested by a vertical Franz diffusion cell method. Physiological saline was selected as the receiving medium, and suckling pig skin with a skin thickness of 0.7-0.8 mm was selected. The pig skin was fixed between the supply pool and the receiving pool, and 0.5±0.01 g of the samples prepared in Example 3, Comparative Example 4 and Comparative Example 5 were taken and placed in the supply pool, and the receiving samples were diluted with squalane or water to a carnosine content of 0.1%. The rotor speed in the receiving pool was adjusted to 300 rpm, and the transdermal test was carried out at 32°C. The receiving medium absorbed from the receiving pool in 24 h was filtered through a 0.22µm filter membrane and the carnosine content was detected by HPLC, and the 24 h skin permeation was calculated.

[0171] Wipe the remaining sample on the surface of the suckling pig skin with absorbent cotton, cut out the part in contact with the sample and cut into pieces, put them into 2 mL grinding tubes respectively, grind them with a tissue grinder, add 1 ml of pure water, ultrasonicate for 30 min, centrifuge at 5000 rpm for 15 min, take the supernatant and filter it through a 0.22 µm filter membrane, and use HPLC to detect the carnosine content, and calculate the 24 h skin retention amount.

[0172] The liquid phase detection conditions were as follows: chromatographic column AQ-C18 C18 (4.6 mm × 250 mm, 5 μm), flow rate 1.0 ml / min, column temperature 35 °C, mobile phase A: 0.1% TFA water, mobile phase B: 0.1% TFA acetonitrile, gradient elution, and detection wavelength 210 nm.

[0173] The test results are as follows Figure 3 As shown: the water-in-oil nanoemulsion has a better ability to penetrate the stratum corneum than the aqueous solution. The 24-hour skin penetration and skin retention of the water-in-oil nanoemulsions of Comparative Example 5 and Example 3 are significantly higher than those of the aqueous solution of Comparative Example 4.

[0174] After the active substance passes through the stratum corneum, in order to make it active, another necessary condition is required: it is retained in the target part / target cell for a long time. The oil-in-water nanoemulsion prepared by nonionic surfactant in Comparative Example 5 will carry the active substance through the living epidermis and dermis after passing through the stratum corneum, and cannot be retained in the target part for a long time. This phenomenon shows that this oil-in-water nanoemulsion has certain safety hazards and cannot reach the expected bioavailability. This may be due to the efflux effect of the cell, which will expel the active substance entering the cell, thereby affecting the absorption and final efficacy of the active substance. The difference is that the tocosyl modified oil-in-water nanoemulsion prepared in Example 3 is still retained in the target part to a large extent after passing through the stratum corneum. The 24-hour skin retention of Example 3 is significantly greater than that of Comparative Example 5, indicating that the oil-in-water nanoemulsion modified by tocosyl can not only improve the skin penetration ability of the active substance, but also inhibit the efflux effect of the cell, and increase the retention of the active substance in the cell, thereby truly improving the skin bioavailability of the hydrophilic active substance.

[0175] Example 9 Testing the efficacy and cellular uptake of tocosolone modified water-in-oil nanoemulsion

[0176] Experimental model: α-MSH-induced mouse melanoma cell model (B16F10).

[0177] The cells were grown at a density of 2×10 6 Inoculate in a 100 mm culture dish and culture for 24 hours. Add 100 nM α-MSH for induction (except the normal control group), and then add 2 ml of DMEM complete medium containing different concentrations of water-in-oil nanoemulsions or solutions prepared in Example 3, Comparative Example 4 and Comparative Example 5. After 48 hours of culture, discard the supernatant, wash with PBS three times, collect the cells in a centrifuge tube, add 1 ml of cell lysis solution to each tube, ultrasonically lyse the cells at 60°C for 30 minutes, centrifuge at 12000 rpm for 30 minutes, take the supernatant liquid, filter it through a 0.22 µm filter membrane, and use HPLC to detect the carnosine content, and calculate the cell uptake of each group.

[0178] The supernatant was discarded, 200 μl DMSO was added and dissolved by ultrasound for 30 min, and the absorbance was measured at a wavelength of 405 nm. The relative melanin content in each group of cells was calculated based on the absorbance of each group.

[0179] The results of cellular uptake were as follows Figure 4 The relative melanin content in the cells is shown in Figure 5 As shown. Figure 4 , 5The results show that under the same active substance concentration conditions, the cellular uptake of Example 3 is significantly higher than that of Comparative Examples 4 and 5, and the relative melanin content of Example 3 is significantly lower than that of Comparative Examples 4 and 5, close to the blank control group. It is proved that the water-in-oil nanoemulsion modified with tocosolan can significantly increase the retention of active substances in cells, and can significantly improve the effect of inhibiting melanin synthesis in B16F10 cells, and significantly improve the bioavailability of hydrophilic active substances.

[0180] In addition, the comparison between Comparative Example 4 and Example 3 shows that the simple dissolved tocopherol aqueous solution cannot achieve the same effect as the tocopherol modified water-in-oil nanoemulsion of the present invention.

[0181] The above results indicate that the water-in-oil nanoemulsion modified with tocosolan can not only increase the early uptake of cells, but also inhibit the efflux of cells in the later stage, thereby increasing the retention of hydrophilic active substances in cells and improving bioavailability.

[0182] Example 10 Human patch test

[0183] Volunteers: The total effective number of people is 30, including 17 females and 13 males, with an age range of 20-40 years old.

[0184] The blank control well is physiological saline. The water-in-oil nanoemulsion prepared in Examples 1 to 5 is diluted 10 times with squalane. The test solution is placed in a waterproof patch chamber, and the dosage is about 0.020 mg-0.025 mg; the test area of ​​the inner forearm skin is cleaned, and a suitable test site is selected and the skin state before patching is recorded. The patch is directly attached to the skin on the inner side of the forearm. The test site cannot contact water and cannot be directly exposed to sunlight. After keeping it for 24 hours, the skin reaction is observed according to the standards of Table 3 30 minutes (after the indentation disappears), 24 hours and 48 hours after the test patch is removed, and the observation results are recorded, photographed and recorded, and observations are scored. When adverse reactions occur, wound erythema and edema can be observed. The method for evaluating edema is to observe the edge of the skin at the adverse reaction site and the adjacent normal skin.

[0185] The results of the human skin patch test are shown in Table 4.

[0186] Table 3. Skin closure patch test skin reaction grading standards

[0187]

[0188] Table 4. Human skin patch test results

[0189]

[0190]

[0191] The test results in Table 4 show that the water-in-oil nanoemulsion sample of the present invention has no erythema, redness or swelling after being in contact with human skin for 24 hours, indicating that the tocosolone-modified water-in-oil nanoemulsion prepared by the present invention is mild and low irritating to the skin and has high safety.

[0192] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A water-in-oil nanoemulsion, characterized in that: It is prepared by dropping an aqueous solution containing tocosolan into an oily solution; The aqueous phase solution is composed of tocopherol, a co-surfactant, a hydrophilic active substance, a preservative and water; the oil phase solution is composed of liquid oil and a surfactant; The mass percentage of each component in the water-in-oil nanoemulsion is: Liquid oil 50~64% Surfactant 10~17% Tocosolan 5~8% Co-surfactant 5~12% Hydrophilic active matter 0.1~6% Preservatives 0.2~0.6% Water 8~15%; The hydrophilic active substance is a hydrophilic functional ingredient used in cosmetics; The surfactant is selected from one or more of sorbitan monooleate, lecithin, hydrogenated lecithin and polyglycerol-3 polyricinoleate; The co-surfactant is one or more of 1,3-butylene glycol, 2,3-butylene glycol, dipropylene glycol, glycerol and ethoxydiglycol.

2. The water-in-oil nanoemulsion according to claim 1, characterized in that The mass percentage of each component in the water-in-oil nanoemulsion is: Liquid oil 53~62% Surfactant 12~15% Tocosolan 5~7% Co-surfactant 6~10% Hydrophilic active ingredients 1~5% Preservatives 0.3~0.5% Water 9~14%.

3. The water-in-oil nanoemulsion according to claim 1, characterized in that The hydrophilic active ingredient is selected from one or more of carnosine, decarboxylated carnosine, nonapeptide-1, hexapeptide-2, acetyl hexapeptide-8, arginine / lysine polypeptide, dipeptide diaminobutyryl benzylamide diacetate, acetyl tetrapeptide-5, tripeptide-1, oligopeptide-1, biotin tripeptide-1, myristoyl pentapeptide-17, hexapeptide-9, pentapeptide-3, acetyl hexapeptide-1, glutathione, bosine, ectoine, ergothioneine and dipotassium glycyrrhizinate.

4. The water-in-oil nanoemulsion according to claim 1, characterized in that The liquid oil is one or more of isohexadecane, isopropyl myristate, caprylic capric triglyceride, isopropyl palmitate and isononyl isononanoate.

5. The water-in-oil nanoemulsion according to claim 1, characterized in that The preservative is 1,2-hexanediol and / or 1,2-pentanediol.

6. The water-in-oil nanoemulsion according to any one of claims 1 to 5, characterized in that Its particle size is 40nm~150nm.

7. Use of the water-in-oil nanoemulsion according to any one of claims 1 to 6 in the preparation of cosmetics.

8. The use according to claim 7, characterized in that: The cosmetics are essence oil, lipstick and make-up remover oil.

9. A method for preparing the water-in-oil nanoemulsion according to any one of claims 1 to 6, characterized in that: The steps include: S1. The liquid oil and surfactant are mixed and heated until completely dissolved to obtain an oil phase; S2. The tocosolan and co-surfactant are mixed and heated until completely dissolved to obtain the tocosolan phase; S3. The hydrophilic active substance and the preservative are added to water and heated until completely dissolved to obtain an aqueous phase; S4. The tocosolan phase prepared in S2 was added to the aqueous phase prepared in S3 and mixed to obtain an aqueous solution; S5. The aqueous phase solution prepared in S4 is dropped into the oil phase prepared in S1, stirred for 5 min to 20 min, and then sheared and emulsified at a rotation speed of 5000 rpm to 7000 rpm for 5 min to 10 min to obtain the water-in-oil nanoemulsion.

10. The method for preparing a water-in-oil nanoemulsion according to claim 9, characterized in that: The heating temperature in S1 is 60°C to 70°C; and / or, The heating temperature in S2 is 45°C to 55°C; and / or, The heating temperature in S3 is 35°C to 40°C; and / or, In S4, before adding the tocosolan phase prepared in S2 to the aqueous phase prepared in S3, the tocosolan phase and the aqueous phase are cooled to 30° C. to 35° C.; and / or, The mixing in S4 is carried out by stirring at a temperature of 30°C to 35°C for 3min to 8min; and / or, In S5, before dripping the aqueous phase solution prepared in S4 into the oil phase prepared in S1, the oil phase is cooled to 30°C to 35°C; and / or, The stirring temperature in S5 is 30° C. to 35° C., and the stirring time is 8 min to 12 min.

Images