Ceramide nanoemulsion as well as preparation method and application thereof

Abstract

The invention relates to a ceramide nanoemulsion as well as a preparation method and application thereof, in particular to the ceramide nanoemulsion which comprises the following components: meadowfoam seed ceramide NP, ceramide AP and ceramide EOP, and further comprises an oil phase and a water phase, the oil phase is prepared from meadowfoam seed oil, tocopheryl acetate, caprylic / capric triglyceride, hexyldecanol and polyglycerol-10 myristate, the water phase is prepared from water, glycerol, hydrogenated lecithin, 1, 2-hexanediol and p-hydroxyacetophenone, and the ceramide nanoemulsion has multiple excellent skin care effects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and specifically relates to a ceramide nanoemulsion, a preparation method thereof and application thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, and the outermost stratum corneum is a natural barrier that maintains skin health, prevents excessive water loss, and resists external stimuli. This barrier function mainly depends on the "brick wall structure" composed of stratum corneum cells and intercellular lipids. Ceramide is the most abundant and important component of intercellular lipids, accounting for about 40-50% of the total amount of intercellular lipids. It plays a core role in maintaining skin barrier integrity, moisturizing, anti-inflammatory, and anti-aging. However, despite the great application potential of ceramide in the field of skin care, it has been facing three major technical challenges that have not been effectively solved for a long time in actual product formulations and applications: ① Poor solubility: Ceramide is a lipid-soluble substance that is extremely difficult to dissolve in water. This makes it difficult to uniformly disperse and stably exist in the mainstream cosmetic formulations based on water (such as emulsions, serums), severely limiting its formulation form and bioavailability. ② Low stability: The molecular structure of ceramide contains unstable amide bonds and other groups, which are sensitive to light, heat, and oxidation. During storage and use, it is prone to degradation, leading to a decrease in product efficacy, which poses a serious challenge to the shelf life and actual effect of the product. ③ Insufficient skin permeability: Due to its large molecular weight and strong hydrophobicity, ceramide alone is difficult to effectively penetrate the stratum corneum of the skin and reach its target location where it exerts physiological functions. Most externally applied ceramide stays on the surface of the skin and cannot be truly supplemented into the missing intercellular lipids, thereby affecting the exertion of its core efficacy such as barrier repair.

[0003] In order to overcome the above problems, the skilled person in the art has made various attempts. For example: using ordinary emulsions, traditional emulsions (such as emulsions, creams) can wrap ceramide in the oil phase, but their particle size is usually large (micron level), and their stability and transdermal absorption efficiency are limited, and they are prone to problems such as separation and precipitation during long-term storage. Or use a single type of ceramide, many existing products only use one or two common synthetic or plant-derived ceramides (such as ceramide AP, EOP, etc.). However, there are at least 12 different subtypes of ceramides in human skin, which work synergistically in a specific ratio and combination. A single or simple combination of ceramides cannot fully simulate and repair the complex "brick wall structure" in the skin, resulting in incomplete and unsatisfactory skin care effects.

[0004] Patent document CN117285435A discloses a method for synthesizing ceramide from white pool flower seed oil and points out its potential skin care efficacy. However, this patent does not solve the three major technical challenges mentioned above. SUMMARY

[0005] Based on the background art and the deficiencies in the prior art, the present application aims to solve the following technical problems: how to provide a formulation form that can simultaneously solve the problems of poor solubility, low stability and insufficient skin permeability of ceramides in application, and can exert the best skin care efficacy (such as barrier repair, anti-inflammatory, moisturizing and anti-aging) through the synergistic compounding of specific ceramides.

[0006] The present application provides a ceramide nanoemulsion, which comprises white pool flower seed ceramide NP, ceramide AP and ceramide EOP.

[0007] Further, the mass ratio of the white pool flower seed ceramide NP, ceramide AP and ceramide EOP is 1:(0.25-0.75):(0.25-0.75).

[0008] Further, the components further comprise an oil phase and an aqueous phase, the oil phase comprises white pool flower seed oil, tocopheryl acetate, caprylic / capric triglyceride, hexyldecanol and polyglyceryl-10 myristate, and the aqueous phase comprises water, glycerol, hydrogenated lecithin, 1,2-hexanediol and p-hydroxyacetophenone.

[0009] Further, the components are compounded in a weight ratio of 0.8-1.5 parts of white pool flower seed ceramide NP, 0.25-0.75 parts of ceramide AP, 0.25-0.75 parts of ceramide EOP, 1-5 parts of white pool flower seed oil, 1-5 parts of tocopheryl acetate, 1-5 parts of caprylic / capric triglyceride, 1-5 parts of hexyldecanol, 1-4 parts of polyglyceryl-10 myristate, 50-70 parts of water, 10-30 parts of glycerol, 1-5 parts of hydrogenated lecithin, 0.1-1 parts of 1,2-hexanediol and 0.1-1 parts of p-hydroxyacetophenone.

[0010] Further, the initial particle size of the nanoemulsion is less than 90 nm, and the particle size change is less than 10% after storage for one month.

[0011] The present application also provides a preparation method of the above-mentioned ceramide nanoemulsion, comprising the following steps: S1: mixing the oil phase ingredients white pool flower seed oil, tocopheryl acetate, caprylic / capric triglyceride, hexyldecanol, polyglyceryl-10 myristate, white pool flower seed ceramide NP, ceramide AP and ceramide EOP according to weight ratio, and heating to dissolve; S2: mixing the aqueous phase ingredients water, glycerol, hydrogenated lecithin, 1,2-hexanediol and p-hydroxyacetophenone according to weight ratio, and heating to dissolve; S3: adding the mixture prepared in step S1 into the aqueous phase in step 2 for emulsification, and stirring uniformly; S4: high pressure homogenization is performed on the mixture emulsified in step S3.

[0012] Preferably, in step S1, the temperature of the heating is 80-90℃; in step S2, the temperature of the heating is 80-85℃.

[0013] Preferably, in step S4, the high pressure is 100-140 MPa.

[0014] The application also provides a use of the ceramide nanoemulsion as described above in the preparation of a cosmetic product for skin care.

[0015] Further, the cosmetic product is used for moisturizing, anti-inflammatory or anti-aging.

[0016] Further, the cosmetic product is a lotion, a cream or a serum.

[0017] Compared with the prior art, the ceramide nanoemulsion provided by the application has the following remarkable beneficial effects: The ceramide nanoemulsion of the application has multiple excellent skin care effects, the application can effectively enhance the activity of HaCaT human keratinocytes, indicating that it has a positive repair and activation effect on skin cells, the application can significantly reduce the mRNA expression amount of the inflammatory factor IL-6 in LPS-induced mouse macrophage RAW 264.7, and exhibit strong anti-inflammatory potential. The application can significantly improve the expression of AQP3 water channel protein in human keratinocytes, thereby enhancing the transport capacity of skin to water and glycerol, and realizing deep moisturizing from the inside out. The application can significantly improve the expression of COL1A1 factor (type I collagen) in human foreskin fibroblasts (HFF-1), and type I collagen is the key to maintaining skin elasticity and toughness, which indicates that the application has obvious anti-aging and collagen regeneration promoting effects.

[0018] The nanoemulsion prepared by the application has an initial particle size of less than 90 nm and a uniform distribution. After one month of accelerated stability test at different temperatures, the particle size change is less than 10%, the application has excellent physical stability, and can ensure the quality and shelf life of the product during storage and transportation. After the ceramide composition of the application is wrapped in the nanoemulsion, the permeability is increased by nearly 50 times compared with the pure ceramide mixture without wrapping. This is due to the extremely small particle size of the nanoemulsion, which can effectively carry active ingredients to penetrate the skin barrier, ensure that they reach the target site and play a role, and fundamentally solve the problem of difficult transdermal penetration of traditional ceramide preparations.

[0019] The preparation method of the application adopts the conventional "phase separation heating-emulsification-high pressure homogenization" process, the steps are simple, the conditions are mild, the requirements for equipment are low, no complex post-treatment is needed, and it is very suitable for large-scale industrial production, and has very high commercial conversion value. Attached Figure Description

[0020] Figure 1 Schematic diagram of thickening and agglomeration during the preparation process of Comparative Example 2; Figure 2 Image of HaCaT human keratinocyte viability test results; Figure 3 Anti-inflammatory effect test results (image); Figure 4 Results of moisturizing efficacy test; Figure 5 Image showing the results of collagen expression test; Figure 6 Image showing the NR test results on the surface of the nematode-free area; Figure 7 The sample prepared in Experiment Example 1 was observed and photographed under a fluorescence microscope during the online insect permeability test; Figure 8 Images of uncoated ceramide samples observed and photographed under a fluorescence microscope during an online insect permeability experiment. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0023] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0024] Example 1 This embodiment provides a ceramide nanoemulsion, the components of which include meadowfoam seed ceramide NP, ceramide AP and ceramide EOP.

[0025] As a further preferred embodiment, the components also include an oil phase and an aqueous phase, wherein the oil phase comprises meadowfoam seed oil, tocopheryl acetate, caprylic / capric triglyceride, hexyldecyl alcohol and polyglycerol-10 myristate, and the aqueous phase comprises water, glycerol, hydrogenated lecithin, 1,2-hexanediol and p-hydroxyacetophenone.

[0026] As a further preferred embodiment, the components are proportioned by weight parts to include 0.8-1.5 parts of the white pool flower seed ceramide NP, 0.25-0.75 parts of ceramide AP, 0.25-0.75 parts of ceramide EOP, 1-5 parts of white pool flower seed oil, 1-5 parts of tocopheryl acetate, 1-5 parts of caprylic / capric triglyceride, 1-5 parts of hexyldecanol, 1-4 parts of polyglyceryl-10 myristate, 50-70 parts of water, 10-30 parts of glycerol, 1-5 parts of hydrogenated lecithin, 0.1-1 part of 1,2-hexanediol, 0.1-1 part of p-hydroxyacetophenone.

[0027] As a further preferred embodiment, the mass ratio of the white pool flower seed ceramide NP, ceramide AP and ceramide EOP is 1:(0.25-0.75):(0.25-0.75).

[0028] Ceramide AP, with a shorter fatty acid chain (C16-C18), is mainly distributed in the upper layer of the stratum corneum, responsible for "quickly filling the barrier gap", improving the skin's instantaneous water retention capacity, and reducing the evaporation of surface water (TEWL).

[0029] Ceramide EOP, with a long fatty acid chain and containing a hydroxyl group (-OH), is mainly distributed in the middle-lower layer of the stratum corneum, and can form a more compact lipid stacking structure through "hydroxyl hydrogen bonds" between molecules, strengthening the "physical defense" of the barrier (resisting the invasion of external pollutants and irritants).

[0030] White pool flower seed ceramide NP (hereinafter referred to as BCH) is a self-developed product of the company (CN 117285435 A), which is obtained by reacting white pool flower seed oil fatty acid and sphingosine compounds. White pool flower seed oil fatty acid belongs to naturally formed fatty acids, the main component of which is monounsaturated fatty acid cis-5-eicosenoic acid, in addition to which it also contains erucic acid, cis-5,13-docosadienoic acid, cis-5-docosenoic acid, etc. It reacts with sphingosine compounds naturally present in the skin under mild room temperature reaction conditions, and the condensation of fatty acids and amino groups is highly active, which can ensure that various types of fatty acids participate in the reaction, and the obtained ceramide can maintain the proportion of fatty acids in the original oil. The fatty acid chain is mainly super-long chain (C20-C22), which can form a "synergistic stacking" with the long chain of ceramide EOP, enhancing the stability of the middle layer barrier; at the same time, a small amount of unsaturated bonds (such as double bonds) can break through "overly tight stacking", avoiding the loss of flexibility of the lipid bilayer due to the structure being too rigid, which can reduce the dryness and desquamation of the skin caused by a rigid barrier, especially suitable for dry or sensitive skin.

[0031] The differences in fatty acid chain length among the three (AP short chain, EOP / BCH long chain) can match the natural fatty acids in the stratum corneum (short chain C16, long chain C24), avoiding the "abnormal fluidity" of the lipid bilayer caused by a single chain length (too many short chains make the barrier easily permeable, too many long chains make the barrier easily rigid). Under the combined action of the three, the lipid bilayer forms a leak-free structure from "upper layer (AP water-locking) → middle layer (EOP defense) → full layer (BCH bonding)," repairing the integrity of the "brick wall structure."

[0032] In addition to fatty acids (or oils), meadowfoam seed oil also contains active ingredients such as α-tocopherol, β-tocopherol, γ-tocopherol, and phytosterols. These nutrients have effects such as moisturizing the skin and providing a skin barrier. Ceramides synthesized from meadowfoam seed oil have a synergistic effect with other active ingredients contained in meadowfoam seed oil, and have better effects than ceramides compounded in a similar proportion.

[0033] As a further preferred embodiment, the initial particle size of the nanoemulsion is less than 90 nm, and the particle size change is less than 10% after one month of storage.

[0034] The small particle size (<90nm) ensures high permeability (easily penetrates the skin barrier) and kinetic stability of the system (not easily aggregated or delaminated). The minimal change in particle size after storage directly demonstrates the outstanding physical stability of the nanoemulsion of this invention, which guarantees the product's shelf life and effectiveness.

[0035] Example 2 This embodiment provides a method for preparing the ceramide nanoemulsion described in Example 1, characterized by comprising the following steps: S1: Mix the oil phase components, meadowfoam seed oil, tocopheryl acetate, caprylic / capric triglyceride, hexyldecyl alcohol, polyglycerol-10 myristate, and meadowfoam seed ceramide NP, ceramide AP, and ceramide EOP in the specified weight proportions; heat until dissolved; S2: Mix the aqueous phase components, including water, glycerin, hydrogenated lecithin, 1,2-hexanediol and p-hydroxyacetophenone, in the indicated weight proportions and heat until dissolved; S3: Add the mixture prepared in step S1 to the aqueous phase described in step 2 and emulsify, stirring until homogeneous; S4: High-pressure homogenization of the emulsified mixture from step S3.

[0036] As a further preferred embodiment, in step S1, the heating temperature is 80-90℃; in step S2, the heating temperature is 80-85℃.

[0037] As a further preferred embodiment, in step S4, the high pressure is 100-140 MPa.

[0038] Example 3 The application of the ceramide nanoemulsion described in Example 1 in the preparation of a cosmetic for skin care.

[0039] As a further preferred embodiment, the cosmetic is used for moisturizing, anti-inflammatory or anti-aging.

[0040] As a further preferred embodiment, the cosmetic is a lotion, cream or serum.

[0041] Example 4 Test Example 1 A ceramide nanoemulsion, the components of which include 62 g of water, 20 g of glycerol, 3 g of white pool flower seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 1 g of white pool flower seed ceramide NP, 0.5 g of ceramide AP, 0.5 g of ceramide EOP, 0.5 g of 1,2-hexanediol, and 0.5 g of p-hydroxyacetophenone.

[0042] The preparation method thereof is as follows: S1: Mix the oil phase ingredients white pool flower seed oil, tocopheryl acetate, caprylic / capric triglyceride, hexyldecanol, polyglyceryl-10 myristate, and white pool flower seed ceramide NP, ceramide AP, and ceramide EOP according to the above weights; heat to X ℃ and heat to dissolution; S2: Mix the water phase ingredients water, glycerol, hydrogenated lecithin, 1,2-hexanediol, and p-hydroxyacetophenone according to the above weights, heat to X ℃, and heat to dissolution; S3: Add the mixture prepared in step S1 to the water phase described in step 2 and emulsify, stirring uniformly; S4: Homogenize the mixture emulsified in step S3 under a pressure of X MPa.

[0043] Test Example 2 A ceramide nanoemulsion, the components of which include 50 g of water, 10 g of glycerol, 1 g of white pool flower seed oil, 1 g of tocopheryl acetate, 1 g of caprylic / capric triglyceride, 1 g of hydrogenated lecithin, 1 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 0.8 g of white pool flower seed ceramide NP, 0.25 g of ceramide AP, 0.25 g of ceramide EOP, 0.1 g of 1,2-hexanediol, and 0.1 g of p-hydroxyacetophenone.

[0044] The preparation method thereof is the same as that of Test Example 1.

[0045] Test Example 3 A ceramide nanoemulsion, the components of which include 70 g of water, 30 g of glycerin, 5 g of white pool seed oil, 5 g of tocopheryl acetate, 5 g of caprylic / capric triglyceride, 5 g of hydrogenated lecithin, 5 g of hexyldecanol, 4 g of polyglyceryl-10 myristate, 1.5 g of white pool seed ceramide NP, 0.75 g of ceramide AP, 0.75 g of ceramide EOP, 1 g of 1,2-hexanediol, and 1 g of p-hydroxyacetophenone.

[0046] The preparation method is the same as that of Test Example 1.

[0047] Comparative Example 1 A ceramide nanoemulsion, the components of which include 63.3 g of water, 20 g of glycerin, 3 g of white pool seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 0.5 g of white pool seed ceramide NP, 0.1 g of ceramide AP, 0.1 g of ceramide EOP, 0.5 g of 1,2-hexanediol, and 0.5 g of p-hydroxyacetophenone. The preparation method is the same as that of Test Example 1.

[0048] Comparative Example 2 A ceramide nanoemulsion, the components of which include 60 g of water, 20 g of glycerin, 3 g of white pool seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 2 g of white pool seed ceramide NP, 1 g of ceramide AP, 1 g of ceramide EOP, 0.5 g of 1,2-hexanediol, and 0.5 g of p-hydroxyacetophenone. The preparation method is the same as that of Test Example 1, and the thickening and clumping during the preparation process are as shown in Figure 1 .

[0049] Comparative Example 3 A ceramide nanoemulsion, the components of which include 65 g of water, 20 g of glycerin, 0 g of white pool seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 1 g of white pool seed ceramide NP, 0.5 g of ceramide AP, 0.5 g of ceramide EOP, 0.5 g of 1,2-hexanediol, and 0.5 g of p-hydroxyacetophenone. The preparation method is the same as that of Test Example 1.

[0050] Comparative Example 4 A ceramide nanoemulsion whose components include 62 g of water, 20 g of glycerin, 3 g of white pool flower seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 1 g of ceramide 3B, 0.5 g of ceramide AP, 0.5 g of ceramide EOP, 0.5 g of 1,2-hexanediol, 0.5 g of p-hydroxyacetophenone. Its preparation method is the same as that of Test Example 1.

[0051] Comparative Example 5 A ceramide nanoemulsion whose components include 63 g of water, 20 g of glycerin, 3 g of white pool flower seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 1 g of white pool flower seed ceramide NP, 0 g of ceramide AP, 0 g of ceramide EOP, 0.5 g of 1,2-hexanediol, 0.5 g of p-hydroxyacetophenone. Its preparation method is the same as that of Test Example 1.

[0052] Comparative Example 6 A ceramide nanoemulsion whose components include 62.5 g of water, 20 g of glycerin, 3 g of white pool flower seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 1 g of white pool flower seed ceramide NP, 0.5 g of ceramide AP, 0 g of ceramide EOP, 0.5 g of 1,2-hexanediol, 0.5 g of p-hydroxyacetophenone. Its preparation method is the same as that of Test Example 1.

[0053] Comparative Example 7 A ceramide nanoemulsion whose components include 62.5 g of water, 20 g of glycerin, 3 g of white pool flower seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 1 g of white pool flower seed ceramide NP, 0 g of ceramide AP, 0.5 g of ceramide EOP, 0.5 g of 1,2-hexanediol, 0.5 g of p-hydroxyacetophenone. Its preparation method is the same as that of Test Example 1.

[0054] Comparative Example 8 A ceramide nanoemulsion whose components include 63 g of water, 20 g of glycerin, 3 g of white pool flower seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 myristate, 0 g of white pool flower seed ceramide NP, 0.5 g of ceramide AP, 0.5 g of ceramide EOP, 0.5 g of 1,2-hexanediol, 0.5 g of p-hydroxyacetophenone. Its preparation method is the same as that of Test Example 1.

[0055] Comparative Example 9 A ceramide nanoemulsion, the components of which include 62 g of water, 20 g of glycerol, 3 g of white pool flower seed oil, 3 g of tocopheryl acetate, 3 g of caprylic / capric triglyceride, 3 g of hydrogenated lecithin, 2 g of hexyldecanol, 1 g of polyglyceryl-10 laurate, 1 g of white pool flower seed ceramide NP, 0.5 g of ceramide AP, 0.5 g of ceramide EOP, 0.5 g of 1,2-hexanediol, and 0.5 g of p-hydroxyacetophenone. The preparation method is the same as that of Test Example 1.

[0056] Effect Example 1 Stability Test Method: 1) Centrifugal stability test: Take 10 mL of each of the above samples and place them in a 15 mL centrifuge tube. Centrifuge at a speed of 4000 rpm for 30 minutes. Observe and record whether the sample has stratification, flocculation, or precipitation.

[0057] 2) Long-term observation at room temperature: Place each sample in a transparent glass bottle and store it at room temperature (25°C ± 2°C) and normal humidity for 30 days.

[0058] 3) Evaluation criteria: Uniform emulsion: The sample remains milky white, has good flowability, and has no visible stratification, oil separation, or precipitation.

[0059] Cannot form a uniform emulsion / demulsification: The sample has oil-water separation, clumping, cream rupture, or other phenomena after preparation or during storage.

[0060] The test results are shown in Table 1.

[0061] Table 1 Stability of ceramide nanoemulsion

[0062] Effect Example 2 After storing the ceramide nanoemulsion samples 1, 2, and 3 prepared in Test Example 1 at 45°C, -15°C, 5°C, and room temperature (25°C) for one month, their particle size, PdI value, Zeta potential, and ceramide content were tested. The particle size, PdI value, and Zeta potential of the initial state of the above samples were tested as a control group for reference. The specific test results are shown in Table 2.

[0063] Table 2 Performance test results of Test Example 1 samples after storage at different temperatures for one month

[0064] Effect Example 3 HaCaT human keratinocyte viability test Assay Method: MTT assay for cell proliferation: HaCaT cells were seeded at a density of 1×10⁴ cells / well in 96-well plates and incubated overnight. After 24 hours, the supernatant was discarded, and 100 μL of culture medium containing different concentrations of samples (products obtained from Experimental Examples 1-3 and Comparative Examples 1-9) or a blank was added. After incubation for another 24 hours, the culture medium was removed, and 100 μL of thiazolyl blue (MTT) was added to each well. The absorbance at 450 nm was measured, and the cell viability was calculated as (Adrug-treated well / Ablank well) × 100%.

[0065] Test results are as follows Figure 2 As shown.

[0066] Conclusion: Figure 2 As shown, compared with the blank control group, samples 1-3 in experiments significantly promoted the proliferation of HaCaT cells at the tested concentrations, with cell survival rates reaching over X%, demonstrating excellent cell repair capabilities. In contrast, the cell-promoting effects of Comparative Example 1 (low ceramide content), Comparative Example 4 (using ordinary ceramide 3B instead of BCH), and Comparative Examples 5-8 (lacking any one or two of the three ceramides) were significantly lower than those of Experiment 1. In particular, Comparative Example 8 (without meadowfoam seed ceramide NP) showed no difference in effect from ordinary emulsions. These results fully demonstrate the synergistic effect of the combination of meadowfoam seed ceramide NP, ceramide AP, and ceramide EOP, which is crucial for promoting skin barrier repair.

[0067] Example 4: Anti-inflammatory effect test Assay Method: RAW macrophages were seeded at a density of 1×10^4 cells / well in 96-well plates and incubated overnight. After 24 hours, the supernatant was discarded, and 100 μL of samples diluted with DMEM medium at different concentrations were added. The negative control group received DMEM medium without samples, and the positive control group received 40 μM quercetin. Each group was in triplicate, and the plates were incubated at 37°C with 5 wt% CO2. Two hours after drug administration, 10 μg / mL LPS was added to both the lipopolysaccharide model group and the experimental group, and they were co-incubated for 24 hours. After the reaction, 50 μL of cell supernatant was collected, and the relative expression level of IL-6 mRNA was measured by RT-PCR.

[0068] Test results are as follows Figure 3 As shown.

[0069] Conclusion: Figure 3As shown, in the LPS-induced inflammation model, samples 1-3 significantly reduced the mRNA expression level of the inflammatory cytokine IL-6 in RAW 264.7 macrophages, and their anti-inflammatory effects were close to or even superior to the positive control quercetin at some concentrations. However, the anti-inflammatory effects of samples 1, 4, 5, and 8 were significantly weaker than those of sample 1. This indicates that the specific ceramide composition provided by this invention, especially when containing meadowfoam seed ceramide NP, can exhibit unexpectedly excellent anti-inflammatory activity.

[0070] Example 5: Moisturizing Efficacy Test Test method: Aquaporin 3 (AQP3) is a transporter protein factor on the cell membrane responsible for the transport of substances such as water, glycerol, and urea. It is mainly expressed in keratinocytes and skin fibroblasts. AQP3 not only participates in skin moisturizing and barrier function, but also plays an important role in skin damage and repair, healing, and anti-aging. It is an important guarantee for maintaining the normal morphology and function of the skin.

[0071] Human keratinocytes (HaCat cells) were seeded at a density of 1 × 10⁴ cells / well in 96-well plates and incubated overnight. After 24 hours, the supernatant was discarded. 100 μL of different concentrations of the products obtained in Experiments 1-3 and Comparative Examples 1-9 were added to DMEM medium. The blank control group was prepared with drug-free DMEM medium. Each group had three replicates. The experimental and blank control groups were dried in a clean bench at a fan speed of 0.4 m / s for 20 min, and then incubated at 37°C with 5% CO₂ for 24 h. After the reaction, 50 μL of the cell supernatant was collected, and the expression of AQP3 cytokines was detected using an AQP3 assay kit.

[0072] Test results are as follows Figure 4 As shown.

[0073] Conclusion: Figure 4 As shown, samples 1-3 significantly upregulated the expression of aquaporin AQP3 in HaCaT cells, indicating that it can fundamentally enhance the hydration capacity of skin cells and achieve deep moisturization. In contrast, the upregulation effect of all comparative samples (Comparative Examples 1-9) on AQP3 was less than that of Sample 1. Among them, Comparative Example 5 (without AP and EOP) and Comparative Example 8 (without BCH) showed the worst effects. This further confirms the functional complementarity and synergy of the three ceramides, which together constitute a complete skin moisturizing network.

[0074] Example 6: Collagen Expression Test Test Methods: Collagen Col-1 is mainly found in the skin, and its content is closely related to the skin's elasticity and resilience. In addition, it also has functions such as skin repair and wound healing. Therefore, the effect of Col-1 expression in human foreskin fibroblasts (HFF-1) on improving skin elasticity and promoting wound healing can be studied.

[0075] HFF-1 cells were seeded at a density of 1×10^6 cells / well in 6-well plates and incubated overnight in a 5% CO2 incubator at 37°C. After 24 h, the supernatant was discarded, and 100 μL of culture medium containing different concentrations of the sample was added. The blank control group was DMEM medium without the drug, and the positive control was 0.025 mM EGCG. After 24 h of drug administration, each group was divided into 3 replicates, and the expression level of Col-1 was detected using a Col-1 kit.

[0076] Test results are as follows Figure 5 As shown.

[0077] Conclusion: Figure 5 As shown, samples 1-3 significantly promoted the expression of type I collagen (COL1A1) in human fibroblasts HFF-1, exhibiting a dose-dependent effect within a certain concentration range, and their promoting effect was superior to the positive control EGCG. The comparative samples, however, showed weaker promoting effects on COL1A1 than those in sample 1. These experimental results demonstrate that the ceramide nanoemulsion of this invention can effectively stimulate collagen regeneration, exhibiting clear anti-aging and skin elasticity-enhancing effects, and that this efficacy depends on the complete combination of the three ceramides.

[0078] Example 7: Nematode Permeability Test (Nile Red) Test sample 1: Nanoemulsion prepared in Experiment Example 1.

[0079] Test Sample 2: A mixture of ceramides alone, without excipients, namely, a mixture of meadowfoam seed ceramide NP, ceramide AP, and ceramide EOP in a mass ratio of 1:0.5:0.5.

[0080] Test method: 1. Nematodes synchronized to the L4 stage (48h).

[0081] 2. Preparation of Nile Red Working Solution: Dilute NR working solution with M9 (1 0.5 0.25 0 ppm), with 3 replicates for each concentration. Store in the dark and prepare fresh before use. Staining solution: Mix NR working solution with 0.1% Triton X-100 at a 1:1 ratio.

[0082] 3. Nematode staining: 30-50 nematodes were transferred to a centrifuge tube, 500 uL of staining solution was added and mixed well; incubated at room temperature for 45 min; after incubation, washed with M9 and centrifuged (3000 rpm, 1 min) for 3 times, and the surface NR of the nematodes was completely removed.

[0083] 4. Fluorescence imaging: the stained nematodes were transferred to a glass slide, anesthetized with 10 mM NaN3, and then observed and photographed under a fluorescence microscope for permeability.

[0084] The test results are shown in Figure 6 、 Figure 7 、 Figure 8

[0085] Conclusion: The permeability experiment results show that, after the ceramide composition of the present application is wrapped in the form of nanoemulsion, although the permeability of the nanoemulsion is not as good as that of the unwrapped ceramide at 0.25 ppm and 0.5 ppm, there is no obvious difference in permeability between the two at 1 ppm, and in the nanoemulsion sample, the ceramide content is only 2%, which indicates that, when the same permeability effect is achieved, the total amount of ceramide contained in the nanoemulsion of the present application is much lower than the amount required by the pure ceramide mixture. It is calculated that the permeability efficiency of the present nanoemulsion preparation is about 50 times higher than that of the pure ceramide mixture without wrapping.

[0086] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A ceramide nanoemulsion, characterized in that, Its components include meadowfoam seed ceramide NP, ceramide AP and ceramide EOP.

2. The ceramide nanoemulsion according to claim 1, characterized in that, The mass ratio of ceramide NP, ceramide AP and ceramide EOP in the meadowfoam seeds is 1:(0.25-0.75):(0.25-0.75).

3. The ceramide nanoemulsion according to claim 1, characterized in that, Its components also include an oil phase and an aqueous phase, wherein the oil phase comprises meadowfoam seed oil, tocopheryl acetate, caprylic / capric triglyceride, hexyldecyl alcohol and polyglycerol-10 myristate, and the aqueous phase comprises water, glycerol, hydrogenated lecithin, 1,2-hexanediol and p-hydroxyacetophenone.

4. The ceramide nanoemulsion according to claim 3, characterized in that, Its components, in parts by weight, include 0.8-1.5 parts meadowfoam seed ceramide NP, 0.25-0.75 parts ceramide AP, 0.25-0.75 parts ceramide EOP, 1-5 parts meadowfoam seed oil, 1-5 parts tocopheryl acetate, 1-5 parts caprylic / capric triglyceride, 1-5 parts hexyldecyl alcohol, 1-4 parts polyglycerol-10 myristate, 50-70 parts water, 10-30 parts glycerol, 1-5 parts hydrogenated lecithin, 0.1-1 part 1,2-hexanediol, and 0.1-1 part p-hydroxyacetophenone.

5. The ceramide nanoemulsion according to claim 1, characterized in that, The initial particle size of the nanoemulsion is less than 90 nm, and after one month of storage, the particle size change is less than 10%.

6. A method for preparing a ceramide nanoemulsion according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Mix the oil phase components, meadowfoam seed oil, tocopheryl acetate, caprylic / capric triglyceride, hexyldecyl alcohol, polyglycerol-10 myristate, and meadowfoam seed ceramide NP, ceramide AP, and ceramide EOP in the specified weight proportions; heat until dissolved; S2: Mix the aqueous phase components, including water, glycerin, hydrogenated lecithin, 1,2-hexanediol and p-hydroxyacetophenone, in the indicated weight proportions and heat until dissolved; S3: Add the mixture prepared in step S1 to the aqueous phase described in step 2 and emulsify, stirring until homogeneous; S4: Homogenize the emulsified mixture from step S3 under high pressure.

7. The preparation method according to claim 6, characterized in that, In step S1, the heating temperature is 80-90℃; in step S2, the heating temperature is 80-85℃. Preferably, in step S4, the high pressure is 100-140 MPa.

8. The use of any one of the ceramide nanoemulsions according to claims 1-5 in the preparation of cosmetics for skin care.

9. The application according to claim 8, characterized in that, The cosmetics are used for moisturizing, anti-inflammatory, or anti-aging purposes.

10. The application according to claim 8, characterized in that, The cosmetic product is a lotion, cream, or serum.

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