Dual ceramide essence inclusion as well as preparation method and application thereof

Abstract

The invention provides a dual ceramide essence inclusion. The dual ceramide essence inclusion is prepared from the following components in parts by weight: 1-6 parts of an active ingredient, 2-8 parts of an amphiphilic inclusion material and 40-70 parts of a microbe-derived vesicle material, wherein the active ingredients comprise ceramide NS / ceramide NG and ceramide AS; the amphiphilic inclusion material comprises the active component to form nanoparticles; the nanoparticle is wrapped by the microbe-derived vesicle material to form a ceramide / amphiphilic inclusion material / vesicle compound. The invention further provides a preparation method and application of the dual ceramide essence inclusion. The dual ceramide essence inclusion provided by the invention is high in stability and good in skin permeability, and has good repairing and anti-aging effects.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of cosmetic technology, in particular to a dual ceramide essence package, a preparation method and application thereof. BACKGROUND

[0002] Skin, as the largest organ of the human body, its barrier function is the key guarantee to maintain the stability of the body environment and resist external stimuli. The integrity of the skin barrier mainly depends on the structure and function of the stratum corneum, and Filaggrin (FLG) is an indispensable key protein in the stratum corneum, which not only provides a scaffold for the extracellular lipid matrix and maintains the mechanical integrity of the stratum corneum, but also plays a core role in regulating the water-retaining capacity of the epidermis and maintaining the acidic pH value. When the FLG content is missing or functionally abnormal, it will cause the skin barrier function to be damaged, and problems such as dryness, desquamation, sensitivity, itching, etc. will occur, and long-term even cause more serious skin conditions such as skin aging and inflammation.

[0003] Ceramide, as an important component of the skin stratum corneum lipid, is a natural active ingredient for maintaining the skin barrier function. It stimulates the synthesis of FLG by promoting the expression of caspase 14, and realizes the repair and homeostasis regulation of the skin barrier. Therefore, ceramide is widely used in skin care products, medical dressings and other fields to improve dry, sensitive and aging skin problems.

[0004] However, the ceramide products applied in the prior art have many limitations: (1) poor stability: the unsaturated bond in the molecular structure of ceramide is easily affected by environmental factors such as light, temperature and oxygen, which leads to oxidative degradation and loss of activity, thereby affecting the efficacy stability of the product; (2) insufficient solubility: ceramide is a fat-soluble ingredient, and its oil-water amphiphilic chemical structure makes it not completely soluble in water or oil, so there are certain difficulties in formula application, and the amount is also limited; (3) single component efficacy limitation: single type ceramide has limited effect on repairing skin barrier and increasing FLG content, and it is difficult to meet the repair needs of complex skin problems (such as multiple aging and barrier damage combined with sensitivity); (4) encapsulation technology defects: although the existing technology uses liposome, nanoemulsion and other encapsulation technologies to improve the stability and permeability of ceramide, there are problems such as low encapsulation rate (usually less than 60%), limited drug loading capacity, uncontrollable release rate (such as burst release leading to local concentration too high to cause irritation, or insufficient sustained release affecting immediate efficacy), and poor biocompatibility of some encapsulation materials, which may cause skin allergy risk.

[0005] With the increasing demand of consumers for the efficacy, safety and stability of skin care products, the development of a ceramide preparation with high stability, excellent skin permeability and the ability to synergistically increase FLG content has become a research hotspot in the field. SUMMARY

[0006] In view of the deficiencies in the prior art, the present disclosure provides a dual ceramide essence package, which has high stability and good skin permeability and good repair and anti-aging effects.

[0007] In a first aspect, the present disclosure provides a dual ceramide essence package, comprising the following components by weight: 1-6 parts of active ingredients, 2-8 parts of amphiphilic inclusion materials, and 40-70 parts of microbial-derived vesicle materials.

[0008] The active ingredients include ceramide NS / ceramide NG and ceramide AS.

[0009] The amphiphilic inclusion material includes the active ingredients to form nanoparticles.

[0010] The microbial-derived vesicle material includes the nanoparticles to form a ceramide / amphiphilic inclusion material / vesicle complex.

[0011] The ceramide / amphiphilic inclusion material / vesicle complex of the present disclosure is a complex formed by using the amphiphilic inclusion material to include the active ingredients (ceramide NS / ceramide NG and ceramide AS) to form nanoparticles, and then using the vesicle material to include the nanoparticles. The use of the amphiphilic inclusion material to include the ceramide improves the amphiphilicity of the ceramide, and improves the water solubility, dispersibility and stability of the ceramide. The vesicle material is a biological membrane vesicle structure formed by microbial fermentation, which can solve the problems of the solubility and stability of the ceramide, and has a very similar structure to the human cell membrane. Compared with the traditional liposome inclusion method, the vesicle material can help to improve the transdermal and intracellular ability of the ceramide, so that the ceramide can play a better repair and anti-aging role in the skin care product system.

[0012] Different subtypes of ceramides and fatty acids aggregate in a highly ordered crystalline phase at physiological temperatures, but the interactions of the head groups are very different. The transverse hydrogen bonds of ceramide AS model increase the cohesion within the bilayer plane. In contrast, the transverse hydrogen bonds between ceramide NS / ceramide NG molecules can increase the significant integrity of the barrier structure through the bilayer. There is a strong bonding between the head groups of ceramide NS / ceramide NG, in addition to having a transverse orthogonal chain organization, there is also a transverse organization. However, the head group interactions collapse with the melting of the ceramide NS / ceramide NG chains. In contrast, ceramide AS maintains strong head group hydrogen bonds between the transverse organizations of ceramide AS even after the chain conformation is disordered.

[0013] Based on this, the ceramide NS / ceramide NG and ceramide AS in the present disclosure are selected to form a dual ceramide. After the two subtypes are compounded, the interaction between the head groups is strengthened, a more stable complex structure is formed, the problem of easy loss of activity caused by the single structure of single ceramide is reduced, and the foundation for subsequent efficacy is laid. Therefore, when the dual ceramide is used in cosmetics, the function of the skin barrier can be synergistically enhanced, and the stability of the skin structure can be improved.

[0014] In some preferred embodiments of the present disclosure, the microorganism comprises a strain of Saccharomyces and a strain of Bacillus. Further, in some embodiments, the strain of Saccharomyces is selected from any one or a combination of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Saccharomyces diastaticus. In other embodiments, the strain of Bacillus is selected from Bacillus subtilis.

[0015] In the present disclosure, the vesicle material can be commercially available or prepared by conventional methods. In some embodiments, the microorganism-derived vesicle material is obtained by fermentation of microorganisms through a fermentation process, for example, the fermentation product obtained after fermentation culture, wall breaking, purification, decolorization and odor removal, and concentration of yeast is the vesicle material. A representative method for preparing a vesicle material is disclosed in patent WO2022055250.

[0016] In some preferred embodiments of the present disclosure, the amphiphilic inclusion material includes, but is not limited to, at least one of lecithin, sphingomyelin, and cholesterols. Further, in some embodiments, the amphiphilic inclusion material is lecithin.

[0017] In the present disclosure, the ratio of ceramide NS / ceramide NG and ceramide AS in the active ingredient is preferably (1-2):(1-2), for example, it can be 1:1, 1:1.5, 1:2, 1.5:1, 2:1, etc. In preferred embodiments, the ratio of ceramide NS / ceramide NG and ceramide AS is 1:1.

[0018] Further, in some preferred embodiments, the dual ceramide essence package comprises, by weight parts, the following components: 2-4 parts of active ingredient, 3-6 parts of amphiphilic inclusion material, and 50-60 parts of microorganism-derived vesicle material.

[0019] Further, the dual ceramide essence package comprises, by weight parts, the following components: 3 parts of active ingredient, 5 parts of amphiphilic inclusion material, and 52 parts of microorganism-derived vesicle material.

[0020] Further, the ratio of ceramide NS / ceramide NG and ceramide AS in the active ingredient is 1:1.

[0021] In a second aspect, the present disclosure provides a preparation method of the dual ceramide essence package, comprising the following steps:

[0022] S1. providing the active ingredient, the amphiphilic inclusion material, and the microbial-derived vesicle material;

[0023] S2. mixing the active ingredient, the amphiphilic inclusion material, and the microbial-derived vesicle material to obtain the dual ceramide essence package.

[0024] In some preferred embodiments of the present disclosure, the vesicle material is prepared by the following steps:

[0025] S3. culturing the yeast in a culture medium, and obtaining a precipitate after centrifugation;

[0026] S4. crushing the precipitate obtained in step S3 to obtain the vesicle material.

[0027] In some preferred embodiments of the present disclosure, the culture medium is a liquid culture medium. Further preferably, the liquid culture medium is selected from a nutrient broth medium.

[0028] In some preferred embodiments of the present disclosure, the crushing is performed by homogenization using a homogenizer.

[0029] In some preferred embodiments of the present disclosure, in step S4, the vesicle material is further separated and purified, and the separation and purification is performed using a solid chromatographic column.

[0030] In a third aspect, the present disclosure provides use of the dual ceramide essence package in the preparation of a cosmetic product.

[0031] In some preferred embodiments of the present disclosure, the dual ceramide essence package is used to increase the filaggrin (FLG) content and / or promote the migration of human keratinocyte (HaCaT) cells.

[0032] In some preferred embodiments of the present disclosure, the dual ceramide essence package is used for skin repair and / or anti-aging.

[0033] In a fourth aspect, the present disclosure provides a cosmetic product comprising: the dual ceramide essence package described above; and a cosmetically acceptable carrier or excipient.

[0034] In some preferred embodiments of the present disclosure, in the cosmetic product, the amount of the dual ceramide essence package added is 1-10 wt%, for example, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, etc.

[0035] In some preferred embodiments of the present disclosure, the aforementioned dual ceramide essence package can be prepared into various cosmetic compositions, such as emulsions, liquids, ointments, creams, pastes, cakes, powders, and the like, by using conventional methods.

[0036] In the present disclosure, other ingredients commonly used in cosmetics, such as solvents, film formers, humectants, preservatives, antibacterial agents, fragrances, antioxidants, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, skin conditioning ingredients (whitening agents, cell activity agents, skin roughness improvement agents, blood circulation promoters, skin astringents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, and the like, can be added without affecting the effects of the present disclosure.

[0037] The cosmetic of the present disclosure includes, but is not limited to, skin care cosmetics, color cosmetics, and ultraviolet protection cosmetics. For example, base skin care cosmetics such as emulsions, creams, lotions, sunscreens, mask materials, facial cleansers, and essences; color cosmetics such as foundations, white powders, and blushes; and the like.

[0038] The cosmetic of the present disclosure is not particularly limited in form and can be in a liquid, emulsion, cream, solid, paste, gel, powder, multi-layer, mousse, spray, or the like.

[0039] Compared with the prior art, the present disclosure has the following beneficial effects:

[0040] 1. The dual ceramide essence package provided by the present disclosure uses amphiphilic inclusion materials and vesicle materials to encapsulate ceramides, making the ceramides have better amphiphilicity, solving the problem of poor solubility of ceramides in water, thereby greatly reducing the difficulty of application of ceramides and facilitating the use of ceramides in various cosmetic product formulations. Moreover, the dual ceramide essence package has good transdermal efficiency and can promote active ingredients to penetrate into the dermis of the skin, thereby better exerting the efficacy thereof.

[0041] 2. The dual ceramide essence package provided by the present disclosure has good repair and anti-aging effects and can be applied to cosmetics.

[0042] 3. The preparation method of the dual ceramide essence package provided by the present disclosure is simple in operation, low in cost, high in efficiency, simple in process, and suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Toxicity of the dual ceramide-human immortalized keratinocyte to Example 1-3, Comparative Example 2-6 samples;

[0044] Figure 2 Toxicity of the dual ceramide-human immortalized keratinocyte to Example 1-3, Comparative Example 2-6 samples;

[0045] Figure 3 Figure 1 is a graph showing the FLG content of each group after sample processing for Examples 1-3 and Comparative Examples 1-6;

[0046] Figure 4 Figure 2 is a graph showing the cell migration rate of each group after sample processing for Examples 1-3 and Comparative Examples 1-6;

[0047] Figure 5 Figure 3 is a graph showing the results of the cell migration experiment; a: blank control group; b: positive control group; c: double ceramide essence wrapper group of Example 1; d: double ceramide group of Comparative Example 1. DETAILED DESCRIPTION

[0048] The technical solutions of the present disclosure will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present disclosure. Any technology implemented based on the above description of the present disclosure is encompassed within the scope intended to be protected by the present disclosure.

[0049] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0050] Example 1

[0051] The present example provides a double ceramide essence wrapper, which is a semi-liquid to paste, light yellow, with a pH of 5.5-7.5. The raw materials of the double ceramide essence wrapper include the following components by mass percentage:

[0052] Yeast-derived vesicle material 52%, glycerol 30%, hexyldecanol 10%, lecithin 5%, ceramide NS / ceramide NG 1.5%, ceramide AS 1.5%.

[0053] The preparation method of the double ceramide essence wrapper of the present example is as follows:

[0054] (1) A suitable amount of lecithin is weighed and ground in a colloid mill. Then, ceramide NS / ceramide NG and ceramide AS are weighed and added to the colloid mill, and are thoroughly mixed and ground. A large amount of deionized water is added to fully dissolve the mixed lecithin and ceramide powders. The solution is then homogenized by a high-pressure microjet homogenizer at a pressure of 30 MPa. After several times of homogenization, the solution is collected and placed in a freeze dryer to remove the solvent at -80°C to obtain lecithin-encapsulated ceramide nanoparticles.

[0055] (2) Dissolve the freeze-dried yeast powder in a nutrient broth liquid (NB) medium. Transfer the dissolved bacteria solution to a sterile test tube containing a certain amount of liquid medium and mix well. The activated bacteria are inoculated into a conical flask containing NB medium and cultured under appropriate conditions with shaking. After the culture is completed, the fermentation broth is centrifuged to obtain a bacterial precipitate. The precipitate is resuspended in a certain volume ratio of phosphate buffer. The buffer solution containing the bacterial fragments is homogenized by a high-pressure microjet homogenizer at a pressure of 30 MPa. The solution containing the bacterial fragments is passed through a solid chromatography column to separate, purify, and collect the vesicle fraction.

[0056] (3) A certain amount of vesicle fraction is diluted with phosphate buffer. A certain amount of ceramide nanometer particles coated with lecithin is weighed. The two are mixed and placed in an ultrasonic instrument for ultrasonic treatment. The liquid is then placed in a high-pressure extruder with nitrogen as the pressure source, extruded through a polycarbonate membrane, and then dried and powdered in the bottom of a centrifugal collection. The resulting product is a double ceramide essence wrapper.

[0057] Example 2

[0058] The present example provides a double ceramide essence wrapper, which is prepared from the following components by mass percentage: 60% of vesicle material from yeast, 25% of glycerol, 6% of hexyldecanol, 6% of lecithin, 2% of ceramide NS / ceramide NG, and 1% of ceramide AS.

[0059] Example 3

[0060] The present example provides a double ceramide essence wrapper, which is prepared from the following components by mass percentage: 45% of vesicle material from Bacillus, 30% of glycerol, 19% of hexyldecanol, 3% of lecithin, 1% of ceramide NS / ceramide NG, and 2% of ceramide AS.

[0061] Comparative Example 1

[0062] The present comparative example provides a double ceramide, which includes ceramide NS / ceramide NG and ceramide AS in a ratio of 1:1.

[0063] Comparative Example 2

[0064] The present comparative example provides a double ceramide liposome, which is prepared from the following components by mass percentage: 1.5% of ceramide NS / ceramide NG, 1.5% of ceramide AS, 7% of lecithin, 1.5% of cholesterol, 0.05% of vitamin C palmitate, 25% of ethanol, and the balance of deionized water.

[0065] The preparation method of the double ceramide liposome of the present comparative example is as follows:

[0066] (1) Take ceramide, lecithin, cholesterol, vitamin C palmitate, add anhydrous ethanol, 50℃ water bath stirring until completely dissolved, to obtain a lipid ethanol solution;

[0067] (2) The water phase is preheated to 55℃, and the lipid ethanol solution is slowly injected into the water phase under magnetic stirring with a constant flow pump at a flow rate of 1 mL / min. Continue stirring for 30 minutes to form the colostrum;

[0068] (3) Transfer the mixed solution to a rotary evaporator, rotate at 40℃, vacuum degree 0.08MPa for 15 minutes to remove ethanol;

[0069] (4) Cool the colostrum to room temperature, and process it with a high-pressure homogenizer: pressure 800bar, cycle 3 times, to obtain double ceramide liposomes with a particle size of 100-200nm. Sterilize with a 0.22μm filter membrane for standby.

[0070] Comparative Example 3

[0071] This comparative example provides a ceramide essence package, the raw materials of which include the following components by mass percentage: yeast-derived vesicle material 52%, glycerol 30%, hexyldecanol 10%, lecithin 5%, ceramide NS / ceramide NG 3%.

[0072] Comparative Example 4

[0073] This comparative example provides a ceramide essence package, the raw materials of which include the following components by mass percentage: yeast-derived vesicle material 52%, glycerol 30%, hexyldecanol 10%, lecithin 5%, ceramide AS 3%.

[0074] Comparative Example 5

[0075] This comparative example provides a double ceramide essence package, the raw materials of which include the following components by mass percentage: yeast-derived vesicle material 52%, glycerol 30%, hexyldecanol 10%, lecithin 5%, ceramide NS / ceramide NG 2.5%, ceramide AS 0.5%.

[0076] Comparative Example 6

[0077] This comparative example provides a double ceramide essence package, the raw materials of which include the following components by mass percentage: yeast-derived vesicle material 52%, glycerol 30%, hexyldecanol 10%, lecithin 5%, ceramide NS / ceramide NG 0.5%, ceramide AS 2.5%.

[0078] Test Example 1: Determination of FLG index in HaCaT cell aging model based on TNF-α and IFN-γ stimulation

[0079] 1. Experimental consumables and instruments

[0080] 60mm cell culture dish (purchased from NEST), human immortalized keratinocytes HaCaT (purchased from the Chinese Academy of Sciences Cell Bank), FLG detection kit (purchased from Nanjing Jianshen Biological Engineering Institute), PBS (cell culture grade, purchased from biosharp), 1640 basal medium (purchased from Gibco), Australian fetal bovine serum protein (purchased from Gibco), trypsin protein (purchased from Wuhan Punuo Sai Biological Technology Co., Ltd.), JTC-801 (purchased from Shanghai Yuanye Biological Technology Co., Ltd.), TNF-α (purchased from Biyun Tian), IFN-γ (purchased from Biyun Tian).

[0081] fluorescence inverted microscope (ICX41, Shunyu Optical Technology (Group) Co., Ltd.), enzyme marker (CMax Plus, American Meigu Molecular Instrument (Shanghai) Co., Ltd.

[0082] 2. Test method

[0083] 2.1 Cytotoxicity experiment

[0084] (1) MTT cytotoxicity test grouping and concentration gradient: blank control group (1640 basal medium), comparative example 1 group (1 ppm, 5 ppm, 10 ppm, 20 ppm), examples 1-3 and comparative examples 2-6 group (33.3 ppm, 166.67 ppm, 333.33 ppm, 666.67 ppm), correction group (PBS without cells).

[0085] (2) HaCaT cells were passaged to T25 cell culture bottles and cultured to a density of more than 80%, trypsinized, centrifuged to collect cells, resuspended with cell culture medium, and diluted with the medium to a cell number of 10×10 4 cells / mL.

[0086] (3) The medium containing 10×10 4 cells / mL HaCaT cells was evenly added to the 96-well plate, 100 μL per well, and the cells were cultured in a cell incubator at 37°C and 5% CO2 for 24 h; the original culture medium of the 96-well plate was discarded, 100 μL of 1640 basal medium was added to each well of the blank group, and 6 parallel duplicate wells were set; the blank control group, examples 1-3 group, comparative examples 1-6 group were added with gradient concentration medium containing double ceramide, double ceramide liposome or double ceramide essence package in each well, and 6 parallel duplicate wells were set for each concentration; the correction group was added with 100 μL PBS in the wells without cells to set 6 parallel duplicate wells; and the culture was continued for 24 h.

[0087] (4) After the completion, discard the original culture medium, wash each well with PBS three times, add 120 μL of 1 mg / mL MTT diluted in culture medium to each well, and continue to culture for 4 h; after the completion, discard the original culture medium of the 96-well plate, wash each well with PBS three times, add 150 μL of DMSO to each well to fully dissolve the crystals, detect OD490 nm with a microplate reader, and calculate the cell survival rate.

[0088] Cell survival rate (%) = (Ax-A) / (A0-A) × 100%

[0089] Ax: absorbance of incubated double ceramide, double ceramide liposome, and double ceramide essence package at 490 nm; A: absorbance of calibration well at 490 nm; A0: absorbance of blank well at 490 nm.

[0090] 2.2 Detection of filaggrin content

[0091] (1) Cell culture and model construction

[0092] Immortalized human keratinocytes were digested and passaged into 60 mm culture dishes in a cell culture incubator at 37°C, 5% CO2, until the cells reached a density of over 70%. The culture medium was discarded, the cells were washed with PBS, and culture medium containing 0.15 ppm of the dual-ceramide sample and 5 ppm of dual-ceramide liposomes or dual-ceramide essence was added, respectively, for a pre-incubation of 24 hours. The culture medium was then replaced with 10 ng / mL TNF-α and 10 ng / mL INF-γ, and the cells were incubated for 48 hours. After completion, the culture medium was discarded, the cells were trypsinized, centrifuged at 2000 g, resuspended in PBS, centrifuged, and washed twice. Finally, the pellet was collected by centrifugation at 2000 g, 0.5 mL of cell lysis buffer was added, and the cells were thoroughly lysed by pipetting. The cells were then briefly stored at 4°C for subsequent testing; triplicates were set up for each group.

[0093] (2) Detection of filaggrin content

[0094] Filaggrin content assay: The supernatant after centrifugation was diluted with PBS to the appropriate multiple for subsequent testing. The assay was performed according to the instructions for the Human FLG ELISA kit from the Nanjing Jiancheng Bioengineering Institute. The standard curve was fitted with a logistic curve, and data were analyzed using SPSS statistical software. Results are expressed as mean ± standard deviation (X ± SD). One-way analysis of variance was used for comparisons between groups; p < 0.05 was considered significant. **, p < 0.01, n = 3, vs. blank control; ##, p < 0.01, n = 3, vs. model group.

[0095] Calculate protein increase rate:

[0096] Improvement rate (%) = (Bx-B) / B×100%

[0097] B: Model group protein content; Bx: Sample group protein content.

[0098] 3. Test results

[0099] 3.1 Cell toxicity test results

[0100] Table 1-2, Figures 1-2 Table 1-2 and Table 2 respectively show the results of the effect of the samples of Examples 1-3 and Comparative Examples 1-6 on the survival rate of human immortalized keratinocytes.

[0101] Table 1-2 and Table 2 respectively show the results of the effect of the samples of Examples 1-3 and Comparative Examples 1-6 on the survival rate of human immortalized keratinocytes.

[0102] Double ceramide concentration (ppm) 0 1 5 10 20 Cell survival rate (%) 100.00 101.18 99.20 94.90 87.07 Standard deviation (%) 1.94 5.20 2.58 4.34 5.29

[0103] Table 2 shows the results of the effect of the samples of Examples 1-3 and Comparative Examples 2-6 on the survival rate of human immortalized keratinocytes.

[0104]

[0105]

[0106] Table 1-2 and Table 2 respectively show the results of the effect of the samples of Examples 1-3 and Comparative Examples 1-6 on the survival rate of human immortalized keratinocytes. Figures 1-2 Table 1-2 and Table 2 respectively show the results of the effect of the samples of Examples 1-3 and Comparative Examples 1-6 on the survival rate of human immortalized keratinocytes.

[0107] 3.2 FLG detection test results

[0108] Table 3 shows the results of the FLG content.

[0109]

[0110]

[0111] Table 1-2 and Table 2 respectively show the results of the effect of the samples of Examples 1-3 and Comparative Examples 1-6 on the survival rate of human immortalized keratinocytes. Figure 3 Table 1-2 and Table 2 respectively show the results of the effect of the samples of Examples 1-3 and Comparative Examples 1-6 on the survival rate of human immortalized keratinocytes.

[0112] Compared with the model group, the FLG content of the dual ceramide essence package of Examples 1-3 increased significantly, with an increase rate of more than 67%, and the dual ceramide essence package of Example 1 had the highest increase rate of 74.29%.

[0113] In Comparative Example 1, the FLG content of the 5 ppm dual ceramide group also increased significantly, with an increase rate of 47.00%, but was significantly lower than that of the dual ceramide essence package of Example 1. In Comparative Example 2, the FLG content of the dual ceramide liposome also increased significantly, reaching 56.01%, but the increase rate was also lower than that of Example 1. This is because the dual ceramide essence package of Example 1 has better transdermal efficiency, which can promote the penetration of active ingredients into the dermis of the skin and better exert its efficacy.

[0114] Compared with the model group, the FLG content of the ceramide essence package of Comparative Examples 3 and 4 also increased significantly, with an increase rate of 40.84% and 49.69%, respectively, but was significantly lower than that of the dual ceramide essence package of Example 1. This indicates that the ratio of the use of ceramide NS / ceramide NG and ceramide AS is significantly better than that of a single ceramide for the increase of FLG content.

[0115] FLG is a key protein in the stratum corneum, and its content directly affects the skin barrier function, while ceramide stimulates the synthesis of FLG by promoting the expression of caspase 14. A single ceramide (such as only ceramide NS / NG or only ceramide AS) has a single stimulation pathway for FLG synthesis and is limited by its range of action in the stratum corneum; in the dual ceramide, ceramide NS / NG provides a physical scaffold for FLG synthesis by enhancing the barrier structure, and ceramide AS provides microenvironment support for FLG synthesis by maintaining hydrogen bond stability, and the use of both can synergistically activate the expression pathway of caspase 14, thereby amplifying the stimulation effect on FLG synthesis, so the increase rate of FLG of the dual ceramide essence package in Example 1 is significantly higher than that of the ceramide essence package of Comparative Examples 3-4.

[0116] Compared with the model group, the FLG content of the ceramide essence package of Comparative Examples 5 and 6 also increased significantly, with an increase rate of 54.19% and 56.58%, respectively, but was significantly lower than that of the dual ceramide essence package of Example 1. This indicates that the ratio of the use of ceramide NS / ceramide NG and ceramide AS needs to be controlled at (1-2):(1-2), and exceeding this ratio will result in a significant decrease in the increase of FLG content.

[0117] Test Example Two: Determination of Relative Migration Rate Index Based on HaCaT Cell Migration

[0118] 1. Experimental consumables and instruments

[0119] HaCaT cells were purchased from the Chinese Academy of Sciences. PBS (cell culture grade, purchased from biosharp), special fetal bovine serum (purchased from Wuhan Punuo Sai Biological Technology Co., Ltd.), DMEM medium (purchased from Gibco), DMSO (biological grade, purchased from Sigma).

[0120] Fluorescence inverted microscope (ICX41, Shunyu Optics Technology (Group) Co., Ltd.).

[0121] 2. Test method

[0122] (1) HaCaT cells were cultured to more than 80% density, then digested and passaged into 60mm cell culture dishes, and continued to be cultured to more than 95% cell density. 200μL pipette gun was vertically drawn in the culture dish to form a straight line, and PBS was used to wash off the detached cells.

[0123] (2) The sample group was added with 5ppm double ceramide, 166.67ppm double ceramide liposome or double ceramide essence package diluted with serum-free medium; the blank control group was added with 3mL of serum-free medium; the positive control group was added with 3mL of 10% special fetal bovine serum medium; and then placed in the incubator for 24h at 37℃ and 5% CO2.

[0124] (3) After the end, PBS was used for washing, and the cells were observed under the microscope to observe the migration state, and the relative migration rates of each group were compared.

[0125] Relative migration rate (%) = (S0-Sn) / S0x100% Formula 1

[0126] In formula 1:

[0127] S0 is the migration area at 0h;

[0128] Sn is the migration area of the blank control group and the sample group at 18h.

[0129] Migration promotion rate (%) = (Am-A0) / A0x100% Formula 2

[0130] In formula 2:

[0131] Am: migration rate of the sample group;

[0132] A0: migration rate of the blank control group.

[0133] The data were analyzed by SPSS statistical software, and the results were expressed as mean ± standard deviation (X ± SD). One-way ANOVA was used for comparison between groups, and p < 0.05 was considered statistically significant. **, p < 0.01, n = 3, Vs blank control group; ****, p < 0.0001, n = 3, Vs blank control group.

[0134] 3. Test results

[0135] Table 4 Summary of cell migration test results

[0136] Group Sample name Sample concentration Relative migration rate (%) Migration enhancement rate (%) Blank control group / / 18.19±4.58 / Positive control group Premium fetal bovine serum 10％ 55.92±2.22**** 207.42％ Example 1 group Double ceramide essence wrap 166.67 ppm 39.34±2.33**** 116.27％ Example 2 group Double ceramide essence wrap 166.67 ppm 39.81±2.17**** 118.86％ Example 3 group Double ceramide essence wrap 166.67 ppm 38.74±1.82**** 112.97％ Comparative example 1 group Double ceramide 5 ppm 28.55±1.86** 56.95％ Comparative example 2 group Double ceramide liposome 166.67 ppm 26.71±2.03** 46.84％ Comparative example 3 group Ceramide essence wrap 166.67 ppm 29.86±2.10** 64.16％ Comparative example 4 group Ceramide essence wrap 166.67 ppm 27.78±1.15** 52.72％ Comparative example 5 group Double ceramide essence wrap 166.67 ppm 31.28±1.62** 71.96％ Comparative example 6 group Double ceramide essence wrap 166.67 ppm 32.80±1.46** 80.32％

[0137] See Table 4 and Figure 4 Compared with the blank control group, the relative migration rate of the cells in the positive control group was significantly increased, indicating that the positive control was effective.

[0138] Compared with the blank control group, the cell migration rate of the double ceramide essence encapsulates of Examples 1-3 was significantly increased, reaching more than 110%.

[0139] In Comparative Example 1, the relative migration rate of the cells in the 5 ppm double ceramide group was also significantly increased compared with the blank control group, with an increase of 56.95%, but the migration rate was much lower than that of the double ceramide essence encapsulate of Example 1. In Comparative Example 2, the cell migration rate of the double ceramide liposome group reached 46.84%, which was also much lower than that of the double ceramide essence encapsulate of Example 1.

[0140] In Comparative Examples 3-4, the relative migration rates of the cells in the ceramide essence encapsulate groups were 64.16% and 52.72%, respectively, which were still much lower than that of the double ceramide essence encapsulate of Example 1. This indicates that the single ceramide essence encapsulate has a lower effect on promoting the migration of HaCaT cells than the double ceramide essence encapsulate.

[0141] Cell migration is a key process in skin repair. Single ceramide can only affect cell migration through a single pathway of improving the barrier or providing local signals, with limited effect. In double ceramide, ceramide NS / NG reduces the interference of external stimuli on cell migration by enhancing barrier integrity, and ceramide AS provides a stable matrix environment for cell migration by maintaining the cohesion of the stratum corneum. The two ceramides synergistically promote the migration signal pathway of keratinocytes, and thus can significantly promote the migration of HaCaT cells.

[0142] In Comparative Examples 5-6, the cell relative migration rates of the ceramide essence package groups were 71.96% and 80.32%, respectively, which were still much lower than that of the double ceramide essence package of Example 1. This indicates that the ratio of ceramide NS / ceramide NG and ceramide AS needs to be controlled at (1-2):(1-2) when used in combination, and exceeding this ratio will result in a significant decrease in the promotion effect on cell migration rate.

[0143] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present disclosure and are not limiting. Although the present disclosure has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present disclosure can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present disclosure, and they should be covered in the scope of the claims of the present disclosure.

Claims

1. A double ceramide essence package, characterized in that: The invention comprises the following components in parts by weight: 1 to 6 parts of active ingredient, 2 to 8 parts of amphiphilic inclusion material and 40 to 70 parts of vesicle material derived from microorganisms; Wherein, the active ingredients include ceramide NS / ceramide NG and ceramide AS; The amphiphilic inclusion material includes the active ingredient to form nanoparticles; The microbial-derived vesicle material wraps the nanoparticles to form a ceramide / amphiphilic inclusion material / vesicle complex.

2. The double ceramide essence package according to claim 1, characterized in that: The microorganisms include strains of the genus Saccharomyces and strains of the genus Bacillus.

3. The double ceramide essence package according to claim 1, characterized in that: The amphiphilic inclusion material includes at least one of lecithin, sphingomyelin, and cholesterol.

4. The double ceramide essence package according to claim 1, characterized in that: In the active ingredient, the ratio of ceramide NS / ceramide NG to ceramide AS is (1-2):(1-2).

5. The double ceramide essence package according to claim 1, characterized in that: The double ceramide essence package comprises the following components in parts by weight: 2 to 4 parts of active ingredients, 3 to 6 parts of amphiphilic inclusion materials and 50 to 60 parts of vesicle materials derived from microorganisms.

6. A method for preparing a double ceramide essence package according to claims 1 to 5, characterized in that: The following steps are involved: S1. Providing the active ingredient, amphiphilic inclusion material and microbial-derived vesicle material; S2. Mixing the active ingredient, the amphiphilic inclusion material and the microbial-derived vesicle material to obtain the dual ceramide essence package.

7. Use of the double ceramide essence package according to any one of claims 1 to 5 in the preparation of cosmetics.

8. A cosmetic, characterized in that: The invention comprises: a double ceramide essence package according to any one of claims 1 to 5; and a cosmetically acceptable carrier or excipient.

9. The cosmetic according to claim 8, characterized in that In the cosmetic, the added amount of the double ceramide essence package is 1 to 10%.

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