DEJ layer repairing composition, anti-aging cream containing DEJ layer repairing composition and preparation method of anti-aging cream

Abstract

The invention relates to the technical field of daily cosmetics, in particular to a DEJ layer repairing composition, anti-aging cream containing the DEJ layer repairing composition and a preparation method of the anti-aging cream. The DEJ layer repairing composition is prepared from ceramide NP, phytosterol oleate and fatty acid. Through compounding of the components, expression of type 7 collagen, type 17 collagen and laminin is effectively promoted, and the effect of strengthening the anti-aging ability of the skin is achieved by repairing a DEJ layer of the skin and improving epidermal communication and skin homeostasis.

Description

Technical Field

[0001] The present application relates to the technical field of daily cosmetics, and more specifically, to a DEJ layer repairing composition, an anti-aging cream containing the DEJ layer repairing composition, and a preparation method thereof. Background Art

[0002] The skin is mainly divided into the epidermis and dermis. The dermis is divided into papillary area and reticular area. The epidermis and dermis are inlaid with finger-like protrusions. The papillary dermis protrudes into the epidermis to form dermal papillae. The part of the epidermis extending into the papillary dermis forms the epidermal foot. The connection and transition area between the epidermis and dermis is the dermal junction zone (DEJ), also known as the basement membrane zone (BMZ).

[0003] DEJ acts as a complex signaling platform that controls dermal-epidermal signaling and plays an important role in maintaining healthy epidermis and dermis. Studies have shown that during the intrinsic aging process, not only does DEJ undergo morphological changes, but more importantly, the protein composition also changes.

[0004] As the skin ages intrinsically, the DEJ becomes significantly thinner and takes on a flattened appearance with reduced surface area due to the loss of ridges. Studies have shown that the DEJ of aged skin flattens by approximately 35%. As a result, aged skin is less resistant to shear forces and more susceptible to injury. Protein reduction occurs in the four regions that make up the DEJ. These changes in protein composition lead to a loss of DEJ function.

[0005] In a young state, the DEJ is intact and has a healthy wave shape. But as we age, it gradually flattens out. If the connection becomes completely flat, signal communication and nutrient exchange will stop, the skin will lose elasticity and firmness, and wrinkles will begin to form. Therefore, the DEJ layer is crucial in the skin aging process, and it is urgent to provide a DEJ layer repair composition, an anti-aging cream containing the DEJ layer repair composition, and a preparation method thereof. Summary of the invention

[0006] In order to achieve targeted repair of the DEJ layer of the skin and effectively fill the gap in the field of deep skin anti-aging, the present application specifically provides a DEJ layer repair composition, an anti-aging cream containing the DEJ layer repair composition and a preparation method thereof.

[0007] In a first aspect, the present application provides a DEJ layer repair composition, which is compounded by ceramide NP, phytosterol oleate and fatty acid.

[0008] Preferably, the mass ratio of ceramide NP, phytosterol oleate and fatty acid is (0.49-1.26):(0.29-1.06):(0.23-0.92).

[0009] Preferably, the mass ratio of ceramide NP, phytosteryl oleate and fatty acid is 0.49:1.02:0.29.

[0010] Preferably, the fatty acid is behenic acid, stearic acid or palmitic acid.

[0011] In a second aspect, the present application provides an application of a DEJ layer repair composition in the preparation of an anti-aging product and / or an anti-aging method, and the product includes a cream, a gel, an ointment, and a patch.

[0012] In a third aspect, the present application provides an anti-aging cream containing a DEJ layer repair composition, including any one of the above-mentioned DEJ layer repair compositions.

[0013] Preferably, it is composed of the following components in mass percentage: Ammonium acryloyldimethyltaurate / VP copolymer 0.1-0.3%, glycerol 5-10%, caprylic / capric triglyceride 20-25%, DEJ layer repair composition 1.5-2.0%, emulsifier 4-6%, and the balance is water.

[0014] Preferably, ammonium acryloyldimethyltaurate / VP copolymer 0.2%, glycerol 7%, caprylic / capric triglyceride 23.2%, DEJ layer repair composition 1.8%, emulsifier 5%, and the balance is water.

[0015] Preferably, the emulsifier is composed of the following components in mass percentage: Arachidyl alcohol 55 wt%, behenyl alcohol 30 wt%, arachidyl glucoside 15 wt%.

[0016] In a fifth aspect, the present application provides a preparation method of an anti-aging cream containing a DEJ layer repair composition, and the specific preparation steps are as follows: 1) Add ammonium acryloyldimethyltaurate / VP copolymer, glycerol and water into the water phase pot according to the corresponding mass parts, stir and disperse evenly, and heat to 85-90 °C to obtain a mixture A for later use; 2) Add caprylic / capric triglyceride, the DEJ layer repair composition, and the emulsifier into the oil phase pot, heat to 85-90 °C, and stir evenly to obtain a mixture B for later use; 3) Add the mixture B into the mixture A, and homogenize at 5000-7000 rpm for 4-6 min; 4) Stir and cool down to 40 °C, and discharge to obtain an anti-aging cream for repairing the DEJ layer of the skin.

[0017] In summary, the present application has the following beneficial effects: 1. Through the compounding of ceramide NP + phytosteryl oleate + fatty acid, the present application effectively promotes the expression of type VII and type XVII collagens and laminin, thereby achieving the effect of repairing the dermal-epidermal junction (DEJ) layer of the skin; 2. The DEJ layer repair composition composed of the compounding of ceramide NP + phytosteryl oleate + behenic acid, when applied to the anti-aging cream system, benefits from the use of special emulsifiers and (lyotropic liquid crystal) emulsification technology, significantly enhancing the stability and bioavailability of each component, and strengthening the original repair effect by forming a lamellar liquid crystal structure similar to that of the healthy stratum corneum; 3. The DEJ layer repair composition in the present application has broad application prospects. Taking the relatively mature anti-aging product - cream at the present stage as an example, while the preparation method is simple, due to the specific component selection and morphology of the DEJ repair composition, its application effect is significantly improved, and the product form of the cream is more conducive to its attachment and use and the retention of the stability of each component. Description of the Drawings

[0018] Figure 1 It is a microscopic image of HE staining of an in vitro reconstructed human skin model; Among them, 1-a is the microscopic image of HE staining of the in vitro reconstructed human skin model in blank group A; Figure 1 -b is the microscopic image of HE staining of the in vitro reconstructed human skin model in model group A; Figure 1 -c is the microscopic image of HE staining of the in vitro reconstructed human skin model in Example 1; Figure 1 -d is the microscopic image of HE staining of the in vitro reconstructed human skin model in Example 2; Figure 1 -e is the microscopic image of HE staining of the in vitro reconstructed human skin model in Example 3; Figure 1 -f is the microscopic image of HE staining of the in vitro reconstructed human skin model in Comparative Example 1; Figure 1 -g is the microscopic image of HE staining of the in vitro reconstructed human skin model in Comparative Example 2; Figure 1 -h is the microscopic image of HE staining of the in vitro reconstructed human skin model in Comparative Example 3; Figure 1 -i is the microscopic image of HE staining of the in vitro reconstructed human skin model in Comparative Example 4; Figure 1 -j is the microscopic image of HE staining of the in vitro reconstructed human skin model in blank group B; Figure 1 -k is the microscopic image of HE staining of the in vitro reconstructed human skin model in model group B; Figure 1 -l is the microscopic image of HE staining of the in vitro reconstructed human skin model in Example 4; Figure 1 -m is the microscopic image of HE staining of the in vitro reconstructed human skin model in Example 5; Figure 2 is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model; Among them Figure 2 -a is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in blank group A; Figure 2 -b is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in model group A; Figure 2 -c is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Example 1; Figure 2 -d is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Example 2; Figure 2 -e is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Example 3; Figure 2 -f is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Comparative Example 1; Figure 2 -g is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Comparative Example 2; Figure 2 -h is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Comparative Example 3; Figure 2 -i is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Comparative Example 4; Figure 2 -j is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in blank group B; Figure 2 -k is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in model group B; Figure 2 -l is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Example 4; Figure 2 -m is the microscopic image of COL17A1 immunofluorescence of the in vitro reconstructed human skin model in Example 5; Figure 3 is the column statistical chart of the relative fluorescence intensity of COL17A1 immunofluorescence of the in vitro reconstructed human skin model; Among them Figure 3-a is a bar chart of the relative fluorescence intensity of COL17A1 immunofluorescence in the in vitro reconstructed human skin model for the blank group A, model group A, Examples 1-3, and Comparative Examples 1-4; Figure 3 -b is a bar chart of the relative fluorescence intensity of COL17A1 immunofluorescence in the in vitro reconstructed human skin model for the blank group B, model group B, and Examples 4-5; Figure 4 It is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model; Among them, 4-a is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of the blank group A; Figure 4 -b is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of the model group A; Figure 4 -c is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Example 1; Figure 4 -d is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Example 2; Figure 4 -e is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Example 3; Figure 4 -f is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Comparative Example 1; Figure 4 -g is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Comparative Example 2; Figure 4 -h is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Comparative Example 3; Figure 4 -i is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Comparative Example 4; Figure 4 -j is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of the blank group B; Figure 4 -k is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of the model group B; Figure 4 -l is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Example 4; Figure 4 -m is a micrograph of COL7A1 immunofluorescence in the in vitro reconstructed human skin model of Example 5; Figure 5 It is a bar chart of the relative fluorescence intensity of COL17A1 immunofluorescence in the in vitro reconstructed human skin model; Among them, 5-a is a column statistical chart of the relative fluorescence intensity of COL17A1 immunofluorescence in the in vitro reconstructed human skin model in blank group A, model group A, Examples 1-3, and Comparative Examples 1-4; Figure 5 -b is a column statistical chart of the relative fluorescence intensity of COL17A1 immunofluorescence in the in vitro reconstructed human skin model in blank group B, model group B, and Examples 4-5; Figure 6 It is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model; Among them, 6-a is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of blank group A; Figure 6 -b is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of model group A; Figure 6 -c is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Example 1; Figure 6 -d is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Example 2; Figure 6 -e is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Example 3; Figure 6 -f is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Comparative Example 1; Figure 6 -g is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Comparative Example 2; Figure 6 -h is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Comparative Example 3; Figure 6 -i is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Comparative Example 4; Figure 6 -j is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of blank group B; Figure 6 -k is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of model group B; Figure 6 -l is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Example 4; Figure 6 -m is a micrograph of Laminin-5 immunofluorescence in the in vitro reconstructed human skin model of Example 5; Figure 7 It is a bar chart of the relative fluorescence intensity of COL17A1 immunofluorescence in an in vitro reconstructed human skin model; Among them, 7-a is a bar chart of the relative fluorescence intensity of COL17A1 immunofluorescence in the in vitro reconstructed human skin model in blank group A, model group A, Examples 1-3, and Comparative Examples 1-4; Figure 7 -b is a bar chart of the relative fluorescence intensity of COL17A1 immunofluorescence in the in vitro reconstructed human skin model in blank group B, model group B, and Examples 4-5; Figure 8 It is the 400-fold microstructural image of the anti-aging cream in Application Example 1 (scale unit: 10um); Figure 9 It is the 400-fold microstructural image of the anti-aging cream in Application Example 2 (scale unit: 10um); Figure 10 It is the 400-fold microstructural image of the anti-aging cream in Application Example 3 (scale unit: 10um); Figure 11 It is the 400-fold microstructural image of the anti-aging cream in Application Example 4 (scale unit: 10um); Figure 12 It is the 400-fold microstructural image of the anti-aging cream in Application Example 5 (scale unit: 10um); Figure 13 It is the 400-fold microstructural image of the anti-aging cream in Comparative Application Example 1 (scale unit: 10um); Figure 14 It is the 400-fold microstructural image of the anti-aging cream in Comparative Application Example 2 (scale unit: 10um); Figure 15 It is the 400-fold microstructural image of the anti-aging cream in Comparative Application Example 3 (scale unit: 10um); Figure 16 It is the 400-fold microstructural image of the anti-aging cream in Comparative Application Example 4 (scale unit: 10um). Detailed implementation manners

[0019] The following further elaborates on this application in combination with examples and Figure 1-16 provides more detailed descriptions.

[0020] Performance detection test I Select the prepared DEJ layer repair compositions in each example and comparative example for detection. The specific detection items, reference standards, and steps are as follows: Establishment of the SLS damage model and processing of test samples Tested with a three-dimensional reconstructed full-thickness skin model produced by Skinology (Shanghai). The groups are as follows: Blank group A: PBS, Blank group B: PBS; Model group A: 0.2 wt% sodium lauryl sulfate (SLS), Model group B: 0.2 wt% sodium lauryl sulfate (SLS); Test sample group: 0.2 wt% SLS + Example 1, 0.2 wt% SLS + Example 2, 0.2 wt% SLS + Example 3, 0.2 wt% SLS + Comparative Example 1, 0.2 wt% SLS + Comparative Example 2, 0.2 wt% SLS + Comparative Example 3, 0.2 wt% SLS + Comparative Example 4, 0.2 wt% SLS + Example 4, 0.2 wt% SLS + Example 5; After the three-dimensional reconstructed full-thickness skin model is processed according to the standard operating procedure, the test is carried out. The drug treatment time is 24 h. After 24 h, the three-dimensional reconstructed full-thickness skin model is taken for fixation, paraffin sectioning, in order to complete subsequent HE staining, COL17A1, COL7A1, Laminin-5 immunohistochemistry and immunofluorescence experiments.

[0021] 1) HE staining of in vitro reconstructed human skin model Table 1. Main detection equipment for the experiment Name Model Manufacturer Upright fluorescence microscope MF52-N Mshot Table 2. Main reagents used in the experiment Name Specification Manufacturer In vitro reconstructed full-thickness skin model 1 skin model / well Skinol 10% Formalin 500 mL / bottle Solarbio, Beijing Modified hematoxylin and eosin (HE) staining kit 4 * 100 mL Solarbio Neutral balsam 100 mL / bottle Solarbio Staining method of HE for in vitro reconstructed human skin model: Fix with 10% formalin for more than 24 h, embed the tissue, perform pathological sectioning, dewax with clearing and dewaxing solution (I) for 15 min, dewax with clearing and dewaxing solution (II) for 15 min, dewax with clearing and dewaxing solution (Ⅲ) for 15 min, 5 min with absolute ethanol (I), 3 min with absolute ethanol (II), 3 min with 95% ethanol, 3 min with 85% ethanol, 1 min with 75% ethanol, wash with water for 2 min, stain with hematoxylin for 5 min, wash with water for 5 s, differentiate with 1% hydrochloric acid ethanol for 1 s, wash with water for 15 s, 1% ammonia water for 1 min, wash with water for 1 min, stain with eosin for 3 min, wash with water for 15 s, 95% ethanol for 1 s, absolute ethanol (I) for 10 s, absolute ethanol (II) for 1 min, clear with clearing and dewaxing solution (I) for 10 min, clear with clearing and dewaxing solution (II) for 10 min, clear with clearing and dewaxing solution (Ⅲ) for 10 min, seal with neutral gum, take pictures and observe under an upright microscope. The staining results are shown in Figures 1-a, 1-b, 1-c, 1-d, 1-e, 1-f, 1-g, 1-h, 1-i, 1-j, 1-k, 1-l, 1-m in the attached instructions. Figure 1 in Figures 1-a, 1-b, 1-c, 1-d, 1-e, 1-f, 1-g, 1-h, 1-i, 1-j, 1-k, 1-l, 1-m in the attached instructions.

[0022] 2) Immunofluorescence of COL17A1 in in vitro reconstructed human skin model Table 3. Main detection equipment for the experiment Name Model Manufacturer Upright fluorescence microscope MF52-N Mshot Table 4. Main reagents used in the experiment Name Specification Manufacturer In vitro reconstructed full-thickness skin model 1 skin model / well Skinol 10% Formalin 500 mL / bottle Solarbio, Beijing Recombinant Anti-Collagen XVII antibody 50 μL / tube abcam Alexa Fluor 488-conjugated goat anti-rabbit IgG 100 μL / tube Beyotime Anti-fluorescence quenching mounting medium 25 mL Beyotime Staining method for immunofluorescence of COL17A1 in in vitro reconstructed human skin model: Fix with 10% formalin for more than 24 h, embed the tissue, perform pathological sectioning, use Clear Dewaxing Solution I for 15 min, Clear Dewaxing Solution II for 15 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 95% ethanol solution for 3 min, 80% ethanol solution for 3 min, wash with tap water for 3 min × 3 times, wash with distilled water for 3 min × 3 times; Antigen retrieval: Immerse the slides in 2% EDTA buffer solution, boil in a water bath at 100 °C for about 10 min, keep slightly boiling at 95 °C for about 15 min, cool naturally to room temperature, wash with distilled water for 3 min × 3 times, wash with PBS solution for 3 min × 3 times, add endogenous peroxidase blocker, incubate at room temperature for 10 min, wash with PBS for 3 min × 3 times, permeabilize with 0.1% TritonX-100 for 10 min, wash with PBS for 3 min × 3 times, add 50% goat serum (diluted with PBS) for blocking, incubate at room temperature for 30 min, pour off the serum without washing, add recombinant Anti-Collagen XVII antibody working solution (diluted 1:100 with blocking solution), incubate overnight at 4 °C, wash with PBS for 3 min × 3 times, add Alexa Fluor 488-labeled goat anti-rabbit IgG (diluted 1:200 with PBS) and incubate at room temperature for 1 h, wash with PBS for 3 min × 3 times, add anti-fluorescence quenching mounting medium (containing DAPI) for mounting, take pictures and observe under an upright microscope. The results are shown in Appendix Figure 2 2-a, 2-b, 2-c, 2-d, 2-e, 2-f, 2-g, 2-h, 2-i, 2-j, 2-k, 2-l, 2-m in the manual. Analyze the fluorescence intensity of the image data using imageJ, referring to Figure 3 3-a and 3-b in the manual.

[0023] 3) Immunofluorescence of COL7A1 in in vitro reconstructed human skin model Table 5. Main detection equipment for the experiment Name Model Manufacturer Upright fluorescence microscope MF52-N Mshot Table 6. Main reagents used in the experiment Staining method for immunofluorescence of COL7A1 in in vitro reconstructed human skin model: Fix with 10% formalin for more than 24 h, embed the tissue, perform pathological sectioning, use Clear Dewaxing Solution Ⅰ for 15 min, Clear Dewaxing Solution Ⅱ for 15 min, absolute ethanol Ⅰ for 5 min, absolute ethanol Ⅱ for 5 min, 95% ethanol solution for 3 min, 80% ethanol solution for 3 min, wash with tap water for 3 min × 3 times, and wash with distilled water for 3 min × 3 times; Antigen retrieval: Immerse the glass slides in 2% EDTA buffer solution, boil in a water bath at 100 °C for about 10 min, keep boiling gently at 95 °C for about 15 min, cool naturally to room temperature, wash with distilled water for 3 min × 3 times, wash with PBS solution for 3 min × 3 times, add endogenous peroxidase blocker dropwise, incubate at room temperature for 10 min, wash with PBS for 3 min × 3 times, permeabilize with 0.1% TritonX-100 for 10 min, wash with PBS for 3 min × 3 times, add 50% goat serum (diluted with PBS) dropwise for blocking, incubate at room temperature for 30 min, pour off the serum, do not wash, add recombinant Anti-Collagen VII antibody working solution (diluted 1:100 with blocking solution) dropwise, incubate overnight at 4 °C, wash with PBS for 3 min × 3 times, add Alexa Fluor 488-labeled goat anti-rabbit IgG (diluted 1:200 with PBS) and incubate at room temperature for 1 h, wash with PBS for 3 min × 3 times, add anti-fluorescence quenching mounting medium (containing DAPI) for mounting, and take pictures and observe under an upright microscope. The results are shown in Appendix Figure 4 4-a, 4-b, 4-c, 4-d, 4-e, 4-f, 4-g, 4-h, 4-i, 4-j, 4-k, 4-l, 4-m in the instruction manual. Analyze the fluorescence intensity of the image data using imageJ, referring to Appendix Figure 5 5-a and 5-b in the instruction manual.

[0024] 4) Immunofluorescence of Laminin-5 in in vitro reconstructed human skin model Table 7. Main detection equipment for the experiment Name Model Manufacturer Upright fluorescence microscope MF52-N Mshot Table 8. Main reagents used in the experiment Name Specification Manufacturer In vitro reconstructed full-thickness skin model 1 skin model / well Skinol 10% Formalin 500 mL / bottle Solarbio, Beijing Anti-Laminin 5 antibody 50 μL / tube abcam Alexa Fluor 488-conjugated goat anti-rabbit IgG 100 μL / tube Beyotime Anti-fluorescence quenching mounting medium 25 mL Beyotime Staining method for immunofluorescence of Laminin-5 in in vitro reconstructed human skin model: Fix with 10% formalin for more than 24 h, embed the tissue, perform pathological sectioning, use Clear Dewaxing Solution Ⅰ for 15 min, Clear Dewaxing Solution Ⅱ for 15 min, absolute ethanol Ⅰ for 5 min, absolute ethanol Ⅱ for 5 min, 95% ethanol solution for 3 min, 80% ethanol solution for 3 min, wash with tap water for 3 min × 3 times, and wash with distilled water for 3 min × 3 times; Antigen retrieval: Soak the glass slides in 2% EDTA buffer solution, boil them in a water bath at 100 °C for about 10 minutes, keep them boiling gently at 95 °C for about 15 minutes after the water temperature reaches 95 °C, cool them naturally to room temperature, wash them with distilled water for 3 minutes × 3 times, wash them with PBS solution for 3 minutes × 3 times, add endogenous peroxidase blocker dropwise, incubate at room temperature for 10 minutes, wash with PBS for 3 minutes × 3 times, permeabilize with 0.1% TritonX-100 for 10 minutes, wash with PBS for 3 minutes × 3 times, add 50% goat serum (diluted with PBS) for blocking, incubate at room temperature for 30 minutes, pour off the serum without washing, add the working solution of Anti-Laminin 5 antibody (diluted 1:200 with the blocking solution), incubate overnight at 4 °C, wash with PBS for 3 minutes × 3 times, add Alexa Fluor488-labeled goat anti-rabbit IgG (diluted 1:200 with PBS) and incubate at room temperature for 1 hour, wash with PBS for 3 minutes × 3 times, add anti-fluorescence quenching mounting medium (containing DAPI) for mounting, take pictures and observe under an upright microscope. The results are shown in Appendix Figure 6 in 6-a, 6-b, 6-c, 6-d, 6-e, 6-f, 6-g, 6-h, 6-i, 6-j, 6-k, 6-l, 6-m. Analyze the fluorescence intensity of the image data using imageJ, referring to Appendix Figure 7 in 7-a and 7-b of the specification.

[0025] Examples 1-3 A DEJ layer repair composition, the components and their corresponding masses (g) are shown in the following table. Among them, the fatty acid is specifically selected from behenic acid; The trade name of the phytosterol oleate is: HYY-98 (95% phytosterol oleate, 5% phytosterols), manufacturer: Hunan Heyiyuan Biotechnology Co., Ltd.

[0026] Table 9: Components and their masses (g) in Examples 1-3 Comparative Examples 1-4 A DEJ layer repair product, different from Example 1 in that each component is replaced by an equal amount of a single or partial component. The specific selection and its corresponding mass are as follows in the table.

[0027] Table 10: Components and their masses (g) in Comparative Examples 1-4 Extract the repair compositions or products in Examples 1-3 and Comparative Examples 1-4 above, and observe the relative fluorescence intensities of HE staining, COL17A1 immunofluorescence, COL7A1 immunofluorescence, and Laminin-5 immunofluorescence of the in vitro reconstructed human skin model according to the above measurement steps. The test results are recorded in the following table.

[0028] Table 11: Performance test results of Examples 1-3 and Comparative Examples 1-4 From the above table and Figure 1-4 it can be seen that the DEJ layer repair composition in Examples 1-3 effectively achieved the repair of the skin DEJ layer, with the relative fluorescence intensity of COL17A1 being 0.47±0.11 - 0.65±0.07, the relative fluorescence intensity of COL7A1 being 0.57±0.06 - 0.72±0.15, and the relative fluorescence intensity of Laminin-5 being 0.96±0.13 - 1.12±0.13; In summary, combining the data of each example, it can be seen that through the compounding of ceramide NP + phytosteryl oleate + behenic acid in this application, the expression of type VII collagen (COL7A1), type XVII collagen (COL17A1), and laminin 5 (Laminin-5) was effectively promoted. Referring to Figure 2-7 and from the Figure 1 skin model constructed therein, it can also be seen that the expression sites are basically completely consistent with those of normal skin; However, from the comparison between Comparative Examples 1-4 and Examples 1-3, it can be seen that a single or partial component cannot achieve a similar effect. The expression sites of the skin models constructed are quite different from those of normal skin, and the relative fluorescence intensity of COL17A1 is only 0.34±0.03 - 0.42±0.10, the relative fluorescence intensity of COL7A1 is only 0.25±0.06 - 0.40±0.06, and the relative fluorescence intensity of Laminin-5 is only 0.54±0.14 - 0.67±0.21; Therefore, it can be concluded that the DEJ layer repair composition in this application effectively fills the gap in the existing products or technologies for repairing DEJ layer damage and anti-aging of the deep skin mechanism, and has extremely high market value, especially when applied to anti-aging products. Taking anti-aging cream as an example, in addition, due to the specific component selection and form of the DEJ repair composition, its application effect is significantly improved, and the product form of the cream is more conducive to its attachment and use and the retention of the stability of each component.

[0029] Examples 4-5 A DEJ layer repair composition, which is different from Example 1 in that the fatty acid is specifically selected from stearic acid or palmitic acid, and its specific selection and corresponding mass (g) are shown in the following table.

[0030] Table 12: Components and their masses (g) in Examples 4-5 Extract the repair composition or product in Examples 4-5 above, and observe the relative fluorescence intensities of HE staining, COL17A1 immunofluorescence, COL7A1 immunofluorescence, and Laminin-5 immunofluorescence of the in vitro reconstructed human skin model according to the above measurement steps. Record the test results in the following table.

[0031] Table 13: Performance test results of Examples 4-5 As can be seen from the above table and Figure 1-7 It can be seen that the DEJ layer repair composition in Examples 4-5 effectively achieved the repair of the skin DEJ layer. The relative fluorescence intensity of COL17A1 was 0.56±0.11 - 0.74±0.05, the relative fluorescence intensity of COL7A1 was 0.56±0.01 - 0.74±0.14, and the relative fluorescence intensity of Laminin-5 was 0.58±0.05 - 0.79±0.09; In summary, combining the data of each example, it can be seen that the fatty acid can be selected from any one of behenic acid, stearic acid or palmitic acid, and all of them can effectively promote the expression of collagen type 7 (COL7A1), collagen type 17 (COL17A1) and laminin 5 (Laminin-5). Refer to Figure 2-7 and from Figure 1 it can also be seen from the skin model constructed therein that the expression sites are basically completely consistent with those of normal skin.

[0032] It should be particularly noted here that: the selection of the fatty acid from any one of behenic acid, stearic acid or palmitic acid is an other example derived from subsequent derivation and optimization. Therefore, it is not from the same batch as Examples 1-3 and Comparative Examples 1-4 before. Its model group and blank group were reconstructed by the same method. For the convenience of distinction, they are denoted as model group B and blank group B, and their statistical charts were made separately.

[0033] Performance test II Select the anti-aging creams prepared in each application example and comparative application example for testing. Specifically, observe the anti-aging cream under a polarizing microscope at 400 times magnification to see if a Maltese cross is formed to judge whether it is a liquid crystal emulsion system. If so, record it as ◎, if not, record it as X.

[0034] Application Examples 1-5 An anti-aging cream, the components thereof and their corresponding masses (g) are shown in the following table, and it is prepared by the following preparation method, wherein the DEJ layer repair composition is prepared from Examples 1-3 respectively: 1) Add ammonium acryloyldimethyltaurate / VP copolymer, glycerol and water to the water phase pot according to the corresponding mass parts, stir and disperse evenly, and heat to 90 °C to obtain the mixture A for use; 2) Add triglyceride caprylate / caprate, DEJ layer repair composition, and emulsifier into the oil phase pot, heat to 90 °C, and stir evenly to obtain mixture B for later use; 3) Add mixture B into mixture A and homogenize at 6000 rpm for 5 min; 4) Stir and cool down to 40 °C, then discharge to obtain the anti-aging cream for repairing the skin DEJ layer.

[0035] Table 14: Components and their masses (g) in Application Examples 1 - 5 Among them, the acryloyldimethyltaurate / VP copolymer has the brand name Aristoflex AVC and is purchased from Clariant Chemical Technology (Shanghai) Co., Ltd. The triglyceride caprylate / caprate has the brand name RADIA 7104 and is purchased from Oriane Industry (Shanghai) Co., Ltd. The emulsifier is composed of 55 wt% of arachidyl alcohol, 30 wt% of behenyl alcohol, and 15 wt% of arachidyl glucoside.

[0036] Comparative Application Examples 1 - 4 A cream, different from Application Example 1 in that the specific usage of its DEJ layer repair composition is as shown in the following table, and the usage of other components and the preparation method are the same: Table 15: Comparison table of the usage of DEJ layer repair composition in Comparative Application Examples 1 - 4 Group Usage of DEJ layer repair composition Comparative application example 1 Prepared from comparative example 1 Comparative application example 2 Prepared from comparative example 2 Comparative application example 3 Prepared from comparative example 3 Comparative application example 4 Prepared from comparative example 4 Extract the anti-aging creams in the above Application Examples 1 - 5 and Comparative Application Examples 1 - 4, observe their structures under 400 times magnification, and judge whether they are a liquid crystal emulsion system. If so, record it as ◎ and take a photo. If not, directly record it as X. The test results are shown in the following table.

[0037] Table 16: Performance test results of Application Examples 1 - 3 and Comparative Application Examples 1 - 4 As can be seen from the above table and Figure 8-16 it can be seen that only the anti-aging creams in Application Examples 1 - 5, due to the use of special emulsifiers and (liquid crystal) emulsification technology, have better component stability and bioavailability, and can enhance the original anti-aging effect by forming a lamellar liquid crystal structure similar to the healthy stratum corneum, while Comparative Application Examples 1 - 4 that obviously cannot form a liquid crystal emulsion structure do not have similar effects.

[0038] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A DEJ layer repair composition, characterized in that: It is compounded by ceramide NP, phytosteryl oleate and fatty acids.

2. The DEJ layer repair composition according to claim 1, characterized in that: The mass ratio of the ceramide NP, phytosterol oleate and fatty acid is (0.49-1.26):(0.29-1.06):(0.23-0.92).

3. The DEJ layer repair composition according to claim 2, characterized in that: The mass ratio of the ceramide NP, phytosterol oleate and fatty acid is 0.49:1.02:0.

29.

4. The DEJ layer repair composition according to claim 1, characterized in that: The fatty acid is behenic acid, stearic acid or palmitic acid.

5. Use of the DEJ layer repair composition according to any one of claims 1 to 4 in the preparation of anti-aging products and / or skin anti-aging methods.

6. An anti-aging cream containing a DEJ layer repair composition, characterized in that: The invention comprises the DEJ layer repairing composition according to any one of claims 1 to 4.

7. The anti-aging cream containing the DEJ layer repair composition according to claim 6, characterized in that: It is composed of the following components in percentage by mass: Ammonium acryloyldimethyltaurate / VP copolymer 0.1-0.3%, glycerin 5-10%, caprylic / capric triglyceride 20-25%, the DEJ layer repair composition 1.5-2.0%, emulsifier 4-6%, and the balance is water.

8. The anti-aging cream containing the DEJ layer repair composition according to claim 7, characterized in that: It is composed of the following components in percentage by mass: 0.2% of ammonium acryloyldimethyltaurate / VP copolymer, 7% of glycerin, 23.2% of caprylic / capric triglyceride, 1.8% of the DEJ layer repair composition, 5% of an emulsifier, and the balance of water.

9. The anti-aging cream containing the DEJ layer repair composition according to claim 7, characterized in that: The emulsifier is composed of the following components in percentage by mass: Arachidyl alcohol 55wt%, behenyl alcohol 30wt%, arachidyl glucoside 15wt%.

10. A method for preparing an anti-aging cream containing a DEJ layer repairing composition as claimed in any one of claims 7 to 9, characterized in that: The specific preparation steps are as follows: 1) Add ammonium acryloyldimethyltaurate / VP copolymer, glycerol and water according to the corresponding mass parts into the water phase pot, stir and disperse evenly, heat to 85-90°C, and obtain mixed material A for standby use; 2) Add caprylic acid / capric acid triglyceride, the DEJ layer repair composition, and an emulsifier into an oil phase pot, heat to 85-90° C., and stir evenly to obtain a mixture B for standby use; 3) Add mixture B into mixture A and homogenize at 5000-7000rpm for 4-6min; 4) Stir and cool to 40°C, then discharge the mixture to obtain an anti-aging cream for repairing the DEJ layer of the skin.

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