ECM (extracellular matrix) protein composition with anti-wrinkle, firming, repairing and moisturizing effects and application of ECM protein composition

Abstract

The invention relates to the technical field of cosmetics, in particular to the field of IPCA61K8, and more particularly relates to an ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects and application thereof. The composition is prepared from collagen, second protein and sodium coralate. The weight ratio of the collagen to the second protein to the sodium currate is (1-5): (10-25): (1-5), and the anti-wrinkle, firming, repairing, soothing and moisturizing mask has good permeability and has excellent anti-wrinkle, firming, repairing, soothing and moisturizing effects.

Description

Technical Field

[0001] The present invention relates to the field of cosmetic technology, in particular to the field of IPCA61K8, and more specifically to an ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects and an application thereof. Background Art

[0002] The extracellular matrix (ECM) is the largest component of the dermis of the skin. The extracellular matrix (ECM) is a colloid-like network structure secreted by cells, containing a variety of proteins and polysaccharides. In addition to serving as a scaffold and structural support for skin cells, giving the skin strength and elasticity, the extracellular matrix (ECM) also regulates cell function through cell adhesion, associates with a variety of growth factors and proteins, promotes cell proliferation, and provides a transport system for nutrients, making the components of the extracellular matrix play an important role in maintaining the normal function of cells, the structure and firmness of the skin, etc. Some existing technologies have the effect of promoting ECM, but the mechanism of their use is too single.

[0003] CN114053168A discloses an anti-aging composition and a cosmetic composition. The anti-aging composition comprises hydroxypropyl tetrahydropyranol, carnosine or a carnosine derivative, and a vitamin C derivative, which are a group of enhancers of the extracellular matrix (ECM) of the skin. When combined, they are sufficient to synergistically promote the production of glycosaminoglycan (GAG) in the dermis, inhibit the activity of hyaluronidase, and jointly promote the production of type IV collagen, thereby enhancing the tightness of basement membrane proteins and the junction of the dermis and epidermis, and playing a significant synergistic role in skin anti-aging, but the invention does not have the effects of anti-wrinkle, firming, repairing, moisturizing, etc. Summary of the invention

[0004] The first aspect of the present invention provides an ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects, the composition comprising: a first protein, a second protein and sodium colanate, the first protein comprising collagen, and the second protein comprising elastin and fibronectin.

[0005] The weight ratio of the collagen, the second protein and sodium colanate is (1-10): (5-30): (1-10).

[0006] The weight ratio of the collagen, the second protein and sodium colanate is (1-5): (10-25): (1-5).

[0007] The weight ratio of the elastin to the fibronectin is 1:(2-8).

[0008] The weight ratio of the elastin to the fibronectin is 1:(3-6).

[0009] Preferably, the weight ratio of elastin to fibronectin is 1:4, and the second protein supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0010] The molecular weight of the collagen is 1000-6000Da.

[0011] Preferably, the molecular weight of the collagen is 2000-5000Da.

[0012] The molecular weight of the sodium colanate is 1000-2000 KDa.

[0013] The preparation method of the ECM protein composition is as follows: collagen, a second protein and sodium colanate are uniformly mixed to obtain the ECM protein composition.

[0014] The second aspect of the present invention provides an application of an ECM protein composition having anti-wrinkle, firming, repairing and moisturizing effects, which is applied to the preparation of cosmetics.

[0015] The composition is added in an amount of 0.05-5 wt % in cosmetics.

[0016] The applicant has found that the selection of collagen with a specific molecular weight can improve the moisturizing and penetration performance. Due to its unique triple helix structure, the orderly arrangement of the molecular structure achieves high moisturizing performance. At the same time, the lower molecular weight is conducive to its penetration into the surface cells. Further research has found that the composite use of collagen with a specific molecular weight, a second protein and sodium colanate can balance the penetration and absorption performance, effectively inhibit basement membrane damage, strengthen the basement membrane structure, restore the true epidermis communication, and tighten the skin. The weight ratio of the collagen, the second protein and sodium colanate is (1-5): (10-25): (1-5), which has excellent anti-wrinkle, firming, repair and moisturizing effects.

[0017] Beneficial Effects

[0018] 1. The combination of the second protein and sodium colanate can balance the permeability and absorption properties, effectively inhibit basement membrane damage, strengthen the basement membrane structure, restore the communication between the dermis and the epidermis, and tighten the skin.

[0019] 2. Selecting collagen with a specific molecular weight can improve moisturizing penetration performance.

[0020] 3. The weight ratio of the collagen, the second protein and sodium colanate is (1-5): (10-25): (1-5), which has excellent anti-wrinkle, firming, repairing and moisturizing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The relative expression of adhesion proteins after HSF cells were treated according to Examples 1-3.

[0022] Figure 2 The relative expression SEM images of adhesion proteins after HSF cells were treated in Examples 1-3.

[0023] Figure 3 This is the relative expression of adhesion proteins after HSF cells were treated with Comparative Example 2.

[0024] Figure 4 This is the relative expression of type I collagen after HSF cells were treated with comparative example 3.

[0025] Figure 5 The relative expression of type I collagen in HSF cells after being treated in Example 1-3.

[0026] Figure 6 This is the effect of Example 1-3 on the migration rate of HaCaT cells after 72 hours of treatment.

[0027] Figure 7 The photographs are of the healing effect of the scratch wound on HaCaT cells after 72 hours of treatment in Example 1-3.

[0028] Figure 8 This is the effect of Example 1 on the survival rate of HSF cells.

[0029] Fig. 9 This is the effect of Example 2 on the survival rate of HSF cells.

[0030] Fig.10 This is the effect of Example 3 on the survival rate of HSF cells.

[0031] Fig.11 The following is an example of the effect of hEGF on the survival rate of HSF cells.

[0032] Fig.12 The electron microscope image shows the effect of Example 1 on the survival rate of HSF cells.

[0033] Fig.13 The electron microscope image shows the effect of Example 2 on the survival rate of HSF cells.

[0034] Fig.14 The electron microscope image shows the effect of Example 3 on the survival rate of HSF cells.

[0035] Fig.15 The electron micrographs show the effect of hEGF on the survival rate of HSF cells.

[0036] Fig.16 is the relative expression of type I collagen in HSF cells after UVA treatment.

[0037] Fig.17 This is the fluorescence graph of the relative expression of type I collagen in HSF cells after UVA treatment.

[0038] Fig.18 The relative expression level of type I collagen in HSF cells after UVA treatment in Example 1-3.

[0039] Fig.19 This is a fluorescence graph of the relative expression of type I collagen in HSF cells after UVA treatment in Example 1-3.

[0040] Fig. 20 is the relative expression rate of type III collagen in HSF cells after UVA treatment.

[0041] Fig.21 This is the fluorescence graph of the relative expression of type III collagen after HSF cells were treated with UVA.

[0042] Fig. 22 The relative expression rate of type III collagen after HSF cells were treated in Example 1-3.

[0043] Fig.23 This is a fluorescence graph of the relative expression of type III collagen after HSF cells were treated in Example 1-3.

[0044] Fig.24 This is the effect of UVB treatment on reactive oxygen free radicals (ROS) in HaCaT cells.

[0045] Fig.25 1% The effect of the treatment of Examples 1-3 on the reactive oxygen free radicals (ROS) in HaCaT cells.

[0046] Fig.26 The effect of 1% of Example 1-3 on the relative expression level of HAS1 gene in HaCaT cells.

[0047] Fig. 27 The effect of 1% of Example 1-3 on the relative expression level of HAS2 gene in HaCaT cells.

[0048] Fig.28 The effect of 1% of Example 1-3 on the relative expression level of HAS3 gene in HaCaT cells.

[0049] Fig.29 The effect of 5% Example 1-3 on the relative expression level of zebrafish Eln1 gene. DETAILED DESCRIPTION

[0050] Example 1

[0051] An ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects, comprising, by weight, 1 part of collagen, 10 parts of a second protein and 1 part of sodium colanate.

[0052] The collagen has a molecular weight of 4988Da and its supplier is Jiangsu Chuangshi Medical Technology Co., Ltd.

[0053] The molecular weight of the sodium colanate is 1000-2000KDa, and the supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0054] The second protein supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0055] The second protein is elastin and fibronectin, and the weight ratio of the elastin to the fibronectin is 1:4.

[0056] The preparation method of the ECM protein composition is as follows: collagen, a second protein and sodium colanate are uniformly mixed to obtain the ECM protein composition.

[0057] Example 2

[0058] An ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects, comprising, by weight, 1 part of collagen, 25 parts of a second protein and 5 parts of sodium colanate.

[0059] The collagen has a molecular weight of 4988Da and its supplier is Jiangsu Chuangshi Medical Technology Co., Ltd.

[0060] The molecular weight of the sodium colanate is 1000-2000KDa, and the supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0061] The second protein supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0062] The second protein is elastin and fibronectin, and the weight ratio of the elastin to the fibronectin is 1:4.

[0063] The preparation method of the ECM protein composition is as follows: collagen, a second protein and sodium colanate are uniformly mixed to obtain the ECM protein composition.

[0064] Example 3

[0065] An ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects, comprising, by weight, 1 part of collagen, 5 parts of a second protein and 1 part of sodium colanate.

[0066] The collagen has a molecular weight of 4988Da and its supplier is Jiangsu Chuangshi Medical Technology Co., Ltd.

[0067] The molecular weight of the sodium colanate is 1000-2000KDa, and the supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0068] The second protein supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0069] The second protein is elastin and fibronectin, and the weight ratio of the elastin to the fibronectin is 1:4.

[0070] The preparation method of the ECM protein composition is as follows: collagen, a second protein and sodium colanate are uniformly mixed to obtain the ECM protein composition.

[0071] Comparative Example 1

[0072] Blank control group: water.

[0073] Comparative Example 2

[0074] An ECM protein is: a second protein.

[0075] The second protein supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0076] The second protein is elastin and fibronectin, and the weight ratio of the elastin to the fibronectin is 1:4.

[0077] Comparative Example 3

[0078] An ECM protein composition comprises, by weight, 5 parts of a second protein and 35 parts of sodium colanate.

[0079] The molecular weight of the sodium colanate is 1000-2000KDa, and the supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0080] The second protein supplier is Shenzhen Boyin Biotechnology Co., Ltd.

[0081] The second protein is elastin and fibronectin, and the weight ratio of the elastin to the fibronectin is 1:4.

[0082] The preparation method of the ECM protein composition is as follows: the second protein and sodium colanate are mixed evenly to obtain the ECM protein composition.

[0083] Performance Testing Methods and Results

[0084] The performance tests were conducted in the examples and comparative examples, wherein the blank control group corresponds to comparative example 1, and the biomimetic poly-ECM protein groups 1-3 correspond to examples 1-3, respectively.

[0085] 1. Repair-Fibroblast adhesion (immunofluorescence).

[0086] Research objectives

[0087] The effects of Examples 1-3 and Comparative Examples 2-3 on the expression of adhesion proteins in HSF fibroblasts were studied to demonstrate the repairing efficacy of the Examples.

[0088] Research content

[0089] Within the range of maximum tolerated concentration, whether the examples can increase the relative expression of fibroblast HSF cell adhesion proteins.

[0090] Research Methods

[0091] Cell culture: The frozen HSF cells were revived and inoculated into cell culture flasks at a suitable density, and DMEM medium (containing FBS) was added and cultured in a cell culture incubator at 37°C and 5% CO2.

[0092] Cell seeding plate: Wash HSF cells in the logarithmic growth phase twice with PBS, add trypsin to digest, add DMEM medium (containing FBS) to terminate digestion, centrifuge, discard the supernatant, add DMEM medium (containing FBS) to make a cell suspension. Add 2mL of cell suspension to a small dish with a coverslip and place it in a cell culture incubator for culture.

[0093] Modeling and sample addition treatment: discard the culture medium in the small dish, add PBS, cover the surface with the sample mixed with PBS, and irradiate the model under the UV crosslinker. Replace the culture medium and continue to culture in a cell culture incubator at 37°C and 5% CO2.

[0094] Immunofluorescence slide processing: each dish was fixed with paraformaldehyde at room temperature, TritonX-100 was prepared, and the membrane was permeabilized at room temperature. After washing with PBS, the slides were blocked at room temperature and the blocking solution was discarded. The primary antibody was incubated at a certain temperature, and the fluorescent secondary antibody was incubated at a certain temperature in the dark. After washing with PBS, DAPI was incubated at room temperature in the dark. The slides were removed, blocked and fixed with the blocking solution overnight, and pictures were taken under a fluorescence microscope for data analysis.

[0095] Data statistics: GraphPad Prism 8.0 software was used to statistically process the data. The experimental data were expressed as mean ± SEM, and the data were statistically analyzed using statistical methods such as t-test or one-way analysis of variance.

[0096] Experimental Results

[0097] The effects of Example 1, Example 2 and Example 3 on the relative expression rate of adhesion proteins in HSF cells are shown in Figure 1-2 After treatment with Example 1, Example 2 and Example 3, the relative expression of HSF cell adhesion protein was 284.32±6.50%, 145.09±9.46% and 144.36±3.44%, respectively, which increased by 184.32% (p<0.001), 45.09% (p<0.01) and 44.36% (p<0.01) respectively compared with the blank control group, indicating that Example 1, Example 2 and Example 3 can promote the expression of adhesion protein (very significantly), as shown in FIG. Figure 3-4As shown, comparative examples 2-3 had no significant effect on the expression of type I collagen in HSF fibroblasts.

[0098] Therefore, after 72 hours of treatment of HSF cells with 1% of Examples 1-3, the relative expression rates of adhesion proteins increased by 184.32% (p<0.001), 45.09% (p<0.01) and 44.36% (p<0.01) respectively compared with the blank control group, indicating that 1% of Examples 1-3 treatment can promote the expression of adhesion proteins in HSF cells (very significantly). Under this experimental condition, 1% of Examples 1-3 has a repairing function.

[0099] 2. Anti-wrinkle and firming - promoting type Ⅰ collagen content

[0100] Research objective: To study the effect of the test substance on the expression of type I collagen in HSF fibroblasts (immunofluorescence experiment), so as to evaluate the anti-wrinkle and firming effect of the test substance.

[0101] Research content: To study whether the test substance can promote the expression of collagen I in fibroblast HSF cells within the maximum tolerated concentration range.

[0102] Research Methods

[0103] Cell culture: The frozen HSF cells were revived and inoculated into cell culture flasks at a suitable density, and DMEM medium (containing FBS) was added and cultured in a cell culture incubator at 37°C and 5% CO2.

[0104] Cell seeding plate: Wash HSF cells in the logarithmic growth phase twice with PBS, add trypsin to digest, add DMEM medium (containing FBS) to terminate digestion, centrifuge, discard the supernatant, add DMEM medium (containing FBS) to make a cell suspension. Add 2mL of cell suspension to a small dish with a coverslip and place it in a cell culture incubator for culture.

[0105] Modeling and sample addition treatment: discard the culture medium in the small dish, add PBS, cover the surface with the sample mixed with PBS, and irradiate the model under the UV crosslinker. Replace the culture medium and continue to culture in a cell culture incubator at 37°C and 5% CO2.

[0106] Immunofluorescence slide processing: each dish was fixed with paraformaldehyde at room temperature, TritonX-100 was prepared, and the membrane was permeabilized at room temperature. After washing with PBS, the slides were blocked at room temperature and the blocking solution was discarded. The primary antibody was incubated at a certain temperature, and the fluorescent secondary antibody was incubated at a certain temperature in the dark. After washing with PBS, DAPI was incubated at room temperature in the dark. The slides were removed, blocked and fixed with the blocking solution overnight, and pictures were taken under a fluorescence microscope for data analysis.

[0107] Data statistics: GraphPad Prism 8.0 software was used to statistically process the data. The experimental data were expressed as mean ± SEM, and the data were statistically analyzed using statistical methods such as t-test or one-way analysis of variance.

[0108] The effects of Example 1, Example 2 and Example 3 on the relative expression level of type I collagen gene in HSF cells are shown in Figure 5 As shown. The relative expression level of type I collagen gene in the blank control group was 100.00±2.34%. After the HSF cells were treated with Example 1, Example 2 and Example 3 at a concentration of 1% for 24 hours, the relative expression levels of type I collagen gene were 113.49±1.21%, 132.47±2.93% and 109.62±4.00%, respectively, which were increased by 13.49% (p<0.01), 32.47% (p<0.001) and 9.62% (p<0.01) respectively compared with the blank control group. The results show that Example 1, Example 2 and Example 3 all promoted the expression of type I collagen gene in HSF cells (very significantly).

[0109] 3. Repair-Cell Migration (Cell Scratching)

[0110] Research objectives

[0111] A cell scratch model of HaCaT cells was constructed to study the effect of the test substances on the cell migration of HaCaT cells and to evaluate the repair activity of the test substances.

[0112] Research content

[0113] Within the maximum tolerated concentration range, whether the test substance can increase the cell migration rate of HaCaT cells is studied.

[0114] Research Methods

[0115] Inoculate HaCaT cells in good growth state in a 6-well plate, and use a marker pen to evenly draw a straight line on the upper and lower sides of the middle of each well. Add 2ml of cell suspension to each well and place it in a cell culture incubator to culture until the cells just cover the entire well plate. Then use a 200μl gun tip to scratch the horizontal line behind the vertical well plate, wash it twice with PBS, and add 2ml of culture medium containing different drug concentrations to each well. Take pictures and record the same field of view for each concentration for 24 hours, and set up a blank control at the same time. Finally, measure the scratch spacing at three positions at each time point and calculate the cell migration rate according to the following formula.

[0116]

[0117] The experimental data are expressed as mean ± SEM data.

[0118] Data processing

[0119] Graphpad 8.0 software was used for data processing, and the experimental data were expressed as mean ± SEM.

[0120] Test results

[0121] The in vitro scratch test is used to evaluate the migration ability of cells. The ability of the sample to promote cell migration is evaluated according to the degree of healing of the scratch wound. Figure 6-7 As shown. After 72 hours of treatment, the migration rate of HaCaT cells in the blank control group was 35.18±.85%. After 72 hours of treatment with 0.125ng / mL hEGF, the migration rate of cells in the positive control group was 56.15±0.46%, which was 20.97% higher than that of the blank control group (p<0.001), indicating that hEGF has a repair effect (very significant). Under the same conditions, the cell migration rates of Example 1, Example 2 and Example 3 with a concentration of 1% after treating HaCaT cells for 72 hours were 65.72±2.47%, 55.18±0.89% and 42.92±0.15%, respectively, which were 30.54% (p<0.001), 20.00% (p<0.001) and 7.74% (p>0.05) higher than those of the blank control group, respectively, all of which promoted the migration of HaCaT cells. Under this experimental condition, Example 1 and Example 2 with a concentration of 1% have a repair effect (very significant). After treatment for 72 hours with 1% concentration of Example 3, the cell migration rate had no significant difference compared with the blank control group, and had no repair effect under this experimental condition.

[0122] 4. Anti-wrinkle and firming-fibroblast cell viability / proliferation

[0123] Research objectives

[0124] The effects of the test substances on the activity of human skin fibroblasts were studied, and the anti-wrinkle efficacy of the test substances was evaluated.

[0125] Research content

[0126] Within the maximum tolerated concentration range, the test substance was investigated to determine whether it could increase the viability of fibroblasts (MTT).

[0127] Research Methods

[0128] MTT cell viability assay Human keratinocytes HaCaT were inoculated in a 96-well plate (5×103 cells / well) and cultured in DMEM medium containing fetal bovine serum. Sample groups and blank control groups were set up and cultured. MTT (5 mg / mL) was then added to each well for incubation, the supernatant was discarded, DMSO was added to the 96-well plate to dissolve formazan, and the absorbance was measured at 570 nm. Cell viability was calculated.

[0129] Cell counting: Human keratinocytes HaCaT were seeded in 6-well plates (105 cells / well) and cultured in DMEM medium containing 10% fetal bovine serum. Sample groups with different concentrations were set up, and a blank control group was set up at the same time. After culturing for 72 hours, the cell culture supernatant was collected for counting.

[0130] Data processing

[0131] Graphpad8.0 software was used for data processing. The experimental data were expressed as mean±SEM and statistical methods such as t-test analysis or variance analysis were used to analyze the data.

[0132] Test results

[0133] MTT cell viability assay results

[0134] The results of Example 1, Example 2 and Example 3 and the effect of hEGF on the survival rate of HSF cells are shown in Figure 8-15 The survival rates of HSF cells in the blank control groups of Example 1, Example 2, Example 3 and hEGF were 100.00±1.01%, 100.00±1.57%, 100.00±2.39% and 100.00±4.04, respectively. The survival rates of HSF cells after culture for 72 hours with 1% of Example 1, Example 2, Example 3 and 0.1250 ng / mL of hEGF were 115.9±3.84%, 117.9±2 .98%, 94.08±3.74% and 115.50±6.52, compared with the blank control group, Example 1 and Example 2 and hEGF increased by 15.9% (p<0.05), 17.9% (p<0.001) and 15.5%, and Example 3 decreased by 5.92%, indicating that treatment with 1% biomimetic poly-ECM protein groups 1 and 2 and 0.1250ng / mL hEGF can promote the proliferation of HSF cells (significantly).

[0135] 5. Anti-wrinkle and firming-Type I collagen expression test (immunofluorescence test)

[0136] Research objectives

[0137] The effect of the test substance on the expression of type I collagen in HSF fibroblasts (immunofluorescence experiment) was studied to evaluate the anti-wrinkle and firming effect of the test substance.

[0138] Research content

[0139] Within the maximum tolerated concentration range, study whether the test substance can promote the expression of collagen I in fibroblast HSF cells.

[0140] Research Methods

[0141] Cell culture: The frozen HSF cells were revived and inoculated into cell culture flasks at a suitable density, and DMEM medium (containing FBS) was added and cultured in a cell culture incubator at 37°C and 5% CO2.

[0142] Cell seeding plate: Wash HSF cells in the logarithmic growth phase twice with PBS, add trypsin to digest, add DMEM medium (containing FBS) to terminate digestion, centrifuge, discard the supernatant, add DMEM medium (containing FBS) to make a cell suspension. Add 2mL of cell suspension to a small dish with a coverslip and place it in a cell culture incubator for culture.

[0143] Modeling and sample addition treatment: discard the culture medium in the small dish, add PBS, cover the surface with the sample mixed with PBS, and irradiate the model under the UV crosslinker. Replace the culture medium and continue to culture in a cell culture incubator at 37°C and 5% CO2.

[0144] Immunofluorescence slide processing: each dish was fixed with paraformaldehyde at room temperature, TritonX-100 was prepared, and the membrane was permeabilized at room temperature. After washing with PBS, the slides were blocked at room temperature and the blocking solution was discarded. The primary antibody was incubated at a certain temperature, and the fluorescent secondary antibody was incubated at a certain temperature in the dark. After washing with PBS, DAPI was incubated at room temperature in the dark. The slides were removed, blocked and fixed with the blocking solution overnight, and pictures were taken under a fluorescence microscope for data analysis.

[0145] Statistics

[0146] GraphPad Prism 8.0 software was used to statistically process the data. The experimental data were expressed as mean ± SEM, and the data were analyzed using statistical methods such as t-test or one-way analysis of variance.

[0147] The effects of Example 1, Example 2 and Example 3 on the relative expression rate of type I collagen in HSF cells are shown in Figure 16-17 The relative expression rate of type I collagen in HSF cells of the model control group after UVA irradiation was 58.15±0.78%, which was 41.85% lower than that of the blank control group (100.00±0.98%) (p<0.001), indicating that the relative expression of type I collagen in HSF cells decreased (very significantly) after UVA modeling, and the model was successfully constructed.

[0148] like Figure 18-19As shown, the relative expression rates of type I collagen in HSF cells treated with 0.125 mg / mL recombinant humanized type III collagen (positive control group) and 1% of Example 1, Example 2 and Example 3 were 116.68±0.85%, 115.83±4.48%, 97.52±3.77% and 134.65±1.40%, respectively. The relative expression rates of type I collagen in HSF cells treated with 0.125 mg / mL recombinant humanized type III collagen and 1% of Example 1 and Example 3 were significantly different from those in the model control group (100.00±1.34 %) increased by 16.68% (p<0.01), 15.83% (p<0.01) and 34.65% (p<0.001) respectively, while 1% of Example 2 decreased by 2.48% (p>0.05) compared with the model control group (100.00±1.34%), indicating that recombinant type III humanized collagen and 1% of Examples 1 and 3 can promote the expression of type I collagen in HSF cells (very significantly), and 1% of Example 2 has no obvious effect on the expression of type I collagen in HSF cells.

[0149] 6. Anti-wrinkle and firming-relative expression of type III collagen

[0150] Research objectives

[0151] The effect of the test substance on the expression of type III collagen in HSF fibroblasts (immunofluorescence experiment) was studied to evaluate the anti-wrinkle and firming efficacy of the test substance.

[0152] Research content

[0153] Within the maximum tolerated concentration range, the test substance was investigated to determine whether it could increase the relative expression of collagen III in fibroblast HSF cells.

[0154] Research Methods

[0155] Cell Culture The cryopreserved HSF cells were revived and inoculated into cell culture flasks at a suitable density, and DMEM medium (containing FBS) was added and cultured in a cell culture incubator at 37° C. and 5% CO 2 .

[0156] Cell plate: Wash HSF cells in the logarithmic growth phase twice with PBS, add trypsin to digest, add DMEM medium (containing FBS) to terminate digestion, centrifuge, discard the supernatant, add DMEM medium (containing FBS) to make a cell suspension. Add 2mL of cell suspension to a small dish with a coverslip and place it in a cell culture incubator for culture.

[0157] Modeling and sample treatment: Discard the culture medium in the small dish, add PBS, cover the surface with a sample mixed with 2PBS, and irradiate the model under a UV crosslinker. Replace the culture medium and continue to culture in a cell culture incubator at 37°C and 5% CO2.

[0158] Each dish was fixed with paraformaldehyde at room temperature, TritonX-100 was prepared, and the membrane was permeabilized at room temperature. After washing with PBS, the slides were blocked at room temperature and the blocking solution was discarded. The primary antibody was incubated at a certain temperature, and the fluorescent secondary antibody was incubated at a certain temperature in the dark. After washing with PBS, DAPI was incubated at room temperature in the dark. The slides were removed, blocked and fixed with the sealing solution overnight, and the images were taken under a fluorescence microscope for data analysis.

[0159] Statistics

[0160] GraphPad Prism 8.0 software was used to statistically process the data. The experimental data were expressed as mean ± SEM, and the data were analyzed using statistical methods such as t-test or one-way analysis of variance.

[0161] Test results

[0162] The effects of Example 1, Example 2 and Example 3 on the relative expression rate of type III collagen in human fibroblasts are shown in Figure 20-21 The relative expression rate of type III collagen in HSF cells of the model control group treated with UVA was 85.31±0.45%, which was 14.69% lower than that of the blank control group (p<0.001), indicating that the type III collagen model was successfully constructed (very significantly).

[0163] like Figure 22-23 As shown, under the same experimental conditions, after adding 0.125 mg / ml recombinant humanized type III collagen to treat HSF cells, the relative expression rate of type III collagen was 110.57±3.26%, which was 10.57% higher than that of the model control group (p<0.05), indicating that 0.125 mg / ml recombinant humanized type III collagen treatment can promote the relative expression of type III collagen in HSF cells (significant). After treatment with 1% of Example 1, Example 2 and Example 3, the relative expression rates of type III collagen were 113.08±3.22%, 118.03±1.51% and 112.79±2.21%, respectively, which were increased by 13.08% (p<0.05), 18.03% (p<0.01) and 12.79% (p<0.05) respectively compared with the model control group, indicating that treatment with 1% of Example 1, Example 2 and Example 3 promoted the relative expression of type III collagen in HSF cells (significantly).

[0164] 7. Anti-wrinkle and firming - reactive oxygen species expression test (HaCaT cell ROS expression)

[0165] Research objectives

[0166] The effect of the test substance on the ROS fluorescence intensity of HaCaT cells was studied to evaluate the anti-wrinkle and firming effect of the test substance.

[0167] Research content

[0168] Within the maximum tolerated concentration range, study whether the test substance can inhibit the ROS fluorescence intensity.

[0169] Research Methods

[0170] Cell Culture The cryopreserved HaCaT cells were revived and seeded into cell culture flasks at an appropriate density, and DMEM medium (10% FBS) was added and cultured in a cell culture incubator at 37° C. and 5% CO 2 .

[0171] Cell plate: Wash HaCaT cells in logarithmic growth phase twice with PBS, add trypsin to digest, add DMEM medium (10% FBS) to terminate digestion, centrifuge, discard the supernatant, add DMEM medium (10% FBS) to prepare cell suspension. Add 2 mL of cell suspension to a small dish with a cover glass, and place in a cell culture incubator for culture.

[0172] ROS detection

[0173] Blank control group: HaCaT cells were not treated in any way;

[0174] Negative control group: HaCaT cells treated with 0.003% (v / v) H2O2;

[0175] Model control group: HaCaT cells were treated with 0.003% (v / v) H2O2 and then irradiated with UVB; sample treatment group: HaCaT cells were treated with 0.003% (v / v) H2O2 and then irradiated with UVB. The cells were washed 3 times with PBS, digested with trypsin, and digested with DMEM. The cells were collected and washed 3 times with PBS, and DCFH-DA (1:1000) dye was prepared and incubated at 37°C in the dark. The cells were washed with PBS and the ROS content was detected.

[0176] The calculation formula of ROS inhibition rate is as follows:

[0177]

[0178] Statistics

[0179] GraphPad Prism 8.0 software was used to statistically process the data. The experimental data were expressed as mean ± SEM, and the data were analyzed using statistical methods such as t-test or one-way analysis of variance.

[0180] Effects of UVB on the production of reactive oxygen species (ROS) in HaCaT cells Fig.25The relative expression of reactive oxygen free radicals (ROS) in the HaCaT cells of the UVB-treated model control group was 489.93±12.75%, which was significantly increased by 389.93% (p<0.001) compared with the blank control group (100.00±12.10), indicating that the oxidative stress model was successfully established (very significantly).

[0181] The effect of 1% of Example 1, Example 2 and Example 3 on the relative expression rate of reactive oxygen species ROS in HaCaT cells is as follows: Fig.26 As shown. Under the same experimental conditions, the ROS inhibition rate of HaCaT cells after treatment with 1% of Example 1, Example 2 and Example 3 was 82.44±0.23, 80.38±0.52 and 80.04±2.70%, which was increased by 82.44% (p<0.001), 80.38% (p<0.001) and 80.04% (p<0.001) compared with the model control group (0.00±2.60). It shows that 1% of Example 1, Example 2 and Example 3 treatment can improve the ROS inhibition rate of HSF cells after UVB irradiation (very significant).

[0182] 8. Moisturizing-keratinocytes (HAS1HAS2HAS3)

[0183] Research objective: To study the effect of the test substance on the relative expression levels of HAS1 / HAS2 / HAS3 genes in HaCaT cells, so as to evaluate the moisturizing effect of the test substance.

[0184] The research content uses the HacaT cell model to study whether the test substance can promote the expression of HAS1 / HAS2 / HAS3 genes within the maximum tolerated concentration range.

[0185] Research Methods

[0186] For cell culture, cryopreserved HSF cells were revived and seeded into cell culture flasks at an appropriate density, and DMEM medium (10% FBS) was added and cultured in a cell culture incubator at 37° C. and 5% CO 2 .

[0187] Cell seeding plate, wash HSF cells in logarithmic growth phase with PBS twice, add trypsin to digest, add DMEM medium (containing FBS) to terminate digestion and centrifuge, discard the supernatant, add DMEM medium (containing FBS) to make a cell suspension, evenly inoculate in cell culture dishes, and culture in a cell culture incubator.

[0188] Add drugs and add 3 mL of DMEM medium (10% FBS) containing samples of corresponding concentrations, and culture in a cell culture incubator at 37°C and 5% CO2.

[0189] Detection of relative gene expression of HAS1 / HAS2 / HAS3: RNA of each group of cells was extracted and reverse transcribed into cDNA, and the gene expression level of HAS1 / HAS2 / HAS3 of each group of cells was detected by RT-qPCR.

[0190] Statistics

[0191] GraphPad Prism 8.0 software was used to statistically process the data. All experimental data were expressed as mean ± SEM, and statistical methods such as t-test or one-way analysis of variance were used to analyze the data. 5. Experimental results

[0192] The effect of 1% of Example 1, Example 2 and Example 3 on the relative expression level of HAS1 gene in HaCaT cells is shown in Figure 27-29 As shown. After the 0.125ng / mL positive control group (hEGF) treated HaCaT cells, the relative expression level of the HAS1 gene was 121.90±2.11%. After 1% of Example 1, Example 2 and Example 3 treated HaCaT cells, the relative expression levels of the HAS1 gene were 112.25±5.10%, 68.92±1.32% and 125.88±6.07%, respectively, compared with the blank control group (100.00±3.78), they increased by 12.25% (p<0.05), decreased by 31.08% (p<0.001) and increased by 25.88% (p<0.001), respectively. The results show that 1% of Example 1 and Example 3 promoted the expression of the HAS1 gene in HaCaT cells (significantly), and 1% of Example 2 inhibited the expression of the HAS1 gene in HaCaT cells (very significantly).

[0193] After the 0.125ng / mL positive control group (hEGF) was used to treat HaCaT cells, the relative expression level of HAS2 gene was 121.90±2.11%. 328.47±3.82%. After 1% of Example 1, Example 2 and Example 3 were used to treat HaCaT cells, the relative expression levels of HAS2 gene were 153.30±1.94%, 164.59±1.73% and 234.64±9.03%, respectively, which were increased by 53.30% (p<0.001), 64.59% (p<0.001) and 134.64% (p<0.001) respectively compared with the blank control group (100.00±0.73%). The results show that 1% of Example 1, Example 2 and Example 3 promoted the expression of HAS2 gene in HaCaT cells (very significantly).

[0194] 9. Expression of firming-elastin (expression of zebrafish Eln1 gene)

[0195] The research objective is to explore the firming effect of the samples by detecting the changes in the relative expression of the zebrafish Eln1 gene.

[0196] Research content: Elastin exists in the extracellular matrix. Although elastin only accounts for 2% of the total dermal protein, it plays an important role in skin elasticity. The reduction of elastin is the main reason for the loosening, sagging and fine wrinkles of skin aging. Zebrafish have genes that regulate elastin that are highly conserved with humans, such as the Eln1 gene. Its expression level changes differently with the age of the fish. Among them, the expression level of the Eln1 gene in 7dpf zebrafish is relatively the highest. Therefore, 7dpf zebrafish are used as the test object to study the effects of different samples on the expression of the Eln1 gene. The firming effect of the sample is reflected by detecting the relative expression of the zebrafish elastin gene.

[0197] Research Methods

[0198] Grouping and Control

[0199] Sample dosage grouping: 5% Example 1, 5% Example 2, 5% Example 3

[0200] Control information: Blank control group: standard dilution water

[0201] Main steps

[0202] Wild-type zebrafish larvae at 4 dpf were collected and placed in a 6-well plate, with 25 larvae in each well.

[0203] The 25 fry in the last well were treated with 5 mL of sample solution, and the 25 fry in the last well were treated with standard diluted water as the blank control group. After treatment, all the fry were collected and washed twice with sterile double distilled water, and then the fry were transferred to marked sterile 1.5 mL EP tubes, zebrafish RNA was extracted, and then the RNA was reverse transcribed into cDNA, and the changes in the mRNA level of the Eln1 gene were detected on a fluorescent quantitative PCR instrument.

[0204] Statistics

[0205] Excel and GraphPad Prism 8.0 software were used to statistically process the data. The experimental data were expressed as mean ± SD, and the data were analyzed using statistical methods such as one-way analysis of variance.

[0206] Effect of Examples 1-3 on the relative expression level of zebrafish Eln1 gene: After zebrafish were treated with Examples 1-3 at a concentration of 5% for 72 hours, the relative expression levels of Eln1 gene were 62.96±1.97%, 134.90±6.54% and 153.08±1.54%, respectively. Compared with the blank control group (100.00±7.30%), the treatment with Example 1 at a concentration of 5% reduced the relative expression level of zebrafish Eln1 gene by 37.04% (p<0.001), while the treatment with Examples 2-3 at a concentration of 5% increased the relative expression level of zebrafish Eln1 gene by 34.90% (p<0.001) and 53.08% (p<0.001), respectively, indicating that Examples 2 and 3 have the effect of promoting the expression of Eln1 gene (very significant).

[0207] 10. Firming-two-photon experiment (improvement of human collagen fibers and elastic fibers)

[0208] Materials and methods

[0209] Research purpose: To verify the effect of the test product on improving collagen fibers and elastic fibers.

[0210] Trial product: Bionic poly-ECM protein group

[0211] How to use the product:

[0212] After cleansing every morning and evening, take an appropriate amount in your hands and pat your face until it is fully absorbed.

[0213] Notice:

[0214] During the test, the subjects are not allowed to use any treatments for skin improvement that may affect the test results.

[0215] Cosmetics, quasi-drugs, and health products are prohibited. Medical treatment, massage, and beauty salon care are also prohibited.

[0216] Seven days before the test, the subjects must stop using skin care products of the same category and with the same efficacy as the test samples on their faces.

[0217] During the test period, maintain the same daily routine as before the test, reduce outdoor activities in strong sunlight as much as possible, and refrain from traveling, vacationing, or having a seriously irregular daily routine.

[0218] During the test, other skin care steps and skin care products used should remain the same as before the test. You cannot change / stop using any other type of facial skin care products you were using before taking the test, nor can you add any facial skin care products that were not used before taking the test.

[0219] On the day of the test, subjects were not allowed to use any skin care products on their faces.

[0220] Subjects: The effective sample size was 4 persons, including 4 females, aged 49 to 53 years old, with an average age of 52.00±2.00 years old; the subjects' skin type was Asian skin, which met the voluntary inclusion criteria.

[0221] Inclusion criteria: a) healthy Chinese women aged 18 to 55 years old; b) those with loose and inelastic facial skin; c) no allergic diseases, no history of allergies to cosmetics and other topical preparations; d) no history of photosensitivity diseases and no recent use of drugs that affect photosensitivity; e) the skin of the test area should be free of birthmarks, inflammation, scars, hirsutism, etc.; f) can cooperate well with the experimenters and maintain a regular life during the test; g) can read and understand all the contents of the informed consent form and voluntarily sign the informed consent form; h) agree not to use any cosmetics, drugs and health products that may affect the results during the trial; i) agree to authorize the use of portrait rights; j) other corresponding inclusion criteria.

[0222] Exclusion criteria: a) Those with obvious scars, birthmarks, etc. in the test area that may affect the test results; b) Those with highly allergic constitutions; c) Women who are pregnant, breastfeeding, or planning to become pregnant during the test period; d) Those with severe heart, liver, and kidney damage and severe immunodeficiency; e) Those with mental illness, severe endocrine diseases, and those taking oral contraceptives; f) Those who rely on hormones or antihistamines for long-term treatment or physical condition maintenance; g) Those who participated in drug clinical trials or other experiments within 60 days before the test, or those who have used drugs that may affect the test results in the past 2 weeks; h) Those who have received medical care, beauty salon care, or treatment for their facial skin within 90 days before the test; i) Those who started taking or applying cosmetics, health products, etc. with similar functions that may affect the test results within 30 days before the test; j) Those who are considered unsuitable for participating in this trial by the researchers; k) Other corresponding exclusion criteria.

[0223] Exit or dropout criteria: a) Not selected (e.g., not meeting the inclusion criteria / or excluded by the exclusion criteria, etc.); b) Subjects are determined to be ineligible after being selected (including but not limited to changes in other lifestyle, diet, skin care habits and skin care products during the evaluation period); c) Subjects choose to withdraw; d) Adverse events or serious adverse events occur; e) Subjects are lost to follow-up; f) Force majeure; g) Others.

[0224] Statistics

[0225] Human efficacy evaluation test data; descriptive statistics: quantity, mean, standard deviation, minimum value, maximum value, etc.; statistical analysis software, SPSS26.0; test environment, the temperature and humidity environment of this test environment is 21±1℃, 50±10%RH, which meets the requirements of the plan.

[0226] Test data

[0227] Skin two-photon microscopy - elastic fiber fluorescence intensity:

[0228] As an energy source to excite the internal fluorescent substances of the specimen, two-photon light is used to excite the internal fluorescent substances of the specimen under the microscope, causing them to emit fluorescence and present a fluorescence phenomenon. Since skin tissue has abundant autofluorescence sources, multiphoton microscopy is particularly suitable for imaging skin tissue without fluorescent materials. Elastic fibers can emit green TPEF signals; collagen fibers can emit red SHG signals. The morphological images of elastic fibers and collagen fibers taken by two-photon light are analyzed by IPP software to analyze the fluorescence intensity of the TPEF signal related to elastic fibers and the fluorescence intensity of the SHG signal related to collagen fibers.

[0229] Skin two-photon microscopy - collagen fiber fluorescence intensity:

[0230] Experimental Conclusion

[0231] Compared with before use, after using the sample of Example 3 for 14 days and 28 days, the fluorescence intensity of the elastic fibers of the subjects' facial skin increased by 49.45% and 85.04%; after using the sample of Example 3 for 14 days and 28 days, the fluorescence intensity of the collagen fibers of the subjects' facial skin increased by 39.50% and 55.46%.

Claims

1. An ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects, characterized in that: The composition comprises: a first protein, a second protein and sodium colanate, wherein the first protein comprises collagen, and the second protein comprises elastin and fibronectin.

2. The ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to claim 1, characterized in that: The weight ratio of the collagen, the second protein and sodium colanate is (1-10): (5-30): (1～10)。 3. The ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to claim 2, characterized in that: The weight ratio of the collagen, the second protein and sodium colanate is (1-5): (10-25): (1～5)。 4. The ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to claim 3, characterized in that: The weight ratio of the elastin to the fibronectin is 1:(2-8).

5. The ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to claim 1 or 4, characterized in that: The weight ratio of the elastin to the fibronectin is 1:(3-6).

6. The ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to claim 5, characterized in that: The molecular weight of the collagen is 1000-6000Da.

7. The ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to claim 6, characterized in that: The molecular weight of the collagen is 2000-5000Da.

8. The ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to claim 7, characterized in that: The molecular weight of the sodium colanate is 1000-2000 KDa.

9. An application of the ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to any one of claims 1 to 8, characterized in that: Used in the preparation of cosmetics.

10. The use of the ECM protein composition with anti-wrinkle, firming, repairing and moisturizing effects according to claim 9, characterized in that: The composition is added in an amount of 0.05-5 wt % in cosmetics.

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