A complex protein composition for improving skin condition, products and uses thereof

Abstract

This invention provides a complex protein composition, product, and application for improving skin condition. The complex protein composition includes type IV collagen, type VII collagen, type XVII collagen, laminin 332, and fibronectin. This complex protein composition helps to improve skin condition, delay the appearance of aging, reduce wrinkles, and improve sagging. Simultaneously, it plays a positive role in repairing and supporting the skin barrier and extracellular matrix structure, which is beneficial for maintaining the stability and orderly remodeling of the skin microenvironment.

Description

Technical Field

[0001] This invention relates to the field of skincare, and more specifically, to a complex protein composition, product, and application for improving skin condition. Background Technology

[0002] Skin aging is a complex process resulting from the combined effects of intrinsic natural aging and extrinsic photoaging. Natural aging is accompanied by a slowdown in cell renewal and a decline in fibroblast activity, leading to a gradual reduction in the synthesis of key extracellular matrix components such as collagen and elastin. This typically manifests as thinning, dryness, fine lines, and sagging due to the reduction of subcutaneous tissue. Meanwhile, extrinsic photoaging caused by prolonged exposure to ultraviolet (UV) radiation significantly accelerates this process. UV radiation not only directly damages cellular DNA and induces oxidative stress, but also continuously activates the matrix metalloproteinase family of enzymes. These enzymes excessively degrade collagen and elastin fibers in the dermis, resulting in deep wrinkles, abnormal pigmentation, telangiectasia, and a significant decrease in elasticity and firmness.

[0003] At the junction of the dermis and epidermis, various structural proteins work together to maintain the integrity and function of the skin. Type VII collagen, forming anchoring fibers, acts as a crucial anchor point connecting the papillary layer of the dermis to the overlying basement membrane; type IV collagen forms the core scaffold of the three-dimensional network of the basement membrane, providing stable attachment for epidermal cells and participating in signal transduction; and type XVII collagen, as a transmembrane protein, directly mediates the connection between basal cells and components such as adhesion proteins in the middle layer of the basement membrane. Based on the key roles of these molecules, current technologies attempt to address skin structural defects caused by aging by supplementing with single collagen components or combining two or three of them.

[0004] However, existing solutions still have certain limitations. The maintenance and repair of skin structure depends on a highly synergistic microenvironment, and the supplementation of a single ingredient often has a limited scope of action, making it difficult to fully rebuild the complex multi-layered skin structure. Even combinations of several ingredients may affect the integrity and stability of the overall structure if they fail to cover all key functional proteins at the basement membrane and junctions and mimic their natural proportions and spatial assembly relationships, thus preventing the repair effect from reaching its optimal level. Summary of the Invention

[0005] The purpose of this invention is to provide a complex protein composition, product and application for improving skin condition. This complex protein composition helps to improve skin condition, delay the appearance of aging, reduce wrinkles and improve sagging. At the same time, it can play a positive role in repairing and supporting the skin barrier and extracellular matrix structure, which is conducive to maintaining the stability and orderly remodeling of the skin microenvironment.

[0006] According to one aspect of the present invention, a complex protein composition for improving skin condition is provided, comprising type IV collagen, type VII collagen, type XVII collagen, laminin 332, and fibronectin.

[0007] This approach utilizes a specific combination of type IV collagen, type VII collagen, type XVII collagen, laminin 332, and fibronectin to construct a synergistic protein network that works together at key levels of skin structure, thereby achieving multi-layered anti-aging effects. Specifically, type IV collagen forms the core reticular scaffold of the basement membrane, providing structural support and signal transduction for upper epidermal cells. Building upon this, type VII collagen forms anchoring fibers that firmly fix the basement membrane to the papillary dermis, enhancing overall skin firmness and anti-sagging ability. Type XVII collagen, as a transmembrane connection point for epidermal basal cells, mediates the physical anchoring and information transmission between epidermal basal cells and the basement membrane. Laminin 332 plays a crucial bridging role in the aforementioned structure, binding to type XVII collagen and cytokines at one end and connecting to type VII collagen at the other, thereby establishing a coherent and stable structural link between the epidermis and dermis, bridging the cell membrane and dermal matrix, and enhancing interfacial stability and resistance to external damage. Fibronectin is widely distributed throughout this network, interacting with various protein components and cell surfaces to assist in guiding cell migration and attachment, guiding collagen remodeling, promoting the organization and repair of the microenvironment, and further enhancing the structural integrity of the dermis. These proteins do not operate in isolation, but rather, through the aforementioned interconnection and functional synergy, collectively strengthen the physiological support system of the skin's extracellular matrix, more effectively activating fibroblast activity at the cellular level, promoting the synthesis and orderly deposition of key collagen proteins, thereby improving the overall integrity and elasticity of the skin structure, contributing to improved skin condition, delayed aging appearance, reduced wrinkles, and improved sagging. Furthermore, this complex protein composition also exhibits a positive regulatory effect on the skin barrier and extracellular matrix homeostasis. By synergistically supplementing key functional proteins in the basement membrane and dermal-epidermal junction, it helps support the gradual repair of the damaged microenvironment and promotes the orderly remodeling of components such as collagen in the extracellular matrix. This multi-protein synergistic approach can, to some extent, improve the loosening of the matrix structure caused by aging or external stimuli, enhance the skin's own repair potential, and thus more stably maintain the support and connectivity functions of skin tissue.

[0008] Preferably, the mass ratio of type IV collagen: type VII collagen: type XVII collagen: laminin 332: fibronectin is 2.8~3.8: 9.8~10.8: 4.8~5.8: 0.8~1.2: 7.2~8.3.

[0009] By optimizing the mass ratio of each component within the aforementioned specific range, the composite system composed of five proteins more closely resembles the components required by the extracellular matrix during skin aging. When the protein ratio in the complex protein composition for improving skin condition is within the above range, it not only helps maintain the stable support of the type IV collagen scaffold but also enhances the bonding strength between laminin 332 and type XVII collagen, resulting in a tighter connection between the epidermis and dermis. Simultaneously, the appropriate amount of type VII collagen strengthens the anchoring effect beneath the basement membrane, while the proportion of fibronectin promotes the coordinated migration of fibroblasts and matrix remodeling activities. Under optimized ratios, the proteins form a more synergistic and balanced functional network, thereby more effectively promoting the synthesis and orderly assembly of key structural proteins at the cellular level, enhancing the integrity and self-repair potential of the dermal structure, and ultimately exhibiting a more positive and stable effect in improving skin elasticity, firmness, and overall appearance.

[0010] Preferably, the mass ratio is calculated as follows: Type IV collagen: Type VII collagen: Type XVII collagen: Laminin 332: Fibronectin = 3~3.5: 10~10.5: 5~5.5: 0.8~1.2: 7.5~8.0.

[0011] Preferably, the mass ratio of type IV collagen: type VII collagen: type XVII collagen: laminin 332: fibronectin is 3.1~3.2: 10.3~10.4: 5.2~5.3: 1: 7.7~7.8.

[0012] According to another aspect of the present invention, a product for improving skin condition is provided, the active ingredient of which includes the above-described complex protein composition for improving skin condition.

[0013] This skin-improving product uses the aforementioned complex protein composition as its active ingredient, providing structurally similar and proportionally balanced protein network support to the skin during use. By mimicking and synergistically strengthening the connections and support relationships between key components in the skin's extracellular matrix, it helps promote the activity and function of the skin's own fibroblasts, supporting the maintenance and orderly renewal of the dermal structure. Therefore, this product is expected to exhibit a coordinated and positive auxiliary effect in improving skin elasticity, firmness, and overall appearance.

[0014] Preferably, the concentration of the compound protein composition for improving skin condition is 0.005~0.03 mg / mL.

[0015] By regulating the concentration of the complex protein composition for improving skin condition, a more stable and efficient bioactive response can be achieved at the cellular level. This concentration range encompasses the experimentally validated optimal action point, allowing the complex protein network to interact with cells at an appropriate dose—sufficient to activate fibroblast proliferation and migration signaling pathways while avoiding metabolic burden or signal saturation that may result from excessive concentration. Mechanistically, this concentration range supports the synthesis and orderly assembly of extracellular matrix proteins, promoting the expression and deposition of key collagen proteins. In terms of indicators, it corresponds to the optimized range for cell proliferation rate and migration capacity, contributing to enhanced repair potential and elasticity maintenance of the dermal structure. By controlling the concentration within this range, the complex protein composition for improving skin condition exhibits a more consistent positive effect in promoting skin health and improving the appearance of aging.

[0016] Preferably, the concentration of the compound protein composition for improving skin condition is 0.005~0.015 mg / mL.

[0017] Preferably, the concentration of the compound protein composition for improving skin condition is 0.01 mg / mL.

[0018] According to another aspect of the present invention, the use of the above-described complex protein composition for improving skin condition is provided in the preparation of products for improving skin condition.

[0019] Using the aforementioned complex protein composition for improving skin condition in the preparation of products allows for formulation designs that more closely resemble the skin's own supporting structure. This application enables the protein components in the product to act synergistically on the skin, helping to support skin cells in maintaining vitality, promoting their self-repair and renewal, thereby helping the skin appear firmer and more elastic. Products obtained through this application are expected to provide a more coordinated and stable supportive effect in helping to improve skin condition.

[0020] Preferably, improving skin condition includes at least one of: promoting collagen expression, promoting skin collagen production, improving skin elasticity, and improving skin firmness. Attached Figure Description

[0021] Figure 1 This is a schematic diagram showing the cell proliferation rate of experimental group 4A at different concentrations. Figure 2 This is a schematic diagram showing the cell proliferation rate of experimental group 1A at different concentrations. Figure 3 This is a schematic diagram showing the cell proliferation rate of control group 1 at different concentrations. Figure 4 This is a schematic diagram showing the cell proliferation rate of experimental group 4A under different irradiation intensities. Figure 5 This is a schematic diagram showing the results of the scratch test for experimental group 1A, experimental group 4A, and control group 1. Figure 6 This is a schematic diagram of the cell migration area in experimental group 1A, experimental group 4A, and control group 1. Figure 7 This is a schematic diagram showing the results of the scratch test between experimental group 4A and a single protein. Figure 8 This is a schematic diagram showing the cell migration area of ​​experimental group 4A and a single protein. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0023] Experiment 1 (1) Preparation of a complex protein composition for improving skin condition This experiment used commercially available type IV collagen, type VII collagen, type XVII collagen, laminin 332, and fibronectin as raw materials to prepare a compound protein composition for improving skin condition.

[0024] The above materials were mixed according to different component ratios to prepare the compound protein compositions for each experimental group according to the component composition recorded in Table 1. In this experiment, the concentration of each experimental group was 0.01 mg / mL.

[0025] The preparation method of this complex protein composition includes the following steps: Each raw protein was tested for endotoxin content, protein concentration, and purity before the preparation of the compound protein composition. All preparation steps were performed in a biosafety laminar flow hood to ensure aseptic operation. The laminar flow hood was sterilized internally with ultraviolet light 30 minutes before operation.

[0026] Subsequently, based on the initial concentration of each raw protein, the volume of each protein solution required to prepare the target composition was calculated. Using a sterile pipette, the corresponding protein solutions were measured and added sequentially to sterile centrifuge tubes. Then, preheated DMEM complete culture medium (37°C) was added to the centrifuge tubes, and the mixture was gently mixed until homogeneous. The prepared compound protein composition was stored under appropriate conditions for later use.

[0027] In this experiment, the experimental groups were labeled as experimental group 1A, experimental group 2A, experimental group 3A, experimental group 4A, experimental group 5A, experimental group 6A, and experimental group 7A, respectively; the control groups were labeled as control group 1, control group 2, control group 3, control group 4, control group 5, control group 6, control group 7, control group 8, and control group 9, respectively.

[0028] Table 1. Weight composition of the complex protein composition in Experiment 1

[0029] Test Example 1 1. Test subjects: Each composition in Experiment 1 was tested, with three replicates for each experimental group. Cell proliferation rate was detected using a CCK-8 assay kit, and the results are expressed as a percentage (%) relative to the control group.

[0030] 2. Test items and test methods: Cell proliferation rate: Cell proliferation rate is a key indicator for evaluating the bioactivity of biological materials (such as collagen). The CCK-8 assay, as a sensitive and reliable in vitro detection method, is widely used to assess the cytotoxicity and proliferation-stimulating effects of compounds. To investigate the proliferation-stimulating effects of different compositions on human dermal fibroblasts (HFF-1 cells), a CCK-8 experiment was conducted.

[0031] Cell proliferation rate was calculated by comparing cell proliferation in each experimental group with that in the control group and the blank group. The formula for calculating cell proliferation rate is: Cell proliferation rate (%) = (Experimental group - Blank group) / (Control group - Blank group) × 100%.

[0032] The main meanings of the three groupings are as follows: Experimental group: cells + culture medium + formulations for each group (or UVA treatment) + detection reagent CCK-8.

[0033] Control group: cells + culture medium (without formulation or any treatment) + test reagent CCK-8.

[0034] Blank group: culture medium + detection reagent CCK-8.

[0035] The culture and passage of human dermal fibroblasts (HFF-1 cells) includes the following procedures: 1. Cell resuscitation: (1) Preheat the water bath to 37°C in advance. (2) Take the cells out of the liquid nitrogen tank or -80°C freezer, immerse them in the water bath, shake them appropriately to accelerate melting, and it is best to melt them within 1 minute.

[0036] The following procedures are performed in a clean bench: (1) Irradiate the ultra-clean workbench with UV light 30 minutes in advance, turn on the fan, and wipe the workbench clean; (2) Take a 15mL centrifuge tube, add 1mL of complete culture medium, which includes 90% DMEM + 10% fetal bovine serum FBS (containing 1% penicillin and antibiotics); then add all the melted cells to the centrifuge tube (1:1) and mix gently; (3) Centrifuge: centrifuge at 1000rpm / 4min and discard the culture medium; (4) Take a fresh 1mL of complete culture medium to resuspend the cells (gently pipette); (5) After resuspending, add the cells to a T25 culture flask or culture dish for culture.

[0037] 2. Cell medium change and passage: (1) Observe the cell state and growth density under an inverted microscope; (2) If the cell culture medium turns yellow or there are floating cells, wash with PBS buffer 2-3 times and then replace with fresh complete culture medium; (3) If the cell density reaches 80%-90% of the exponential growth phase, add trypsin containing EDTA for digestion; (4) Centrifuge: centrifuge at 1000 rpm / 4 min and discard the culture medium; (5) Resuspend the cells in fresh complete culture medium, gently blowing to avoid generating bubbles and damaging the cells; (6) After resuspending, add the cells to a T25 culture flask or culture dish for culture.

[0038] Test results are shown in Table 2.

[0039] Table 2. Test results of the complex protein composition for improving skin condition in Experiment 1

[0040] Results analysis: The cell proliferation results of the experimental and control groups showed a clear regularity in the promoting effect of different complex protein compositions on the proliferation of human dermal fibroblasts. Experimental groups 1A to 7A all contained five key components: type IV, type VII, type XVII collagen, laminin 332, and fibronectin. With changes in the proportions of each group, cell proliferation initially showed an upward trend, reaching its optimal level in experimental group 4A before gradually declining. Among these, the proportions of experimental groups 2A to 6A all fell within the optimal mass ratio range, and these groups generally showed higher cell proliferation promoting effects than experimental groups 1A and 7A. This indicates that when the ratio of type IV collagen: type VII collagen: type XVII collagen: laminin 332: fibronectin is 2.8~3.8: 9.8~10.8: 4.8~5.8: 0.8~1.2: 7.2~8.3, a more coordinated synergistic effect can be formed, and the complex protein composition that improves skin condition has a more significant supporting effect on fibroblast activity.

[0041] In control groups 1 through 3, neither using only three types of collagen nor using only the combination of laminin 332 and fibronectin was as effective as the combination of five proteins used in experimental group 4A. This indicates that the three types of collagen, laminin 332, and fibronectin form a synergistic relationship that enhances each other; the absence of any one of them will affect the overall function.

[0042] Comparisons with groups 4 and 5, and experimental group 4A, show that in the absence of type VII collagen, even with the presence of the other four proteins in the composition, the proliferative effect did not reach the level of the five-protein combination in experimental group 4A. Furthermore, the effect was even more limited when type VII collagen was used alone.

[0043] Comparisons with groups 6 and 7, and experimental group 4A, show that when the composition lacks type XVII collagen, its proliferative effect is lower than that of experimental group 4A, even though the other four proteins are present. Furthermore, the effect of using type XVII collagen alone is relatively limited.

[0044] The comparisons with groups 8 and 9, and experimental group 4A, show that when fibronectin is absent in the composition, its proliferative effect is still lower than that of experimental group 4A, even though the other four proteins are present. Furthermore, the proliferative effect of fibronectin alone is relatively limited.

[0045] Experiment 2 1. Preparation of a complex protein composition for improving skin condition In this experiment, compositions were prepared according to the preparation methods and raw material compositions described in Experimental Group 1A, Experimental Group 4A, and Control Group 1, respectively. Different products were prepared with concentration as the variable. The product numbers and corresponding variables are shown in Table 3. Except for the variables shown in Table 3, the other raw material ratios and preparation methods used in the batteries prepared in this experiment were strictly consistent with those in Example 1.

[0046] 2. The cell proliferation rate test method described in Test Example 1 was used to test the above groups.

[0047] 3. Test Results: The test results for experimental group 4A are shown in Table 3. Figure 1 As shown; the test results for experimental group 1A are as follows: Figure 2 As shown, the test results for control group 1 are as follows: Figure 3 As shown in the figure. Based on the results, a hypothetical significance analysis was conducted on the proliferation rates of different formulations at the same concentration level, as well as the proliferation rates among different concentrations within the same formulation. One-way ANOVA was used, with a significance level set at p < 0.05.

[0048] Table 3. Cell proliferation rate corresponding to different concentrations of experimental group 4A

[0049] Results analysis: like Figures 1-3 As shown, the complex protein composition for improving skin condition provided by experimental group 4A exhibited the highest cell proliferation rate at a concentration of 0.01 mg / mL, approaching 200%, with a significant difference. Furthermore, at the same concentration, the complex protein composition for improving skin condition provided by experimental group 4A showed an advantage in cell proliferation rate compared to the complex protein compositions provided by experimental group 1A and control group 1.

[0050] Experiment 3 Cell Proliferation Experiment This experiment used the complex protein composition for improving skin condition from experimental group 4A, at a concentration of 0.01 mg / mL.

[0051] To investigate the effects of different UV irradiation doses on HSF cells, the irradiation intensity of a UV phototherapy device (365nm UVA) was first measured. Then, based on the formula that UV irradiation dose (D) = average UV irradiation intensity (Iaverage) and irradiation time (T), the required UVA irradiation time for different UV irradiation doses was calculated. Human dermal fibroblasts were treated in groups, and HFF-1 proliferation was detected using a CCK-8 assay kit.

[0052] Test results are as follows Figure 4 As shown, the proliferation inhibition rate of human dermal fibroblasts (HFF-1) gradually increased with increasing UV irradiation dose. At an irradiation dose of 4 J / cm², the proliferation inhibition rate reached approximately 50%, meaning cell viability decreased to half, a statistically significant difference compared to the control group. This result demonstrates that irradiation at 4 J / cm² can produce a clear biological effect, establishing a suitable model for subsequent research and facilitating further investigation of the protective or repairing effects of the complex protein composition on photodamaged cells under controlled conditions.

[0053] Experiment 4 Cell migration assay / scratch assay Experimental subjects: The compositions of experimental group 1A, experimental group 4A, and control group 1, all at a concentration of 0.01 mg / mL. Additionally, experiments were conducted using type I protein, type IV collagen, type XVII collagen, and BPOXY as individual components, and all were performed concurrently.

[0054] Experimental Procedure: When cells reached 90% confluence, an artificial "scratch" (cell-free region) was created on the monolayer of adherent cells. The culture medium was washed twice with PBS (phosphate-buffered saline). The solution was then used to clean the cells.2 After UVA treatment, various compound formulations and single-component collagen were added at 0.01 mg / mL. A Ctrl group (no UVA irradiation) served as the negative control, while a group irradiated with UVA but with the same volume of DMEM medium (serum-free medium to avoid affecting migration rate observation due to cell proliferation) served as the positive control. Cell migration in each culture dish was observed at 0 and 24 hours after the addition of each formulation using a 10x optical microscope. The ability of cells to migrate to the scratched area was recorded to assess the migration and repair capabilities of human dermal fibroblasts.

[0055] Experimental results: Based on the results shown in the figure ( Figures 5-8 Compared to the single protein, β-xylene, and control group 1, the complex protein compositions for improving skin condition in experimental groups 1A and 4A showed significant differences in cell migration area after 24 hours, but the complex protein composition for improving skin condition in experimental group 4A had a larger cell migration area. The combined results of cell proliferation and migration experiments indicate that this complex protein composition has a positive effect on promoting the activity and migration ability of human dermal fibroblasts, which is related to its coordinated regulation of the extracellular matrix microenvironment and helps support the structural repair process of skin tissue after aging or photodamage.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A complex protein composition for improving skin condition, characterized in that, Including type IV collagen, type VII collagen, type XVII collagen, laminin 332, and fibronectin.

2. The complex protein composition for improving skin condition as described in claim 1, characterized in that, The mass ratio of type IV collagen to type VII collagen to type XVII collagen to laminin 332 to fibronectin is 2.8~3.8: 9.8~10.8: 4.8~5.8: 0.8~1.2: 7.2~8.

3.

3. The complex protein composition for improving skin condition as described in claim 1, characterized in that, The mass ratio of type IV collagen to type VII collagen to type XVII collagen to laminin 332 to fibronectin is 3~3.5: 10~10.5: 5~5.5: 0.8~1.2: 7.5~8.

0.

4. The complex protein composition for improving skin condition as described in claim 1, characterized in that, The mass ratio of type IV collagen to type VII collagen to type XVII collagen to laminin 332 to fibronectin is 3.1-3.2: 10.3-10.4: 5.2-5.3: 1: 7.7-7.

8.

5. A product for improving skin condition, characterized in that, Its active ingredients include the complex protein composition for improving skin condition as described in any one of claims 1 to 4.

6. The product for improving skin condition as described in claim 5, characterized in that, The concentration of the compound protein composition for improving skin condition is 0.005~0.03 mg / mL.

7. The product for improving skin condition as described in claim 5, characterized in that, The concentration of the compound protein composition for improving skin condition is 0.005~0.015 mg / mL.

8. The product for improving skin condition as described in claim 5, characterized in that, The concentration of the complex protein composition for improving skin condition is 0.01 mg / mL.

9. The use of the complex protein composition for improving skin condition as described in any one of claims 1 to 4 in the preparation of products for improving skin condition.

10. The application as described in claim 9, characterized in that, The improvement in skin condition includes at least one of the following: promoting collagen expression, promoting skin collagen production, improving skin elasticity, and improving skin firmness.

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