A hair loss prevention and repair composition containing recombinant collagen and application thereof

Abstract

The application provides a recombined collagen-containing anti-hair loss repair composition and application thereof, and relates to the technical field of cosmetics.The recombined collagen-containing anti-hair loss repair composition comprises 1-4 parts of recombined type I collagen, 1-3 parts of recombined type III collagen and 2-5 parts of recombined type XI I collagen according to weight fraction.The recombined type I collagen, the recombined type III collagen and the recombined type XI I collagen are combined according to the weight ratio of (1-4):(1-3):(2-5) to form a multi-dimensional effect of "scalp structure support-hair follicle nutrient supply-hair follicle stem cell regulation", and the anti-hair loss repair effect is significantly better than that of single collagen or two collagen combinations, thereby providing a safe, efficient and stable systematic anti-hair loss repair scheme for people with hair loss.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to a hair loss prevention and repair composition containing recombinant collagen and its application. Background Technology

[0002] With increasing life pressures, irregular sleep patterns, and environmental factors, hair loss is becoming increasingly common and affecting younger people, creating an urgent market demand for safe and effective anti-hair loss products based on bioactive ingredients. Healthy hair growth is a complex biological process, rooted in a healthy scalp microenvironment and properly functioning hair follicles. An ideal scalp microenvironment should possess the following characteristics: Structural integrity: A healthy scalp dermis has a dense and elastic extracellular matrix (ECM), mainly composed of collagen and elastin, providing a stable physical anchor for hair follicles and preventing them from loosening and falling out due to external traction; Adequate nutrition: The capillary network surrounding the hair follicles must be unobstructed, continuously supplying the dermal papilla cells and hair matrix cells with the oxygen and nutrients necessary for growth; Normal functional regulation: The activity and differentiation cycle of hair follicle stem cells (HFSCs) are precisely regulated by multiple signaling pathways. Imbalance in any of these pathways can lead to premature entry of hair follicles into the regression and resting phases, or even miniaturization, ultimately resulting in hair loss and the inability to regrow. Hair loss is essentially a comprehensive manifestation of imbalances in one or more of the above pathways. Therefore, single-dimensional intervention often only treats the symptoms and not the root cause. Existing products cannot simultaneously cover multiple targets such as scalp structural support, hair follicle nutrition supply, and hair follicle degeneration, and cannot achieve a multi-dimensional and systematic anti-hair loss effect. Summary of the Invention

[0003] The problem addressed by this invention is how to achieve multi-dimensional, systematic hair loss prevention.

[0004] To address the above problems, the present invention provides a hair loss prevention and repair composition containing recombinant collagen and its application.

[0005] In a first aspect, the present invention provides a hair loss prevention and repair composition containing recombinant collagen, comprising, by weight, 1 to 4 parts of recombinant type I collagen, 1 to 3 parts of recombinant type III collagen, and 2 to 5 parts of recombinant type XVII collagen.

[0006] Optionally, the weight ratio of recombinant type I collagen, recombinant type III collagen, and recombinant type XVII collagen is 2:1:5.

[0007] Optionally, it may also include collagen component stabilizers, which include humectants, thickeners, pH adjusters, and preservatives.

[0008] Optionally, the collagen stabilizer, by weight, includes 8 to 35 parts of moisturizer, 0.5 to 8 parts of thickener, 0.1 to 3 parts of pH adjuster, and 0.1 to 0.5 parts of preservative.

[0009] Optionally, the humectant includes one or more of trehalose, beta-glucan, hydrolyzed sclerotium gum, mannose, glyceryl polyether-7, glyceryl polyether-26, glycerol, and 1,3-propanediol.

[0010] Optionally, the humectant, by weight, consists of 3 to 10 parts glycerin, 1 to 8 parts trehalose, 3 to 12 parts glyceryl polyether-26 and 2 to 10 parts 1,3-propanediol.

[0011] Optionally, the thickener includes one or more of xanthan gum, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, carbomer 980, and carbomer 940.

[0012] Optionally, the pH adjuster includes one or more of triethanolamine, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0013] Alternatively, the preservative may include one or more of phenoxyethanol, methylparaben, and propylparaben.

[0014] Secondly, the present invention provides the application of the hair loss prevention and repair composition containing recombinant collagen as described above in the preparation of hair loss prevention and repair products.

[0015] The beneficial effects of the hair loss prevention and repair composition containing recombinant collagen of the present invention and its application are as follows: recombinant type I collagen, recombinant type III collagen, and recombinant type XVII collagen are combined in a weight ratio of (1-4):(1-3):(2-5) to form a multi-dimensional effect of "scalp structure support - hair follicle nutrition supply - hair follicle stem cell regulation". The hair loss prevention and repair effect is significantly better than that of single collagen or two collagen combinations, providing a safe, efficient and stable systemic hair loss prevention and repair solution for people with hair loss. Attached Figure Description

[0016] Figure 1 This is a line graph comparing the collagen secretion-promoting experiments of the anti-hair loss and repair composition of the present invention with those of the comparative examples; Figure 2 This is a bar chart comparing the average VEGF secretion amount of dermal papilla cells in hair follicles with that of the anti-hair loss and repair composition of the present invention, in both examples and comparative examples. Figure 3 This is a bar chart comparing the average cell survival rate of the anti-hair loss and repair composition of the present invention with that of the comparative examples; Figure 4 This is a line graph comparing the collagen retention rate of the anti-hair loss and repair composition of the present invention with that of the comparative examples. Detailed Implementation

[0017] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention's description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "comprising" and its variations are open-ended, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the description below. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0019] An embodiment of the present invention provides a hair loss prevention and repair composition containing recombinant collagen, which, by weight, comprises 1 to 4 parts of recombinant type I collagen, 1 to 3 parts of recombinant type III collagen, and 2 to 5 parts of recombinant type XVII collagen.

[0020] Specifically, recombinant collagen, as a bioactive component with an amino acid sequence highly consistent with human collagen, possesses excellent biocompatibility and targeting function. Recombinant type I collagen (COL1A1) is the main structural collagen in the dermis (accounting for 80-90%), forming a dense fiber network around hair follicles. Its high tensile strength can enhance the toughness of the scalp dermis and reduce loosening or displacement of hair follicles due to mechanical forces. Recombinant type III collagen (COL3A1) forms a loose network structure and is mainly distributed around blood vessels (accounting for 10-15% of skin collagen). Utilizing its high cell adhesion and three-dimensional porous structure, it can promote the proliferation and migration of vascular cells, possessing excellent tissue repair and nutrient delivery functions, and delaying hair follicle atrophy. Recombinant type XVII collagen (COL17A1) is a transmembrane protein specifically expressed at the epidermal-dermal junction (DEJ) and the follicular bulge area. It can stabilize the ecological niche of hair follicle stem cells (HFSCs) through integrin α6β4-mediated adhesion signals, activate the Wnt / β-catenin pathway, inhibit the miniaturization process of hair follicles, and prolong the hair follicle growth phase. Type XVII collagen can further enhance the synthesis of type I and III collagen by upregulating the TGF-β signaling pathway. After type III collagen binds to α1β1 / α2β1 integrins, it can regulate angiogenesis, acting on hair follicle-related cells through a dual pathway: on the one hand, it optimizes the microenvironment of hair follicle nutrient supply, promoting the penetration of type XVII collagen into the bulge area; on the other hand, it activates vascularized dermal papilla cells, mediating their signal transduction to hair follicle stem cells. After type XVII collagen activates hair follicle stem cells, the hair follicle epithelial cells differentiated from the stem cells need to rely on the structural space provided by type I collagen and the nutrients transported by type III collagen to successfully proliferate and differentiate, forming a healthy hair shaft.

[0021] In this embodiment, recombinant type I collagen, recombinant type III collagen, and recombinant type XVII collagen are combined in a weight ratio of (1-4):(1-3):(2-5) to form a multi-dimensional effect of "scalp structure support - hair follicle nutrition supply - hair follicle stem cell regulation". The anti-hair loss and repair effect is significantly better than that of single collagen or two collagen combinations. The triple collagen core system composed of recombinant type I collagen, recombinant type III collagen, and recombinant type XVII collagen provides a safe, efficient and stable systemic anti-hair loss and repair solution for people with hair loss.

[0022] Optionally, the weight ratio of recombinant type I collagen, recombinant type III collagen, and recombinant type XVII collagen is 2:1:5.

[0023] In this embodiment, controlling the mass ratio of recombinant type I collagen, recombinant type III collagen, and recombinant type XVII collagen can prevent the content of any single component from being too high or too low, which could affect the anti-hair loss and repair effect.

[0024] Optionally, it may also include collagen component stabilizers, which include humectants, thickeners, pH adjusters, and preservatives.

[0025] Specifically, the solvent is purified water.

[0026] In this optional embodiment, the collagen stabilizer has good compatibility with the triple collagen core system. It can be stored for more than 90 days at 4℃, 25℃, and 40℃, with a collagen content retention rate of ≥91%. Moreover, the system does not exhibit stratification, turbidity, or precipitation, and the viscosity fluctuation is ≤10%, ensuring the long-term effectiveness of the product. This solves the problems of existing collagen-based anti-hair loss products having a single dimension of action and poor compatibility with excipients.

[0027] Optionally, the collagen stabilizer, by weight, includes 8 to 35 parts of moisturizer, 0.5 to 8 parts of thickener, 0.1 to 3 parts of pH adjuster, and 0.1 to 0.5 parts of preservative.

[0028] Specifically, moisturizers build a solid foundation for scalp hydration, replenishing and locking in moisture to prevent dryness and tightness. They improve the scalp's microenvironment, providing a moist and comfortable growth environment for hair follicles, indirectly aiding in hair loss prevention and repair. They also enhance the moisturizing feel of the product during use, reducing dryness and pulling sensations on the scalp.

[0029] Thickeners optimize the product's user experience by adjusting its viscosity, resulting in a smooth, non-dripping formula that allows for even coverage of the scalp. They enhance the adhesion of ingredients to the scalp, prolonging the effectiveness of active ingredients such as recombinant collagen. They also improve the smoothness and stability of the product texture, preventing separation and sedimentation.

[0030] pH adjusters ensure formula stability and scalp compatibility, adjusting the product's pH value to near the scalp's slightly acidic range (typically 5.5-7.0) to reduce scalp irritation. They maintain the structural stability of ingredients such as recombinant collagen in the formula, preventing them from becoming ineffective due to pH imbalance. They help maintain the scalp's natural barrier function, reducing the risk of scalp sensitivity and inflammation, indirectly aiding in repair.

[0031] Preservatives safeguard the product's safety and shelf life, inhibiting the growth of bacteria, mold, and other microorganisms in the formula and preventing product spoilage. They also prevent scalp infections, allergies, and other problems caused by microbial contamination, ensuring safe use. Extending the product's shelf life ensures that the efficacy and safety of the ingredients are unaffected when consumers use it within the expiration date.

[0032] In this optional embodiment, the recombinant collagen is assisted in exerting its anti-hair loss and repair effects from four dimensions: moisturizing, texture, stability, and safety, making the product both easy to use and durable.

[0033] Optionally, the humectant includes one or more of trehalose, beta-glucan, hydrolyzed sclerotium gum, mannose, glyceryl polyether-7, glyceryl polyether-26, glycerol, and 1,3-propanediol.

[0034] In this optional embodiment, glycerin, 1,3-propanediol, glyceryl polyether-7, and glyceryl polyether-26 are small-molecule moisturizers that can quickly penetrate the scalp stratum corneum to replenish deep moisture and form a breathable moisturizing film on the skin surface to reduce moisture loss. Trehalose and mannose are sugar moisturizers that not only absorb and lock in moisture but also relieve the tightness caused by scalp dryness and improve the smoothness and hydration during use. β-glucan and hydrolyzed sclerotium gum have both moisturizing and gentle repairing properties, helping to maintain the scalp barrier function, reduce scalp sensitivity caused by dryness, and create a stable environment for hair follicles. All ingredients are low-irritant and highly compatible, and will not react with recombinant collagen, ensuring the stability of the core active ingredients while avoiding burdening the scalp.

[0035] Optionally, the humectant, by weight, consists of 3 to 10 parts glycerin, 1 to 8 parts trehalose, 3 to 12 parts glyceryl polyether-26 and 2 to 10 parts 1,3-propanediol.

[0036] In this optional embodiment, glycerol and 1,3-propanediol have small molecular weights, allowing them to quickly penetrate the superficial layer of the stratum corneum, replenish moisture promptly, and prevent collagen from shrinking and deforming due to dehydration of the surface layer; the long-chain structure of glycerol polyether-26 forms a moisture-releasing system in the middle layer of the stratum corneum, continuously providing a moist environment for collagen; trehalose can penetrate into the deep layer of the stratum corneum, regulate intracellular osmotic pressure, protect cell structure, and prevent conformational changes caused by dehydration of the deep layer of collagen.

[0037] Optionally, the thickener includes one or more of xanthan gum, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, carbomer 980, and carbomer 940.

[0038] Specifically, the thickener adjusts the viscosity of the composition to 2800-5000 mPa. s.

[0039] In this optional embodiment, xanthan gum, hydroxyethyl cellulose, and ethyl hydroxyethyl cellulose have both thickening and suspending effects, which can enable the formula to form a uniform gel or emulsion, avoid component separation, and improve the smoothness of application, making it easier to spread on the scalp.

[0040] The thixotropic gel system formed by Carbopol 980 and Carbopol 940 can encapsulate collagen molecules, reducing their contact with the external environment, minimizing activity loss, and providing high thickening efficiency. A small amount is sufficient to achieve the ideal viscosity, while also giving the product a refreshing feel, preventing stickiness or suffocation on the scalp. This aligns with the "easy absorption, no residue" requirements of anti-hair loss products. All ingredients are highly compatible with moisturizers, pH adjusters, preservatives, and recombinant collagen, ensuring no reactions that could render the formula ineffective and guaranteeing overall stability. It enhances the product's film-forming properties and adhesion on the scalp, prolonging the residence time of core ingredients such as recombinant collagen, allowing for more effective repair and anti-hair loss treatments. The texture is gentle and non-irritating, meeting the gentle requirements of scalp care products and avoiding scalp sensitivity and discomfort caused by thickeners.

[0041] Optionally, the pH adjuster includes one or more of triethanolamine, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0042] Specifically, it is used to adjust the pH of the composition to 5.5-7.0.

[0043] In this optional embodiment, the pH adjuster can efficiently and gently adjust the pH value of the formulation to a range suitable for the scalp, while being compatible with other ingredients, ensuring the stability and safety of the recombinant collagen anti-hair loss repair composition.

[0044] Specifically, triethanolamine is an alkaline regulator that quickly neutralizes acidic components in the formula, adjusting the pH to a slightly acidic level (4.5-5.5), matching the scalp's natural pH environment. Sodium dihydrogen phosphate (acidic) and disodium hydrogen phosphate (alkaline) are often used together to form a buffer system, which not only regulates the pH but also stabilizes the formula's pH, preventing pH fluctuations during subsequent use or storage. This avoids scalp irritation due to excessively high or low pH levels, reducing redness, itching, and other discomfort, and protecting the scalp barrier function. All three pH regulators are highly compatible with moisturizers, thickeners, preservatives, and recombinant collagen, and will not cause chemical reactions that lead to ingredient degradation or formula separation and precipitation. Triethanolamine can also neutralize carbomer-based thickeners, further enhancing the product's thickening effect and smoothness, achieving two benefits at once.

[0045] Alternatively, the preservative may include one or more of phenoxyethanol, methylparaben, and propylparaben.

[0046] Specifically, phenoxyethanol is a broad-spectrum antibacterial agent, inhibiting bacteria and fungi, and has good water solubility, allowing it to quickly integrate into the formulation. Methylparaben and propylparaben, belonging to the paraben class, have strong specific antibacterial activity, effectively inhibiting Gram-positive, Gram-negative bacteria, and fungi; their combination enhances the antibacterial range and efficiency. This prevents product deterioration due to microbial contamination during production, storage, and use, avoiding scalp infections, allergies, and other problems. The three preservatives are compatible with the previously mentioned moisturizers, thickeners, pH adjusters, and recombinant collagen, and will not react to cause formula failure or abnormal texture. With low irritation, within the recommended dosage of 0.1-0.5 parts, it meets the mild standards for scalp care products and will not significantly burden the scalp barrier. Phenoxyethanol also has mild moisturizing properties, enhancing the product's hydration while inhibiting bacteria, creating a synergistic effect with the moisturizing system. When used alone, methylparaben or propylparaben can achieve a basic antibacterial effect at a dosage of 0.1-0.3 parts. When used alone, phenoxyethanol can be used at a slightly higher dosage (0.3-0.5 parts). When used in combination (such as phenoxyethanol + propylparaben), the total dosage should be controlled within 0.1-0.5 parts. This reduces the amount of a single preservative used, lowers the risk of irritation, and improves antibacterial stability, making it suitable for long-term storage.

[0047] In this optional embodiment, the preservative effectively inhibits microbial growth at low dosages, ensuring the safety and shelf life of the recombinant collagen anti-hair loss and repair composition. At the same time, it has strong compatibility, low irritation, and does not affect the performance of the core efficacy.

[0048] Another embodiment of the present invention provides the application of the hair loss prevention and repair composition containing recombinant collagen as described above in the preparation of hair loss prevention and repair products.

[0049] Specifically, anti-hair loss and hair repair products include one or more hair care products such as anti-hair loss shampoo, conditioner, anti-hair loss serum, hair mask, and hair oil.

[0050] The present invention will be further described below with reference to specific embodiments.

[0051] Example 1 The preparation method of the anti-hair loss and repair composition containing recombinant collagen is as follows: (1) Collagen pre-dissolution: Weigh the above-mentioned recombinant type I, type III and type XVII collagen, mix them, add 1,3-propanediol and glycerol, and stir to form a transparent collagen encapsulation solution; (2) Carbomer-glycerol polyether mixture: Carbomer 980 was added to 50 ml of purified water and fully swollen. Then glycerol polyether-26 was added and stirred evenly to obtain carbomer-glycerol polyether-26 mixture; (3) System fusion: Slowly add collagen encapsulation solution to glycerol polyether-26 mixture, then add trehalose and triethanolamine, stir until pH=6.1, add methylparaben and propylparaben, add water to 100g, stir for 35 minutes; (4) Post-processing: Cool the mixed composition to room temperature to obtain a colorless and transparent recombinant collagen anti-hair loss repair composition, put it into a clean container and seal it for storage.

[0052] Detailed Implementation Plan First, we screened the synergistic ratio of the triple recombinant collagen system, and then verified the stability of collagen content by the collagen component stabilizer.

[0053] I. Experimental Verification of the Anti-Hair Loss Effect and Synergistic Effect of the Triple Recombinant Collagen System Experimental objective: To verify the synergistic effect of a system composed of recombinant type I + type III + type XVII collagen in a specific ratio in the dimensions of "scalp structural support - hair follicle nutrition supply - hair follicle regulation", and its advantages in preventing hair loss and repairing hair loss compared with single collagen and the combination of two types of collagen.

[0054] Samples tested: (1) Examples 1-7 and Comparative Examples 1-18, the amount of recombinant type I collagen, recombinant type III collagen and recombinant type XVII collagen added to the triple recombinant collagen core system is shown in Table 1.

[0055] (2) Example and Comparative Example Design: Example 1 is the target preferred ratio; Examples 2-3 cover the boundary of the range; Examples 4-5 supplement extreme combinations to verify the influence weight of the ratio of each component; Examples 6-7 balance the intermediate values ​​to reflect the continuity of the ratio gradient. Comparative Examples 1-12 verify the independent effect of a single collagen component at different concentrations and clarify the effective concentration boundary of each component; Comparative Examples 13-15 verify the synergistic effect of two components when combined in the target ratio; Comparative Examples 16-18 verify the efficacy when the ratio exceeds the patent limit range, highlighting the necessity of the target range.

[0056] (3) Composition and preparation method: The collagen component stabilizers and preparation methods of Examples 1-7 and Comparative Examples 1-18 are the same.

[0057] Table 1. Proportion of each component in the triple-recombinant collagen core system

[0058] Experiment 1: Collagen secretion promotion experiment (detecting the scalp's structural support capacity).

[0059] Experimental Objective: This experiment aims to verify the promoting effect of the triple collagen system on the secretion of type I and type III collagen by human skin fibroblasts (HSF) and to evaluate the structural support capacity of the scalp.

[0060] Test samples: Examples 1-7 and Comparative Examples 1-18; The main instruments and reagents used in the detection were: ELISA reader, UVA irradiator, real-time quantitative PCR instrument, HSF cells (human skin fibroblasts), DMEM culture medium, fetal bovine serum, penicillin-streptomycin antibody, and human type I and type III collagen ELISA kits.

[0061] Testing steps: Sample preparation: Each test sample was diluted to 0.1% (total collagen concentration) using HSF complete cell culture medium as the test solution.

[0062] Cell preparation: HSF cells in logarithmic growth phase were digested with trypsin, and digestion was terminated by adding complete culture medium. The cell density was adjusted to 6 × 10⁶ cells / year. 4 Cells were seeded per well in a 24-well plate with 1 mL of cell suspension added to each well. The cells were then incubated at 37°C in a 5% CO2 incubator for 24 hours until the cell confluence reached 70%-80%.

[0063] Grouping and Modeling: Each group has at least 6 replicates, and the culture time is 48 hours. The grouping is as follows: Normal group: HSF cells were cultured in serum-free DMEM and were not irradiated with UVA.

[0064] Model group: HSF cells were irradiated with UVA for 30 min to establish the model, and then cultured in serum-free medium; Experimental group: After HSF cells were modeled by UVA irradiation, the original culture medium was removed, and 1 mL of the test solution was added to each well for culture.

[0065] Collagen detection: After 48 hours of culture, the supernatant from each well was collected. Following the ELISA kit instructions, the OD value at 450 nm was measured using a microplate reader. The result was then substituted into the standard curve equation (R0). 2 ≥0.99) Calculate collagen concentration.

[0066] Data processing: Calculation of collagen secretion per unit cell: Use the following formula.

[0067] Where Q: Total amount of collagen secreted per unit number of HSF cells, ng / cell; Xs: Collagen concentration in the experimental group, ng / mL; Xc: Collagen concentration in the model group, ng / mL; V: Culture medium volume, mL; ρ: Number of cells at termination.

[0068] Promotion rate calculation: Promotion rate (%) = [(Collagen concentration in experimental group - Collagen concentration in model group) / (Collagen concentration in normal group - Collagen concentration in model group)] × 100%; Statistical analysis: Data are expressed as mean. SPSS software was used for t-tests and one-way ANOVA. P < 0.05 was considered statistically significant.

[0069] The experimental results are shown in Table 2. Table 2 Statistical data for the examples and comparative examples

[0070] Plot the data in Table 2 as a line chart, see... Figure 1 Table 2 and Figure 1 It is evident that single-component groups can only promote the secretion of a specific type of collagen at specific concentrations, resulting in uneven effects. Two-component combinations showed better promotion rates than most single-component groups, but significantly lower than three-component combinations. Non-target three-component combinations, deviating from the patented ratio range, exhibited significantly insufficient efficacy. The target three-component combinations (Examples 1-7) showed much higher promotion rates for type I and type III collagen secretion than other groups, with Example 1 showing the highest rate. This confirms that the triple collagen compounded according to the proportions of this invention can achieve synergistic effects in the "scalp structure support" dimension, and the effect is significantly better than systems using single collagen or two-collagen combinations.

[0071] Experiment 2: Vascular endothelial growth factor (VEGF) content experiment (detecting hair follicle nutrient supply).

[0072] Experimental objective: To verify the promoting effect of the triple collagen system on VEGF secretion by human dermal papilla cells (DPCs) and to evaluate the nutrient supply capacity of hair follicles.

[0073] Test samples: Examples 1-7 and Comparative Examples 1-18; The main instruments and reagents used in the detection were: human dermal papilla cells, MSCM culture medium, PBS, MTT, DMSO, minoxidil, dihydrotestosterone, and Human VEGF ELISA kit.

[0074] Testing steps: Cell preparation: Resuscitate dermal papilla cells and culture them in MSCM medium until the plating rate reaches 60%, then adjust the cell density to 5 × 10⁶ cells / year. 4 Inoculate one well per well in a 6-well plate and incubate overnight at 37°C with 5% CO2.

[0075] Solution preparation: Prepare the working solution of the test substance according to the experimental design shown in Table 3.

[0076] Table 3 Working solutions of test substances

[0077] Drug administration: According to the experimental design shown in Table 3, when the cell layering rate in the 6-well plate reached 40%~60%, the cells were administered to groups, with 2 mL of drug per well and 3 replicates per group. The cells were incubated in an incubator (37℃, 5% CO2) for 24 h. Collect cell supernatant: After culturing for 24 hours, collect the cell culture supernatant in EP tubes. After collection, it will be used for VEGF secretion content detection. The sample is stored frozen at -80℃. Detection and Data Processing: Follow the ELISA kit instructions, measure the OD value at 450nm wavelength using a microplate reader, and substitute it into the standard curve equation (R0). 2 VEGF concentration was calculated using a ≥0.99 g / L scale, plotted using GraphPadPrism 9.0 software, and statistically analyzed using the t-test method (P<0.05 was considered statistically significant). The experimental results are shown in Table 4.

[0078] Table 4. Average VEGF concentration in each group

[0079] The results of the examples and comparative examples are plotted in a bar chart based on Table 4, as shown below. Figure 2 As shown.

[0080] From Table 4 and Figure 2 As can be seen, the concentration of the target triple collagen combination (Examples 1-7) was 730.6~780.3 pg / mL, significantly higher than that of PC and Comparative Examples 1-18. The synergistic effect of the multiple components was significant, and the advantage of hair follicle nutrient supply far exceeded that of single or two-combination collagen. This confirms that the triple collagen compounded according to the proportions of this invention can achieve synergistic effects in the dimension of "hair follicle nutrient supply", and the effect is significantly better than that of single collagen and two-combination collagen systems.

[0081] Experiment 3: Hair follicle stem cell proliferation activity experiment (detecting cell regulatory function).

[0082] Experimental objective: This experiment aims to verify the promoting effect of the triple collagen system on the proliferation of rat hair follicle stem cells (HFSCs) and to evaluate the regulatory capacity of hair follicle stem cells.

[0083] Test samples: Examples 1-7 and Comparative Examples 1-18; The main instruments and reagents used in the detection were: microplate reader, CO2 cell culture incubator, rat hair follicle stem cells, stem cell culture medium, CCK8 reagent kit, serum-free DMEM medium, and minoxidil.

[0084] Testing steps: Cell preparation: Rat hair follicle stem cells were cultured in stem cell-specific culture medium, and the cell density was adjusted to 3 × 10⁻⁶ cells / year. 3 Cells were seeded per well in a 96-well plate with 100 μL of cell suspension added to each well. The cells were then incubated at 37°C and 5% CO2 for 24 h until they adhered to the plate. Grouping and drug administration: Each group has no fewer than 6 replicates, and the culture time is 48 hours. The grouping is as follows: Negative control group (NC group): Only stem cell culture medium was added, without any active ingredients; Positive control group (PC group): Added stem cell culture medium containing 500 μM minoxidil; Experimental group: Add stem cell-specific culture medium containing 0.1% of each test sample; CCK8 assay: After 48 h of culture, discard the original culture medium, add 100 μL of culture medium containing 5 μL CCK8 reagent to each well, and continue to culture in a 37℃, 5% CO2 incubator for 4 h. Measure the OD value at 490 nm wavelength using an ELISA reader. Data processing: Cell viability was calculated using the following formula: Cell viability = [(As-Ab) / (An-Ab)] × 100%. Where Ab is the absorbance of the blank group, An is the absorbance of the negative control group, and As is the absorbance of the positive control group or experimental group.

[0085] Statistical analysis: t-tests and one-way ANOVA were performed using SPSS 26.0 software. P < 0.05 was considered statistically significant.

[0086] The experimental results are plotted as a bar chart, such as... Figure 3 As shown.

[0087] Figure 3 As can be seen, the cell survival rate of the single-component group was slightly higher than or close to that of the positive control, but the promoting effect was limited; the cell survival rate of the two-component combination group was significantly higher than that of the single-component group, but still far lower than that of the target three-component combination group; the cell survival rate of the non-target three-component combination group was close to that of the two-component combination group, showing no obvious advantage; the survival rate of hair follicle stem cells in Examples 1-7 was significantly higher than that in Comparative Examples 1-18. This confirms that the triple collagen compounded according to the proportions of this invention can achieve synergistic enhancement in the dimension of "hair follicle stem cell regulation", and the effect is significantly better than the systems of single collagen and two collagen combinations.

[0088] In summary, the collagen secretion, VEGF expression, and HFDPC cell survival rate of the embodiments were significantly better than those of the parallel ratios, indicating that the system composed of recombinant type I + type III + XVII collagen in a specific ratio of (1-4):(1-3):(2-5) achieves synergistic effects in multiple dimensions of "scalp structure support - hair follicle nutrition supply - hair follicle stem cell regulation", and its synergistic effect in preventing hair loss and repairing hair loss is significantly better than that of single collagen and the system of two collagen combinations.

[0089] II. The effect of collagen stabilizers on the stability of collagen content.

[0090] Experimental objective: To verify the protective effect of the collagen component stabilizer screened in this invention on the content stability of a triple collagen system (recombinant type I, type III, and type XVII collagen, weight ratio 2:1:5), and to evaluate the impact of missing / incompatible excipients.

[0091] Tested samples: Examples 1, 8-9 and 19-25: All samples have the same triple collagen core system (recombinant type I 0.2%, type III 0.1%, type XVII 0.5%), only the collagen component stabilizer is different, as shown in Table 5. The preparation method is the same as in Example 1.

[0092] Table 5 Collagen component stabilizer formulation

[0093] Main instruments and reagents used in the test: microplate reader (560nm), QuickZyme collagen test kit, 10N NaOH, 10N HCl; Testing steps: (1) Sample pretreatment: Take 50 μL of sample and centrifuge to remove particulate matter; if the concentration is too high, dilute with deionized water to the detection range.

[0094] (2) Collagen hydrolysis: Take 50 μL of pretreated sample, add 50 μL of 10N NaOH and mix, heat at 100℃ for 1 h (hydrolyze collagen to hydroxyproline); after cooling to 25℃, add 50 μL of 10N HCl to neutralize (adjust pH to 7.0), add 150 μL of deionized water to dilute 1:1, centrifuge at 4℃ and 10000 rpm for 2 min, and take the supernatant for later use; (3) Preparation of standard: Take 20 μL of 1 mg / mL hydroxyproline standard, add 380 μL of deionized water to prepare a 50 μg / mL stock solution, and then serially dilute to prepare a series of standard solutions (covering the detection range of the kit).

[0095] (4) 96-well plate operation: Add 20 μL of standard / sample supernatant to each well, and set up 3 replicates; add 8 μL of reagent A (oxidation reagent) + 90 μL of oxidation buffer, mix well and incubate at room temperature for 10 min; add 90 μL of reagent B (colorimetric reagent), transfer the liquid up and down until the turbidity disappears, and incubate at 37℃ for 90 min; measure the OD value at 560 nm wavelength using an ELISA reader. Data Calculation: Fitting the Standard Curve (R) 2 ≥0.99), calculate the collagen concentration in the sample, and calculate the collagen retention rate using the following formula: Collagen retention rate (%) = (Collagen concentration after storage / Initial collagen concentration) × 100%; System status observation: Record the changes in appearance (color, clarity) and viscosity (25℃, 60rpm) of each sample during storage at 4℃, 25℃, and 40℃ for 90 days.

[0096] The results of the collagen retention rate experiment are shown in Table 6.

[0097] Table 6. Results of Collagen Retention Rate Experiment

[0098] Draw a line chart based on the data in Table 6, such as... Figure 4 As shown.

[0099] Table 6 and Figure 4 The experimental results of observing the system state are as follows: Example 1, 8-12: The gel remained colorless and transparent under all storage conditions within 90 days, without layering, turbidity, or precipitation; the viscosity remained between 2800-4800 mPa. s, with initial viscosity (3200 mPa) The difference is ≤10% (approximately 10 seconds), and there is no significant change in the skin feel after application (good spreadability and absorption).

[0100] Comparative Examples 19-21 (not compatible with humectant): After storage at 25℃ for 60 days, no obvious stratification was observed, and the viscosity decreased to 2000-2600 mPa. s, a dried film appeared on the surface of some samples.

[0101] Comparative Example 22 (thickener not compatible): Particulate precipitation appeared after 60 days of storage at 4℃; viscosity fluctuated greatly after storage at 25℃ and 40℃ (it decreased to 2000 mPa after 90 days at 40℃). (Below s), it is prone to clumping when applied.

[0102] Comparative Example 23 (preservative not compatible): After 60 days of storage at 40℃, a slight odor and turbidity appeared in the solution. After 90 days of storage at 40℃, collagen precipitation led to sedimentation and a significant decrease in content retention.

[0103] Comparative Examples 24-25 (pH adjuster incompatible): Slight pH drift occurred under all storage conditions (initial 6.1-90 days 4.8-5.2), and viscosity decreased to 2500 mPa. Below s.

[0104] Experimental data analysis: The example groups (Examples 1, 8-12), thanks to their suitable combination of collagen stabilizers, maintained an average collagen retention rate exceeding 91% under different storage conditions (4℃, 25℃, and 40℃), and the system remained stable throughout. In contrast, the comparative groups, due to the absence / replacement of humectants and incompatibility of thickeners / pH adjusters / preservatives, exhibited an average collagen retention rate of only 70.03%-84.35%, and problems such as turbidity and abnormal viscosity were observed.

[0105] This indicates that the collagen component stabilizer screened in this invention has excellent compatibility with the triple collagen system, and its stability protection effect on the triple collagen system is significantly better than that of stabilizer systems with missing or incompatible components.

[0106] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A hair loss prevention and repair composition containing recombinant collagen, characterized in that, Based on weight parts, it includes 1 to 4 parts of recombinant type I collagen, 1 to 3 parts of recombinant type III collagen, and 2 to 5 parts of recombinant type XVII collagen.

2. The anti-hair loss and repair composition containing recombinant collagen according to claim 1, characterized in that, The weight ratio of the recombinant type I collagen, the recombinant type III collagen, and the recombinant type XVII collagen is 2:1:

5.

3. The anti-hair loss and repair composition containing recombinant collagen according to claim 1, characterized in that, It also includes collagen component stabilizers, which include humectants, thickeners, pH adjusters, and preservatives.

4. The anti-hair loss and repair composition containing recombinant collagen according to claim 3, characterized in that, The collagen stabilizer comprises, by weight, 8 to 35 parts of moisturizer, 0.5 to 8 parts of thickener, 0.1 to 3 parts of pH adjuster and 0.1 to 0.5 parts of preservative.

5. The anti-hair loss and repair composition containing recombinant collagen according to claim 3, characterized in that, The humectant includes one or more of trehalose, β-glucan, hydrolyzed sclerotium gum, mannose, glyceryl polyether-7, glyceryl polyether-26, glycerol, and 1,3-propanediol.

6. The anti-hair loss and repair composition containing recombinant collagen according to claim 5, characterized in that, The humectant, by weight, comprises 3 to 10 parts glycerin, 1 to 8 parts trehalose, 3 to 12 parts glyceryl polyether-26 and 2 to 10 parts 1,3-propanediol.

7. The anti-hair loss and repair composition containing recombinant collagen according to claim 3, characterized in that, The thickener includes one or more of xanthan gum, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, carbomer 980, and carbomer 940.

8. The anti-hair loss and repair composition containing recombinant collagen according to claim 3, characterized in that, The pH adjuster includes one or more of triethanolamine, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

9. The anti-hair loss and repair composition containing recombinant collagen according to claim 3, characterized in that, The preservatives include one or more of phenoxyethanol, methylparaben, and propylparaben.

10. The use of the anti-hair loss and repair composition containing recombinant collagen as described in any one of claims 1-9 in the preparation of anti-hair loss and repair hair products.

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