Anti-hair loss and hair growth composition based on cyclic polypeptide c8DSS as well as preparation method and application of anti-hair loss and hair growth composition

Abstract

The invention provides an anti-hair loss and hair growth composition based on cyclic polypeptide c8DSS as well as a preparation method and application of the anti-hair loss and hair growth composition, and relates to the technical field of anti-hair loss and hair growth compositions. The hair loss preventing and hair growing composition based on the cyclic polypeptide c8DSS is prepared from the following components in percentage by mass: 0.45 percent to 1.36 percent of the cyclic polypeptide c8DSS, 0.45 percent of a stable protective agent, 2.73 percent of a penetration enhancer, 2.912 percent of a moisturizing stabilizer and the balance of purified water. The straight-chain 8DSS polypeptide is cyclized into c8DSS, so that the stability and the biological activity of the c8DSS polypeptide are remarkably improved; meanwhile, the annular structure keeps the optimal spatial configuration, the binding capacity with a cell receptor is enhanced, the hair follicle growth cycle can be regulated and controlled through hair follicle sebaceous gland deep penetration, and hair follicles are promoted to enter a growth period from a resting period in advance. The traditional Chinese medicine composition is scientific and reasonable in formula, can be prepared into various dosage forms, is convenient to use, good in safety, free of obvious side effects and wide in application range, and has good market application prospects.

Description

Technical Field

[0001] This invention relates to the field of anti-hair loss and hair growth composition technology, and in particular to an anti-hair loss and hair growth composition based on cyclic polypeptide c8DSS, its preparation method and application. Background Technology

[0002] Hair is an important indicator of human health, and its normal growth is closely related to the overall health of the body. However, with the fast pace of modern life, nutritional imbalances, air pollution, increased work and life stress, and various pathological factors can all lead to increasingly common hair loss problems. These adverse factors can shorten or abnormally terminate the hair growth cycle, resulting in damaged hair follicle function, insufficient nutrient supply to the hair, and consequently, hair follicle mutation, atrophy, or even necrosis, ultimately causing hair growth obstruction and hair loss.

[0003] Currently, hair loss prevention and hair regrowth products on the market mainly fall into three categories. The first category includes products with added natural plant extracts or synthetic growth promoters. These primarily improve microcirculation around hair follicles through blood-activating ingredients, promoting the absorption of nutrients by hair follicle cells. However, this method is slow to take effect and has limited efficacy. The second category uses drug therapy, with representative drugs including minoxidil and finasteride. While these have some effect, long-term use has significant side effects, limiting their clinical application. The third category is based on tissue engineering technology. This method takes a cellular approach, regulating the differentiation and directed proliferation of hair follicle stem cells to transform aging and atrophied hair follicles into healthy hair follicle cells, enabling hair regrowth and fundamentally solving the problem of hair loss. This represents the future direction of hair loss treatment.

[0004] According to cell and molecular biology research, signal regulation in hair follicle stem cells involves multiple signaling pathways, among which the Wnt signaling pathway plays a decisive role. Key proteins involved in this pathway include Wnt protein, β-catenin, and GSK-3β, which effectively promote hair follicle cell differentiation and proliferation. Studies have found that the polypeptide 8DSS, composed of eight repeats of the aspartic-serine-serine tripeptide and containing 24 amino acids, not only plays a regulatory role in tooth calcification but also significantly promotes hair regeneration in shaved areas of experimental animals. 8DSS can activate the expression levels of GSK-3β and β-catenin in the cell nucleus, thereby inducing hair follicle development and regeneration by activating the Wnt signaling pathway.

[0005] However, the linear 8DSS peptide exhibits significant stability issues. The N-terminus of this linear peptide is asparagine, and the C-terminus is serine. In acidic solutions, asparagine readily undergoes deamidation, leading to peptide bond breakage and the release of aspartic acid. In alkaline environments, the hydroxyl group on the β-carbon atom of the serine residue readily undergoes β-elimination, forming an unsaturated structure. Furthermore, once inside the skin, the linear peptide is easily hydrolyzed by proteases in the cellular fluid, severely impacting the persistence and stability of its efficacy. These problems limit the effectiveness and widespread application of linear 8DSS peptides in practical applications. Summary of the Invention

[0006] To overcome the problems of poor stability and easy degradation of existing linear 8DSS peptides, this invention provides a hair loss prevention and hair growth composition based on cyclic peptide c8DSS, its preparation method, and its application. The composition of this invention exhibits higher stability, bioactivity, and clinical efficacy, and has promising application prospects.

[0007] The first aspect of this invention is to provide a hair loss prevention and hair growth composition based on the cyclic polypeptide c8DSS, the composition comprising the following components by weight percentage: Cyclic polypeptide c8DSS 0.45%-1.36%, stabilizer 0.45%, penetration enhancer 2.73%, moisturizing stabilizer 2.912%, balance is purified water.

[0008] Preferably, the c8DSS is obtained by first- and last-chain 8DSS peptides through cyclization. This invention does not strictly limit the specific method of first- and last-chain cyclization; any method commonly used in the art to achieve cyclization can be used in this invention. For example, 8DSS is synthesized using a fully automated peptide synthesizer (Changsha Chutian Technology E820), with the reaction time and reagent amount pre-set. The instrument automatically completes sample addition and cleaning operations, and the reaction flask is rotated 180° up and down (60 times / min). Unless otherwise specified, the reaction temperature is room temperature and the pH is natural. The 8DSS is then cyclized, purified, and lyophilized. The specific steps are as follows: ① Resin activation: 10 g of 2-CTC-Resin resin was soaked in 80 ml of DCM (dichloromethane) for 20 min to activate the resin by swelling; ② Synthesis of Fmoc-Ser(tBu)-2-CTC Resin peptide resin: Add Fmoc-Ser(tBu)-OH, DIEA (N,N-diisopropylethylamine), and DCM (dichloromethane) reagent (13 g / 12 ml / 100 ml) and react for 2 h, then wash with 100 ml isopropanol and 100 ml DMF; ③ Remove the protecting groups on the Fmoc-Ser(tBu)-2-CTC Resin peptide resin: Add 100ml of 20% piperidine DMF solution for 20 min to remove the 9-fluorene methoxycarbonyl group (Fmoc). ④ Synthesis of Ser(tBu)-Ser(tBu)-2-CTC Resin peptide resin: Add Fmoc-Ser(tBu)-OH, HOBT (hydroxybenzotriazole), DIC (N,N'-diisopropylcarbodiimide), and DMF (dimethylformamide) reagents (12.5 g / 5.5 ml / 8.5 g / 100 ml) and react for 2 h; after the reaction is completed, remove the protecting group (Fmoc) with 20% piperidine in DMF solution, and wash with 100 ml isopropanol and 100 ml DMF; ⑤ Synthesize Asp(tBu)-Ser(tBu)-Ser(tBu)-2-CTC Resin peptide resin; add Fmoc-Asp(tBu)-OH, HOBT (hydroxybenzotriazole), DIC (N,N'-diisopropylcarbodiimide), and DMF (dimethylformamide) reagents (12.5 g / 5.5 ml / 8.5 g / 100 ml) and react for 2 h; after the reaction is completed, deprotect the group (Fmoc) with 20% piperidine in DMF solution, and wash with 100 ml isopropanol and 100 ml DMF. ⑥ Then, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH and Fmoc-Asp(tBu)-OH are added in sequence to participate in the peptide resin condensation reaction. Repeat the operation according to steps ④ and ⑤ to obtain 8DSS peptide resin. ⑦ Peptide cleavage resin: Add 200 ml of cleavage solution (triisopropylsilane:trifluoroacetic acid:anisyl sulfide:water = 5:4:88:3 combination), stir for 1.5 h, separate 8DSS from 8DSS peptide resin, filter to remove resin, pour the filtrate into 400 ml of frozen ether, centrifuge, wash and collect the precipitate. ⑧ Cyclation of 8DSS: Dissolve approximately 0.2 M 8DSS solution in DMF, add HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) as a peptide condensing agent with an equimolar concentration of 8DSS, catalyze the reaction at pH 8.5, stir for 2 h, and the amino and carboxyl groups at the beginning and end of the peptide will condense to form amide bonds, thus obtaining cyclic c8DSS; ⑨ After the cyclization reaction was completed, all amino acid protecting groups (tBu) of c8DSS were deprotected by stirring a mixture of 85% trifluoroacetic acid, 10% triisopropylsilane, and 5% water at 5°C for 1 h. The product was precipitated by adding frozen acetone, centrifuging to collect the precipitate, and obtaining unprotected c8DSS. The organic solvent was removed under vacuum, and the product was freeze-dried to a weight of 9.78 g, yielding 54.5%. ⑩ Further purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The stationary phase was C18, and the mobile phase A was 0.1% TFA aqueous solution; the mobile phase B was acetonitrile / water / TFA = 90 / 10 / 0.1 (v / v / v). Detection was performed using 215 nm UV, the column temperature was 35℃, and gradient elution was carried out at a flow rate of 5 ml / min. The main peak component was collected, and after rotary evaporation of the organic solvent, it was lyophilized to obtain 5.22 g of c8DSS lyophilized powder with a purity of 98%, yielding 29.1%.

[0009] This invention employs a cyclic c8DSS structure, which significantly strengthens intramolecular hydrogen bonds and reduces peptide polarity, thereby greatly improving peptide stability. c8DSS not only effectively avoids the problem of linear peptides being easily hydrolyzed or degraded by proteases, but also better maintains the peptide's spatial conformation, facilitating receptor binding and greatly enhancing the peptide's biological activity.

[0010] Preferably, the stabilizing agent is bovine serum albumin.

[0011] Preferably, the penetration enhancer is propylene glycol and / or plant volatile oil.

[0012] Preferably, the moisturizing stabilizer is a polysaccharide alcohol. More preferably, the moisturizing stabilizer is at least one selected from sorbitol, trehalose, β-glucan, glycerin, and sodium hyaluronate.

[0013] The penetration enhancer and moisturizing stabilizer of this invention can ensure the high efficiency and stability of the product, promote the penetration of active ingredients through the skin barrier, maintain product stability, and improve the user experience.

[0014] Preferably, the hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS can be an oil, water, suspension, emulsion, gel or lyophilized powder.

[0015] A second aspect of the present invention is to provide a method for preparing the hair loss prevention and hair growth composition based on the cyclic polypeptide c8DSS, specifically comprising the following steps: S1. Dissolve c8DSS lyophilized powder in purified water, add a stabilizer, and obtain c8DSS solution after sterile filtration. S2. Dissolve the moisturizer and penetration enhancer in purified water, heat and stir until completely dissolved, then cool to obtain the hair growth agent base solution; S3. Mix the c8DSS solution from step S1 with the base solution from step S2 in a specific ratio to obtain a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS.

[0016] Preferably, in step S1, the concentration of bovine serum albumin in the c8DSS solution is 5%, and the concentration of c8DSS is 5%-15%; the aseptic filtration is performed through a 0.2 μm microporous membrane.

[0017] Preferably, in step S2, the concentration of the penetration enhancer in the hair growth agent base solution is 3%; the concentration of the moisturizing stabilizer is 3.2%; the heating and stirring temperature is 85℃-95℃, the heating and stirring time is 15 min; and the temperature after cooling is 42℃-48℃.

[0018] Preferably, in step S3, the weight ratio of the c8DSS solution to the hair growth agent base solution is 1:(5-15).

[0019] A third aspect of the present invention is to provide a method for applying the hair loss prevention and hair growth composition based on the cyclic polypeptide c8DSS, comprising the following steps: applying the composition to the hair loss area of ​​the scalp and massaging for 1-2 minutes, once in the morning and once in the evening.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention achieves a significant improvement in stability by cyclizing the linear 8DSS peptide into c8DSS. Protease tolerance experiments show that after 15 hours of hydrolysis, the amino peptide nitrogen content of the c8DSS solution is only 0.11%, while that of the linear 8DSS is as high as 0.29%, demonstrating a significantly enhanced resistance to protease degradation. In a 50°C high-temperature tolerance experiment, after 10 days, the amino peptide nitrogen content of c8DSS is 0.31%, while that of the linear 8DSS is as high as 0.96%. Electrophoresis patterns also confirm that c8DSS has significantly fewer degradation products, fully demonstrating that the cyclic structure can effectively resist the destructive effects of acidic and alkaline environments, high temperatures, and enzymatic hydrolysis.

[0021] Meanwhile, the cyclic structure maintains the optimal spatial configuration of the polypeptide, enhances its binding ability to cell receptors, and significantly improves its biological activity. c8DSS can effectively penetrate deep into the hair follicle through the sebaceous glands, promote the secretion of signaling molecules by dermal papilla cells, regulate the hair follicle growth cycle, and cause the hair follicle to enter the growth phase earlier from the resting phase. Clinical application studies on 124 patients with different types of hair loss have confirmed that the product of this invention has significant efficacy for hereditary hair loss, seborrheic alopecia, chemical alopecia, pathological alopecia, and endocrine-related hair loss, with an overall effective rate of 82.1%. Among them, the effective rate for chemical alopecia is as high as 92.31%, the effective rate for seborrheic alopecia is 80.95%, and the effective rate for pathological alopecia is 83.33%. Typical cases show that patients can observe significant improvements such as reduced scalp sebum secretion, the growth of fine new hair, harder and darker hair, and a significant increase in hair volume after 1-3 months of use.

[0022] Furthermore, the formula of this invention is scientifically sound and reasonable. It not only optimizes the structure of the core active ingredients, but also scientifically combines penetration enhancers and moisturizing stabilizers to form a synergistic formula system. The product can be formulated into various dosage forms as needed, is convenient to use, has good safety, no obvious side effects, and a wide range of applications. It provides an effective treatment option for different hair loss patients and has good market application prospects. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings.

[0024] Figure 1 This is a schematic diagram of the structure of the linear polypeptide 8DSS cyclized into the cyclic polypeptide c8DSS in Test Example 1 of the present invention; Figure 2 This is an electrophoretic graph comparing the stability of c8DSS and 8DSS under high temperature conditions in Test Example 1 of this invention; Figure 3 This is a head photograph of a patient with seborrheic alopecia (typical case 1) in test example 2 of this invention before treatment with the product of this invention; Figure 4 This is a head photograph of a patient with seborrheic alopecia (typical case 1) after treatment with the product of this invention (Example 2 of this invention). Figure 5 This is a head photograph of a patient with hereditary alopecia (typical case 2) before treatment with the product of this invention, as shown in Test Example 2 of this invention. Figure 6 This is a head photograph of a patient with hereditary alopecia (typical case 2) after treatment with the product of this invention in test example 2 of this invention; Figure 7 This is a head photograph of a patient with endocrine-related hair loss (typical case 3) before treatment with the product of this invention, as shown in Test Example 2 of this invention. Figure 8This is a head photograph of a patient with endocrine-related hair loss (typical case 3) after treatment with the product of this invention (Example 2 of this invention). Figure 9 This is a flowchart illustrating the preparation method of the anti-hair loss and hair growth composition based on the cyclic polypeptide c8DSS of the present invention. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] The c8DSS used in this invention was provided by the peptide synthesis laboratory of Hangzhou Gutuo Biotechnology Co., Ltd., and is in lyophilized powder form. The remaining raw materials are pharmaceutical-grade cosmetic raw materials.

[0027] Unless otherwise specified, all experiments were repeated three times, and the results are expressed as averages.

[0028] Example 1: A method for preparing a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS, comprising the following steps: S1. Weigh the purified water according to the ratio, pour it into a mixing tank, add bovine serum albumin, stir evenly, add c8DSS lyophilized powder, stir evenly, and filter aseptically with a 0.2 μm microporous membrane to obtain a c8DSS solution; the c8DSS solution has the following composition by mass percentage: bovine serum albumin 5%, c8DSS 5%, and the remainder is purified water. S2. Pour glycerin, propylene glycol, β-glucan, and sodium hyaluronate into a mixing tank, add purified water, start stirring, heat to 90°C, continue stirring for 15 minutes until all substances dissolve, turn on cooling water, and cool the solution to 45°C to obtain the hair growth agent base solution; the hair growth agent base solution, by mass percentage, is composed of the following: glycerin 3%, propylene glycol 3%, β-glucan 0.1%, sodium hyaluronate 0.1%, and the balance is purified water; S3. Mix the c8DSS solution from step S1 and the hair growth agent base solution from step S2 at a ratio of 1:10 until homogeneous. This will yield the final product, a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS.

[0029] Example 2: A method for preparing a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS, comprising the following steps: S1. Weigh the purified water according to the ratio, pour it into a mixing tank, add bovine serum albumin, stir evenly, add c8DSS lyophilized powder, stir evenly, and filter aseptically with a 0.2 μm microporous membrane to obtain a c8DSS solution; the c8DSS solution has the following composition by mass percentage: bovine serum albumin 5%, c8DSS 10%, and the remainder is purified water. S2. Pour glycerin, propylene glycol, β-glucan, and sodium hyaluronate into a mixing tank, add purified water, start stirring, heat to 90°C, continue stirring for 15 minutes until all substances dissolve, turn on cooling water, and cool the solution to 45°C to obtain the hair growth agent base solution; the hair growth agent base solution, by mass percentage, is composed of the following: glycerin 3%, propylene glycol 3%, β-glucan 0.1%, sodium hyaluronate 0.1%, and the balance is purified water; S3. Mix the c8DSS solution from step S1 and the hair growth agent base solution from step S2 at a ratio of 1:10 until homogeneous. This will yield the final product, a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS.

[0030] Example 3: A method for preparing a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS, comprising the following steps: S1. Weigh the purified water according to the ratio, pour it into a mixing tank, add bovine serum albumin, stir evenly, add c8DSS lyophilized powder, stir evenly, and filter aseptically with a 0.2 μm microporous membrane to obtain a c8DSS solution; the c8DSS solution has the following composition by mass percentage: bovine serum albumin 5%, c8DSS 15%, and the remainder is purified water. S2. Pour glycerin, propylene glycol, β-glucan, and sodium hyaluronate into a mixing tank, add purified water, start stirring, heat to 85-95℃, continue stirring for 15 minutes until all substances are dissolved, turn on cooling water, and cool the solution to 45℃ to obtain the hair growth agent base solution; the hair growth agent base solution, by mass percentage, is composed of the following: glycerin 3%, propylene glycol 3%, β-glucan 0.1%, sodium hyaluronate 0.1%, and the balance is purified water; S3. Mix the c8DSS solution from step S1 and the hair growth agent base solution from step S2 at a ratio of 1:10 until homogeneous. This will yield the final product, a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS.

[0031] Test Example 1: Stability Comparison of Cyclic c8DSS and Linear 8DSS (1) Tests on the tolerance of c8DSS and 8DSS to protease hydrolysis The purpose of this experiment is to observe the hydrolysis of c8DSS and 8DSS by proteases. The number of free amino acids after peptide hydrolysis can determine the degree of peptide hydrolysis.

[0032] Sample preparation: Dissolve c8DSS and 8DSS in purified water at a weight ratio of 5%.

[0033] Trypsin (activity: 50,000 U / g, dosage: 0.1%) was added to both c8DSS and 8DSS solutions, mixed well, and placed in a water bath at 37℃. Samples were taken at different hydrolysis times to detect the amino peptide nitrogen content (%) of the solution. The detection method was based on the pH meter method in GB / T5009.39.

[0034] The results are shown in Table 1.

[0035] Table 1 Results of c8DSS and 8DSS protease tolerance tests

[0036] As can be seen from the data in Table 1, there are significant differences between c8DSS and 8DSS in terms of protease degradation tolerance: (1) Comparison of degradation rates: After 15 hours of hydrolysis treatment, the amino peptide nitrogen content of c8DSS solution was only 0.11%, while that of linear 8DSS was as high as 0.29%, which is 2.64 times that of the former, indicating that c8DSS has a significantly better resistance to protease degradation than 8DSS.

[0037] (2) Degradation kinetics: In the 0-3 hour stage, c8DSS hardly degraded (amino peptide nitrogen content was 0), while 8DSS reached 0.02% at 3 hours; in the rapid degradation stage of 3-9 hours, the degradation rate of 8DSS increased significantly, while c8DSS maintained a low degradation level; after 9-15 hours, the degradation rates of both slowed down, but the difference was still significant.

[0038] (3) Explanation of structural stability: The cyclic structure of c8DSS eliminates the N-terminus and C-terminus of the linear polypeptide, avoiding the deamidation reaction of asparagine and the enzymatic attack site of the terminal group. At the same time, the cyclic structure enhances the intramolecular hydrogen bond, which significantly improves the tolerance to protease hydrolysis.

[0039] The protease degradation experiments of the two peptides showed that the amino peptide nitrogen in the aqueous solution of c8DSS peptide was significantly less than that of 8DSS peptide, indicating that the c8DSS structure is more stable than 8DSS and can protect the peptide chain from hydrolysis in the trypsin environment, thus maintaining the structure of the c8DSS peptide molecule.

[0040] (2) High-temperature resistance test of c8DSS and 8DSS Peptides degrade more rapidly in high-temperature water environments, especially asparagine, whose peptide bonds are prone to deamidation, releasing asparagine.

[0041] Sample preparation: Dissolve c8DSS and 8DSS in purified water at a weight ratio of 50%.

[0042] The high-temperature test for the product stability influencing factors involved placing the product in a constant temperature chamber at 50±2℃ for 10 days. Samples were taken on the 5th and 10th days to compare the degree of peptide degradation. The results are shown in Table 2. Electrophoresis results of the samples are shown in [Table 2]. Figure 2 .

[0043] Table 2. Results of high-temperature tolerance tests for c8DSS and 8DSS

[0044] Table 2 shows a comparison of the stability of c8DSS and 8DSS at a high temperature of 50℃. The results indicate that: (1) High temperature stability comparison: After 10 days of storage, the amino peptide nitrogen content of c8DSS was 0.31%, while that of 8DSS was as high as 0.96%, which is 3.1 times that of c8DSS, indicating that the cyclic structure significantly improves the thermal stability of the peptide.

[0045] (2) Degradation process analysis: On day 5, the degradation degree of c8DSS was only 0.04%, while that of 8DSS had reached 0.23% (5.75-fold difference); on day 10, the difference further increased to 3.1-fold. This indicates that the degradation of linear peptides is accelerated more significantly with the extension of high-temperature treatment time.

[0046] (3) Electrophoretic pattern verification ( Figure 2 The electrophoresis results clearly show that 8DSS produced a large number of degradation products after high-temperature treatment (the number of low molecular weight bands increased significantly), while the main band of c8DSS remained relatively intact and the number of degradation products was significantly less, which is consistent with the amino peptide nitrogen data.

[0047] (4) Practical application significance: The results show that c8DSS has better stability under both ambient and high temperature storage conditions, which is beneficial for the long-term preservation and transportation of the product, reduces the dependence on cold chain storage, and improves the practicality of the product.

[0048] The high-temperature degradation tests of the two peptides showed that the c8DSS peptide had significantly stronger high-temperature tolerance than the 8DSS peptide. On day 10, nearly 10% of the 8DSS peptide was hydrolyzed. Electrophoresis showed that after the hydrolysis of the 8DSS peptide, a large number of small peptide molecules or amino acids were released, while the c8DSS peptide had significantly fewer small molecules released after hydrolysis. This demonstrates that the c8DSS peptide has stronger structural stability than the 8DSS peptide.

[0049] Test Example 2: Cytotoxicity assay of cyclic c8DSS Experimental objective: To evaluate the toxic effects of the cyclic polypeptide c8DSS on normal cells and ensure its safety as a hair loss prevention and hair regrowth product.

[0050] Assay Principle: Cytotoxicity was detected using the CCK-8 (Cell Counting Kit-8) reagent kit. The main component of CCK-8 is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt, which, in the presence of an electron coupling reagent, can be reduced by dehydrogenases in the mitochondria of cells to produce a water-soluble orange-yellow formazan product. The intensity of the color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at a wavelength of 450 nm using a microplate reader, indirectly reflecting the number of viable cells.

[0051] Materials and Methods: Cell line: 3T3 fibroblasts; Culture conditions: DMEM medium, 37℃, 5% CO2 incubator.

[0052] Experimental steps: (1) Cell preparation: After passaged twice, 3T3 fibroblasts were completely digested with trypsin and diluted to an appropriate concentration with DMEM culture medium. (2) Cell seeding: at 8 × 10 3 The cells were seeded at a density of 1 cell per well into 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to adhere and grow. (3) Sample preparation: Prepare solutions containing different concentrations of c8DSS (0 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL), replace the original culture medium, and set up 3 replicates for each concentration; (4) Continue incubation: Incubate at 37℃ and 5% CO2 for 24 hours; (5) CCK-8 detection: Add 10 μL of CCK-8 staining solution to each well and continue incubation for 40 minutes; (6) Results determination: The OD value of each well was measured and recorded using an ELISA reader at a wavelength of 450 nm.

[0053] Reference settings: Negative control: blank culture medium treatment; Positive control: 0.2% Triton X-100 treatment.

[0054] Test results Table 3. Effects of different concentrations of c8DSS on the viability of 3T3 fibroblasts.

[0055] Note: Relative cell viability = (OD value of treatment group / OD value of blank control group) × 100%.

[0056] Table 3 shows that the cyclic polypeptide c8DSS exhibited no significant cytotoxicity against 3T3 fibroblasts at concentrations ≤20 mg / mL (2.0 g / L), with relative cell viability remaining above 99%. The actual application concentrations of c8DSS in the product of this invention (0.45%-1.36%) demonstrate good biocompatibility, making it suitable as a core active ingredient in hair loss prevention and hair regrowth products. These results provide important safety data support for the clinical application of c8DSS and also verify the advantage of the cyclic structure design in maintaining bioactivity without increasing cytotoxicity.

[0057] Test Case 3: Clinical Application Effects in Hair Loss Patients The c8DSS hair loss prevention and hair regrowth agent was used in a clinical trial on 124 patients, including those with seborrheic alopecia, hereditary alopecia, and pathological alopecia. The application method was to apply the agent to the affected area twice daily, massaging for 2 minutes without rinsing. Specific data are shown in Table 4.

[0058] Table 4. Statistical analysis of clinical application effects in patients with hair loss

[0059] Table 4 summarizes the clinical application effects of 124 patients with different types of hair loss. The results show that the product of this invention has significant efficacy for multiple types of hair loss: (1) Overall efficacy evaluation: 102 out of 124 patients were effective, with a total effective rate of 82.1%, which proves that the c8DSS anti-hair loss and hair growth composition has good clinical application value.

[0060] (2) Analysis of differences in treatment efficacy among different types of hair loss: Chemical alopecia has the best therapeutic effect (92.31%), possibly because the hair follicle damage in this type of hair loss is relatively reversible, and c8DSS can effectively promote the hair follicle to enter the growth phase from the resting phase. The efficacy rates were also high for pathological alopecia (83.33%) and seborrheic alopecia (80.95%), indicating that c8DSS can improve the hair follicle microenvironment and promote hair follicle regeneration by regulating the Wnt signaling pathway. The efficacy rates for endocrine-related hair loss (78.95%) and hereditary hair loss (75.0%) were relatively low, which is related to the complexity of the etiology and the severity of hair follicle damage in these two types of hair loss, but they still achieved high efficacy rates.

[0061] (3) Treatment cycle analysis: The treatment time for different types of hair loss is 4-6 months. Among them, chemical hair loss and endocrine disorder hair loss can be effective in as little as 4 months, while hereditary and seborrheic hair loss usually require 5-6 months, indicating that the repair and regeneration of hair follicles requires a long cycle.

[0062] (4) Gender distribution characteristics: Hereditary hair loss and seborrheic alopecia are mainly concentrated in male patients, while chemical hair loss and pathological hair loss are distributed in both male and female patients. Endocrine disorder hair loss is more common in female patients, which is consistent with the pathogenesis and gender characteristics of different types of hair loss.

[0063] (5) Clinical application value: The overall effective rate of up to 82.1% proves the success of the c8DSS cyclic structure design. The high stability and bioactivity of the cyclic peptide translate into actual clinical efficacy, providing a safe and effective treatment option for hair loss patients.

[0064] The data above shows that the longest treatment time for hair loss patients is 6 months, and the average overall effectiveness rate is 82.1%.

[0065] Case 1: Mr. He, male, 37 years old, suffers from seborrheic alopecia, with thin and sparse hair on the top of his head and oily scalp. Figure 3 After using this product for one month, scalp oiliness decreased and fine hairs began to grow. After three months, hair became thicker, and after six months, hair volume was significantly restored, transforming from soft and fine hair to hard and dark hair. Figure 4 ).

[0066] Typical Case 2: Mr. Ma, male, 45 years old. This patient has hereditary alopecia; his father is bald, and the patient's hair on the top of his head gradually decreased after the age of 35. Figure 5 After using the hair growth product for two months, fine new hairs began to grow, and after six months, new hairs gradually grew in the balding areas on the top of the head. Figure 6 ).

[0067] Typical Case 3: Mr. Liang, male, 30 years old, suffered from hair loss due to high work pressure and lack of sleep. Figure 7 The doctor diagnosed her with an endocrine disorder. After using hair growth products for 3 months, her hair quantity improved significantly; after 3 months, her hair became thicker; and after 6 months, her hair volume significantly recovered. Figure 8 ).

[0068] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS, characterized in that, The composition comprises the following components by weight percentage: Cyclic polypeptide c8DSS 0.45%-1.36%, stabilizer 0.45%, penetration enhancer 2.73%, moisturizing stabilizer 2.912%, balance is purified water.

2. The hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS according to claim 1, characterized in that, The c8DSS is obtained by cyclizing a linear 8DSS polypeptide from both ends.

3. The hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS according to claim 1, characterized in that, The stabilizing agent is bovine serum albumin.

4. The hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS according to claim 1, characterized in that, The penetration enhancer is propylene glycol and / or plant volatile oil.

5. The hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS according to claim 1, characterized in that, The moisturizing stabilizer is a polysaccharide alcohol.

6. The hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS according to claim 5, characterized in that, The moisturizing stabilizer is at least one of sorbitol, trehalose, β-glucan, glycerin, and sodium hyaluronate.

7. The hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS according to claim 1, characterized in that, The hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS can be an oil, water, suspension, emulsion, gel or lyophilized powder.

8. A method for preparing the anti-hair loss and hair growth composition based on the cyclic polypeptide c8DSS according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Dissolve c8DSS lyophilized powder in purified water, add a stabilizer, and obtain c8DSS solution after sterile filtration. S2. Dissolve the moisturizer and penetration enhancer in purified water, heat and stir until completely dissolved, then cool to obtain the hair growth agent base solution; S3. Mix the c8DSS solution from step S1 with the base solution from step S2 in a specific ratio to obtain a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS.

9. The preparation method according to claim 8, characterized in that, In step S3, the weight ratio of the c8DSS solution to the hair growth agent base solution is 1:

10.

10. A method for applying a hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS, characterized in that, The steps are as follows: Apply the composition to the balding area of ​​the scalp and massage for 1-2 minutes, once in the morning and once in the evening; the hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS is the hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS according to any one of claims 1-7 or the hair loss prevention and hair growth composition based on cyclic polypeptide c8DSS prepared by the method according to any one of claims 8 or 9.

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