A shell membrane hydrolysate, a method for preparing the same, a peptide derived therefrom, uses thereof, and a medicament for promoting hair growth

Abstract

The present application provides a kind of shell membrane hydrolysate, its preparation method, its derived peptide, its purposes and a kind of drug for promoting hair growth, belong to the technical field of bioactive peptide preparation and application.The shell membrane hydrolysate of the present application is obtained by enzymolysis of eggshell membrane in aqueous solution containing cysteine and alkaline protease, and the amino acid sequence of the derived peptide is shown in SEQ ID NO:1 or SEQ ID NO:3.The present application studies a high-efficiency preparation method for shell membrane hydrolysate, and the shell membrane hydrolysate prepared by the present application has high protein content and has the effect of treating androgenetic alopecia.In addition, the present application obtains shell membrane derived peptides FPVTPN and WGDLGA from shell membrane hydrolysate, and research finds that these two kinds of shell membrane derived peptides can promote human dermal hair papilla cell proliferation and inhibit DHT toxicity, thereby promoting hair growth, and are expected to be used for preparing a drug for preventing and / or treating alopecia, especially androgenetic alopecia, and have good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation and application technology, specifically relating to a shell membrane hydrolysate, its preparation method, its derived peptides, its uses, and a drug for promoting hair growth. Background Technology

[0002] Androgenetic alopecia (AGA) is one of the most common types of hair loss in clinical practice. Its core mechanism involves dihydrotestosterone (DHT) acting on the dermal papilla cells of the hair follicle, leading to follicle miniaturization and a shortened anagen phase. Epidemiological surveys show that the prevalence is approximately 21.3% in men and 6.0% in women, meaning a significant number of people are affected. Therefore, developing more effective treatments and studying its mechanisms is of great importance. The main pathological feature of this disease is follicle miniaturization, where terminal hairs gradually transform into fine, soft vellus hairs, resulting in thinner hair diameter and decreased density. Hair structure consists of the hair shaft located on the skin surface and the hair follicle located deep within the skin. As a complex micro-organ, the hair follicle periodically undergoes three phases: anagen (growth) phase, catagen (transitional) phase, and telogen (resting) phase. The core mechanism of AGA lies in the action of dihydrotestosterone (DHT) on the dermal papilla cells (DPCs) of the hair follicle, leading to follicle miniaturization and a shortened anagen phase. Furthermore, studies have shown that the expression levels of certain inflammatory markers in the scalp of patients with AGA (aerobically relapsed hair loss) are significantly higher than in non-alopecia areas. These abnormal gene expression and signaling pathway disorders ultimately lead to follicular cycle dysregulation, follicular miniaturization, and progressive hair loss. Therefore, regulating the follicular cycle, maintaining the anagen (growth) phase, and shortening the catagen (transitional) and telogen (resting) phases to promote hair growth is of great significance in improving hair thinning caused by AGA.

[0003] Currently, the clinical management of androgenetic alopecia mainly relies on three strategies: drug intervention, surgery, and physical therapy. Drug treatment primarily includes topical minoxidil and oral 5α-reductase inhibitors (such as finasteride and dutasteride), requiring long-term or even lifelong use to maintain efficacy. However, it also faces challenges such as relapse after discontinuation, significant individual variability in efficacy, and potential adverse reactions. Surgical treatments, such as hair transplantation, can improve appearance relatively quickly, but are limited by high costs, insufficient donor follicle resources, potential postoperative scarring, long recovery periods, and the inability to fundamentally halt the hair loss process. While adjunctive therapies such as low-dose laser therapy, platelet-rich plasma injection, and microneedling have shown some potential, current clinical evidence is insufficient, and standardized treatment protocols and guidelines are lacking. In recent years, emerging therapies such as hair follicle-activating peptides, mRNA liposomes, and hair follicle bioengineering have gained attention; however, their technical processes are complex, and clinical translation still faces technical bottlenecks. In summary, existing therapies have shortcomings in long-term safety, efficacy durability, and accessibility. Therefore, finding safe, effective, and well-defined treatment methods is crucial.

[0004] In the research of naturally derived hair growth active substances, eggshell membrane, as a major byproduct of poultry egg processing, is rich in keratin, collagen, hyaluronic acid, and various bioactive proteins, offering advantages such as wide availability, low cost, and good biocompatibility. Existing research shows that enzymatic hydrolysis of the shell membrane yields bioactive peptide fragments, which exhibit good biological activity in promoting cell proliferation, anti-oxidation, and anti-inflammation. However, research on the application of shell membrane hydrolysates in hair regeneration is still in its early stages. Current technologies lack systematic optimization of the shell membrane hydrolysate preparation process, as well as the identification of derived peptide sequences and in-depth mechanistic studies of their hair growth activity. Furthermore, how to efficiently apply shell membrane hydrolysates and their specific derived peptides to promote hair growth, and how to clarify their targets and signaling pathways, remain pressing technical problems to be solved in this field.

[0005] Therefore, developing a method to obtain shell membrane hydrolysates with clear hair growth activity through optimized enzymatic hydrolysis, and screening and identifying specific derivative peptides that promote hair growth, is of great scientific significance and application value for expanding the high-value utilization of shell membrane resources and providing a safe and effective new treatment strategy for androgenetic alopecia. Summary of the Invention

[0006] The purpose of this invention is to provide a shell membrane hydrolysate, its preparation method, its derived peptides, its uses, and a drug for promoting hair growth.

[0007] This invention provides a shell membrane hydrolysate, which is obtained by enzymatic hydrolysis of eggshell membrane in an aqueous solution containing cysteine ​​and protease.

[0008] Furthermore, the liquid-to-solid ratio of the eggshell membrane to the solvent water in the solution is 1g:(10-100)mL; And / or, the concentration of cysteine ​​in the solution is 0.01-0.1 mol / L; And / or, the concentration of the protease in the solution is 0.1-1%.

[0009] Furthermore, the pH value of the enzymatic hydrolysis is 8.5-9.5, the temperature of the enzymatic hydrolysis is 55-60℃, and the time of the enzymatic hydrolysis is 5-10h; And / or, after enzymatic hydrolysis, enzyme inactivation treatment is performed, followed by centrifugation, dialyzing of the supernatant, and freeze-drying; the enzyme inactivation treatment includes the following steps: adjusting the pH of the system to 6.5-7.5, heating to 90-100℃ to inactivate enzymes for 15-20 min.

[0010] The present invention also provides a method for preparing the aforementioned shell membrane hydrolysate, comprising the following steps: (1) Dissolve the eggshell membrane and cysteine ​​in water to obtain a mixed solution; (2) Adjust the pH of the mixed solution to 8.5-9.5, add the protease, mix well, and then carry out enzymatic hydrolysis; (3) The enzymatic hydrolysate is purified to obtain the shell membrane hydrolysate.

[0011] Furthermore, In step (1), the liquid-to-solid ratio of the eggshell membrane and water is 1g:(10-100)mL; And / or, in step (1), the concentration of cysteine ​​in the mixed solution is 0.01-0.1 mol / L; And / or, in step (2), the concentration of the protease is 0.1% to 1%; And / or, in step (2), the pH value of the enzymatic hydrolysis is 8.5-9.5, the temperature of the enzymatic hydrolysis is 55-60℃, and the time of the enzymatic hydrolysis is 5-10h; And / or, in step (3), the purification method includes the following steps: inactivating the enzyme in the enzymatic hydrolysis system, then centrifuging, dialyzing the supernatant and freeze-drying; the inactivation of the enzyme includes the following steps: adjusting the pH of the system to 6.5-7.5, heating to 90-100℃ to inactivate the enzyme for 15-20 min.

[0012] The present invention also provides a shell membrane-derived peptide, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:3.

[0013] The present invention also provides the use of the aforementioned shell membrane hydrolysate or the aforementioned shell membrane-derived peptide in the preparation of a medicament for promoting hair growth.

[0014] Furthermore, the drug is a drug for the prevention and / or treatment of hair loss.

[0015] Furthermore, the drug is a drug for the prevention and / or treatment of androgenetic alopecia.

[0016] The present invention also provides a medicament for promoting hair growth, comprising the aforementioned shell membrane hydrolysate or the aforementioned shell membrane-derived peptide.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: To address the limitations of existing treatments for androgenetic alopecia, such as significant side effects and high treatment costs, this invention is proposed. This invention develops an efficient method for preparing shell membrane hydrolysates. The shell membrane hydrolysates prepared by this invention have a high protein content and are effective in treating androgenetic alopecia. Furthermore, this invention yields shell membrane-derived peptides FPVTPN and WGDLGA from the shell membrane hydrolysates. Studies have shown that these two shell membrane-derived peptides can promote the proliferation of human dermal papilla cells and inhibit DHT toxicity, thereby promoting hair growth. They hold promise for use in the preparation of drugs for the prevention and / or treatment of hair loss, particularly androgenetic alopecia, and have excellent application prospects.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 The figure shows the results of shell membrane dissolution rate in the preparation of shell membrane hydrolysates by cysteine-assisted enzymatic hydrolysis and ordinary enzymatic hydrolysis according to the present invention. In the figure, a and b represent significant differences between data. Two sets of data with the same letter indicate no statistical difference, and two sets of data with different letters indicate statistical differences.

[0021] Figure 2 The graph shows the protein content of the shell membrane hydrolysate prepared by cysteine-assisted enzymatic hydrolysis and the shell membrane hydrolysate prepared by ordinary enzymatic hydrolysis according to the present invention. In the graph, a and b represent significant differences between the data. Two sets of data with the same letter indicate no statistical difference, and two sets of data with different letters indicate a statistical difference.

[0022] Figure 3Macroscopic images of skin color changes and hair growth on the backs of mice in each group on days 0, 7, 14, 21, and 28 after drug administration.

[0023] Figure 4 The graph shows the relative area (%) of hair growth on the back of mice in each group after 28 days of drug administration. In the graph, a, b, c, and d represent significant differences between data. Data with the same letter indicate no statistical difference between two groups, and data with different letters indicate statistical differences between two groups.

[0024] Figure 5 HE staining results of skin tissue in the hairless area of ​​mice in each group on day 28 after drug administration.

[0025] Figure 6 The graph shows the DHT content in the skin tissue of the hairless area of ​​mice in each group after 28 days of drug administration. In the graph, a, b, c, and d represent the significant differences between the data. Two groups of data with the same letter indicate no statistical difference, and two groups of data with different letters indicate a statistical difference.

[0026] Figure 7 The graph shows the TGF-β content in the skin tissue of the hairless area of ​​mice in each group after 28 days of drug administration. In the graph, a, b, and c represent significant differences between data. Data with the same letter indicate no statistical difference between two groups, and data with different letters indicate statistical differences between two groups.

[0027] Figure 8 The graph shows the effects of three shell-derived peptides, FPVTPN, WGGPAG, and WGDLGA, on the proliferation of HDPCs. In the graph, a, b, and c represent significant differences between data. Data sets with the same letter indicate no statistical difference, while data sets with different letters indicate a statistical difference.

[0028] Figure 9 The graph shows the inhibitory effects of three shell-derived peptides, FPVTPN, WGGPAG, and WGDLGA, on DHT toxicity in HDPCs. In the graph, a, b, and c represent significant differences between data points. Data points with the same letter indicate no statistical difference, while data points with different letters indicate a statistical difference. Detailed Implementation

[0029] Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0030] Example 1: Preparation of shell membrane hydrolysate This embodiment uses cysteine-assisted enzymatic hydrolysis to efficiently prepare shell membrane hydrolysates.

[0031] Using dried eggshell membranes as raw material, the membranes were pulverized. At a material-to-liquid ratio of 1:20 (g:mL), 10 g of eggshell membrane powder was added to 200 mL of deionized water. 1.05 g of cysteine ​​hydrochloride monohydrate was weighed and dissolved in the eggshell membrane mixture to achieve a cysteine ​​concentration of 0.03 mol / L. The pH of the mixture was adjusted to 9.0 using 1 mol / L sodium hydroxide and 1 mol / L hydrochloric acid solutions. Then, 1 g of protease (Anzhanmei) was added. ® FF104AN (protease) was mixed thoroughly and placed in a shaking water bath at 55 °C for 6 h. During the enzymatic hydrolysis, the pH was adjusted to 9.0 every h using 1 mol / L sodium hydroxide aqueous solution and 1 mol / L hydrochloric acid aqueous solution. After the enzymatic hydrolysis was completed, the pH was adjusted to 7.0 using 1 mol / L sodium hydroxide aqueous solution and 1 mol / L hydrochloric acid aqueous solution, and the temperature was raised to 90 °C for 15 min to inactivate the enzyme. After cooling to room temperature, the mixture was centrifuged (4000 xg, 15 min). The supernatant was then dialyzed and desalted for 24 h using a dialysis bag with a molecular weight cutoff of 200 Da. The dialysate was deionized water. After dialysis, the shell membrane hydrolysate, i.e., eggshell membrane polypeptide powder, was obtained by vacuum freeze-drying.

[0032] Example 2: Preparation of shell membrane hydrolysate-derived peptides The eggshell membrane polypeptide powder obtained in Example 1 was sequenced by HPLC-MS / MS. The identified peptides were selected, and their water solubility was predicted using the online prediction tool Peptide Property Calculator (pH 7.4). ToxinPred (SVM algorithm, default threshold) was used for toxicity prediction. The bioinformatics tool AutodocVina software was used for peptide function prediction, and molecular docking was performed with type II 5-α reductase SRD5A2 and androgen receptor protein AR, respectively. Three water-soluble and non-toxic polypeptide fragments (shell membrane hydrolysate-derived peptides) were finally obtained: ① the polypeptide fragment FPVTPN with a molecular weight of 674.35 Da, ② the polypeptide fragment WGGPAG with a molecular weight of 544.25 Da, and ③ the polypeptide fragment WGDLGA with a molecular weight of 618.29 Da. The water solubility, toxicity, and molecular docking results of the three polypeptide fragments are shown in Table 1.

[0033] Table 1. Water solubility, toxicity, and molecular docking results of shell membrane hydrolysate-derived peptides Table 1 shows that FPVTPN, WGDLGA, and WGGPAG exhibit good binding energies to type II 5-α reductase SRD5A2 and androgen receptor protein AR during molecular docking. To verify their actual efficacy, they were applied to hair follicle-associated cells (HDPCs) to observe their proliferative effects.

[0034] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0035] Experimental Example 1: Comparison of the effects of cysteine-assisted enzymatic hydrolysis and conventional enzymatic hydrolysis on the preparation of shell membrane hydrolysates 1. Experimental Methods (1) Detection of shell membrane dissolution rate Cysteine-assisted enzymatic hydrolysis was used to prepare shell membrane hydrolysates according to the method described in Example 1. The conventional enzymatic hydrolysis method for preparing shell membrane hydrolysates is as follows: Dried eggshell membranes were used as raw material and pulverized. At a material-to-liquid ratio of 1:20 (g:mL), 10 g of eggshell membrane powder was added to 200 mL of deionized water. The pH of the mixed solution was adjusted to 9.0 using a 1 mol / L sodium hydroxide aqueous solution and a 1 mol / L hydrochloric acid aqueous solution. Then, 1 g of protease (Anzhanmei) was added. ® FF104AN (protease) was mixed thoroughly and placed in a shaking water bath at 55 °C for 4 h. During the enzymatic hydrolysis, the pH was adjusted to 9.0 every 1 h using 1 mol / L sodium hydroxide aqueous solution and 1 mol / L hydrochloric acid aqueous solution. After the enzymatic hydrolysis was completed, the pH was adjusted to 7.0 using 1 mol / L sodium hydroxide aqueous solution and 1 mol / L hydrochloric acid aqueous solution, and the temperature was raised to 90 °C for 15 min to inactivate the enzyme. After cooling to room temperature, the mixture was centrifuged (4000 xg, 15 min). The supernatant was then dialyzed and desalted for 24 h using a dialysis bag with a molecular weight cutoff of 200 Da. The dialysate was deionized water. After dialysis, the shell membrane hydrolysate was obtained by vacuum freeze-drying.

[0036] The shell membrane solubility was measured for both the shell membrane hydrolysate prepared by the method in Example 1 and the shell membrane hydrolysate prepared by conventional enzymatic hydrolysis. The specific detection method and calculation formula for the shell membrane solubility are shown below: After enzymatic hydrolysis, the solution was removed, cooled to room temperature, and centrifuged (4000 x g, 15 min). The precipitate was then dried in an oven at 105°C until constant weight, weighed, and compared with the initial 10 g of shell membrane material to obtain the shell membrane solubility rate. The formula for calculating the shell membrane solubility rate is as follows: (2) Protein content detection Cysteine-assisted enzymatic hydrolysis was used to prepare shell membrane hydrolysates according to the method described in Example 1. Conventional enzymatic hydrolysis was used to prepare shell membrane hydrolysates according to the method described in "Shell Membrane Solubility Detection" above.

[0037] The protein content of the shell membrane hydrolysate prepared by the method in Example 1 and the shell membrane hydrolysate prepared by conventional enzymatic hydrolysis were determined separately. The specific detection method and calculation formula for protein content are shown below: The protein content of the samples was determined using the Kjeldahl method as specified in GB 5009.5-2016, "National Food Safety Standard - Determination of Protein in Food," with units of g / 100 g. An appropriate amount of the sample to be tested (accurate to 0.0001 g) was accurately weighed and placed in a digestion tube. Copper sulfate and potassium sulfate were added as catalysts, and concentrated sulfuric acid was used for digestion, converting the organic nitrogen in the sample into inorganic ammonium salts. After complete digestion, distillation was performed using a fully automated Kjeldahl nitrogen analyzer (or a semi-automatic distillation titration device). Ammonia gas was absorbed in boric acid absorption solution, and titration was performed with standard hydrochloric acid or sulfuric acid titration solution. The volume of standard titration solution consumed was recorded. A blank control experiment was also performed. The protein content in the sample was calculated using the following formula: In the formula: X — The mass fraction of protein in the sample, expressed as a percentage (%). V1—Volume of standard titration solution consumed in sample titration, in milliliters (mL). V0—The volume of standard titration solution consumed in the blank titration, in milliliters (mL); c — Concentration of the standard titration hydrochloric acid solution, in moles per liter (mol / L). 14.01 — Molar mass of nitrogen, in grams per mole (g / mol). F – Conversion factor for nitrogen to protein; F is 6.25 for this sample. m — Mass of the sample, in grams (g); 1000 – Conversion factor for milligrams to grams; 100 – the coefficient for converting to percentages.

[0038] 2. Experimental Results Figure 1 The graph shows the shell membrane solubility results for the preparation of shell membrane hydrolysates using cysteine-assisted enzymatic hydrolysis and conventional enzymatic hydrolysis, as presented in this invention. Figure 1 As shown, compared with the ordinary enzymatic hydrolysis method, cysteine-assisted enzymatic hydrolysis is more efficient. The shell membrane dissolution rate of this method is 91.6%, while the dissolution rate of the shell membrane of the ordinary enzymatic hydrolysis method is 44.1%.

[0039] Figure 2 The graph shows the protein content of the shell membrane hydrolysate prepared by cysteine-assisted enzymatic hydrolysis according to this invention and the shell membrane hydrolysate prepared by ordinary enzymatic hydrolysis. (See figure.) Figure 2 As shown, compared with the protein content of 34.39% in the shell membrane hydrolysate prepared by conventional enzymatic hydrolysis, the protein content in the hydrolysate obtained by enzymatic hydrolysis in this invention is 78.93%, which is a significant increase.

[0040] Experimental Example 2: Application of the shell membrane hydrolysate prepared in this invention to treat androgenetic alopecia in mice. 1. Experimental Methods The shell membrane hydrolysate used in this experiment was prepared by the method described in Example 1, and was dissolved in physiological saline as a solvent.

[0041] Eight-week-old male C57 / 6J mice were weighed and administered 1.25% tribromoethanol solution at a concentration of 10 mL / kg. The backs of the mice were shaved using a pet hair removal device, covering an area of ​​approximately 3 cm × 4 cm. Hair removal cream was then evenly applied to the experimental area. Cotton balls were moistened with physiological saline, and the cream was left on for 2-3 minutes. The cream and any remaining hair were then wiped away with the moistened cotton balls. Mice whose back hair follicles were in the resting phase (i.e., the skin on their backs was pink and without scars) were selected for the experiment.

[0042] Experimental grouping scheme: control group (applied with 0.3 mL of physiological saline), model group (applied with 0.3 mL of physiological saline), minoxidil group (applied with 0.3 mL of 5% minoxidil), high-dose shell membrane hydrolysate group (applied with 0.3 mL of 5% shell membrane hydrolysate), and low-dose shell membrane hydrolysate group (applied with 0.3 mL of 2% shell membrane hydrolysate). During the administration period, except for the control group mice, each mouse was injected with testosterone propionate at 25 mg / kg / day to establish androgenetic alopecia model. Each group consisted of 6 mice.

[0043] Each group was administered the drug once daily, along with a single injection of testosterone propionate, for a total of 28 days. Starting from day 0 of treatment, changes in skin color and hair growth on the backs of mice were recorded every 7 days, and images were taken for comparison. Quantitative analysis was performed using ImageJ image analysis software to calculate the relative area (%) of hair growth on the backs of mice. The formula for calculating the relative area (%) of hair growth on the backs of mice is as follows: Twenty-eight days after drug administration, skin tissue from the hairless areas of mice was collected for HE staining. Simultaneously, skin tissue from the hairless areas of mice was collected, and the concentrations of DHT and TGF-β in the mouse skin tissue were detected using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Equal mass of skin tissue was weighed from each group, and a 1:9 mixture of sample (g) and PBS (mL) was prepared and thoroughly homogenized on ice. The homogenate was then centrifuged at 5000×g for 5–10 minutes, and the supernatant was collected for analysis. The final results were presented as the DHT and TGF-β content per unit volume of skin tissue homogenate supernatant.

[0044] 2. Experimental Results Figure 3 Macroscopic images of skin color changes and hair growth on the backs of mice in each group on days 0, 7, 14, 21 and 28 after drug administration are shown. There are 6 mice in each group. Figure 4This image shows the relative area (%) of hair growth on the back of mice in each group after day 28 of drug administration. Figure 3 and Figure 4 It was found that, compared with the blank control group, the model group mice still had significantly sparse hair on their backs 28 days after hair removal, and their skin remained pink, indicating that testosterone propionate successfully inhibited hair growth and the model was effectively established. In contrast, each treatment group showed varying degrees of hair regrowth promotion, and all showed statistically significant differences compared with the model group. P <0.05). Among them, the high-dose group of shell membrane hydrolysate showed the most significant effect, with hair coverage density and growth status second only to the blank control group, significantly better than the model group. Moreover, compared with minoxidil, shell membrane hydrolysate takes effect faster and is more effective.

[0045] Figure 5 HE staining results of skin tissue from the hair-removed areas of mice in each group on day 28 after drug administration. Figure 5 It can be seen that after 28 days, the untreated control group and the model group had fewer hair follicles in their skin, and these were all located in the epidermis, where the follicles were in the resting or regression phase. However, most hair follicles in the model group showed vacuolation, indicating that the hair follicles were damaged. In contrast, the HE staining results of the low- and high-dose shell membrane hydrolysate groups showed large and robust hair follicles, with a number similar to the minoxidil group. Furthermore, compared to the minoxidil group, there were more anagen follicles located in the lower dermis, and the hair follicles in the low- and high-dose shell membrane hydrolysate groups were larger and robust. This indicates that the shell membrane hydrolysate prepared in this invention can effectively improve androgenetic alopecia, shorten the regression and resting phases of hair follicles, and promote the entry of hair follicles into the anagen phase, thereby promoting hair growth.

[0046] Figure 6 and Figure 7 The figures show the DHT and TGF-β levels in the skin tissue of the hair-loss area in mice of each group after day 28 of drug administration. In the pathogenesis of androgenetic alopecia, DHT is converted from testosterone by 5α-reductase, binds to androgen receptors in hair follicles, shortens the anagen phase of the hair follicle, gradually causes it to atrophy, and ultimately leads to thinning and loss of hair. Figure 6 As shown, compared with the blank group, the DHT content in the model group and the drug group was significantly increased, indicating that the modeling successfully induced an abnormal increase in local DHT levels. Under the treatment of different drugs, the DHT content in mouse skin was significantly reduced, with the high-dose shell membrane hydrolysate group showing the most significant effect. TGF-β is a key negative regulatory signal leading to hair follicle miniaturization and premature entry into the regression phase, such as... Figure 7As shown, compared with the blank control group, the TGF-β concentration in the model group was significantly increased. Under minoxidil treatment, the TGF-β concentration in mouse skin tissue significantly decreased, approaching the level of the blank group. The high-dose shell membrane hydrolysate group achieved the same therapeutic effect as minoxidil, exhibiting an inhibitory effect on TGF-β concentration. These results indicate that the shell membrane hydrolysate prepared in this invention can significantly reduce the levels of DHT and TGF-β in the skin tissue of mice with androgenetic alopecia, suggesting that it exerts its anti-hair loss effect by regulating androgen metabolism and the negative signaling pathway in hair follicles.

[0047] Example 3: The polypeptide prepared in this invention has the effect of promoting the proliferation of human dermal papilla cells and inhibiting androgenetic alopecia. According to the sequences described in SEQ ID NO:1-3, different shell membrane-derived peptide fragments (FPVTPN, WGGPAG, WGDLGA) were artificially synthesized and the following cell experiments were performed.

[0048] 1. Experimental Methods Passaged human dermal papilla cells (HDPCs) were cultured at 3 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated for 24 h to reach the logarithmic growth phase. Then, 100 μL of 100 μg / mL FPVTPN, WGGPAG, or WGDLGA solutions (using culture medium as the solvent) were added, and the cells were incubated for 48 h. Cells treated with culture medium only served as a control (pure cell group), while unseeded culture medium served as a blank. After 48 h of culture, cell viability was measured using a CCK-8 assay kit. Absorbance (OD) values ​​at 450 nm were measured using a microplate reader, and cell viability was calculated.

[0049] Passaged human dermal papilla cells (HDPCs) were cultured at 3 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well into 96-well plates and incubated for 24 h to reach the logarithmic growth phase. Then, 50 μL each of 200 μg / mL FPVTPN, WGGPAG, WGDLGA solutions and 100 μM DHT solution were added, for a total of 100 μL (final single peptide concentration 100 μg / mL, DHT concentration 50 μM, solvent: culture medium). Incubation was continued for 48 h. Cells treated with only DHT (50 μM) served as the model group (DHT group), cells treated with only culture medium served as the control group (pure cell group), and unseeded culture medium served as the blank. After 48 h of culture, cell viability was measured using a CCK-8 assay kit. Absorbance (OD) values ​​at 450 nm were measured using a microplate reader, and cell viability was calculated.

[0050] The formula for calculating cell viability is as follows: In the formula: A1 – Absorbance of the sample group.

[0051] A0 – Absorbance of the blank group.

[0052] A x —Absorbance of pure cell group.

[0053] 2. Experimental Results Figure 8 This image shows the effects of three shell-derived peptides, FPVTPN, WGGPAG, and WGDLGA, on the proliferation of HDPCs. Figure 8 It was observed that the cell viability of both peptide fragments FPVTPN and WGDLGA remained above 100% at a concentration of 100 μg / mL, demonstrating a significant effect on promoting HDPC proliferation; while the effect of peptide fragment WGGPAG on promoting HDPC proliferation was poor, with no effect on promoting HDPC proliferation.

[0054] Figure 9 This study demonstrated the protective effects of three shell-derived peptides, FPVTPN, WGGPAG, and WGDLGA, on human dermal papillary cells (HDPCs) in a model of androgenetic alopecia. After treatment with 50 μM DHT, cell viability decreased to 83.92%, while FPVTPN at a concentration of 100 μg / mL maintained cell viability above 100%. WGDLGA at the same concentration showed cell viability comparable to the untreated control group, indicating that these two peptides effectively counteracted DHT-induced cell damage. In contrast, WGGPAG failed to significantly improve the DHT-induced decrease in cell viability and did not promote HDPC proliferation.

[0055] Experimental results show that the peptide fragments FPVTPN and WGDLGA can effectively promote the proliferation of human dermal papilla cells, thereby promoting hair growth, with WGDLGA showing the best effect. Both FPVTPN and WGDLGA can exert their potential to treat androgenetic alopecia by promoting the proliferation of HDPCs. FPVTPN showed the best performance in inhibiting DHT toxicity, while WGDLGA was outstanding in maintaining cell viability; conversely, WGGPAG did not show a significant therapeutic effect in this model.

[0056] In summary, this invention has developed an efficient method for preparing shell membrane hydrolysates. The shell membrane hydrolysates prepared by this invention have a high protein content and are effective in treating androgenetic alopecia. Furthermore, this invention yields shell membrane-derived peptides FPVTPN and WGDLGA from the shell membrane hydrolysates. Studies have shown that these two shell membrane-derived peptides can promote the proliferation of human dermal papilla cells and inhibit DHT toxicity, thereby promoting hair growth. They hold promise for use in the preparation of drugs for the prevention and / or treatment of hair loss, particularly androgenetic alopecia, and have excellent application prospects.

Claims

1. A shell-membrane-derived peptide, characterized in that: The amino acid sequence of the shell membrane-derived peptide is shown in SEQ ID NO:1 or SEQ ID NO:

3.

2. Use of the shell membrane-derived peptide of claim 1 in the preparation of a medicament for promoting the proliferation of human dermal papilla cells.

3. The use according to claim 2, characterized in that: The drug in question is a drug that inhibits DTH toxicity.

4. A drug for promoting the proliferation of human dermal papilla cells, characterized in that: It includes the shell-derived peptide as described in claim 1.

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