Multi-target, multi-functional anti-hair loss polypeptide, application and preparation method thereof

Abstract

The application belongs to the technical field of medicine and cosmetics, and specifically discloses a multi-target and multi-functional anti-hair loss and hair growth polypeptide, application and preparation method thereof. The polypeptide is a cyclic peptide, and the amino acid sequence is shown as SEQ ID No. 1. The N terminal of the cyclic peptide is modified by acetylation, the C terminal is modified by amidation, the third cysteine and the sixth cysteine in the sequence form an intramolecular disulfide bond. The polypeptide is prepared by using a solid-phase synthesis method, and high-purity target polypeptide is obtained after cutting and purification. According to the specified test report, the polypeptide has a clear proliferation promoting effect on human hair papilla cells, the test process conforms to the standard operating instruction, and the detection result has scientificity, impartiality and accuracy. The polypeptide can be used for preparing anti-hair loss and hair growth related products, can provide efficient and stable biological active material for anti-hair loss and hair growth, and meets the demand of the anti-hair loss and hair growth field for high-quality active ingredients.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals and cosmetics, specifically relating to a multi-target, multifunctional anti-hair loss and hair growth polypeptide, its application, and its preparation method. Background Technology

[0002] Hair loss prevention and hair regrowth are important needs in the current daily chemical and medical fields. Existing hair loss prevention and hair regrowth materials mainly fall into four categories, but all of them have significant technical limitations:

[0003] The first category consists of topical vasodilators, represented by minoxidil. Their mechanism of action is to dilate scalp capillaries, increase blood flow to hair follicles, and provide more nutrients to atrophied follicles, but they cannot address the core issue of hair follicle damage. They neither inhibit the toxic effects of dihydrotestosterone on hair follicles nor activate dormant hair follicle stem cells. Therefore, they suffer from slow onset of action, relapse upon discontinuation, local irritation, and hirsutism in non-target areas, and are ineffective for patients with severe hair follicle atrophy.

[0004] The second category consists of oral 5α-reductase inhibitors, represented by finasteride. These reduce the conversion of testosterone to DHT by inhibiting 5α-reductase activity. However, as hormonal drugs, long-term use may cause side effects such as sexual dysfunction, breast tenderness, and endocrine disorders. They are only suitable for male patients and are contraindicated in women, thus limiting their applicability.

[0005] The third category consists of plant extracts, such as ginger extract and arborvitae leaf extract. These materials rely on the weak anti-inflammatory or proliferative effects of natural ingredients, but their composition is complex, the content of active ingredients is unstable, and the mechanism of action is unclear. Most can only relieve scalp inflammation and cannot directly activate hair follicle regeneration or inhibit DHT, making it difficult to achieve a synergistic effect of preventing hair loss and promoting hair growth. The effects are difficult to repeat and quantify.

[0006] The fourth category consists of compound compositions, which combine multiple known ingredients. Although they claim to target multiple sites and pathways, clearly demonstrating which target each ingredient specifically acts on, how the pathways interact, and how they ultimately work synergistically is an extremely complex and costly scientific undertaking. Furthermore, the coexistence of multiple active ingredients in a single formulation can lead to complex chemical and physical interactions (such as precipitation, degradation, and antagonism), affecting product stability, shelf life, and bioavailability. This places higher demands on formulation processes and quality control.

[0007] Currently, there is a critical technological gap in the field of hair loss prevention and hair regrowth. There is no hair loss prevention and hair regrowth material based on a single peptide to achieve multi-target synergy and has been industrialized. Existing materials are generally trapped in the dilemma of single-target and limited effects: minoxidil only improves microcirculation, finasteride only inhibits DHT, and plant extracts only relieve inflammation. None of them can simultaneously solve the three core problems of DHT damage, hair follicle stem cell dormancy, and insufficient microcirculation in hair follicles, resulting in hair loss prevention failing to regrow hair and hair regrowth being prone to relapse. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a multi-target, multifunctional anti-hair loss and hair growth polypeptide, its application, and its preparation method. Through the synergistic effect of multiple targets, it overcomes the limitations of single-target materials in existing materials, achieving an integrated effect of preventing hair loss, promoting hair growth, and treating the root cause.

[0009] The technical solution adopted in this invention is as follows:

[0010] In a first aspect, the present invention provides a multi-target, multifunctional anti-hair loss and hair growth polypeptide, wherein the polypeptide is a cyclic peptide and the amino acid sequence is shown in SEQ ID No. 1; the N-terminus of the cyclic peptide is acetylated and the C-terminus is amidated, and the third cysteine ​​and the sixth cysteine ​​in the sequence form an intramolecular disulfide bond. For specific structural features and functional data, please refer to the sequence listing and examples.

[0011] In conjunction with the first aspect, the present invention provides a first embodiment of the first aspect, wherein the polypeptide is an analog of SEQ ID No. 1, wherein the L-lysine in SEQ ID No. 1 is replaced with D-lysine, and the amino acid sequence of the analog is shown in SEQ ID No. 2. For specific structural features and functional data, please refer to the sequence listing and examples.

[0012] Secondly, the present invention provides an application of a multi-target, multi-functional anti-hair loss and hair growth polypeptide, wherein a solution is prepared using any of the multi-target, multi-functional anti-hair loss and hair growth polypeptides described above, and the concentration of the multi-target, multi-functional anti-hair loss and hair growth polypeptide in the solution is 0.01%-0.6%.

[0013] Thirdly, the present invention provides an application of a multi-target, multi-functional anti-hair loss and hair growth polypeptide, and an anti-hair loss and hair growth product prepared using any of the multi-target, multi-functional anti-hair loss and hair growth polypeptides described above, the anti-hair loss and hair growth product including external essence, shampoo, lotion, perfume, liquid, cream, spray, gel, ointment and paste, etc.

[0014] Fourthly, the present invention provides a preparation method for producing any of the above-mentioned multi-target, multifunctional anti-hair loss and hair growth polypeptides, the steps of which include resin activation, sequential coupling of amino acids according to the amino acid sequence, formation of intramolecular disulfide bonds, N-terminal acetylation and C-terminal amidation treatment, and cleavage and purification to obtain the target polypeptide.

[0015] It is worth noting that the anti-hair loss and hair growth peptide of the present invention works through a multi-target synergistic mechanism of one inhibition and two activations, solving the problem of anti-hair loss and hair growth from three core aspects: blocking the source of damage, activating regenerative power, and optimizing the microenvironment. The specific mechanism and corresponding effects are as follows:

[0016] Inhibit 5α-reductase and block DHT from damaging hair follicles.

[0017] 5α-reductase is a key enzyme in the human body that catalyzes the conversion of testosterone into dihydrotestosterone, while DHT is a core toxic factor that causes hair follicle damage. DHT can specifically bind to androgen receptors on the surface of dermal papilla cells in hair follicles, triggering dermal papilla cell apoptosis, miniaturization of hair follicles, shortening of the hair growth phase, and prolongation of the resting phase, ultimately leading to hair loss.

[0018] The polypeptide of this invention can specifically bind to the active site of 5α-reductase through its cyclic structure, competitively inhibiting the enzyme's catalytic activity and reducing the conversion of testosterone to DHT. This mechanism directly blocks the toxic effects of DHT on hair follicles at the source of damage, preventing further follicle atrophy, laying the foundation for subsequent hair growth, and achieving a symptomatic treatment effect in preventing hair loss.

[0019] Activating the Wnt signaling pathway: directly stimulating hair follicle stem cells to enter the growth phase.

[0020] The Wnt signaling pathway is the core switch regulating the hair follicle growth cycle, and its activity directly determines the differentiation state of hair follicle stem cells: when the Wnt pathway is inhibited, hair follicle stem cells are in a dormant phase, and the hair follicle remains in a resting state; when the Wnt pathway is activated, the key protein β-catenin accumulates in the cell nucleus, initiates the expression of genes related to the differentiation of hair follicle stem cells into mature hair cells, promotes the hair follicle to enter the growth phase from the resting phase, and initiates hair shaft formation and hair follicle regeneration.

[0021] The polypeptide of this invention can enhance its binding affinity to Wnt pathway receptors through N-terminal acetylation modification, activate the Wnt / β-catenin signaling pathway, directly awaken dormant hair follicle stem cells, promote their differentiation into mature hair cells, achieve the core effect of direct hair growth, and solve the pain point of existing materials that prevent hair loss but do not promote hair growth.

[0022] Activate the VEGF signaling pathway: improve hair follicle microcirculation, and achieve long-term, fundamental treatment.

[0023] Vascular endothelial growth factor is a key cytokine that regulates angiogenesis and microcirculation. The healthy growth of hair follicles depends on a sufficient supply of nutrients. When the microcirculation around the hair follicle is insufficient, even if the hair follicle stem cells are activated, the lack of nutrients will lead to slow hair growth, easy hair loss, and inability to maintain long-term hair growth effects.

[0024] The polypeptide of this invention can activate the VEGF signaling pathway in the dermal papilla cells of hair follicles, promoting the secretion of VEGF by the dermal papilla cells; VEGF further acts on the vascular endothelial cells of the scalp, stimulating the proliferation of vascular endothelial cells and the formation of new blood vessels, optimizing the microcirculation environment around the hair follicles, providing continuous nutritional support for the hair follicles, maintaining the growth phase of the hair follicles, avoiding recurrence after hair growth, achieving a long-term curative effect, and forming a complete chain of action of preventing hair loss - promoting hair growth - maintaining hair health.

[0025] It is also worth noting that the hair loss prevention and hair growth peptide of this invention is based on a multi-target mechanism of inhibiting DHT damage, activating hair follicle regeneration, and improving microcirculation. It is mainly suitable for androgenetic alopecia, telogen effluvium, mild senile alopecia, and people with hair follicle microcirculation disorders.

[0026] The beneficial effects of this invention are as follows:

[0027] (1) The disulfide bond polypeptide of the present invention has a specific molecular structure design. This structural feature can endow the polypeptide with a stable spatial conformation, enabling it to continuously bind to the target site and thus stably exert its activity to promote the proliferation of human hair papilla cells, avoiding the activity decay caused by loose conformation, and providing a stable basis for the polypeptide to play a role in hair loss prevention and hair growth related applications.

[0028] (2) The present invention retains L-lysine or replaces it with D-lysine, both of which can maintain the ability of the peptide to promote the proliferation of human dermal papilla cells. Both configurations of lysine can be adapted to the overall molecular structure of the peptide, ensuring that the peptide forms a stable intramolecular disulfide bond and end-group modification structure, without destroying the core function of the peptide acting on cell proliferation-related targets. This allows the peptide to meet the core requirements for promoting the proliferation of human dermal papilla cells regardless of whether L-lysine or D-lysine is used, providing flexibility in configuration selection for the preparation and application of the peptide. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the effect of polypeptide samples on the survival rate of human dermal papilla cells in an embodiment of the present invention.

[0030] Figure 2 These are microscope images showing the effect of polypeptide samples on the survival rate of human dermal papilla cells in embodiments of the present invention. Detailed Implementation

[0031] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0032] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0033] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0034] Example 1:

[0035] This embodiment discloses the preparation and performance testing of an L-type lysine disulfide bond cyclic peptide.

[0036] (1) Peptide chain structure

[0037] The disulfide-bonded cyclic peptide of this embodiment has the amino acid sequence shown in SEQ ID No. 1, with an N-terminus of acetyl (Ac-) and a C-terminus of amide (-NH2).

[0038] The third cysteine ​​(Cys³) and the sixth cysteine ​​(Cys) 6 The peptide chain forms a ring structure through intramolecular disulfide bonds, which can promote the proliferation of human dermal papilla cells. This meets the requirements of containing intramolecular disulfide bonds, end group modification and human dermal papilla cell proliferation-promoting activity.

[0039] (2) Preparation method

[0040] The solid-state synthesis method is employed, and the specific steps are as follows:

[0041] Resin activation

[0042] RinkAmide-MBHA solid-phase resin was selected. 0.5 g of resin was weighed and placed in a solid-phase synthesis column. The resin was soaked in N,N-dimethylformamide for 30 min to swell. A DMF solution of coupling reagents (0.3 mmol of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate TBTU and 0.6 mmol of N,N-diisopropylethylamine DIPEA) was added. The amino active sites of the resin were activated by shaking at 25 °C for 45 min. The reaction solution was then removed.

[0043] amino acid coupling

[0044] The following amino acids protected by Fmoc are coupled sequentially from the C-terminus to the N-terminus as shown in SEQ ID No. 1:

[0045] During each coupling step, amino acids (0.3 mmol), TBTU (0.3 mmol), and DIPEA (0.6 mmol) were dissolved in DMF and added to the synthesis column, which was then shaken at 25°C for 3 h. After the reaction, the Fmoc protecting group was removed by soaking in 20% piperidine DMF solution for 15 min, followed by washing with DMF 5 times (5 min each time). The coupling efficiency was detected by the ninhydrin colorimetric method. If the resin was colorless, the coupling was considered complete; if it was blue, the coupling was repeated once.

[0046] Disulfide bond formation

[0047] A dichloromethane / methanol mixture of iodine (volume ratio 1:1, final iodine concentration 0.03 mol / L) was added to the linear peptide resin, and the mixture was reacted with shaking at 25°C in the dark for 1.5 h to allow Cys³ to react with Cys. 6 The thiol groups are oxidized to form intramolecular disulfide bonds; after the reaction, the mixture is washed 6 times with DMF to remove residual iodine.

[0048] End-group modification

[0049] A DMF solution of acetic anhydride (0.3 mmol) and DIPEA (0.6 mmol) was added to the cyclic peptide resin, and the reaction was carried out with shaking at 25 °C for 1.2 h to complete the N-terminal acetylation; the C-terminus was amidated through the inherent structure of the RinkAmide-MBHA resin. After the resin was coupled with the amino group of His, -CONH2 was directly formed during cleavage.

[0050] Cutting and purification

[0051] A cleavage reagent was added to the cyclic peptide resin. The cleavage reagent was trifluoroacetic acid (TFA): triisopropylsilane (TIS): water = 95:2.5:2.5. The cleavage was then performed at 25°C with shaking for 2.5 h. The cleavage solution was filtered into diethyl ether (pre-cooled to -20°C), and the precipitate was collected by centrifugation to obtain crude peptide. The crude peptide was purified by high performance liquid chromatography (HPLC) using a C18 column, an acetonitrile-0.1% TFA aqueous solution as the mobile phase, and a detection wavelength of 220 nm. The main peak component was collected. The main peak solution was freeze-dried to obtain L-type lysine disulfide bond cyclic peptide with a purity ≥95%.

[0052] (3) Proliferation experiment test

[0053] Test Principles

[0054] In the presence of an electron coupling agent, WST-8 in the CCK-8 reagent can be reduced by dehydrogenases in the mitochondria of living cells to a highly water-soluble orange-yellow formazandye product. Dead cells do not have this function. The color intensity of the product is directly proportional to the number of living cells. The OD value is measured at a wavelength of 450 nm using a TECAN multi-functional microplate reader (model SPARK10M) to calculate cell viability and proliferation rate, and to quantitatively assess peptide chain activity.

[0055] Human dermal papilla cells (source: Yizefeng, generation P3, survival rate >95%), complete culture medium (GIBCO®), sterile phosphate-buffered saline (meilune), trypsin-EDTA solution (GIBCO®), CCK-8 kit (MedChemExpress). All materials were consistent with the report to avoid deviations in results due to differences in materials.

[0056] Cell seeding and grouping

[0057] Logarithmic growth phase human dermal papilla cells were digested with trypsin-EDTA solution and seeded into 96-well plates at a density of 5 × 10³ cells / well. The plates were then pre-cultured in a CO2 incubator at 37°C and 5% CO2 for 24 h. Subsequently, the L-type lysine disulfide cyclic peptide prepared in this example was diluted with complete culture medium to four test concentrations: 0.1 μM, 5 μM, 10 μM, and 25 μM. Three replicates were set for each concentration to reduce random error.

[0058] Simultaneously set:

[0059] Blank control group, complete culture medium only;

[0060] Positive control group: 0.1% positive control and 1 μM minoxidil. Minoxidil is a known hair growth-related proliferative substance used to verify the effectiveness of the test system.

[0061] Cultivation and Observation

[0062] After adding the sample, the cells were cultured under the same conditions for another 48 hours. During this period, the changes in cell morphology and number were observed regularly using an inverted fluorescence microscope. The cells were recorded to ensure that there were no abnormal states such as shrinkage or apoptosis, and to ensure that the peptide chains were not cytotoxic.

[0063] OD value detection

[0064] After 48 hours of culture, the CCK-8 reagent was diluted 10-fold with serum-free medium, and 110 μL of the diluent was added to each well. The mixture was incubated at 37°C for 0.5 hours. After incubation, the OD value of each well was measured at 450 nm using a TECAN multi-mode microplate reader, and the proliferation rate was calculated according to the formula.

[0065] Cell proliferation rate = (OD test - OD white) / OD white × 100%

[0066] Where ODtest is the OD value of the test well, and ODwhite is the OD value of the blank well.

[0067] Core data results

[0068]

[0069] The data above show that the L-type lysine disulfide bond cyclic peptide exhibited higher cell viability and positive proliferation rates than the blank control group within the concentration range of 0.1 μM-25 μM. Furthermore, all concentrations except the 10 μM group met the criteria for significant difference from the blank control group (p<0.05), demonstrating its clear proliferative effect on human dermal papilla cells. The 0.1 μM concentration showed the best proliferation effect, approaching the activity of 1 μM minoxidil and superior to the 0.1% positive control, further validating its high efficiency.

[0070] Simultaneously refer to Figure 1 It was directly observed that the bar heights of all L-cyclic peptide concentration groups were higher than those of the blank control group, and all were marked with an asterisk (*), consistent with the quantitative data, demonstrating a significant improvement in survival rate. The bar height of the 0.1 μM L-cyclic peptide was second only to that of the 1 μM minoxidil group, and slightly higher than that of the 5 μM and 25 μM concentration groups, clearly reflecting that 0.1 μM is within the optimal proliferation concentration range. The small difference between the L-cyclic peptide concentration groups and the positive control demonstrated that its proliferation activity is comparable to that of known active substances, further corroborating its effectiveness.

[0071] Reference Figure 2 The activity effect can be visually verified. In the blank control group, the number of cells was small, the gaps between cells were large, and some cells showed spindle-shaped shrinkage (characteristic of weak activity). In the photos of the 0.1% positive control and 1μM minoxidil groups, the number of cells increased significantly, the gaps between cells decreased, and the cells showed full polygonal shapes (characteristic of strong activity). In the photos of the L-cyclic peptide groups at all concentrations, the cell morphology and density were close to those of the positive control group. The 0.1μM and 5μM groups had the highest cell density, completely filling the field of view and showing full morphology; the 10μM and 25μM groups had slightly lower densities, but were still significantly higher than the blank group, with no shrinking or apoptotic cells. This morphological evidence and quantitative data... Figure 1 The results provide triple confirmation, fully demonstrating the proliferation-promoting effect of L-type lysine disulfide cyclic peptide, without cytotoxicity.

[0072] Example 2:

[0073] This embodiment discloses the preparation and performance testing of another D-type lysine disulfide bond cyclic peptide.

[0074] (1) Definition of peptide chain structure

[0075] The disulfide cyclic peptide of this embodiment is shown in SEQ ID No. 2. The disulfide cyclic peptide of this embodiment is an analog of SEQ ID No. 1, with an N-terminus of acetyl (Ac-) and a C-terminus of amide (-NH2).

[0076] The third cysteine ​​(Cys³) and the sixth cysteine ​​(Cys) 6 It forms a ring structure through intramolecular disulfide bonds.

[0077] (2) Preparation method

[0078] The solid-phase synthesis method was adopted, and the experimental materials and procedures strictly followed the standardized process associated with the test report, as detailed below:

[0079] Resin activation

[0080] Weigh 0.5g of RinkAmide-MBHA resin and place it in a solid-phase synthesis column. Soak it in N,N-dimethylformamide (DMF) for 30min to swell it. Add DMF solution of coupling reagent, shake at 25℃ to activate the amino active sites of the resin for 45min, and remove the reaction solution to ensure that the active sites of the resin can bind to the subsequent amino acids.

[0081] amino acid coupling

[0082] Fmoc-protected amino acids were coupled sequentially from C-terminus to N-terminus. During each coupling step, the corresponding amino acid (0.3 mmol), TBTU (0.3 mmol), and DIPEA (0.6 mmol) were dissolved in DMF, added to the synthesis column, and reacted with shaking at 25°C for 3 h.

[0083] After the reaction was completed, the Fmoc protecting group was removed by soaking in 20% piperidine DMF solution for 15 min, and then washed 5 times with DMF. The coupling efficiency was detected by ninhydrin colorimetric method. If the resin was colorless, the coupling was considered complete. If it was blue, the reagent was added and the coupling was repeated once to ensure that the peptide sequence was accurate.

[0084] Disulfide bond formation

[0085] Iodine-containing dichloromethane / methanol mixture was added to the linear peptide resin, and the reaction was carried out at 25°C with shaking in the dark for 1.5 h to allow Cys³ to react with Cys. 6 The thiol groups are oxidized to form intramolecular disulfide bonds; after the reaction, the iodine is washed 6 times with DMF to remove residual iodine and avoid iodine from interfering with the subsequent peptide chain activity.

[0086] End-group modification

[0087] A DMF solution of acetic anhydride and DIPEA was added to the cyclic peptide resin, and the reaction was carried out with shaking at 25°C for 1.2 h to complete the N-terminal acetylation. The C-terminus was amidated through the inherent structure of the RinkAmide-MBHA resin. After the resin was coupled with the amino group of His, -CONH2 was directly formed during cleavage without the need for additional modification steps.

[0088] Cutting and purification

[0089] A cleavage reagent was added to the cyclic peptide resin, and the mixture was cleaved at 25°C with shaking for 2.5 h to remove the side chain protecting groups and separate the cyclic peptide from the resin. The cleavage solution was filtered into pre-cooled diethyl ether, centrifuged at 3000 rpm for 10 min, and the precipitate was collected to obtain the crude peptide. The crude peptide was purified by high performance liquid chromatography (HPLC) using a C18 column, an acetonitrile-0.1% TFA aqueous solution as the mobile phase, and a detection wavelength of 220 nm. The main peak component was collected. The main peak solution was freeze-dried to obtain a D-type lysine disulfide bond cyclic peptide with a purity ≥95%.

[0090] (3) Human hair papilla cell proliferation performance test

[0091] Test basis and methods

[0092] Following the instructions in MY-SOP-158 "Cell Proliferation Assay", the human dermal papilla cell proliferation assay (CCK-8 method) was used. Human dermal papilla cells in the logarithmic growth phase (passage P3, survival rate >95%) were seeded in 96-well plates (5 × 10³ cells / well) and cultured at 25°C for 24 h.

[0093] The D-type lysine disulfide bond cyclic peptide prepared in this example was diluted with complete culture medium, and the following settings were made:

[0094] Blank control group, complete culture medium only;

[0095] Linear peptide control group, linear peptides with homologous acyclic composition, without Cys³-Cys 6 Disulfide bonds;

[0096] Each group had 3 replicates. After culturing for 48 hours, 110 μL of CCK-8 reagent diluted 10 times was added to each well, and the cells were incubated at 37°C for 0.5 hours. The OD value was measured at 450 nm using a microplate reader, and the cell proliferation rate was calculated.

[0097] Test Results

[0098]

[0099] The results show that the D-type lysine disulfide bond cyclic peptide in this embodiment has a significantly better effect on promoting the proliferation of human dermal papilla cells than the linear peptide of the same sequence, and is statistically significant compared with the blank control group. This proves that the cyclic structure is the key to the peptide chain's proliferation-promoting activity, and also verifies the effectiveness of the peptide chain under the D-type lysine configuration.

[0100] Example 3

[0101] This embodiment is based on the preparation and effect testing of the hair growth solution and essence of the above-mentioned disulfide bond cyclic peptide. Specifically, it includes a hair growth solution, namely a liposome-encapsulated preparation containing L-type lysine disulfide bond cyclic peptide.

[0102] (1) Formula composition

[0103]

[0104] (2) Preparation method

[0105] Liposome preparation:

[0106] Hydrogenated lecithin, cholesterol, and phosphatidylcholine were dissolved in 20 mL of anhydrous ethanol in a specific ratio and heated in a water bath at 60 °C until completely dissolved. The ethanol was removed by vacuum evaporation in a rotary evaporator (50 °C, 100 rpm) to form a uniform lipid film. Phosphate buffer (pH 7.4, 20 mL) containing an L-type lysine disulfide bond cyclic peptide was added and the mixture was shaken in a water bath at 60 °C for 30 min to form multilayer liposomes. The liposomes were ultrasonically treated with a probe sonicator (300 W power, 3 s working time, 7 s interval) for 10 min to prepare small single-chamber liposomes with a particle size of approximately 100-200 nm. The suspension was filtered through a 0.22 μm filter membrane to obtain the polypeptide liposome suspension.

[0107] Preparation of hair growth solution:

[0108] Dissolve glycerol in 50 mL of deionized water and stir until transparent. Add polypeptide liposome suspension and stir magnetically for 30 min (200 rpm) to mix evenly. Add phenoxyethanol and continue stirring for 15 min. Add deionized water to make up to 100 mL. Filter through a 0.22 μm filter membrane for sterilization and dispense to obtain the hair growth solution.

[0109] (3) Effect test

[0110] Hair follicle tissue, 0.2-0.3 mm in diameter, was taken from the back of a mouse and divided into:

[0111] Control group: physiological saline;

[0112] Free polypeptide group, 0.2 μM, without liposomes;

[0113] Liposome polypeptide group, solution in this example, 0.2 μM;

[0114] Ten hair follicles were cultured in each group at 37°C and 5% CO2 for 7 days. The growth status of the hair follicles was observed and the hair shaft length was measured daily.

[0115] In the control group, the average hair shaft length was 0.8 mm after 7 days, and 3 hair follicles showed atrophy. In the free peptide group, the average hair shaft length was 1.0 mm after 7 days, and 2 hair follicles showed atrophy. The hair shaft diameter increased by 5% compared to the control group. In the hair growth solution group, the average hair shaft length was 1.8 mm after 7 days, with no hair follicle atrophy, and the hair shaft diameter increased by 22% compared to the control group. This demonstrates that the hair growth solution can promote hair follicle growth, reflecting the hair growth support effect of peptide chains. Furthermore, liposome encapsulation is a means to achieve transdermal absorption of water-soluble peptides, effectively solving the problem of difficult transdermal absorption of peptide drugs.

[0116] Furthermore, the inhibitory effect of the L-lysine disulfide bond cyclic peptide on 5α-reductase activity was verified.

[0117] Test sample: L-type lysine disulfide bond cyclic peptide of the present invention (prepared in Example 1, purity ≥95%, diluted with PBS buffer to four concentrations of 0.1 μM, 5 μM, 10 μM and 25 μM).

[0118] Positive control: 1 μM finasteride (a known 5α-reductase inhibitor);

[0119] Blank control: PBS buffer;

[0120] 5α-Reductase Activity Assay Kit (purchased from Solarbio, containing 5α-reductase, testosterone substrate, and DHT assay reagent);

[0121] Microplate reader (TECANSPARK10M).

[0122] Following the kit instructions, add 5α-reductase and testosterone substrate sequentially to a 96-well plate, followed by the test sample, positive control, and blank control, with three replicates per group. Incubate at 37°C for 2 hours to allow the enzymatic reaction to proceed fully. Add the DHT detection reagent from the kit and incubate at 37°C in the dark for 30 minutes. Measure the OD value of each well using a microplate reader at 450 nm. Calculate the DHT production in each group based on the kit's standard curve, and calculate the 5α-reductase inhibition rate using the following formula:

[0123] Inhibition rate (%) = (DHT production in the control group - DHT production in the experimental group) / DHT production in the control group × 100%

[0124] The results are as follows:

[0125]

[0126] Note: This experiment presents preliminary results using the in vitro reagent kit system (Solarbio kit), and non-specific inhibitory effects have not been ruled out.

[0127] The L-lysine disulfide cyclic peptide of this invention can significantly reduce DHT production within a concentration range of 0.1 μM-25 μM, achieving an inhibition rate of 46.8%-65.7% against 5α-reductase. The inhibition rate increases with increasing concentration. In the in vitro kit system, a decreasing trend in DHT production was observed with the L-lysine disulfide cyclic peptide, and the inhibition rate plateaued with increasing concentration, suggesting a regulatory effect on 5α-reductase activity. However, subsequent experiments with increasing concentrations showed that the 10 μM group only increased by 4.2% compared to 5 μM, and the 25 μM group only increased by 0.7% compared to 10 μM, indicating that the pathway activation efficiency slows down or plateaus slightly after the concentration exceeds 5 μM.

[0128] Then, the activation effect of L-lysine disulfide cyclic peptide on the Wnt signaling pathway was verified.

[0129] Test sample: L-type lysine disulfide bond cyclic peptide of the present invention (prepared in Example 1, purity ≥95%, diluted with complete culture medium to 0.1 μM and 5 μM);

[0130] Positive control: 100 ng / mL Wnt3a protein (a known Wnt pathway activator).

[0131] Blank control: Complete culture medium;

[0132] Experimental cells: human hair follicle stem cells (purchased from Yizefeng, passage P3, survival rate >95%).

[0133] Western blot assay reagent (containing β-catenin primary antibody, GAPDH internal control primary antibody, HRP-labeled secondary antibody, and ECL chemiluminescence solution);

[0134] Protein extraction kit, electrophoresis apparatus, transfer apparatus, chemiluminescence imaging system.

[0135] The experimental method is as follows:

[0136] Human hair follicle stem cells were used at a rate of 2×10 5 Inoculate 1 cell / well into a 6-well plate and incubate at 37°C and 5% CO2 for 24 hours;

[0137] Add the test sample, positive control, and blank control respectively, and continue culturing for 48 hours;

[0138] Total protein was extracted from cells in each group using a protein extraction kit, and the protein concentration was measured.

[0139] SDS-PAGE electrophoresis was performed (20 μg / well), and the sample was transferred to a PVDF membrane and blocked with 5% skim milk for 1 h.

[0140] Add β-catenin primary antibody (1:1000 dilution) and GAPDH internal control primary antibody (1:5000 dilution), and incubate overnight at 4°C;

[0141] HRP-labeled secondary antibody (1:3000 dilution) was added and incubated at room temperature for 1 h. ECL chemiluminescence was then applied for color development. Images were acquired using an imaging system, and the relative expression level of β-catenin protein was analyzed. GAPDH was used as an internal control, and the β-catenin / GAPDH grayscale ratio was calculated. The experimental results are as follows:

[0142]

[0143] The above results demonstrate that L-lysine disulfide bond cyclic peptides can increase the relative expression of β-catenin in human hair follicle stem cells in the concentration range of 0.1 μM-5 μM, with the 5 μM group showing a 2.64-fold increase, suggesting that it may activate the Wnt / β-catenin signaling pathway.

[0144] Then, the activation effect of the L-type lysine disulfide cyclic peptide on the VEGF signaling pathway was further verified.

[0145] Test sample: L-type lysine disulfide bond cyclic peptide of the present invention (prepared in Example 1, purity ≥95%, diluted with complete culture medium to 0.1μM, 5μM, 25μM);

[0146] Positive control: 10 ng / mL bFGF (a known pro-angiogenic factor);

[0147] Blank control: Complete culture medium;

[0148] Experimental cells: human dermal papilla cells (purchased from Yizefeng, passage P3, survival rate >95%).

[0149] Human VEGFELISA assay kit (purchased from R&D Systems);

[0150] Microplate reader (TECANSPARK10M);

[0151] Human umbilical vein endothelial cells (HUVEC, purchased from ATCC, used for auxiliary verification of angiogenesis).

[0152] VEGF secretion was detected using the ELISA method.

[0153] Human dermal papilla cells were divided into 5×10 4 Cells were seeded per well in 24-well plates and incubated at 37°C with 5% CO2 for 24 hours. Test samples, positive controls, and blank controls were added, and the plates were incubated for another 48 hours. The supernatant from each group was collected and centrifuged at 12000 rpm for 10 minutes at 4°C to remove impurities. Following the ELISA kit instructions, supernatant (100 μL / well) was added to 96-well plates and incubated at 37°C for 1 hour. Detection antibodies and enzyme conjugates were added, followed by incubation. Substrate chromogenic solution was then added, and the plates were incubated at 37°C in the dark for 15 minutes. Stop solution was added, and the OD value was measured at 450 nm using a microplate reader. The VEGF secretion level (pg / mL) for each group was calculated based on the standard curve. The VEGF secretion results are as follows:

[0154]

[0155] Endothelial cell proliferation assay (CCK-8 assay, to assist in verifying VEGF activity)

[0156] Collect the supernatant (containing secreted VEGF) of dermal papilla cells from each group above as conditioned medium; add HUVECs at 1×10⁻⁶. 4 HUVECs were seeded per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The original culture medium was discarded, and conditioned medium (100 μL / well) was added to each well, with three replicates per well. The plates were incubated for another 24 h, then CCK-8 reagent (10 μL / well) was added and incubated at 37°C for 1 h. The OD value was measured at 450 nm using a microplate reader, and the HUVEC proliferation rate was calculated (based on HUVECs cultured in the blank control group). The HUVEC proliferation rate results are as follows:

[0157]

[0158] The L-type lysine disulfide bond cyclic peptide of this invention can significantly promote the secretion of VEGF by human dermal papilla cells, and the secreted VEGF has biological activity and can promote angiogenesis. The HUVEC proliferation rate results show that 0.1 μM is the optimal dose, achieving a HUVEC proliferation rate of 172.6%; as the concentration increases to 5 μM and 25 μM, the HUVEC proliferation rate gradually decreases to 145.8% and 128.4%, respectively, showing a clear dose-dependent decreasing trend.

[0159] This result is highly consistent with the findings of dermal papilla cell proliferation experiments, both indicating that a low dose (0.1 μM) of the L-lysine disulfide cyclic peptide exhibits optimal biological activity, while excessively high concentrations reduce its promoting effect. The presumed reason is that high concentrations of the peptide may lead to overactivation of cell signaling pathways, triggering a negative feedback regulatory mechanism.

[0160] In summary, this polypeptide can effectively activate the VEGF signaling pathway and improve hair follicle microcirculation by promoting VEGF secretion, thus verifying the mechanism of action of VEGF pathway activation.

[0161] Furthermore, this embodiment also provides a hair growth essence.

[0162] (1) The formula is as follows:

[0163]

[0164] (2) The preparation method is as follows:

[0165] Equipment preparation includes a probe-type ultrasonic instrument, a dynamic light scattering instrument, an analytical balance (accuracy 0.0001g), and a magnetic stirrer;

[0166] Then, accurately weigh the hydrogenated lecithin and cholesterol using an analytical balance, place them in a beaker, add 50g of ethanol, place the beaker in a 60℃ constant temperature water bath, and stir magnetically until completely dissolved to form a transparent oil phase solution.

[0167] Add L-type lysine disulfide bond cyclic peptide to the oil phase solution, and gently shake for 3 minutes to ensure that the active peptide is uniformly dispersed in the oil phase;

[0168] Keep the solution temperature ≤40℃, slowly add deionized water while sonicating with a probe-type ultrasonic instrument (power 200W, time 12 minutes, intermittent sonication: work for 30 seconds, pause for 10 seconds) until a milky white liposome suspension is formed;

[0169] Liposome particle size should be measured using a DLS instrument: the particle size should be 100-200nm. If it does not meet the standard, sonicate for 1-2 minutes and retest until it meets the requirements.

[0170] Next is the preparation of the aqueous phase, which requires the preparation of equipment such as a mixer, heating mantle, and pH meter.

[0171] Add deionized water to the mixing tank, then add glycerol, panthenol, and dipotassium glycyrrhizate in sequence. Turn on the heating mantle to raise the temperature to 60°C and stir at 300 rpm for 15 minutes until the solution is completely transparent.

[0172] Add 5g of phenoxyethanol and 1g of vitamin E, and continue stirring at 60℃ for 10 minutes to ensure that the preservatives and antioxidants are evenly dispersed.

[0173] Once the temperature of the aqueous phase drops below 45°C, slowly pump the qualified liposome suspension into the aqueous phase stirring tank and stir at 500 rpm for 30 minutes, avoiding the generation of bubbles during the stirring process.

[0174] Add azone and continue stirring at 500 rpm for 10 minutes to ensure that the transdermal penetration enhancer is fully incorporated into the system;

[0175] Adjust the pH of the system to 5.5-6.0 with a 10% citric acid solution (monitor in real time with a pH meter) to make the pH of the essence suitable for the physiological environment of the scalp (normal scalp pH is 4.5-6.5).

[0176] Add deionized water to a total weight of 1000g, turn on the homogenizer and homogenize at 2000rpm for 5 minutes to obtain a uniform and delicate hair growth essence.

[0177] Fill the serum into 30ml brown glass bottles, and seal the bottles with nitrogen during the filling process to isolate them from oxygen. After labeling, store them in a cool, dry place (temperature 10-25℃, relative humidity ≤60%), avoiding light and high temperature.

[0178] (3) Effect test

[0179] Test subjects: Thirty volunteers with mild hair loss (15 men and 15 women, aged 25-45, with 50-100 hairs lost daily, and no scalp inflammation or skin disease) were randomly divided into two groups:

[0180] Experimental group: Using the hair growth essence containing liposomes from this embodiment;

[0181] Control group: Hair growth serum without liposomes and with the same formula as the rest (active peptides added directly, without encapsulation).

[0182] Instructions for use: Two groups of volunteers applied the corresponding serum to the scalp area with hair loss (1mL / time) once in the morning and once in the evening, massaging for 3 minutes to promote absorption, for 12 consecutive weeks; during the test period, avoid using other hair loss prevention and hair growth products, and maintain a consistent work and rest schedule and diet.

[0183] Testing indicators: Transdermal efficiency: Before the experiment, an in vitro porcine skin transdermal test (Franz diffusion cell method) was used to detect the amount of active peptides transdermally within 24 hours; Hair loss: The daily hair loss of volunteers was recorded weekly (hair loss was collected and counted by combing method), and the weekly average was taken; Hair growth: At weeks 4, 8, and 12, the number of new vellus hairs, hair diameter, and blackness in the hair loss area were observed using a hair analyzer, and the average of the three tests was taken; Scalp tolerance: Whether redness, swelling, itching, or other discomfort symptoms occurred on the scalp during use was recorded.

[0184] Test Results

[0185] Transdermal efficiency: The transdermal amount of active peptides in the experimental group was 2.3 times that of the control group after 24 hours, which proves that liposome encapsulation and binding of azone can significantly enhance the skin penetration ability of L-type lysine disulfide bond cyclic peptides and ensure that the active ingredients reach the hair follicles in the dermis.

[0186] Improvement in hair loss:

[0187] Experimental group: After 12 weeks, the average daily hair loss decreased to 15-25 strands, a reduction of 70%-80% compared to the baseline (50-100 strands);

[0188] Control group: After 12 weeks, the average daily hair loss decreased to 30-45 strands, a reduction of 30%-50% compared to baseline;

[0189] The experimental group showed a significantly greater reduction in hair loss than the control group, demonstrating that liposomes enhance the effectiveness of active ingredients.

[0190] Improved hair growth:

[0191] Number of new vellus hairs: After 12 weeks, the number of new vellus hairs in the balding area of ​​the experimental group increased by 65% ​​compared with the baseline, while that in the control group increased by 35%.

[0192] Hair diameter: The average diameter of newly grown hair in the experimental group increased by 20% compared to the baseline, while that in the control group increased by 8%.

[0193] Hair blackness: The blackness value of newly grown hair in the experimental group (detected by spectrophotometry) increased by 18% compared with the baseline, while that in the control group increased by 6%.

[0194] The experimental group showed better results than the control group in terms of the number, diameter, and blackness of new hair growth, demonstrating that liposomes can enhance the activation effect of active peptides on hair follicles.

[0195] Scalp tolerance: No discomfort such as scalp redness, swelling or itching was observed in either group of volunteers during the use period. Moreover, due to the encapsulation of active peptides by liposomes, the experimental group experienced less direct contact with the scalp and achieved a scalp comfort score (out of 5) of 4.8, which was higher than the control group's 4.2.

[0196] This embodiment contains a hair growth essence containing L-type lysine disulfide bond cyclic peptides encapsulated in liposomes. Through the synergistic effect of liposomes and azone, the transdermal efficiency of active ingredients is significantly improved. It is more effective than essences without liposomes in reducing hair loss and promoting hair growth. Moreover, it has good scalp tolerance and can effectively solve the problem of active ingredients being difficult to penetrate the skin, providing a better formula and process solution for anti-hair loss and hair growth products.

[0197] Furthermore, this embodiment also provides a shampoo formula and preparation method. Taking the production of 500g of shampoo as an example, the core ingredients are:

[0198] 0.4 g of L-lysine disulfide bond cyclic peptide encapsulated in liposomes, 50 g of peptide chain from Example 1 encapsulated in liposomes;

[0199] Sodium lauroyl glutamate 30g, a mild surfactant;

[0200] Cocamidopropyl betaine 15g, conditioning agent;

[0201] Panthenol 5g, a soothing agent;

[0202] Sodium hyaluronate 2g, humectant;

[0203] Dipotassium glycyrrhizate 1g, an anti-inflammatory component;

[0204] Phosphatidylcholine 2g, liposome stabilizer;

[0205] Adjust the pH to 5.5-6.0 with citric acid, and make up the rest with deionized water.

[0206] Preparation method:

[0207] Dissolve surfactants and moisturizers in 60°C deionized water and stir until transparent; cool to 40°C and add liposome-encapsulated active peptides, stir for 15 minutes; add soothing agents and anti-inflammatory ingredients, adjust the pH, add water to the total weight, and stir until homogeneous.

[0208] Effect test

[0209] Twenty volunteers with moderate hair loss were selected. These volunteers experienced hair loss of 60-120 strands per day and were randomly divided into two groups: the experimental group used the shampoo described in this example, 5 ml three times a week, massaging the scalp for 2 minutes before rinsing; the control group 1 used the same basic shampoo without active peptides for 12 consecutive weeks; and the control group 2 used commercially available minoxidil solution at a concentration of 20 mg / mL, 0.5 mL once daily, applying evenly to the hair loss area and massaging for 30 seconds for 12 weeks.

[0210] After 12 weeks, the daily hair loss in the experimental group decreased to 35-40 strands, a 50% reduction from baseline, and the number of new vellus hairs in the balding area increased by 50%. In control group 1, hair loss decreased by 20%, and new vellus hairs increased by 10%. The daily hair loss in control group 2 after 12 weeks was also generally within the range of 40 strands. There was no difference between the experimental group and control group 2 using minoxidil. Compared to the dosage of minoxidil, the dosage used in this example was smaller and had no side effects associated with minoxidil.

[0211] This invention is not limited to the optional embodiments described above, and anyone can derive other various forms of products based on the inspiration of this invention. The specific embodiments described above should not be construed as limiting the scope of protection of this invention; the scope of protection of this invention should be determined by the claims, and the specification can be used to interpret the claims.

Claims

1. A multi-target, multi-functional anti-hair loss polypeptide, characterized in that, The polypeptide is a cyclic peptide with an amino acid sequence as shown in SEQ ID No.

1. The N-terminus of the cyclic peptide is modified by acetylation, and the C-terminus is modified by amidation. The cysteine ​​residues at the third and sixth positions in the sequence form an intramolecular disulfide bond.

2. Multi-target, multi-functional anti-hair loss polypeptide, characterized in that, The polypeptide is an analogue of SEQ ID No. 1, wherein the L-lysine in SEQ ID No. 1 is replaced with D-lysine, and the amino acid sequence of the analogue is shown in SEQ ID No.

2.

3. The application of a multi-target, multi-functional anti-hair loss and hair growth polypeptide, wherein the multi-target, multi-functional anti-hair loss and hair growth polypeptide described in claim 1 or 2 is used in the preparation of anti-hair loss and hair growth products, characterized in that, The hair loss prevention and hair growth products include topical shampoos, serums, lotions, creams, sprays, gels, and ointments.

4. A method for preparing the multi-target, multi-functional hair loss prevention polypeptide according to claim 1 or 2, characterized in that, The steps include resin activation, sequential coupling of amino acids according to the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2, formation of intramolecular disulfide bonds, N-terminal acetylation and C-terminal amidation treatment, and cleavage and purification to obtain the target polypeptide.

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