Polypeptide composition for treating skin inflammation related diseases and repairing skin

Abstract

The invention discloses a polypeptide composition for treating skin inflammation related diseases and repairing skin, and particularly relates to application of acetyl dipeptide-1 in preparation of drugs, health care products and cosmetics for treating the skin inflammation related diseases, promoting healing, resisting inflammation, resisting bacteria, resisting oxidation and repairing tissue. The water-soluble acetyl dipeptide-1 and the specific polypeptide are compounded and have a synergistic effect, so that the obvious effect of treating the skin inflammation related diseases by the acetyl dipeptide-1 is realized; besides, the inclusion compound obtained by adding the hydroxypropyl-beta-cyclodextrin-chitosan compound into the polypeptide composition greatly improves the skin permeability of the polypeptide, the permeation rate and the permeation amount are remarkably increased, and the resident dosage of the skin is remarkably increased while the usage amount of the polypeptide is small.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide application, and in particular relates to the use of a polypeptide composition in treating skin inflammatory related diseases. Background Art

[0002] Under external stimulation such as ultraviolet rays, cosmetics, unhealthy diet or lifestyle habits, the skin will have a series of problems, such as dull skin, spots, excessive sebum secretion, acne, bacterial infection and inflammation.

[0003] Acne is a chronic inflammatory skin disease of the pilosebaceous gland unit, which is mainly common in adolescents and has a great impact on their psychological and social development. The occurrence of acne is closely related to factors such as excessive sebum secretion, blockage of the pilosebaceous gland duct, bacterial infection and inflammatory response. After entering puberty, the level of androgens in the human body, especially testosterone, rises rapidly, promoting the development of sebaceous glands and producing a large amount of sebum. At the same time, the abnormal keratinization of the pilosebaceous gland duct causes blockage of the duct, which hinders the discharge of sebum and forms keratin plugs, i.e. micro-acne. The secretion and discharge of a large amount of sebum are blocked, which leads to bacterial infection. There are many microorganisms in the hair follicles, such as Propionibacterium acnes, Staphylococcus albus and Malassezia furfur. Among them, Propionibacterium acnes plays the most critical role. It is the dominant flora in the sebaceous glands of acne patients, accounting for 89% of all detected microorganisms. This bacterium is an anaerobic bacterium. The obstruction of sebum discharge just creates a good local anaerobic environment for it, allowing Propionibacterium acnes to multiply in large numbers. The lipase produced by Propionibacterium acnes can decompose triglycerides in sebum and produce free fatty acids, which are the main factors leading to the formation of acne inflammatory lesions. In addition, Propionibacterium acnes can also produce polypeptide substances, which can attract neutrophils, activate complement and cause leukocytes to release various enzymes, inducing or aggravating inflammation.

[0004] The essence of acne is an inflammatory response, which is the earliest event in acne damage. It begins before the keratinization of the hair follicle opening and the formation of microcomedones, and runs through the entire acne. The onset of acne is related to a variety of inflammatory factors. TNF-α, IL-6, IL-8, IL-12, and IL-1α promote excessive keratinization of the hair follicle opening and the characteristic inflammatory damage of acne. When the skin is affected by various external irritants and allergens, it will cause oxidative stress in the skin. Oxidative stress caused by excessive reactive oxygen free radicals (ROS) can also induce skin inflammation. On the one hand, inflammatory cells produce ROS and participate in oxidative stress. On the other hand, ROS can activate NF-κB, leading to enhanced gene expression of multiple inflammatory cytokines (TNF-α, IL-1, IL-6, IL-8), chemotaxis of inflammatory cells such as neutrophils, and enhanced inflammatory response. Therefore, skin acne, inflammatory processes and peroxidation damage are closely related. Reports show that the local antioxidant products of severe acne are 2 to 4 times that of normal or mild acne. Anti-oxidation is a very important task for acne and inflammatory skin.

[0005] Bioactive peptides have a variety of biological activities, such as antioxidant, anti-aging, anti-inflammatory and antibacterial activities, making them ideal anti-inflammatory ingredients. Bioactive peptides participate in a variety of physiological and biochemical activities of the human body, and their biological activity is extremely high. Small doses of peptides can play a biological regulatory role in a variety of cellular physiological functions and metabolic activities. Acetyl dipeptide-1 cetyl ester is an anti-inflammatory and soothing active peptide that helps inhibit the release of inflammatory factors, stimulates the synthesis of collagen, and has anti-inflammatory, relaxing, and anti-allergic effects. However, due to its characteristics of being "poorly soluble in water", it is necessary to add a polyethylene glycol structure solubilizer or polyol for solubilization when using it. The scientific community has found that polyethylene glycol may have a certain risk of sensitization and may cause systemic allergic reactions. In the development of sensitive skin formulas, the use of solubilizers such as polyethylene glycol may cause tingling and itching discomfort. Therefore, through technical research, acetyl dipeptide-1 was prepared using a unique liquid phase synthesis production process. As a new raw material, it can be truly soluble in water, fundamentally solving the development problems brought about by solubilizers. Therefore, the development of acetyl dipeptide-1 products for the treatment of skin inflammatory diseases has great application prospects and value.

[0006] At present, some products on the market that improve skin acne and inflammation mainly kill Propionibacterium acnes as the main means, and the action pathway is relatively single or the effect is poor. There is an urgent need in the art to develop a drug that can effectively prevent and treat the above skin problems. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide an application of acetyl dipeptide-1 in the preparation of medicines, health products and cosmetics for treating skin inflammatory diseases, promoting healing, anti-inflammatory, antibacterial, antioxidant and tissue repair.

[0008] Preferably, the skin inflammatory disease is acne.

[0009] On the other hand, the present invention provides a polypeptide composition for treating skin inflammatory diseases and repairing skin. Specifically, the composition comprises a composite peptide composed of acetyl dipeptide-1 and tripeptide-1 copper.

[0010] Preferably, the mass ratio of acetyl dipeptide-1 to tripeptide-1 in the composite peptide of the composition is 1:1-5, more preferably 1:1-3, and particularly preferably 1:2.

[0011] Preferably, a hydroxypropyl-β-cyclodextrin-chitosan complex is added to the composition, and the composite peptide is encapsulated in the hydroxypropyl-β-cyclodextrin-chitosan complex.

[0012] Preferably, the mass ratio of the composite peptide to the hydroxypropyl-β-cyclodextrin-chitosan complex in the composition is 1:1-10, and more preferably 1:5.

[0013] Preferably, the mass ratio of hydroxypropyl-β-cyclodextrin to chitosan in the complex is 1:0.1-1, particularly preferably 1:0.5.

[0014] In a preferred embodiment, the preparation method of the above polypeptide composition is as follows: Step 1: Add the prescribed amount of complex peptide and an appropriate amount of purified water into a container, stir at room temperature to dissolve, and prepare a complex peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand, centrifuge the mixture, take the supernatant filter to filter out the precipitate, and freeze-dry to obtain the inclusion polypeptide composition.

[0015] The application of the present invention is to prepare any pharmaceutically acceptable external preparation by combining a pharmaceutically effective amount of the polypeptide composition with a pharmaceutically acceptable excipient.

[0016] Preferably, the topical preparation is one of a gel, a cream, a lotion, a facial cream, an ointment, a paste, a lotion, a patch, a dressing, a film coating, a cataplasm, an aerosol or a spray.

[0017] On the other hand, the present invention also provides a polypeptide composition gel for treating skin inflammatory diseases and repairing skin, wherein the gel comprises the polypeptide composition and a pharmaceutically acceptable gel excipient.

[0018] Preferably, the gel auxiliary material comprises a gel matrix, a moisturizer, an antioxidant, an antibacterial agent, a pH regulator, and a water solvent.

[0019] The gel matrix is ​​preferably one or more of carbomer, poloxamer, sodium carboxymethyl cellulose, alginate, chitosan, liquid paraffin-hydrogenated soybean lecithin, colloidal silver-gelatin, and collagen.

[0020] The moisturizing agent is preferably one or more of glycerin, propylene glycol, hyaluronic acid, and butylene glycol, and glycerin is further preferred; the content thereof is selected according to demand and conventional knowledge in the art.

[0021] The antioxidant is preferably one or more of sulfite, cysteine, BHT, vitamin C, vitamin E, BHT, and potassium sorbate.

[0022] The antibacterial agent is preferably one or more of phenoxyethanol, deoxycholic acid, silver ions, parabens, benzoic acid, phenol, PHL, scutellaria baicalensis extract, phellodendron amurense extract, forsythia suspensa extract, dandelion extract and aloe extract; the content thereof is selected according to demand and conventional knowledge in the art, and the antibacterial agent is used at a concentration that is harmless and non-irritating to the human body while achieving a good antibacterial effect.

[0023] The pH adjuster is preferably triethanolamine.

[0024] In a preferred embodiment, the present invention provides a polypeptide composition gel, the components of which are as shown in Table 1: Table 1 Gel formula with 10~20% peptide composition content

[0025] Among them, the pH value of the gel ranges from 6 to 7.

[0026] In a preferred embodiment, the present invention provides a polypeptide composition gel, the component contents of which are as shown in Table 2: Table 2 Gel formula with 15% peptide composition content

[0027] Among them, the pH value of the gel ranges from 6 to 7.

[0028] On the other hand, the present invention provides a method for preparing a polypeptide composition gel, comprising weighing a prescribed amount of a gel matrix and evenly sprinkling it on the surface of an appropriate amount of distilled water, and standing to swell for 24 hours; adding an appropriate amount of purified water to the prescribed amount of the polypeptide composition and stirring to prepare an aqueous solution; adding the aqueous solution of the polypeptide composition to the gel matrix and stirring to mix well, adding a prescribed amount of an antioxidant, an antibacterial agent, and a moisturizer and mixing well, adding an appropriate amount of a pH adjuster to adjust the pH value of the gel to about 7, adding purified water to a sufficient amount, stirring evenly, and obtaining a target product.

[0029] The present invention further provides a use of a polypeptide composition for treating skin inflammatory diseases, which is used to prepare a preparation for treating skin diseases or improving skin discomfort or abnormal symptoms caused by the disease, wherein the composition is administered at least once a day. In some embodiments, the composition is administered once, twice, three times or four times a day.

[0030] Among them, skin inflammatory diseases are skin diseases or conditions such as acne, atopic dermatitis, eczema, rash, urticaria, itching, eczema outbreaks, eosinophilic dermatosis, reactive neutrophilic dermatosis, immune bullous dermatosis, cutaneous fibrohistiocyte proliferation, and keloid.

[0031] Preferably, the skin inflammatory disease is acne.

[0032] It is explained here that in order to fill the gap in the domestic water-soluble soothing agent market and break through the problem of "difficulty in dissolving in water" of peptides such as acetyl dipeptide-1 cetyl ester for sensitive skin, the R&D personnel have made technical breakthroughs and adopted a unique liquid phase synthesis production process to prepare acetyl dipeptide-1 that can truly dissolve in water, fundamentally solving the development problems brought by the solubilizer, and with high purity crystallization, non-hygroscopicity, and easy preservation, and successfully completed the filing as a new raw material; acetyl dipeptide-1, as a neuropeptide, achieves anti-inflammatory, soothing and anti-allergic effects by regulating the key regulatory factors TNF-α, IL-6, IL-1β and PGE2 in skin inflammation. In the product development process, in order to obtain more effective products, the researchers tried to develop a peptide composition containing acetyl dipeptide-1 for the treatment of skin inflammatory diseases; selected a variety of similar peptides with anti-inflammatory functions, and finally found that adding a specific tripeptide-1 copper to acetyl dipeptide-1 can achieve significant synergistic effects.

[0033] Beneficial effects of the present invention: Compared with the prior art, this application overcomes the problem of "poor water solubility" of peptides for sensitive skin, and uses water-soluble acetyl dipeptide-1 and specific polypeptides to work synergistically, thereby achieving the significant effects of acetyl dipeptide-1 in treating acne, promoting skin healing, anti-inflammatory, antibacterial, antioxidant, and tissue repair. In addition, the inclusion compound obtained by adding hydroxypropyl-β-cyclodextrin-chitosan complex to the polypeptide composition greatly improves the skin permeability of the polypeptide, and the penetration rate and penetration amount are significantly improved. While using a small amount of polypeptide, the resident dose of the skin is significantly improved. DETAILED DESCRIPTION

[0034] The present invention is further described below by way of examples. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial sources unless otherwise specified.

[0035] Example 1 Preparation of polypeptide composition Referring to the prescription in Table 3, acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water were added to a container and stirred at room temperature to dissolve to prepare a composite peptide solution; the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan were added with an appropriate amount of purified water to prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution; the composite peptide solution was slowly added to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution and mixed thoroughly, allowed to stand, the mixture was centrifuged using a centrifuge, the supernatant was filtered to remove the precipitate, and freeze-dried to obtain an inclusion polypeptide composition.

[0036] Table 3 Prescription of composition of Example 1

[0037] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0038] Example 2 Preparation of polypeptide composition Table 4 Prescription of composition of Example 2

[0039] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0040] Example 3 Preparation of polypeptide composition Table 5 Prescription of composition of Example 3

[0041] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0042] Example 4 Preparation of polypeptide composition Table 6 Prescription of composition of Example 4

[0043] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0044] Example 5 Preparation of polypeptide composition Table 7 Prescription of composition of Example 5

[0045] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0046] Comparative Example 1 Preparation of polypeptide composition Table 8 Comparative Example 1 Composition Prescription

[0047] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1 and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0048] Comparative Example 2 Preparation of polypeptide composition Table 9 Comparative Example 2 Composition Prescription

[0049] The specific preparation process is as follows: Step 1: Add the prescribed amount of tripeptide-1 copper and an appropriate amount of purified water into a container, stir at room temperature to dissolve, and prepare a complex peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0050] Comparative Example 3 Preparation of polypeptide composition Table 10 Comparative Example 3 Composition Prescription

[0051] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, palmitoyl tetrapeptide-7 and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0052] Comparative Example 4 Preparation of polypeptide composition Table 11 Comparative Example 4 Composition Prescription

[0053] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, palmitoyl tripeptide-8 and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0054] Comparative Example 5 Preparation of polypeptide composition Table 12 Comparative Example 5 Composition Prescription

[0055] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, palmitoyl tripeptide-8 and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0056] Comparative Example 6 Preparation of polypeptide composition Table 13 Comparative Example 6 Composition Prescription

[0057] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin, add an appropriate amount of purified water, heat to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0058] Comparative Example 7 Preparation of polypeptide composition Table 14 Comparative Example 7 Composition Prescription

[0059] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of chitosan, add an appropriate amount of purified water, heat to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a chitosan aqueous solution, and cool to room temperature for use; Step 3: Slowly add the composite peptide solution obtained in step 1 to the chitosan aqueous solution while stirring, mix thoroughly, let stand for 1 hour, centrifuge at 2000 rpm for 30 minutes, filter out the precipitate with a polypropylene filter with a pore size of 5 microns, and freeze-dry to obtain a polypeptide composition.

[0060] Comparative Example 8 Preparation of polypeptide composition Table 15 Comparative Example 8 Composition Prescription

[0061] The specific preparation process is as follows: Step 1: Add the prescribed amount of acetyl dipeptide-1, tripeptide-1 copper and an appropriate amount of purified water into a container, stir to dissolve at room temperature, and prepare a composite peptide solution; Step 2: Weigh the prescribed amount of glycerol and 1,3-propylene glycol, add appropriate amount of purified water, heat and stir to dissolve; Step 3: Slowly add the composite peptide solution obtained in step 1 to the solution in step 2 while stirring, mix thoroughly, filter, and freeze-dry to obtain a polypeptide composition.

[0062] Verification Example: Skin permeability experiment.

[0063] Using Bama miniature pig ex vivo skin as skin model material, Franz diffusion cell and HPLC were used to determine the amount of peptide composition retained on the skin surface and in the dermis at different time points.

[0064] Preparation of ex vivo pig skin: After Bama miniature pigs were killed, the abdominal skin was immediately cut off, and the subcutaneous fat and connective tissue were peeled off. The skin surface was repeatedly wiped with a 95% alcohol cotton ball, and then rinsed with physiological saline. The water was absorbed with filter paper and set aside.

[0065] Transdermal test: Franz transdermal tester was used for transdermal test. Pig skin was placed on the top of the receiving pool, with the stratum corneum facing upwards and in contact with the supply pool, and the dermis in contact with the transdermal receiving solution, which was 5 mL of saline. The sample (calculated as composite peptide, containing about 10 mg of composite peptide) was directly added to the transdermal supply pool (saline solution). The water bath temperature of the diffusion pool was 37±0.5 。 C, stirring speed 450 r·min -1 The sampling time points are 0.5h, 1h, 2h, 4h, 8h and 12h.

[0066] Skin surface samples: Use 3 cotton balls to wipe off the residue on the skin surface, then rinse the skin surface 3 times with normal saline solution, 1 mL each time, then place the cotton balls in normal saline for ultrasonic extraction, cool to room temperature, and set to 5 mL.

[0067] Preparation of dermis samples: Use cotton balls to wipe the residue on the skin surface, and then use the tape sticking method to stick the epidermis 20 times to stick the stratum corneum off. Then cut the remaining skin after treatment into pieces, add 5 mL of physiological saline solution in a homogenizer for homogenization, centrifuge, take the supernatant, and filter it with a 0.22 m microporous filter membrane. Take 0.5 mL of all samples and add 100 g mL -1 0.5 mL of DTNB solution was vortexed for 3 min, filtered through a 0.22 μm microporous membrane and then analyzed by HPLC.

[0068] Method for determination of complex peptide content: Chromatographic conditions: Chromatographic column: Agilent Poroshell 120HILIC-Z (100mm×3.0mm, 2.7μm); Chromatographic column temperature: 30℃; Mobile phase flow rate: 0.3mL / min; Fluidity: Phase A 15mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid), Phase B: acetonitrile (containing 0.1% formic acid); Elution program (0min, Phase A 10%, Phase B 90%; 1min, Phase A 10%, Phase B 90%; 8min, Phase A 50%, Phase B 50%; 12min, Phase A 50%, Phase B 50%; 12.1min, Phase A 10%, Phase B 90%; 17min, Phase A 10%, Phase B 90%).

[0069] Calculation of the amount of retention: The amount of complex peptides (the sum of the contents of each peptide) retained on the skin surface and in the skin per unit area of ​​skin is calculated according to the formula: Q r = C i ×V i / S; where Q r (μg·cm -2 ) represents the amount of complex peptides retained per unit area in the skin at a certain time point; C i (μg·mL -1 ) represents the concentration of the composite peptide; V i (mL) represents the volume of the extraction solution (5.0 mL); S (cm 2 ) represents the area of ​​the diffusion cell exposed to the ex vivo skin (1.76 cm 2 ).

[0070] Table 16 Results of the test on the retention of the composite peptide in the surface layer of pig skin

[0071] Table 17 Test results of the retention of the composite peptide in the dermis of pig skin

[0072] The above results show that the polypeptide composition of the present application can quickly penetrate into the dermis, the permeability is significantly improved, and the release rate is fast; in addition, the retention amount of the polypeptide composition of the present application in the dermis is significantly improved.

[0073] DPPH free radical scavenging ability determination.

[0074] Test method: Weigh a certain amount of DPPH and prepare a 0.1 mg / mL DPPH solution with anhydrous ethanol; take 2 mL of the sample solution of Examples 1-8, Comparative Examples 1-3, and polypeptide compositions (take 2 g of the polypeptide composition and add 1000 g of anhydrous ethanol to dissolve and dilute to prepare the sample solution), add 2 mL of DPPH solution, mix evenly, react at room temperature for 30 minutes, and measure the absorbance Ai at 517 nm; simultaneously measure the absorbance A0 of 2 mL DPPH solution + 2 mL of anhydrous ethanol and the absorbance Aj after mixing 2 mL of sample solution + 2 mL of anhydrous ethanol, repeat the measurement twice, and the calculation formula for the scavenging rate of the sample for DPPH free radicals is: DPPH free radical scavenging rate = (A0-Aj+Ai) / A0×100%; the antioxidant capacity can be reflected by the DPPH free radical scavenging ability measurement. The higher the DPPH free radical scavenging rate, the stronger the antioxidant capacity, and vice versa.

[0075] Table 18 DPPH free radical scavenging rate test results of composite peptides

[0076] The above results show that under the synergistic effect of the polypeptide complex of the present application, the DPPH free radical scavenging ability is significantly improved compared with the use of a single peptide; tripeptide-1 copper is combined with acetyl dipeptide-1 compared with anti-inflammatory functional peptides such as palmitoyl tetrapeptide-7, palmitoyl tripeptide-8, arginine / lysine peptide, and significantly improves its DPPH free radical scavenging ability; the polypeptide composition of the present application has a good antioxidant effect.

[0077] Animal testing of acne treatment effectiveness.

[0078] Drug preparation: Take the lyophilized powder of the polypeptide composition prepared in Example 1 and Comparative Examples 1-5 respectively, and prepare 100g of gel according to the following prescription: Table 19 Gel formulations prepared

[0079] Weigh the prescribed amount of Carbomer-940 and sprinkle it evenly on the surface of an appropriate amount of distilled water, and let it stand to swell for 24 hours; add the prescribed amount of freeze-dried powder of the polypeptide composition to an appropriate amount of purified water and stir to prepare an aqueous solution; add the aqueous solution of the polypeptide composition to the gel matrix and stir to mix, then add the prescribed amount of glycerol, vitamin C, and deoxycholic acid and mix well, add an appropriate amount of triethanolamine to adjust the pH value of the gel to about 7, add purified water to a sufficient amount, stir evenly, and obtain a sample for later use.

[0080] Propionibacterium acnes liquid: Dissolve the freeze-dried Propionibacterium acnes in sodium thioethoxide medium under anaerobic conditions to form a suspended bacterial liquid, culture it in an anaerobic tank, and then inoculate it on an anaerobic blood plate, place it in an anaerobic tank and place it in a 37°C constant temperature incubator for 24 hours. Take out the colony with a sterile cotton swab, wash it three times with physiological saline, and adjust the concentration of Propionibacterium acnes to 6×10 7 cfu / ml, inactivate in 95℃ water bath for 15min and set aside.

[0081] Animal modeling: Several rats were divided into a blank group and a modeling group. According to the classic rat modeling method, Propionibacterium acnes was injected intradermally into the auricle to simulate the external manifestations of acne and the pathogenesis of inflammatory response, and the rat ear acne model was prepared. In the modeling group, each rat was injected intradermally with Propionibacterium acnes liquid (6×10 7 cfu / ml) 50 μl, once a day for 3 consecutive days. On the third day, each rat was intraperitoneally injected with Propionibacterium liquid (6×10 7cfu / ml) 1ml, once a day, for 7 consecutive days. The epidermis of the rat auricle was rough and dry, the skin was red and swollen, the capillaries were invisible, and the touch was hard and thick. According to the experimental acne grading standard, the experimental acne model of the rat auricle has been formed. The epidermis of the auricle of the rats in the blank control group was normal in color, the capillaries were clearly visible, and the touch was soft and thin; the epidermis of the auricle of the rats in the model group was rough and dry, the skin was red and swollen, the capillaries were invisible, and the touch was hard and thick.

[0082] Grouping and administration: Several rats with successful modeling were randomly divided into a model control group and a model administration group, with 6 rats in each group, half of which were male and half were female; among them, the model control group was not administered; one model administration group was selected to apply 0.1% retinoic acid cream 0.1 g, once / d, for 14 consecutive days; the model administration group was selected to apply 0.2 g of the polypeptide composition gel of Example 1 and Comparative Examples 1-5, once / d, for 14 consecutive days.

[0083] ELISA was used to detect the levels of IL-1β, TNF-α and IL-6 in rat serum: after anesthesia, blood was collected from the ear vein and divided into centrifuge tubes. The tubes were allowed to stand for 30 min and then centrifuged at 1 000 r / min for 5 min. The supernatant was collected and divided into centrifuge tubes. The enzyme-linked immunosorbent assay (ELISA) method was used to detect the levels of IL-1β, TNF-α and IL-6 in the supernatant of cell culture in each group.

[0084] Table 20 Measurement results of ear swelling rate of rats in each group

[0085] The ear swelling rate measurement results of each group of rats showed that: compared with the blank control group, the auricles of the rats in the model group were obviously red and swollen, and the auricle swelling rate was significantly increased. The auricle swelling rate of the rats in the model medication group applied with the gel of the present invention was significantly lower than that in the model control group, which had a significant inhibitory effect on the inflammation of the acne model rats.

[0086] Table 21 Test results of serum IL-1β, TNF-α, and IL-6 levels in each group of rat auricle acne model (pg / mL)

[0087] The test results of the inflammatory factor content in the serum of rats in each group showed that: compared with the blank control group, the inflammatory factors in the serum of rats in the model group were significantly increased, and the IL-1β, TNF-α, and IL-6 in the serum of rats in the model control group were significantly higher than those in the blank control group; in the rat model administration group given the gel of Example 1 of the present invention, the IL-1β, TNF-α, and IL-6 were significantly reduced compared with the model control group not administered, which had a significant inhibitory effect on the inflammation of acne model rats.

[0088] It is explained here that the embodiments of the present invention are only used to illustrate the present invention, rather than to limit the present invention. Therefore, simple improvements to the present invention based on the method of the present invention are all within the scope of protection required by the present invention.

Claims

1. A new application of acetyl dipeptide-1, characterized in that: Application in the preparation of medicines, health products and cosmetics for treating skin inflammatory diseases.

2. The use according to claim 1, characterized in that The skin inflammatory related diseases are skin diseases or conditions such as acne, atopic dermatitis, eczema, rash, urticaria, itching, eczema outbreaks, eosinophilic dermatosis, reactive neutrophilic dermatosis, immune bullous dermatosis, cutaneous fibrohistiocyte proliferation, and keloid.

3. The use according to claim 1, characterized in that The skin inflammation-related disease is acne.

4. A polypeptide composition for treating skin inflammatory diseases and repairing skin, characterized in that: The polypeptide composition comprises a composite peptide composed of acetyl dipeptide-1 and tripeptide-1 copper; further, a hydroxypropyl-β-cyclodextrin-chitosan complex is added to the composition to form an inclusion compound.

5. The polypeptide composition according to claim 4, characterized in that: The mass ratio of acetyl dipeptide-1 to tripeptide-1 in the composite peptide of the composition is 1:1-5.

6. The polypeptide composition according to claim 4, characterized in that: The mass ratio of the composite peptide to the hydroxypropyl-β-cyclodextrin-chitosan complex in the composition is 1:1-10.

7. The polypeptide composition according to claim 4, characterized in that: The mass ratio of hydroxypropyl-β-cyclodextrin to chitosan in the composite is 1:0.1~1.

8. A method for preparing the polypeptide composition according to claim 4, characterized in that: The preparation method comprises the following steps: Step 1: Add the prescribed amount of complex peptide and an appropriate amount of purified water into a container, stir at room temperature to dissolve, and prepare a complex peptide solution; Step 2: Weigh the prescribed amount of hydroxypropyl-β-cyclodextrin and chitosan, add appropriate amount of purified water, heat the mixed solution to 60-70°C, add acetic acid dropwise while stirring to adjust the pH value to 4-6, prepare a hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution, and cool to room temperature for use; Step 3: Slowly add the complex peptide solution obtained in step 1 to the hydroxypropyl-β-cyclodextrin-chitosan complex aqueous solution while stirring, mix thoroughly, let stand, centrifuge the mixture, take the supernatant filter to filter out the precipitate, and freeze-dry to obtain the inclusion polypeptide composition.

9. Use of the polypeptide composition according to claim 4 in the preparation of a drug for treating skin inflammatory diseases, characterized in that: A pharmaceutically effective amount of the polypeptide composition is combined with a pharmaceutically acceptable excipient to prepare any pharmaceutically acceptable external preparation, wherein the external preparation is one of a gel, cream, lotion, facial cream, ointment, paste, lotion, patch, dressing, film coating, papule, aerosol or spray.

10. Use of the polypeptide composition according to claim 9 in the preparation of a drug for treating skin inflammatory diseases, characterized in that: The external preparation is a gel, which comprises a polypeptide composition and a pharmaceutically acceptable gel excipient, wherein the gel excipient is selected from one or more of a gel matrix, a moisturizer, an antioxidant, an antibacterial agent, a pH regulator, and an aqueous solvent.

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