Use of polypeptides in the preparation of skin care products

Abstract

The application provides application of a polypeptide in preparation of skin repair products. It is found that the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 can significantly promote the scratch healing of human immortalized epidermal keratinocytes (HaCaT), and then effectively repair the skin barrier, has excellent skin repair efficacy, and is suitable for preparation of a human epidermal cell scratch repair promoter, a skin barrier repair product and a skin repair product.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology. More specifically, it relates to the application of peptides in the preparation of skin repair products. Background Technology

[0002] Skin barrier damage is an abnormal state of skin barrier function characterized by disruption of the stratum corneum structure, loss of the sebum film, increased transepidermal water loss, and decreased skin tolerance. This makes the skin prone to problems such as erythema, itching, stinging, peeling, and recurrent sensitivity when exposed to external stimuli (e.g., ultraviolet radiation, pollutants, temperature changes, cosmetic ingredients), severely impacting the patient's appearance and quality of life. Its occurrence is usually the result of multiple factors, including age-related physiological skin degeneration, environmental aggressors (e.g., over-cleansing, frequent exfoliation, prolonged sun exposure), genetic factors (e.g., atopic constitution), poor skincare habits (e.g., misuse of steroid creams, use of highly irritating products), and the effects of certain diseases and medications (e.g., eczema, rosacea, and long-term use of retinoids).

[0003] Currently, the main methods used for skin repair are: (1) Drug treatment: Symptomatic treatment is often used, such as using weak glucocorticoid ointment to reduce inflammation during the acute phase of inflammation, oral antihistamines to relieve discomfort for those with obvious itching, and topical antibiotic ointment when secondary bacterial infection occurs; however, this symptomatic treatment is difficult to repair the damaged skin barrier structure, and long-term or improper use of hormones may lead to serious side effects such as skin atrophy, telangiectasia and hormone-dependent dermatitis; (2) Cosmetic repair: by using products containing physiological lipids such as ceramides, cholesterol, and fatty acids to supplement intercellular lipids, reduce neurogenic inflammation, and promote orderly collagen regeneration, thereby assisting in the restoration of barrier function; however, most commercially available products are difficult to achieve clinical-level repair depth, and drug intervention is still required for acute severe barrier damage; (3) Traditional Chinese medicine treatment: based on the principle of syndrome differentiation and treatment, traditional Chinese medicines that clear heat and dampness, cool blood and detoxify, or nourish blood and moisten dryness are used for internal or external use to regulate the body's overall condition, but the components of traditional Chinese medicine are complex and the effect is relatively slow, and some topical preparations may cause contact allergies, and quality control is difficult. There are also certain difficulties in the preparation; (4) Physical therapy: such as red light therapy, yellow light therapy, soothing and infusion device, etc., which utilize the photothermal effect or ionization osmosis principle of specific wavelengths to help reduce inflammatory response, promote microcirculation and absorption of nutrients. However, physical therapy usually needs to be performed multiple times according to the course of treatment, and each treatment takes a long time. Moreover, for patients with severe barrier damage, the effect of physical therapy alone is limited; (5) Surgical treatment: mainly for severe scar hyperplasia or vascular hyperplasia caused by barrier damage, laser abrasion or surgical excision can be used. However, the surgical trauma is large, and there is a risk of pigmentation, infection and scar recurrence after surgery. The recovery period is long and it cannot fundamentally improve the sensitive nature of the skin; (6) Basic care: including avoiding excessive cleaning, simplifying skin care, strengthening physical sun protection, and using gentle moisturizers. This is the foundation of all skin repair programs. However, this method is highly dependent on the patient's compliance, and the effect is slow. Moreover, for patients in the acute inflammatory period or with severe barrier dysfunction, basic care alone is often difficult to block the inflammatory cascade reaction and cannot quickly relieve symptoms.

[0004] Peptides are a class of bioactive molecules composed of multiple α-amino acids linked by peptide bonds. They have attracted much attention in the field of dermatological pharmaceuticals and cosmetics due to their significant advantages such as good biocompatibility, low toxicity and side effects, strong targeting, rapid onset of action, diverse physiological activities and low tendency to cause drug resistance. However, the types and applications of peptides that can effectively repair the skin are still relatively scarce. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing the application of polypeptides (amino acid sequences as shown in SEQ ID NO:1) in the preparation of skin repair products. By significantly promoting scratch healing of human immortalized epidermal keratinocytes (HaCaT), it effectively repairs the skin barrier and has excellent skin repair efficacy. It is suitable for the preparation of human epidermal cell scratch repair promoters, skin barrier repair products, and skin repair products.

[0006] The primary objective of this invention is to provide the application of polypeptides in the preparation of skin repair products.

[0007] A second objective of this invention is to provide the application of polypeptide-related biomaterials in the preparation of skin repair products.

[0008] A third objective of this invention is to provide the application of the polypeptide in the preparation of a human epidermal cell scratch repair promoter.

[0009] A fourth objective of this invention is to provide the application of peptides in the preparation of skin barrier repair products.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention discovers that a polypeptide with the amino acid sequence shown in SEQ ID NO:1 can significantly promote scratch healing of human immortalized epidermal keratinocytes (HaCaT), thereby effectively repairing the skin barrier and exhibiting excellent skin repair efficacy. It is suitable for preparing human epidermal cell scratch repair promoters, skin barrier repair products, and skin repair products. Therefore, this invention provides the application of the polypeptide with the amino acid sequence shown in SEQ ID NO:1 and related biomaterials in the preparation of skin repair products, human epidermal cell scratch repair promoters, and skin barrier repair products.

[0011] Preferably, the relevant biological material is a nucleic acid molecule capable of expressing the polypeptide, or an expression cassette, recombinant plasmid, recombinant bacteria, or recombinant cell containing the nucleic acid molecule.

[0012] Preferably, the product is a pharmaceutical and / or cosmetic.

[0013] More preferably, the drug is one or more of the following: oral liquid, tincture, tincture, aerosol, powder, injection, sterile powder for injection, and suppository. The types of the above preparations can be understood according to the relevant definitions in *Pharmaceutics* (6th Edition, People's Medical Publishing House, Cui Fude), and the preparation of the above preparations can be carried out according to the relevant preparation methods in *Pharmaceutics* (6th Edition, People's Medical Publishing House, Cui Fude).

[0014] More preferably, the drug further contains pharmaceutically acceptable excipients.

[0015] Furthermore, the excipients include emulsifiers (such as Tween and / or polyoxyethylene castor oil, used to reduce interfacial tension, promote emulsification of the dispersed phase, and maintain the stability of the emulsion), wetting agents (such as sodium lauryl sulfate, used to help the pharmaceutical ingredients disperse and dissolve better), colorants (used to improve the appearance and color of the pharmaceutical product, making it easier to identify and distinguish), solid lubricants (such as stearic acid and / or magnesium stearate, used to reduce friction and ensure successful pharmaceutical manufacturing), alginic acid (to maintain the physical stability of the pharmaceutical product and prevent the pharmaceutical particles from settling or agglomerating), and flavoring agents (used to improve the taste of the pharmaceutical product). The following are some of the ingredients used in traditional Chinese medicine: (1) To improve patient adherence to medication; (2) Stabilizers (to increase the physical stability of drugs and prevent them from deteriorating or degrading during storage or transportation); (3) Antioxidants (to prevent drugs from deteriorating due to oxidation during storage or use and extend their shelf life); (4) Compressed tablets (to compress loose granular substances into solid tablets, making them convenient for patients to take and carry, and also improving drug stability and bioavailability); (5) Isotonic salt solutions (to adjust the osmotic pressure of drugs to meet human physiological requirements, thereby reducing irritation and adverse reactions); (6) Vegetable oils (such as flower oils). Oils such as vegetable oil, cottonseed oil, sesame oil, olive oil, corn oil, and / or cocoa butter are used as solvents or carriers to help drug ingredients dissolve or disperse better; polyols (such as propylene glycol, glycerin, sorbitol, mannitol, and / or polyethylene glycol) are used to improve drug stability, prevent drug dehydration or deterioration during storage, and also to improve drug taste and solubility; phosphate buffers are used to adjust the pH of drugs to meet human physiological requirements, thereby reducing irritation and adverse reactions; and starches (such as corn starch and / or potato starch) are used to help tablets dissolve or disperse. The excipients are selected from one or more of the following: sugars (such as lactose, glucose, and / or sucrose, which help the drug components disperse and dissolve better, while providing necessary energy support); binders (such as gelatin, which help the drug components bind better); lubricants (such as talc, which reduce friction); and cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and / or methyl cellulose, which improve the compressibility and moldability of the drug, and also control the release rate). The type of excipient can be selected based on the need to improve the stability, activity, and / or bioavailability of the drug.

[0016] More preferably, the cosmetic is an aqueous solution, hydrogel, oil solution, oleogel, emulsion, cream, suspension, powder, compressed powder, stick, aerosol, foam, or film.

[0017] More preferably, the cosmetic also contains cosmetically acceptable excipients.

[0018] Furthermore, the excipients are solid lubricants (such as stearic acid, magnesium stearate, and / or cetearyl alcohol, used to reduce shear resistance, improve material spreadability, and ensure smooth emulsification and filling processes), vegetable oils (such as jojoba oil, olive oil, sweet almond oil, grapeseed oil, and / or shea butter, used as oil phase solvents or emollient carriers to promote the penetration and dispersion of active ingredients), polyols (such as glycerin, butylene glycol, propylene glycol, 1,3-propanediol, and / or sorbitol, used as humectants and low-temperature antifreeze agents to maintain the product's moisture content and improve skin feel and solubility), and gelling materials (such as xanthan gum, carbomer, hydroxyethyl cellulose, and / or sodium alginate, used to construct a three-dimensional network structure and maintain the product's rheological properties and physical stability). The ingredients include: emulsifiers (such as cetearyl glucoside, polysorbate-60, Span-80 and / or PEG-100 stearate, used to reduce the interfacial tension between oil and water and form a stable O / W or W / O emulsion), wetting agents (such as disodium lauryl sulfosuccinate, lecithin, etc., used to improve the wetting and dispersion of powders and oil phases in the aqueous phase), colorants (such as iron oxides, titanium dioxide, carmine and / or food colorings, used to give products a specific hue, enhance visual appeal and product recognition), fragrances (such as rose essential oil, menthol, citral and / or synthetic fragrances, used to mask the odor of raw materials, provide a pleasant sensory experience, and improve user compliance), and fillers (such as lye). Ingredients include: boron nitride, silica, and / or kaolin, used to absorb oils, improve the compactness and smoothness of powders, and facilitate molding; stabilizers (such as disodium EDTA and sodium citrate, used to chelate metal ions to prevent product oxidation and discoloration and system demulsification); antioxidants (such as vitamin E, BHT, BHA, and / or propyl gallate, used to block free radical chain reactions and delay oil rancidity and active ingredient oxidation); pH adjusters (such as triethanolamine, citric acid, sodium hydroxide, and / or arginine, used to precisely control the product's pH to ensure it remains within the skin's tolerance range and maintains ingredient activity); and sugars (such as trehalose, mannose, xylitol, and / or sucrose, used as natural moisturizing factors and humectants to protect the skin). The product contains one or more of the following: cell membrane structure (providing a moisturizing feel), starch and its derivatives (such as corn starch, modified starch, etc., used to absorb excess facial oil, provide a soft-focus matte effect, and assist in powder forming), high molecular polymers (such as polyvinylpyrrolidone, acrylate copolymers, polydimethylsiloxane / vinyl dimethylsiloxane cross-linked polymers, etc., used to adjust the thixotropy, film-forming properties and setting ability of the product), thickeners (such as xanthan gum, magnesium aluminum silicate, etc., used to increase the viscosity of continuous phases, suspend solid particles, and prevent sedimentation), and preservatives (such as phenoxyethanol, potassium sorbate, chlorophenoxyethanol and / or octyl glycol, etc., which prevent product contamination by microorganisms and extend shelf life by destroying the cell structure of microorganisms or inhibiting their enzyme activity).The type of excipient can be selected based on the needs of improving product stability, skin feel, safety, and efficacy.

[0019] The present invention has the following beneficial effects: This invention discovers that a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 can significantly promote scratch healing of human immortalized epidermal keratinocytes (HaCaT), thereby effectively repairing the skin barrier and exhibiting excellent skin repair efficacy. It is suitable for preparing human epidermal cell scratch repair promoters, skin barrier repair products, and skin repair products. Attached Figure Description

[0020] Figure 1 The results show the toxicity test results of the peptide on HaCaT cells.

[0021] Figure 2 The scratch photograph is taken at 0 h, which is the negative control group.

[0022] Figure 3 The scratch image is taken at 22 hours for the negative control group.

[0023] Figure 4 This is a photograph of the scratches on sample group at time 0.

[0024] Figure 5 This is a photograph of the scratches on the sample group at 22 hours. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0026] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0027] Example 1 Synthesis of Polypeptides The company was commissioned to synthesize a polypeptide (amino acid sequence as shown in SEQ ID NO:1: GLDWWQL).

[0028] Example 2: The polypeptide is non-toxic to cells. Sample solution: The polypeptide obtained in Example 1 was dissolved in a solution containing 3% ( v / v Sample solutions with concentrations of 0.005 wt%, 0.004 wt%, 0.003 wt%, 0.002 wt%, and 0.001 wt% were obtained in MEM complete culture medium containing fetal bovine serum.

[0029] Immortalized human epidermal keratinocytes (HaCaT) cultured to the logarithmic growth phase were used at a concentration of 1 × 10⁻⁶. 4The culture medium was seeded at a density of 100 cells / well in 96-well plates and incubated at 37 °C and 5% CO2 for 24 h. The original culture medium was then discarded. 200 μL of sample solution was added to the sample group, and 200 μL of solution containing 3% CO2 was added to the negative control group (NC). v / v Fetal bovine serum was cultured in MEM complete medium. Both groups were cultured at 37℃ and 5% CO2 for 24 h. The original medium was then discarded, and 220 μL of CCK-8 working solution (20 μL CCK8 solution + 200 μL MEM complete medium) was added. After incubation at 37℃ and 5% CO2 for 2 h, the OD values ​​of each group were measured using a microplate reader. 450 The value is calculated according to the formula "Cell viability (%) = OD". 样品组 / OD 阴性对照组 Cell viability was calculated using the formula "×100%" to reflect the toxicity of the peptide to HaCaT cells.

[0030] The results are as follows Figure 1 As shown, compared with the negative control group, there was no significant difference in cell survival rate among the sample groups with added 0.001wt% to 0.005wt% polypeptide, indicating that the polypeptide of the present invention is non-toxic to HaCaT cells in the concentration range of 0.001wt% to 0.005wt% and has high safety.

[0031] Example 3: The polypeptide has skin repair effects This study used immortalized human epidermal keratinocytes (HaCaT) as an in vitro evaluation model, with cell migration as the core target. The specific mechanism of action is as follows: Epidermal keratinocytes are the main constituent cells of the epidermis. They are not only the core building blocks of the skin's physical barrier but also participate in the healing regulation process after skin injury. Their proliferation and migration capabilities directly affect the repair efficiency of micro-damage and the integrity of the barrier. Under normal conditions, cells form the epidermal defense line through tight junctions, maintaining the skin's barrier and hydration. When the skin is stimulated by external factors or the barrier is damaged, cells migrate, proliferate, and rebuild tight junction structures to achieve wound healing and barrier repair. Insufficient migration ability will delay repair and cause problems such as dryness and sensitivity. When the sample acts on the cells, if it has repair activity, it can significantly enhance cell migration ability by regulating intracellular PI3K / Akt, MAPK, and other related signaling pathways. At the same time, it promotes gene transcription and protein synthesis of barrier-related proteins such as keratin and tight junction proteins, enhancing the tightness of intercellular connections. This process not only quickly closes minor skin damage but also rebuilds the complete epidermal physical barrier, enhancing the skin's water retention capacity and resistance to external stimuli, thereby achieving skin repair. This embodiment uses a scratch assay to quantitatively detect the scratch healing rate of human immortalized epidermal keratinocytes (HaCaT), clarifying the regulatory effect of the sample on the repair function of human immortalized epidermal keratinocytes, thus scientifically evaluating its in vitro repair efficacy.

[0032] Sample solution: The polypeptide obtained in Example 1 was dissolved in a solution containing 3% ( v / v A sample solution with a concentration of 0.005 wt% was obtained by culturing fetal bovine serum in MEM complete medium.

[0033] Immortalized human epidermal keratinocytes (HaCaT) cultured to the logarithmic growth phase were used at 2 × 10⁻⁶. 5 Seeds were planted at a density of cells / well in 12-well plates and incubated at 37 ℃ and 5% CO2 for 24 h. After scratching, 2 mL of sample solution was added to the sample group, and 2 mL of solution containing 3% CO2 was added to the negative control group (NC). v / v Fetal bovine serum was cultured in MEM complete medium. Immediately afterward, the scratched area was photographed (considered the result at h0). The culture was continued at 37 ℃ and 5% CO2 for 22 h, and a photograph of the scratched area was taken at h22. The scratch photographs are shown below. Figures 2-5 As shown, where, Figure 2 This is a photograph of the scratches taken at 0 h in the negative control group. Figure 3 This is a photograph of the scratches taken at 22 hours in the negative control group. Figure 4 This is a photograph of the scratches on the sample group at time 0. Figure 5The image shows the scratches in the sample group at 22 hours. Image analysis was performed using ImageJ software, and the results were analyzed according to the formula: "Scratch healing rate (%) = (A0 - A0) / ... 22 ) / A0×100%" where A0 represents the area of ​​the scratched region at time 0 h, A 22 The scratch healing rate is calculated by taking the area of ​​the scratched region at 22 hours.

[0034] The results showed that the scratch healing rate of the negative control group was 63.0%±2.2%, while that of the sample group was 88.3%±3.5%. That is, the scratch healing rate of the sample group was significantly higher than that of the negative control group, indicating that the polypeptide of the present invention can effectively repair the skin barrier and has excellent skin repair effects. It is suitable for preparing human epidermal cell scratch repair promoters, skin barrier repair products and skin repair products.

[0035] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of polypeptides in the preparation of skin repair products, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The application of peptide-related biomaterials in the preparation of skin repair products, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

3. The application according to claim 2, characterized in that, The relevant biological material is a nucleic acid molecule that can express the polypeptide, or an expression cassette, recombinant plasmid, recombinant bacteria, or recombinant cell containing the nucleic acid molecule.

4. The application according to claim 1 or 2, characterized in that, The products mentioned are pharmaceuticals and / or cosmetics.

5. The application according to claim 4, characterized in that, The medicine is one or more of the following: oral liquid, tincture, aerosol, powder, injection, sterile powder for injection, and suppository.

6. The application according to claim 4, characterized in that, The drug also contains pharmaceutically acceptable excipients.

7. The application according to claim 4, characterized in that, The cosmetics mentioned are aqueous solutions, hydrogels, oil solutions, oleogels, emulsions, creams, suspensions, powders, compressed powder cakes, sticks, aerosols, foams, and films.

8. The application according to claim 4, characterized in that, The cosmetic also contains cosmetically acceptable excipients.

9. The application of polypeptides in the preparation of human epidermal cell scratch repair promoters, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

10. The application of peptides in the preparation of skin barrier repair products, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

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