Use of polypeptide-skin in37 and skincare product comprising said polypeptide

Abstract

When the polypeptide in37 of the present invention is applied to the skin as a skin-protecting agent, it is safe and has significant skin-repairing and skin-soothing effects.SOLUTION: The present invention relates to the field of polypeptide skincare, and particularly discloses the use of polypeptide in37 and a skincare product comprising the polypeptide, i.e., the use of polypeptide in37 in any one of (1) the preparation of a composition having a skin repair effect, (2) the preparation of a composition having a skin soothing effect, and (3) a cream product, an emulsion, a gel, a serum, a lotion, a shampoo, a body wash, a pack, and a freeze-dried product. Polypeptide in37 is a chemically synthesized polypeptide with a molecular weight of more than 99%, which is composed of 13 amino acids, has a molecular weight of 1711Da, is lower than the molecular weight of 2000Da, and is suitable for skin absorption.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of polypeptide skin care, and in particular to the use of polypeptide-in37 and skin care products containing said polypeptide. [Background technology]

[0002] Sensitive skin has a significant impact on people's quality of life. Sensitive skin typically presents with skin allergy symptoms and discomfort, such as redness, itching, dryness, stinging, burning, and tightness. While it is difficult to identify specific allergens, the common thread is that the skin barrier is damaged, meaning the surface of the skin loses its normal protective function and moisture, resulting in dry, scaling, or itchy skin. Furthermore, allergens on the surface of the skin, such as pathogens, pollen, and chemicals, can easily penetrate the interior of the skin through the damaged skin barrier, further triggering inflammatory reactions in the skin.

[0003] However, conventional skin care products for alleviating skin sensitivity problems often contain medicinal ingredients, which have drawbacks such as difficulty in controlling the amount used, the possibility of causing discomfort to some people, and insufficient effectiveness, preventing them from being widely used in everyday cosmetics and skin care products.

[0004] Polypeptides are compounds formed by the linking of amino acids through peptide bonds, and are also intermediate products of protein hydrolysis. They are widely involved in the vital activities of the human body, and are safe and reliable substances with clear effects, appropriate activity, well-defined structure, etc. Due to the advantages of polypeptides, such as high safety and strong biological activity, more and more polypeptides are being widely used in the development of cosmetics.

[0005] The applicant filed PCT application PCT / RU2018 / 000037 on January 29, 2018, entered the Chinese national phase on May 22, 2019, and was granted a patent on June 2, 2023. The application number is 201880003993.4. The proposal discloses a total of 16 polypeptide molecules, all of which have antibacterial and antiviral properties. Polypeptide-in37 in the present application is the sixth molecular formula sequence in the proposal. Summary of the Invention [Means for solving the problem]

[0006] One of the objects of the present invention is to provide a method for the preparation of polypeptide-in37, (1) Preparation of a composition having a skin repair effect, (2) Preparation of a composition having a skin soothing effect; (3) Providing for use in any one of cream products, emulsions, gels, serums, lotions, shampoos, body washes, packs, and freeze-dried products; The molecular formula of the polypeptide-in37 is H-Ile-Leu-Pro-D-Phe-Lys-D-Phe-Pro-D-Phe-D-Phe-Pro-D-Phe-Arg-Arg-NH2.

[0007] Polypeptide-in37 is a chemically synthesized polypeptide with a purity of over 99% using current synthesis technology. It was independently developed by the applicant and is composed of 13 amino acids. Its molecular weight is 1711 Da, which is lower than 2000 Da, making it suitable for absorption by the skin.

[0008] Through numerous experiments, the applicant has found that polypeptide-IN37 has novel effects, and when applied to the skin, it is safe and has significant skin repair and soothing functions, and has passed a large number of efficacy and toxicity tests with satisfactory results, making it a valuable raw material for cosmetics.

[0009] With this in mind, we are filing patent applications in the field of cosmetic ingredients to protect future commercial development of our products.

[0010] The specific molecular structure is as follows: [ka]

[0011] Preferably, the concentration of polypeptide-in37 is 0 to 0.0391 mg / mL and exceeds 0.

[0012] Preferably, the concentration of polypeptide-in37 in (1) is 0.0156 to 0.0313 mg / mL.

[0013] Preferably, the concentration of polypeptide-in37 in (2) is 0.00049 to 0.0391 mg / mL.

[0014] Preferably, the form of polypeptide-in37 used in the production of cream products, emulsions, gels, serums, lotions, shampoos, body washes, packs, and freeze-dried products in (3) includes granular, powdery, or water-soluble liquid forms.

[0015] A skin care product, the ingredients of which include polypeptide-in37, wherein the weight of the polypeptide-in37 accounts for 0.005 to 2% of the total weight of the skin care product.

[0016] Preferably, the skin care product comprises an aqueous phase material, an oil phase material, and a thickener.

[0017] The aqueous phase contains polypeptide-in37, glycerin, sorbitol, hydroxyethylcellulose, and purified water, and the water-soluble active ingredient is at least one of hyaluronic acid, ceramide, collagen, niacinamide, carnosine, acetyl hexapeptide, acetyl tetrapeptide, hydroxypropyltetrahydropyrantriol, and sodium alginate.

[0018] The collagen is water-soluble collagen, its molecular weight is 6000, and it is AR grade, and it is purchased from Sichuan Yongyang Biotechnology Co., Ltd.

[0019] The oil phase material comprises at least one of olive oil, almond oil, milk tree oil, palm oil, jojoba oil, lanolin, avocado oil, and vitamin E. The thickening agent includes at least one of spermaceti, potassium laurate, and cocoa butter.

[0020] Preferably, the ingredients of the skin care product include, by mass, 0.005 to 2% polypeptide-in37, 0.01 to 18% water-soluble active ingredient, 1 to 20% glycerin, 1 to 20% sorbitol, 1 to 20% hydroxyethyl cellulose, 0.1 to 80% oil phase material, 0.1 to 30% thickener, and the remainder is water.

[0021] Preferably, the skin care product is at least one of a cream product, a milky lotion, a gel, a serum, a lotion, a shampoo, a body wash, a mask, and a freeze-dried product.

[0022] The method for producing the skin care product includes: (1) adding polypeptide-in37, a water-soluble active ingredient, glycerin, sorbitol, and hydroxyethyl cellulose to water and heating and stirring (stirring at 40 to 80°C for 2 to 8 hours); (2) heating and stirring the oil phase material (stirring at 40 to 80°C for 2 to 8 hours); (3) continuously stirring the aqueous phase material, adding the oil phase material during the stirring process, and homogeneously emulsifying until the two phases are completely emulsified; (4) Adding a thickener and continuing to emulsify homogeneously, heating and stirring until homogeneous (stirring at 40-80°C for 2-8 hours), leaving to stand for 1-5 hours, and molding and stabilizing the mixture.

[0023] The present application has conducted further research on the conventional polypeptide-in37 and discovered a novel use thereof, namely, that polypeptide-in37 has skin repair and skin soothing functions and can be used to manufacture compositions having skin repair and / or skin soothing effects, as well as to manufacture cream products, emulsions, gels, serums, lotions, shampoos, body washes, packs, freeze-dried products, etc. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 is a graph comparing cell viability among groups in Example 2. [Figure 2] This is a comparison diagram of the cell fusion rate of each group in Example 2. In the comparison between the NC group and the BC group, significance is represented by #, P<0.05 is represented by #, and P<0.01 is represented by ##. In the comparison between the PC group, the sample group, and the NC group, significance is represented by *, P<0.05 is represented by *, and P<0.01 is represented by **. [Figure 3] 1 shows comparative photographs of cell migration in each group in Example 2. [Figure 4] FIG. 10 is a comparison diagram of the results of LOR immunofluorescence staining for each group in Example 3. [Figure 5] FIG. 10 is a comparison diagram of the results of FLG immunofluorescence staining for each group in Example 3. [Figure 6] This is a comparison diagram of the LOR relative integrated optical density (IOD) values ​​of each group in Example 3. In the comparison between the NC group and the BC group, significance is represented by #, P<0.05 is represented by #, and P<0.01 is represented by ##, and in the comparison between the PC group, the sample group, and the NC group, significance is represented by *, P<0.05 is represented by *, and P<0.01 is represented by **. [Figure 7] This is a comparison diagram of the FLG relative integrated optical density (IOD) values ​​of each group in Example 3. In the comparison between the NC group and the BC group, significance is represented by #, P<0.05 is represented by #, and P<0.01 is represented by ##. In the comparison between the PC group, the sample group, and the NC group, significance is represented by *, P<0.05 is represented by *, and P<0.01 is represented by **. [Figure 8] FIG. 10 is a graph comparing cell viability among groups in Example 4. [Figure 9]This is a graph comparing the mast cell degranulation rates of each group in Example 4. In the comparison between the NC group and the BC group, significance is indicated by #, with P<0.05 being indicated by # and P<0.01 being indicated by ##, and in the comparison between the PC group, the sample group and the NC group, significance is indicated by *, with P<0.05 being indicated by * and P<0.01 being indicated by **. [Figure 10] 1 shows photographs comparing the morphology of mast cell degranulation in each group in Example 4. [Figure 11] FIG. 10 is a graph comparing cell viability among groups in Example 5. [Figure 12] This is a comparison diagram of the TNF-α content of each group in Example 5. In the comparison between the NC group and the BC group, significance is represented by #, with P<0.05 being represented by # and P<0.01 being represented by ##, and in the comparison between the PC group, the sample group and the NC group, significance is represented by *, with P<0.05 being represented by * and P<0.01 being represented by **. [Figure 13] This is a diagram comparing the IL-6 content of each group in Example 5. In the comparison between the NC group and the BC group, significance is indicated by #, with P<0.05 being indicated by # and P<0.01 being indicated by ##, and in the comparison between the PC group, the sample group and the NC group, significance is indicated by *, with P<0.05 being indicated by * and P<0.01 being indicated by **. [Figure 14] This is a diagram comparing the IL-1α content of each group in Example 5. In the comparison between the NC group and the BC group, significance is indicated by #, with P<0.05 being indicated by # and P<0.01 being indicated by ##, and in the comparison between the PC group, the sample group and the NC group, significance is indicated by *, with P<0.05 being indicated by * and P<0.01 being indicated by **. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will now be further described with reference to specific examples.

[0026] Example 1: Safety verification

[0027] 1. Based on the "Cosmetics Safety Technical Standards" (2015 edition), the safety verification of powdered polypeptide-in37 was conducted and the results are as follows: (1) Microbial measurement: The total number of colonies, mold and yeast counts were all <10 CFU / g, and no heat-resistant coliforms, Staphylococcus aureus, or Pseudomonas aeruginosa were detected. (2) Physical and chemical determination: mercury content <0.002mg / kg, lead content <1.5mg / kg, arsenic content <0.01mg / kg, cadmium content <0.18mg / kg.

[0028] 2. A toxicity test was conducted based on the bacterial recovery mutation test in Chapter 6, Section 8 of the "Cosmetics Safety Technical Standards" (2015 edition). After measuring polypeptide-in37 in five test strains, the results were negative both with and without S9, and it was determined to be mutagenic.

[0029] 3. Skin irritation tests were conducted based on the multiple skin irritation tests in Chapter 6, Section 4 of the "Cosmetics Safety Technical Standards" (2015 edition).

[0030] 3.1 Experimental animals: New Zealand white rabbits; grade: normal grade; quantity: 4; gender: female; weight: 2.158 kg - 2.339 kg; supplier: Zedunshan Animal Breeding Farm Co., Ltd., Songjiang District, Shanghai; Experimental Animal Production Permit Number: SCXK (Shanghai) 2022-0001; Quality Approval Number: 20220001001735.

[0031] 3.2 Breeding environment: Standard-grade animal room, room number: 322, breeding room temperature 16~26℃, relative humidity 40~70%, experimental animal use permit number: SYXK (Shanghai) 2021-0023.

[0032] 3.3 Feed supplier: Shanghai Zhouyu Biotechnology Co., Ltd., production permit number: Shanghai Feed Permit (2021) 04027, no restrictions on drinking water.

[0033] 3.4 Test Method

[0034] 3.4.1 Before the test, the animals should be allowed to adapt to the environment of the laboratory animal room for 3 days to ensure that the animals entering the group are healthy and have no skin damage.

[0035] 3.4.2 Approximately 24 hours before the test, shave both sides of the dorsal spine of the experimental animals, leaving a hair removal area of ​​approximately 3cm x 3cm on the left and right sides, without damaging the skin, and the application area is 2.5cm x 2.5cm.

[0036] 3.4.3 The next day, after weighing, 0.5 g of the test substance (polypeptide-in37) was thoroughly wetted with distilled water and applied to the left side of the skin. The skin was then covered with two layers of gauze (2.5 cm x 2.5 cm) and one layer of cellophane, and secured with non-irritating tape and bandages. The right side served as the blank control. The test substance was applied once a day for 14 consecutive days.

[0037] 3.4.4 Clinical observation: From the second day onwards, the residual test substance was removed with warm water and the results were observed after 1 hour.

[0038] 3.4.5 Judgment criteria and scoring: In accordance with the result evaluation of 5.4.3 of the skin irritation / corrosion test in Chapter 6, Section 4 of the "Cosmetics Safety Technical Standards" (2015 edition), multiple skin irritation response scoring was performed, and the erythema and edema of the sample and control were observed and scored. The average cumulative score for each animal was calculated daily to determine the severity of skin irritation.

[0039] 3.5 Test results Table 1-1. Results of multiple skin irritation tests on New Zealand white rabbits [Table 1-1] NOTE: The mean cumulative score per animal each day (skin irritation index) = total score of erythema and edema over 14 days per animal / (number of animals tested x number of days of multiple skin test irritation).

[0040] In summary, polypeptide-in37 is non-irritating to New Zealand White rabbits after multiple skin irritation tests.

[0041] 4. Skin allergy test was conducted based on the skin allergy test in Chapter 6, Section 6 of the "Cosmetics Safety Technical Standards" (2015 edition).

[0042] 1-Chloro-2,4-dinitrobenzene (manufactured by Tokyo Chemical Industry Co., Ltd., lot number WT6CA-LI) was used as the positive substance. Acetone (manufactured by Sinopharm Chemical Reagent Co., Ltd., lot number 20211022) was used as the solvent. The induction concentration of the positive substance was 0.8%, the induction concentration was 0.2%, and the dosage was 0.2 mL / animal for both.

[0043] 4.1 Experimental animals: Albino guinea pigs; grade: ordinary grade; quantity: 20 animals in the test substance group, 10 animals in the negative control group, 20 animals in the positive control group, body weight: 224 - 295 g. Supplier: Shanghai Songjiang Chedun Animal Breeding Farm Co., Ltd., experimental animal production license number: SCXK(Shanghai)2022 - 0001; quality certificate number: 20220001001734

[0044] 4.2 Breeding environment: Ordinary grade animal room, room number: 327, breeding room temperature 18 - 29 °C, relative humidity 40 - 70%, experimental animal use license number: SYXK(Shanghai)2021 - 0023.

[0045] 4.3 Feed supplier: Wuhan Wanqian Jiaxing Biotechnology Co., Ltd., production license number: SCXK(E)2021 - 0011. There is no restriction on drinking water.

[0046] 4.4 Litter supplier: Pichou Xiaohe Technology Development Co., Ltd., production license number: Su Si Yu (2021) 03006.

[0047] 4.5 Test method

[0048] 4.5.1 Before the test, the animals were adapted to the environment of the experimental animal room for 3 days to ensure the health of the animals in the group and no skin damage.

[0049] 4.5.2 Approximately 24 hours before the start of the test, depilation was performed on the left side of the back of the albino guinea pigs, and the depilation range was 4 - 6 cm 2 and.

[0050] 4.5.3 Induction contact: 0.2 g of the test substance at the induction concentration was thoroughly moistened with distilled water and applied to the left shaved skin (2 cm × 2 cm) of the experimental animals. The area was covered with two layers of gauze and one layer of cellophane, and then fixed with non-irritating tape for 6 hours. The same procedure was repeated on the 7th and 14th days.

[0051] 4.5.4 24 hours before the start of stimulation contact, the hair on the right side of the back of the albino guinea pig was removed. The hair removal area was 4-6 cm. 2 Let's say.

[0052] 4.5.5 Irritation contact: 14 days after the final induction, 0.2 g of the test substance at the induction concentration was taken, thoroughly moistened with distilled water, and applied to a 2 cm x 2 cm area of ​​the depilated skin on the right side of the experimental animals. The area was then covered with two layers of gauze and one layer of cellophane and fixed with non-irritating tape for 6 hours. Skin reactions were observed 24 hours and 48 hours after the end of the irritation contact.

[0053] 4.5.6 Negative control group: Distilled water was used as a control during induction contact, and the test substance was applied at an induction concentration during stimulation contact. The negative control group was treated in the same manner as the test group.

[0054] 4.5.7 Positive control group: At the time of induction contact, a 0.8% solution of 1-chloro-2,4-dinitrobenzene in acetone was applied, and at the time of irritation contact, a 0.2% solution of 1-chloro-2,4-dinitrobenzene in acetone was applied. The positive control group was treated in the same manner as the test group.

[0055] 4.5.8 Clinical Observation: Skin reactions were observed 24 and 48 hours after irritant contact.

[0056] 4.5.9 Judgment criteria and scoring: Skin allergy reactions were scored according to the evaluation method for the skin allergy reaction test, 5.1, local closed application test (Buehler Test, BT), in Chapter 6, Section 6 of the "Cosmetics Safety Technical Standards" (2015 edition). If a reaction score of ≥ 2 was observed in the test substance group, the animal was judged to have a positive skin allergy reaction, and the sensitization intensity of the test substance was graded according to the sensitization intensity control table. If the sensitization rate was 0%, no skin allergy reaction was observed.

[0057] 4.6 Test results Table 1-2 Albino guinea pig skin allergy reaction test results (BT method) [Table 1-2] Note: *1 Both starting and ending weights are expressed as mean ± SD. *2 In the skin reaction intensity column, if the skin reaction score is 0, 1, 2, 3..., enter the percentage of the number of animals that reacted to the number of test animals. Positive control test date: 2023-12-14~2024-01-13

[0058] From the above, no skin allergic reaction was observed in the skin allergic reaction test of polypeptide-in37 in albino guinea pigs.

[0059] Example 2 Repair effect test - keratinocyte migration ability test

[0060] 1. Purpose of this Example: Human immortalized keratinocytes (HaCaT) were used as the research subject, and the repair effect of the sample on skin barrier damage was evaluated by measuring the migration effect of the test sample on the cells. The human immortalized keratinocytes (HaCaT) used in this example were purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd., product number ZQ0044.

[0061] 2. Main reagents used in this example: high-glucose DMEM medium (Gibco), fetal bovine serum (Gibco), PBS (VivaCell), MTT (Sigma), trypsin (Gibco), DMSO (China Pharmaceutical).

[0062] 3. Main equipment used in this example: CO2 incubator (Thermo, 160i), biosafety cabinet (ESCO, LA2-6A1), inverted fluorescence microscope (KEYENCE BZ-X810), microplate reader (Tecan, Spark).

[0063] 4. Test Method for this Example

[0064] 4.1 Cytotoxicity

[0065] (1) Cell inoculation: 1 x 10 cells 4 The cells were seeded onto a 96-well plate at a density of 1000 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0066] (2) Experimental grouping: In the experiment, a blank group, a control group, a positive control group, and a sample group were set up. In the sample group, eight concentration gradients were set up for each sample, and triplicate wells were set up for each concentration gradient.

[0067] (3) Preparation of solutions: Sample working solutions of different concentrations were prepared according to the test concentration setting table. Table 2-1 Test concentration setting table [Table 2-1]

[0068] (4) Treatment: Treatment was performed when the cell seeding rate in the 96-well plate reached 40-60%. For the control group, 200 μL of culture medium containing 10% PBS was added to each well. For the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well. For the sample group, 200 μL of culture medium containing the corresponding sample concentration was added to each well. For the blank group, no cells were seeded; only 200 μL of cell culture medium was added. After treatment, the 96-well plate was placed in an incubator (37°C, 5% CO2) for incubation.

[0069] (5) Measurement: After culturing the cells for 24 hours, the supernatant was discarded, and a medium containing 0.5 mg / mL MTT was added. The cells were cultured at 37°C in the dark for 4 hours. After the culture was completed, the supernatant was discarded, 100 μL of DMSO was added to each well, and the OD value was read at 490 nm.

[0070] (6) Calculation of cell viability: Calculation by formula Cell viability = (sample well OD - blank well OD) ÷ (solvent control well OD - blank well OD) × 100%

[0071] (7) Test results

[0072] The samples were administered at eight different concentrations, and cytotoxicity assays were carried out. The MTT assay results are shown in Table 2-2. Table 2-2 MTT measurement results [Table 2-2] A cell viability diagram (shown in Figure 1) was plotted with the eight concentrations selected for the samples on the horizontal axis and cell viability on the vertical axis. MTT results showed that polypeptide-in37 showed no keratinocyte toxicity over a concentration range of 0.0313% (m / V).

[0073] 4.2 Cell migration assay

[0074] (1) Collect cells in the logarithmic growth phase and measure their cell density at 1.5 x 10 5 The cells were seeded at 100 cells / well into a 24-well culture plate.

[0075] (2) After culturing for 24 hours in an incubator (37°C, 5% CO2), a horizontal scratch was made perpendicular to the 24-well plate using a 200 μL tip, and the cells were washed once with PBS to remove the scratched cells.

[0076] (3) Samples were placed as shown in Table 2-3. They were placed in an incubator (37°C, 5% CO2) and cultured for 24 hours, with three replicate samples for each group. Table 2-3 Experimental design [Table 2-3]

[0077] (4) The migrated cells were photographed using an inverted microscope, and the average scratch area was calculated using Image Pro Plus software.

[0078] Healing rate (%) = (initial scratch area - current scratch area) ÷ initial scratch area × 100%

[0079] (5) Test results The test results are shown in Figure 3, and the analysis of the results is shown in Tables 2-4 and Figure 2. Table 2-4 Summary of cell fusion rate results [Table 2-4] Note: In statistical analysis using two-tailed t-test, when comparing the sample group, PC group, and BC group, significance is indicated by *, P<0.05 is indicated by *, and P<0.01 is indicated by **.

[0080] The results and analysis of the above experiments showed that the cell adhesion level in the PC group was significantly higher than that in the BC group (P<0.01), demonstrating the validity of the positive control assay. Compared with the BC group, the cell adhesion rate was significantly increased after treatment with the 0.0156% and 0.0313% (m / V) samples (P<0.01).

[0081] In summary, using a human immortalized keratinocyte (HaCaT) model, polypeptide-in37 significantly increased the rate of keratinocyte cell fusion after 24 hours of treatment at concentrations of 0.0156% and 0.0313% (m / V), with a statistically significant difference compared to the BC group (P<0.01). This demonstrates that polypeptide-in37 promotes keratinocyte cell fusion and has a repair effect.

[0082] Example 3 Repair Effect Test - Measurement of the Content of UVB-Stimulated Keratinocyte Barrier-Related Proteins

[0083] 1. Purpose of this Example: Human immortalized keratinocytes (HaCaT) were used as the study object, and the repair effect of the samples was evaluated by measuring the protein content of tropolin (LOR) and filaggrin (FLG) using immunofluorescence technology.

[0084] The human immortalized keratinocytes (HaCaT) used in this example were purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd., product number ZQ0044.

[0085] 2. Main reagents used in this example: high-glucose DMEM culture medium (Gibco), PBS (Gibco), trypsin (Gibco), MTT (Sigma), LOR antibody (Abcam), FLG antibody (Proteintech), goat anti-rabbit secondary antibody (Proteintech), BSA (Absin), and DAPI stain.

[0086] 3. Main equipment used in this example: CO2 incubator (Thermo, 160i), 24-well plate cover glass (Solarbio), ultraviolet light therapy device (SIGMA, SS-03), biosafety cabinet (SUZHOU ANTAI AIRTECH, BSC-1604IIA2), inverted fluorescence microscope (KEYENCE BZ-X810), microplate reader (Tecan, Spark).

[0087] 4. Test Method of This Example (Immunofluorescence Test)

[0088] (1) Cell seeding: Place sterile cell culture cover glasses on the bottom of a 24-well plate and seed at an appropriate seeding density (1 × 10 5 The cells were seeded onto a 24-well plate at 1000 x 1000 cells / well and then cultured overnight in an incubator (37°C, 5% CO2).

[0089] (2) Experimental grouping: In the experiment, a blank control group, a negative control group, a positive control group, and a sample group were set up. The sample group was set up with multiple concentration gradients. Table 3-1 Experimental design [Table 3-1]

[0090] (3) Preparation of solutions: Using cell culture medium, prepare working solutions of test substances at different concentrations based on the concentrations listed in the experimental design (Table 3-1).

[0091] (4) Radiation: Irradiation was performed when the cell seeding density in the 24-well plate reached 40-60%. The negative control group, positive control group, and sample group received a total dose of 300 mJ / cm. 2 At the same time, a blank control group was placed in the same environment (UVB irradiation dose 0 J / cm 2 ) was placed.

[0092] (5) Administration: After irradiation, the original cell culture medium was sucked up and then administered to each group. For the experimental group, 1.0 mL of sample working solution was added per well, with triplicate wells set up for each working solution concentration. For the control group (BC group), 1.0 mL of cell culture medium was added per well, with triplicate wells set up. The cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 hours.

[0093] (6) Sample collection: After removing the supernatant, the cells were washed three times with PBS.

[0094] (7) Immunofluorescence staining: Conventional immunofluorescence staining procedures were performed, including fixation, blocking, addition of primary and secondary antibodies, nucleic acid staining with DAPI, mounting, and subsequent imaging under a fluorescence microscope.

[0095] (8) LOR and FLG fluorescence intensities were quantitatively analyzed using Image Pro Plus software.

[0096] 5. Test Results

[0097] 5.1 LOR test results

[0098] After applying the sample to keratinocytes, the results of LOR protein immunofluorescence staining are shown in Figure 4 (blue fluorescence represents the cell nuclei, green fluorescence represents LOR, and the expression level is proportional to the area and intensity of the green fluorescence; the larger the area of ​​green fluorescence and the darker the color, the greater the amount of LOR). The relative IOD values ​​(i.e., integrated optical density values, which reflect the relative LOR content) are shown in Table 3-2 and Figure 6. Table 3-2 Summary of LOR immunofluorescence analysis results [Table 3-2] Note: In statistical analysis using a two-tailed t-test, when comparing the NC group with the BC group, significance is indicated by #, with P<0.05 indicated by # and P<0.01 indicated by ##. When comparing the PC group, sample group, and the NC group, significance is indicated by *, with P<0.05 indicated by * and P<0.01 indicated by **.

[0099] Compared with the BC group, the NC group had a significantly decreased LOR content (P<0.01), indicating that the UVB stimulation model was successfully established in this experiment.Compared with the NC group, the PC group had a significantly increased LOR content at the administration concentration of 100 μg / mL VC + 7 μg / mL VE (P<0.01), indicating the validity of the positive control in this experiment.

[0100] Compared with the NC group, polypeptide-in37 significantly increased the LOR content at the administration concentrations of 0.0078%, 0.0156% and 0.0313% (m / V) (P<0.01).

[0101] 5.2 FLG test results

[0102] After applying the sample to keratinocytes, the results of FLG immunofluorescence staining are shown in Figure 5 (blue fluorescence represents cell nuclei, green fluorescence represents FLG, and their expression levels are proportional to the area and intensity of the green fluorescence; the larger the area of ​​green fluorescence and the darker the color, the greater the amount of FLG. The relative IOD values ​​(i.e., integrated optical density values, which reflect the relative FLG content) are shown in Table 3-3 and Figure 7. Table 3-3 Summary of FLG immunofluorescence analysis results [Table 3-3] Note: In statistical analysis using a two-tailed t-test, when comparing the NC group with the BC group, significance is indicated by #, with P<0.05 indicated by # and P<0.01 indicated by ##. When comparing the PC group, sample group, and the NC group, significance is indicated by *, with P<0.05 indicated by * and P<0.01 indicated by **.

[0103] Compared with the BC group, the FLG content in the NC group was significantly decreased (P<0.01), indicating that the UVB stimulation model was successfully established in this experiment.Compared with the NC group, the FLG content in the PC group was significantly increased (P<0.01) at the administration concentration of 100 μg / mL VC + 7 μg / mL VE, indicating the validity of the positive control in this experiment.

[0104] Compared with the NC group, polypeptide-in37 significantly increased the FLG content at concentrations of 0.0078%, 0.0156%, and 0.0313% (m / V) (P<0.01).

[0105] In summary, in a human immortalized keratinocyte (HaCaT) model, polypeptide-in37 significantly increased the contents of the cell barrier-associated proteins tropolin (LOR) and filaggrin (FLG) at concentrations of 0.0078%, 0.0156%, and 0.0313% (m / V), with statistically significant differences compared to the NC group (P<0.01), demonstrating the repair effect of polypeptide-in37.

[0106] Example 4 Sedative effect - Degranulation measurement of C48 / 80 stimulated mast cells

[0107] 1. The purpose of this example: To establish a degranulation model based on C48 / 80 stimulated mouse mastocytoma cell P815, and evaluate the sedative effect of the test sample by measuring the changes in the degranulation morphology and degranulation rate of the enlarged cells of the sample. The mouse mastocytoma cells P815 used in this example were purchased from the Cell Bank of the Chinese Academy of Sciences, with the product number TCM12.

[0108] 2. Main reagents used in this example: high-glucose DMEM medium (VivaCell), fetal bovine serum (Gibco), PBS (VivaCell), CCK-8 (Biyuntian), DMSO (China Pharmaceuticals), trypsin (Gibco).

[0109] 3. Main equipment used in this example: CO2 incubator (Thermo, 160i), biosafety cabinet (SUZHOU ANTAI AIRTECH, BSC-1604IIA2), inverted fluorescence microscope (KEYENCE Z-X810), microplate reader (Tecan, Spark).

[0110] 4. Test Method for this Example

[0111] 4.1 Cytotoxicity

[0112] (1) Cell inoculation: 1×10 4The cells were seeded into a 96-well plate at a density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0113] (2) Experimental grouping: In the experiment, a blank group, a control group, a positive control group, and a sample group were set up. In the sample group, eight concentration gradients were set up for each sample, and triplicate wells were set up for each concentration gradient.

[0114] (3) Preparation of solutions: Sample working solutions of different concentrations were prepared according to the test concentration setting table. Table 4-1 Test concentration setting table [Table 4-1]

[0115] (4) Treatment: Treatment was performed when the cell seeding rate in the 96-well plate reached 40-60%. For the control group, 200 μL of culture medium containing 10% PBS was added to each well. For the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well. For the sample group, 200 μL of culture medium containing the corresponding sample concentration was added to each well. For the blank group, no cells were seeded; only 200 μL of cell culture medium was added. After treatment, the 96-well plate was placed in an incubator (37°C, 5% CO2) for incubation.

[0116] (5) Measurement: After culturing the cells for 24 hours, CCK-8 working solution was added, and the mixture was incubated at 37°C for 4 hours in the dark. After the incubation was completed, the OD value was read at 450 nm.

[0117] (6) Calculation of cell viability: Calculation by formula Cell viability = (sample well OD - blank well OD) ÷ (solvent control well OD - blank well OD) × 100%

[0118] (7) Test results The samples were administered at eight different concentrations, and cytotoxicity measurements were carried out. The CCK-8 measurement results are shown in Table 4-2. Table 4-2 CCK-8 measurement results [Table 4-2]

[0119] A cell viability diagram (shown in Figure 8) was plotted with the eight concentrations selected for the samples on the horizontal axis and cell viability on the vertical axis.

[0120] According to the CCK-8 assay results, polypeptide-in37 showed no mast cell toxicity in the concentration range of 0.00391% (m / V).

[0121] 4.2 Degranulation test of P815 cells

[0122] (1) Cell inoculation: 1×10 5 The cells were seeded into a 24-well plate at a density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0123] (2) Preparation of solutions: Working solutions of test substances with different concentrations were prepared depending on the experimental group. Table 4-3 Experimental design [Table 4-3]

[0124] (3) Treatment: According to the experimental design, when the cell seeding rate in the 24-well plate reached 40-50%, the cells were divided into groups and treated. Each group had triplicate wells, and 1 mL of medium containing different concentrations of the test sample was added to each well. After treatment was complete, the 24-well plate was placed in an incubator (37°C, 5% CO2) and cultured for 2 hours.

[0125] (4) C48 / 80 stimulation: After incubating the samples for 2 hours, the liquid was discarded. Depending on the experimental group, 1 mL of serum-free high-glucose DMEM culture medium was added to the blank control group, 1 mL of serum-free high-glucose DMEM culture medium containing C48 / 80 was added to the negative control group, 1 mL of serum-free high-glucose DMEM culture medium containing C48 / 80 and sodium cromoglycate was added to the positive control group, and 1 mL of serum-free high-glucose DMEM culture medium containing C48 / 80 and a sample at the corresponding administration concentration was added to the sample group. After administration, C48 / 80 stimulation was maintained for 45 minutes and the reaction was stopped in an ice bath.

[0126] (5) Cell morphology observation: After the culture was completed, the degranulation status of the cells in each group was observed under an inverted microscope and photographed. The photographs were used to calculate the cell degranulation rate using IPP software. Degranulation rate (%) = total number of degranulated cells ÷ (total number of degranulated cells + total number of non-degranulated cells) × 100%

[0127] (6) Test results The test was carried out based on the degranulation model of "C48 / 80 stimulated mast cells", the degranulation rate was calculated, and the morphological results are shown in FIG. 10, and the degranulation rate statistical results are shown in Table 4-4 and FIG. Table 4-4 Mast cell degranulation rate [Table 4-4] Note: In statistical analysis using the t-test method, when comparing the NC group with the BC group, significance is indicated by #, P<0.05 is indicated by #, and P<0.01 is indicated by ##. When comparing the PC group, sample group, and the NC group, significance is indicated by *, P<0.05 is indicated by *, and P<0.01 is indicated by **.

[0128] The mast cell degranulation rate was significantly higher in the NC group than in the BC group (P<0.01), indicating that the degranulation model was successfully established in this experiment. The mast cell degranulation rate was significantly lower in the PC group than in the NC group (P<0.01), indicating that the positive control assay was valid.

[0129] Compared with the NC group, polypeptide-in37 significantly decreased the degranulation rate of mast cells at concentrations of 0.00098%, 0.00195%, and 0.00391% (m / V) (P<0.01).

[0130] In summary, based on the C48 / 80-stimulated mouse mastocytoma cell P815 model, polypeptide-in37 significantly reduced the mast cell degranulation rate at concentrations of 0.00098%, 0.00195%, and 0.00391% (m / V), with statistically significant differences (P<0.01) compared with the NC group, demonstrating the skin soothing effect of polypeptide-in37.

[0131] Example 5 Sedative effect - LPS-stimulated macrophage RAW264.7 inflammatory factor (TNF-α, IL-6, IL-1α) content measurement

[0132] 1. Purpose of this Example: Based on the LPS-stimulated macrophage RAW264.7 model, the sedative effect of the test sample was evaluated by measuring the changes in the content of inflammatory factors (TNF-α, IL-6, IL-1α) in the cells caused by the sample.

[0133] 2. Main reagents used in this example: High-glucose DMEM medium (Gibco), fetal bovine serum (Gibco), PBS (VivaCell), MTT (Sigma), DMSO (Kokuyo), trypsin (Gibco), LPS (Sigma), dexamethasone (Sigma), Mouse TNF-α ELISA Kit (Dr. Toku), Mouse IL-6 Valukine ELISA Kit (Dr. Toku), Mouse IL-1α Valukine ELISA Kit (Dr. Toku)

[0134] 3. Main equipment used in this example: CO2 incubator (Thermo, 160i), biosafety cabinet (SUZHOU ANTAI AIRTECH, BSC-1604IIA2), inverted fluorescence microscope (KEYENCE BZ-X810), microplate reader (Tecan, Spark).

[0135] 4. Test Method for this Example

[0136] 4.1 Cytotoxicity

[0137] (1) Cell inoculation: 1×10 4 The cells were seeded into a 96-well plate at a density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0138] (2) Experimental grouping: In the experiment, a blank group, a control group, a positive control group, and a sample group were set up. In the sample group, eight concentration gradients were set up for each sample, and triplicate wells were set up for each concentration gradient.

[0139] (3) Preparation of solutions: Sample working solutions of different concentrations were prepared according to the test concentration setting table. Table 5-1 Test concentration setting table [Table 5-1]

[0140] (4) Treatment: Treatment was performed when the cell seeding rate per 96-well plate reached 40-60%. For the control group, 200 μL of culture medium containing 10% PBS was added to each well. For the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well. For the sample group, 200 μL of culture medium containing the corresponding sample concentration was added to each well. For the blank group, no cells were seeded; only 200 μL of cell culture medium was added. After treatment, the 96-well plate was placed in an incubator (37°C, 5% CO2) for incubation.

[0141] (5) Measurement: After 24 hours of cell culture, the supernatant was discarded, MTT working solution (0.5 mg / mL) was added, and the cells were cultured in the dark at 37°C for 4 hours. After the culture was completed, the supernatant was discarded, 100 μL of DMSO was added to each well, and the OD value was read at 490 nm.

[0142] (6) Calculation of cell viability: Calculation by formula Cell viability = (sample well OD - blank well OD) ÷ (solvent control well OD - blank well OD) × 100%

[0143] (7) Test results The samples were administered at eight different concentrations, and cytotoxicity assays were carried out. The MTT assay results are shown in Table 5-2. Table 5-2 MTT measurement results [Table 5-2]

[0144] A cell viability diagram (shown in Figure 11) was plotted with the eight concentrations selected for the samples on the horizontal axis and cell viability on the vertical axis.

[0145] Therefore, the MTT results showed that polypeptide-in37 showed no toxicity to macrophages RAW264.7 over the concentration range of 0.00195% (m / V).

[0146] 4.2 Inflammatory factor content test

[0147] (1) Cell inoculation: 1×10 5 The cells were seeded into a 24-well plate at a density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0148] (2) Preparation of solutions: Working solutions of test substances with different concentrations were prepared depending on the experimental group. Table 5-3 Experimental design [Table 5-3]

[0149] (3) Induction and administration: When the cell seeding rate in the 24-well plate reached 40-60%, 100 μL of 10x LPS working solution was added to the well plate based on the experimental design. The well plate was shaken left and right to homogenize the drug in the well plate, and then the sample was added. The final LPS concentration was 1 μg / mL. Triplicate wells were placed in each group. After administration, the wells were placed in an incubator (37°C, 5% CO2) and continued to be cultured for 24 hours.

[0150] (4) Sample collection: After incubation, the cell culture supernatant was collected in an EP tube (Note: the amount of sample collected was determined by the measurement index). After collection, the sample was frozen and stored in a -80°C freezer.

[0151] (5) Measurement of TNF-α content: Measure according to the instructions for the Mouse TNF-α ELISA kit. IL-6 content measurement: Measure according to the instructions of the Mouse IL-6 ELISA kit. IL-1α content measurement: Measure according to the instructions for the Mouse IL-1α ELISA kit.

[0152] (6) Test results 1) Based on the experimental method, the cell supernatant was collected and the TNF-α content was measured. The measurement results are shown in Table 5-4, and the change trend is shown in Figure 12. Table 5-4 Summary of TNF-α data [Table 5-4] Note: In statistical analysis using the t-test method, when comparing the NC group with the BC group, significance is indicated by #, P<0.05 is indicated by #, and P<0.01 is indicated by ##. When comparing the PC group, sample group, and the NC group, significance is indicated by *, P<0.05 is indicated by *, and P<0.01 is indicated by **.

[0153] Compared with the BC group, the NC group had significantly higher levels of the inflammatory factor TNF-α secretion (P<0.01), demonstrating the success of the LPS-stimulated model in this experiment.Compared with the NC group, the PC group had significantly lower levels of the inflammatory factor TNF-α secretion in the 100 μg / mL dexamethasone-treated macrophages (P<0.01), demonstrating the validity of this positive control assay.

[0154] Compared with the NC group, polypeptide-in37 significantly reduced the secretion of the inflammatory factor TNF-α in macrophages RAW264.7 at concentrations of 0.00049%, 0.00098%, and 0.00195% (m / V) (P<0.05).

[0155] 2) Based on the experimental method, the cell supernatant was collected and the IL-6 content was measured. The measurement results are shown in Table 5-5, and the change trend is shown in Figure 13. Table 5-5 IL-6 data summary table [Table 5-5] Note: In statistical analysis using the t-test method, when comparing the NC group with the BC group, significance is indicated by #, P<0.05 is indicated by #, and P<0.01 is indicated by ##. When comparing the PC group, sample group, and the NC group, significance is indicated by *, P<0.05 is indicated by *, and P<0.01 is indicated by **.

[0156] In the NC group, the amount of IL-6 secreted by macrophages in RAW264.7 was significantly increased compared to the BC group (P<0.01), demonstrating the success of the LPS-stimulated model in this experiment.In the PC group, the amount of IL-6 secreted by macrophages in RAW264.7 under 100 μg / mL dexamethasone was significantly decreased compared to the NC group (P<0.01), demonstrating the validity of this positive control assay.

[0157] Compared with the NC group, polypeptide-in37 significantly reduced the secretion of IL-6, an inflammatory factor, from macrophages RAW264.7 at concentrations of 0.00049%, 0.00098%, and 0.00195% (m / V) (P<0.01).

[0158] 3) Based on the experimental method, the cell supernatant was collected and the IL-1α content was measured. The measurement results are shown in Table 5-6, and the change trend is shown in Figure 14. Table 5-6 IL-1α data summary table [Table 5-6] Note: In statistical analysis using the t-test method, when comparing the NC group with the BC group, significance is indicated by #, P<0.05 is indicated by #, and P<0.01 is indicated by ##. When comparing the PC group, sample group, and the NC group, significance is indicated by *, P<0.05 is indicated by *, and P<0.01 is indicated by **.

[0159] Compared with the BC group, the NC group had significantly higher levels of IL-1α secretion (P<0.01), demonstrating the success of the LPS-stimulated model in this experiment.Compared with the NC group, the PC group had significantly lower levels of IL-1α secretion under 100 μg / mL dexamethasone (P<0.01), demonstrating the validity of this positive control assay.

[0160] Compared with the NC group, polypeptide-in37 significantly reduced the secretion of IL-1α, an inflammatory factor, from macrophages RAW264.7 at concentrations of 0.00098% and 0.00195% (m / V) (P<0.01).

[0161] In summary, using the LPS-stimulated RAW264.7 macrophage model, polypeptide-in37 significantly reduced the secretion of inflammatory factors TNF-α and IL-6 in RAW264.7 macrophages at concentrations of 0.00049%, 0.00098%, and 0.00195% (m / V), with statistical significance (P<0.05) compared with the NC group. Polypeptide-in37 significantly reduced the secretion of IL-1α in RAW264.7 macrophages at concentrations of 0.00098% and 0.00195% (m / V), with statistical significance (P<0.01) compared with the NC group. These results demonstrate that polypeptide-in37 inhibits the secretion of inflammatory factors TNF-α, IL-6, and IL-1α in RAW264.7 macrophages, demonstrating its calming effect.

[0162] Example 6 This is a cosmetic serum whose ingredients contain, by mass percentage, 0.1% polypeptide-in37, 0.08% hyaluronic acid, 1% niacinamide, and 0.3% p-hydroxyacetophenone, with the remainder being purified water.

[0163] Manufacturing method of the above beauty serum: The ingredients are stirred until uniformly dissolved, and then bottled.

[0164] Example 7 The pack contains, by mass, 0.2% polypeptide-in37, 0.1% hyaluronic acid, 1% nicotinamide, and 0.3% p-hydroxyacetophenone, with the remainder being purified water.

[0165] The method for producing the pack includes stirring the raw materials to dissolve them uniformly, immersing the mask substrate in the mixture, and packaging the mixture in a bag.

[0166] Example 8 The cream contains, by mass, 0.5% polypeptide-in37, 0.1% acetyl tetrapeptide, 0.05% sodium alginate, 0.05% ceramide, 4% glycerin, 1% sorbitol, 5% hydroxyethyl cellulose, 9% coconut oil, 9% jojoba oil, 2% vitamin E, 10% spermaceti, and 0.3% p-hydroxyacetophenone, with the remainder being purified water.

[0167] The method for producing the cream is as follows: (1) adding polypeptide-in37, acetyl tetrapeptide, sodium alginate, ceramide, glycerin, sorbitol, and hydroxyethyl cellulose to water and stirring at 40°C for 2 hours; (2) Mixing coconut oil, jojoba oil and vitamin E and stirring at 65°C for 2 hours; (3) continuously stirring the aqueous phase material, adding the oil phase material during the stirring process, and homogeneously emulsifying until the two phases are completely emulsified; (4) Subsequently, spermaceti and p-hydroxyacetophenone are added to continue homogeneous emulsification, followed by stirring at 50°C for 4 hours, allowing to stand for 3 hours, and then molding and stabilizing the mixture.

[0168] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereto. Within the disclosed technical scope of the present invention, any equivalent replacements or modifications made by those skilled in the art based on the technical solutions and inventive ideas of the present invention should all be included in the protection scope of the present invention.

Claims

1. Use of polypeptide-in37 in the manufacture of a composition having a skin repair effect, wherein the molecular formula of said polypeptide-in37 is: H-Ile-Leu-Pro-D-Phe-Lys-D-Phe-Pro-D-Phe-D-Phe-Pro-D-Phe-Arg-Arg-NH ２ The use characterized in that

2. The use according to claim 1, characterized in that the concentration of the polypeptide-in37 is 0 to 0.0391 mg / mL and is greater than 0.

3. The use according to claim 1, characterized in that the concentration of the polypeptide-in37 is 0.0156 to 0.0313 mg / mL.

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