Polypeptide with anti-aging, repairing and soothing functions and application thereof

Abstract

The invention relates to the field of biological medicines and cosmetics, in particular to a composition which takes tetrapeptide and bisabolol as core components and has anti-inflammatory and anti-aging activity.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically, to a polypeptide having anti-aging, repairing and soothing effects and its application. Background Art

[0002] High mobility group protein B1 (HMGB1) is a highly conserved nuclear protein widely distributed in mammalian cells. With the discovery of its pro-inflammatory effect, HMGB1 has become one of the hot spots in critical care medicine research in recent years. A review report published by Cui Lixia et al. revealed the important role of HMGB1 in many skin diseases. For example, in atopic dermatitis, the expression levels of HMGB1 in serum and lesions were higher than those in the control group, and were positively correlated with the severity of dermatitis. In the mouse model of psoriasis vulgaris, the expression level of HMGB1 was significantly increased; in addition to its important role in skin diseases, related studies have shown that HMGB1 protein is also involved in physiological processes such as pigment regulation, inhibition of inflammation, regulation of immunity, and improvement of aging; in summary, HMGB1, as an important target, provides researchers with a research object for the treatment of various diseases. MD-2, called myeloid differentiation 2, is an important auxiliary protein of TLR4 and plays an important role in the process of TLR4 recognizing LPS. For example, MD-2 is involved in the HMGB1-dependent TLR4 regulatory system;

[0003] α-Bisabolol, also known as sweet bisabolol and bisabolol, has the ability to protect the skin from environmental stress and has skin soothing and antibacterial effects. In addition, bisabolol is also a fixative with good stability and is mostly used in medicine, spices and cosmetics.

[0004] A tetrapeptide (amino acid sequence: FSSE) derived from natural plants and microorganisms, which has been proven to be a specific MD-2 antagonist, can prevent MD-2-HMGB1 interaction and TLR4 signal transduction.

[0005] The inventors of the present application unexpectedly discovered that the inventors have investigated the efficacy of tetrapeptide alone, and confirmed through a large number of experiments that it has skin anti-aging (anti-wrinkle, firming), skin soothing, UV damage repair and skin barrier repair effects, and has the effects of increasing the content of filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1). As a bioactive peptide, it has low toxicity and good safety, and its safety is significantly better than conventional chemical preparations (for example: pironic acid has a carcinogenic risk and irritates the eyes, respiratory system and skin); at the same time, the combination of tetrapeptide α-bisabolol has significantly better anti-inflammatory and anti-aging effects than the use of tetrapeptide or α-bisabolol alone, and an unexpected synergistic effect is achieved. Summary of the invention

[0006] The present invention relates to a composition containing a biologically active tetrapeptide and / or α-bisabolol;

[0007] In a first aspect, the present invention relates to a biologically active tetrapeptide, the amino acid sequence of which is FSSE (SEQ ID NO. 1).

[0008] In some embodiments, the amino acid side chain groups, amino terminus or carboxyl terminus of the biologically active tetrapeptide can be modified by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation to obtain derivatives of the biologically active peptide without affecting its biological activity.

[0009] In a second aspect, the present invention relates to a pharmaceutical composition comprising the bioactive tetrapeptide and a pharmaceutically acceptable carrier.

[0010] In some embodiments, the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate buffered saline, balanced salt solution and dimethyl sulfoxide (DMSO), lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0011] In other embodiments, the composition may be administered orally, by inhalation, rectally, vaginally, topically, nasally, ophthalmically, or parenterally.

[0012] In other embodiments, the composition is a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, a buccal dosage form, a sublingual dosage form, a nasal dosage form, an inhaler, a nebulizer, a topical dosage form, a transdermal dosage form, and a suppository.

[0013] In a third aspect, the present invention relates to the use of the above-mentioned bioactive tetrapeptide or pharmaceutical composition in the preparation of active peptide drugs, cosmetics or skin care products that can improve the sub-health state of the skin, increase skin gloss, reduce roughness, reduce the area of ​​spots and red areas, reduce crow's feet, nasolabial folds and fine lines under the eyes, and increase skin elasticity and firmness.

[0014] In some embodiments, the present invention relates to the use of a bioactive tetrapeptide in the preparation of medicines, cosmetics or skin care products that can improve the sub-health state of the skin, increase skin gloss and moisture content, reduce roughness, reduce the area of ​​erythema, reduce crow's feet, nasolabial folds and fine lines under the eyes, improve skin elasticity and firmness, have the effects of repairing ultraviolet damage and repairing the skin barrier, and have the effects of increasing the content of filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1), and the amino acid sequence of the bioactive tetrapeptide is FSSE.

[0015] In some embodiments, the N-terminus of the biologically active tetrapeptide is acetylated and the C-terminus is amidated.

[0016] In a fourth aspect, the present invention relates to the use of the bioactive tetrapeptide or pharmaceutical composition and α-bisabolol in the combined preparation of a drug, cosmetic or skin care product having synergistic anti-inflammatory or anti-aging effects.

[0017] In some embodiments, the concentration of the biologically active tetrapeptide is: 0-100 ppm, preferably 1-40 ppm; more preferably: 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm; most preferably 5 ppm or 30 ppm;

[0018] In some embodiments, the concentration of α-bisabolol is: 0-50μM, preferably 1-25μM, more preferably: 2μM, 3μM, 4μM, 5μM, 6μM, 6.25μM, 7μM, 8μM, 9μM, 10μM, 11μM, 12μM, 12.5μM, 13μM, 13.5μM, 14μM, 14.5μM, 15μM, 15.5μM, 16μM, 16.5μM, 17μM, 17.5μM, 18μM, 18.25μM, 18.5μM, 19μM, 20μM, 20μM, 21μM, 22μM, 23μM, 24μM, 25μM; most preferably: 25μM.

[0019] In some embodiments, the present invention relates to the use of a composition in the preparation of a medicine or cosmetic or skin care product with synergistic anti-inflammatory and / or anti-aging effects, wherein the composition comprises 10 ppm of a bioactive tetrapeptide and 25 uM α-bisabolol, and the amino acid sequence of the bioactive tetrapeptide is FSSE.

[0020] In some embodiments, the present invention relates to the use of a composition for preparing a medicine or cosmetic or skin care product having synergistic anti-inflammatory and / or anti-aging effects, wherein the composition consists of 10 ppm of a bioactive tetrapeptide and 25 μM α-bisabolol, and the amino acid sequence of the bioactive tetrapeptide is FSSE.

[0021] As used herein, the term "crow's feet area and volume" refers to fine lines located around the corners of the eyes caused by facial expressions such as smiling or blinking. The area and volume of these fine lines can be measured by specific instruments or software to evaluate the effect of anti-aging products on reducing fine lines around the eyes. A reduction in area and volume generally indicates that the product has a positive effect on improving skin elasticity and reducing wrinkles.

[0022] As used herein, the term "nasolabial fold area, volume" refers to the wrinkles located on both sides of the mouth corners, which usually become more obvious with age. By measuring the area and volume of nasolabial folds, the effectiveness of anti-aging products in smoothing deep facial wrinkles can be evaluated. Reduced area and volume indicate that the product is able to effectively improve skin structure and reduce signs of facial aging.

[0023] As used herein, the term "skin elasticity R2 value" refers to a value obtained by a skin elasticity measuring instrument, which is used to reflect the elasticity of the skin. The R2 value is determined by measuring the ability of the skin to recover to its original shape after mechanical stretching. The higher the R2 value, the better the skin elasticity and the better it can resist deformation during the aging process.

[0024] As used herein, the term "skin firmness F4 value" refers to a quantitative parameter for evaluating skin firmness. The F4 value is measured by a special skin testing instrument and reflects the firmness and elasticity of the skin. The higher the F4 value, the better the skin firmness, which helps to reduce skin sagging and wrinkle formation.

[0025] As used herein, the term "skin radiance" refers to the ability of the skin surface to reflect light, a property that is often associated with skin health and youthfulness. Skin surfaces with higher radiance reflect light more effectively, giving a bright, fresher visual appearance, and are often viewed as a sign of health and vitality. In anti-aging product evaluations, increased radiance is considered a positive indicator of product effectiveness, indicating that the product helps improve the skin's surface properties and enhances the skin's natural radiance.

[0026] As used herein, the term "facial skin brightness (cheek L value)" refers to a parameter used to measure the brightness of skin color. In color science, the L value represents a brightness level from black (0) to white (100), and the higher the L value, the brighter the skin. In skin science research and cosmetic effect evaluation, the cheek L value is often used as a quantitative indicator to evaluate the effect of improving skin brightness and color. Improving the cheek L value usually means that the skin becomes brighter and more even, which is one of the effects that whitening products or anti-aging products hope to achieve.

[0027] As used herein, the term "optical density of facial skin spots" refers to the color depth and distribution density of the spots on the skin. This indicator is measured by a specific instrument and is used to evaluate the effectiveness of whitening or spot reduction products. A higher optical density of spots indicates that the spots are darker and more densely distributed, while a lower optical density indicates that the spots are lighter and more sparsely distributed. Whitening or spot reduction products are designed to reduce the optical density of spots and make the skin tone more even.

[0028] As used herein, the term "skin erythema area" refers to the size of the erythema area on the skin surface due to inflammation or other reasons. The appearance of erythema is usually related to skin inflammation, allergic reactions or other skin problems. When evaluating the effect of anti-inflammatory, sedative or anti-allergic products, a reduction in the erythema area is considered a positive result, indicating that the product can effectively reduce the skin inflammation state and improve skin health.

[0029] As used herein, the term "facial lactic acid stinging score" refers to a subjective score that evaluates the skin irritation (e.g., stinging) caused by a product. When conducting a skin irritation test, participants give a score based on the degree of irritation they feel after using the product (e.g., no, mild, moderate, strong). The lower the score, the less irritating the product is to the skin and the more suitable it is for sensitive skin.

[0030] As used herein, the term "scalp moisture content" refers to the ability of the scalp's surface layer to retain moisture. This indicator is extremely important for assessing the health of the scalp and the skin as a whole. A scalp with adequate moisture content can remain healthier and more vibrant, with less dryness, itchiness and other problems. Improving scalp moisture content is a goal that many hair care and skin care products attempt to achieve.

[0031] As used herein, the term "skin TEWL value" reflects the integrity of the skin barrier function. A lower TEWL value indicates that the skin barrier function is good, effectively preventing excessive evaporation of water and maintaining the moisture balance of the skin. When evaluating the effects of skin care and hair care products, reducing the TEWL value is generally regarded as an indicator of improving skin barrier function and enhancing skin moisturizing ability.

[0032] As used herein, the term "skin erythema index EI value" refers to an indicator of the degree of scalp erythema measured by an instrument, which is used to quantitatively evaluate the inflammation or sensitivity of the scalp. The higher the EI value, the more severe the scalp inflammation or sensitivity. It is an important indicator in evaluating the calming and soothing effects of skin care or hair care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Anti-inflammatory effect (NO content) test results of the combined use of bioactive tetrapeptide and α-bisabolol;

[0034] Figure 2 : Anti-inflammatory effect (TNF-α content) test results of the combined use of bioactive tetrapeptide and α-bisabolol;

[0035] Figure 3: Results of the skin anti-wrinkle, firming and soothing efficacy evaluation experiment;

[0036] Figure 3A : The efficacy of biologically active tetrapeptide-the results of the crow's feet wrinkle accumulation experiment;

[0037] Figure 3B : Bioactive tetrapeptide efficacy-crow's feet length experimental results;

[0038] Figure 3C : Bioactive tetrapeptide efficacy-nasolabial fold area experimental results;

[0039] Figure 3D : Bioactive tetrapeptide efficacy-F4 value experimental results;

[0040] Figure 3E : Bioactive tetrapeptide efficacy-R2 value experimental results;

[0041] Figure 3F : Bioactive tetrapeptide efficacy-water content experimental results;

[0042] Figure 3G : Bioactive tetrapeptide efficacy-erythema area experimental results;

[0043] Figure 3H : Experimental results of the efficacy of bioactive tetrapeptides-EI value;

[0044] Figure 4 :Sunburn cells (sunburn cells) number result bar graph;

[0045] Figure 5 : FLG relative integrated optical density (IOD) value bar graph;

[0046] Figure 6 : LOR relative integrated optical density (IOD) value bar graph;

[0047] Figure 7 : TGM1 relative integrated optical density (IOD) value bar graph; DETAILED DESCRIPTION

[0048] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0049] Although the numerical ranges and parameter approximations shown in the broad scope of the present application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0050] Example 1: Experiment on the synergistic effect of the combined use of biologically active tetrapeptides and α-bisabolol

[0051] Reagents used in the experiment:

[0052] NO detection kit:

[0053] Brand / Manufacturer: Biyuntian

[0054] Product Name: NO Assay Kit

[0055] Application: To quantitatively detect the concentration of nitrite (stable metabolite of NO) in cell culture supernatant.

[0056] TNF-α Detection Kit:

[0057] Brand / Manufacturer: Lianke

[0058] Product Name: Mouse TNF-α ELISA Kit

[0059] Application: Used to quantitatively detect the content of TNF-α protein from mice.

[0060] Samples used in the experiment

[0061] Bioactive tetrapeptide: pure peptide powder with FSSE sequence;

[0062] α-Bisabolol: Bisabolol standard pure liquid (commercially available)

[0063] Group A: 10ppm biologically active tetrapeptide (FE4)

[0064] Group B: 25uM α-Bisabolol

[0065] GroupC:5ppm FE4+12.5uMα-Bisabolol

[0066] GroupD:7.5ppm FE4+6.25uMα-Bisabolol

[0067] GroupE:2.5ppm FE4+18.75uMα-Bisabolol

[0068] 1.1 Cell culture:

[0069] RAW264.7 cells were diluted to a seeding density of 1.8×10^6 cells / mL and seeded into a 12-well plate at 1 mL / well. Incubate at 37°C and 5% CO 2 , 95% RH for 24±2 hours.

[0070] Experimental groups:

[0071] A blank control group, a negative control group, a positive control group and a sample group were set up, and 4 replicate wells were set up in each group.

[0072] 1.2 Liquid preparation:

[0073] Sample: Dissolve the test sample in cell culture medium to prepare a concentration of 0.3%. Positive control: Use dexamethasone stock solution, thaw and dilute to 10 μg / mL.

[0074] 1.3 Add sample:

[0075] When the cell fusion degree reaches more than 80%, add the corresponding reagents to each group:

[0076] Blank control group: DMEM high-glucose medium was added.

[0077] Negative control group: DMEM high-glucose medium containing 200 ng / mL LPS was added.

[0078] Positive control group: DMEM high-glucose medium containing 10 μg / mL dexamethasone and 200 ng / mL LPS was added.

[0079] Sample group: Add DMEM high-glucose medium containing 200 ng / mL LPS and samples of corresponding concentrations, and return the 12-well plate to the incubator for further culture for 24±2 hours.

[0080] 1.4 Content detection:

[0081] The standard samples were detected by ELISA and the standard curve was drawn to detect the NO and TNF-α content in the supernatant of each group. The actual content of NO and TNF-α in each group was calculated according to the standard curve, and the data of each group were compared to evaluate the anti-inflammatory effect of the samples.

[0082] 1.5 Data Recording:

[0083] Record data such as cell count, reagent addition amount, ELISA test results, etc. The experimental results are shown in Table 1, Table 2, Figure 1 and Figure 2 ;

[0084] Table 1: NO content experimental results

[0085]

[0086] Table 2: TNF-α content experimental results

[0087]

[0088] The anti-inflammatory effect of the samples was evaluated based on the inhibition rate of NO and TNF-α. Compared with the negative control group, the NO and TNF-α content of the sample group was significantly reduced, proving that it had a very significant anti-inflammatory effect. In addition, the synergistic effect was calculated using the widely used Burgi formula: q = E〔A / 2+B / 2〕 / EA (or EB), where E〔A / 2+B / 2〕 refers to the effect of half the amount of A and B, and EA and EB are the effects of A and B alone. Calculated by the above formula, q is greater than 1 (the specific process is as follows), proving that the two have a synergistic effect, and the combined effect is significantly better than that of using them alone.

[0089] NO inhibition degree:

[0090] Q=E〔A / 2+B / 2〕 / EA=(12.59-5.47) / (12.59-6.85)>1

[0091] Q=E〔A / 2+B / 2〕 / EB=(12.59-5.47) / (12.59-7.53)>1

[0092] The degree of inhibition of TNF-α:

[0093] Q=E〔A / 2+B / 2〕 / EA=(32.32-13.06) / (32.32-16.56)>1

[0094] Q=E〔A / 2+B / 2〕 / EB=(32.32-13.06) / (32.32-15.25)>1

[0095] Example 2: Clinical Trial on the Efficacy of Bioactive Tetrapeptides

[0096] The purpose of this clinical trial is to confirm the efficacy of bioactive tetrapeptide (amino acid sequence: FSSE, referred to as FE4, C-terminus: amidation modification; N-terminus: acetylation modification) as a core ingredient of cosmetics in terms of anti-wrinkle, firming, and soothing effects.

[0097] 2.1 Sample name and specific concentration

[0098] Blank group - emulsion (without FE4); sample group - emulsion (containing 30ppm FE4); control group - emulsion (containing

[0099] 1.125% 4-tert-butyl cyclohexanol)

[0100] Test product: The original cosmetic final product is the sample group (the difference between the other groups and the sample group is only FE4, the blank group does not contain FE4, and the control group contains 4-tert-butyl cyclohexanol), and its specific ingredients are as follows:

[0101] Raw material name INCI name Theoretical ratio: Deionized water water 94.3％ SEPIGEL 305 Polyacrylamide, C13-14 Isoparaffin, Laureth-7 1.5％ Hydroite 6 Hexanediol 1,2-Hexanediol 0.5％ SymSave H Fresh Ketone p-Hydroxyacetophenone 0.5％ EDTA-2Na Disodium EDTA 0.2％ FE4 3％

[0102] How to use: Apply an appropriate amount of the shaken sample to facial skin in the morning and evening and wait for it to be absorbed. Use twice a day.

[0103] 2. Subjects: 33 subjects were enrolled in the test, 32 subjects were effective in the test, 30 subjects were counted, including 3 males and 27 females, aged from 30 to 56 years old, with an average age of 43.3±8.7 years old.

[0104] 2.2 Experimental procedures

[0105] Test method:

[0106] The samples submitted by the client are tested according to the specific requirements of the HBCR 2023-050 Cosmetic Anti-wrinkle, Firming, Soothing and Repairing Efficacy Test Program, and qualified subjects are recruited for testing to ensure that the final number of effective cases is not less than 30. The subjects use cleaning products to wash their faces under specified environmental conditions, dry them with dandruff-free absorbent paper after washing, sit in the test environment for 30 minutes, and then conduct a skin basal value test (each volunteer has a test group and a control group on the left and right faces respectively). The test indicators include: crow's feet wrinkle accumulation, crow's feet wrinkle length, nasolabial fold area, skin firmness F4 value, skin elasticity R2 value, skin moisture content, skin erythema index (EI value), skin erythema area, etc.

[0107] The experimental results show that:

[0108] 1. Compared with before using the product, after using the product for 4 weeks, the volume of crow's feet in the lotion (containing 30ppm FE4) group decreased by 5.85, with a decrease rate of 18.99%; the area of ​​crow's feet in the lotion (excluding FE4) group decreased by 3.67, with a decrease rate of 11.37%; there was a significant difference in the area of ​​crow's feet between the lotion (containing 30ppm FE4) group and the lotion (excluding FE4) group (P<0.05), see Figure 3A ;

[0109] 2. Compared with before using the product, after using the product for 4 weeks, the length of crow's feet in the lotion (containing 30ppm FE4) group decreased by 16.17, with a decrease rate of 15.98%; the length of crow's feet in the lotion (excluding FE4) group decreased by 1.73, with a decrease rate of 1.95%; there was a significant difference in the length of crow's feet between the lotion (containing 30ppm FE4) group and the lotion (excluding FE4) group (P<0.05), see Figure 3B ;

[0110] 3. Compared with before using the product, after using the product for 4 weeks, the area of ​​nasolabial folds in the lotion (containing 30ppm FE4) group decreased by 6.21, with a decrease rate of 8.91%; the area of ​​nasolabial folds in the lotion (excluding FE4) group decreased by 2.74, with a decrease rate of 3.64%. There was a significant difference in the area of ​​nasolabial folds between the lotion (containing 30ppm FE4) group and the lotion (excluding FE4) group (P<0.05). For details, see Figure 3C ;

[0111] 4. Compared with before using the product, after using the product for 4 weeks, the F4 value of the lotion (containing 30ppm FE4) group was 15.20%; the F4 value of the lotion (excluding FE4) group decreased by 0.23, and the decrease rate was 4.11%. There was a significant difference in the F4 value between the lotion (containing 30ppm FE4) group and the lotion (excluding FE4) group (P<0.05). For details, see Figure 3D ;

[0112] 5. Compared with before use of the product, after using the product for 4 weeks, the R2 value of the lotion (containing 30ppm FE4) group increased by 2.86, with an increase rate of 4.93%; the R2 value of the lotion (excluding FE4) group increased by 0.98, with an increase rate of 1.72%; there was a significant difference in the R2 value between the lotion (containing 30ppm FE4) group and the lotion (excluding FE4) group (P<0.05), see Figure 3E ;

[0113] 6. Compared with before use of the product, after using the product for 4 weeks, the water content of the lotion (containing 30ppm FE4) group increased by 4.77, with an increase rate of 9.74%; the water content of the lotion (excluding FE4) group increased by 0.27, with an increase rate of 0.55%; there was a significant difference in water content between the lotion (containing 30ppm FE4) group and the lotion (excluding FE4) group (P<0.05), see Figure 3F ;

[0114] 7. Compared with before use of the product, after using the product for 4 weeks, the erythema area of ​​the lotion (containing 30ppm FE4) group decreased by 68.56, with a decrease rate of 22.70%; the erythema area of ​​the lotion (excluding FE4) group decreased by 11.34, with a decrease rate of 5.66%; there was a significant difference in the difference in erythema area between the lotion (containing 30ppm FE4) group and the lotion (excluding FE4) group (P<0.05), see Figure 3G ;

[0115] 8. Compared with before use of the product, after using the product for 4 weeks, the EI value of the emulsion (containing 30ppm FE4) group decreased by 30.72, with a decrease rate of 10.85%; the EI value of the emulsion (excluding FE4) group decreased by 1.39, with a decrease rate of 0.50%; the difference in EI values ​​between the pore solution (containing 30ppm FE4) group and the emulsion (excluding FE4) group was significantly different (P<0.05), see Figure 3H .

[0116] Example 3: Repair efficacy test

[0117] Experimental principle: This experiment is based on a 3D epidermal skin model The 3D epidermal model is a "simulated" skin prepared in vitro using tissue engineering technology, with keratinocytes as the main "raw material", and is used for tissue morphology, filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1) content detection. The 3D epidermal model EpiKutis can be used as a substitute for human "skin" for basic research and for safety and efficacy evaluation in the cosmetics, chemicals, medical devices, health foods and other industries. At present, its application in the cosmetics industry is relatively mature, such as safety and efficacy evaluation of cosmetic raw materials, evaluation of finished product efficacy and claim evidence.

[0118] Experimental Materials:

[0119] The model used in this test is a 3D epidermal skin model Batch number: ES240904, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0120] EpiGrowth culture medium (Guangdong Boxi Biotechnology), PBS (Solaibao), WY14643 (Sigma), FLG antibody (Abcam), LOR antibody (Abcam), TGM1 antibody (Abcam), paraformaldehyde (Biosharp), xylene (Sinopharm), hematoxylin (Biyuntian), and eosin (Biyuntian);

[0121] Main equipment:

[0122] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), upright microscope (Olympus, BX53), UVB irradiator (Philips), and fluorescence microscope (Olympus, BX43).

[0123] Experimental steps:

[0124] 1. Experimental groups:

[0125]

[0126] Note: WY14643 is pironic acid

[0127] 2. Model Dosing and Irradiation

[0128] 1) According to the test group, transfer the model to a 6-well plate (add 0.9 mL EpiGrowth culture medium in advance) and mark the test group number on the 6-well plate.

[0129] 2) The corresponding working solution was added to the PC group liquid, and the sample group was administered with surface medication.

[0130] 3) Except for the BC group, the other groups were irradiated with UVB at a dose of 600 mJ / cm2. 2 Incubate in an incubator (37°C, 5% CO2) for 24 h.

[0131] 4) After the incubation, wash the remaining test substance with sterile PBS solution and wipe off the remaining liquid with a sterile cotton swab.

[0132] 3. Tissue morphology test

[0133] The model was fixed with 4% paraformaldehyde for 24 hours, and then stained with H&E. The model was photographed under a microscope, and the images were collected and analyzed.

[0134] 4. Immunofluorescence test

[0135] The model to be tested was fixed with 4% paraformaldehyde for 24 hours, and then immunofluorescence detection was performed. The model was observed and photographed under a microscope, and the images were collected and analyzed.

[0136] 5. Calculation and analysis of results

[0137]

[0138] GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD. The t-test statistical analysis was used for comparison between groups. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different. The following results were obtained:

[0139] Table 3: Experimental results of protective effect against UVB irradiation

[0140]

[0141] Table 4: Summary of FLG immunofluorescence analysis results

[0142]

[0143] Note: The integrated optical density (IOD) reflects the content of FLG. When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, and P-value < 0.05 is indicated by

[0144] Indicated as *, P-value<0.01 as **

[0145] Table 5: Summary of LOR immunofluorescence analysis results

[0146]

[0147] Note: The value of optical density (IOD) reflects the content of LOR. When using the t-test method for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **

[0148] Table 6: Summary of TGM1 immunofluorescence analysis results

[0149]

[0150] Note: The integrated optical density (IOD) reflects the content of TGM1. When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, and P-value < 0.05 is indicated by

[0151] Indicated as *, P-value<0.01 as **

[0152] In summary, based on the 3D epidermal skin model Compared with the control group, the number of Sunburn cells in sample FE4 at a concentration of 10ppm decreased significantly, with an inhibition rate of 45.45%; the levels of filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1) increased significantly, with an increase rate of 86.67%, 105.13%, and 92.31%, respectively. This indicates that the sample at this concentration can improve tissue morphology, increase the levels of filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1), and has very significant effects on repairing UV damage and repairing the skin barrier. Specifically:

[0153] 1. FLG is one of the key factors in maintaining skin barrier function, and it plays an important role in the terminal differentiation of keratinocytes. The degradation product of FLG is the main source of natural moisturizing factor (NMF), which is essential for maintaining the water content of the stratum corneum and participating in skin hydration. Abnormal metabolism or gene mutation of FLG is associated with the occurrence of various skin diseases, such as atopic dermatitis (AD). The up-regulated expression of FLG may be a potential repair method for damaged skin barrier.

[0154] 2. Loricaril protein (LOR) is one of the key proteins that make up the stratum corneum. It is expressed in the granular layer and plays a role in the formation of lamellar bodies. Together with FLG, it maintains the skin barrier function. LOR is equivalent to the "armor" of keratinocytes, making keratinocytes stronger. LOR cross-links with S100 protein, late cornified envelope family protein and involucrin under the action of TGM1 to form a strong cornified envelope as a barrier against the external environment.

[0155] 3. Transglutaminase 1 (TGM1) is a calcium-dependent enzyme that participates in the formation of ε-(γ-glutamyl)lysine cross-links in the keratin membrane and is the material basis of the skin barrier function. TGM1 promotes the formation and maintenance of the stratum corneum by catalyzing the formation of isopeptide bonds between proteins.

[0156] In summary, one of the causes of skin aging is the change in the structure of the dermis, including the reduction or excessive degradation of collagen and elastin. As key structural proteins of the stratum corneum, the normal function of FLG and LOR helps maintain the elasticity and structure of the skin, thereby combating skin aging. TGM1 also contributes to the maintenance of skin structure and elasticity by participating in protein cross-linking, indirectly affecting the skin's anti-aging ability. Therefore, these proteins are not only directly involved in the formation and maintenance of the skin barrier, but are also closely related to the skin's moisturizing, elasticity, and resistance to external damage. By promoting the expression and function of these proteins, the skin barrier can be strengthened, water loss can be reduced, and the skin's toughness and anti-aging ability can be improved.

[0157] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a bioactive tetrapeptide in the preparation of cosmetics with anti-ultraviolet damage, anti-wrinkle, firming and soothing effects, wherein the amino acid sequence of the tetrapeptide is FSSE.

2. The use according to claim 1, wherein the concentration of the biologically active tetrapeptide is preferably 1-40 ppm; more preferably: 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm; most preferably 5 ppm or 30 ppm.

3. The use according to any one of claims 1 to 2, wherein the concentration of the biologically active tetrapeptide is 30 ppm, and the N-terminus of the biologically active tetrapeptide is acetylated and the C-terminus is amidated.

4. A bioactive tetrapeptide for use in the preparation of medicines, cosmetics or skin care products that can improve sub-healthy skin conditions, increase skin gloss and moisture content, reduce roughness, reduce erythema area, reduce crow's feet, nasolabial folds and fine lines under the eyes, improve skin elasticity and firmness, have UV damage repair and skin barrier repair effects, and have the effect of increasing the content of filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1), wherein the amino acid sequence of the bioactive tetrapeptide is FSSE.

5. The use according to claim 4, wherein the concentration of the biologically active tetrapeptide is preferably 1-40 ppm; more preferably: 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm; most preferably 5 ppm or 30 ppm.

6. The use according to any one of claims 4 to 5, wherein the concentration of the biologically active tetrapeptide is 30 ppm.

7. Use of a composition in the preparation of a medicine or cosmetic or skin care product having synergistic anti-inflammatory and / or anti-aging effects, the composition comprising 10 ppm of a bioactive tetrapeptide and 25 uM α-bisabolol, wherein the amino acid sequence of the bioactive tetrapeptide is FSSE.

8. Use of a composition in the preparation of a medicine, cosmetic or skin care product having synergistic anti-inflammatory and / or anti-aging effects, the composition consisting of 10 ppm of a bioactive tetrapeptide and 25 μM α-bisabolol, wherein the amino acid sequence of the bioactive tetrapeptide is FSSE.

9. Use of a composition in the preparation of a medicine or cosmetic or skin care product having synergistic anti-inflammatory and / or anti-aging effects, the composition consisting of 7.5 ppm of a bioactive tetrapeptide and 6.25 μM α-bisabolol, wherein the amino acid sequence of the bioactive tetrapeptide is FSSE.

10. Use of a composition in the preparation of a medicine or cosmetic or skin care product having synergistic anti-inflammatory and / or anti-aging effects, the composition consisting of 2.5 ppm of a bioactive tetrapeptide and 18.75 μM α-bisabolol, wherein the amino acid sequence of the bioactive tetrapeptide is FSSE.

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