Modified peptides such as polypeptides LA-19 and TM-19 with anti-aging and repairing effects and application of modified peptides

Abstract

Relates to the field of molecular biology, in particular to polypeptide and application thereof, and a composition for relieving or treating ultraviolet damage, oxidation, inflammation and aging and application thereof.

Description

Modified peptides such as LA-19 and TM-19 with anti-aging and repair effects and their uses Technical Field

[0001] The present application relates to the field of cosmetics or medicine, and specifically to a polypeptide and its use, a composition for alleviating or treating ultraviolet damage, oxidation, inflammation, or aging and its use. Background Art

[0002] As we age, facial skin ages and wrinkles worsen. As the organ with the most contact with the outside world, the skin will gradually age like other organs, weakening or losing its functions. This not only affects the appearance but also increases the incidence of various skin diseases. Skin aging refers to the aging-related functional degeneration of the skin due to stimulation from the internal and external environment, which reduces the skin's perception and regeneration capabilities, and fails to promptly improve or reduce the damage caused to the body by the internal and external environment. Exogenous skin aging is mainly caused by environmental factors such as ultraviolet (UV) radiation, sun exposure, and exposure to harmful chemicals. Photoaging caused by UV rays can not only cause skin sagging, roughness, yellowish or grayish-yellow skin discoloration, capillary dilation, and pigmentation, but in severe cases it can also lead to skin collagen degradation and accumulation of abnormal elastin fibers, inducing acute inflammatory reactions in the skin and causing skin cancer.

[0003] In recent years, the application of peptide raw materials has become one of the hot spots in the research and development of cosmetic functional products. Due to its good tolerance, high safety and significant efficacy, it has broad development prospects in the application of anti-aging skin. The anti-aging peptides in the existing technology include: acetyl hexapeptide-8, which has the same mechanism as botulinum toxin. Its advantages are relatively safe to use and significant clinical and practical efficacy; palmitoyl pentapeptide, as one of the earliest signal peptides used in skin care products, can effectively improve skin roughness, reduce the number of fine lines, reduce the depth and area of ​​wrinkles, etc., and has the characteristics of being mild and non-irritating; palmitoyl tetrapeptide-7 is famous for its anti-inflammatory effect. It can inhibit inflammatory mediators, reduce the base number of white blood cells, and reduce skin inflammatory damage. It is added more in skin care products.

[0004] Therefore, it is necessary to discover more peptides that are safe and have significant anti-aging effects to further expand the diversity of anti-aging peptides and provide more options for the development of cosmetics, health products, functional foods and medicines.

[0005] It should be noted that the approaches described in this section are not necessarily approaches that have been previously conceived or employed. Unless otherwise indicated, it should not be assumed that any approach described in this section is prior art simply because it is included in this section. Similarly, unless otherwise indicated, the issues mentioned in this section should not be considered to have been recognized in any prior art.

[0006] Summary of the Invention

[0007] Based on this, in order to seek new polypeptides with higher safety and anti-aging activity, the present application provides a polypeptide, characterized in that the polypeptide comprises at least one of the amino acid sequences that are at least 80%, 90%, 95% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1-10.

[0008] The novel polypeptides disclosed in this application possess multiple anti-aging activities, including anti-inflammatory, antioxidant, anti-UV, and skin-protective properties, and are of great significance for the development of cosmetics, health products, foods, and pharmaceuticals with these properties. Specifically, the polypeptides can protect against UV damage to the skin and enhance the body's antioxidant and anti-inflammatory capabilities. Furthermore, the polypeptides are relatively safe, not only not affecting the growth of skin keratinocytes but also promoting their proliferation and skin keratin regeneration.

[0009] In addition, compared with the more widely used antioxidant ingredients retinol and anti-inflammatory peptide palmitoyl tetrapeptide-7 in the prior art, the polypeptide disclosed in this application has higher anti-aging related activities such as anti-ultraviolet, anti-oxidation, and anti-inflammatory.

[0010] According to one embodiment of the present application, a composition for alleviating or treating ultraviolet damage, oxidation, inflammation, or aging can be provided, characterized in that the composition comprises: a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-10; and pharmaceutically, nutritionally or physiologically acceptable excipients.

[0011] According to one embodiment of the present application, the use of the polypeptide described in the present application in the preparation of anti-ultraviolet, antioxidant, anti-inflammatory, or anti-aging products can be provided.

[0012] According to one embodiment of the present application, the use of a polypeptide having an amino acid sequence of TKAAA in the preparation of anti-ultraviolet, anti-oxidation, anti-inflammatory, or anti-skin aging products can be provided.

[0013] According to one embodiment of the present application, the use of the polypeptide described in the present application and the composition described in the present application in alleviating or treating ultraviolet damage, oxidation, inflammation, or aging can be provided.

[0014] According to one embodiment of the present application, a polypeptide having an amino acid sequence of TKAAA may be provided for use in alleviating or treating ultraviolet damage, oxidation, inflammation, or skin aging.

[0015] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings illustrate exemplary embodiments and constitute a part of the specification. Together with the description of the specification, they serve to explain exemplary implementation of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals designate similar, but not necessarily identical, elements.

[0017] Figure 1 shows the results of keratinocyte cytotoxicity assays of the peptides described in Example 1. From left to right, the results for peptides LA-19, TM-19, P3-10, and Acein are shown. Results are expressed as mean ± SEM. *P < 0.05 indicates a significant difference compared to the blank group; **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate highly significant differences.

[0018] Figure 2 shows the results of testing the anti-UV activity of the peptides in Example 2 on keratinocytes. From left to right, the results for peptides LA-19, TM-19, P3-10, and Acein are shown. Results are expressed as mean ± SEM. For comparisons between the model group and the blank group, a significant difference of ####P < 0.0001 indicates successful establishment of the UV damage model. For comparisons with the model group, a significant difference of *P < 0.05 is indicated, while a significant difference of **P < 0.01, ***P < 0.001, and ****P < 0.0001 is indicated.

[0019] Figure 3 shows the results of the antioxidant activity test on keratinocytes by the polypeptides in Example 3. The activity of superoxide dismutase (SOD) in the cells represents the antioxidant capacity. The results are expressed as Mean ± SEM. When comparing the model group with the blank group, #P < 0.05 indicates a significant difference, and ####P < 0.0001 indicates a very significant difference, indicating that the UV-induced oxidative damage model was successfully established. When compared with the model group, *P < 0.05 indicates a significant difference, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate very significant differences.

[0020] Figure 4 shows the results of the antioxidant activity test of the polypeptide in Example 3 on keratinocytes. The catalase CAT in the cells represents the antioxidant capacity. The results are expressed as Mean ± SEM. When comparing the model group with the blank group, #P < 0.05 indicates a significant difference, and ####P < 0.0001 indicates a very significant difference, indicating that the UV-induced oxidative damage model was successfully established. When compared with the model group, *P < 0.05 indicates a significant difference, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate very significant differences.

[0021] Figure 5 shows the results of the antioxidant activity test of the polypeptide in Example 3 on keratinocytes. The activity of malondialdehyde (MDA) in the cells represents the degree of oxidative damage in the cells. The results are expressed as Mean ± SEM. Compared with the blank group, #P < 0.05 indicates a significant difference, and ####P < 0.0001 indicates a very significant difference, indicating that the UV-induced oxidative damage model was successfully established. Compared with the model group, *P < 0.05 indicates a significant difference, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate very significant differences.

[0022] Figure 6 shows the results of the anti-inflammatory activity test of the polypeptide on keratinocytes in Example 4. The inflammatory factor IL-6 represents the level of inflammation in the cells. The results are expressed as Mean ± SEM. Compared with the blank group, the model group showed a significant difference of ###P < 0.001 and ####P < 0.0001, indicating that the UV-induced cellular inflammation model was successfully established. Compared with the model group, *P < 0.05 was a significant difference, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 were very significant differences.

[0023] Figure 7 shows the results of the anti-inflammatory activity test of the polypeptide on keratinocytes in Example 4. The inflammatory factor TNF-α represents the level of inflammation in the cells. The results are expressed as Mean ± SEM. Compared with the blank group, the model group showed a significant difference of ###P < 0.001 and ####P < 0.0001, indicating that the UV-induced cellular inflammation model was successfully established. Compared with the model group, *P < 0.05 showed a significant difference, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 showed a significant difference. DETAILED DESCRIPTION

[0024] Unless otherwise specified or contradicted by the context, the terms or expressions used in this article should be read in conjunction with the entire content of this article and as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0025] As used herein, "polypeptide" and "peptide" are used interchangeably to refer to amino acid polymers of any length. Thus, polypeptides, oligopeptides, proteins, antibodies, and enzymes are all included within the definition of polypeptide.

[0026] As used herein, a "variant" of a polypeptide sequence is a polypeptide that differs from a reference polypeptide but retains essential properties. A typical variant of a polypeptide differs from another reference polypeptide in amino acid sequence. In general, the differences are limited so that the sequences of the reference polypeptide and the variant are very similar overall and, in many regions, identical. The variant polypeptide and the reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residues may or may not be residues encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring, such as allelic variations, or they may be unknown naturally occurring variants. Non-naturally occurring polynucleotide and polypeptide variants may be made by mutagenesis techniques, direct synthesis, and other recombinant methods known to the skilled artisan.

[0027] As used herein, "relieve" and "treat" and their synonyms refer to the improvement of a disease, disorder, and / or condition. "Relieve" and "treat" can be an improvement in at least one measurable physical parameter, which is not necessarily discernible to the patient. "Relieve" and "treat" can also be the inhibition of the progression of a disease, disorder, and / or condition, either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. "Relieve" and "treat" can also be the slowing down of the progression of a disease, disorder, and / or condition or its reversal.

[0028] As used herein, "treatment" is intended to encompass "prevention." "Prevention" and its synonyms refer to delaying the onset of or reducing the risk of acquiring a particular disease, disorder, and / or condition, or symptoms associated with such diseases, disorders, and / or conditions.

[0029] As used herein, a "pharmaceutically acceptable excipient" refers to a carrier, diluent, or adjuvant used in the formulation or administration of a drug, which is not itself an essential active ingredient and is not unduly toxic upon administration. Suitable pharmaceutically acceptable excipients are well known to those of ordinary skill in the art and include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and surfactants, including, for example, polysorbate 20.

[0030] As used herein, "physiologically acceptable excipient" and "nutritionally acceptable excipient" refer to a carrier, diluent, or adjuvant that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered polypeptide.

[0031] peptides

[0032] The present application provides a polypeptide, characterized in that the polypeptide comprises at least one of the amino acid sequences having at least 80%, 90%, 95% or 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-10.

[0033] In some embodiments, the polypeptide comprises a variant sequence of the amino acid sequence shown in any one of SEQ ID NOs: 1-10, and the variant sequence has no less than 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-10, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a value in a range consisting of any two of the above points.

[0034] In some embodiments, the polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-10.

[0035] In some embodiments, the polypeptide comprises a derivative of the amino acid sequence set forth in any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide is a derivative of the amino acid sequence set forth in any one of SEQ ID NOs: 1-10. The derivative is obtained by modifying the amino acid side chains, the amino terminus, or the carboxyl terminus of the polypeptide set forth in any one of SEQ ID NOs: 1-10, wherein the modification includes, but is not limited to, hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification, or glycosylation.

[0036] In some preferred embodiments, the polypeptide is an amino acid sequence shown in any one of SEQ ID NOs: 1-10.

[0037] In some preferred embodiments, the polypeptide further comprises a terminal modification, including acetylation and / or amidation. Adding modifications to the polypeptide termini can further improve the activity and increase the stability of the polypeptide. Terminal modification methods are commonly known in the art, and any suitable terminal modification method can be used with the polypeptides disclosed herein, without limitation.

[0038] Composition

[0039] According to one embodiment of the present application, a composition for alleviating or treating ultraviolet damage, oxidation, inflammation, or aging can be provided, characterized in that the composition comprises: at least one of the polypeptides having an amino acid sequence as shown in any one of SEQ ID NOs: 1-10; and pharmaceutically, nutritionally or physiologically acceptable excipients.

[0040] In some embodiments, the composition can be used to alleviate UV damage, oxidation, inflammation, or aging in any part of the body. In some preferred embodiments, the composition is used to alleviate UV damage, oxidation, inflammation, or aging in the skin.

[0041] Different composition types can be selected according to different uses. In some embodiments, the composition includes a cosmetic composition, a health product composition, a food composition, or a pharmaceutical composition. In some preferred embodiments, the composition for alleviating UV damage, oxidation, inflammation, or aging of the skin is a cosmetic composition.

[0042] Suitable dosage forms are well known to those skilled in the art. In some embodiments, the dosage form of the composition includes injection, lyophilized powder injection, tablet, granule, aqueous solution, oil, cream, gel, emulsion, powder, or aerosol.

[0043] Different dosage forms can be selected depending on the type of composition. In some preferred embodiments, the cosmetic composition may be in the form of an aqueous solution, an oil, a gel, a cream, an emulsion, a powder, or an aerosol. In some preferred embodiments, the pharmaceutical composition or health product composition may be in the form of an injection, a lyophilized powder injection, a tablet, or a granule.

[0044] Suitable adjuvants are well known to those of ordinary skill in the art, such as liquids, gels or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobials, isotonic agents, buffers, antioxidants, suspending agents / dispersants, chelating agents, diluents, adjuvants, vehicles or non-toxic auxiliary substances, other components known in the art or their various combinations. In some embodiments, the adjuvants include diluents, fillers, adhesives, wetting agents, absorption enhancers, surfactants, lubricants, and / or stabilizers.

[0045] Different excipients can be selected depending on the composition and dosage form. In some preferred embodiments, aqueous cosmetic compositions may include excipients such as water, ethanol, humectants, surfactants, softeners, and adhesives. In some preferred embodiments, tablet pharmaceutical compositions or health supplement compositions may include calcium bicarbonate, calcium phosphate, various sugars and starch types, and cellulose derivatives.

[0046] use

[0047] According to one embodiment of the present application, the use of the polypeptide described in the present application in the preparation of anti-ultraviolet, antioxidant, anti-inflammatory, or anti-aging products can be provided.

[0048] According to one embodiment of the present application, the use of a polypeptide having an amino acid sequence of TKAAA in the preparation of anti-ultraviolet, anti-oxidation, anti-inflammatory, or anti-skin aging products can be provided.

[0049] Different product types can be selected according to different uses. In some embodiments, the composition includes cosmetics, health products, foods, or medicines. In some preferred embodiments, the product used to alleviate UV damage, oxidation, inflammation, or aging of the skin is a cosmetic.

[0050] Suitable dosage forms are well known to those skilled in the art. In some embodiments, the dosage form of the product includes injection, lyophilized powder injection, tablet, granule, liquid, oil, cream, gel, emulsion, powder, or aerosol.

[0051] Different dosage forms can be selected according to the different types of products. In some preferred embodiments, the dosage forms available for the cosmetics include, but are not limited to, aqueous solutions, oils, gels, creams, lotions, powders, or aerosols. In some preferred embodiments, the dosage forms available for the medicines or health products include, but are not limited to, injections, lyophilized powder injections, tablets, or granules.

[0052] According to one embodiment of the present application, the use of the polypeptide described in the present application and the composition described in the present application in alleviating or treating ultraviolet damage, oxidation, inflammation, or aging can be provided.

[0053] In some embodiments, the product, polypeptide, or composition can be used to alleviate UV damage, oxidation, inflammation, or aging in any part of the body. In some preferred embodiments, the product, polypeptide, or composition is used to alleviate UV damage, oxidation, inflammation, or aging in the skin.

[0054] According to one embodiment of the present application, a polypeptide having an amino acid sequence of TKAAA may be provided for use in alleviating or treating ultraviolet damage, oxidation, inflammation, or skin aging.

[0055] In some embodiments, the invention can be used to alleviate UV damage, oxidation, and inflammation in any part of the body. In some preferred embodiments, the invention can be used to alleviate UV damage, oxidation, and inflammation in the skin.

[0056] method

[0057] According to one embodiment of the present application, a method for alleviating or treating ultraviolet damage, oxidation, inflammation, or aging can be provided, comprising administering the polypeptide described in the present application, the composition described in the present application, and / or the polypeptide having the amino acid sequence of TKAAA to a subject in need thereof.

[0058] In some embodiments, the product, polypeptide, or composition can be used to alleviate UV damage, oxidation, inflammation, or aging in any part of the body. In some preferred embodiments, the product, polypeptide, or composition is used to alleviate UV damage, oxidation, inflammation, or aging in the skin.

[0059] In the methods described herein, the dosage of the polypeptide or composition may depend on several factors, including the severity and responsiveness of the symptoms, the route of administration, the duration of treatment (several days to several months to several years), and the time to improvement of symptoms. Those skilled in the art can adjust the dosage regimen to provide a therapeutic response according to the patient's specific circumstances. For example, a single administration may be performed, several separate doses may be administered within a predetermined time period, or the dosage may be reduced or increased as indicated by the treatment situation. The dosage specification is determined by the unique characteristics of the active compound and the specific therapeutic effect to be achieved. The dosage value may vary with the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen may be adjusted over time according to individual needs and the professional judgment of the treating clinician.

[0060] The various embodiments and preferences for the present application described above can be combined with each other (as long as they are not inherently contradictory to each other and are applicable to the purposes of the present application), and the various embodiments formed by such combination are considered to be part of the present application.

[0061] Example

[0062] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0063] Unless otherwise stated, all reagents and instruments used in the following examples are commercially available conventional products. Unless otherwise stated, experiments were performed under conventional conditions or conditions recommended by the manufacturer.

[0064] This application utilizes the well-established human keratinocyte photodamage model to screen for peptide structures with anti-aging properties. This model effectively demonstrates whether a peptide possesses efficacy in protecting against UV damage, promoting cell proliferation, and protecting against oxidative damage and inflammatory damage. Therefore, the human keratinocyte photodamage model can comprehensively evaluate the anti-aging efficacy of peptide drugs and their potential for use in cosmetics, health supplements, and other applications.

[0065] Example 1: Detection of cytotoxicity of polypeptides on keratinocytes

[0066] 1.1 Experimental materials and instruments

[0067] Polypeptide LA-19 (sequence shown in SEQ ID NO: 1), concentration 1 pM-10 μM;

[0068] Polypeptide TM-19 (sequence shown in SEQ ID NO: 2), concentration 1 pM-10 μM;

[0069] Polypeptide P3 (sequence shown in SEQ ID NO: 3), concentration 1 nM-100 μM;

[0070] Polypeptide P4 (sequence shown in SEQ ID NO: 4), concentration 1 nM-100 μM;

[0071] Polypeptide P5 (sequence shown in SEQ ID NO: 5), concentration 1 nM-100 μM;

[0072] Polypeptide P6 (sequence shown in SEQ ID NO: 6), with acetylation modification at its N-terminus, at a concentration of 1 nM-100 μM (acetylation method is to remove Fmoc after all coupling is completed, and acetylate the polypeptide using 0.5 ml of acetic anhydride);

[0073] Peptide P7 (sequence shown in SEQ ID NO: 7), with an amidation modification at its carbon terminus, at a concentration of 1 nM to 100 μM (amidation is performed using Rink resin, which causes the peptide to be automatically amidated);

[0074] Polypeptide P8 (sequence shown in SEQ ID NO: 8), concentration 1 nM-100 μM;

[0075] Polypeptide P9 (sequence shown in SEQ ID NO: 9), having an acetylated N-terminus, at a concentration of 1 nM-100 μM;

[0076] Polypeptide P10 (sequence shown in SEQ ID NO: 10), concentration 1 nM-100 μM;

[0077] Acein (sequence: TKAAA), concentration: 1 nM-100 μM;

[0078] Cisplatin injection (Jiangsu Hausen Pharmaceutical Group Co., Ltd.) was used as a positive control at a concentration of 20 μg / mL.

[0079] Human keratinocytes (HaCaT cells, Nanjing Saihongrui Biotechnology Co., Ltd.)

[0080] Cell culture conditions: constant temperature and humidity incubator (37° C., 95% air, 5% carbon dioxide), 90% DMEM high glucose medium + 10% fetal bovine serum (Nanjing Shenghang Biotechnology Co., Ltd.).

[0081] Trypsin-EDTA solution (Nanjing Shenghang Biotechnology Co., Ltd.); PBS solution (containing KH2PO4, Na2HPO4, NaCl, KCl, Nanjing Chemical Reagent Co., Ltd.); CCK-8 cell viability detection kit (Nanjing Enjing Biotechnology Co., Ltd.).

[0082] 96-well plate (Shanghai Jing'an Biotechnology Co., Ltd.); U-Pure ultrapure water manufacturing system (Sichuan U-Pure Ultrapure Technology Co., Ltd.); microplate reader (Thermo Fisher (Shanghai) Instrument Co., Ltd.); analytical balance (Sartorius Scientific Instrument Co., Ltd.); low-speed centrifuge (Changsha Xiangzhi Centrifuge Instrument Co., Ltd.).

[0083] 1.2 Experimental methods

[0084] Remove the cell culture flask from the cell culture incubator and observe under the microscope. The cells are in the logarithmic growth phase. In a clean workbench, discard the culture medium, rinse the cells with 2mL PBS and discard, repeat once, add 2mL of 0.25% trypsin to digest, and stop with the corresponding volume of serum-containing culture medium. Transfer the cells to a 15mL conical bottom centrifuge tube. Centrifuge at 1000rpm for 5 minutes. Resuspend in serum-containing culture medium, the resuspension volume is 2mL. Count and calculate the cells. Dilute the cells. Plate, take the diluted cell suspension, plate 10 wells in a 96-well plate 4 Cells were plated per well, and surrounding wells were sealed with PBS. The plates were removed from the cell culture incubator, the culture medium discarded, and the corresponding drugs were added to the drug-treated wells. The plates were then placed in the incubator and incubated for 48 hours. On the day of testing, the 96-well plates were removed from the cell culture incubator. On a clean bench, 10 μL of CCK-8 reagent was added to each well. The plates were quickly placed in the cell culture incubator and incubated for 60 minutes. The absorbance was measured with a microplate reader at OD 450 nm. The average of the blank group readings was taken, and the value of each group was divided by it to determine the change in cell viability. The results are expressed as mean ± SEM. Compared with the blank group, *P < 0.05 indicates a significant difference, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate a very significant difference.

[0085] 1.3 Experimental Results

[0086] In Table 1 and Figure 1, the blank group received no drug treatment, while the positive group received 20 μg / mL cisplatin. The results showed that compared to the blank group, peptides LA-19, P3, P4, P5, P6, P7, P8, P9, P10, and Acein did not significantly inhibit cell growth in the peptide-treated group, but rather promoted cell proliferation at certain concentrations. Among them, peptide LA-19 showed no significant toxicity to keratinocytes and promoted cell proliferation, with a significant concentration-dependent activity trend. Peptide TM-19 slightly inhibited cell growth at low concentrations but promoted cell proliferation at high concentrations, demonstrating skin repair efficacy, with a significant concentration-dependent activity trend. Furthermore, compared to Acein, peptides LA-19, TM-19, P7, and P9 all significantly promoted keratinocyte growth, with LA-19 showing a superior effect, indicating that these peptides possess superior skin repair efficacy. These results confirm the safety of all eleven peptides in human skin, and the experimental results are statistically significant.

[0087] Table 1 Cytotoxicity test of peptides on keratinocytes Note: The blank group was serum-free medium without drug, and the positive group was 20 μg / mL cisplatin injection; the above results are expressed as Mean ± SEM; compared with the blank group, *P < 0.05 indicates significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicate very significant difference.

[0088] Example 2: Detection of the anti-ultraviolet activity of polypeptides on keratinocytes

[0089] 2.1 Experimental materials and instruments

[0090] UVB lamp (Philips International Group Co., Ltd., Germany); UV irradiator (Shenzhen Baorui Instrument Co., Ltd.); Retinol (Anagy Shanghai Pharmaceutical Chemical Co., Ltd.) Other experimental materials and instruments are the same as those in Example 1.

[0091] 2.2 Experimental methods

[0092] Remove the cell culture flask from the cell culture incubator and observe under the microscope. The cells are in the logarithmic growth phase. In a clean workbench, discard the culture medium, rinse the cells with 2mL PBS and discard, repeat once, add 2mL of 0.25% trypsin to digest, and stop with the corresponding volume of serum-containing culture medium. Transfer the cells to a 15mL conical bottom centrifuge tube. Centrifuge at 1000rpm for 5 minutes. Resuspend in serum-containing culture medium, the resuspension volume is 2mL. Count and calculate the cells. Dilute the cells. Plate, take the diluted cell suspension, plate 10 wells in a 96-well plate 4Cell / well, surrounding wells were sealed with PBS. The wells were removed from the cell culture incubator, the culture medium was discarded, the corresponding drugs were added to the wells of the drug group, and the cells were incubated in the incubator for 48 hours. After 24 hours of incubation, the cells were UV-modeled, the UVB lamp was turned on, and the irradiation intensity was measured using an ultraviolet irradiator. When it reached 500 μW / cm 2 When the 96-well plate was opened, the irradiation was carried out for 1 min, so that the irradiation dose reached 30 mJ / cm 2 . After completion, cover the 96-well plate and place it in the incubator. On the day of the test, remove the 96-well plate from the cell culture incubator, add 10 μL of CCK-8 reagent to each well in the clean workbench, and quickly place it in the cell culture incubator for 60 minutes. The absorbance value is measured by a microplate reader, and the absorbance value is set at OD 450nm. Take the average of the blank group readings and divide the value of each group by it to obtain the change in cell viability. The results are expressed as Mean ± SEM. Compared with the blank group, ####P<0.0001 is a very significant difference; compared with the model group, *P<0.05 is a significant difference, **P<0.01, ***P<0.001, ****P<0.0001 are very significant differences.

[0093] 2.3 Experimental Results

[0094] In Table 2 and Figure 2, the blank group was not UV-irradiated or treated with drugs; the model group was UV-irradiated but not treated with drugs; and the positive group was UV-irradiated and treated with 1μM retinol. The results showed significant differences between the model group and the blank group, indicating successful establishment of the UV-damage model. Compared with the model group, the peptides LA-19, TM-19, P3, P4, P5, P6, P7, P8, P9, P10, and Acein in the drug-treated group all exhibited some UV-damage resistance, mitigating the inhibition of UV-irradiation on cell growth. Their UV-damage resistance showed a significant concentration-dependent trend, with some concentrations restoring damaged keratinocytes to the level of the blank group's activity. Compared with the positive control group, some peptide concentrations in the drug-treated group exhibited higher UV-damage resistance than the positive drug 1μM retinol. In addition, compared with the peptide Acein, the peptides LA-19, TM-19, P6, P7, P8, P9 and P10 can more significantly slow down the inhibition of cell growth caused by ultraviolet radiation. At some concentrations, they can even make the activity of damaged keratinocytes higher than that of the blank group cells, indicating that these peptides have better anti-ultraviolet damage activity.

[0095] Ultraviolet radiation is the primary cause of cellular DNA damage and can lead to cellular senescence (Stem Cells International, 2016, 2016:e7370642. DOI: 10.1155 / 2016 / 7370642.). Alleviating UV-induced damage can effectively improve cellular senescence, particularly skin cell aging. Therefore, the above results demonstrate that the polypeptides provided herein can effectively alleviate UV-induced skin damage and can be used to improve skin aging caused by photooxidation, with statistically significant results.

[0096] Table 2 Detection of anti-ultraviolet activity of peptides on keratinocytes Note: The blank group was serum-free medium without drugs and did not receive UV modeling; the model group was serum-free medium without drugs; the positive group was 1 μM retinol; the above results are expressed as Mean ± SEM; compared with the blank group, ####P<0.0001 indicates a very significant difference; compared with the drug-treated group and the model group, *P<0.05 indicates a significant difference, **P<0.01, ***P<0.001, and ****P<0.0001 indicate very significant differences.

[0097] Example 3: Detection of the Antioxidant Activity of Peptides on Keratinocytes

[0098] 3.1 Experimental materials and instruments

[0099] Superoxide dismutase (SOD) detection kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.); catalase (CAT) detection kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.); cell malondialdehyde (MDA) detection kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.); six-well plate (Wuxi Nice Life Science Co., Ltd.). Other experimental materials and instruments are the same as those in Examples 1 and 2.

[0100] 3.2 Experimental methods

[0101] Remove the cell culture flask from the cell culture incubator and observe under a microscope that the cells are in the logarithmic growth phase. In a clean bench, discard the culture medium, rinse the cells with 2mL PBS and discard, repeat once, add 2mL of 0.25% trypsin to digest, and stop with the corresponding volume of serum-containing culture medium. Transfer the cells to a 15mL conical bottom centrifuge tube. Centrifuge at 1000rpm for 5 minutes. Resuspend in serum-containing culture medium, the resuspension volume is 2mL. Count and calculate the cells. Dilute the cells. Plate, take the diluted cell suspension, and plate 3x10 5Remove the plate from the cell culture incubator, discard the culture medium, add the corresponding drug to the drug group plate, and incubate in the incubator for 48 hours. After 24 hours of incubation, perform cell UV modeling, turn on the UVB lamp, and use a UV irradiator to measure the irradiation intensity. When it reaches 500μW / cm 2 When the irradiation dose reaches 30 mJ / cm 2 . After the test, cover the six-well plate and place it in the incubator. On the day of the test, take out the six-well plate from the cell culture incubator, collect the cells in the clean workbench, and prepare the cell content sample after disruption. According to the instructions of each test kit, the absorbance value was measured by microplate reader, and the antioxidant-related activity value was calculated. The results are expressed as Mean ± SEM. When the model group was compared with the blank group, #P < 0.05 was a significant difference, and ####P < 0.0001 was a very significant difference; when the drug-treated group was compared with the model group, *P < 0.05 was a significant difference, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 were very significant differences.

[0102] 3.3 Experimental Results

[0103] In Table 3 and Figures 3-5, the blank group was not UV-irradiated or treated with drugs; the model group was UV-irradiated but not treated with drugs; and the positive control group was UV-irradiated and treated with 1 μM retinol. The results showed significant differences between the model group and the blank group, indicating that the UV-induced oxidative damage model was successfully established. Compared with the model group, the peptides LA-19, TM-19, P3, P4, P5, P6, P7, P8, P9, P10, and Acein in the drug-treated group all exhibited antioxidant activity and were able to mitigate UV-induced cellular oxidative damage. Compared with the positive control, some peptide concentrations in the drug-treated group exhibited antioxidant activity higher than that of the positive drug 1 μM retinol. Furthermore, compared with Acein, peptides P6, P7, P8, P9, and P10 significantly enhanced the antioxidant capacity of cells, demonstrating their superior antioxidant efficacy.

[0104] Oxidative stress plays an important role in aging, especially skin aging (Antioxidants, 2022, 11(6): 1121. DOI: 10.3390 / antiox11061121.). During the aging process, the emergence of oxidative stress is mainly attributed to the increase in reactive oxygen species and the decrease in the levels of enzymes and non-enzymatic protective agents. Alleviating the damage caused by oxidative stress is conducive to slowing down the aging process. Therefore, the above results show that the polypeptide provided in this application can effectively improve cell oxidative damage and can be used to improve aging caused by oxidative stress, especially skin aging. The experimental results are statistically significant.

[0105] Table 3 Antioxidant activity of peptides on keratinocytes Note: The blank group was serum-free medium without drugs and did not receive UV modeling; the model group was serum-free medium without drugs; the positive group was 1 μM retinol; the above results are expressed as Mean ± SEM; compared with the blank group, #P < 0.05 indicates a significant difference, and ####P < 0.0001 indicates a very significant difference; compared with the drug-treated group and the model group, *P < 0.05 indicates a significant difference, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate very significant differences.

[0106] Example 4: Detection of the anti-inflammatory activity of polypeptides on keratinocytes

[0107] 4.1 Experimental materials and instruments

[0108] Interleukin-6 ELISA kit (Shanghai Biotech Co., Ltd.); tumor necrosis factor α ELISA kit (Shanghai Biotech Co., Ltd.); six-well plate (Wuxi Nice Life Science Co., Ltd.); palmitoyl tetrapeptide-7 (Shenzhen Jianyuan Pharmaceutical Technology Co., Ltd.). Other experimental materials and instruments were the same as in Examples 1, 2, and 3.

[0109] 4.2 Experimental methods

[0110] Remove the cell culture flask from the cell culture incubator and observe under a microscope that the cells are in the logarithmic growth phase. In a clean bench, discard the culture medium, rinse the cells with 2mL PBS and discard, repeat once, add 2mL of 0.25% trypsin to digest, and stop with the corresponding volume of serum-containing culture medium. Transfer the cells to a 15mL conical bottom centrifuge tube. Centrifuge at 1000rpm for 5 minutes. Resuspend in serum-containing culture medium, the resuspension volume is 2mL. Count and calculate the cells. Dilute the cells. Plate, take the diluted cell suspension, and plate 3x10 5 Remove the plate from the cell culture incubator, discard the culture medium, add the corresponding drug to the drug group plate, and incubate in the incubator for 48 hours. After 24 hours of incubation, perform cell UV modeling, turn on the UVB lamp, and use a UV irradiator to measure the irradiation intensity. When it reaches 500μW / cm 2 When the irradiation dose reaches 30 mJ / cm 2. After the test, cover the six-well plate and place it in the incubator. On the day of the test, take out the six-well plate from the cell culture incubator, collect the cell culture medium in the clean workbench, centrifuge and take the supernatant to obtain a cell suspension sample, operate according to the instructions of each ELISA test kit, and finally measure the absorbance value with a microplate reader to calculate the antioxidant-related activity value. The results are expressed as Mean ± SEM. Compared with the blank group, ###P<0.001 indicates a significant difference, and ####P<0.0001 indicates a very significant difference; compared with the drug-treated group and the model group, *P<0.05 indicates a significant difference, and **P<0.01, ***P<0.001, and ****P<0.0001 indicate very significant differences.

[0111] 4.3 Experimental Results

[0112] In Table 4 and Figures 6-7, the blank group was not UV-irradiated or treated with drugs; the model group was UV-irradiated but not treated with drugs; and the positive control group was UV-irradiated and treated with 10 μM palmitoyl tetrapeptide-7. The results showed significant differences between the model group and the blank group, indicating that the UV-induced cellular inflammation model was successfully established. Compared with the model group, the peptides LA-19, TM-19, P3, P4, P5, P6, P7, P8, P9, P10, and Acein in the drug-treated group all exhibited certain anti-inflammatory properties and were able to alleviate the degree of UV-induced cellular inflammation. Compared with the positive control, some concentrations of the peptides in the drug-treated group exhibited higher anti-inflammatory activity than the positive control, 10 μM palmitoyl tetrapeptide-7. Furthermore, compared with Acein, peptides P6, P7, P8, P9, and P10 significantly enhanced the antioxidant capacity of cells, demonstrating their superior antioxidant efficacy.

[0113] Studies have found that chronic inflammation can accelerate the aging process through reactive oxygen species-mediated telomere dysfunction and cellular senescence (Nature Communications, 2014, 5(1):4172.DOI:10.1038 / ncomms5172.). In addition, studies have also found that inflammation caused by ultraviolet radiation can accelerate the aging process of the skin (Inflammation Research, 2022, 71(7):817-831.DOI:10.1007 / s00011-022-01598-8.). Therefore, the above results show that the polypeptide provided in this application has good anti-inflammatory activity and can be used to improve inflammation-related aging, especially skin aging, and the experimental results are statistically significant.

[0114] Table 4 Detection of anti-inflammatory activity of polypeptides on keratinocytes Note: The blank group was a serum-free medium without drugs and did not receive UV modeling; the model group was a serum-free medium without drugs; the positive group was 10 μM palmitoyl tetrapeptide-7; the above results are expressed as Mean ± SEM; compared with the blank group, ###P<0.001 and ####P<0.0001 were very significant differences; compared with the drug-treated group and the model group, *P<0.05 was a significant difference, **P<0.01, ***P<0.001, and ****P<0.0001 were very significant differences.

[0115] It should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Although specific implementation methods have been described, for the applicant or other skilled in the art, there may be or are currently no alternatives, modifications, changes, improvements and substantial equivalents of the above implementation methods. Therefore, the attached claims submitted and the claims that may be amended are intended to cover all such alternatives, modifications, changes, improvements and substantial equivalents. It is important to note that as technology evolves, many of the elements described herein may be replaced by equivalent elements that appear after the present application.

Claims

1. A polypeptide, characterized in that The polypeptide comprises at least one of the amino acid sequences having at least 80%, 90%, 95% or 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 1-10.

2. The polypeptide according to claim 1, wherein the polypeptide is an amino acid sequence shown in any one of SEQ ID NOs: 1-10.

3. The polypeptide according to claim 1, further comprising a terminal modification, wherein the terminal modification comprises acetylation and / or amidation.

4. A composition for alleviating or treating ultraviolet damage, oxidation, inflammation, or aging, characterized in that: The composition comprises: A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-10; and Pharmaceutically, nutritionally or physiologically acceptable excipients.

5. The composition of claim 4, wherein the ultraviolet damage, oxidation, inflammation, or aging comprises ultraviolet damage, oxidation, inflammation, or aging of the skin.

6. The composition according to claim 4, comprising a cosmetic composition, a health product composition, a food composition, or a pharmaceutical composition.

7. The composition according to claim 4, wherein the auxiliary materials include diluents, fillers, adhesives, wetting agents, absorption promoters, surfactants, lubricants, and / or stabilizers.

8. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of anti-ultraviolet, anti-oxidation, anti-inflammatory, or anti-aging products.

9. Use of a polypeptide having an amino acid sequence of TKAAA in the preparation of anti-ultraviolet, anti-oxidant, anti-inflammatory, or anti-skin aging products.

10. The use according to claim 8 or 9, wherein the product comprises cosmetics, health products, food, or medicine.

11. Use of the polypeptide according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 7 in alleviating or treating ultraviolet damage, oxidation, inflammation, or aging.

12. The use according to claim 11, wherein the ultraviolet damage, oxidation, inflammation, or aging comprises ultraviolet damage, oxidation, inflammation, or aging of the skin.

13. Use of a polypeptide having an amino acid sequence of TKAAA in alleviating or treating ultraviolet damage, oxidation, inflammation, or skin aging.

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