Novel polypeptide and use thereof

Abstract

The present disclosure provides a novel polypeptide and a preparation method therefor, a nucleic acid, an expression vector, a host cell, a composition, and a use of the polypeptide. The polypeptide of the present application has various effects on the skin, such as moisturizing, anti-oxidation, soothing, firmness, anti-wrinkle, sebum control and / or whitening, and has broad applications in the preparation of cosmetic products and / or pharmaceuticals for moisturizing, anti-oxidation, soothing, firmness, anti-wrinkle, sebum control, whitening, or any combination thereof.

Description

A novel polypeptide and its uses

[0001] Cross-references

[0002] This disclosure claims priority to Chinese application 202510475802.6, filed on April 16, 2025, the contents of which are considered part of this disclosure and are incorporated herein in their entirety. Technical Field

[0003] This disclosure relates to the fields of biotechnology, cosmetics and pharmaceuticals, and specifically to a novel polypeptide and its uses. Background Technology

[0004] The skin is the largest organ in the human body and acts as a barrier against irritants, bacteria, allergens, etc., making skin care extremely important.

[0005] The appearance of the skin is related to the moisture content of the stratum corneum. Normal skin's stratum corneum typically contains 10-30% water to maintain its softness and elasticity. Dehydration can lead to dry, tight, rough, dull, and flaky skin, as well as fine lines, causing various skin problems and premature aging. In severe cases, it can cause chapped skin, allowing bacteria and viruses to enter the body through these cracks, affecting health. Using moisturizing substances can prevent moisture evaporation, keeping the skin hydrated; it can also improve microcirculation and enhance skin hydration.

[0006] Many factors contribute to skin aging. Besides genetics, environmental stress from sources like ultraviolet radiation and smog causes oxidative stress, leading to cell oxidation and the production of free radicals. Excessive accumulation of free radicals in the stratum corneum can cause age spots, wrinkles, and other skin problems, accelerating skin aging and even leading to skin cancer. Using substances with antioxidant capabilities can reduce the production of free radicals and eliminate aging metabolic products, thereby improving the skin's antioxidant capacity and effectively delaying skin aging.

[0007] Allergic reactions and inflammatory reactions often go hand in hand, leading to sensitive and fragile skin, and even damage to the stratum corneum barrier, resulting in redness, burning, stinging, itching, and / or tightness. Sensitive skin is more susceptible to irritation from internal and external factors and is more prone to allergies. Using products with soothing properties can effectively reduce or alleviate discomfort caused by various factors (such as environmental factors, chemicals, physical irritants, etc.), helping to improve skin irritation and other conditions.

[0008] Collagen is one of the most abundant proteins in the skin, responsible for providing support and keeping it firm and plump; elastin gives the skin its elasticity. Collagen and elastin are key to maintaining youthful and healthy skin. As we age, the body's collagen and elastin levels decrease. This decline affects the skin's ability to retain moisture, causing it to lose elasticity and become more prone to sagging and wrinkles. Supplementing with collagen and elastin can firm the skin and reduce wrinkles.

[0009] Hormones, temperature, and other factors can affect the secretion of sebum by sebaceous glands. For example, when androgen levels are high, sebaceous glands become more active, leading to oily skin. Oxidized sebum that soaks into the stratum corneum can cause dull skin and acne. Using oil-control products can reduce sebum production from sebaceous gland cells, improving problems such as blackheads, enlarged pores, acne, pimples, and folliculitis caused by excessive oil production.

[0010] Melanin is an amino acid derivative synthesized by melanocytes. Melanocytes in the skin are mainly distributed in the basal layer of the epidermis. Melanin then migrates through layers of cell metabolism, eventually reaching the epidermis to form different types of spots such as freckles, sunspots, and dark spots. Additionally, dark acne scars are formed due to increased skin pigment synthesis stimulated by inflammatory responses. Tyrosinase (TYR) is considered the most important key enzyme in melanin synthesis; inhibiting its activity directly affects the rate and quantity of melanin synthesis, and is currently a major research focus in the market for skin whitening products.

[0011] Therefore, it is essential to provide products that are safe and non-irritating, with moisturizing, antioxidant, soothing, firming, anti-wrinkle, oil-controlling, and / or whitening effects.

[0012] Peptides play a vital role in regulating the functional activities of various systems, organs, tissues, and cells in the body, and are frequently used in fields such as functional analysis, antibody research, drug development, and beauty and health care. With the increasing maturity of biotechnology and peptide synthesis technology, more and more peptides are being developed and applied clinically.

[0013] In summary, developing novel peptides with moisturizing, antioxidant, soothing, firming, anti-wrinkle, oil-controlling, and / or whitening activities has significant practical and economic value. Summary of the Invention

[0014] To address the aforementioned technical problems, this application provides a novel polypeptide and its use in the preparation of cosmetic products and / or pharmaceuticals for moisturizing, anti-oxidation, soothing, firming, anti-wrinkle, oil control, whitening, or any combination thereof.

[0015] In a first aspect, this application provides a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70%, at least 75%, and at least 85% identity with SEQ ID NO: 1.

[0016] The peptides in this application have good safety profiles and exhibit good moisturizing, antioxidant, soothing, firming, anti-wrinkle, oil-controlling, and / or whitening effects.

[0017] Secondly, this application provides a method for preparing the polypeptide of the first aspect of this application.

[0018] Thirdly, this application provides a nucleic acid encoding a polypeptide of the first aspect of this application.

[0019] Fourthly, this application provides an expression vector comprising the nucleic acid described in the third aspect of this application.

[0020] Fifthly, this application provides a host cell comprising the nucleic acid of the third aspect of this application and / or the expression vector of the fourth aspect of this application.

[0021] In a sixth aspect, this application provides a composition comprising a polypeptide of the first aspect of this application, a nucleic acid of the third aspect of this application, an expression vector of the fourth aspect of this application, and / or a host cell of the fifth aspect of this application, and one or more acceptable excipients.

[0022] In a seventh aspect, this application provides the use of the polypeptide of the first aspect of this application, the nucleic acid of the third aspect of this application, the expression vector of the fourth aspect of this application, the host cell of the fifth aspect of this application, and / or the composition of the sixth aspect of this application in the preparation of cosmetic products for moisturizing, anti-oxidation, soothing, firming, anti-wrinkle, oil control, whitening, or any combination thereof.

[0023] Eighthly, this application provides the use of the polypeptide of the first aspect of this application, the nucleic acid of the third aspect of this application, the expression vector of the fourth aspect of this application, the host cell of the fifth aspect of this application, or the composition of the sixth aspect of this application in the preparation of a medicine for moisturizing, anti-oxidation, soothing, firming, anti-wrinkle, oil control, whitening, or any combination thereof.

[0024] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0025] It should be understood that these accompanying drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0026] Figure 1 shows the results of the HSF cytotoxicity test of palmitoyl peptide and myristoyl peptide in Example 2.1.

[0027] Figure 2 shows the effect of palmitoyl peptide and myristoyl peptide on the expression level of type I collagen in HSF cells in Example 2.2.

[0028] Figure 3 shows the results of the toxicity test of palmitoyl polypeptide and myristoyl polypeptide on human primary sebaceous gland cells in Example 3.1.

[0029] Figure 4 shows an inverted fluorescence microscope image of the human primary sebaceous gland cell oleic acid-induced model in Example 3.2 after treatment with palmitoyl polypeptide, myristoyl polypeptide and positive control, and staining with BDPY 493 / 503.

[0030] Figure 5 shows a statistical graph of the average fluorescence intensity of the inverted fluorescence microscope images in Example 3.2.

[0031] Figure 6 shows the results of the HaCat cytotoxicity test of palmitoyl peptide and myristoyl peptide in Example 4.1.

[0032] Figure 7 shows the effect of palmitoyl polypeptide and myristoyl polypeptide on AQP3 expression in HaCaT cells in Example 4.2.

[0033] Figure 8 shows the total cellular fluorescence statistics after TRPV1 immunofluorescence cytochemical staining correction in Example 5. Detailed Implementation

[0034] Unless otherwise stated, the terminology used herein has the common meaning understood by one of ordinary skill in the art. It may vary for those skilled in the art depending on the desired properties and effects sought through this application, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or as understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the experimental procedures in organic chemistry, medicinal chemistry, and biology described herein are well-known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Where multiple definitions exist for terms used herein, the definitions in this section shall prevail unless otherwise stated.

[0035] Unless otherwise stated, all figures used in this specification and claims to indicate content, concentration, proportion, weight, particle size, percentage, technical effect, etc., shall in any event be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. “About” or “approximately” can be understood to mean a range of plus or minus 10%, 20%, 30%, 40%, or 50% of the indicated values.

[0036] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0037] When used in this document, the terms “including,” “contains,” or “comprising” mean that other elements are not excluded in addition to the elements listed.

[0038] As used herein, the terms “polypeptide,” “protein,” “peptide,” and “amino acid sequence” are used interchangeably and refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, may contain modified amino acids or amino acid analogs, and may be broken down by non-amino acid chemical motifs. The term also includes amino acid polymers that have been naturally or artificially modified (e.g., disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a label or bioactive component). The term “peptide” includes two or more naturally occurring or synthetic amino acids linked by covalent bonds (e.g., amide bonds).

[0039] When used in this article, the term "N-terminus of a polypeptide" refers to the start of the amino acid sequence, which is the end of the polypeptide with a free amino group.

[0040] When used in this article, the term "C-terminus of a polypeptide" refers to the termination end of the amino acid sequence, which is the end of the polypeptide with a free carboxyl group.

[0041] When used in this article, the term "amino acid residue side chain" refers to the characteristic functional group (generally referred to as the R group) attached to the α-carbon of an amino acid residue.

[0042] As used herein, the term "identity" is typically determined using sequence analysis software. Protein analysis software employs similarity metrics tailored to various substitutions, deletions, and other modifications to align similar sequences. For example, GCG software includes programs such as "Gap" and "Bestfit" to determine sequence homology or identity between closely related peptides, such as homologous peptides from different species, or wild-type proteins and their mutants. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA software (with default or recommended parameters) and programs in GCG version 6.1. FASTA software (such as FASTA2 and FASTA3) provides alignment results for the best overlapping region between the query and retrieved sequences, as well as the percentage of sequence identity (Pearson method). Another preferred algorithm is the computer program BLAST, particularly BLASTP and BLASTN, which uses default parameters when aligning sequences of this disclosure with databases containing a large number of sequences from different organisms. (For example, see Altschul et al. (1990) J. Mol. Biol. 215:403410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389402). In some embodiments, "sequence identity" is determined by comparing two optimally aligned sequences, wherein, to achieve optimal alignment, a portion of the polynucleotide or polypeptide sequence may contain 20% or less of insertions or deletions (i.e., gaps) compared to a reference sequence (which does not contain insertions or deletions). The percentage is calculated by determining the number of identical nucleic acid bases or amino acid residues in the two sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence, and then multiplying the result by 100 to obtain the percentage of sequence identity.

[0043] When used in this document, the term "chemical modification" refers to the method of modifying proteins or nucleic acids by introducing or removing chemical groups.

[0044] When used herein, the term "amino acid residue side chain has chemical modification" means that the amino acid residue side chain can be substituted by any conventional substituents known in the art.

[0045] As used in this article, the term "palmitoylation modification" refers to palmitic acid (C4N ... 16 H 32 O2) with acyl groups (CH3(CH2) 14 The -CO-) form is attached to the peptide chain or amino acid.

[0046] When used in this article, the term "myristic modification" refers to myristic acid (C... 14 H 28 O2) with acyl groups (CH3(CH2) 12 The -CO-) form is attached to the peptide chain or amino acid.

[0047] When used in this document, the term "acyl group" refers to a monovalent group formed by removing the hydroxyl group from an organic or inorganic oxyacid, usually with the general formula RC(O)-, where R is a hydrocarbon group or other organic group.

[0048] When used herein, the term "salt form" refers to a salt that retains the desired biological activity of the peptide of this application and exhibits lower undesirable toxicological effects. Specifically, a peptide can form a salt when a free amino group (e.g., Arg, Lys, and His) is present at the N-terminus of its peptide sequence or on its side chain.

[0049] As used herein, the term "nucleic acid" refers to a polymer of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues. In this application, nucleic acids and polynucleotides are used interchangeably.

[0050] As used in this article, the term "vector" refers to a self-replicating DNA molecule, often in the form of a circular double-stranded DNA molecule, that transfers a foreign target gene into a host organism. Typical vectors include plasmids, viruses, bacteriophages, kinases, and mini-chromosomes. Among these, plasmids are the most common form of vector, referring to circular double-stranded DNA that can accept foreign nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0051] As used herein, the term "expression vector" refers to the connection of multiple nucleic acid fragments in a functional relationship. When one nucleic acid is functionally connected to another nucleic acid sequence, it is considered "operably connected." For example, if a promoter or other transcriptional regulatory sequence affects the transcription of a coding sequence (or gene), then the coding sequence is operably connected in a manner that allows the promoter to induce transcription of the gene. Operable connection means that the connected nucleotide sequences may be contiguous or discontinuous. In this application, expression vectors and recombinant vectors are used interchangeably.

[0052] As used herein, the term "acceptable excipient" refers to an acceptable raw material, component, or carrier that imparts morphology or consistency to a polypeptide composition. When mixed, each excipient is compatible with the polypeptide and other components of the polypeptide composition, thereby avoiding significant reduction in the potency of the polypeptide at application and avoiding interactions that would lead to unacceptable components.

[0053] As used herein, the term "subject" refers to a primate (e.g., a human), a dog, a rabbit, a guinea pig, a pig, a rat, and a mouse. In some embodiments, the subject is a primate. In some specific embodiments, the subject is a human.

[0054] When used in this article, a subject is considered "needing" administration if the subject would benefit from it biologically, medically, or in terms of quality of life.

[0055] As used herein, the term "therapeutic effective dose" or "effective dose" refers to an amount that results in benefit compared to a corresponding subject who did not receive that amount, but is sufficiently low within the range of reasonable (medical) judgment to avoid serious side effects. The therapeutic effective dose or effective dose of a peptide will vary depending on factors such as the chosen route of administration; associated symptoms or disease; the severity of the associated symptoms or disease; the subject's age, body type, weight, and physical condition; the duration of administration; and the nature of concurrent administration, but can still be determined in a conventional manner by someone skilled in the art.

[0056] In this article, the term "AQP-3 protein" or "AQP3 protein" refers to an aquaporin (AQP), also known as aquaporin, a protein located on the cell membrane (intrinsic membrane protein) that forms "pores" in the cell membrane, controlling the entry and exit of water into and out of the cell. AQP3, as an important functional aquaporin in keratinocytes of the epidermis, is widely involved in the rapid transmembrane transport of water between the intracellular and extracellular spaces. It also mediates the uptake and transport of small molecule moisturizing substances such as glycerol. Its expression level is closely related to cellular water retention capacity, stratum corneum water homeostasis, and overall skin moisturizing function. When the test sample is applied to keratinocytes, if it exhibits moisturizing activity, it can upregulate the gene transcription and protein expression levels of AQP3 by regulating related intracellular signaling pathways, thereby enhancing the cell's efficiency in adsorbing, transporting, and storing water, improving the stratum corneum's water reserve, and ultimately improving skin moisturizing efficacy. Human immortalized keratinocytes (HaCaT cells) are immortalized cell lines of normal human skin keratinocytes derived from non-tumor sources. They can synthesize skin moisturizing-related biomolecules such as hyaluronic acid (HA), AQP, TJP, and filaggrin (FLG). By detecting the effect of peptides on the levels of moisturizing-related proteins such as AQP, Claudin-1, and / or FLG, their moisturizing capabilities can be reflected, and the moisturizing efficacy of peptides can be evaluated.

[0057] In this article, the term "collagen" refers to a biological macromolecule, a key protein for maintaining healthy and strong skin. It gives skin structure, firmness, and elasticity, making it appear firm and plump. Fibroblasts synthesize and secrete collagen and elastin, forming collagen fibers, reticular fibers, and elastic fibers, as well as synthesizing and secreting matrix components such as glycosaminoglycans and glycoproteins. Collagen and elastin significantly influence skin elasticity and are important indicators for evaluating firming and anti-wrinkle effects.

[0058] When used in this article, the term "TRPV-1 protein" (transient receptor potential-Vanilloid-1) refers to an important protein involved in the transmission of pain and itch sensations. It functions as a molecular receptor and can be activated by a range of physical and chemical stimuli (capsaicin, vanilloid), thereby causing Ca2+ irritation. 2+ Intravenous flow transmits the sensations of pain / itching and promotes the release of neurogenic inflammatory factors, further inducing inflammation and leading to local capillary dilation. TRPV-1 primarily mediates inflammatory pain, neuropathic pain, and visceral pain, and is also involved in the pathways of acute and chronic pruritus. When the skin is stimulated by external factors, the skin's immune system is activated, releasing various inflammatory factors and histamine, causing increased blood flow and cell permeability, resulting in sensations of tightness, stinging, burning, tingling, pain, and persistent itching on the face. Ultraviolet (UV) radiation is an important environmental factor; skin exposure to UV radiation can cause various skin damage, including sunburn, premature aging, cancer, and inflammation. The effects of UV radiation may be mediated by various cellular changes, including autophagy, cell cycle control, and cell death, as well as molecular changes, including signal transduction and gene expression. UV radiation can also act as a broad activator of cell surface growth factor receptors and cytokine receptors. Literature shows that TRPV1 protein expression is significantly increased in young buttock skin exposed to UV radiation. Furthermore, ultraviolet radiation has been found to increase TRPV1 expression in HaCaT cells and human skin. When TRPV-1 expression is inhibited, the skin can be effectively soothed.

[0059] polypeptide

[0060] In a first aspect, this application provides a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70%, at least 75%, and at least 85% identity with SEQ ID NO: 1.

[0061] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 73%, 75%, 77%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1. In some embodiments, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1.

[0062] The amino acid sequence shown in SEQ ID NO: 1 is VHIALNP.

[0063] In some embodiments, the polypeptide is modified. In some embodiments, the modification includes chemical modification.

[0064] In some embodiments, the N-terminus, C-terminus, and / or amino acid residue side chains of the peptide are chemically modified.

[0065] In some embodiments, the N-terminus of the polypeptide is chemically modified.

[0066] In some embodiments, the chemical modification includes at least one of palmitoylation and myristoylation. In some embodiments, the chemical modification includes palmitoylation. In some embodiments, the chemical modification includes myristoylation.

[0067] In some embodiments, palmitoyl modification is introduced at the N-terminus. Palmitic acid reacts with the free amino group at the N-terminus of the peptide, and the modified peptide facilitates the formation of a micelle structure, achieving sustained release and enhanced transdermal absorption. In some embodiments, the peptide modified with palmitoyl at the N-terminus is (CH3(CH2)). 14 -CO)-VHIALNP has good biocompatibility and moisturizing, soothing, firming, anti-wrinkle and oil-controlling effects. It is suitable for all skin types and can meet the oil control, moisturizing, soothing and / or firming and anti-wrinkle needs of oily skin.

[0068] In some embodiments, myristic acid is introduced at the N-terminus as a modifier. Myristic acid reacts with the free amino group at the N-terminus of the peptide, increasing the lipophilicity of the modified peptide and enhancing its penetration into the stratum corneum. In some embodiments, the peptide modified with myristic acid at the N-terminus is (CH3(CH2)). 12 -CO)-VHIALNP has good biocompatibility and moisturizing, soothing, firming, anti-wrinkle and oil-controlling effects. It is suitable for all skin types and can meet the needs of oily skin for oil control, moisturizing, soothing and / or firming and anti-wrinkle.

[0069] In some implementations, the polypeptide includes a salt form.

[0070] In some embodiments, the salt form includes acetate, trifluoroacetate, hydrochloride, sulfate, citrate, or salicylate. In some embodiments, the salt form includes acetate. In some embodiments, the salt form includes trifluoroacetate. In some embodiments, the polypeptide trifluoroacetate form can be converted into a polypeptide molecular form, a polypeptide acetate form, and / or a polypeptide hydrochloride form using conventional techniques in the art.

[0071] Preparation method

[0072] Secondly, this application provides a method for preparing the polypeptide of the first aspect of this application.

[0073] In some implementations, the polypeptide is prepared by biosynthesis.

[0074] In some embodiments, the polypeptide is prepared by chemical synthesis.

[0075] In some embodiments, the chemical synthesis method may be a solid-phase (e.g., Fmoc solid-phase synthesis) or a liquid-phase synthesis method. In some embodiments, the chemical synthesis method includes a solid-phase method (Stewart, JM, Young, JD, "Solid Phase Peptide Synthesis, 2nd Edition", (1984), Pierce Chemical Company, Rockford, IL (1984); Bodanzsky M., Bodanzsky A., "The practice of Peptide Synthesis", (1994), Springer Verlag, Berlin; Lloyd Williams P. et al., "Chemical Approaches to the Synthesis of Peptides and Proteins", (1997), CRC, Boca Raton, FL, USA). In some embodiments, chemical synthesis methods include solution synthesis or enzymatic synthesis (Kullmann W. "Proteases as catalysts for enzymic syntheses of opioid peptides", (1980), J. Biol. Chem., 255(17), 8234-8238).

[0076] In some embodiments, the peptide is prepared by Fmoc solid-phase synthesis.

[0077] The preparation method of the peptides in this application conforms to the concept of green synthesis, taking into account low cost, low toxicity and high scalability, laying a solid foundation for subsequent industrialization.

[0078] Nucleic acid, expression vector, host cell

[0079] Thirdly, this application provides a nucleic acid encoding a polypeptide of the first aspect of this application.

[0080] Fourthly, this application provides an expression vector comprising the nucleic acid described in the third aspect of this application.

[0081] Fifthly, this application provides a host cell comprising the nucleic acid of the third aspect of this application and / or the expression vector of the fourth aspect of this application.

[0082] In some embodiments, the host cell described herein may be a prokaryotic cell, fungal cell, yeast cell, or eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, Enterobacteriaceae bacteria, such as Escherichia coli (E. coli). In some embodiments, the cell is a human cell. In some embodiments, the host cell includes, for example, human immortalized keratinocytes (HaCat cells), human skin fibroblasts (HSF cells), or human sebaceous gland cells.

[0083] Composition

[0084] In a sixth aspect, this application provides a composition comprising a polypeptide of the first aspect of this application, a nucleic acid of the third aspect of this application, an expression vector of the fourth aspect of this application, and / or a host cell of the fifth aspect of this application, and one or more acceptable excipients.

[0085] In some embodiments, acceptable excipients include, but are not limited to, diluents, fillers, binders, disintegrants, lubricants, flow aids, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, or buffers. Those skilled in the art may selectively use the above-mentioned acceptable excipients depending on the dosage form of the composition.

[0086] In some embodiments, the composition includes the form of cream, lotion, solution, ointment, paste, spray, suspension, gel, mask, tablet, capsule, granules or powder.

[0087] In some embodiments, the composition comprises a second active ingredient.

[0088] In some embodiments, the second active ingredient has uses including but not limited to moisturizing, anti-aging, anti-wrinkle, anti-allergy, anti-inflammatory, repairing, sun protection, antioxidant, soothing, firming, oil control, whitening, spot removal, or acne treatment.

[0089] use

[0090] In a seventh aspect, this application provides the use of the polypeptide of the first aspect of this application, the nucleic acid of the third aspect of this application, the expression vector of the fourth aspect of this application, the host cell of the fifth aspect of this application, and / or the composition of the sixth aspect of this application in the preparation of cosmetic products for moisturizing, anti-oxidation, soothing, firming, anti-wrinkle, oil control, whitening, or any combination thereof.

[0091] In some embodiments, this application provides the use of the polypeptide of the first aspect of this application, the nucleic acid of the third aspect of this application, the expression vector of the fourth aspect of this application, the host cell of the fifth aspect of this application, and / or the composition of the sixth aspect of this application in the preparation of cosmetic products for skin moisturizing, skin anti-oxidation, skin soothing, skin firming, skin anti-wrinkle, skin oil control, skin whitening, or any combination thereof.

[0092] In some embodiments, beauty products include, but are not limited to, beauty product ingredients, toners, lotions, hydrating sprays, emulsions, face creams, body lotions, serums, face masks, pressed powders, liquid foundations, cream foundations, makeup bases, sunscreens, sunscreen sprays, skin cleansers, makeup remover balms, makeup remover liquids, makeup remover milks, makeup remover sheets, facial cleansers, shampoos, conditioners, or shower gels.

[0093] In some embodiments, the polypeptides of this application are used to develop moisturizing, soothing, firming, anti-wrinkle, and / or oil-controlling products such as toners, lotions, hydrating sprays, emulsions, creams, body lotions, serums, masks, pressed powders, liquid foundations, cream foundations, makeup bases, sunscreens, sunscreen sprays, skin cleansers, makeup remover balms, makeup remover liquids, makeup remover milks, makeup remover sheets, and / or facial cleansers.

[0094] In some embodiments, the peptides of this application are used to develop lotion and / or cream products that provide long-lasting moisturizing, soothing, firming, anti-wrinkle, and / or oil-controlling effects.

[0095] Eighthly, this application provides the use of the polypeptide of the first aspect of this application, the nucleic acid of the third aspect of this application, the expression vector of the fourth aspect of this application, the host cell of the fifth aspect of this application, and / or the composition of the sixth aspect of this application in the preparation of a medicine for moisturizing, anti-oxidation, soothing, firming, anti-wrinkle, oil control, whitening, or any combination thereof.

[0096] In some embodiments, this application provides the use of the polypeptide of the first aspect of this application, the nucleic acid of the third aspect of this application, the expression vector of the fourth aspect of this application, the host cell of the fifth aspect of this application, and / or the composition of the sixth aspect of this application in the preparation of a medicine for skin moisturizing, skin anti-oxidation, skin soothing, skin firming, skin anti-wrinkle, skin oil control, skin whitening, or any combination thereof.

[0097] In some implementations, the medicine includes, but is not limited to, ointments for use on the skin surface.

[0098] Methods of prevention, treatment, relief or improvement

[0099] This application also provides a method for preventing, alleviating, or improving symptoms associated with dry skin, skin oxidation, skin allergies, skin inflammation, skin laxity, skin wrinkles, oily skin, and / or skin pigmentation, the method comprising administering to a subject in need an effective dose of a polypeptide, nucleic acid, expression vector, host cell, or composition as described in the first aspect of this application, a third aspect of this application, an expression vector, a fourth aspect of this application, a fifth aspect of this application, and / or a sixth aspect of this application.

[0100] This application also provides a method for preventing, treating, alleviating, or improving diseases and / or conditions related to dry skin, skin oxidation, skin allergies, skin inflammation, skin laxity, skin wrinkles, oily skin, and / or skin pigmentation, said method comprising administering to a subject in need a therapeutically effective dose of a polypeptide, nucleic acid, expression vector, host cell, or composition as described in the first aspect of this application, the third aspect of this application, the fourth aspect of this application, or the sixth aspect of this application.

[0101] The various embodiments and options described above for this application can be combined with each other (as long as they are not inherently contradictory) and are applicable to the technical solutions of this application. All embodiments formed by such combinations are considered part of this application.

[0102] The following describes exemplary embodiments of this application, including various details of the embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0103] Example

[0104] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0105] Example 1: Synthesis of Polypeptides

[0106] 1.1 Synthesis of Palmitoyl Peptide

[0107] The free amino group of the N-terminal valine in the amino acid sequence shown in SEQ ID NO: 1 reacts with the carboxyl group of palmitic acid to form an amide bond. The palmitoyl polypeptide is shown in the following formula: (CH3(CH2)) 14 -CO)-VHIALNP.

[0108] Palmitoyl peptides were prepared by Fmoc solid-phase synthesis using 2-chlorotriphenylmethyl resin with a substitution degree of 1.1 mmol / g as the carrier. The resin swelled after shaking with DMF (15 mL / g) for 60 min, the solvent was filtered, and 3 molar excess of Fmoc-Pro-OH and 10 molar excess of DIEA were added. The mixture was dissolved in DMF and shaken for 30 min, followed by methanol capping for 30 min. Deprotection was performed by treating with 20% piperidine / DMF solution (15 mL / g) for 5 min and 15 min respectively. A deep blue color on Kaiser's reagent test indicated a positive result. The peptides were then washed twice with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g). During the condensation stage, add 3 times molar excess of Fmoc-Asn(Trt)-OH, 3 times molar excess of HBTU, and 10 times molar excess of DIEA. Dissolve in DMF and shake for 45 min. Kaiser reagent detection shows no color, indicating a negative result (detection of free amino groups ensures complete reaction). Wash once with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g). Repeat the above deprotection and washing process, connecting Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, and palmitic acid sequentially from right to left. After the connection was completed, the resin was washed twice with DMF (10 mL / g), three times with DCM (10 mL / g), and four times with methanol (10 mL / g) and dried for 10 min. The resin was then cut for 180 min with a cutting solution (10 mL / g) containing 95% trifluoroacetic acid (TFA), 2% water, 2% ethylenedithiol (EDT), and 1% triisopropylsilane (TIS). The lysate was dried with nitrogen, and the precipitate was precipitated with diethyl ether and washed six times. The resin was then evaporated at room temperature. The crude product was dissolved in H2O / ACN, and the target peak was analyzed by HPLC. It was then purified using a C18 reversed-phase chromatography system (wavelength: 220 nm; flow rate: 15 mL / min; injection volume: 20 mL; column temperature: 25℃; eluent A: 0.1% TFA / H2O; eluent B: 0.1% TFA / ACN). The target peak solution was collected, and after passing mass spectrometry and purity tests, it was lyophilized (LYO-0.5 laboratory lyophilizer). The finished product was identified by MS molecular weight and HPLC purity. The molecular weight determined by ESI-MS was 1001.34 (ESI-MS does not reflect the molecular weight of the counterion). The product content was ≥91.0%, the HPLC purity was 95.08%, and the palmitic acid conjugation rate was ≥95%. After passing the identification, it was sealed and packaged, and stored at -20℃.

[0109] The palmitoyl polypeptide prepared in this embodiment is in the form of trifluoroacetate.

[0110] 1.2 Synthesis of Myristoyl Peptide

[0111] The free amino group of the N-terminal valine in the amino acid sequence shown in SEQ ID NO: 1 reacts with the carboxyl group of myristic acid to form an amide bond. The myristic yl polypeptide is shown in the following formula: (CH3(CH2)) 12 -CO)-VHIALNP.

[0112] Myristoyl peptides were synthesized using the Fmoc solid-phase method with 2-chlorotriphenylmethyl resin (1.1 mmol / g). The resin was first swollen by shaking with DMF (15 mL / g) for 60 min, filtered, and then 3 molar excess of Fmoc-Pro-OH and 10 molar excess of DIEA were added. The mixture was dissolved in DMF and shaken for 30 min, followed by methanol capping for 30 min. Deprotection was performed by first treating with a 20% piperidine / DMF solution (15 mL / g) for 5 min and discarding the solution, then treating with the same concentration solution for 15 min. A deep blue color change on Kaiser reagent was a positive result. Washing was performed twice with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g). During condensation, add 3 times molar excess of Fmoc-Asn(Trt)-OH, 3 times molar excess of HBTU, and 10 times molar excess of DIEA. Dissolve in DMF and shake for 45 min. A colorless test with Kaiser reagent indicates a negative result. Wash once with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g). Repeat the deprotection and washing steps, connecting Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, and myristic acid sequentially from right to left. The resin was then washed twice with DMF (10 mL / g), three times with DCM (10 mL / g), and four times with methanol (10 mL / g) and dried for 10 min. It was then cut for 180 min with a cutting solution (10 mL / g) of 95% trifluoroacetic acid (TFA), 2% water, 2% ethylenedithiol (EDT), and 1% triisopropylsilane (TIS). The pyrolysis solution was dried with nitrogen, and the precipitate was precipitated with diethyl ether and washed six times before being evaporated at room temperature. The crude product was dissolved in H2O / ACN, and the target peak was determined by HPLC analysis. It was then purified using a C18 reversed-phase chromatography system (wavelength: 220 nm; flow rate: 15 mL / min; injection volume: 20 mL; column temperature: 25℃; eluent A: 0.1% TFA / H2O; eluent B: 0.1% TFA / ACN). The target peak solution was collected, and after passing mass spectrometry and purity tests, it was lyophilized (LYO-0.5 laboratory lyophilizer). The finished product was then subjected to MS molecular weight identification and HPLC purity identification. The molecular weight determined by ESI-MS was 973.28 (ESI-MS does not reflect the molecular weight of the counterion). The product content was ≥92.0%, the HPLC purity was 96.86%, and the single impurity content was ≤1.5%. Finally, it was sealed and stored at -20℃.

[0113] The myristoyl polypeptide prepared in this embodiment is in the form of trifluoroacetate.

[0114] Example 2: Safety evaluation and firming and anti-wrinkle activity evaluation of peptides

[0115] 2.1 Cytotoxicity test of peptides

[0116] The cytotoxicity doses were designed with six concentration gradients: 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM. Human skin fibroblast (HSF) complete culture medium was also set up as a negative control (NC).

[0117] Experimental methods: Human skin fibroblasts (HSF cells, Shanghai Boson Biotechnology Co., Ltd., catalog number BES23113HC) were used at a concentration of 1×10⁻⁶. 4 Cells were seeded in 96-well plates and cultured at 37°C / 5% CO2 for 24 h. The old culture medium was discarded, and medium containing different concentrations of palmitoyl peptide or myristoyl peptide prepared in Example 1 was added. A negative control was also included. The plates were cultured for another 48 h. The supernatant was discarded, and medium containing 10% MTT (MP Biomedicals, 102227) was added. The plates were incubated at 37°C / 5% CO2 for another 4 h. The liquid in the wells was carefully aspirated, and an appropriate amount of DMSO was added to each well. The plates were shaken well, and the absorbance at 490 nm was measured using a microplate reader. The experiment was performed in triplicate.

[0118] Cell viability = (OD value of sample group / average OD value of negative control group) × 100%

[0119] The experimental data were imported into statistical analysis software to calculate the mean, standard deviation and coefficient of variation for each treatment group. The results are shown in Table 1 and Figure 1. When the sample concentration was <1mM, the HSF cell survival rate was >80%, indicating that the palmitoyl peptide and myristoyl peptide of this application have good biosafety.

[0120] Table 1: Results of acyl peptide cytotoxicity assays against HSF cells

[0121] 2.2 Evaluation of the firming and anti-wrinkle activity of the peptides

[0122] This embodiment evaluates the firming and anti-wrinkle effects of the peptides in this application by detecting the expression level of type I collagen in human skin fibroblasts (HSF).

[0123] Experimental methods: Human skin fibroblasts (HSF cells, Shanghai Boson Biotechnology Co., Ltd., catalog number BES23113HC) were used at a concentration of 3 × 10⁻⁶ cells / year. 4Cells were seeded in 24-well plates and cultured at 37°C / 5% CO2 for 24 h. The old culture medium was discarded, and culture medium containing different concentrations of palmitoyl peptide or myristoyl peptide prepared in Example 1 (0.0625 mM, 0.125 mM, 0.25 mM) was added. A negative control (NC, HSF complete cell culture medium) and a positive control (PC, TGF-β1 (NOVOPROTEIN / nearshore protein, CA59), 100 ng / mL) were also set up. The cells were cultured for another 72 h, and the cell culture supernatant was collected into sterile centrifuge tubes and centrifuged at 4°C and 12,000 rpm. The supernatant was collected, and the expression level of type I collagen in the cells was detected using an enzyme-linked immunosorbent assay kit for human type I collagen α1 (COL1α1) (Elabscience, E-EL-H0869).

[0124] Using Curve Expert 1.4 standard curve analysis software, a regression equation for the fitted standard curve was calculated based on the concentration of type I collagen standard and the OD450nm value. The sample OD450nm value was then substituted into the equation to calculate the type I collagen content of the sample. The average value of the three replicates for each group was taken as the final type I collagen result.

[0125] The results are shown in Table 2 and Figure 2. Compared with the negative control group, the expression levels of type I collagen in the 0.125 mM and 0.25 mM palmitoyl peptide or myristoyl peptide groups were significantly increased (P<0.01). The increase rates in the palmitoyl peptide group were 7.41% and 17.34%, respectively, and the increase rates in the myristoyl peptide group were 6.78% and 14.63%, respectively. This indicates that the palmitoyl peptide and myristoyl peptide of this application have a promoting effect on the expression of type I collagen and have a firming and anti-wrinkle effect.

[0126] Table 2: Results of Type I Collagen Expression Levels

[0127] Note: P<0.01 indicates a highly significant difference compared to the negative control group (NC); P>0.05 indicates no significant difference compared to the negative control group (NC).

[0128] Example 3: Safety evaluation and oil-controlling activity evaluation of peptides

[0129] 3.1 Cytotoxicity test of peptides

[0130] The cytotoxicity doses were designed with six concentration gradients: 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM. Human primary sebaceous gland cell complete culture medium was set up as a negative control (NC).

[0131] Experimental methods: Human primary sebaceous gland cells (from Zhejiang Meisen Cell Technology Co., Ltd.) were used at a concentration of 1×10⁻⁶ cells / cells. 4 Cells were seeded in 96-well plates and cultured at 37°C / 5% CO2 for 24 h. The old culture medium was discarded, and medium containing different concentrations of palmitoyl peptide or myristoyl peptide prepared in Example 1 was added. A negative control was also included. The plates were cultured for another 48 h. The supernatant was discarded, and medium containing 10% MTT (MP Biomedicals, 102227) was added. The plates were incubated at 37°C / 5% CO2 for another 4 h. The liquid in the wells was carefully aspirated, and an appropriate amount of DMSO was added to each well. The plates were shaken well, and the absorbance at 490 nm was measured using a microplate reader. The experiment was performed in triplicate.

[0132] Cell viability = (OD value of sample group / average OD value of negative control group) × 100%

[0133] The experimental data were imported into statistical analysis software to calculate the mean, standard deviation, and coefficient of variation for each treatment group. The results are shown in Table 3 and Figure 3. When the sample concentration was <0.5mM, the survival rate of human primary sebaceous gland cells was about 80%, indicating that the palmitoyl polypeptide and myristoyl polypeptide of this application have good biocompatibility.

[0134] Table 3: Results of toxicity tests of acyl peptides on human primary sebaceous gland cells

[0135] 3.2 Oil-controlling activity test of peptides

[0136] During differentiation, primary human sebaceous gland cells synthesize and accumulate large amounts of triglycerides and cholesterol esters, forming lipid droplets. This embodiment uses a 300 μM oleic acid-induced human sebaceous gland cell model. A lipid droplet fluorescence staining kit (BDPY 493 / 503 has a high affinity for neutral lipids and can label lipid droplets as bright green fluorescence) was used to compare the amount of sebum secreted by sebaceous gland cells and evaluate the oil-controlling efficacy of the peptides in this application.

[0137] Experimental methods: Human primary sebaceous gland cells (derived from Zhejiang Meisen Cell Technology Co., Ltd.) were used at a concentration of 3 × 10⁻⁶ cells / year. 4Cells were seeded in 24-well plates and cultured at 37°C / 5% CO2 for 24 h. The old culture medium was discarded. Except for the negative control group (NC), 300 μM oleic acid was added to the other groups to induce lipid secretion and construct cell models. After successful modeling, the model control group (Model) was left untreated, the positive control group (PC) was treated with 50 μM isotretinoin, and the sample groups were cultured in medium containing different concentrations of palmitoyl peptide or myristoyl peptide prepared in Example 1 (0.0625 mM, 0.125 mM, 0.25 mM), and cultured for another 48 h. After culture, the cells were washed with PBS, fixed with 4% paraformaldehyde, and stained using a lipid droplet fluorescence staining kit (Beyotime, BDPY493 / 503). Images were taken using an inverted fluorescence microscope (Olympus, CKX53) (Ex / Em≈493 / 503 nm), as shown in Figure 4. Image J analysis was used to analyze the average fluorescence intensity of each group, as shown in Table 4 and Figure 5.

[0138] Table 4: Detection results of average fluorescence intensity

[0139] Note: P<0.001 indicates an extremely significant difference compared to the model control group (M); P<0.01 indicates a highly significant difference compared to the model control group (M); P<0.05 indicates a significant difference compared to the model control group (M); P>0.05 indicates no significant difference compared to the model control group (M).

[0140] Statistical results are expressed as mean ± SD, and statistical analysis was performed using SPSS software. This experiment employed independent samples t-tests between two groups and one-way ANOVA for statistical analysis among multiple groups. The statistical results were defined with α = 0.05 as the test limit, and P < 0.05 (compared to the model control group) was considered statistically significant.

[0141] The average fluorescence intensity of the model control group was significantly enhanced compared with that of the negative control group, indicating that the stimulation conditions in this test were effective.

[0142] Compared with the model control group, the average fluorescence intensity of the 0.125mM palmitoyl peptide group was significantly reduced, and the average fluorescence intensity of the 0.25mM palmitoyl peptide group, 0.125mM myristoyl peptide group and 0.25mM myristoyl peptide group was extremely significantly reduced, indicating that the palmitoyl peptide and myristoyl peptide of this application can inhibit the content of lipid droplets in human primary sebaceous gland cells and have oil control effect.

[0143] Example 4: Safety and moisturizing activity evaluation of peptides

[0144] 4.1 Cytotoxicity test of peptides

[0145] The cytotoxicity doses were designed as eight concentration gradients: 0.5 mM, 0.35 mM, 0.25 mM, 0.125 mM, 0.0625 mM, 0.0313 mM, 0.0156 mM, and 0.01 mM. MEM complete culture medium was also set up as a negative control (NC).

[0146] Experimental methods: Human immortalized keratinocytes (HaCaT cells, passage 20, sourced from the China Center for Type Culture Collection (CCTCC)) were seeded in 96-well plates and cultured at 37℃ / 5% CO2 for 24-48 h. When the cell confluence was about 80%, culture medium containing different concentrations of palmitoyl polypeptide or myristoyl polypeptide prepared in Example 1 was added. A negative control was also set up. The cells were cultured for another 48 h. The absorbance was then measured at 450 nm using the CCK8 assay after an incubation period of 2 h.

[0147] Cell viability = (OD value of sample group / average OD value of negative control group) × 100%

[0148] The experimental data were imported into statistical analysis software to calculate the mean, standard deviation, and coefficient of variation for each treatment group (if the coefficient of variation (CV) value is less than 20%, the parallelism of the experiment is considered valid).

[0149] The results are shown in Table 5 and Figure 6. The survival rate of HaCaT cells reached 80% or above at all concentrations, indicating that the palmitoyl polypeptide and myristoyl polypeptide of this application have good biosafety.

[0150] Table 5: Results of acyl peptide cytotoxicity assays against HaCaT cells

[0151] 4.2 Moisturizing Activity Test of Peptides

[0152] This embodiment is based on a human keratinocyte model. By detecting the expression level of intracellular aquaporin 3 (AQP3), the regulatory role of AQP3 expression is clarified, and the moisturizing efficacy of the peptides in this application is evaluated.

[0153] Experimental Methods: Immortalized human keratinocytes (HaCaT cells, passage 26, sourced from the China Center for Type Culture Collection (CCTCC)) were seeded in 96-well plates and cultured at 37℃ / 5% CO2 for 24-48 h. When the cell confluence reached approximately 80%, culture medium containing different concentrations of palmitoyl polypeptide or myristoyl polypeptide prepared in Example 1 (0.125 mM, 0.0625 mM, 0.0313 mM) was added. A negative control (MEM complete medium) and a positive control (PC, TGF-α, 1 μg / mL) were also provided. The cells were cultured for another 48 h. The expression level of aquaporin 3 in the cells was detected using the Human AQP3 (Aquaporin 3) ELISA Kit (Wuhan Huamei Biotechnology Co., Ltd., batch number: F03027442).

[0154] AQP3 improvement rate = (BA) / A

[0155] In the formula: A represents the average AQP3 expression level in the negative control group; B represents the average AQP3 expression level in the sample group or the positive control group. Experimental data were imported into statistical analysis software, and the mean, standard deviation, and coefficient of variation for each treatment group were calculated. Statistical tests were performed using one-way ANOVA, the Levene test was used for homogeneity of variance, and the Fisher LSD test was used for multiple comparisons.

[0156] The results are shown in Table 6 and Figure 7. Compared with the negative control group, the expression levels of AQP3 in the 0.125 mM and 0.0625 mM palmitoyl peptide or myristoyl peptide groups were significantly increased (P<0.01). The increase rates in the palmitoyl peptide group were 39.28% and 30.30%, respectively, and the increase rates in the myristoyl peptide group were 39.88% and 30.18%, respectively. This indicates that the palmitoyl peptide and myristoyl peptide of this application promote the expression of AQP3, thereby enhancing the water retention capacity of cells and having a moisturizing effect.

[0157] Table 6: AQP3 Expression Level Results Note: P<0.01 indicates a highly significant difference compared to the negative control group (NC); P>0.05 indicates no significant difference compared to the negative control group (NC).

[0158] Example 5: Evaluation of the soothing activity of the peptide

[0159] This embodiment uses a UV-induced dedifferentiation HaCaT cell model (TRPV1 protein expression upregulated) to evaluate the soothing efficacy of the peptides in this application by detecting the intracellular TRPV1 content in blast cells using immunofluorescence staining. HaCaT cells (purchased from the Kunming Cell Bank of the Chinese Academy of Sciences) were dedifferentiated prior to the experiment to make their biological characteristics closer to those of basal skin cells, thus more accurately mimicking the TRPV1 protein upregulation mechanism.

[0160] Experimental methods:

[0161] (1) HaCaT cells were cultured in calcium-free, glutamine-free DMEM high-glucose medium combined with calcium ion adsorption resin (Chelex 100, GIBCO) for half a month to obtain dedifferentiated HaCaT cells.

[0162] (2) Take dedifferentiated HaCaT cells that are in good growth condition and in the mid-to-late logarithmic growth stage, digest the cells, count them, adjust the cell density, and seed the cells into 24-well cell culture plates (with sterile cell spreaders added to the culture plates beforehand), and place them in a cell culture incubator for culture.

[0163] (3) Discard the culture medium in the wells, add the corresponding culture medium to each group according to the experimental group (Table 7), and place them in a cell culture incubator to continue culturing;

[0164] (4) Remove the cell plate, aspirate the supernatant, wash with PBS, and expose the cells to UVA for irradiation (UV340B ultraviolet irradiator, Xinbao Scientific Instruments).

[0165] (5) After irradiation, discard the PBS, add the corresponding culture medium to each group, and place them in a cell culture incubator for continued culture.

[0166] (6) Remove the culture plate, discard the culture medium in the wells, wash with PBS and then discard;

[0167] (7) Add paraformaldehyde (Beyotime) fixative to fix the cells, wash with PBS and discard;

[0168] (8) Permeabilize with Triton X-100 solution (Shanghai Sangon Biotech), wash with PBS and discard;

[0169] (9) Add bovine serum albumin solution (Roche) for blocking treatment, wash with PBS and discard;

[0170] (10) Add Rabbit Anti-TRPV1 antibody (Boster) and Goat Anti-Rabbit IgG H&L (Alexa) respectively. Staining and incubating with 488)(Abcam) solution, followed by washing with PBS and discarding the solution;

[0171] (11) Counterstain with DAPI staining solution (Beyotime), wash with PBS and discard;

[0172] (12) Add a drop of anti-fluorescence quencher (Beyotime) to a glass slide, cover it with a cell cover, and observe and photograph it under an inverted fluorescence microscope (Nikon).

[0173] Table 7: Experimental Groups in Example 5

[0174] Fluorescence intensity was measured using ImageJ 1.53t software, and corrected total cell fluorescence (CTCF) was calculated using the following formula:

[0175] CTCF = Cumulative optical density – Area of ​​the cell region being tested × Average background optical density

[0176] Data were statistically analyzed using GraphPadPrism 7.00 software and independent samples t-test. A p-value < 0.05 was considered statistically significant.

[0177] The results are shown in Table 8 and Figure 8. Under the experimental conditions, the TRPV1 CTCF of the negative control group (NC) was 167,375,582.27, and the TRPV1 CTCF of the model control group (M) was 330,115,277.41. Compared with the negative control group (NC), the TRPV1 CTCF of the model control group (M) was significantly increased (p<0.05). The TRPV1 CTCF of the positive control group (PC) was 140,553,790.00. Compared with the model control group, the TRPV1 CTCF of the positive control group (PC) was significantly decreased, indicating that the experimental system was working normally.

[0178] Compared with the model control group (M), 0.125 mM and 0.0625 mM palmitoyl peptide or myristoyl peptide can significantly reduce the overexpression of TRPV1 protein in keratinocytes irradiated by UVA, indicating that the palmitoyl peptide or myristoyl peptide provided in this application can reduce the content of TRPV1 in keratinocytes after UV damage modeling and has the potential to soothe skin sensitivity reactions caused by ultraviolet radiation.

[0179] Table 8: Analysis data of TRPV1 immunofluorescence cytochemical staining results Note: P<0.001 indicates an extremely significant difference compared to the model control group (M); P<0.01 indicates a highly significant difference compared to the model control group (M); P<0.05 indicates a significant difference compared to the model control group (M); P>0.05 indicates no significant difference compared to the model control group (M).

[0180] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A polypeptide, wherein, The polypeptide comprises an amino acid sequence having at least 70%, at least 75%, and at least 85% identity with SEQ ID NO:

1.

2. The polypeptide according to claim 1, wherein, The polypeptide has the amino acid sequence shown in SEQ ID NO:

1.

3. The polypeptide according to claim 1 or 2, wherein, The polypeptide is modified; optionally, the N-terminus, C-terminus, and / or amino acid residue side chains of the polypeptide are chemically modified; more preferably, the chemical modification includes at least one of palmitoylation and myristoylation.

4. The polypeptide according to any one of claims 1-3, wherein, The polypeptide comprises a salt form, optionally including at least one of acetate, trifluoroacetate, hydrochloride, sulfate, citrate, and salicylate.

5. A method for preparing the polypeptide according to any one of claims 1-4.

6. A nucleic acid encoding the polypeptide of any one of claims 1-4.

7. An expression vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 and / or the expression vector of claim 7.

9. A composition, characterized in that, The composition comprises the polypeptide of any one of claims 1-4, the nucleic acid of claim 6, the expression vector of claim 7, and / or the host cell of claim 8, and one or more acceptable excipients.

10. The composition according to claim 9, wherein, The composition includes creams, lotions, solutions, ointments, pastes, sprays, suspensions, gels, masks, tablets, capsules, granules, or powders.

11. The composition according to claim 9 or 10, wherein, The composition contains a second active ingredient.

12. Use of the polypeptide of any one of claims 1-4, the nucleic acid of claim 6, the expression vector of claim 7, the host cell of claim 8, and / or the composition of any one of claims 9-11 in the preparation of cosmetic products for moisturizing, anti-oxidation, soothing, firming, anti-wrinkle, oil control, whitening, or any combination thereof.

13. Use of the polypeptide of any one of claims 1-4, the nucleic acid of claim 6, the expression vector of claim 7, the host cell of claim 8, and / or the composition of any one of claims 9-11 in the preparation of a pharmaceutical product for moisturizing, anti-oxidation, soothing, firming, anti-wrinkle, oil control, whitening, or any combination thereof.

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