New polypeptide and use thereof

Abstract

Provided are a new polypeptide and a preparation method therefor, a nucleic acid, an expression vector, a host cell, a composition and the use of the polypeptide. The polypeptide has many benefits for the skin. For example, the polypeptide has moisturizing, antioxidant, soothing, firming, anti-wrinkle, oil-control and / or whitening effects. Thus, the polypeptide has broad applications in the preparation of cosmetics for a moisturizing, antioxidant, soothing, firming, anti-wrinkle, oil-control or whitening purpose or any functional combination thereof, and / or in the preparation of a drug for a moisturizing, antioxidant, soothing, firming, anti-wrinkle, oil controlling or whitening purpose or any functional 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 and elastin are key to maintaining youthful, healthy skin. As we age, the body produces less collagen and elastin, which 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 moisturizing, antioxidant, soothing, firming, anti-wrinkle, oil-controlling and / or whitening abilities.

[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 peptide cytotoxicity test on HaCaT cells in Example 2.1.

[0027] Figure 2 shows the effect of peptides and positive controls on AQP3 expression in HaCaT cells in Example 2.2, where "**" indicates P<0.01.

[0028] Figure 3 shows an inverted fluorescence microscope image of the HaCaT cell UV damage model in Example 4 after treatment with peptides and a positive control.

[0029] Figure 4 shows the total cellular fluorescence statistics after TRPV1 immunofluorescence cytochemical staining correction in Example 4. In the figure, "*" indicates a significant difference compared with the model control group (M) (P<0.05), "**" indicates an extremely significant difference compared with the model control group (M) (P<0.01), and "***" indicates an extremely significant difference compared with the model control group (M) (P<0.001).

[0030] Figure 5 shows the results of the peptide cytotoxicity test on FB cells in Example 5.1.

[0031] Figure 6 shows the results of the effect of peptides and positive controls on elastin expression in FB cells in Example 5.2, where "*" indicates P<0.05 and "**" indicates P<0.01.

[0032] Figure 7 shows the effect of peptides and positive controls on the expression level of type I collagen in FB cells in Example 6, where "**" indicates P<0.01.

[0033] Figure 8 shows an inverted fluorescence microscope image of the SZ95 cell dihydrotestosterone-induced model in Example 7.2 after treatment with peptides and a positive control.

[0034] Figure 9 shows the results of the peptide's inhibition of tyrosinase in vitro in Example 8.2.

[0035] Figure 10 shows the effect of peptides and positive controls on the relative melanin content in B16-F10 cells in Example 8.3, where "**" indicates P<0.01. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] When used in this document, the terms “including,” “contains,” or “comprising” mean that other elements are not excluded in addition to the elements listed.

[0040] 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).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] When used in this document, the term "chemical modification" refers to the method of modifying a protein or nucleic acid by introducing or removing chemical groups.

[0046] 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.

[0047] As used herein, the term "acetylation modification" refers to modification involving the addition of an acetyl group (-C=OCH3). As 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, the peptide can form a salt when a free amino group (e.g., Arg, Lys, and His) is present at the N-terminus of the peptide sequence or on its side chain.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] When used in this article, the term "Claudin-1 protein" refers to a tight junction protein (TJP), which is one of the main components of tight junctions.

[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 movement of water in and out of the cell. 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 ability can be reflected, and the moisturizing efficacy of peptides can be evaluated.

[0057] In this article, the term "NRF2 protein" refers to nuclear factor (erythrocyte-derived 2)-associated factor 2 protein, with the gene name NFE2L2. NRF2 protein is a key transcription factor primarily involved in regulating cellular antioxidant responses. It helps cells resist oxidative stress and damage from toxic substances by binding to antioxidant response elements (AREs) and activating various protective genes. Under normal conditions, NRF2 protein binds to Keap1 protein (Kelch ECH-associated protein 1) and is degraded. However, under oxidative stress, NRF2 protein is activated and translocated to the nucleus, promoting the expression of antioxidant genes.

[0058] 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, and also synthesize and secrete matrix components such as glycosaminoglycans and glycoproteins. Collagen and elastin significantly affect skin elasticity and are important indicators for evaluating anti-wrinkle efficacy.

[0059] 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.

[0060] When used in this article, the term "Nile Green staining solution" refers to a lipophilic staining solution. Nile Green fluoresces strongly in lipid-rich environments but exhibits little fluorescence in aqueous media. It is an important staining agent for detecting intracellular lipid droplets using fluorescence microscopy, flow cytometry, or fluorescence microplate readers. Unlike Nile Red, which has a broad fluorescence spectrum, Nile Green stains intracellular lipid droplets only with green fluorescence.

[0061] When used in this article, the term "Nile Red" refers to a lipophilic staining solution. Nile Red exhibits environmentally sensitive fluorescence, showing strong fluorescence in lipid-rich environments and weak fluorescence in aqueous media. Nile Red is an excellent and important staining agent for detecting intracellular lipid droplets using fluorescence microscopy and flow cytometry, staining intracellular lipid droplets red at wavelengths of 559 / 635 nm.

[0062] As used in this application, the term "BCA method" refers to the biquinoline carboxylic acid assay. BCA analysis relies primarily on two reactions. First, under alkaline conditions, the peptide bonds in protein molecules react with Cu... 2+ Complexation forms a complex, while Cu 2+ Reduced to Cu + Diquinoline carboxylic acid and its sodium salt are water-soluble compounds that, under alkaline conditions, can react with Cu. + They combine to form a stable purple-blue complex with a high light absorption value at 562 nm, and the intensity of the compound's color is directly proportional to the protein concentration, which can be used to determine the protein concentration.

[0063] polypeptide

[0064] 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.

[0065] 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.

[0066] In some embodiments, the polypeptide is modified. In some embodiments, the modification includes chemical modification.

[0067] In some embodiments, the N-terminus, C-terminus, and / or amino acid residue side chains of the peptide are chemically modified.

[0068] In some embodiments, the N-terminus of the polypeptide is chemically modified.

[0069] In some implementations, chemical modifications include, but are not limited to, acetylation modifications.

[0070] In some implementations, the polypeptide includes a salt form.

[0071] In some embodiments, the salt form includes acetate, trifluoroacetate, hydrochloride, sulfate, citrate, or salicylate. In some embodiments, the salt form includes acetate.

[0072] Acetic acid has low biotoxicity and is a weak acid, so it has little effect on peptide activity.

[0073] Preparation method

[0074] Secondly, this application provides a method for preparing the polypeptide of the first aspect of this application.

[0075] In some implementations, the polypeptide is prepared by biosynthesis.

[0076] In some embodiments, the polypeptide is prepared by chemical synthesis.

[0077] 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).

[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 (SZ95 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] 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.

[0094] 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.

[0095] In some implementations, the medicine includes, but is not limited to, ointments for use on the skin surface.

[0096] Methods of prevention, treatment, relief or improvement

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] Example

[0102] 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.

[0103] Example 1: Synthesis of Polypeptides

[0104] The sequence of the polypeptide is shown in Table 1 below, synthesized and provided by Chengdu Kaijie Peptide Technology Co., Ltd. The N-terminus of the polypeptide has a 1-equivalent acetylation (-C=OCH3) modification.

[0105] Table 1: Peptide amino acid sequence

[0106] Example 2: Safety and moisturizing activity evaluation of peptides

[0107] 2.1 Cytotoxicity test of peptides

[0108] The cytotoxicity doses were designed as eight concentration gradients: 0.5000%, 0.3500%, 0.2500%, 0.1250%, 0.0625%, 0.0313%, 0.0156%, and 0.0100%. MEM complete culture medium was also set up as a negative control (NC).

[0109] Experimental Methods: Immortalized human 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 hours. When cell confluence reached approximately 80%, culture medium containing different concentrations of peptides was added, along with a negative control. Culture was continued for another 48 hours. Cell viability was then measured using the CCK8 assay at 450 nm after a 2-hour incubation period. Cell viability = (OD value of sample group / average OD value of negative control group) × 100%

[0110] 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).

[0111] The results are shown in Table 2 and Figure 1. The survival rate of HaCaT cells reached 80% or higher at all concentrations, indicating that the peptides of this application have good biosafety.

[0112] Table 2: Results of peptide cytotoxicity assays against HaCaT cells

[0113] 2.2 Moisturizing Activity Test of Peptides

[0114] This embodiment is based on a human keratinocyte model. The moisturizing efficacy of the peptides in this application is evaluated by detecting the expression level of aquaporin 3 (AQP3) in the cells.

[0115] 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 peptides (0.1250%, 0.0625%, and 0.0313%) was added. A negative control (MEM complete medium) and a positive control (PC, TGF-α, 1 μg / mL) were also included. The cells were cultured for another 48 h. The expression level of aquaporin 3 was detected using the Human AQP3 (Aquaporin 3) ELISA Kit (Wuhan Huamei Biotechnology Co., Ltd., batch number: F03027442). AQP3 enhancement rate = (BA) / A

[0116] 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.

[0117] The results are shown in Table 3 and Figure 2. Compared with the negative control group, the expression levels of AQP3 in the 0.1250% and 0.0625% sample groups were significantly increased (P<0.01), with an increase rate of 39.5% and 30.4%, respectively. This indicates that the peptide promotes the expression of AQP3 and has a moisturizing effect.

[0118] Table 3: AQP3 Expression Levels

[0119] 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).

[0120] Example 3: Evaluation of the antioxidant activity of peptides

[0121] In this embodiment, the antioxidant efficacy of the peptides in this application was evaluated by stimulating HaCaT cells with H2O2 and measuring the NRF2 protein content using Western blotting.

[0122] Sample preparation: Calculate the appropriate volume of cell culture medium according to the peptide mass and purity to prepare a 10% (w / v) sample solution. Dilute the solution with culture medium to concentrations of 1%, 0.1%, and 0.01% for later use.

[0123] Experimental Methods: HaCaT cells were seeded in 35 mm Petri dishes and cultured at 37℃ / 5% CO2 for 48 h. When cell confluence reached 85%, H2O2 was used to stimulate cell modeling. Culture media containing different concentrations of peptides were added. Untreated cells served as a negative control, while H2O2-treated cells without peptides served as a model control. Cells were cultured for another 4 h, and cytosolic proteins were extracted and stored for later use. Protein concentration was determined using the BCA method. NRF2 protein content was determined by Western blotting, and statistical data were output using ImageJ.

[0124] The results are shown in Table 4. Compared with the model control, the addition of 1% and 0.1% concentrations of the peptide of this application increased the expression level of NRF2 protein in cells in a dose-dependent manner. This result indicates that the peptide of this application has antioxidant effects.

[0125] Table 4: NRF2 protein expression levels

[0126] Example 4: Evaluation of the soothing activity of the peptide

[0127] 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.

[0128] Experimental methods:

[0129] (1) HaCaT cells were cultured in calcium-free and glutamine-free medium with serum treated with Chelex 100 resin for half a month to obtain dedifferentiated HaCaT cells.

[0130] (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.

[0131] (3) Discard the culture medium in the wells, add the corresponding culture medium to each group according to the experimental group (Table 5), and place them in a cell culture incubator to continue culturing;

[0132] (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).

[0133] (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.

[0134] (6) Remove the culture plate, discard the culture medium in the wells, wash with PBS and then discard;

[0135] (7) Add paraformaldehyde (Beyotime) fixative to fix the cells, wash with PBS and discard;

[0136] (8) Permeabilize with Triton X-100 solution (Shanghai Sangon Biotech), wash with PBS and discard;

[0137] (9) Add bovine serum albumin solution (Roche) for blocking treatment, wash with PBS and discard;

[0138] (10) Add RabbitAnti-TRPV1 antibody (Boster) and GoatAnti-Rabbit IgG H&L (Alexa) respectively. Staining and incubating with 488)(Abcam) solution, followed by washing with PBS and discarding the solution;

[0139] (11) Counterstain with DAPI staining solution (Beyotime), wash with PBS and discard;

[0140] (12) Add a drop of anti-fluorescence quencher (Beyotime) to the glass slide, cover it with the cell climbing slide, and observe and photograph it under an inverted fluorescence microscope (Nikon) (Figure 3).

[0141] Table 5: Experimental Groups in Example 4

[0142] Fluorescence intensity was measured using ImageJ 1.53t software, and corrected total cell fluorescence (CTCF) was calculated using the following formula: CTCF = Cumulative optical density – Area of ​​the cell region being measured × Average background optical density

[0143] Data were statistically analyzed using GraphPadPrism 7.00 software and independent samples t-test. A p-value < 0.05 was considered statistically significant.

[0144] The results are shown in Table 6 and Figure 4. 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. The peptide provided in this application showed that the CTCF of cells at concentrations of 0.125%, 0.0625%, and 0.0313% were 172,760,844.60, 197,380,115.00, and 275,150,125.13, respectively. Compared with the model control group (M), the reduction rates were 47.67%, 40.21%, and 16.65%, respectively, all of which were significantly lower than those of the model control group (M). This indicates that the peptide provided in this application can reduce the TRPV1 content in keratinocytes after UV damage modeling at concentrations of 0.125%, 0.0625%, and 0.0313%.

[0145] In summary, in this embodiment, 0.125%, 0.0625%, and 0.0313% of the peptides can significantly reduce the TRPV1 content in keratinocytes after UV damage modeling (P<0.05), demonstrating a soothing effect.

[0146] Table 6: Analysis data of TRPV1 immunofluorescence cytochemical staining results

[0147] Example 5: Safety and firming activity evaluation of peptides

[0148] 5.1 Cytotoxicity test of peptides

[0149] The cytotoxicity doses were designed as eight concentration gradients: 0.4000%, 0.2800%, 0.2000%, 0.1000%, 0.0500%, 0.0250%, 0.0125%, and 0.0080%. FB cell complete culture medium was also set up as a negative control (NC).

[0150] Experimental Methods: Human primary fibroblasts (FB cells, passage 16, purchased from Guangdong Boxi Biotechnology Co., Ltd.) were seeded in 96-well plates and cultured at 37℃ / 5% CO2 for 24-48 hours. When the cell confluence reached approximately 80%, culture medium containing different concentrations of peptides was added, along with a negative control. The cells were cultured for another 48 hours. The absorbance was then measured at 450 nm using the CCK8 assay after a 2-hour incubation period. Cell viability was calculated as: (OD value of sample group / average OD value of negative control group) × 100%.

[0151] 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 7 and Figure 5. The survival rate of FB cells reached 88% or above at each concentration, indicating that the peptide of this application has good biosafety.

[0152] Table 7: Results of peptide cytotoxicity assays against FB cells

[0153] 5.2 Tests on the firming activity of peptides

[0154] This embodiment evaluates the firming effect of the peptides in this application by detecting the expression level of elastin in human primary fibroblasts.

[0155] Experimental Methods: Human primary fibroblasts (FB cells, passage 16, purchased from Guangdong Boxi Biotechnology Co., Ltd.) 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 peptides (0.1250%, 0.0625%, and 0.0313%) was added. A negative control (FB cell complete culture medium) and a positive control (PC, TGF-β1, 100 ng / mL) were also provided. The cells were cultured for another 48 h. The expression level of elastin in the cells was detected using the Human Elastin (ELN) ELISA Kit (Wuhan Huamei Biotechnology Co., Ltd., batch number: Y07022763). Elastin enhancement rate = (BA) / A

[0156] In the formula: A represents the average elastin expression level in the negative control group; B represents the average elastin expression level in the positive control group or sample 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.

[0157] The results are shown in Table 8 and Figure 6. Compared with the negative control group, the elastin expression levels in the 0.1250% and 0.0625% sample groups were significantly increased (P<0.01), with increases of 42.1% and 27.1%, respectively; the elastin expression level in the 0.0313% sample group was significantly increased (P<0.05), with an increase of 16.0%, indicating that the peptide promoted elastin expression and had a firming effect. Specifically, the firming activity at a concentration of 0.1250% was comparable to that of the positive control.

[0158] Table 8: Results of Elastin Expression Levels

[0159] Note: P<0.05 indicates a significant difference compared to the negative control group (NC); P<0.01 indicates a highly significant difference compared to the negative control group (NC).

[0160] Example 6: Evaluation of the anti-wrinkle activity of peptides

[0161] This embodiment evaluates the anti-wrinkle efficacy of the peptides in this application by detecting the expression level of type I collagen in human primary fibroblasts.

[0162] Experimental Methods: Human primary fibroblasts (FB cells, passage 16, purchased from Guangdong Boxi Biotechnology Co., Ltd.) 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 peptides (0.1250%, 0.0625%, and 0.0313%) was added. A negative control (FB cell complete culture medium) and a positive control (PC, TGF-β1, 100 ng / mL) were also provided. The cells were cultured for another 48 h. The expression level of type I collagen in the cells was detected using the Human Collagen Type I (Col I) ELISA Kit (Wuhan Huamei Biotechnology Co., Ltd., batch number: Y06022762). Collagen enhancement rate = (BA) / A

[0163] In the formula: A represents the average expression level of type I collagen in the negative control group; B represents the average expression level of type I collagen in the positive control group or sample 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.

[0164] The results are shown in Table 9 and Figure 7. Compared with the negative control group, the expression levels of type I collagen in the 0.1250% and 0.0625% sample groups were significantly increased (P<0.01), with an increase rate of 47.7% and 38.4%, respectively. This indicates that the peptide in this application promotes the expression of type I collagen and has anti-wrinkle effects.

[0165] Table 9: Results of Type I Collagen Expression Levels

[0166] 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).

[0167] Example 7: Safety evaluation and oil-controlling activity evaluation of peptides

[0168] 7.1 Cytotoxicity test of peptides

[0169] The cytotoxic doses were designed to be 0.0625%, 0.03125%, 0.0156%, and 0.0100%, for a total of four concentration gradients. DMEM complete medium was also set up as a negative control (NC).

[0170] Experimental methods: Human sebaceous gland cells (SZ95 cells, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) were seeded in 96-well plates (5×10⁶ cells / wells). 3Cells were cultured at 37℃ / 5% CO2 for 24 hours. When cell confluence reached approximately 80%, culture medium containing different concentrations of peptides was added, along with a negative control. This was repeated three times biologically, and cultured for another 24 hours. After the initial culture, MTT solution was added to each well, and the cells were cultured for another 4 hours. The culture medium was then removed, DMSO solution was added, and the mixture was shaken to mix. The absorbance at 490 nm was measured using a microplate reader.

[0171] The experimental data were imported into statistical analysis software to calculate the mean, standard deviation, and p-value for each treatment group.

[0172] The results are shown in Table 10. The cell viability of SZ95 cells reached about 85% or more at concentrations of 0.0625% and below, indicating that the peptides of this application have good biosafety.

[0173] Table 10: Results of peptide activity assay for SZ95 cells

[0174] Note: Statistical results are expressed as mean ± SD, and statistical analysis was performed using SPSS software. One-way ANOVA was used for statistical analysis among multiple groups. The statistical results were compared with the control group using α = 0.05 as the test limit, and P < 0.05 was considered statistically significant.

[0175] 7.2 Oil-controlling activity test of peptides

[0176] Sebaceous glands are the source and target tissue of androgens. Dehydroepiandrosterone (DHEA) is produced in sebaceous glands, which, through a series of enzymatic catalysis, is converted into the most active forms, testosterone and 5α-dihydrotestosterone (5α-DHT). These stimulate lipid synthesis and the proliferation and differentiation of sebaceous gland cells. During this proliferation and differentiation process, undifferentiated juvenile cells proliferate first, with some daughter cells forming lipid droplets and migrating towards the center of the gland cell, gradually developing and differentiating into mature sebaceous gland cells capable of storing and secreting lipids. This embodiment uses a human sebaceous gland cell model induced by dihydrotestosterone (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D413176-25mg). The amount of sebum secreted by sebaceous gland cells was compared using Nile Red (Sigma-Aldrich, catalog number: 7385-67-3) staining to evaluate the oil-controlling efficacy of the peptides in this application.

[0177] Experimental method: Cells were seeded in 12-well plates (1×10⁻⁶ cells / well). 5 (samples / well), incubated at 37℃ with 5% CO2 for 24 hours. Experimental groups are as follows:

[0178] After incubation, the culture medium was removed, and the cells were washed 1-2 times with D-Hanks buffer. The normal control group was replaced with fresh culture medium, the model control group with fresh culture medium containing dihydrotestosterone (DHT), the positive control group with fresh culture medium containing isotretinoin and DHT, and the peptide group with fresh culture medium containing different concentrations of peptide (0.015625%, 0.03125%, and 0.0625%) and DHT. Three biological replicates were performed, and the cells were incubated at 37°C with 5% CO2 for 24 hours. After incubation, the culture medium was removed, the cells were pre-cooled, washed 3 times with D-Hanks buffer, fixed with 4% paraformaldehyde, washed again with D-Hanks buffer, stained with Nile Red, and photographed using an inverted fluorescence microscope (Nikon), as shown in Figure 8. Image J analysis was used to determine the average fluorescence intensity of each group, as shown in Table 11.

[0179] In the formula, A represents the average fluorescence intensity.

[0180] Table 11: Detection Results of Average Fluorescence Intensity

[0181] 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 of multiple groups. The statistical results were defined with α = 0.05 as the test cutoff, and P < 0.05 (compared to the model control group) was considered statistically significant. The mean fluorescence intensity of the model control group was significantly enhanced compared to the normal control group, indicating that the stimulation conditions in this test were effective.

[0182] The average fluorescence intensity of the model control group was significantly enhanced compared with that of the normal control group, indicating that the stimulation conditions in this experiment were effective. Compared with the model control group, the average fluorescence intensity of the peptide group was significantly reduced (significantly reduced by 34%, 42%, and 32% at concentrations of 0.015625%, 0.03125%, and 0.0625%, respectively), indicating that the peptide of this application has oil-controlling effects.

[0183] 7.3 Assay for peptide inhibition of 5α-reductase activity

[0184] This embodiment evaluates the oil-controlling efficacy of the peptide by detecting its inhibitory effect on 5α-reductase. [Please provide the experimental procedures for the peptide's inhibition of 5α-reductase activity.]

[0185] Experimental Method: Weigh 0.0500 g of peptide, add primary water to a final volume of 10 mL to prepare a 0.500% solution, then dilute with primary water to prepare 0.250% and 0.125% solutions. The peptide solutions at concentrations of 0.125%, 0.250%, and 0.500% were used for testing. A 1 g / L finasteride solution was used as a positive control.

[0186] Place 30 2mL centrifuge tubes on a centrifuge rack, dividing them into 10 groups, with 3 replicates per group. Add reagents and incubate according to the following steps:

[0187] OD 340 The differences are shown in the table below:

[0188] Note: P<0.05 indicates a significant difference compared to the negative control. 5α-Reductase inhibition rate (%) = [(T0-T) / T0] × 100%

[0189] In the formula, T0: negative control OD 340 The average of the differences (N1-N2); T: OD of the peptide or positive control. 340 Difference (T1-T2 or P1-P2).

[0190] Table 12: Results of 5α-reductase inhibition rate

[0191] Note: P<0.05 indicates a significant difference compared to the negative control.

[0192] The results are shown in Table 12. The inhibition rates of 5α-reductase by the peptides at concentrations of 0.125%, 0.250%, and 0.500% were 7.3% ± 0.6%, 9.8% ± 0.8%, and 15.1% ± 0.8%, respectively. This indicates that the peptides of this application have a good inhibitory effect on 5α-reductase activity, reducing lipid synthesis and achieving oil control.

[0193] Example 8: Evaluation of the skin-whitening activity of the peptide

[0194] 8.1 Cytotoxicity test of peptides

[0195] The cytotoxic doses were designed to be 0.2500%, 0.1250%, 0.0625%, 0.0313%, 0.0156%, and 0.0100%, for a total of six concentration gradients. DMEM complete medium was also set up as a negative control (NC).

[0196] Experimental Methods: Mouse skin melanoma cells (B16-F10 cells, passage 18, purchased from Wuhan Pronosei Life Sciences Co., Ltd.) were seeded in 96-well plates and cultured at 37℃ / 5% CO2 for 24-48 hours. When the cell confluence reached approximately 80%, culture medium containing different concentrations of peptides was added, along with a negative control. The cells were cultured for another 48 hours. The absorbance was then measured at 450 nm using the CCK8 assay after a 2-hour incubation period. Cell viability was calculated as: (OD value of sample group / average OD value of negative control group) × 100%.

[0197] The experimental data were imported into statistical analysis software to calculate the mean, standard deviation, and coefficient of variation for each treatment group.

[0198] The results are shown in Table 13. The survival rate of B16-F10 cells at concentrations of 0.2500% and below reached 75% or higher, indicating that the peptides of this application have good biosafety at concentrations of 0.2500% and below.

[0199] Table 13: Results of peptide cytotoxicity assays against B16-F10 cells

[0200] 8.2 Assay for the in vitro inhibition of tyrosinase activity by peptides

[0201] Tyrosinase controls the melanin formation process, and its activity level plays a major role in the amount and deposition of melanin. By inhibiting tyrosinase activity or blocking the oxidation reaction from tyrosine to melanin, melanin production can be reduced, achieving a skin whitening effect. This embodiment evaluates the skin whitening efficacy of the peptide by detecting its inhibitory effect on tyrosinase.

[0202] Experimental method: Weigh the polypeptide, dissolve it in primary water, and bring the volume to 50 mL to prepare a 0.1250% solution. Then dilute it to 0.0313% and 0.0100% solutions, and measure out three concentration solutions for testing. Kojic acid (0.007 mg / mL) was used as a positive control.

[0203] Add the reagents and incubate in the following order, with three replicates per group: Tyrosinase inhibition rate (%) = [(T0-T) / T0] × 100%

[0204] In the formula, T0: negative control OD 475 The average of the differences (N1-N2); T: OD of the peptide or positive control. 475 Difference (T1-T2 or P1-P2).

[0205] The experimental data were imported into statistical analysis software to calculate the mean and standard deviation for each group. A significance analysis was then performed with a significance level of α = 0.05.

[0206] The results are shown in Tables 14 and 15 and Figure 9, indicating that the OD of the 0.1250% concentration of the peptide... 475 The difference (T1-T2) was significantly lower than (P<0.05) in the OD of the negative control. 475 The difference (N1-N2) and the tyrosinase inhibition rate were 13.86% ± 1.41%, indicating that the polypeptide of this application has a good whitening effect at a concentration of 0.1250%.

[0207] Table 14: OD 475 Difference

[0208] Note: P<0.05 indicates a significant difference compared to the negative control.

[0209] Table 15: Results of Tyrosinase Inhibition Rate

[0210] 8.3 Tests on the Inhibition of Melanin Synthesis by Peptides

[0211] This embodiment is based on a mouse skin melanoma cell (B16-F10) model. The whitening efficacy of the peptide in this application is evaluated by detecting the inhibitory effect of the peptide on melanin synthesis in the cells.

[0212] Experimental methods: Mouse skin melanoma cells (B16-F10 cells, passage 13, purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were seeded in 96-well plates and cultured at 37℃ / 5% CO2 for 24-48 h. When the cell confluence was about 80%, medium containing different concentrations of peptides (0.1250%, 0.0625%, 0.0313%) was added. A negative control (DMEM complete medium) and a positive control (PC, AA2G, 2-O-α-D-glucopyranosyl-L-ascorbic acid, 1%) were also set up. The cells were cultured for another 48 h, and the absorbance was read at 475 nm using a multi-functional microplate reader to calculate the relative melanin content of the cells.

[0213] The relative melanin content of cells is calculated using the following formula: Relative melanin content = (As / Ac) × 100%

[0214] As represents the absorbance value of the sample group or positive control group; Ac represents the absorbance value of the negative control group.

[0215] The amount of melanin reduction in cells is calculated using the following formula:

[0216] The decrease in cellular melanin content = relative melanin content of cells in the negative control group - relative melanin content of cells in the sample group or positive control group

[0217] The experimental data were imported into statistical analysis software, and the mean, standard deviation (SD), and coefficient of variation (CV) of 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.

[0218] The results are shown in Table 16 and Figure 10. Compared with the negative control group, the relative melanin content of cells in the 0.1250% polypeptide group was significantly reduced (P<0.01), with a melanin reduction of 19.71%. That is, the polypeptide of this application has a whitening effect by inhibiting melanin synthesis at a concentration of 0.1250%.

[0219] Table 16: Results of Relative Melanin Content Detection

[0220] 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).

[0221] 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 acetylation modification.

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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