A synthetic peptide and its composition and use

Abstract

Peptides of general formula (I), stereoisomers thereof, mixtures of stereoisomers, or cosmetically acceptable salts thereof or pharmaceutically acceptable salts thereof, or cosmetic or pharmaceutical compositions thereof, and their use for the treatment or care of skin or mucosal conditions, disorders or diseases.

Description

Technical Field

[0001] The present invention relates to peptides capable of promoting the proliferation of fibroblasts in the skin or mucosa, and cosmetic or pharmaceutical compositions containing these peptides and their use in treating or caring for the skin or mucosa. Background Art

[0002] Skin is the largest organ in the human body, covering the body surface. It plays an important barrier role for the body. It can resist mechanical damage, buffer external impact, prevent damage to tissues and organs in the body, prevent the invasion of harmful substances from the outside world, resist oxidation, resist ultraviolet damage, and prevent the loss of various nutrients, water, and electrolytes in the body. It plays an important role in maintaining the stability of the human body's internal environment. The skin is composed of the epidermis, dermis, and subcutaneous tissue. The outermost layer is the epidermis, which is mainly composed of keratinocytes, melanocytes, Langerhans cells, etc. The dermis is located below the epidermis and contains dermal cells, collagen fibers, elastic fibers, matrix, hair, sebaceous glands, sweat glands, blood vessels, etc. The dermis is tightly connected to the epidermis through the basement membrane.

[0003] The most important cells in the dermis are fibroblasts. Fibroblasts are large in size and diverse in shape, mostly in the form of protruding spindles or star-shaped flat structures. The cytoplasm contains rich and well-developed rough endoplasmic reticulum, Golgi apparatus and free ribosomes, so they have a strong ability to synthesize and secrete proteins, and can secrete extracellular matrix components such as procollagen, fibronectin and collagenase. Fibroblasts use amino acids such as proline and lysine to synthesize proalpha polypeptide chains on the ribosomes of the rough endoplasmic reticulum. After the polypeptide chains are transported to the Golgi apparatus, they form procollagen molecules. Procollagen molecules are released outside the cell through exocytosis. Under the catalysis of procollagen peptidase, the tail of the proalpha polypeptide chain of the procollagen molecule is removed to become tropocollagen molecules. Tropocollagen molecules are arranged in parallel rows and combined into collagen fibrils with periodic horizontal stripes. Collagen fibrils combine with each other to form collagen fibers. In addition to producing collagen fibers, fibroblasts can also produce elastic fibers. The main component of collagen fibers is type I collagen, which has strong tensile strength and gives the skin toughness. Elastic fibers are composed of elastin and filamentous protein, which give the skin elasticity. Collagen fibers, elastic fibers, and the extracellular matrix filling between fibers and cells provide support for the skin.

[0004] The morphology, physiology and mechanical properties of the skin change significantly with age. During the natural aging of the skin, the function of fibroblasts declines, collagen fibers and elastic fibers are damaged and broken, and the balance between the production and degradation of important biomolecules such as collagen in the skin tends to the degradation process with age, resulting in progressive thinning and tissue destruction of the dermis, and causing dermal relaxation and subsequent wrinkle formation. Prolonged exposure to sunlight or environmental pollutants accelerates the aging process of the skin. Ultraviolet rays inhibit the synthesis of collagen and fibronectin in fibroblasts, and ultraviolet radiation can cause high expression of matrix metalloproteinases (MMPs), causing serious damage to collagen in the dermis, resulting in the destruction of dermal integrity, reduced skin strength and elasticity, and accelerated skin aging.

[0005] Therefore, by promoting the proliferation of fibroblasts, enhancing their activity, and increasing the synthesis and secretion of components such as collagen, elastin, and glycosaminoglycans, the skin's mechanical properties such as flexibility, elasticity, and firmness that are lost with aging or exposure to sunlight and / or environmental pollutants can be improved, damaged skin can be repaired, and wrinkles can be reduced.

[0006] As the first line of defense of the human body, the skin is easily damaged by the outside world, which causes the skin to lose its barrier function, leading to weakened resistance. Non-pathogenic bacteria or conditional pathogens can invade the skin, induce infection, and even have a significant impact on physical health. Therefore, accelerating the repair of skin wounds is of great significance in diseases with difficult-to-heal wounds and postoperative repair.

[0007] Wound repair is a precise, complex, and phased dynamic process involving network regulation of multiple cells such as cell proliferation, migration, extracellular matrix degradation, angiogenesis, and epithelial tissue remodeling. Fibroblasts are the main cells for wound repair. Mature fibroblasts are in a quiescent state, with reduced cell volume, a long spindle shape, and reduced rough endoplasmic reticulum and Golgi bodies. Mature fibroblasts are called fibrocytes. Fibroblasts and fibrocytes can transform into each other under certain conditions. Under the stimulation of trauma and other factors, some fibrocytes are transformed back into immature fibroblasts, and their synthesis and secretion functions are restored, stimulating the production of new extracellular matrix. A large amount of extracellular matrix and new capillaries form granulation tissue to fill the defective tissue. In the repair process, the proliferation and migration of fibroblasts are crucial to the formation of granulation tissue and the repair of wounds. Studies have shown that fibroblasts are mainly involved in the proliferation and tissue remodeling phases of wound repair. In the proliferation phase, fibroblasts proliferate, migrate, and form granulation tissue in large quantities, which mainly play a role in resisting infection and filling wounds. During the tissue remodeling period, myofibroblasts, endothelial cells and macrophages differentiated from fibroblasts undergo apoptosis, and ultimately the dermis is filled with extracellular matrix proteins, achieving the purpose of wound repair and maintaining the structural and functional integrity of the skin.

[0008] In summary, fibroblasts play an important role in skin aging and wound repair. It is of great significance to develop products that can promote fibroblast proliferation, enhance fibroblast activity, increase the synthesis and secretion of collagen, elastin, glycosaminoglycans and other components, so as to compensate for the loss of extracellular matrix such as collagen and elastin due to aging or exposure to sunlight and / or environmental pollutants, delay aging, fill the gaps in necrotic tissue areas after skin damage, and promote wound repair. Summary of the invention

[0009] The inventors of the present invention have unexpectedly found through a large number of experimental studies that the proliferation and activity of fibroblasts can be regulated by certain synthetic peptides. The present invention aims to provide a peptide that can promote the proliferation of fibroblasts in the skin or mucous membranes. These peptides and cosmetic compositions or pharmaceutical compositions containing these peptides can improve or prevent the treatment or care of skin or mucous membrane conditions, disorders or diseases by promoting the proliferation of fibroblasts, thereby improving the skin barrier function or treating, preventing or repairing skin aging or photoaging, or for re-epithelialization or healing of the skin or mucous membranes.

[0010] In view of this, the present invention provides a peptide represented by general formula (I), or a stereoisomer thereof, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, characterized in that:

[0011]

[0012] In formula (I),

[0013] R2 is selected from: H, alkyl, alkenylalkyl, carboxyalkyl, esteralkyl, alkylthiol, carboxyalkylthiol, or

[0014]

[0015] In formula (I) and formula (i),

[0016] n is selected from: 1 or 2;

[0017] R1 is selected from: H or R4-CO-, wherein R4 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

[0018] R3 is selected from: -NR5R6 or -OR5, wherein each R5 and R6 are independently selected from: H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

[0019] The groups R1 and R3 are bound to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of these peptide sequences, respectively.

[0020] The alkyl group in the “alkyl”, “alkenylalkyl”, “carboxyalkyl”, “esteralkyl”, “alkylthiol” and “carboxyalkylthiol” refers to a saturated aliphatic straight or branched chain alkyl group having 1 to 24 carbon atoms (optionally having 1 to 16 carbon atoms; optionally having 1 to 14 carbon atoms; optionally having 1 to 12 carbon atoms; optionally having 1, 2, 3, 4, 5, or 6 carbon atoms); may be selected from: methyl, ethyl, isopropyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 2-ethylhexyl, 2-methylbutyl, or 5-methylhexyl;

[0021] The alkenyl in the "alkenyl" and "alkenylalkyl" refers to a straight or branched alkenyl having 2-24 carbon atoms (optionally having 2-16 carbon atoms; optionally having 2-14 carbon atoms; optionally having 2-12 carbon atoms, optionally having 2, 3, 4, 5, or 6 carbon atoms), the "alkenyl" having one or more carbon-carbon double bonds, optionally having 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the "alkenyl" is bonded to the rest of the molecule through a single bond; it can be selected from: vinyl, oleyl, or linoleyl;

[0022] The ester group in the "ester alkyl" is a R(C=O)OR' group, wherein R and R' are each independently a single bond, an alkyl group or an alkylene group;

[0023] As is understood in the art, there may be a degree of substitution in the above-mentioned groups. Thus, there may be substitution in any group in the present invention. The substituted groups mentioned in this document in the groups of the present invention indicate that the specified group can be substituted with one or more substituents at one or more available positions, preferably at 1, 2, or 3 positions, more preferably at 1 or 2 positions, and more preferably at 1 position. These substituents include, for example, and are not limited to: C1-C4 alkyl; hydroxyl; C1-C4 alkoxy; amino; C1-C4 aminoalkyl; C1-C4 carbonyloxy; C1-C4 oxycarbonyl; halogen (such as fluorine, chlorine, bromine, and iodine); cyano; nitro; azide; C1-C4 alkylsulfonyl; thiol; C1-C4 alkylthio; C6-C 30 Aryloxy such as phenoxy; -NR b (C=NR b )NR b R c , where R b and R c are independently selected from: H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C 10 Cycloalkyl, C6-C 18 Aryl, C7-C 17an aralkyl group, a heterocyclic group having three to ten members, or a protecting group for an amino group.

[0024] Optionally, in formula (I), when n is 2, R2 is selected from: H, alkyl, alkenyl alkyl, carboxyalkyl, esteralkyl, alkylthiol or carboxyalkylthiol.

[0025] Optionally, R1 is selected from: H, acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl or linoleoyl.

[0026] Optionally, R1 is selected from H, acetyl, lauroyl, myristoyl or palmitoyl.

[0027] Alternatively, R1 is H, acetyl or palmitoyl.

[0028] Optionally, R2 is selected from: H, -CH3, -CH2CH3, -CH2CH=CH2, -CH2COOH, -CH2COOC(CH3)3, -S-CH2CH3, -S-(CH2)5CH3, -S-CH2COOH or

[0029]

[0030] Optionally, when n is 2, R2 is selected from: H, -CH3, -CH2CH3, -CH2CH=CH2, -CH2COOH, -CH2COOC(CH3)3, -S-CH2CH3, -S-(CH2)5CH3 or -S-CH2COOH.

[0031] Optionally, R5 and R6 are independently selected from: H, methyl, ethyl, hexyl, dodecyl or hexadecyl;

[0032] Optionally, R5 is H and R6 is selected from: H, methyl, ethyl, hexyl, dodecyl and hexadecyl;

[0033] Optionally, R3 is -OH or -NH2.

[0034] The peptide represented by formula (I), or its stereoisomer, or mixture of stereoisomers, or cosmetically acceptable salt thereof, or pharmaceutically acceptable salt thereof, is selected from the following peptides (1)-(46):

[0035] (1)

[0036] (2)H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH;

[0037] (3)

[0038] (4)

[0039] (5)

[0040] (6)

[0041] (7)

[0042] (8)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH;

[0043] (9)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH2;

[0044] (10)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH;

[0045] (11)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH2;

[0046] (12)

[0047] (13)

[0048] (14)

[0049] (15)

[0050] (16)

[0051] (17)

[0052] (18)

[0053] (19)

[0054] (20)

[0055] (21)

[0056] (22)

[0057] (23)

[0058] (24)

[0059] (25)

[0060] (26)

[0061] (27)

[0062] (28)

[0063] (29)

[0064] (30)

[0065] (31)

[0066] (32)

[0067] (33)

[0068] (34)

[0069] (35)

[0070] (36)

[0071] (37)

[0072] (38)

[0073] (39)

[0074] (40)

[0075] (41)

[0076] (42)H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2;

[0077] (43)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH;

[0078] (44)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2;

[0079] (45)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH;

[0080] (46) Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2.

[0081] The peptides of formula (I) of the present invention may exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids contained therein may have L-, D-configurations, or may be racemic independently of one another. Thus, it is possible to obtain isomeric mixtures as well as racemic mixtures or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and what isomers or isomeric mixtures are present. The preferred structure of the peptides of formula (I) of the present invention is a pure isomer, i.e., an enantiomer or a diastereomer.

[0082] For example, when -Phe- is described in the present invention, it should be understood that -Phe- is selected from -L-Phe-, -D-Phe-, or a mixture of the two, and is racemic or non-racemic. The preparation methods described in this document enable a person skilled in the art to obtain each stereoisomer of the peptide of the present invention by selecting amino acids with the correct configuration.

[0083] The term "cosmetically acceptable salt or pharmaceutically acceptable salt" refers to a salt approved for use in animals, and more specifically in humans, including metal salts of the peptide shown in formula (I), the metal including, but not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, etc.; including salts formed by the peptide shown in formula (I) and organic bases, the organic bases including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine, etc.; including salts formed by the peptide shown in formula (I) and inorganic acids or organic acids, the organic acids including, but not limited to: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoate or gluconic acid, etc.; the inorganic acids including, but not limited to: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0084] The synthesis of the peptide of formula (I) of the present invention, or its stereoisomer or its cosmetically acceptable salt, or pharmaceutically acceptable salt can be carried out according to conventional methods known in the prior art, such as using the method of solid phase peptide synthesis [Stewart J.M. and Young JD, "Solid Phase Peptide Synthesis", 2nd edition, (1984), Pierce Chemical Company, Rockford, Illinois; Bodanzsky M., Bodanzsky A. "The practice of peptide synthesis", (1984), Springer Verlag, New York; Lloyd-Williams P., Albericio F., Giralt E. "Chemical methods for the synthesis of peptides and proteins" . Approaches to the Synthesis of Peptides and Proteins", (1997), CRC, Boca Raton, Florida, USA], synthesis in solution, a combination of solid phase synthesis and methods of synthesis in solution or enzymatic synthesis [Kullmann W., "Proteases as catalysts for enzymatic syntheses of opioid peptides", (1980), Journal of Biological Chemistry, 255, 8234-8238]. The peptides can also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins of animal, fungal or, preferably, plant origin, which release peptide fragments containing at least the desired sequence.

[0085] For example, a method for obtaining a peptide having the general formula (I) comprises the following steps:

[0086] - coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a C-terminus that is protected or bound to a solid support;

[0087] - Elimination of the group protecting the N-terminus;

[0088] - repeating this coupling sequence and eliminating the group protecting the N-terminus until the desired peptide sequence is obtained;

[0089] - Elimination of the group protecting the C-terminus or cleavage from the solid support.

[0090] Preferably, the C-terminus is bound to a solid support and the method is carried out on a solid phase, comprising coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a C-terminus bound to a polymer support; eliminating the group protecting the N-terminus; and repeating this sequence as many times as required in order to thereby obtain a peptide of the desired length, followed by cleavage of the synthesized peptide from the initial polymer support.

[0091] The functional groups of the amino acid side chains remain fully protected with temporary or permanent protecting groups throughout the synthesis and can be deprotected simultaneously or orthogonally to the process of cleavage of the peptide from the polymer support.

[0092] Alternatively, solid phase synthesis can be carried out by a convergent strategy of coupling a dipeptide or tripeptide to a polymer support or to a dipeptide or amino acid previously bound to a polymer support. Convergent synthesis strategies are well known to those skilled in the art and are described in Lloyd-Williams P., Albericio F. and Giralt E. "Convergent solid phase peptide synthesis", (1993), Tetrahedron 49: 11065-11133.

[0093] The method may include the additional steps of deprotecting the N-terminus and C-terminus and / or cleaving the peptide from the polymer support in an undefined order using standard conditions and methods known in the art, and subsequently modifying the functional groups at the termini. The optional modification of the N-terminus and C-terminus may be performed on the peptide of general formula (I) bound to the polymer support, or after the peptide has been cleaved from the polymer support.

[0094] The peptides of the present invention can be administered to promote fibroblast proliferation and increase collagen production by any means that causes contact between the peptides and the site of action in mammals, preferably in humans, and in the form of a composition containing them.

[0095] To this end, another aspect of the present invention provides a cosmetic composition or pharmaceutical composition, comprising an effective amount of the peptide of formula (I) above, or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, and at least one excipient or optional cosmetically or pharmaceutically acceptable adjuvant. These compositions can be prepared by conventional methods known to those skilled in the art ["Harry's Cosmeticology", Seventh Edition, (1982), edited by Wilkinson JB, Moore RJ, Longman House, Essex, UK].

[0096] Optionally, the adjuvant is selected from: collagen synthesis stimulators, agents that regulate PGC-1α synthesis, agents that regulate the activity of PPARγ, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic agents or agents that stimulate fat decomposition, lipolytic agents, lipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulators or inhibitors, whitening agents or depigmenting agents, pigmentation promoting agents, self-tanning agents, anti-aging agents, NO-synthase inhibitors, 5α-reductase inhibitors, inhibitors of lysyl hydroxylase and / or prolyl hydroxylase, antioxidants, free radical scavengers and / or anti-atmospheric pollution agents, Reactive carbonyl scavengers, anti-glycation agents, antihistamines, antivirals, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioning agents, moisturizers, moisture retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolases, vitamins, amino acids, proteins, pigments or colorants, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating under-eye bags, exfoliants, keratolytic agents, keratolytic agents, antimicrobial agents, antifungal agents, antifungal agents, bacteriostatic agents, bacteriostatic agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and / or are capable of inhibiting or preventing their degradation, agents that stimulate elastin synthesis, stimulating agents that stimulate the synthesis of decorin, agents that stimulate the synthesis of laminin, agents that stimulate the synthesis of defensins, agents that stimulate the synthesis of chaperone proteins, agents that stimulate the synthesis of cAMP, heat shock proteins, agents that stimulate the synthesis of HSP70, agents that stimulate the synthesis of heat shock proteins, agents that stimulate the synthesis of hyaluronic acid, agents that stimulate the synthesis of fibronectin, agents that stimulate the synthesis of sirtuins, agents that stimulate the synthesis of lipids and stratum corneum components, ceramides, fatty acids, agents that inhibit collagen degradation, agents that inhibit elastin degradation, agents that inhibit serine proteases, agents that stimulate fibroblast proliferation, agents that stimulate keratinocyte proliferation, agents that stimulate adipocyte proliferation, agents that stimulate melanocyte proliferation, agents that stimulate keratinocyte differentiation, agents that inhibit acetylcholinesterase, skin skin relaxants, agents that stimulate glycosaminoglycan synthesis, anti-hyperkeratotic agents, comedolytic agents, anti-psoriatic agents, anti-dermatitis agents, anti-eczema agents, DNA repair agents, DNA protective agents, stabilizers, antipruritic agents, agents for treating and / or caring for sensitive skin, firming agents, firming agents, reconstructing agents, anti-stretch mark agents, adhesives, agents for regulating sebum production, antiperspirants, agents that stimulate healing, agents that assist healing, agents that stimulate re-epithelialization, agents that assist re-epithelialization, cytokine growth factors, sedatives, anti-inflammatory agents, anesthetics, agents that act on capillary circulation and / or microcirculation, agents that stimulate angiogenesis, agents that inhibit vascular permeability, venous tension agents, agents that act on cell metabolism, agents for improving dermal-epidermal junction, agents that induce hair growth,Hair growth inhibitors or retardants, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents obtained from biological fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotectants effective against A and / or B ultraviolet rays, or mixtures thereof, provided that they are physically and chemically compatible with the components of the composition, and in particular compatible with the peptides of general formula (I) contained in the composition of the present invention. In addition, the nature of these additional ingredients should not unacceptably change the benefits of the peptides of the present invention. The nature of these additional ingredients can be synthetic or natural, such as plant extracts, or from a biotechnological process, or from a combination of a synthetic process and a biotechnological process. Additional examples can be found in the "CTFA International Cosmetic Ingredient Dictionary & Handbook", 12th edition (2008). In the context of the present invention, a biotechnological process should be understood as any method for producing the active ingredient, or a portion of the active ingredient, in an organism, or in a part of an organism. ,

[0097] Alternatively, the above-mentioned cosmetic composition or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by a biotechnological process, which is a collagen synthesis stimulator, such as, but not limited to, ascorbic acid and its derivatives such as ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate and ascorbic acid α- and β-glucoside, retinol and retinol derivatives such as retinoic acid, retinal, retinyl acetate or retinyl palmitate, vegetable extracts such as extracts of Aloe and Centella, carnitine, carnosine, creatine, asiatic acid, madecassoside, asiatic saponin, Centella extract, niacinamide, astaxanthin, glucans such as genistein Soybean extract and soy isoflavones of ketones and daidzein, rutin, chrysin, morin, betel nut alkaloids, forskolin, betulinic acid, plantago extract, TGF-β, ginkgo extract, glutamine, glycolic acid, [INCI: Palmitoyl Pentapeptide-4], Matrixyl [INCI: Palmitoyl Tetrapeptide-7, Palmitoyl Oligopeptide] or [INCI: C12-15 Alkyl Benzoate, Tribehenin, Ceramide 2, PEG10 Rapeseed Sterol, Palmitoyl Oligopeptide] sold by Sederma / Croda, -Coll [INCI: Palmitoyl Tripeptide-5], [INCI: Locust Bean (Locust Bean) Bean) (Ceratonia siliqua) gum] or BeauActive™ MTP [INCI: Hydrolyzed Milk Protein], Thalassine™ [INCI: Algae Extract] sold by Biotechmarine, EquiStat™ [INCI: Apple Fruit Extract, Glycine Soja Seed Extract] or Juvenesce [INCI: Ethoxydiglycol and Caprylic Triglyceride, Retinol, Ursolic Acid, Vitamin K1, Ilomastat] sold by Coletica / Engelhard / BASF, SMS Anti- [INCI: Purified Oligopeptides from Annona Squamosa] or Papilactyl [INCI: Water, Cyperus Esculentus Tuber Extract] sold by Silab, ChroNOIine™ [INCI: Glycerin, Water (Aqua), Dextran, Caprooyl Tetrapeptide-3 (Cyperus Esculentus) Tuber Extract) sold by Atrium Innovations / Unipex Group Tetrapeptide-3)], [INCI: Tripeptide-1] or 4 [INCI: Water, Dextran, Acetyl Tetrapeptide-2], BioLyse TA sold by Solabia[INCI: Pro-oligopeptide, composed of L-lysine and L-arginine] or [INCI: Arginine / Lysine Polypeptide], In-Tense [INCI: Caprylic / Capric Triglyceride, Spilanthes Acmella Flower Extract] sold by Gattefosse, PhytoCellTecTM Malus Domestica [INCI: Apple (Malus Domestica) Fruit Cell Culture, Xanthan Gum, Glycerin, Lecithin, Phenoxyethanol, Water] sold by Mibelle Biochemistry, Phytosphingosine SLC [INCI: Salicyloyl Phytosphingosine] sold by Evonik Goldschmidt, Dakaline [INCI: Prunus Amygdalus Dulcis (Prunus Armeniaca) Flower Extract] sold by Soliance Amygdalus kernel extract, Anogeissus Leiocarpus bark extract], [INCI: Hexapeptide-9], GP4G [INCI: Artemia extract], D'OrientineTM [INCI: Phoenix Dactylifera (Date) seed extract] or EderlineTM [INCI: Pyrus Malus (Apple) fruit extract] sold by Vincience, or Homeostatine [INCI: Enteromorpha Compressa, Caesalpinia Spinosa] sold by Provital.

[0098] Alternatively, the above-mentioned cosmetic or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by a biotechnological process, which is an elastase inhibitor, such as, but not limited to, [INCI: Glycine Soja (Soybean) Protein], [INCI: Glycine Soja (Soybean) Protein, Oxidoreductase] or -Age [INCI: Hydrolyzed Rice Bran Protein, Glycine Soja (Soybean) Protein, Oxidoreductase] sold by Pentapharm / DSM, Juvenesce [INCI: Ethoxydiglycol and Caprylic Triglyceride, Retinol, Ursolic Acid, Vitamin K1, Ilomastat], MicromerolTM [INCI: Apple (Pyrus Malus) Extract], Heather Extract [INCI: Calluna Vulgaris Extract], [INCI: Hydrolyzed Potato Protein] or Flavagrum TMPEG [INCI: PEG-6 Isostearate, Hesperidin Laurate], TP LS8657 [INCI: Pisum Sativum Extract] sold by Laboratoires Sérobiologiques / Cognis, Sepilift DPHP [INCI: Dipalmitoyl Hydroxyproline] sold by SEPPIC, [INCI: Alcohol, Water, Glycerin, Hydrolyzed Rice Protein, Ilex Aquifolium Extract, Sodium Ursolic Acid, Sodium Oleanolic Acid] sold by Rahn, Age Defense 2 [INCI: Juglans Regia (Walnut) Seed Extract] sold by Gattefosse, IP 2000 [INCI: Glucan, Trifluoroacetyl Tripeptide-2], Radicaptol [INCI: Propylene Glycol, Water, Passiflora Incarnata Flower Extract, Ribes Nigrum (Black Currant) Leaf Extract, Vitis Vinifera (Grape) Leaf Extract] sold by Solabia or ViaPure™ Boswellia [INCI: Olivanum (Boswellia Serrata) Extract] sold by Soliance, among others.

[0099] Optionally, the above-mentioned cosmetic composition or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by a biotechnological method, which is an active carbonyl scavenger, free radical scavenger and / or anti-glycation agent, such as and not limited to carnosine and its derivatives, GHK [INCI: Tripeptide-1] and its salts and / or derivatives, or [INCI: Hydrolyzed Wheat Protein, Hydrolyzed Soy Protein, Tripeptide-1] or PreventheliaTM [INCI: Diaminopropionyl Tripeptide-33] sold by Lipotec, etc.

[0100] Optionally, the above-mentioned cosmetic or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one extract, which is an anti-wrinkle and / or anti-aging agent, such as, but not limited to, extracts of grapes, Rosa canina, Curcuma longa, Iris pallida, Theobroma cacao, Ginkgo, Leontopodium Alpinum or Dunaliella salina, or at least one synthetic compound or product, which is an anti-wrinkle or anti-aging agent, such as, but not limited to, [INCI: Palmitoyl Pentapeptide-3], Matrixyl [INCI: Palmitoyl Tetrapeptide-7, Palmitoyl Oligopeptide], EssenskinTM [INCI: Calcium Hydroxymethionine], Renovage [INCI: Teprenone] or [INCI: Palmitoyl Oligopeptide] sold by Sederma / Croda, [INCI: Pentapeptide-3], Syn [INCI: Dipeptide diaminobutyroyl benzylamide diacetate], -Coll [INCI: Palmitoyl tripeptide-5], Phytaluronate [INCI: Locust bean (Carob) gum] or [INCI: Glycine soja (soybean) protein, oxidoreductase] sold by SM, MyoxinolTM [INCI: Hydrolyzed Hibiscus syringae flavescentis (Hibiscus syringae flavescentis) sold by Laboratoires Sérobiologiques / Cognis Esculentus Extract], SyniorageTM [INCI: Acetyl Tetrapeptide-11], DermicanTM [INCI: Acetyl Tetrapeptide-9] or DN-AGETMLS [INCI: Cassia Alata Leaf Extract], Algisum [INCI: Methylsilanol Mannouronate] or Hydroxyprolisilane [INCI: Methylsilanol Hydroxyproline Aspartate] sold by Exsymol,Sold by Lipotec [INCI: Acetyl Hexapeptide-8], SNAP-7 [INCI: Acetyl Heptapeptide-4], SNAP-8 [INCI: Acetyl Octapeptide-3], [INCI: Pentapeptide-18], InylineTM [INCI: (Proposed) Acetyl Hexapeptide-30], [INCI: Hydrolyzed Wheat Protein, Hydrolyzed Soy Protein, Tripeptide-1], PreventheliaTM [INCI: Diaminopropionyl Tripeptide-33], DecorinylTM [INCI: Tripeptide-10 Citrulline], TrylagenTM [INCI: Pseudoalteromonas Ferment Extract, Hydrolyzed Wheat Protein, Hydrolyzed Soy Protein, Tripeptide-10 Citrulline, Tripeptide-1]; [INCI: Acetyl Tetrapeptide-5], Peptide AC29 [INCI: Acetyl Tripeptide-30 Citrulline], Lipochroman-6 [INCI: Dimethylmethoxychromanol], Chromabright™ [INCI: Dimethylmethoxychromanyl palmitate], Vilastene™ [INCI: Lysine HCl, Lecithin, Tripeptide-10 Citrulline], dGlyAGE™ [INCI: Lysine HCl, Lecithin, Tripeptide-9 Citrulline] or [INCI: Pseudoalteromonas ferment extract], [INCI: Tripeptide-1, Glucan] marketed by Institut Europeen de Biologie Cellulaire, IS [INCI: Hexapeptide-9] marketed by Vincience, Orsirtine™ GL [INCI: Oryza Sativa (Rice) Extract], D′Orientine™ IS [INCI: Date Palm (Date Palm) Seed Extract], PhytoquintescineTM [INCI: Einkorn (Triticum monococcum) Extract] or QuintescineTM IS [INCI: Dipeptide-4], BONT-L Peptide [INCI: Palmitoyl Hexapeptide-19] sold by InfinitecActivos, DeepalineTM PVB [INCI: Palmitoyl Hydrolyzed Wheat Protein] or DPHP [INCI: Dipalmitoyl Hydroxyproline] sold by Seppic, Expression [INCI: Acmella oleracea Extract], In-Tense [INCI: Buttonwood Flower Extract] or Age Defense 2 [INCI: Juglans Regia (Walnut) Seed Extract] sold by Gattefossé,ThalassineTM [INCI: Algae Extract] marketed by Biotechmarine, ChroNOlineTM [INCI: Hexanoyl Tetrapeptide-3] or Thymulen-4 [INCI: Acetyl Tetrapeptide-2] marketed by AtriumInnovations / Unipex Group, EquiStat [INCI: Apple Fruit Extract, Glycine Soja Seed Extract] or Juvenesce [INCI: Ethoxydiglycol and Caprylic Triglyceride, Retinol, Ursolic Acid, Vitamin K1, Ilomastat] marketed by Coletica / Engelhard / BASF, Ameliox [INCI: Carnosine, Tocopherol, Silybum Marianum Fruit Extract] or PhytoCellTec Malus Domestica [INCI: Apple Fruit Cell Culture] marketed by Mibelle Biochemistry, Bioxilift [INCI: Pimpinella Anisum Extract] or SMSAnti- [INCI: Annona Fruit Extract] marketed by Silab, Ca, 2+ Channel antagonists include, but are not limited to, alverine, manganese or magnesium salts, certain secondary or tertiary amines, retinol and its derivatives, idebenone and its derivatives, coenzyme Q10 and its derivatives, boswellic acid and its derivatives, GHK and its derivatives and / or salts, carnosine and its derivatives, DNA repair enzymes include, but are not limited to, photolyase or T4 endonuclease V, or chloride channel antagonists, etc.

[0101] Alternatively, the above-mentioned cosmetic or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by a biotechnological process, which is an agent that stimulates healing, an agent that assists healing, an agent that stimulates re-epithelialization and / or an agent that assists re-epithelialization, such as, but not limited to, Centella asiatica extract, Rosa moschata extract, Echinacea angustifolia extract, Symphytum officinal extract, Equisetum arvense extract, Hypericum perforatum extract, Mimosa tenuiflora extract, Aloe vera extract, Epitheliizing sold by Provital [INCI: Calendula officinalis, Hypericum perforatum, Chamomilla recutita]. recutita), rosemary (Rosmarinusofficinalis)], LS9028 sold by Laboratories Serobiologiques / Cognis [INCI: hydrolyzed casein, hydrolyzed yeast protein, lysine hydrochloride], [INCI: Zea mays (corn) grain extract] sold by Coletica / Engelhard / BASF, cadherins, integrins, selectins, hyaluronan receptors, immunoglobulins, fibroblast growth factor, connective tissue growth factor, platelet-derived growth factor, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor, keratinocyte growth factor, colony stimulating factor, transforming growth factor-β, tumor necrosis factor-α, interferons, interleukins, matrix metalloproteinases, protein tyrosine phosphatase receptors, [INCI: Pseudoalteromonas ferment extract] or [INCI: Tripeptide-10 citrulline] sold by Lipotec, or mixtures thereof.

[0102] Furthermore, the above-mentioned cosmetic or pharmaceutical composition of the invention may contain or may be co-administered with analgesic and / or anti-inflammatory compounds in order to reduce swelling and irritation associated with sensitive skin and the healing process, or for the treatment of excessive scars or keloids. Among the compounds, emphasis may be placed on steroidal compounds such as hydrocortisone, non-steroidal compounds such as acetaminophen or acetylsalicylic acid, or natural extracts or essential oils having inherent analgesic and anti-inflammatory activity.

[0103] Optionally, the formulation of the cosmetic or pharmaceutical composition is selected from the group consisting of: cream, oil, milk, balm, foam, lotion, gel, liniment, serum, soap, shampoo, conditioner, serum, ointment, mousse, pomade, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, syrup, elixir, polysaccharide film, jelly or gelatin;

[0104] Optionally, the capsule includes: a soft capsule, a hard capsule, and may be a gelatin capsule;

[0105] Optionally, the tablet comprises: a sugar-coated tablet.

[0106] The cosmetic or pharmaceutical composition of the present invention may include a transdermal enhancer that increases the absorption of the peptide of the present invention, such as, but not limited to, dimethyl sulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone (1-dodecylazacycloheptan-2-one), alcohol, urea, ethoxydiglycol, acetone, propylene glycol or polyethylene glycol, etc.

[0107] In addition, the peptide shown in formula (I) of the present invention, or its stereoisomer, or mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the cosmetic or pharmaceutical composition, can be applied to the local area to be treated by iontophoresis, sonophoresis, electroporation, micro-electric patch, mechanical compression, osmotic pressure gradient, bandage therapy, microinjection or needle-free injection by pressure (e.g., injection by oxygen pressure), or any combination thereof, so as to achieve greater penetration of the peptide of the present invention. The application area will be determined by the nature of the condition, disorder and / or disease to be treated and / or cared for.

[0108] In addition, the peptides of formula (I) of the present invention, or their stereoisomers, or mixtures of stereoisomers, or their cosmetically acceptable salts, or their pharmaceutically acceptable salts, or the above-mentioned cosmetic or pharmaceutical compositions can be incorporated into any form of functional food or fortified food, such as and not limited to dietary bars or compressed or non-compressed powders. These powders can be dissolved in water, juice, soda water, dairy products, soy derivatives, or can be incorporated into dietary bars. These peptides of the present invention can be formulated with common excipients or adjuvants for oral compositions or food supplements, such as and not limited to, fat components, aqueous components, humectants, preservatives, thickeners, flavoring agents, fragrances, antioxidants, or colorants commonly found in the food industry.

[0109] The peptides of the present invention have variable solubility in water, depending on the nature of their sequence or any possible modifications in the N-terminus and / or C-terminus. The peptides of the present invention can therefore be incorporated into the composition via an aqueous solution, and those that are insoluble in water can be dissolved in conventional cosmetically or pharmaceutically acceptable solvents, such as, but not limited to, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol or polyethylene glycol, or any combination thereof.

[0110] The cosmetically or pharmaceutically effective amount of the peptides of the invention to be administered, and their dosage, will depend on many factors, including the age, condition of the patient, severity of the disorder or disease, route and frequency of administration, and the specific nature of the peptide to be used.

[0111] "Cosmetically or pharmaceutically effective amount" means an amount of one or more peptides of the present invention that is non-toxic but sufficient to provide the desired effect. The peptides of the present invention are used in cosmetic or pharmaceutical compositions of the present invention at cosmetically or pharmaceutically effective concentrations to obtain the desired effect; in a preferred form, between 0.00000001% (by weight) and 20% (by weight), preferably between 0.000001% (by weight) and 15% (by weight), more preferably between 0.0001% (by weight) and 10% (by weight), and even more preferably between 0.0001% (by weight) and 5% (by weight), relative to the total weight of the composition.

[0112] In another aspect, the present invention provides a cosmetically or pharmaceutically acceptable delivery system or sustained-release system to achieve better penetration of the active ingredient and / or improve its pharmacokinetic and pharmacodynamic properties, which comprises an effective amount of the peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the above cosmetic or pharmaceutical composition;

[0113] The term "delivery system" refers to a diluent, adjuvant, excipient or carrier used together with the peptide of the present invention, which is selected from: water, oil or surfactant, including those of petroleum origin, animal origin, plant origin, or synthetic origin, such as and not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitan ester, ether sulfate, sulfate, betaine, glucosides, maltosides, fatty alcohols, nonoxynol ether, poloxamer, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin and the like. Those of ordinary skill in the art are known to diluents that can be used in different delivery systems that can administer the peptide of the present invention.

[0114] The term "sustained release" is used in its conventional sense to refer to a delivery system that provides for gradual release of a compound over a period of time, and preferably, but not necessarily, with a relatively constant level of compound released over the entire period of time.

[0115] Examples of delivery systems or sustained release systems are liposomes, oleosomes, nonionic surfactant liposome vesicles, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, lipid vesicles, micelles, millispheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, milliparticles, microparticles or nanoparticles. Preferred delivery systems or sustained release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions with an internal structure of reverse micelles.

[0116] Sustained-release system can be prepared by methods known in the prior art, and can be given, for example, in the following manner: by topical or transdermal administration, including adhesive patch, non-adhesive patch, closure patch, and microelectronic patch; or by systemic administration, for example and not limited to, oral or parenteral route, including nose, rectum, subcutaneous implantation or injection or directly implanted or injected into a specific body part, and preferably these peptides of the present invention of a relatively constant amount should be released. The amount of the peptide included in the sustained-release system will depend on the release kinetics and duration of the position, peptide of the present invention, and the condition to be treated and / or the property of nursing, illness and / or disease to be given, for example, the composition.

[0117] In another aspect, the present invention provides a cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support, which comprises an effective amount of the peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the above cosmetic or pharmaceutical composition; which is selected from, but not limited to: talc, bentonite, silicon dioxide, starch, or maltodextrin, etc.

[0118] In another aspect, the present invention provides a cosmetic comprising an effective amount of the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the above cosmetic or pharmaceutical composition;

[0119] Optionally, the cosmetics are selected from concealer, foundation, makeup remover, makeup remover milk, eye shadow, lip stick, lip gloss, lip balm or lip powder.

[0120] In another aspect, a fabric, nonwoven fabric or medical device is provided, which comprises an effective amount of the peptide shown in the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the above cosmetic or pharmaceutical composition. The peptide of the present invention is released due to biodegradation of the anchoring system of the fabric, nonwoven fabric or medical device or by friction with the body, by body moisture, by pH of the skin or by body temperature. In addition, fabrics and nonwoven fabrics can be used to make clothes that are in direct contact with the body.

[0121] Examples of fabrics, nonwoven fabrics, clothing, medical devices and means for fixing peptides thereto include the above-mentioned delivery systems and / or sustained-release systems, which can be found in the literature and are known in the prior art [Schaab CK (1986) "Impregnating Fabrics With Microcapsules", HAPPI May 1986; Nelson G. (2002) "Application of microencapsulation in textiles" Int. J. Pharm. 242: 5562; "Biofunctional Textiles and the Skin" (2006) Curr. Probl. Dermatol. v. 33, Hipler UC and Elsner P. eds.]. S. Karger AG, Basel, Switzerland; Malcom RK; McCullagh SD, Woolfson AD, Gorman SP, Jones DS and Cuddy J. (2004) "Controlled release of a model antibacterial drug from a novel self lubricating silicone biomaterial" J. Cont. Release 97: 313320]. Preferred fabrics, nonwoven fabrics, garments or medical devices are bandages, gauze, T-shirts, socks, pantyhose, underwear, belts, gloves, diapers, sanitary napkins, dressings, bedspreads, wipes, adhesive patches, non-adhesive patches, occlusive patches, microelectronic patches or facial masks.

[0122] Another aspect of the present invention provides a peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the above cosmetic or pharmaceutical composition, or the above cosmetically or pharmaceutically acceptable delivery system or sustained-release system, or the above cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support, or the above cosmetic, or the above fabric, nonwoven fabric or medical device for use in preparing a cosmetic composition or pharmaceutical composition for treating or caring for skin or mucous membranes.

[0123] Another aspect of the present invention provides a peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the above cosmetic or pharmaceutical composition, or the above cosmetically or pharmaceutically acceptable delivery system or sustained-release system, or the above cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support, or the above cosmetic, or the above fabric, nonwoven fabric or medical device for use in preparing a cosmetic composition or pharmaceutical composition for treating or caring for body surface trauma, burns, skin ulcers, reducing scar formation, or accelerating scar repair.

[0124] Another aspect of the present invention provides a peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the above cosmetic or pharmaceutical composition, or the above cosmetically or pharmaceutically acceptable delivery system or sustained-release system, or the above cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support, or the above cosmetic, or the above fabric, non-woven fabric or medical device for use in the preparation of a cosmetic composition or pharmaceutical composition for promoting fibroblast proliferation, increasing collagen production, improving skin barrier function, or for re-epithelialization or healing of skin or mucous membranes.

[0125] In one aspect of the present invention, there is provided a peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the above cosmetic or pharmaceutical composition, or the above cosmetically or pharmaceutically acceptable delivery system or sustained-release system, or the above cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support, or the above cosmetic, or the above fabric, nonwoven fabric or medical device for use in preparing a cosmetic composition or pharmaceutical composition for treating, preventing or repairing skin aging or photoaging.

[0126] Optionally, the treatment, prevention or repair of skin aging or photoaging is the reduction, prevention or treatment of facial wrinkles.

[0127] In another aspect, the present invention provides a method for treating or caring for skin or mucous membranes, which comprises administering a cosmetically or pharmaceutically effective amount of the peptide represented by the above formula (I), or its stereoisomers, or a mixture of stereoisomers, or its cosmetically acceptable salts, or its pharmaceutically acceptable salts, or the above cosmetic or pharmaceutical compositions.

[0128] In another aspect, the present invention provides a method for treating or caring for body surface trauma, burns, skin ulcers, reducing scar formation, and accelerating scar repair, which comprises administering a cosmetically or pharmaceutically effective amount of the peptide represented by the above formula (I), or its stereoisomers, or a mixture of stereoisomers, or its cosmetically acceptable salts, or its pharmaceutically acceptable salts, or the above cosmetic or pharmaceutical compositions.

[0129] In another aspect, the present invention provides a method for promoting fibroblast proliferation, increasing collagen production, improving skin barrier function, or re-epithelialization or healing of skin and / or mucous membranes, the method comprising administering a cosmetically or pharmaceutically effective amount of the peptide represented by formula (I) above, or a stereoisomer thereof, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the cosmetic or pharmaceutical composition above.

[0130] In another aspect, the present invention provides a method for treating, preventing or repairing skin aging or photoaging, which comprises administering a cosmetically or pharmaceutically effective amount of the peptide represented by the above formula (I), or its stereoisomers, or a mixture of stereoisomers, or its cosmetically acceptable salts, or its pharmaceutically acceptable salts, or the above cosmetic or pharmaceutical compositions.

[0131] Treatment, prevention or repair of skin aging or photoaging is the reduction, prevention or treatment of facial wrinkles.

[0132] The composition containing the peptide represented by the above formula (I) of the present invention, or its stereoisomer, or mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the above cosmetic or pharmaceutical composition can be applied to the skin and / or mucous membrane, or orally or parenterally administered as needed to treat and / or care for a condition, disorder and / or disease.

[0133] The frequency of administration or dosing can vary widely, depending on the needs of each subject, with suggested administration or dosing ranging from once a month to 10 times a day, preferably from once a week to 4 times a day, more preferably from 3 times a week to 3 times a day, and even more preferably once or twice a day.

[0134] definition

[0135] In order to facilitate the understanding of the present invention, the meanings of some terms and expressions used in the present invention are explained as follows:

[0136] In the present invention, the term "skin" is understood to mean the multiple layers that make it up, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both ends are included. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes, etc. In the present invention, the term "skin" includes the scalp.

[0137] The term "treatment" refers to administering a peptide according to the invention to alleviate or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with the disease or condition. The term "treatment" also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or condition.

[0138] The term "care" includes the prevention of diseases and / or conditions.

[0139] The term "prevent" refers to the ability of the peptides of the present invention to prevent, delay, or hinder the onset or development of a disease or condition before it occurs.

[0140] The term "aging" refers to the changes that the skin undergoes with age (natural aging) or through exposure to sunlight (photoaging) or to environmental pollutants, such as chemical dirt or pollution, tobacco smoke, etc., and includes all changes that are outwardly visible and / or perceptible by touch, such as and not limited to: the development of discontinuities in the skin (such as wrinkles, fine lines, expression lines, stretch lines, stripes, grooves, unevenness or roughness, increase in pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of deformation recovery ability, loss of resilience), sagging of the skin (such as sagging cheeks, bags under the eyes, or the appearance of a double chin, etc.), changes in skin color (such as scarring, redness, bags under the eyes, or the appearance of areas of hyperpigmentation such as age spots or freckles, etc.), abnormal differentiation, hyperkeratinization, elastosis, keratosis, hair loss, orange peel skin, loss of collagen structure, and other histological changes in the stratum corneum, dermis, epidermis, vascular system (such as the appearance of spider veins or telangiectasias) or those tissues close to the skin.

[0141] The term "photoaging" refers to the premature aging of the skin due to long-term exposure to ultraviolet radiation, which presents the same physiological characteristics as natural aging, such as but not limited to: loosening, sagging, color changes or irregular pigmentation, abnormalities and / or excessive keratinization.

[0142] In the present specification, the abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Commission of Biochemical Nomenclature in the European Journal of Biochemistry (Eur. J. Biochem. 1984, 138: 9-37).

[0143] Thus, for example, Ala represents NH2-CH(CH3)-COOH, Ala- represents NH2-CH(CH3)-CO-, -Ala represents -NH-CH(CH3)-COOH, and -Ala- represents -NH-CH(CH3)-CO-. Thus, the hyphen representing a peptide bond eliminates the OH in the 1-carboxyl group of an amino acid (represented here in conventional non-ionized form) when located on the right side of the symbol, and eliminates the H in the 2-amino group of an amino acid when located on the left side of the symbol; both modifications can be applied to the same symbol (see Table 1).

[0144] Table 1 Structures of amino acid residues and their one-letter and three-letter abbreviations

[0145]

[0146]

[0147] The abbreviation "Ac-" is used in the present invention to represent the acetyl group (CH3-CO-), and the abbreviation "Palm-" is used to represent the palmitoyl group (CH3-(CH2) 14 -CO-).

[0148] The beneficial effects achieved by the present invention compared with the prior art include:

[0149] The peptides described in the present invention are artificially designed and easy to synthesize. They can promote fibroblast proliferation, improve skin barrier function, promote wound healing, treat or care for body surface trauma, burns, skin ulcers, reduce scar formation, accelerate scar repair, and can also be used to treat, prevent and / or repair skin aging and / or photoaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 The graphs show the effects of peptides (1) to (5) on HaCaT cell activity. * indicates that the drug-treated group has a statistically significant difference compared with the blank control group, p<0.05 (n=4). ** indicates that the drug-treated group has a significant difference compared with the blank control group, p<0.01 (n=4).

[0151] Figure 2The figure shows the effect of peptides (1) to (5) on the activity of NIH3T3 cells. * indicates that the drug group has a statistical difference compared with the blank control group, p<0.05 (n=4). ** indicates that the drug group has a significant difference compared with the blank control group, p<0.01 (n=4).

[0152] Figure 3 The figure is the effect of peptide (1) and peptide (2) on HaCaT cell adhesion. * indicates that the difference between the drug group and the blank control group is statistically significant, p < 0.05 (n = 4). ** indicates that the difference between the drug group and the blank control group is significant, p < 0.01 (n = 4). *** indicates that the difference between the drug group and the blank control group is extremely significant, p < 0.001 (n = 4).

[0153] Figure 4 This is the result of HaCaT cell scratch experiment.

[0154] Figure 5 The repair effect of peptides (1) to (5) on photodamaged HaCaT cells. ### indicates that the difference is extremely significant compared with the non-irradiated normal control group, p<0.001 (n=4). * indicates that there is a statistical difference compared with the irradiated control group, p<0.05 (n=4). ** indicates that there is a significant difference compared with the irradiated control group, p<0.01 (n=4). *** indicates that there is an extremely significant difference compared with the irradiated control group, p<0.001 (n=4).

[0155] Figure 6 The repair effect of peptide (1) and peptide (2) on photodamaged NIH3T3 cells. ### indicates that the difference is extremely significant compared with the non-irradiated normal control group, p<0.001 (n=4). * indicates that there is a statistical difference compared with the irradiated control group, p<0.05 (n=4). ** indicates that there is a significant difference compared with the irradiated control group, p<0.01 (n=4). *** indicates that there is an extremely significant difference compared with the irradiated control group, p<0.001 (n=4).

[0156] Figure 7 This is a graph showing the effect of peptide (2) on TNF-α secretion. ### indicates that the difference is extremely significant compared with the control group, p<0.001 (n=4). *** indicates that the difference is extremely significant compared with the LPS group, p<0.001 (n=4).

[0157] Figure 8 This is a graph showing the effect of peptide (2) on IL-6 secretion. ### indicates that the difference is extremely significant compared with the control group, p<0.001 (n=4). *** indicates that the difference is extremely significant compared with the LPS group, p<0.001 (n=4).

[0158] Fig. 9 This is a graph showing the effect of peptide (2) on IL-8 secretion. ## indicates a significant difference compared with the control group, p<0.01 (n=4). ** indicates a significant difference compared with the LPS group, p<0.01 (n=4). DETAILED DESCRIPTION

[0159] In order to better understand the present invention, the present invention is described in detail below in conjunction with embodiments and drawings. However, it should be understood that these embodiments and drawings are only used for illustrative purposes and are not intended to limit the scope of the present invention.

[0160] abbreviation

[0161] The abbreviations used for amino acids follow the rules specified by the Biochemical Nomenclature Commission of the IUPAC-IUB in Eur J. Biochem. (1984) 138:9-37 and J. Chem (1989) 264:633-673.

[0162] Wang Resin: Wang Resin; DMF: N,N-dimethylformamide; DCM: dichloromethane; DIC: diisopropylcarbodiimide; Ac2O: acetic anhydride; DIPEA: diisopropylethylamine; Fmoc: 9-fluorenylmethoxycarbonyl; piperidine: piperidine; HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid; TIS: triisopropylsilane; EDT: 1,2-ethanedithiol; DMSO: dimethyl sulfoxide; Ac: acetyl; Palm: palm Acyl; Ala: Alanine; Pro: Proline; Phe: Phenylalanine; Arg: Arginine; Met: Methionine; Gly: Glycine; Ile: Isoleucine; Cys: Cysteine; Thr: Threonine; Asn: Asparagine; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Trt: triphenylmethyl or trityl; tBu: tert-butyl; Amide Resin: a starting resin for peptide synthesis; Fmoc-linker: 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetic acid.

[0163] Example 1

[0164] Preparation of Fmoc-Ala-Pro-Phe-Arg(Pbf)-Met-Gly-Ile-Cys(Trt)-Thr(tBu)-Thr(tBu)-Asn(Trt)-Wang Resin.

[0165] 1.1 Swelling of resin

[0166] Weigh 35g (49.7mmol) of Wang Resin into a 2L solid phase synthesis reaction column. Add 100ml of DMF to completely submerge the resin, then introduce nitrogen, start nitrogen stirring and bubbling, 5min, and then drain the solvent for 5min. Continue to add 100ml of DMF, completely submerge the resin, then introduce nitrogen, 30min, the resin is completely swollen, and the solvent is drained. Add another 100ml of DMF, completely submerge the resin, then introduce nitrogen, start nitrogen stirring and bubbling, and drain the solvent for 5min, and the swelling is complete.

[0167] 1.2 Removal of Fmoc

[0168] The swollen resin was de-Fmoced twice with 500 ml 20% piperidine / DMF, the first reaction lasted 5 min, the second reaction lasted 8 min, and the solvent was drained for 3 min. The resin was washed with DMF 7-8 times and the solvent was drained.

[0169] 1.3 Feeding reaction

[0170] Weigh 53.38g of Fmoc-Asn(Trt)-OH (89.46mmol; 1.8 equivalents) and 14.51g of HOBt into a dry 500ml round-bottom ground flask. Add DMF to dissolve it and place it in a ±5℃ refrigerator for 10min. Add 20.78ml DIC to activate for 10min, avoiding moisture. Add the activated amino acid to the resin after Fmoc protection and react for 2h. Control the reaction temperature at 25℃~35℃ and draw off the reaction solution. Wash the resin with DMF, 100ml each time. After the resin and solvent are evenly mixed, stir for 2min and drain the solvent. Wash the resin with DMF several times until the filtered solvent is clear and transparent.

[0171] The resin substitution degree was detected to be 0.5, and the calculated synthesis scale was 24.9 mmol.

[0172] The N-terminal Fmoc group was deprotected and 24.74 g of activated Fmoc-Thr(tBu)-OH (62.25 mmol; 2.5 equivalents) was coupled to the peptidyl resin using DMF as solvent in the presence of 10.09 g of HOBt and 14.46 ml of DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the coupling of the next amino acid. In each coupling, 24.74 g of Fmoc-Thr(tBu)-OH (62.25 mmol; 2.5 equiv.); 36.46 g of Fmoc-Cys(Trt)-OH (62.25 mmol; 2.5 equiv.); 22 g of Fmoc-Ile-OH (62.25 mmol; 2.5 equiv.); and 18.51 g of Fmoc-Gly-OH (62.25 mmol; 2.5 equiv.) were coupled sequentially using DMF as solvent in the presence of 10.09 g of HOBt and 14.46 ml of DIC.

[0173] The N-terminal Fmoc group was deprotected and 32.37 g of Fmoc-Met-OH (87.15 mmol; 3.5 equivalents) after activation was coupled to the peptidyl resin using DMF as solvent in the presence of 14.13 g of HOBt and 20.24 ml of DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the coupling of the next amino acid. In each coupling, 56.54 g of Fmoc-Arg(Pbf)-OH (87.15 mmol; 3.5 eq.); 33.76 g of Fmoc-Phe-OH (87.15 mmol; 3.5 eq.); 29.40 g of Fmoc-Pro-OH (87.15 mmol; 3.5 eq.) and then 27.13 g of Fmoc-Ala-OH (87.15 mmol; 3.5 eq.) were coupled sequentially in the presence of 14.13 g of HOBt and 20.24 ml of DIC using DMF as solvent.

[0174] After the synthesis, the resin was washed with DMF, 100 ml each time, the resin and the solvent were uniformly mixed, stirred for 2 minutes, and the solvent was drained. The resin was washed with DMF several times until the filtered solvent was clear and transparent.

[0175] Example 2

[0176] Preparation of Fmoc-Ala-Pro-Phe-Arg(Pbf)-Met-Gly-Ile-Cys(Trt)-Thr(tBu)-Thr(tBu)-Asn(Trt)-Linker-Amide Resin.

[0177] 2.1 Preparation of Fmoc-Linker-Amide Resin

[0178] Weigh 50 g (60 mmol) of Amide Resin resin into a 2 L solid phase synthesis reaction column. Wash the resin once with 400 ml DMF, swell the resin with 500 ml DCM for 20 min, and remove the solvent. Wash the resin 3 times with 400 ml DMF.

[0179] Weigh 80.9g (150mmol) of Fmoc-linker and 24.3g (180mmol) of HOBt into a dry 500ml wide-mouthed conical flask. Add about 150ml of DMF solvent to dissolve and place in an ice-water bath to cool for 2min. Add 30ml (195mmol) of DIC to activate for 3min, avoiding moisture. Add the activated Fmoc-linker to the swollen resin and react for 2h, then remove the reaction solution. Wash the resin 3 times with 400ml of DMF, 2min each time.

[0180] After adding 28 ml (300 mmol) of Ac2O, 10.7 ml (60 mmol) of DIPEA and 400 ml of DMF, the mixture was sealed for 3 h, and then the resin was washed four times with 500 ml of DMF and twice with 500 ml of DCM. The solvent was removed.

[0181] 2.2 Removal of Fmoc

[0182] Fmoc-Linker-Amide Resin was de-Fmoced twice with 500 ml 20% piperidine / DMF, the first reaction lasted 5 min, the second reaction lasted 8 min, and the solvent was drained for 3 min. The resin was washed 4 times with 500 ml DMF and 2 times with 500 ml DCM. The solvent was drained.

[0183] 2.3 Feeding reaction

[0184] Weigh 52.21g of Fmoc-Asn(Trt)-OH (87.5mmol) and 14g of HOBt into a dry 500ml wide-mouthed conical flask. Add DMF to dissolve and cool in an ice-water bath. Add 20ml of DIC to activate for 3min, avoiding moisture. Add the activated amino acid to the deprotected resin and react for 2h, then remove the reaction solution. Wash the resin twice with 500ml of DMF, 2min each time, and 400ml of DCM, then remove the solvent.

[0185] The N-terminal Fmoc group was deprotected and 34.78 g of Fmoc-Thr(tBu)-OH (87.5 mmol) after activation was coupled to the peptidyl resin using DMF as solvent in the presence of 14 g of HOBt and 20 ml of DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated to couple the next amino acid. In each coupling, 14 g of HOBt and 20 ml of DIC were used. In the case of DIC, using DMF as solvent, 34.78 g of Fmoc-Thr(tBu)-OH (87.5 mmol); 51.25 g of Fmoc-Cys(Trt)-OH (87.5 mmol); 30.92 g of Fmoc-Ile-OH (87.5 mmol); 26.01 g of Fmoc-Gly-OH (87.5 mmol); 32.50 g of Fmoc-Met-OH (87.5 mmol); 56.77 g of Fmoc-Arg(Pbf)-OH (87.5 mmol); 33.90 g of Fmoc-Phe-OH (87.5 mmol); 29.52 g of Fmoc-Pro-OH (87.5 mmol) and then 27.24 g of Fmoc-Ala-OH (87.5 mmol) were coupled sequentially.

[0186] After the synthesis, the resin was washed 4 times with 600 ml DMF for 2 min each time and 2 times with 500 ml DCM, and the solvent was pumped off.

[0187] Example 3

[0188] The N-terminal Fmoc protecting group was removed.

[0189] The N-terminal Fmoc group of the peptidyl resins obtained in Examples 1 and 2 was deprotected by using 500 ml of 20% piperidine / DMF for two times, the first reaction lasted for 5 min, the second reaction lasted for 8 min, and the solvent was drained for 3 min. The resin was washed 4 times with 600 ml of DMF for 2 min each time, and the resin was washed twice with 500 ml of DCM, and the solvent was drained.

[0190] Example 4

[0191] The R1 palmitoyl group was introduced onto the peptidyl resin obtained in Example 3.

[0192] In the presence of 1.53 g of HOBt (10 mmol; 10 equivalents) and 1.54 mL of DIC (10 mmol; 10 equivalents), 2.56 g of palmitic acid (10 mmol; 10 equivalents) previously dissolved in 1 ml of DMF were added to 1 mmol of the peptidyl resin obtained in Example 3. They were allowed to react for 15 h, after which the resin was washed 4 times with 600 ml of DMF for 2 min each time, 2 times with 500 ml of DCM, and the solvent was pumped off.

[0193] Example 5

[0194] An R1 acetyl group was introduced onto the peptidyl resin obtained in Example 3.

[0195] 1 mmol of the peptidyl resin obtained in Example 3 was treated with 25 equivalents of Ac2O in the presence of 25 equivalents of DIPEA using 5 mL of DMF as solvent. They were allowed to react for 30 min, after which the resin was washed 4 times with 600 ml of DMF for 2 min each time and 2 times with 500 ml of DCM, and the solvent was pumped off.

[0196] Example 6

[0197] The peptidyl resins obtained in Examples 3, 4, and 5 were cleaved from the polymer support.

[0198] The peptidyl resins obtained in Examples 3, 4, and 5 were shrunk three times with 500 ml, 300 ml, and 200 ml of methanol and dried under vacuum.

[0199] Weigh 1 g of the dried peptidyl resins of Examples 3, 4, and 5, and treat with 7 ml of cleavage solution (TFA: EDT: anisole: thioanisole: TIS: H2O = 87.5: 2.5: 2.5: 2.5: 2.5: 2.5) for 2.5 h. Filter with a sand core funnel, wash the resin with 1 ml of trifluoroacetic acid, and slowly drop the filtrate into 64 ml of ice-cold isopropyl ether. A large amount of white solid precipitates, which is washed 6 times with isopropyl ether after centrifugation. Dry the final precipitate under vacuum.

[0200] Example 7

[0201] The preparation and purification were carried out by high performance liquid chromatography.

[0202] The crude peptide obtained in Example 6 was dissolved in methanol and water, and filtered through a filter membrane with a pore size of 0.45 μm.

[0203] Purification preparation conditions:

[0204] Mobile phase A: pure acetonitrile; Mobile phase B: 0.1% acetic acid aqueous solution;

[0205] Detection wavelength: 215nm;

[0206] Chromatographic column: C18-20*150mm;

[0207] Elution gradient:

[0208] Time (min) Flow rate (ml / min) A％ B％ 0 40 10 90 3 40 12 88 63 40 37 63 64 40 10 90 75 40 10 90

[0209] The filtered sample was injected for purification, and the fractions were collected, concentrated, and lyophilized to obtain the following peptides with a purity of more than 90%:

[0210] 1. H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH;

[0211] 2. H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH2;

[0212] 3. Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH, that is, peptide (8);

[0213] 4. Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH2, that is, peptide (9);

[0214] 5. Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH, that is, peptide (10);

[0215] 6. Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH2, that is, peptide (11).

[0216] Example 8

[0217] The peptide obtained in Example 7 was subjected to oxidation treatment.

[0218] The peptide obtained in Example 7 was added to a 250 ml pear-shaped bottle, dissolved in 80 ml purified water, and 8 g of guanidine hydrochloride and 20 ml of DMSO were added respectively. The mixture was stirred for 30 h in an open state, and the reaction process was monitored by HPLC. After the oxidation was completed, 4 ml of acetic acid was added to dissolve the precipitate, and vacuum filtered using a 0.22 μm filter membrane.

[0219] Example 9

[0220] The filtrate obtained in Example 8 was purified using high performance liquid chromatography.

[0221] Purification preparation conditions:

[0222] Mobile phase A: pure acetonitrile; Mobile phase B: 0.1% acetic acid aqueous solution;

[0223] Detection wavelength: 215nm;

[0224] Chromatographic column: C18-20*150mm;

[0225] Elution gradient:

[0226] Time (min) Flow rate (ml / min) A％ B％ 0 40 8 92 5 40 8 92 50 40 53 47 53 40 8 92 65 40 8 92

[0227] The filtrate was injected for purification, and the fractions were collected, concentrated and lyophilized to obtain the following peptides with a purity of more than 95%:

[0228] 7. namely peptide (1);

[0229] 8. namely peptides (12);

[0230] 9. namely peptides (13);

[0231] 10. namely peptides (14);

[0232] 11. namely peptide (15);

[0233] 12. namely peptide (16).

[0234] Example 10

[0235] The side chain thiol group of Cys8 of the peptide obtained in Example 7 was modified with an allyl group.

[0236] 120 mg of the peptide obtained in Example 7 was dissolved in 40 ml of water, and 5 times the amount of allyl bromide was added under nitrogen protection. The reaction was allowed to react at room temperature. The reaction process was monitored by HPLC until the reaction was complete, and the following crude peptide was obtained:

[0237] 13. namely peptides (3);

[0238] 14. namely peptide (17);

[0239] 15. namely peptide (18);

[0240] 16. namely peptides (19);

[0241] 17. namely peptide (20);

[0242] 18. That is, peptide (21).

[0243] Embodiment 11

[0244] The side chain thiol group of Cys8 of the peptide obtained in Example 7 was modified with tert-butyl bromoacetate.

[0245] 120 mg of the peptide obtained in Example 7 was dissolved in 40 ml of water, and 5 times the amount of tert-butyl bromoacetate was added under nitrogen protection. The reaction was allowed to proceed at room temperature. The reaction process was monitored by HPLC until the reaction was complete, and the following crude peptide was obtained:

[0246] 19. namely peptide (4);

[0247] 20. namely peptides (22);

[0248] twenty one, namely peptides (23);

[0249] twenty two, namely peptide (24);

[0250] twenty three, namely peptide (25);

[0251] twenty four, namely peptide (26).

[0252] Example 12

[0253] The side chain thiol group of Cys8 of the peptide obtained in Example 7 was modified with mercaptoethane.

[0254] 120 mg of the peptide obtained in Example 7 was weighed and dissolved in 40 ml of water. Under nitrogen protection, 5 times the amount of ethanethiosulfate was added and reacted at room temperature. The reaction process was monitored by HPLC until the reaction was complete to obtain the following crude peptide:

[0255] 25. namely peptide (5);

[0256] 26. namely peptide (27);

[0257] 27. namely peptides (28);

[0258] 28. namely peptides (29);

[0259] 29. namely peptide (30);

[0260] 30. namely peptide (31).

[0261] Example 13

[0262] The side chain thiol group of Cys8 of the peptide obtained in Example 7 was modified with hexanethiol.

[0263] 120 mg of the peptide obtained in Example 7 was weighed and dissolved in 40 ml of water. Under nitrogen protection, 5 times the amount of hexanethiol was added and reacted at room temperature. The reaction process was monitored by HPLC until the reaction was complete to obtain the following crude peptide:

[0264] 31. namely peptide (6);

[0265] 32. namely peptides (32);

[0266] 33. namely peptides (33);

[0267] 34. namely peptides (34);

[0268] 35. namely peptides (35);

[0269] 36. namely peptide (36).

[0270] Embodiment 14

[0271] The side chain thiol group of Cys8 of the peptide obtained in Example 7 was modified with bromoacetic acid.

[0272] 120 mg of the peptide obtained in Example 7 was weighed and dissolved in 40 ml of water. Under nitrogen protection, 5 times the amount of bromoacetic acid was added and reacted at room temperature. The reaction process was monitored by HPLC until the reaction was complete to obtain the following crude peptide:

[0273] 37. namely peptide (7);

[0274] 38. namely peptides (37);

[0275] 39. namely peptides (38);

[0276] 40. namely peptides (39);

[0277] 41. namely peptide (40);

[0278] 42. That is, peptide (41).

[0279] Embodiment 15

[0280] The products obtained in Examples 10-14 were purified using high performance liquid chromatography.

[0281] Purification preparation conditions:

[0282] Mobile phase A: pure acetonitrile; Mobile phase B: 0.1% acetic acid aqueous solution;

[0283] Detection wavelength: 215nm;

[0284] Chromatographic column: C18-20*150mm;

[0285] Elution gradient:

[0286] Time (min) Flow rate (ml / min) A％ B％ 0 40 11 89 48 40 35 65 56 40 38 62 59 40 48 52 60 40 80 20 70 40 80 20

[0287] The sample was purified, and the fractions were collected, concentrated, and lyophilized to obtain a peptide with a purity of more than 80%.

[0288] Example 16

[0289] Preparation of Fmoc-Ala-Pro-Phe-Arg(Pbf)-Met-Gly-Ile-Met-Thr(tBu)-Thr(tBu)-Asn(Trt)-Wang Resin.

[0290] 16.1 Resin Swelling

[0291] Weigh 35g (49.7mmol) of Wang Resin into a 2L solid phase synthesis reaction column. Add 100ml of DMF to completely submerge the resin, then introduce nitrogen, start nitrogen stirring and bubbling, 5min, and then drain the solvent for 5min. Continue to add 100ml of DMF, completely submerge the resin, introduce nitrogen, 30min, the resin is completely swollen, and the solvent is drained. Add another 100ml of DMF, completely submerge the resin, introduce nitrogen, start nitrogen stirring and bubbling, drain the solvent for 5min, and the swelling is complete.

[0292] 16.2 Removal of Fmoc

[0293] The swollen resin was de-Fmoced twice with 500 ml 20% piperidine / DMF, the first reaction lasted 5 min, the second reaction lasted 8 min, and the solvent was drained for 3 min. The resin was washed with DMF 7-8 times and the solvent was drained.

[0294] 16.3 Feeding reaction

[0295] Weigh 53.38g of Fmoc-Asn(Trt)-OH (89.46mmol; 1.8 equivalents) and 14.51g of HOBt into a dry 500ml round-bottom ground flask. Add DMF to dissolve it and place it in a ±5℃ refrigerator for 10min. Add 20.78ml DIC to activate for 10min, avoiding moisture. Add the activated amino acid to the resin after Fmoc protection and react for 2h. Control the reaction temperature at 25℃~35℃ and draw off the reaction solution. Wash the resin with DMF, 100ml each time. After the resin and solvent are evenly mixed, stir for 2min and drain the solvent. Wash the resin with DMF several times until the filtered solvent is clear and transparent.

[0296] The resin substitution degree was detected to be 0.5, and the calculated synthesis scale was 24.9 mmol.

[0297] The N-terminal Fmoc group was deprotected and 24.74 g of Fmoc-Thr(tBu)-OH (62.25 mmol; 2.5 equivalents) after activation was coupled to the peptidyl resin using DMF as solvent in the presence of 10.09 g of HOBt and 14.46 ml of DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the coupling of the next amino acid. In each coupling, 24.74 g of Fmoc-Thr(tBu)-OH (62.25 mmol; 2.5 equiv.); 23.12 g of Fmoc-Met-OH (62.25 mmol; 2.5 equiv.); 22 g of Fmoc-Ile-OH (62.25 mmol; 2.5 equiv.); 18.51 g of Fmoc-Gly-OH (62.25 mmol; 2.5 equiv.) were coupled sequentially in the presence of 10.09 g of HOBt and 14.46 ml of DIC using DMF as solvent.

[0298] The N-terminal Fmoc group was deprotected and 32.37 g of Fmoc-Met-OH (87.15 mmol; 3.5 equivalents) after activation was coupled to the peptidyl resin using DMF as solvent in the presence of 14.13 g of HOBt and 20.24 ml of DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the coupling of the next amino acid. In each coupling, 56.54 g of Fmoc-Arg(Pbf)-OH (87.15 mmol; 3.5 eq.); 33.76 g of Fmoc-Phe-OH (87.15 mmol; 3.5 eq.); 29.40 g of Fmoc-Pro-OH (87.15 mmol; 3.5 eq.) and then 27.13 g of Fmoc-Ala-OH (87.15 mmol; 3.5 eq.) were coupled sequentially in the presence of 14.13 g of HOBt and 20.24 ml of DIC using DMF as solvent.

[0299] After the synthesis, the resin was washed with DMF, 100 ml each time, the resin and the solvent were uniformly mixed, stirred for 2 minutes, and the solvent was drained. The resin was washed with DMF several times until the filtered solvent was clear and transparent.

[0300] Embodiment 17

[0301] Preparation of Fmoc-Ala-Pro-Phe-Arg(Pbf)-Met-Gly-Ile-Met-Thr(tBu)-Thr(tBu)-Asn(Trt)-Linker-Amide Resin.

[0302] 17.1 Preparation of Fmoc-Linker-Amide Resin

[0303] Weigh 50 g (60 mmol) of Amide Resin resin into a 2 L solid phase synthesis reaction column. Wash the resin once with 400 ml DMF, swell the resin with 500 ml DCM for 20 min, and remove the solvent. Wash the resin 3 times with 400 ml DMF.

[0304] Weigh 80.9g (150mmol) of Fmoc-linker and 24.3g (180mmol) of HOBt into a dry 500ml wide-mouthed conical flask. Add about 150ml of DMF solvent to dissolve and place in an ice-water bath to cool for 2min. Add 30ml (195mmol) of DIC to activate for 3min, avoiding moisture. Add the activated Fmoc-linker to the swollen resin and react for 2h, then remove the reaction solution. Wash the resin 3 times with 400ml of DMF, 2min each time.

[0305] After adding 28 ml (300 mmol) of Ac2O, 10.7 ml (60 mmol) of DIPEA and 400 ml of DMF, the mixture was sealed for 3 h, and then the resin was washed four times with 500 ml of DMF and twice with 500 ml of DCM. The solvent was removed.

[0306] 17.2 Removal of Fmoc

[0307] Fmoc-Linker-Amide Resin was de-Fmoced twice with 500 ml 20% piperidine / DMF, the first reaction lasted 5 min, the second reaction lasted 8 min, and the solvent was drained for 3 min. The resin was washed 4 times with 500 ml DMF and 2 times with 500 ml DCM. The solvent was drained.

[0308] 17.3 Feeding reaction

[0309] Weigh 52.21g of Fmoc-Asn(Trt)-OH (87.5mmol) and 14g of HOBt into a dry 500ml wide-mouthed conical flask. Add DMF to dissolve and cool in an ice-water bath. Add 20ml of DIC to activate for 3min, avoiding moisture. Add the activated amino acid to the deprotected resin and react for 2h, then remove the reaction solution. Wash the resin twice with 500ml of DMF, 2min each time, and 400ml of DCM, then remove the solvent.

[0310] The N-terminal Fmoc group was deprotected and 34.78 g of Fmoc-Thr(tBu)-OH (87.5 mmol) after activation was coupled to the peptidyl resin using DMF as solvent in the presence of 14 g of HOBt and 20 ml of DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for coupling the next amino acid. In each coupling, 34.78 g of Fmoc-Thr(tBu)-OH (87.5 mmol); 32.50 g of Fmoc-Met-OH (87.5 mmol); 30.92 g of Fmoc-Ile-OH (87.5 mmol); 26.01 g of Fmoc-Gly-OH (87.5 mmol); 32.50 g of Fmoc-Met-OH (87.5 mmol); 56.77 g of Fmoc-Arg(Pbf)-OH (87.5 mmol); 33.90 g of Fmoc-Phe-OH (87.5 mmol); 29.52 g of Fmoc-Pro-OH (87.5 mmol) and then 27.24 g of Fmoc-Ala-OH (87.5 mmol) were coupled sequentially in the presence of 14 g of HOBt and 20 ml of DIC using DMF as solvent.

[0311] After the synthesis, the resin was washed 4 times with 600 ml DMF for 2 min each time and 2 times with 500 ml DCM, and the solvent was pumped off.

[0312] Embodiment 18

[0313] The N-terminal Fmoc protecting group was removed.

[0314] The N-terminal Fmoc group of the peptidyl resins obtained in Examples 16 and 17 was deprotected by using 500 ml of 20% piperidine / DMF for two times, the first reaction lasted for 5 min, the second reaction lasted for 8 min, and the solvent was drained for 3 min. The resin was washed 4 times with 600 ml of DMF for 2 min each time, and the resin was washed twice with 500 ml of DCM, and the solvent was drained.

[0315] Embodiment 19

[0316] An R1 palmitoyl group was introduced onto the peptidyl resin obtained in Example 18.

[0317] In the presence of 1.53 g of HOBt (10 mmol; 10 equivalents) and 1.54 mL of DIC (10 mmol; 10 equivalents), 2.56 g of palmitic acid (10 mmol; 10 equivalents) previously dissolved in 1 ml of DMF were added to 1 mmol of the peptidyl resin obtained in Example 18. They were allowed to react for 15 h, after which the resin was washed 4 times with 600 ml of DMF for 2 min each time, 2 times with 500 ml of DCM, and the solvent was pumped off.

[0318] Embodiment 20

[0319] An R1 acetyl group was introduced onto the peptidyl resin obtained in Example 18.

[0320] 1 mmol of the peptidyl resin obtained in Example 18 was treated with 25 equivalents of Ac2O in the presence of 25 equivalents of DIPEA using 5 mL of DMF as solvent. They were allowed to react for 30 min, after which the resin was washed 4 times with 600 ml of DMF for 2 min each time and 2 times with 500 ml of DCM, and the solvent was pumped off.

[0321] Embodiment 21

[0322] The peptidyl resins obtained in Examples 18, 19 and 20 were cleaved from the polymer support.

[0323] The peptidyl resins obtained in Examples 18, 19 and 20 were shrunk three times with 500 ml, 300 ml and 200 ml of methanol and dried under vacuum.

[0324] Weigh 1 g of the dried peptidyl resins of Examples 18, 19 and 20, and treat with 7 ml of cleavage solution (TFA: EDT: anisole: thioanisole: TIS: H2O = 87.5: 2.5: 2.5: 2.5: 2.5: 2.5) for 2.5 h. Filter with a sand core funnel, wash the resin with 1 ml of trifluoroacetic acid, and slowly drop the filtrate into 64 ml of ice-cold isopropyl ether. A large amount of white solid precipitates, and after centrifugation, wash with isopropyl ether 6 times. Dry the final precipitate under vacuum.

[0325] Embodiment 22

[0326] The preparation and purification were carried out by high performance liquid chromatography.

[0327] The crude peptide obtained in Example 21 was dissolved in methanol and water, and filtered through a filter membrane with a pore size of 0.45 μm.

[0328] Purification preparation conditions:

[0329] Mobile phase A: pure acetonitrile; Mobile phase B: 0.1% acetic acid aqueous solution;

[0330] Detection wavelength: 215nm;

[0331] Chromatographic column: C18-20*150mm;

[0332] Elution gradient:

[0333] Time (min) Flow rate (ml / min) A％ B％ 0 40 10 90 4 40 12 88 64 40 32 68 65 40 10 90 75 40 10 90

[0334] The filtered sample was injected for purification, and the fractions were collected, concentrated, and lyophilized to obtain the following peptides with a purity of more than 90%:

[0335] 43. H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH, that is, peptide (2);

[0336] 44. H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2, that is, peptide (42);

[0337] 45. Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH, that is, peptide (43);

[0338] 46. ​​Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2, that is, peptide (44);

[0339] 47. Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH, that is, peptide (45);

[0340] 48. Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2, that is, peptide (46).

[0341] Embodiment 23

[0342] Other compounds of the general formula (I) of the present invention can be prepared by similar methods.

[0343] The molecular weights of the peptides obtained were determined by ESI-MS, and the test results of some compounds are shown in Table 2 below.

[0344] Table 2 Molecular weight determination by mass spectrometry

[0345]

[0346]

[0347] Example 24 Cell proliferation assay

[0348] 24.1 Reagents and Materials

[0349] Phosphate buffered saline (PBS) (Gibco), MTT (MTT) (Sigma), dimethyl sulfoxide (DMSO) (Sigma), DMEM (DMEM) (Gibco), and fetal bovine serum (Gibco).

[0350] 24.2 Instruments

[0351] Microplate reader (MD, USA), CO2 incubator (Shanghai Yiheng), clean bench (Suzhou Purification), biological inverted microscope (Chongqing Optoelectronics).

[0352] 24.3 Cell lines

[0353] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences, and mouse skin fibroblasts (NIH3T3) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0354] 24.4 Samples to be tested

[0355] Administration groups: peptide (1), peptide (2), peptide (3), peptide (4), and peptide (5), with concentrations of 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm, respectively.

[0356] Control group: PBS blank control.

[0357] 24.5 Experimental Methods

[0358] a) Take cells in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count 1-4×10 5 / mL, and made into cell suspension;

[0359] b) Appropriately dilute and inoculate 2000 cells / well of the cell suspension onto a 96-well plate, 200 μL / well, and culture in a constant temperature CO2 incubator for 24 hours.

[0360] c) Replace the medium, add the sample to be tested, 20 μL / well, and culture for 24 hours;

[0361] d) Add MTT reagent to a 96-well plate, 20 μL / well, and react in an incubator for 4 hours;

[0362] e) Remove the supernatant, add DMSO, 150 μL / well, and shake on a plate shaker for 5 minutes;

[0363] f) After 5 minutes, use a microplate reader to read the reference OD values ​​at 490nm and 630nm wavelengths, and calculate the cell viability: Cell viability = (OD of the dosing well - OD of the zeroing well) / (OD of the control well - OD of the zeroing well) × 100%

[0364] 24.6 Results

[0365] According to the parallel design principle, the experiment was repeated at least three times, and the experimental data were expressed as mean ± SD. SPSS 17.0 statistical software was used for data analysis.

[0366] The MTT assay is a method for detecting cell survival and growth. The measured OD value is proportional to the cell activity. The larger the OD value, the higher the cell activity.

[0367] Effects of peptides (1) to (5) on HaCaT cell activity Figure 1 The results showed that peptides (1) to (5) could improve the activity of HaCaT cells to a certain extent, among which peptides (2) and (3) had a greater effect on the activity of HaCaT cells. They could improve the activity of HaCaT cells and promote their proliferation at doses of 12.5ppm to 100ppm.

[0368] Effects of peptides (1) to (5) on NIH3T3 cell activity Figure 2 The results showed that peptides (1) to (5) could improve the activity of NIH3T3 cells to a certain extent, among which peptide (3) had a greater effect on the activity of NIH3T3 cells. It could improve the activity of NIH3T3 cells and promote their proliferation at doses of 12.5ppm-100ppm.

[0369] Example 25 Test for promoting cell adhesion

[0370] 25.1 Reagents and Materials

[0371] Phosphate buffered saline (PBS) (Gibco), MTT (MTT) (Sigma), dimethyl sulfoxide (DMSO) (Sigma), DMEM (DMEM) (Gibco), and fetal bovine serum (Gibco).

[0372] 25.2 Instruments

[0373] Microplate reader (MD, USA), CO2 incubator (Shanghai Yiheng), clean bench (Suzhou Purification), biological inverted microscope (Chongqing Optoelectronics).

[0374] 25.3 Cell lines

[0375] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of Type Culture Collection of the Chinese Academy of Sciences.

[0376] 25.4 Samples to be tested

[0377] Administration group: peptide (1) and peptide (2) at concentrations of 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm, respectively. Dissolved in PBS solution.

[0378] Control group: PBS blank control.

[0379] 25.5 Experimental Methods

[0380] Cryopreserved HaCaT human keratinocytes were cultured and passaged at a ratio of 1:2 until about the fifth generation, and cells with better growth were selected as experimental subjects.

[0381] The sample to be tested was added to a 96-well plate at 20 μL / well and dried in a constant temperature oven at 37°C overnight. On the second day, the HaCaT cells with good growth were digested and plated at a density of 10,000 HaCaT cells / well, and the culture medium was supplemented to 200 μL, and incubated in a 37°C, 5% CO2 incubator for 3 hours. After the culture was completed, the culture plate was removed and the culture medium was continued to be supplemented until the liquid surface just overflowed, and then sealed with a sealing film to ensure that there were no bubbles. Flipped under the action of a three-dimensional force for 20 minutes. Discard the original culture medium, add 90 μL of fresh culture medium and 10 μL of 5 mg / mL MTT to each well, and incubate in a 37°C, 5% CO2 incubator for 3 hours. Discard the solution and add 150 μL of DMSO. Set the detection wavelength to 490 nm and the reference wavelength to 630 nm, and use an enzyme reader to read the OD value.

[0382] 25.6 Results

[0383] By selecting HaCaT cells, plating them in a drug-coated 96-well plate, incubating and subjecting them to a three-dimensional force, cells with strong adhesion can remain on the 96-well plate, and by performing MTT quantitative analysis on the live cells on the plate, the effect of the drug on cell adhesion can be evaluated to determine whether the peptide of the present invention can improve cell elasticity. After the three-dimensional force, the more live cells remain on the 96-well plate, the greater the measured OD value, indicating that the cell adhesion is stronger.

[0384] The experimental results are as follows Figure 3 As shown, compared with the normal control group, peptide (1) and peptide (2) can both enhance the adhesion ability of HaCaT cells, thereby helping to improve their elasticity.

[0385] Example 26 Scratch Test

[0386] 26.1 Reagents and Materials

[0387] Phosphate buffered saline (PBS) (Gibco), Dulbecco's modified Eagle's medium (DMEM) (Gibco), and fetal bovine serum (Gibco).

[0388] 26.2 Instruments

[0389] Optical microscope, CO2 incubator.

[0390] 26.3 Cell lines

[0391] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of Type Culture Collection of the Chinese Academy of Sciences.

[0392] 26.4 Samples to be tested

[0393] Administration group: 100 ppm of peptide (1), 100 ppm of peptide (2), and 50 U / mL of epidermal growth factor (EGF).

[0394] Control group: PBS blank control.

[0395] 26.5 Experimental Methods

[0396] Take the frozen HaCaT cells and culture them, subculture them at a ratio of 1:2 to about 5 generations, and select cells with good growth as experimental subjects. Inoculate the cells in a 12-well plate at a density of 200,000 cells / well, with 2 mL of cell suspension per well. After the cells are attached to the wall and fully grown, replace the culture medium containing 0.5% to 1% fetal bovine serum to maintain the cells for 24 hours to synchronize them. Scratch with a sterile pipette tip, wash off the fallen cells with PBS, add the sample to be tested, and continue to incubate in a 37°C, 5% CO2 incubator. After 24 hours, analyze the wound healing area under an optical microscope.

[0397] 26.6 Results

[0398] The scratch migration experiment of cells can reflect the proliferation and repair ability of cells. Figure 4 As shown, the cell scratches in the normal control group were still obvious, and the scratch spacing was normal; compared with the control group, the cells in the EGF-positive group proliferated and migrated significantly, and the spacing was significantly shortened; and the scratch spacing of peptide (1) and peptide (2) was shorter than that of the cells in the normal control group, and cell migration tracks appeared. It can be seen that the peptides of the present invention can promote the proliferation and migration of HaCaT human keratinocytes and have a skin repair effect.

[0399] Example 27 Photodamage repair experiment

[0400] 27.1 Reagents and Materials

[0401] Phosphate buffered saline (PBS) (Gibco), MTT (MTT) (Sigma), dimethyl sulfoxide (DMSO) (Sigma), DMEM (DMEM) (Gibco), and fetal bovine serum (Gibco).

[0402] 27.2 Instruments

[0403] Microplate reader (MD, USA), CO2 incubator (Shanghai Yiheng), clean bench (Suzhou Purification), biological inverted microscope (Chongqing Optoelectronics).

[0404] 27.3 Cell lines

[0405] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences, and mouse skin fibroblasts (NIH3T3) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0406] 27.4 Samples to be tested

[0407] The concentrations of peptide (1), peptide (2), peptide (3), peptide (4), and peptide (5) are 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm, respectively.

[0408] 27.5 Experimental Methods

[0409] Take cells in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count 1-4×10 5 / mL to prepare a cell suspension.

[0410] 10,000 cells were seeded into a 96-well plate. When the cells were grown to about 80%, a UVB light damage model was established. 50 μL of culture medium was added, and no UVB irradiation was performed, which served as the blank control group. The experimental group was repeatedly washed with an appropriate amount of PBS until colorless, and then 50 μL of PBS was added to irradiate at 80 J / cm 3 Irradiate under UVB light, with the light source and culture bottle 15 cm apart. After irradiation, the experimental group discarded PBS, added culture medium and multiple dilution drugs to 200 μL, and continued to incubate in a 37°C, 5% CO2 incubator for 24 h.

[0411] After 24 hours, add 22 μL of 5 mg / mL MTT to each well, incubate in an incubator for 4 hours, discard the original solution, add 150 μL / well of DMSO, and read the reference OD values ​​at 490 nm and 630 nm using a microplate reader after 5 minutes.

[0412] 27.6 Results

[0413] A UVB light-damaged cell model was established, and the drug was administered 24 hours after ultraviolet light irradiation. The cell proliferation activity was detected using the MTT method to evaluate the repair ability of the peptide of the present invention on light-damaged cells.

[0414] The results of HaCaT cell light damage repair are shown in Figure 5 . The results showed that compared with the non-irradiated normal control group, the cell activity was significantly reduced after UVB irradiation, indicating that the light damage model was successfully established. After administration, peptides (1) to (5) were able to increase the activity of light-damaged HaCaT cells to a certain extent, among which peptides (2) and (3) had a better effect on increasing cell activity. This shows that the peptides of the present invention can repair light-damaged HaCaT cells, increase their activity, and have good skin repair ability.

[0415] The results of NIH3T3 cell light damage repair are shown in Figure 6 . The results showed that compared with the normal group without radiation, the activity of NIH3T3 cells after radiation was significantly reduced, indicating that the photodamage model was successfully established. After administration, peptide (1) at 50 and 100 ppm was able to significantly increase cell activity; peptide (2) at doses of 12.5 ppm-100 ppm was able to significantly increase NIH3T3 cell activity. This indicates that peptide (1) and peptide (2) can repair photodamaged NIH3T3 cells within a certain concentration range, increase their activity, and have good skin repair capabilities.

[0416] Example 28 Anti-inflammatory repair experiment

[0417] 28.1 Reagents and Materials

[0418] LPS, dexamethasone (DXM), ELISA kit.

[0419] 28.2 Instruments

[0420] Microplate reader (MD, USA), CO2 incubator (Yiheng, Shanghai), clean bench (Suzhou Purification).

[0421] 28.3 Cell lines

[0422] Mouse mononuclear macrophages (RAW264.7 cells).

[0423] 28.4 Samples to be tested

[0424] Peptide (2), tested at a concentration of 100 ppm.

[0425] 28.5 Experimental Methods

[0426] a) Take RAW264.7 cells in the exponential growth phase and add 0.25% trypsin digestion solution to digest and remove the adherent cells. Count 1-4×10 6 / mL, and made into cell suspension;

[0427] b) Appropriately dilute 100,000 cells / well of the cell suspension and inoculate it on a 6-well plate. When the cells grow to about 80%, establish an LPS-stimulated inflammation model. Set up a blank control group (control), an LPS group, a dexamethasone group (DXM), and a polypeptide group. Add 1000 μL of culture medium to the blank control group; add 200 ng / mL LPS to the LPS group, and add culture medium to 1000 μL; add 200 ng / mL LPS and 50 ng / mL dexamethasone to the dexamethasone group, and add culture medium to 1000 μL; add 200 ng / mL LPS and peptide (2) to the polypeptide group, and add culture medium to 1000 μL (the final test concentration of the polypeptide is 100 ppm). Continue to incubate in a 37°C, 5% CO2 incubator for 48 hours;

[0428] c) centrifuging at 3000 rpm for 10 min to collect the cell supernatant to obtain the sample solution;

[0429] d) Operate according to the ELISA operating instructions: Move all reagents to room temperature for at least 30 minutes, prepare reagents according to the instructions, and set aside. Set up standard wells and test sample wells respectively, add 100μL of standard or test sample to each well, shake gently, cover with plate stickers, and incubate at 37℃ for 2h. Set up 4 replicate wells for each sample. Discard the liquid, spin dry, and wash the plate 5 times. Add 100μL of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37℃ for 1h. Discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1min each time, 200μL / well, spin dry. Add 100μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37℃ for 1h. Discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1min each time, 200μL / well, and spin dry. Add 100 μL of substrate solution to each well in sequence, and color develop at 37°C in the dark for 15-30 minutes. Add 100 μL of stop solution to each well in sequence to terminate the reaction. Measure the OD value of each well in sequence at 450 nm using an ELISA reader within 15 minutes after the reaction is terminated.

[0430] 28.6 Experimental Results

[0431] Inflammatory factors such as TNF-α, IL-6 and IL-8 can cause excessive immune response in the body, leading to skin ulceration, redness and swelling, etc. Therefore, inhibiting inflammatory factors such as TNF-α, IL-6 and IL-8 can achieve anti-inflammatory and repair effects. The inflammation model was established by stimulating RAW264.7 cells with 200ng / mL LPS to explore the anti-inflammatory and repair effects of the peptides of the present invention.

[0432] The experimental results are as follows Figure 7-9As shown, after LPS stimulation, TNF-α, IL-6 and IL-8 cytokines were significantly upregulated; after administration, 100 ppm of peptide (2) significantly downregulated the expression of TNF-α, IL-6 and IL-8, and its anti-inflammatory effect was equivalent to that of the positive drug dexamethasone, indicating that peptide (2) has a significant anti-inflammatory and repair effect.

[0433] Example 29 Stability Test Study

[0434] 1. The peptide H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH (hereinafter referred to as reference peptide A) sample and the peptide (1) and peptide (2) samples of the present invention were dissolved in water respectively, and placed at room temperature. The purity changes of each sample at 0 days, 8 days and 18 days were examined by HPLC. The results are shown in Table 3 below:

[0435] Table 3 Stability study of reference peptide A aqueous solution and peptide (1) and peptide (2) aqueous solutions

[0436] Test conditions Reference peptide A aqueous solution (purity) Peptide (1) aqueous solution (purity) Peptide (2) aqueous solution (purity) Solution 0 day 88.030％ 98.443％ 99.291％ 8 days at room temperature 80.880％ 98.362％ 99.122％ 18 days at normal temperature 72.693％ 96.703％ 98.045％

[0437] From the results in Table 3, it can be seen that the purity of the reference peptide A aqueous solution was greatly reduced on day 0 after exposure to air, and the purity dropped to 72.693% after being placed in the aqueous solution at room temperature for 18 days. Compared with the reference peptide A aqueous solution, the purity of the peptide (1) and peptide (2) aqueous solutions did not change significantly after being placed at room temperature for 18 days.

[0438] 2. Stability study of reference peptide A powder, peptide (1) powder, and peptide (2) powder

[0439] The reference peptide A powder, peptide (1) powder, and peptide (2) powder were placed at 25°C and 40°C, respectively, and the change of their purity over time was investigated by HPLC. The results are shown in Table 4 below:

[0440] Table 4 Stability study of reference peptide A powder, peptide (1) powder, and peptide (2) powder

[0441]

[0442] From the results in Table 4, it can be seen that the purity of the reference peptide A powder decreased significantly after being placed at 40°C for 5 days, and was only 57.503%. In contrast, the purity of the peptide (1) powder and the peptide (2) powder did not change significantly after being placed at 40°C for 20 days.

[0443] 3. Study on the stability of peptide (3)-(7) powder

[0444] The powder samples of peptides (3)-(7) were placed under high humidity (RH92.5%, 25°C) conditions for 10 days, and the purity changes of each sample were examined by HPLC. The results are shown in Table 5 below:

[0445] Table 5 Stability study of peptide (3)-(7) powders

[0446] Sample No. 0 days (purity) High humidity 10 days (purity) Peptides (3) 97.605％ 96.251％ Peptides (4) 96.017％ 93.962％ Peptides (5) 97.482％ 94.603％ Peptides (6) 96.235％ 95.719％ Peptides (7) 98.541％ 97.863％

[0447] From the results in Table 5, it can be seen that the purity of peptides (3)-(7) did not change significantly after being placed under high humidity (RH92.5%, 25°C) conditions for 10 days.

[0448] In this Example 29, the HPLC chromatographic separation conditions are as follows:

[0449] Chromatographic column: Octadecyl bonded silica as filler 4.6*250mm, 5μm

[0450] Mobile phase A: acetonitrile (containing 0.09% trifluoroacetic acid)

[0451] Mobile phase B: 0.1% trifluoroacetic acid

[0452] Diluent: Water

[0453] Flow rate: 1.0ml / min;

[0454] Column temperature: 35°C;

[0455] Detection wavelength: 215nm;

[0456] Injection volume: 10 μl;

[0457] Run time: 32 minutes

[0458] Gradient elution table:

[0459]

[0460]

[0461] Test solution: Take an appropriate amount of the product and dissolve in water to make a solution containing 1 mg per 1 ml.

[0462] Example 30 Preparation of liposomes containing peptide (1)

[0463]

[0464] Dipalmitoylphosphatidylcholine was weighed and dissolved in chloroform. The solvent was evaporated under vacuum until a thin layer of phospholipids was obtained, which was treated with an aqueous peptide solution of the desired concentration at 55°C to hydrate and obtain multilamellar liposomes. The multilamellar liposomes were treated by high pressure homogenization to obtain smaller and more uniform unilamellar liposomes.

[0465] Example 31 Preparation of a microemulsion composition containing peptide (2)

[0466]

[0467] According to the prescribed amount, weigh the ingredients of phase B and add them to the container. Next, add phase D to phase B and homogenize under continuous stirring. Then add phase A to the mixture. Finally, add phase C to obtain a microemulsion composition containing peptide (2).

[0468] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, but it does not mean that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. The following peptide (1)-peptide (2), peptide (12)-peptide (16), peptide (42)-peptide (46), or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof: (1) (2)H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH; (12) (13) (14) (15) (16) (42)H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2; (43)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH; (44)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2; (45)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH; (46) Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH2.

2. The peptide according to claim 1, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, characterized in that: The cosmetically acceptable salts or pharmaceutically acceptable salts include metal salts of the peptides, the metals including lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.

3. The peptide according to claim 1, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, characterized in that: The cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by the peptide and an organic base, and the organic base includes ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine.

4. The peptide according to claim 1, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, characterized in that: The cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by the peptide and an inorganic acid or an organic acid, wherein the organic acid includes acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; The inorganic acid includes hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

5. A cosmetic or pharmaceutical composition, characterized in that: The composition comprises an effective amount of the peptide according to any one of claims 1 to 4, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, and at least one excipient.

6. The cosmetic or pharmaceutical composition according to claim 5, characterized in that Cosmetically or pharmaceutically acceptable adjuvants are also included.

7. A cosmetically or pharmaceutically acceptable delivery system or sustained-release system, characterized in that: A composition comprising an effective amount of a peptide according to any one of claims 1 to 4, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to claim 5 or 6; The cosmetically or pharmaceutically acceptable delivery system or sustained-release system is selected from the group consisting of liposomes, oleosomes, nanostructured lipid carriers, sponges, lipid vesicles, micelles, microemulsions or nanoemulsions.

8. The cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to claim 7, characterized in that: The cosmetically or pharmaceutically acceptable delivery system or sustained-release system is a liposome or a microemulsion.

9. The cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to claim 7, characterized in that: The cosmetically or pharmaceutically acceptable delivery system or sustained-release system is a water-in-oil microemulsion having an internal structure of reverse micelles.

10. A cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support, characterized in that: A composition comprising an effective amount of a peptide according to any one of claims 1 to 4, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to claim 5 or 6; The cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support is selected from the group consisting of talc, bentonite, silicon dioxide, starch or maltodextrin.

11. A cosmetic, characterized in that: A cosmetic or pharmaceutical composition comprising an effective amount of the peptide according to any one of claims 1 to 4, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to claim 5 or 6.

12. The cosmetic according to claim 11, characterized in that The cosmetics are selected from: foundation, makeup remover, eye shadow, lip gloss, lip balm or lip powder.

13. A fabric, nonwoven fabric or medical device, characterized in that: A cosmetic or pharmaceutical composition comprising an effective amount of the peptide according to any one of claims 1 to 4, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to claim 5 or 6.

14. The fabric, nonwoven fabric or medical device according to claim 13, characterized in that The fabric, nonwoven fabric or medical device is selected from the group consisting of a bandage, gauze, t-shirt, socks, pantyhose, underwear, a belt, gloves, a diaper, a sanitary napkin, a bed cover, a wipe, an adhesive patch, a non-adhesive patch or a facial mask.

15. Use of the peptide according to any one of claims 1 to 4, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the cosmetic or pharmaceutical composition according to claim 5 or 6, or the cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to any one of claims 7 to 9, or the cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support according to claim 10, or the cosmetic according to claim 11 or 12, or the fabric, nonwoven fabric or medical device according to claim 13 or 14 in the preparation of a cosmetic composition or pharmaceutical composition for treating or caring for the skin or mucous membranes.

16. Use of the peptide according to any one of claims 1 to 4, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the cosmetic or pharmaceutical composition according to claim 5 or 6, or the cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to any one of claims 7 to 9, or the cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support according to claim 10, or the cosmetic according to claim 11 or 12, or the fabric, nonwoven fabric or medical device according to claim 13 or 14 in the preparation of a cosmetic composition or pharmaceutical composition for treating or caring for body surface trauma, burns, skin ulcers, reducing scar formation, or accelerating scar repair.

17. Use of the peptide according to any one of claims 1 to 4, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the cosmetic or pharmaceutical composition according to claim 5 or 6, or the cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to any one of claims 7 to 9, or the cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support according to claim 10, or the cosmetic according to claim 11 or 12, or the fabric, nonwoven fabric or medical device according to claim 13 or 14 in the preparation of a cosmetic composition or pharmaceutical composition for improving skin barrier function, or for re-epithelialization or healing of skin or mucous membranes.

18. Use of the peptide according to any one of claims 1 to 4, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the cosmetic or pharmaceutical composition according to claim 5 or 6, or the cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to any one of claims 7 to 9, or the cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support according to claim 10, or the cosmetic according to claim 11 or 12, or the fabric, nonwoven fabric or medical device according to claim 13 or 14 in the preparation of a cosmetic composition or pharmaceutical composition for treating, preventing or repairing skin aging or photoaging.

19. The use according to claim 18, wherein Treatment, prevention or repair of skin aging or photoaging is the reduction, prevention or treatment of facial wrinkles.

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