Peptides with anti-aging effects, compositions and uses thereof

Abstract

The present application provides a peptide having the formula (I) R1-Ser-Pro-X1-Gln-R2 or a cosmetically or pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition thereof, and their use in the preparation of a cosmetic or pharmaceutical composition for treating, preventing or repairing skin aging or photoaging, promoting collagen production, increasing skin elasticity and / or skin firmness, promoting PER1 protein expression, regulating skin circadian rhythm and / or repairing damaged skin cells. The peptide of formula (I) regulates the expression of skin circadian cycle regulation genes and related proteins, regulates skin circadian rhythm and resynchronizes skin cell biological clock, enhances cell vitality, repairs damaged cells, thereby alleviating signs of skin aging, and can be used in the field of cosmetics or medicine to resist skin aging, and can also be used to prepare a PER1 protein activator.

Description

Technical Field

[0001] This invention relates to the fields of medicine and cosmetics, and in particular to a peptide with anti-aging effects, its cosmetic or pharmaceutical compositions, and uses. Background Technology

[0002] As the body's first line of physiological defense, the skin protects tissues and organs from chemical, mechanical, physical, and pathogenic microorganism attacks, while also constantly enduring the challenges of the external environment. In recent years, due to environmental aggressors such as UV radiation and pollution, the number of patients with skin diseases has been increasing year by year, accelerating skin aging. Environmental aggressors can damage the DNA of skin cells and affect their circadian rhythm.

[0003] Circadian rhythms refer to the 24-hour cycle of bodily activities, also known as the biological clock, which regulates many of our metabolic, physiological, and behavioral processes. Extensive evidence suggests that circadian rhythm disorders and imbalances can have significant negative effects on health, including jet lag, fatigue syndrome caused by working night shifts, sleep disturbances, and skin diseases. Furthermore, many serious health threats, such as cardiovascular diseases and cancer, are also related to circadian rhythm disruptions. However, with the development of modern civilization, more and more people are deviating from the lifestyle of working at sunrise and resting at sunset, leading to increasingly widespread circadian rhythm disorders.

[0004] Independent biological clock systems also exist in keratinocytes, melanocytes, and fibroblasts of the skin. Circadian rhythm proteins are expressed not only in cultured human cells but also in human skin tissues, influencing skin function. Skin is easily exposed to external environmental factors such as light, temperature, humidity, ultraviolet radiation, and pathogens, which change on a daily cycle. To adapt to this environment, the skin regulates various physiological functions by activating its own biological clock. Experiments have shown that skin cell proliferation and differentiation, water loss, sebum production, temperature, pH, and wrinkle formation all change on a 24-hour cycle. For example, skin cell proliferation and differentiation occur on a 24-hour cycle through a series of processes. During the day, the skin is easily damaged by ultraviolet radiation, so skin cell differentiation mainly occurs between evening and early morning. By temporally separating these processes, the skin is protected from harmful environments. However, when this rhythm is disrupted, the necessary physiological responses cannot occur at the appropriate time, thus disrupting the skin's circadian rhythm and accelerating skin damage.

[0005] Recent studies have identified genes associated with the body's natural circadian rhythm, including the Clock and PER1 genes, both of which encode proteins that regulate this rhythm (Clock and PER1 proteins). PER1 is a core component of the biological clock that regulates the skin's circadian rhythm and is a member of the Period gene family, which consists of PER1, PER2, and PER3. These genes are involved in metabolism, movement, and behavior, with PER1 playing a central role. The Clock and PER1 genes are also present in skin cells. PER1 gene expression induces cellular activity programs related to biological processes that occur at night, such as repair. Epidermal progenitor cells / stem cells exhibit distinct circadian rhythm characteristics in their DNA replication, DNA repair mechanisms, and cell division. Skin cells chronically exposed to environmental aggressors often show reduced, irregular, or asynchronous Clock or PER1 gene expression, disrupting the normal circadian rhythm in skin cells. Over time, the normal circadian rhythm and synchronicity of cells are disrupted, the skin's balance is damaged, thus accelerating the skin's natural aging process, causing skin damage, and resulting in problems such as wrinkles, fine lines, loose skin, uneven pigmentation, age spots, and freckles.

[0006] Analysis of the Clock and PER1 genes revealed that they are core genes and negative feedback genes regulating the skin's circadian rhythm. These genes participate in regulating the skin's response to environmental stress. Furthermore, the Clock and PER1 genes, operating on an approximately 24-hour cycle, interfere with the expression and activity regulation of genes and proteins involved in various physiological processes. This biological clock adapts to the external environment to regulate the activity of physiological responses. By regulating various skin cell activities according to the circadian rhythm, the skin is protected from various environmental damages such as UV radiation, temperature, chemical and physical harm, and microbial infections.

[0007] Therefore, it is necessary to study an active ingredient that can improve the skin's circadian rhythm, activate skin circadian rhythm regulatory genes and related proteins, based on the mechanism of the skin's circadian rhythm, and apply it to the fields of medicine and cosmetics to alleviate skin aging. Summary of the Invention

[0008] The purpose of this invention is to provide a stable, efficient, safe, and non-irritating active ingredient that can repair damaged cells and achieve anti-aging effects by activating the expression of genes and related proteins that regulate the skin's circadian rhythm. At the same time, it can promote collagen production, increase skin elasticity and / or skin firmness, and prevent skin sagging, making it widely applicable in the fields of medicine and cosmetics.

[0009] In view of this, the present invention provides a peptide of formula (I), or a stereoisomer thereof, or a mixture thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof.

[0010] R1-Ser-Pro-X1-Gln-R2(I)

[0011] In formula (I),

[0012] X1 is selected from: -Gly-, -Asn-, -Ala-, -Ile-, -Val-, -Ser-, -Thr-, or -Met-;

[0013] R1 is selected from: H or R3-CO-, and R3 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

[0014] R2 is selected from: -NR4R5 or -OR4, wherein R4 and R5 are independently selected from: H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

[0015] The alkyl group refers to a saturated aliphatic straight-chain or branched alkyl group having 1-24 carbon atoms (optionally having 1-16 carbon atoms; optional having 1-14 carbon atoms; optional having 1-12 carbon atoms; optional having 1, 2, 3, 4, 5, or 6 carbon atoms); and 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;

[0016] The alkenyl group refers to a straight-chain or branched alkenyl group having 2-24 carbon atoms (optionally 2-16 carbon atoms; optional 2-14 carbon atoms; optional 2-12 carbon atoms; optional 2, 3, 4, 5, or 6 carbon atoms); the alkenyl group has one or more carbon-carbon double bonds, optionally having 1, 2, or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bonded to the rest of the molecule by a single bond; and may be selected from: vinyl, oleyl, or linoleyl.

[0017] Optionally, the substituents in "substituted alkyl" and "substituted alkenyl" are selected from C1-C4 alkyl; hydroxyl; C1-C4 alkoxy; amino; C1-C4 aminoalkyl; C1-C4 carbonyloxy; C1-C4 oxycarbonyl; halogens (such as fluorine, chlorine, bromine, and iodine); cyano; nitro; azide; C1-C4 alkylsulfonyl; thiol; C1-C4 alkylthio; C6-C 30 Aryl groups, such as phenoxy groups; -NR b (C=NR) b )NR b Rc , where R b and R c It is independently selected from: H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C 10 cycloalkyl, C6-C 18 Aryl, C7-C 17 Aryl groups, heterocyclic groups with three to ten members, or amino groups as protecting groups.

[0018] Optionally, R1 is selected from: H, acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, or linoleoyl; R4 and R5 are independently selected from: H, methyl, ethyl, hexyl, dodecyl, or hexadecyl.

[0019] Optionally, R1 is selected from H, acetyl, myristoyl, or palmitoyl; R4 is H and R5 is selected from H, methyl, ethyl, hexyl, dodecyl, or hexadecyl.

[0020] Specifically, R1 is H or acetyl; R2 is -OH or -NH2.

[0021] Optionally, the peptide represented by formula (I), or a stereoisomer thereof, a mixture thereof, a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, is selected from the following peptides (1)-(32):

[0022] (1)H-Ser-Pro-Gly-Gln-OH;

[0023] (2)H-Ser-Pro-Gly-Gln-NH2;

[0024] (3)Ac-Ser-Pro-Gly-Gln-OH;

[0025] (4)Ac-Ser-Pro-Gly-Gln-NH2;

[0026] (5)H-Ser-Pro-Ala-Gln-OH;

[0027] (6)H-Ser-Pro-Ala-Gln-NH2;

[0028] (7)Ac-Ser-Pro-Ala-Gln-OH;

[0029] (8)Ac-Ser-Pro-Ala-Gln-NH2;

[0030] (9)H-Ser-Pro-Asn-Gln-NH2;

[0031] (10)H-Ser-Pro-Asn-Gln-OH;

[0032] (11)Ac-Ser-Pro-Asn-Gln-OH;

[0033] (12)Ac-Ser-Pro-Asn-Gln-NH2;

[0034] (13)H-Ser-Pro-Ile-Gln-OH;

[0035] (14)H-Ser-Pro-Ile-Gln-NH2;

[0036] (15)Ac-Ser-Pro-Ile-Gln-OH;

[0037] (16)Ac-Ser-Pro-Ile-Gln-NH2;

[0038] (17)H-Ser-Pro-Val-Gln-OH;

[0039] (18)H-Ser-Pro-Val-Gln-NH2;

[0040] (19)Ac-Ser-Pro-Val-Gln-OH;

[0041] (20)Ac-Ser-Pro-Val-Gln-NH2;

[0042] (21)H-Ser-Pro-Ser-Gln-OH;

[0043] (22)H-Ser-Pro-Ser-Gln-NH2;

[0044] (23)Ac-Ser-Pro-Ser-Gln-OH;

[0045] (24)Ac-Ser-Pro-Ser-Gln-NH2;

[0046] (25)H-Ser-Pro-Thr-Gln-OH;

[0047] (26)H-Ser-Pro-Thr-Gln-NH2;

[0048] (27)Ac-Ser-Pro-Thr-Gln-OH;

[0049] (28)Ac-Ser-Pro-Thr-Gln-NH2;

[0050] (29)H-Ser-Pro-Met-Gln-OH;

[0051] (30)H-Ser-Pro-Met-Gln-NH2;

[0052] (31)Ac-Ser-Pro-Met-Gln-OH;

[0053] (32)Ac-Ser-Pro-Met-Gln-NH2.

[0054] Optionally, it is selected from peptide (1), peptide (2), peptide (3), peptide (4), peptide (9), peptide (10), peptide (11), and peptide (12); specifically,

[0055] (1)H-Ser-Pro-Gly-Gln-OH;

[0056] (2)H-Ser-Pro-Gly-Gln-NH2;

[0057] (3)Ac-Ser-Pro-Gly-Gln-OH;

[0058] (4)Ac-Ser-Pro-Gly-Gln-NH2;

[0059] (9)H-Ser-Pro-Asn-Gln-NH2;

[0060] (10)H-Ser-Pro-Asn-Gln-OH;

[0061] (11)Ac-Ser-Pro-Asn-Gln-OH;

[0062] (12)Ac-Ser-Pro-Asn-Gln-NH2.

[0063] The peptide represented by formula (I) of the present invention can exist as a stereoisomer or a mixture of stereoisomers; for example, the amino acids contained therein can have L-, D-configurations, or be racemic independently of each other. Therefore, it is possible to obtain isomeric mixtures and racemic mixtures or diastereomeric mixtures, or pure diastereomeric or enantiomers, depending on the number of asymmetric carbons and the presence of any isomers or isomeric mixtures. The preferred structure of the peptide represented by formula (I) of the present invention is a pure isomer, i.e., an enantiomer or diastereomeric isomer.

[0064] For example, when -Pro- is mentioned in this invention, it should be understood that -Pro- is selected from -L-Pro-, -D-Pro-, or a mixture of both, and is racemic or non-racemic. The preparation methods described in this document enable those skilled in the art to obtain each stereoisomer of the peptide of this invention by selecting amino acids having the correct configuration.

[0065] The present invention also includes all suitable isotopic variants of the peptide represented by formula (I). These isotopic variants of the peptides of the present invention are understood herein to refer to compounds in which at least one atom within the peptide of the present invention is replaced by another atom of the same atomic number, but said other atom has an atomic mass different from that of atoms commonly or predominantly found in nature. Examples of isotopes that can be incorporated into the peptides of the present invention are those of hydrogen, carbon, nitrogen, oxygen, or sulfur, for example… 2 H (deuterium) 3 H (tritium) 13 C 14 C 15 N、 17 O、 18 O、 33 S, 34 S, 35 S or 36 S. Specific isotopic variants of the peptides of the present invention (especially those incorporating one or more radioactive isotopes) may be advantageous, for example, in examining mechanisms of action or distribution of active compounds in vivo; due to their relatively simple prepareability and detectability, especially with 3 H or 14 Compounds labeled with the C isotope are suitable for this purpose. Furthermore, the incorporation of isotopes (e.g., deuterium) can produce specific therapeutic benefits, such as prolonged in vivo half-life or reduced required active dose, due to the enhanced metabolic stability of the compounds; therefore, in certain cases, such modification of the peptides of the present invention may also constitute a preferred embodiment of the invention. Isotopic variants of the peptides of the present invention can be prepared by methods known to those skilled in the art, such as those further described below and those described in the examples, using respective reagents and / or isotope modifiers of the starting materials.

[0066] Furthermore, the present invention also includes prodrugs of the peptides of the present invention. The term "prodrug" herein refers to a compound that may be biologically active or inactive on its own, but which, during its residence time in the body, undergoes a reaction (e.g., metabolism or hydrolysis) to generate the peptides of the present invention.

[0067] The term "cosmetically acceptable salt or pharmaceutically acceptable salt" refers to a salt approved for use in animals, and more precisely in humans, comprising a metal salt of a peptide represented by formula (I), wherein the metal includes, but is not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum; comprising a salt formed by a peptide represented by formula (I) and an organic base, wherein the organic base includes, but is not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine; comprising a salt formed by a peptide represented by formula (I) and an inorganic or organic acid, wherein the organic acid includes, but is 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; optionally, the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

[0068] The synthesis of the peptide represented by formula (I) of the present invention, or its stereoisomers, or its cosmetically acceptable salts, or its pharmaceutically acceptable salts, can be carried out according to conventional methods known in the art, such as solid-phase synthesis, liquid-phase synthesis, or a combination of solid and liquid methods. It can also be prepared by biotechnological methods aimed at producing a desired sequence, or by controlled hydrolysis of proteins of animal, fungal, or plant origin.

[0069] For example, a method for obtaining the peptide represented by formula (I) includes the following steps:

[0070] - Couple amino acids with a protected N-terminus and a free C-terminus to amino acids with a free N-terminus and a protected C-terminus or a C-terminus bound to a solid support.

[0071] - Eliminate the groups protecting the N-terminus;

[0072] - Repeat this coupling sequence and remove the group protecting the N-terminus until the desired peptide sequence is obtained;

[0073] - Eliminate the groups protecting the C-terminus or cleave them from the solid support.

[0074] 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 to an amino acid having a free N-terminus and a C-terminus bound to a polymer support; removing the group protecting the N-terminus; and repeating this sequence a number of times as required to thus obtain a peptide of the desired length, followed by cleaving the synthesized peptide from the original polymer support.

[0075] Throughout the synthesis, the functional groups of the side chains of these amino acids are adequately protected with temporary or permanent protecting groups and can be deprotected simultaneously or orthogonally with the process of cleaving the peptide from the polymer carrier.

[0076] Alternatively, solid-phase synthesis can be carried out using a convergent strategy, which involves coupling dipeptides or tripeptides to a polymer support or to dipeptides or amino acids previously bound to a polymer support.

[0077] The N-terminus and C-terminus are deprotected and / or the peptide is cleaved from the polymer support in an indeterminate order using standard conditions and methods known in the art, after which the functional groups at the ends may be modified. Optional N-terminal and C-terminal modifications may be made to the peptide of formula (I) bound to the polymer support, or optional N-terminal and C-terminal modifications may be made after the peptide has been cleaved from the polymer support.

[0078] In another aspect of the present invention, a cosmetic or pharmaceutical composition is provided, comprising an effective amount of the peptide represented by formula (I) above, or a stereoisomer thereof, or a mixture thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, and at least one excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant.

[0079] Optionally, the adjuvant is selected from: collagen synthesis stimulants, agents regulating PGC-1α synthesis, agents regulating PPARγ activity, agents increasing or decreasing triglyceride content in adipocytes, agents stimulating or delaying adipocyte differentiation, lipolytic agents or agents stimulating lipolysis, lipolytic agents, lipogenic agents, inhibitors of acetylcholine receptor aggregation, agents inhibiting muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulants or inhibitors, whitening agents or depigmenting agents, pigmentation promoters, self-tanning agents, anti-aging agents, NO-synthesizers, 5α-reductase inhibitors, lysyl hydroxylase and / or prolyl hydroxylase inhibitors, antioxidants, free radical scavengers and / or anti-air pollution agents, and active agents. Carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioning agents, moisture-retaining substances, alpha-hydroxy acids, beta-hydroxy acids, humectants, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents that reduce or treat under-eye bags, exfoliants, antimicrobial agents, sterilizing agents, bacteriostatic agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and / or inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate core proteoglycan synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, and stimulants. Agents that stimulate chaperone protein synthesis, cAMP synthesis, HSP70 synthesis, heat shock protein synthesis, hyaluronic acid synthesis, fibronectin synthesis, deacetylase synthesis, lipid and stratum corneum component synthesis, ceramides, fatty acids, collagen degradation inhibitors, elastin degradation inhibitors, serine protease inhibitors, fibroblast proliferation stimulators, keratinocyte proliferation stimulators, adipocyte proliferation stimulators, melanocyte proliferation stimulators, keratinocyte differentiation stimulators, acetylcholinesterase inhibitors, skin relaxants, glycosaminoglycan synthesis stimulators, anti-hyperkeratosis agents, comedolytic agents, anti-psoriasis agents, anti-eczema agents, DNA. Repairing agents, DNA protectants, stabilizers, antipruritics, agents for treating and / or caring for sensitive skin, hardening agents, firming agents, reconstructing agents, anti-stretch mark agents, agents that regulate sebum production, antiperspirants, agents that stimulate healing, agents that assist healing, agents that stimulate re-epithelialization, agents that assist re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents acting on capillary circulation and / or microcirculation, agents that stimulate angiogenesis, agents that inhibit vascular permeability, venous tension agents, agents acting on cell metabolism, agents for improving dermal-epidermal junction, agents that induce hair growth, agents that inhibit or delay hair growth, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents derived from bio-fermentation processes, inorganic salts, cell extracts, sunscreens.And organic or inorganic photoprotective agents or mixtures thereof that effectively resist UVA and / or UVB rays.

[0080] Optionally, the formulation of the cosmetic or pharmaceutical composition is selected from: creams, oils, balms, foams, lotions, gels, liniments, serums, ointments, mousses, powders, sticks, pens, sprays, aerosols, capsules, tablets, granules, chewing gum, solutions, suspensions, emulsions, elixirs, polysaccharide films, gels, or gelatin.

[0081] Optionally, the capsules include: soft capsules, hard capsules, and optionally gelatin capsules;

[0082] Optionally, the tablet comprises a sugar-coated tablet.

[0083] The peptides of the present invention have variable solubility in water depending on the nature of their sequence or any possible modifications to the N-terminus and / or C-terminus. Therefore, the peptides of the present invention can be incorporated into compositions by means of aqueous solutions, and those insoluble in water are soluble in conventional solvents acceptable for cosmetic or pharmaceutical purposes, such as, and not limited to, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol, or polyethylene glycol, or any combination thereof.

[0084] The amount of the peptides of the present invention to be applied for cosmetic or pharmaceutical purposes, and their dosage, will depend on many factors, including age, patient condition, severity of symptoms or disease, route and frequency of application, and the specific nature of the peptide to be used.

[0085] "Cosmetic or pharmaceutically effective amount" means an amount of one or more peptides of the invention that is non-toxic but sufficient to provide the desired effect. The peptides of the invention are used in cosmetic or pharmaceutical compositions of the invention at a cosmetic or pharmaceutically effective concentration to obtain the desired effect; in a preferred form, the concentration is 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.

[0086] In another aspect, the present invention provides a cosmetically or pharmaceutically acceptable delivery system or sustained-release system for achieving better penetration of the active ingredient and / or improving its pharmacokinetic and pharmacodynamic properties, comprising an effective amount of the peptide represented by formula (I) above, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the cosmetic or pharmaceutical composition described above.

[0087] The term "delivery system" refers to a diluent, adjuvant, excipient, or carrier applied with the peptides of the present invention, selected from water, oil, or surfactants, including those of petroleum, animal, plant, or synthetic origin, such as and not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitol ester, ether sulfate, sulfate, betaine, glucosinolate, maltodextrin, fatty alcohol, nonyl alcohol ether, poloxamer, polyoxyethylene, polyethylene glycol, dextran, glycerol, digitalis saponins, and the like. Diluents that can be used in various delivery systems for administering the peptides of the present invention are known to those skilled in the art.

[0088] The term “sustained release” is used in its conventional sense to refer to a compound delivery system that provides the gradual release of a compound over a period of time, and preferably, but not necessarily, has a relatively constant level of compound release throughout the time period.

[0089] Examples of delivery systems or sustained-release systems include liposomes, oil bodies, nonionic surfactant liposome vesicles, liposomes, milligrams, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, lipid vesicles, micelles, milligrams, microspheres, nanospheres, lipospheres, microemulsions, nanoemulsions, milligram particles, microparticles, or nanoparticles. Preferred delivery systems or sustained-release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions having an antimicelle internal structure.

[0090] Sustained-release systems can be prepared by methods known in the art and can be administered, for example, by topical or transdermal administration, including adhesive patches, non-adhesive patches, occlusive patches, and microelectronic patches; or by systemic administration, for example, but not limited to, oral or parenteral routes, including nasal, rectal, subcutaneous implantation or injection, or direct implantation or injection into a specific body site, and preferably should release a relatively constant amount of the peptides of the invention. The amount of peptides contained in the sustained-release system will depend, for example, on the site where the composition will be administered, the release kinetics and duration of the peptides of the invention, and the nature of the symptoms, conditions, and / or diseases to be treated and / or cared for.

[0091] In another aspect of the present invention, there is provided the use of the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture thereof, 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, in the preparation of a cosmetic composition or pharmaceutical composition for treating, preventing or repairing skin aging and / or photoaging.

[0092] In another aspect of the present invention, there is provided the use of the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture thereof, 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, in the preparation of a cosmetic composition or pharmaceutical composition for increasing skin elasticity and / or skin firmness.

[0093] In another aspect, the present invention provides the use of a peptide of formula (I) above, or a stereoisomer thereof, or a mixture thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the above-described cosmetic or pharmaceutical composition, or the above-described cosmetically or pharmaceutically acceptable delivery system or sustained-release system, in the preparation of a cosmetic composition or pharmaceutical composition for promoting collagen production.

[0094] In another aspect of the present invention, there is provided the use of the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture thereof, 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, in the preparation of cosmetic or pharmaceutical compositions for promoting PER1 protein expression, regulating skin circadian rhythms and / or repairing damaged skin cells.

[0095] In another aspect of the present invention, there is provided the use of the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture thereof, 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, in the preparation of a PER1 protein activator.

[0096] To facilitate understanding of this invention, the meanings of some terms and expressions used in this invention are explained below:

[0097] In this invention, the term "skin" should be understood as the multiple layers that constitute it, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, including both ends. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes. In this invention, the term "skin" includes the scalp.

[0098] The term "treatment" refers to administering the peptide according to the invention to alleviate or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with such disease or condition. The term "treatment" also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or condition.

[0099] The term "prevention" refers to the ability of the peptides of the present invention to prevent, delay, or hinder the occurrence or development of a disease or condition before it occurs.

[0100] The term "repair" refers to the ability of the peptides of this invention to improve, alleviate, or restore a disease or condition to its original state after its onset.

[0101] The term "aging" refers to changes that the skin undergoes with age (natural aging), or changes that occur through exposure to sunlight (photoaging) or environmental pollutants such as chemical dirt or contaminants, tobacco smoke, etc., and includes all externally visible and / or tactilely perceptible changes, such as but not limited to: the development of discontinuities in the skin (e.g., wrinkles, fine lines, expression lines, stretch marks, lines, grooves, unevenness or roughness, increased pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of resilience, loss of elasticity), sagging skin (e.g., drooping cheeks, under-eye bags, or double chin), changes in skin color (e.g., scars, redness, under-eye bags, or areas of hyperpigmentation such as age spots or freckles), abnormal differentiation, excessive keratinization, degeneration of elastic tissue, keratosis, hair loss, orange peel-like skin, loss of collagen structure, and other histological changes in the stratum corneum, dermis, epidermis, vascular system (e.g., the appearance of spider veins or telangiectasia) or those tissues close to the skin.

[0102] The term "photoaging" refers to premature aging of the skin due to prolonged exposure to ultraviolet radiation. It exhibits the same physiological characteristics as natural aging, such as, but not limited to: sagging, drooping, color changes or irregular, abnormal and / or excessive keratinization of the skin.

[0103] In this 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).

[0104] Therefore, for example, Gly represents NH2-CH2-COOH, Gly- represents NH2-CH2-CO-, -Gly represents -NH-CH2-COOH, and -Gly- represents -NH-CH2-CO-. Thus, the hyphen representing the peptide bond eliminates the OH group in the 1-carboxyl group of the amino acid (represented here in its conventional non-ionic form) when it is on the right side of the symbol, and eliminates the H group in the 2-amino group of the amino acid when it is on the left side of the symbol; both modifications can be applied to the same symbol (see Table 1).

[0105] Table 1. Structures of amino acid residues and their single-letter and three-letter abbreviations.

[0106]

[0107] The abbreviation "Ac-" is used in this invention to represent acetyl (CH3-CO-).

[0108] The beneficial effects of this invention compared to the prior art include:

[0109] 1. The peptides described in this invention are easy to synthesize, have high safety, and can increase the activity of skin fibroblasts and promote collagen production, thereby increasing skin elasticity and / or skin firmness. They can be used to prevent or even treat skin laxity, and to treat, prevent and / or repair skin aging or photoaging.

[0110] 2. The peptides of the present invention can promote PER1 protein expression, increase PER1 protein content, participate in the regulation of the skin's circadian rhythm cycle, thereby restoring the circadian rhythm and resynchronizing the skin cell biological clock, and can be used to repair damaged skin cells and alleviate signs of skin aging; they can also be used to prepare PER1 protein activators. Attached Figure Description

[0111] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the present invention will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0112] Figure 1 It is peptide (2)H-Ser-Pro-Gly-Gln-NH2 (molecular formula C 15 H 26 Mass spectrum of N6O6, [M+H] + The mass-to-charge ratio (m / z) of the quasi-molecular ion peak was 387.2045, and the molecular weight measured by mass spectrometry was 386.20.

[0113] Figure 2 It is a peptide (9)H-Ser-Pro-Asn-Gln-NH2 (molecular formula C 17 H 29 Mass spectrum of N7O7, [M+H] + The mass-to-charge ratio (m / z) of the quasi-molecular ion peak was 444.2271, and the molecular weight measured by mass spectrometry was 443.23.

[0114] Figure 3 This is a graph showing the effect of the test samples on the activity of HaCaT cells.

[0115] Figure 4This is a graph showing the effect of the test samples on HSF cell activity.

[0116] Figure 5 This is a graph showing the effect of the test samples on the activity of HSF cells after UV treatment.

[0117] Figure 6 This is a graph showing the effect of the test sample on collagen content.

[0118] Figure 7 This is a graph showing the effect of the test samples on the PER1 protein content. Detailed Implementation

[0119] To make the objectives, features, and advantages of this invention more apparent and understandable, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0120] It should be noted that, in this invention, the abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Biochemical Nomenclature Committee in Eur. J. Biochem. (1984) 138: 9-37 and J. Chem. (1989) 264: 633-673.

[0121] Amide Resin: A starting resin for peptide synthesis; Fmoc-Linker: 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetic acid; DMF: N,N-dimethylformamide; HOBt: 1-hydroxybenzotriazole; DIC: diisopropylcarbodiimide; DIPEA: diisopropylethylamine; Ac2O: acetic anhydride; piperidine: piperidine; TFA: trifluoroacetic acid; TIS: triisopropylsilane; Ser: serine; Pro: proline; Gly: glycine; Asn: asparagine; Gln: glutamine; Fmoc: 9-fluorenylmethoxycarbonyl; tBu: tert-butyl; Trt: triphenylmethyl.

[0122] Example 1: Preparation of H-Ser-Pro-Gly-Gln-NH2

[0123] 1.1 Preparation of Fmoc-Linker-Amide Resin

[0124] Weigh 5g of Amide Resin into a solid-phase synthesis reaction column, swell it with DMF, wash the resin, and remove the solvent.

[0125] Weigh 7.1g of Fmoc-Linker and 4.4g of HOBt into a dry Erlenmeyer flask. Dissolve in DMF solvent and cool in an ice-water bath for 10 minutes. Activate with DIC for 10 minutes, avoiding moisture.

[0126] The activated Fmoc-Linker was added to the swollen resin and reacted for 2.5 hours. The reaction solution was then removed, the resin was washed, and the solvent was removed.

[0127] Continue adding Ac2O and DIPEA for end-capping treatment for 1.5 hours. Wash the resin and remove the solvent.

[0128] 1.2 De-Fmoc

[0129] Fmoc-Linker-Amide Resin was used to remove Fmoc twice with 20% piperidine / DMF, 10 min each time. Samples were taken for K testing; the color development was deep blue. The resin was then washed 7 times with DMF, and the solvent was removed.

[0130] 1.3 Feeding and Reaction

[0131] Weigh 11.7 g of Fmoc-Gln(Trt)-OH and 4.4 g of HOBt into a dry Erlenmeyer flask, add DMF to dissolve them, seal and place in a -18°C freezer for 30 min. Add 6.3 mL of DIC to activate for 3 min, avoiding moisture. Add the activated amino acid to the deprotected resin and react for 2 h, then remove the reaction solution. A colorless and transparent K test indicates complete reaction.

[0132] The N-terminal Fmoc group was deprotected, and 5.7 g of activated Fmoc-Gly-OH was coupled to a peptide resin in the presence of 4.4 g HOBt and 6.3 mL DIC using DMF as a solvent, with the reaction time lasting 2 h. The resin was then washed, and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. In each coupling, 7.3 g of Fmoc-Pro-OH and subsequently 8.3 g of Fmoc-Ser(tBu)-OH were sequentially coupled in the presence of 4.4 g HOBt and 6.3 mL DIC using DMF as a solvent; after complete reaction, the resin was washed, and the solvent was removed.

[0133] The N-terminal Fmoc group of the peptide resin was deprotected by using 20% ​​piperidine / DMF twice, 10 min each time. A sample was taken for K-test, and the color development was deep blue. The resin was washed 6 times with DMF, the solvent was removed, and after shrinkage and drying, 12.2 g of Ser(tBu)-Pro-Gly-Gln(Trt)-Linker-Amide Resin was obtained.

[0134] 1.4 Pyrolysis

[0135] Measure 95 mL of TFA, 2.5 mL of TIS and 2.5 mL of water, mix and stir well to obtain the lysis buffer, seal and store at -18℃ for later use; store isopropyl ether at -18℃ for later use.

[0136] Weigh 12.2 g of Ser(tBu)-Pro-Gly-Gln(Trt)-Linker-Amide Resin and add it to a round-bottom flask. Add the frozen lysis buffer and stir for 2 h. Filter the mixture, collect the filtrate, concentrate it to 15 mL, add isopropyl ether, stir, centrifuge, and wash 6 times until the pH reaches 3-4. Dry under vacuum to obtain 4.8 g of crude H-Ser-Pro-Gly-Gln-NH2 peptide.

[0137] 1.5 Purification

[0138] 4.8 g of crude peptide was weighed and dissolved in 90 mL of methanol:water (V:V = 1:2). The solution was filtered through a 0.22 μm microporous membrane to obtain a clear and transparent solution. The solution was then purified by reversed-phase HPLC. The purification gradient is shown in the table below.

[0139]

[0140]

[0141] The filtered sample was purified by injection, the fraction was collected, concentrated and freeze-dried to obtain peptide (2)H-Ser-Pro-Gly-Gln-NH2 with a purity of 98.726%.

[0142] Example 2: Preparation of H-Ser-Pro-Asn-Gln-NH2

[0143] 2.1 Preparation of Fmoc-Linker-Amide Resin

[0144] Weigh 5g of Amide Resin into a solid-phase synthesis reaction column, swell it with DMF, wash the resin, and remove the solvent.

[0145] Weigh 7.1g of Fmoc-Linker and 4.4g of HOBt into a dry Erlenmeyer flask. Dissolve in DMF solvent and cool in an ice-water bath for 10 minutes. Activate with DIC for 10 minutes, avoiding moisture.

[0146] The activated Fmoc-Linker was added to the swollen resin and reacted for 2.5 hours. The reaction solution was then removed, the resin was washed, and the solvent was removed.

[0147] Continue adding Ac2O and DIPEA for end-capping treatment for 1.5 hours. Wash the resin and remove the solvent.

[0148] 2.2 De-Fmoc

[0149] Fmoc-Linker-Amide Resin was used to remove Fmoc twice with 20% piperidine / DMF, 10 min each time. Samples were taken for K testing; the color development was deep blue. The resin was then washed 7 times with DMF, and the solvent was removed.

[0150] 2.3 Feeding and Reaction

[0151] Weigh 11.7 g of Fmoc-Gln(Trt)-OH and 4.4 g of HOBt into a dry Erlenmeyer flask, add DMF to dissolve them, seal and place in a -18°C freezer for 30 min. Add 6.3 mL of DIC to activate for 3 min, avoiding moisture. Add the activated amino acid to the deprotected resin and react for 2 h, then remove the reaction solution. A colorless and transparent K test indicates complete reaction.

[0152] The N-terminal Fmoc group was deprotected, and 11.5 g of activated Fmoc-Asn(Trt)-OH was coupled to a peptide resin in the presence of 4.4 g HOBt and 6.3 mL DIC using DMF as a solvent, with the reaction lasting 2 h. The resin was then washed, and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. In each coupling, 7.3 g of Fmoc-Pro-OH and subsequently 8.3 g of Fmoc-Ser(tBu)-OH were sequentially coupled in the presence of 4.4 g HOBt and 6.3 mL DIC using DMF as a solvent; after the reaction was complete, the resin was washed, and the solvent was removed.

[0153] The N-terminal Fmoc group of the peptide resin was deprotected by using 20% ​​piperidine / DMF twice, 10 min each time. A sample was taken for K-test, and the color development was deep blue. The resin was washed 6 times with DMF, the solvent was removed, and after shrinkage and drying, 16.2 g of Ser(tBu)-Pro-Asn(Trt)-Gln(Trt)-Linker-Amide Resin was obtained.

[0154] 2.4 Pyrolysis

[0155] Measure 95 mL of TFA, 2.5 mL of TIS and 2.5 mL of water, mix and stir well to obtain the lysis buffer, seal and store at -18℃ for later use; store isopropyl ether at -18℃ for later use.

[0156] Weigh 16.2 g of Ser(tBu)-Pro-Asn(Trt)-Gln(Trt)-Linker-Amide Resin and add it to a round-bottom flask. Add the previously frozen lysis buffer and stir for 2 h. Filter the mixture, collect the filtrate, concentrate it to 15 mL, add isopropyl ether, stir, centrifuge, and wash 6 times until the pH reaches 3-4. Dry under vacuum to obtain 5.2 g of crude H-Ser-Pro-Asn-Gln-NH2 peptide.

[0157] 2.5 Purification

[0158] 5.2 g of crude peptide was weighed and dissolved in 120 mL of methanol:water (V:V = 1:2). The solution was filtered through a 0.22 μm microporous membrane to obtain a clear and transparent solution. The solution was then purified by reversed-phase HPLC. The purification gradient is shown in the table below.

[0159] Time (min) Flow rate (mL / min) A% (acetonitrile) B% (0.1% acetic acid + pure water) 0 15 0 100 10 15 0 100 20 15 1 99

[0160] The filtered sample was purified by injection, the fraction was collected, concentrated and freeze-dried to obtain peptide (9)H-Ser-Pro-Asn-Gln-NH2 with a purity of 99.696%.

[0161] Example 3

[0162] Other peptides in formula (I) of this invention can be prepared by a similar method.

[0163] The molecular weights of the obtained peptides were determined by ESI-MS. The test results for some peptides are shown in Table 2 below. Figure 1-2 :

[0164] Table 2. Results of molecular weight determination by mass spectrometry

[0165] serial number sequence Molecular weight mass spectrometry analysis results (2) <![CDATA[H-Ser-Pro-Gly-Gln-NH2]]> 386.20 (9) <![CDATA[H-Ser-Pro-Asn-Gln-NH2]]> 443.23

[0166] Example 4 Cell Proliferation Experiment

[0167] 4.1 Reagents and Materials

[0168] Thiazole blue (MTT), dimethyl sulfoxide (DMSO), high glucose medium (DMEM), fetal bovine serum, PBS.

[0169] 4.2 Instruments

[0170] Microplate reader, CO2 incubator, clean bench.

[0171] 4.3 Cell lines

[0172] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee, and human skin fibroblasts (HSF) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0173] 4.4 Samples to be tested

[0174] The drug administration groups were peptide (2) and peptide (9), with test concentrations of 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, 100 ppm, and 200 ppm.

[0175] Blank control group: PBS.

[0176] Positive control group: 2% DMSO.

[0177] 4.5 Experimental Methods

[0178] HaCaT keratinocytes and HSF fibroblasts in the exponential growth phase were taken separately and digested with 0.25% trypsin solution to detach the adherent cells. The cells were counted at 1–4 × 10⁻⁶. 5 Cells were prepared as a suspension at a density of 1 cell per mL.

[0179] The cell suspension was seeded into 96-well plates at 200 μL / well and incubated in a constant temperature CO2 incubator for 24 h.

[0180] After changing the medium, add 20 μL of samples from the drug administration group, blank control group, and positive control group to each well, and incubate at 37°C and 5% CO2 for 72 h.

[0181] Then, 20 μL of 5 mg / ml MTT was added to each well, and the mixture was incubated at 37°C in a 5% CO2 incubator for 4 h. The original solution was discarded, and 150 μL of DMSO was added to each well. The wells were then shaken on a plate for 5 min, and the OD value of each well was measured at 570 nm using a microplate reader. Cell viability was also calculated.

[0182] Cell viability = (OD of drug delivery well - OD of zeroing well) / (OD of blank control well - OD of zeroing well) × 100%

[0183] 4.6 Experimental Results

[0184] The MTT assay is a method for detecting cell viability and growth, and the measured OD value is directly proportional to cell activity.

[0185] The results of the effect of the test samples on HaCaT cell viability are shown in the figure. Figure 3The results showed that, compared with the blank control group, the activity of HaCaT cells in the positive control group was significantly reduced, indicating that 2% DMSO had a toxic effect on HaCaT cells. The drug-treated group had no toxic effect on HaCaT cells in the range of 200 ppm. Moreover, the peptides (2) and (9) of the present invention could significantly improve cell activity and promote HaCaT cell proliferation at a low concentration of 6.25 ppm, and the cell proliferation-promoting effect was enhanced with increasing concentration.

[0186] The results of the effect of the test samples on HSF cell viability are shown in the figure. Figure 4 The results showed that the HSF cell activity of the positive control group was significantly reduced compared with the blank control group, indicating that 2% DMSO has a toxic effect on HSF cells. The drug-treated group had no toxic effect on HSF fibroblasts in the range of 200 ppm. Moreover, the peptides (2) and (9) of the present invention can significantly improve cell activity and promote HSF fibroblast proliferation at a low concentration of 6.25 ppm. The cell proliferation-promoting effect is enhanced with increasing concentration.

[0187] Therefore, the peptides of the present invention not only have no toxic effect on HaCaT keratinocytes and HSF fibroblasts, but can also enhance cell activity and promote their proliferation, thereby increasing skin elasticity and / or skin firmness, and can be used to prevent or even treat skin laxity.

[0188] Example 5: Photoaging Experiment

[0189] 5.1 Reagents and Materials

[0190] Fetal bovine serum, high glucose medium (DMEM), penicillin, streptomycin, and thiazolyl blue (MTT).

[0191] 5.2 Instruments

[0192] Microplate reader, CO2 incubator, clean bench.

[0193] 5.3 Cell lines

[0194] Human skin fibroblasts (HSF) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0195] 5.4 Samples to be tested

[0196] Drug administration group:

[0197] Reference 1 (Palm-Lys-Thr-Thr-Lys-Ser) was tested at concentrations of 12.5 ppm, 25 ppm, and 50 ppm.

[0198] Reference 2 (Ser-Pro-Leu-Gln-NH2) was tested at concentrations of 12.5 ppm, 25 ppm, and 50 ppm.

[0199] Peptide (2), tested at concentrations of 12.5 ppm, 25 ppm, and 50 ppm;

[0200] Peptide (9) was tested at concentrations of 12.5 ppm, 25 ppm and 50 ppm.

[0201] Blank control group: PBS.

[0202] UV group: UV radiation, plus PBS.

[0203] 5.5 Experimental Methods

[0204] HSF fibroblasts in good exponential growth phase were collected, and 0.25% trypsin digestion solution was added to digest and detach the adherent cells. The cells were counted at 1–4 × 10⁻⁶. 5 Cells were prepared as a suspension at a density of 1 cell per mL.

[0205] A cell suspension of 10,000 cells / well was appropriately diluted and seeded into 96-well plates. When the cells reached approximately 80% confluence, a UV photoaging model was established. The blank control group was treated with 50 μL PBS, and the culture medium was supplemented to 200 μL; no UV irradiation was performed. For the UV group and the drug-treated group, after repeated washing with appropriate amounts of PBS until colorless, 50 μL PBS was added, and the cells were treated at 80 mJ / cm². 2 Irradiate under a UV lamp with a 15cm distance between the lamp source and the culture flask. After irradiation, discard the PBS. Add PBS solution and culture medium to 200μL for the UV group, and add culture medium and serially diluted drug to 200μL for the drug-treated group. The blank control group, UV group, and drug-treated group were incubated at 37℃ in a 5% CO2 incubator for 24h.

[0206] Then, add 20 μL of 5 mg / mL MTT to each well and incubate at 37°C in a 5% CO2 incubator for 4 h. Discard the original solution, add 150 μL of DMSO per well, and shake on a plate shaker for 5 min. Then, read the reference OD values ​​at 490 nm and 630 nm wavelengths using a microplate reader.

[0207] 5.6 Experimental Results

[0208] Skin aging is influenced by both endogenous and exogenous factors, such as genetics, environmental exposure, ultraviolet radiation, and hormonal changes. The accumulation of these factors, especially ultraviolet radiation, leads to changes in skin structure, function, and appearance. This experiment used 80mJ / cm²... 2 UV energy is used to establish a skin photoaging model.

[0209] Experimental results are as follows Figure 5 As shown, after UV radiation, the activity of HSF fibroblasts decreased significantly, indicating that a photoaging model was successfully established. The drug-treated groups showed increased cell activity within the 50 ppm range, thereby improving cell aging and producing a significant anti-photoaging effect. Compared with reference substances 1 and 2, peptides (2) and (9) of the present invention significantly improved cell activity and significantly promoted HSF fibroblast proliferation. Specifically, peptide (2) at a low concentration of 12.5 ppm could greatly enhance the activity of HSF fibroblasts and produce a significant anti-photoaging effect. Therefore, the peptides of the present invention can be used to treat, prevent, and / or repair skin aging or photoaging, and have superior anti-aging effects compared to existing technologies.

[0210] Example 6: Collagen Content Test

[0211] 6.1 Reagents and Materials

[0212] Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, BCA protein kit, and collagen IELISA kit.

[0213] 6.2 Instruments

[0214] Microplate reader, CO2 incubator, clean bench, incubator.

[0215] 6.3 Cell lines

[0216] Human skin fibroblasts (HSF) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0217] 6.4 Sample to be tested

[0218] Drug administration group:

[0219] Reference 1 (Palm-Lys-Thr-Thr-Lys-Ser) was tested at concentrations of 12.5 ppm and 25 ppm.

[0220] Reference 2 (Ser-Pro-Leu-Gln-NH2) was tested at concentrations of 12.5 ppm and 25 ppm.

[0221] Peptide (2), tested at concentrations of 12.5 ppm and 25 ppm respectively;

[0222] Peptide (9) was tested at concentrations of 12.5 ppm and 25 ppm.

[0223] Blank control group: PBS.

[0224] UV group: UV radiation, plus PBS.

[0225] 6.5 Experimental Methods

[0226] HSF fibroblasts in good exponential growth phase were collected, and 0.25% trypsin digestion solution was added to digest and detach the adherent cells. The cells were counted at 1–4 × 10⁻⁶. 6 Cells were prepared as a suspension at a density of 1 cell per mL.

[0227] A cell suspension of 100,000 cells / well was appropriately diluted and seeded into 6-well plates. Cells were cultured to approximately 80% confluence before modeling. The blank control group was treated with 200 μL PBS, and the culture medium was adjusted to 800 μL; no UV irradiation was performed. For the UV group and the drug-treated group, after repeated washing with PBS until colorless, 200 μL PBS was added, and the mixture was incubated at 80 mJ / cm². 2 Irradiate under a UV lamp with a 15cm distance between the lamp source and the culture flask. After irradiation, discard the PBS. Add PBS solution and culture medium to 800μL for the UV group, and add culture medium and serially diluted drug to 800μL for the drug-treated group. The blank control group, UV group, and drug-treated group were incubated at 37℃ in a 5% CO2 incubator for 48h.

[0228] After culture, the cells in well 1 were digested, counted, and diluted to 0.5 × 10⁻⁶. 6 Cells were collected from the remaining wells using a cell scraper. After resuspending the cells in 500 μL of the resuspended cells, 50 μL of the resuspended cells were taken from each well and sonicated for 30 seconds. Total protein was then determined using the BCA method. The cells in the other wells were diluted according to the protein concentration of the first well to bring the total cell suspension concentration to 0.5 × 10⁻⁶. 6 Cells / mL. The adjusted cell suspension was sonicated for 30 seconds, centrifuged at 1500 x g for 15 minutes, and the cell supernatant was collected to obtain the sample solution. The procedure was followed according to the Collagen I ELISA kit instructions. The OD values ​​of each well were measured sequentially at 450 nm using a microplate reader within 15 minutes.

[0229] 6.6 Experimental Results

[0230] Skin aging is closely related to collagen, the most abundant protein found in connective tissue, which plays a vital role in skin plumpness and firmness. Collagen production is inhibited in environments with excessive ultraviolet (UV) radiation. This experiment used test samples to treat cells exposed to UV radiation and measured the collagen I content in the corresponding cells to determine whether the peptides of this invention can promote collagen production.

[0231] The results of the test samples' effect on collagen content are shown in the figure. Figure 6The results showed that, compared with the blank control group, the collagen content in the UV group was significantly reduced; compared with the UV group, reference 1 and reference 2 could not increase the collagen content in cells after ultraviolet radiation, while the peptides (2) and (9) of the present invention could significantly increase the collagen content and promote collagen expression in the range of 12.5-25 ppm, and had excellent collagen-promoting effects.

[0232] Therefore, the peptides of the present invention can promote collagen production and increase collagen content, thereby increasing skin elasticity and / or skin firmness. They can be used to prevent or even treat skin laxity, treat, prevent and / or repair skin aging or photoaging, and can be used in the cosmetic or pharmaceutical fields to combat skin aging.

[0233] Example 7: PER1 protein content test

[0234] 7.1 Reagents and Materials

[0235] Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, and human circadian rhythm protein 1 (PER1) ELISA kit.

[0236] 7.2 Instruments

[0237] Microplate reader, CO2 incubator, clean bench, incubator.

[0238] 7.3 Cell lines

[0239] Human skin fibroblasts (HSF) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0240] 7.4 Sample to be tested

[0241] Drug administration group:

[0242] Reference 2 (Ser-Pro-Leu-Gln-NH2), with a test concentration of 25 ppm;

[0243] Peptide (2), with a test concentration of 25 ppm.

[0244] Blank control group: PBS.

[0245] 7.5 Experimental Methods

[0246] HSF fibroblasts in good exponential growth phase were collected, and 0.25% trypsin digestion solution was added to digest and detach the adherent cells. The cells were counted at 1–4 × 10⁻⁶. 6 Cells were prepared as a suspension at a density of 1 cell per mL.

[0247] A cell suspension of 100,000 cells / well was appropriately diluted and seeded into 6-well plates. Cells were cultured to approximately 80% confluence to form a model. For the blank control group, 200 μL of PBS was added, and the culture medium was supplemented to 800 μL. For the drug-treated group, an appropriate amount of PBS was added, and the cells were washed repeatedly until colorless. Then, culture medium and the drug were added to 800 μL. All groups were incubated at 37°C in a 5% CO2 incubator for 48 h.

[0248] After culture, scrape off the cells using a cell scraper, mix thoroughly by pipetting, sonicate the cell suspension for 30 seconds, centrifuge at 1500 x g for 15 minutes, and collect the cell supernatant to obtain the sample solution; follow the instructions for the PER1 protein ELISA kit. Within 15 minutes, measure the OD value of each well sequentially at 450 nm using a microplate reader.

[0249] 7.6 Experimental Results

[0250] Circadian rhythm protein 1 (PER1) is a core component of the biological clock that regulates the skin's circadian rhythm. It participates in regulating the skin's response to environmental stressors, protecting the skin from various environmental damages such as UV radiation, temperature, chemical and physical damage, and microbial infections. Furthermore, PER1 protein expression is correlated with aging, and increasing PER1 protein levels can, to some extent, improve skin aging. This experiment determined whether the peptide of this invention can promote PER1 protein expression by detecting the PER1 protein content in relevant cells.

[0251] The results of the effect of the test samples on PER1 protein content are shown in the figure. Figure 7 The results showed that, compared with the blank control group, 25 ppm of peptide (2) could significantly increase the content of PER1 protein and promote the expression of PER1 protein. Moreover, compared with reference 2, peptide (2) of the present invention had a better effect on promoting the expression of PER1 protein.

[0252] Therefore, the peptides of the present invention can promote PER1 protein expression, increase PER1 protein content, participate in the regulation of the skin's circadian rhythm, thereby restoring the circadian rhythm and resynchronizing the skin cell biological clock. They can be used to repair damaged skin cells and alleviate signs of skin aging; they can also be used to prepare PER1 protein activators.

[0253] Example 8: Preparation of a peptide (2)-containing essence

[0254]

[0255] Add the prescribed amount of sodium hyaluronate to water, stir to mix evenly, then heat to 80-85℃, keep warm and stir to disperse evenly. When the temperature drops below 40℃, add glycerin, jujube gum, peptide (2), vitamin C, caprylyl glycol, and 1,2-hexanediol, and stir evenly. Adjust the pH of the solution to approximately 5.5 with 15% triethanolamine.

[0256] Example 9: Preparation of liposomes containing peptide (9)

[0257]

[0258] Dipalmitoylphosphatidylcholine was weighed and dissolved in chloroform. The solvent was evaporated under vacuum until a thin phospholipid layer was obtained. This layer was then hydrated at 55°C with a peptide aqueous solution of the desired concentration to obtain multicompartment liposomes. The multicompartment liposomes were homogenized under high pressure to obtain smaller and more uniform singlecompartment liposomes.

[0259] The above description, in conjunction with specific preferred embodiments, provides a further detailed account of the present invention, but does not imply that the specific implementation of the invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the inventive concept, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. The peptide represented by formula (I) or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, R1-Ser-Pro-X1-Gln-R2(I) In formula (I), X1 is -Asn-; R1 is H; R2 is -OH or -NH2.

2. The peptide represented by formula (I) according to claim 1, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, characterized in that, It is H-Ser-Pro-Asn-Gln-NH2.

3. The peptide of formula (I) according to any one of claims 1-2, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The cosmetically acceptable or pharmaceutically acceptable salts include metal salts of peptides represented by formula (I), wherein the metal includes lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum.

4. The peptide of formula (I) according to any one of claims 1-2, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The cosmetically acceptable or pharmaceutically acceptable salts include salts formed from peptides of formula (I) and organic bases, wherein the organic bases include: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine.

5. The peptide of formula (I) according to any one of claims 1-2, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The cosmetically acceptable or pharmaceutically acceptable salts include salts formed by peptides of formula (I) with inorganic or organic acids, wherein the organic acids include: 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, pyric acid, or gluconic acid. Alternatively, the inorganic acid may include hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

6. A cosmetic or pharmaceutical composition, characterized in that, It includes an effective amount of the peptide of formula (I) as described in any one of claims 1-5, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, as well as at least one excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant.

7. The cosmetic or pharmaceutical composition according to claim 6, characterized in that, The adjuvants are selected from: collagen synthesis stimulants, agents that regulate PGC-1α synthesis, agents that regulate PPARγ activity, agents that increase or decrease triglyceride content in adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic agents or agents that stimulate fat breakdown, lipolytic agents, lipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulants or inhibitors, whitening agents or bleaching agents, pigmentation promoters, self-tanning agents, anti-aging agents, NO-synthesizers, 5α-reductase inhibitors, lysyl hydroxylase and / or prolyl hydroxylase inhibitors, antioxidants, free radical scavengers and / or anti-air pollution agents, and active agents. Carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, alpha-hydroxy acids, beta-hydroxy acids, humectants, epidermal hydrolases, vitamins, amino acids, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents that reduce under-eye bags, exfoliants, sterilizing agents, bacteriostatic agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and / or inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate core proteoglycan synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, agents that stimulate chaperone protein synthesis, agents that stimulate cAMP synthesis, and other stimulants. Agents that stimulate heat shock protein synthesis, hyaluronic acid synthesis, fibronectin synthesis, deacetylase synthesis, lipid and stratum corneum component synthesis, ceramides, fatty acids, collagen degradation inhibitors, elastin degradation inhibitors, serine protease inhibitors, fibroblast proliferation stimulators, keratinocyte proliferation stimulators, adipocyte proliferation stimulators, melanocyte proliferation stimulators, keratinocyte differentiation stimulators, acetylcholinesterase inhibitors, skin relaxants, glycosaminoglycan synthesis stimulators, anti-hyperkeratosis agents, comedolytics, anti-psoriasis agents, anti-eczema agents, DNA repair agents, DNA protectants, stabilizers, antipruritics, and for the care of sensitive skin. Agents, curing agents, firming agents, reconstructing agents, anti-stretch mark agents, agents that regulate sebum production, antiperspirants, agents that stimulate healing, agents that assist healing, agents that stimulate re-epithelialization, agents that assist re-epithelialization, cytokines, 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, agents that inhibit or delay hair growth, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents derived from bio-fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents or mixtures thereof that are effective against UVA and / or UVB.

8. The cosmetic or pharmaceutical composition according to claim 6 or 7, characterized in that, The formulation of the cosmetic or pharmaceutical composition is selected from: creams, oils, balms, foams, lotions, gels, liniments, slurries, ointments, mousses, powders, rods, pens, sprays, aerosols, capsules, tablets, granules, chewing gum, solutions, suspensions, emulsions, elixirs, polysaccharide films, gels, or gelatin.

9. The cosmetic or pharmaceutical composition according to claim 8, characterized in that, The capsules include: soft capsules and hard capsules.

10. The cosmetic or pharmaceutical composition according to claim 8, characterized in that, The capsules are gelatin capsules.

11. The cosmetic or pharmaceutical composition according to claim 8, characterized in that, The tablets include: sugar-coated tablets.

12. A cosmetically or pharmaceutically acceptable delivery or sustained-release system, characterized in that, Contains an effective amount of the peptide of formula (I) as described in any one of claims 1-5, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition as described in any one of claims 6-11; The cosmetically or pharmaceutically acceptable delivery or sustained-release system is selected from: liposomes, oleosomes, nonionic surfactant liposome vesicles, liposomes, milli-capsules, micro-capsules, nano-capsules, nanostructured lipid carriers, sponges, cyclodextrins, lipid vesicles, micelles, milli-spheres, micro-spheres, nano-spheres, lipospheres, micro-emulsions, or nanoemulsions.

13. The cosmetically or pharmaceutically acceptable delivery or sustained-release system according to claim 12, characterized in that, The cosmetically or pharmaceutically acceptable delivery or sustained-release system is a liposome or a microemulsion.

14. The cosmetically or pharmaceutically acceptable delivery or sustained-release system according to claim 12, characterized in that, The cosmetically or pharmaceutically acceptable delivery or sustained-release system is a water-in-oil microemulsion with an internal structure of reverse micelles.

15. Use of the peptide of formula (I) according to any one of claims 1-5, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6-11, or a cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to any one of claims 12-14, in the preparation of a cosmetic composition or pharmaceutical composition for treating, preventing or repairing skin aging and / or photoaging.

16. Use of the peptide of formula (I) according to any one of claims 1-5, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6-11, or a cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to any one of claims 12-14, in the preparation of a cosmetic composition or pharmaceutical composition for increasing skin elasticity and / or skin firmness.

17. Use of the peptide of formula (I) according to any one of claims 1-5, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6-11, or a cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to any one of claims 12-14, in the preparation of a cosmetic composition or pharmaceutical composition for promoting collagen production.

18. Use of the peptide of formula (I) according to any one of claims 1-5, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6-11, or a cosmetically or pharmaceutically acceptable delivery system or sustained-release system according to any one of claims 12-14, in the preparation of a cosmetic or pharmaceutical composition for regulating the skin’s circadian rhythm and / or repairing damaged skin cells.

19. The use according to claim 18, characterized in that, The regulation of skin circadian rhythms includes promoting PER1 protein expression.

Images