WO2024109878A1 - 具有抗衰老作用的肽及其组合物和用途 - Google Patents

具有抗衰老作用的肽及其组合物和用途 Download PDF

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Publication number
WO2024109878A1
WO2024109878A1 PCT/CN2023/133632 CN2023133632W WO2024109878A1 WO 2024109878 A1 WO2024109878 A1 WO 2024109878A1 CN 2023133632 W CN2023133632 W CN 2023133632W WO 2024109878 A1 WO2024109878 A1 WO 2024109878A1
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Prior art keywords
agents
ser
pro
gln
acceptable salt
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PCT/CN2023/133632
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English (en)
French (fr)
Inventor
丁文锋
肖玉
赵文豪
孙新林
陈雪
观富宜
彭晏
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深圳市维琪科技股份有限公司
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Publication of WO2024109878A1 publication Critical patent/WO2024109878A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the technical field of medicine and cosmetics, in particular to a peptide with anti-aging effect and a composition and use thereof.
  • the skin As the first physiological defense line of the human body, the skin protects tissues and organs from chemical, mechanical, physical and pathogenic microorganisms, while also being constantly tested by the external environment.
  • environmental invasions such as UV radiation and environmental pollution
  • the number of patients with skin diseases has increased year by year, which has also accelerated skin aging.
  • Environmental invasions can damage the DNA of skin cells and affect their circadian rhythms.
  • Circadian rhythm refers to the changes in life activities in a 24-hour cycle, also known as the biological clock, which regulates many of our metabolism, physiological activities and behaviors.
  • circadian rhythm disorders can have a great negative effect on human health, including jet lag, fatigue syndrome caused by night shifts, sleep disorders, skin diseases, etc.
  • diseases that seriously threaten human health such as cardiovascular and cerebrovascular diseases and tumors, are also related to circadian rhythm disorders.
  • circadian rhythm disorders are also related to circadian rhythm disorders.
  • Circadian rhythm proteins are expressed not only in cultured human cells, but also in human skin biological tissues, affecting skin function.
  • the skin is easily exposed to external environmental factors such as light, temperature, humidity, ultraviolet rays, and pathogens, which change in a cycle of one day.
  • the skin regulates various physiological functions by activating the biological clock in the skin.
  • skin cell proliferation and differentiation, water loss, sebum production, temperature, pH, and wrinkle formation all change in a 24-hour cycle. For example, the proliferation and differentiation of skin cells occur through a series of processes in a 24-hour cycle.
  • the skin is easily damaged by ultraviolet exposure during the day, so the differentiation of skin cells mainly occurs between dusk and dawn.
  • the skin is protected from harmful environments.
  • the rhythm of this process is disrupted, the required physiological response cannot be produced at the appropriate time. In this way, the circadian rhythm of the skin is disrupted, which will accelerate skin damage.
  • genes related to the natural body's circadian rhythm include the Clock gene and the PER1 gene, both of which encode proteins that regulate the circadian rhythm (Clock protein and PER1 protein).
  • PER1 is one of the core components of the biological clock that regulates the skin's circadian rhythm cycle and is a member of the Period gene family.
  • the Period gene family consists of PER1, PER2, and PER3. These genes are involved in the body's metabolism, motor activity, and behavior, among which PER1 plays a core role.
  • the Clock gene and PER1 gene are also present in skin cells, and the expression of the PER1 gene induces a program of cellular activity related to biological processes (such as repair) that take place at night.
  • the DNA replication, DNA repair mechanism and cell division of epidermal progenitor cells/stem cells show obvious circadian rhythm characteristics.
  • Skin cells that are exposed to environmental invasion for a long time usually show reduced, irregular or asynchronous expression of the Clock gene or PER1 gene, disrupting the normal circadian rhythm in skin cells. Over time, the normal cellular circadian rhythm and synchronization are disrupted, and the balance of the skin is destroyed, thereby accelerating the natural aging process of the skin, causing skin damage, wrinkles, fine lines, sagging skin, uneven pigmentation, age spots, spots and other problems.
  • Clock gene and PER1 gene are the core genes and negative feedback genes of biological clock that regulate the circadian rhythm cycle of the skin, and participate in regulating the response process of the skin to environmental stress. Moreover, Clock gene and PER1 gene interfere with the expression and activity regulation of genetic genes and proteins in various physiological processes with a cycle of about 24 hours, so that the biological clock caters to the external environment to regulate the activity of physiological reactions. According to the circadian rhythm, the various activities of skin cells are regulated to protect the skin from various environmental damages such as UV radiation, temperature, chemical and physical damage, and microbial infection.
  • the purpose of the present invention is to provide a stable, efficient, safe, and non-irritating active substance to the skin, which can repair damaged cells by activating the expression of skin circadian rhythm cycle regulatory genes and related proteins, achieve anti-aging effects, and at the same time promote collagen production, increase skin elasticity and/or skin firmness, and prevent skin sagging, so that it has a wide range of applications in the fields of medicine and cosmetics.
  • the present invention provides a peptide represented by formula (I), or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, R 1 -Ser-Pro-X 1 -Gln-R 2 (I)
  • X1 is selected from: -Gly-, -Asn-, -Ala-, -Ile-, -Val-, -Ser-, -Thr- or -Met-;
  • R 1 is selected from: H or R 3 -CO-, R 3 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
  • R 2 is selected from: -NR 4 R 5 or -OR 4 , wherein R 4 and R 5 are independently selected from: H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
  • the alkyl group refers to a saturated aliphatic straight chain 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).
  • alkyl can 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;
  • the alkenyl group refers to a straight or branched alkenyl group 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 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 through a single bond; and can be selected from: vinyl, oleyl, or linoleyl;
  • R 1 is selected from: H, acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl or linoleoyl;
  • R 4 and R 5 are independently selected from: H, methyl, ethyl, hexyl, dodecyl or hexadecyl;
  • R 1 is selected from H, acetyl, myristoyl or palmitoyl;
  • R 4 is H and
  • R 5 is selected from: H, methyl, ethyl, hexyl, dodecyl or hexadecyl;
  • R 1 is H or acetyl
  • R 2 is -OH or -NH 2 .
  • the peptide represented by formula (I), or its stereoisomer, or its mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt is selected from the following peptides (1)-(32):
  • peptide (1) selected from peptide (1), peptide (2), peptide (3), peptide (4), peptide (9), peptide (10), peptide (11), peptide (12); specifically,
  • 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.
  • -Pro- is selected from -L-Pro-, -D-Pro-, or a mixture of the two, and is racemic or non-racemic.
  • the preparation method described in this document enables 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.
  • the present invention also includes all suitable isotopic variants of the peptides shown in formula (I).
  • Isotopic variants of these peptides of the present invention are understood here to mean compounds in which at least one atom in the peptide of the present invention is replaced by another atom of the same atomic number, but the atomic mass of the other atom is different from the atomic mass usually or mainly present in nature.
  • isotopes that can be incorporated into the peptides of the present invention are those of hydrogen, carbon, nitrogen, oxygen or sulfur, such as 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.
  • isotopic variants of the peptides of the present invention may be useful, for example, for examining the mechanism of action or the distribution of the active compound in vivo; compounds labeled with 3 H or 14 C isotopes are particularly suitable for this purpose due to their relatively simple preparability and detectability.
  • an isotope e.g., deuterium
  • Isotopic variants of the peptides of the present invention can be prepared by methods known to those skilled in the art, such as by the methods further described below and in the examples, by using the respective isotopic modifications of the reagents and/or starting materials.
  • the present invention also includes prodrugs of the peptides of the present invention.
  • prodrug herein means such compounds: which themselves may be biologically active or inactive, but during their residence time in the body, they react (e.g., metabolize or hydrolyze) to generate the peptides of the present invention.
  • 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 represented by 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 represented by formula (I) and organic bases, the organic bases including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histamine Acid or piperazine, etc.; including salts formed by the peptide represented by formula (I) and inorganic acids or organic acids, the organic acids include, but are 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, trifluoroace
  • 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 prior art, such as solid phase synthesis, liquid phase synthesis or a combination of solid phase and liquid phase, and can also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins having animal, fungal, or plant origin.
  • a method for obtaining a peptide represented by formula (I) comprises the following steps:
  • 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.
  • 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.
  • solid phase synthesis can be performed by a convergent strategy of coupling the dipeptide or tripeptide to a polymer support or to a dipeptide or amino acid previously bound to a polymer support.
  • the functional groups at the terminals may be modified by 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.
  • the optional modification of the N-terminus and C-terminus may be performed on the peptide of formula (I) bound to the polymer support, or after the peptide has been cleaved from the polymer support.
  • Another aspect of the present invention provides a cosmetic or pharmaceutical composition, 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, and at least one excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant;
  • the adjuvant is selected from: collagen synthesis stimulators, agents that regulate PGC-1 ⁇ synthesis, agents that regulate the activity of PPAR ⁇ , 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, 5 ⁇ -reductase inhibitors, inhibitors of lysyl hydroxylase and/or prolyl hydroxylase, antioxidants, free radical scavengers and/or anti-air pollution agents, active carbonyl scavengers , anti
  • the formulation of the cosmetic or pharmaceutical composition is selected from the group consisting of: cream, oil, balm, foam, lotion, gel, liniment, slurry, ointment, mousse, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, elixir, polysaccharide film, jelly or gelatin;
  • the capsule includes: a soft capsule, a hard capsule, and may be a gelatin capsule;
  • the tablet comprises: a sugar-coated tablet.
  • the peptides of the present invention have variable solubility in water, depending on the nature of their sequence or any possible modifications at 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.
  • 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.
  • 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.
  • Another aspect of 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 its stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the above cosmetic or pharmaceutical composition.
  • 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.
  • 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
  • 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.
  • 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 having an internal structure of reverse micelles.
  • 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.
  • topical or transdermal administration including adhesive patch, non-adhesive patch, closure patch, and microelectronic patch
  • 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, at which the composition will be given, for example.
  • Another aspect of the present invention provides a use of the peptide represented by the above formula (I), or its stereoisomer, or a mixture of its 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 in the preparation of a cosmetic composition or pharmaceutical composition for treating, preventing or repairing skin aging and/or photoaging.
  • Another aspect of the present invention provides a peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers 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.
  • Another aspect of the present invention provides a peptide represented by the above formula (I), or its stereoisomer, or a mixture of its 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 in the preparation of a cosmetic composition or pharmaceutical composition for promoting collagen production.
  • Another aspect of the present invention provides a use of the peptide represented by formula (I) above, or its stereoisomer, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the cosmetic or pharmaceutical composition above, or the cosmetically or pharmaceutically acceptable delivery system or sustained-release system above in the preparation of a cosmetic composition or pharmaceutical composition for promoting PER1 protein expression, regulating skin circadian rhythm and/or repairing damaged skin cells.
  • Another aspect of the present invention provides a use of the peptide represented by the above formula (I), or its stereoisomer, or a mixture of its stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, 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.
  • Another aspect of the present invention provides a peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the above cosmetic or pharmaceutical composition, Or use of the above-mentioned cosmetically or pharmaceutically acceptable delivery system or sustained-release system in the preparation of a composition for promoting Clock gene expression and/or resynchronizing the biological clock of skin cells.
  • Another aspect of the present invention provides a use of the peptide represented by formula (I) above, or its stereoisomer, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the cosmetic or pharmaceutical composition, or the cosmetically or pharmaceutically acceptable delivery system or sustained-release system in the preparation of a Clock protein activator.
  • Another aspect of the present invention provides a peptide represented by the above formula (I), or its stereoisomer, or a mixture of its 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 for use in the preparation of cosmetics.
  • Another aspect of the present invention provides a cosmetic comprising the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers 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;
  • the dosage form of the cosmetic includes cream, lotion, gel, powder, tablet, film, aerosol or spray.
  • 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.
  • 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.
  • treatment also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or condition.
  • 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.
  • repair refers to the ability of the peptides of the present invention to improve, alleviate or restore a disease or condition to its original state after it occurs.
  • aging refers to the changes that the skin undergoes with age (chronological 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 marks, striae, furrows, unevenness or roughness, increase in pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of ability to recover from deformation, loss of resilience), sagging of the skin
  • the present invention may include changes in the skin color (such as scarring, redness, eye bags, or the appearance of areas of hyperpigmentation such as age spots or freckles), abnormal differentiation, hyperkeratinization, elastosis, keratosis, hair loss, orange peel skin, loss of collagen structure, and other histological changes in the stratum corneum, dermis, epider
  • 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.
  • Gly represents NH2 - CH2 -COOH
  • Gly- represents NH2 - CH2 -CO-
  • -Gly represents -NH- CH2- COOH
  • -Gly- represents -NH- CH2 -CO-.
  • the hyphen representing a peptide bond eliminates the OH from 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 from 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).
  • Ac- is used herein to represent an acetyl group ( CH3 -CO-).
  • the peptide of the present invention is easy to synthesize and has high safety. It can increase the activity of skin fibroblasts and promote collagen production, thereby increasing skin elasticity and/or skin firmness. It can be used to prevent or even treat skin sagging, and treat, prevent and/or repair skin aging or photoaging.
  • the peptide of the present invention can promote the expression of PER1 protein, increase the content of PER1 protein, participate in the regulation of the skin circadian rhythm cycle, thereby restoring the circadian rhythm and resynchronizing the biological clock of skin cells. It can be used to repair damaged skin cells and alleviate signs of skin aging; it can also be used to prepare PER1 protein activators.
  • the peptide of the present invention can activate the expression of skin circadian rhythm cycle regulatory genes and Clock and other related proteins, regulate the skin circadian rhythm, resynchronize the biological clock of skin cells, enhance cell vitality, repair damaged cells, and alleviate signs of skin aging. It has a wide range of applications in the field of cosmetics or medicine, and can also be used to prepare Clock protein activators.
  • FIG1 is a mass spectrum of peptide (1) H-Ser-Pro-Gly-Gln-OH (molecular formula C 15 H 25 N 5 O 7 ), the mass-to-charge ratio (m/z) of the [M+H] + quasi-molecular ion peak is 388.40, and the molecular weight measured by mass spectrometry is 387.40.
  • Figure 2 is a mass spectrum of peptide (2) H-Ser-Pro-Gly-Gln- NH2 (molecular formula C15H26N6O6 ), the mass-to-charge ratio (m/z) of the [M+H] + quasi -molecular ion peak is 387.2045, and the molecular weight measured by mass spectrometry is 386.20.
  • Figure 3 is a mass spectrum of peptide (9) H-Ser-Pro-Asn-Gln-NH 2 (molecular formula C 17 H 29 N 7 O 7 ), [M+H] + quasi-molecular ion
  • the mass-to-charge ratio (m/z) of the sub-peak is 444.2271, and the molecular weight measured by mass spectrometry is 443.23.
  • FIG. 4 is a graph showing the effects of the test samples on the activity of HaCaT cells.
  • FIG. 5 is a graph showing the effect of the test samples on the activity of HSF cells.
  • FIG. 6 is a graph showing the effect of the test samples on the activity of HSF cells after UV treatment.
  • FIG. 7 is a graph showing the effect of the test samples on the collagen content.
  • FIG. 8 is a graph showing the effect of the test samples on the PER1 protein content.
  • Amide Resin a starting resin for peptide synthesis; Wang Resin: Wang Resin; Fmoc-Linker: 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetic acid; DMF: N,N-dimethylformamide; DCM: dichloromethane; DMAP: 4-dimethylaminopyridine; HOBt: 1-hydroxybenzotriazole; DIC: diisopropylcarbodiimide; DIPEA: diisopropylethylamine; Ac 2 O: 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: trity
  • Fmoc-Gln(Trt)-Wang Resin was de-Fmoced twice with 20% piperidine/DMF, each time for 10 min, and the sample was taken for K test, and the color was dark blue. The resin was washed with DMF 7 times and the solvent was removed.
  • the N-terminal Fmoc group was deprotected and 7 g of activated Fmoc-Pro-OH was coupled to the peptidyl resin in the presence of 3 g HOBt and 5 mL DIC using DMF as solvent for 1 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the next amino acid.
  • 9 g of Fmoc-Ser(tBu)-OH was coupled to the peptidyl resin in the presence of 3 g HOBt and 5 mL DIC using DMF as solvent for 1.2 h; after the reaction was complete, the resin was washed and the solvent was removed.
  • the N-terminal Fmoc group of the peptidyl resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 minutes, and the sample was taken for K inspection, and the color was dark blue.
  • the resin was washed with DMF for 6 times, the solvent was removed, and 14g of H-Ser(tBu)-Pro-Gly-Gln(Trt)-WangResin was obtained after shrinkage and drying.
  • the activated Fmoc-Linker was added to the swollen resin and reacted for 2.5 h. The reaction solution was removed, the resin was washed, and the solvent was removed.
  • Fmoc-Linker-Amide Resin was de-Fmoced twice with 20% piperidine/DMF, each time for 10 min, and the sample was taken for K test, which showed dark blue color. The resin was washed with DMF 7 times and the solvent was removed.
  • the N-terminal Fmoc group was deprotected and 5.7 g of activated Fmoc-Gly-OH was coupled to the peptidyl resin in the presence of 4.4 g HOBt and 6.3 mL DIC using DMF as solvent 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, 7.3 g of Fmoc-Pro-OH and then 8.3 g of Fmoc-Ser(tBu)-OH were coupled sequentially in the presence of 4.4 g HOBt and 6.3 mL DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was removed.
  • the N-terminal Fmoc group of the peptide resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 minutes, and the sample was taken for K test, and the color was dark blue.
  • the resin was washed with DMF for 6 times, the solvent was removed, and 12.2g of Ser(tBu)-Pro-Gly-Gln(Trt)-Linker-Amide Resin was obtained after shrinkage and drying.
  • the activated Fmoc-Linker was added to the swollen resin and reacted for 2.5 h. The reaction solution was removed, the resin was washed, and the solvent was removed.
  • Fmoc-Linker-Amide Resin was de-Fmoced twice with 20% piperidine/DMF, each time for 10 min, and the sample was taken for K test, which showed dark blue color. The resin was washed with DMF 7 times and the solvent was removed.
  • the N-terminal Fmoc group was deprotected and 11.5 g of activated Fmoc-Asn(Trt)-OH was coupled to the peptidyl resin in the presence of 4.4 g HOBt and 6.3 mL DIC using DMF as solvent 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.
  • the N-terminal Fmoc group of the peptide resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 minutes, and the sample was taken for K test, and the color was dark blue.
  • the resin was washed with DMF for 6 times, the solvent was removed, and 16.2g of Ser(tBu)-Pro-Asn(Trt)-Gln(Trt)-Linker-Amide Resin was obtained after shrinkage and drying.
  • MTT dimethyl sulfoxide (DMSO), DMEM (deoxynucleotidylserine medium), fetal bovine serum, and PBS.
  • DMSO dimethyl sulfoxide
  • DMEM deoxynucleotidylserine medium
  • fetal bovine serum and PBS.
  • HaCaT Human keratinocytes
  • HSF human skin fibroblasts
  • Drug administration groups peptide (2) and peptide (9), the test concentrations were 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, 100 ppm, and 200 ppm.
  • Blank control group PBS.
  • Positive control group 2% DMSO.
  • HaCaT keratinocytes and HSF fibroblasts in good exponential growth phase were taken respectively, and 0.25% trypsin digestion solution was added to digest and make the adherent cells fall off, and 1-4 ⁇ 10 5 cells/mL were counted to prepare cell suspension.
  • the cell suspension was inoculated on a 96-well plate at 200 ⁇ L/well and cultured in a constant temperature CO 2 incubator for 24 h.
  • the medium was changed, and samples of the drug-treated group, blank control group and positive control group were added at 20 ⁇ L/well, respectively, and the cells were incubated in a 37°C, 5% CO 2 incubator for 72 h.
  • the MTT assay is a method for detecting cell survival and growth.
  • the measured OD value is proportional to the cell activity.
  • the results of the effects of the test samples on the activity of HaCaT cells are shown in FIG4 .
  • the results show that, compared with the blank control group, the activity of HaCaT cells in the positive control group was significantly decreased, indicating that 2% DMSO had a toxic effect on HaCaT cells.
  • the drug administration group had no toxic effect on HaCaT cells within the range of 200 ppm.
  • the peptides (2) and (9) of the present invention were able to significantly increase cell activity and promote HaCaT cell proliferation at a low concentration of 6.25 ppm, and the cell proliferation promoting effect increased with increasing concentration.
  • the results of the effects of the test samples on the activity of HSF cells are shown in FIG5 .
  • the results show that compared with the blank control group, the activity of HSF cells in the positive control group was significantly decreased, indicating that 2% DMSO was toxic to HSF cells.
  • the drug-treated group had no toxic effect on HSF fibroblasts within the range of 200 ppm.
  • the peptides (2) and (9) of the present invention were able to significantly increase cell activity and promote the proliferation of HSF fibroblasts at a low concentration of 6.25 ppm, and the cell proliferation promoting effect increased with increasing concentration.
  • the peptides of the present invention not only have no toxic effect on HaCaT keratinocytes and HSF fibroblasts, but also can improve the activity of cells and promote their proliferation, thereby increasing skin elasticity and/or skin firmness, and can be used to prevent or even treat skin sagging.
  • Fetal bovine serum, DMEM, penicillin, streptomycin, MTT Fetal bovine serum, DMEM, penicillin, streptomycin, MTT.
  • HSF Human skin fibroblasts
  • Reference substance 1 (Palm-Lys-Thr-Thr-Lys-Ser), tested at concentrations of 12.5 ppm, 25 ppm, and 50 ppm;
  • Reference substance 2 (Ser-Pro-Leu-Gln-NH 2 ), tested at concentrations of 12.5 ppm, 25 ppm, and 50 ppm;
  • peptide (2) tested at concentrations of 12.5 ppm, 25 ppm, and 50 ppm;
  • Peptide (9) was tested at concentrations of 12.5 ppm, 25 ppm, and 50 ppm.
  • Blank control group PBS.
  • UV group UV radiation, plus PBS.
  • HSF fibroblasts in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, count 1-4 ⁇ 10 5 cells/mL, and make a cell suspension.
  • the PBS was discarded, and the UV group was added with PBS solution and culture medium to 200 ⁇ L, and the drug-treated group was added with culture medium and multiple dilution drugs to 200 ⁇ L.
  • the blank control group, UV group, and drug-treated group continued to be incubated in a 37°C, 5% CO 2 incubator for 24 hours.
  • Skin aging is affected by endogenous and exogenous factors, such as genetics, environmental exposure, ultraviolet radiation, hormone changes, etc. The accumulation of these factors, especially ultraviolet radiation, leads to changes in skin structure, function and appearance.
  • 80mJ/ cm2 UV energy was selected for radiation to establish a skin photoaging model.
  • HSF Human skin fibroblasts
  • Reference substance 1 (Palm-Lys-Thr-Thr-Lys-Ser), tested at concentrations of 12.5 ppm and 25 ppm;
  • Reference substance 2 (Ser-Pro-Leu-Gln-NH 2 ), tested at concentrations of 12.5 ppm and 25 ppm;
  • Peptide (9) was tested at concentrations of 12.5 ppm and 25 ppm.
  • Blank control group PBS.
  • UV group UV radiation, plus PBS.
  • HSF fibroblasts in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, count 1-4 ⁇ 10 6 cells/mL, and make a cell suspension.
  • Appropriate dilution was performed to inoculate 100,000 cells/well of the cell suspension on a 6-well plate, and the model was established when the cells grew to about 80%.
  • the blank control group was added with 200 ⁇ L PBS, and the culture medium was supplemented to 800 ⁇ L, and no UV irradiation was performed; the UV group and the drug-treated group were added with an appropriate amount of PBS and repeatedly washed until colorless, and then 200 ⁇ L PBS was added and placed under an 80 mJ/cm 2 UV lamp for irradiation, with a distance of 15 cm between the lamp source and the culture bottle.
  • the PBS was discarded, and the UV group was added with PBS solution and culture medium to 800 ⁇ L, and the drug-treated group was added with culture medium.
  • the blank control group, UV group and drug administration group were incubated in a 37°C, 5% CO 2 incubator for 48 h.
  • the cells in the first well were digested and counted, and diluted to 0.5 ⁇ 10 6 cells/mL.
  • the cells in the remaining wells were scraped off with a cell scraper, resuspended in 500 ⁇ L, and 50 ⁇ L was taken from all wells for 30 seconds of ultrasound.
  • the total protein was determined using the BCA method.
  • the other wells were diluted according to the protein concentration of the first well to make the concentration of all cell suspensions 0.5 ⁇ 10 6 cells/mL.
  • the cell suspension after the concentration was adjusted was ultrasonically disrupted for 30 seconds, centrifuged at 1500x ⁇ for 15 minutes, and the cell supernatant was collected to obtain the sample solution; the operation was carried out according to the collagen I ELISA kit operating instructions.
  • the OD value of each well was measured in sequence at 450nm using an enzyme reader within 15 minutes.
  • the peptides of the present invention can promote collagen production, increase collagen content, thereby increasing skin elasticity and/or skin firmness, and can be used to prevent or even treat skin sagging, treat, prevent and/or repair skin aging or photoaging, and can be used in the cosmetics or medical fields to resist skin aging.
  • HSF Human skin fibroblasts
  • Peptide (2) tested at a concentration of 25 ppm.
  • Blank control group PBS.
  • HSF fibroblasts in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, count 1-4 ⁇ 10 6 cells/mL, and make a cell suspension.
  • Appropriate dilution was performed and 100,000 cells/well of the cell suspension was inoculated on a 6-well plate.
  • the model was established when the cells grew to about 80%.
  • the blank control group was added with 200 ⁇ L PBS and the culture medium was supplemented to 800 ⁇ L; the drug group was repeatedly washed with an appropriate amount of PBS until colorless, and then the culture medium and drug were added to 800 ⁇ L.
  • Each group was placed in a 37°C, 5% CO 2 incubator for 48 h.
  • the cells were scraped off with a cell scraper, mixed by blowing, and the cell suspension was ultrasonically disrupted for 30 seconds, centrifuged at 1500x ⁇ for 15 minutes, and the cell supernatant was collected to obtain the sample solution; the operation was performed according to the operating instructions of the PER1 protein ELISA kit.
  • the OD value of each well was measured in sequence at 450nm using an enzyme reader within 15 minutes.
  • Circadian rhythm protein 1 is one of the core components of the biological clock that regulates the circadian rhythm cycle of the skin. It is involved in regulating the skin's response to environmental stress and protects the skin from various environmental damages such as UV radiation, temperature, chemical and physical damage, and microbial infection.
  • the expression of PER1 protein has a certain correlation with aging. Increasing the content of PER1 protein can improve skin aging to a certain extent. This experiment detects the content of PER1 protein in the corresponding cells to determine whether the peptide of the present invention can promote the expression of PER1 protein.
  • the peptide of the present invention can promote the expression of PER1 protein, increase the content of PER1 protein, participate in the regulation of the skin circadian rhythm cycle, thereby restoring the circadian rhythm and resynchronizing the biological clock of skin cells, and can be used to repair damaged skin cells and alleviate signs of skin aging; it can also be used to prepare PER1 protein activators.
  • PBS Phosphate buffered saline
  • DMSO dimethyl sulfoxide
  • DMEM high glucose medium
  • fetal bovine serum Gibco
  • RNA extract Wuhan Sevier Biotechnology Co., Ltd.
  • SweScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR One-Step gDNA Remover
  • 2 ⁇ SYBR Green qPCR Master Mix None ROX
  • primers Wuhan Sevier Biotechnology Co., Ltd.
  • Carbon dioxide incubator (Shanghai Yiheng), clean bench (Sujing), biological inverted microscope (Chongqing Optoelectronics), microplate reader (MD, USA), fluorescence upright microscope (Guangzhou Mingmei), desktop high-speed refrigerated microcentrifuge (DragonLab), vortex mixer (Wuhan Sevier Biotechnology Co., Ltd.), handheld centrifuge (Wuhan Sevier Biotechnology Co., Ltd.), fluorescence quantitative PCR instrument (Bio-rad), PCR instrument (Beijing Dongsheng Innovation Biotechnology Co., Ltd.), dry constant temperature metal bath (Wuhan Sevier Biotechnology Co., Ltd.), ultra-micro spectrophotometer (Thermo), film sealing instrument (intelligent) (Wuhan Sevier Biotechnology Co., Ltd.).
  • HSF Human dermal fibroblasts
  • Peptide group Peptide (1), Peptide (2), the test concentration of the above samples was 25 ppm.
  • Control group PBS.
  • Collecting cells Collect cells at different times after adding samples. When collecting cells, pour out the culture medium, add trypsin to digest, terminate digestion and centrifuge to obtain cell pellets. Then pour out the pellet, add 500 ⁇ L PBS to each well to wash the cells, centrifuge, discard the supernatant, and store in a -80°C refrigerator.
  • B CT (target gene, control sample) - CT (internal standard gene, control sample)
  • Biological rhythm refers to the law of changes in the body's activities over time, and is one of the important characteristics of life activities.
  • the most studied biological rhythm at present is the circadian rhythm.
  • the Clock gene is the main gene for studying circadian rhythm-related actions.
  • the biological clock system in the human body is disturbed by external factors such as ultraviolet rays, which will cause circadian rhythm disorders, thereby affecting metabolic activities, making the repair function worse, and then showing signs of aging, which is manifested at the genetic level as abnormal expression of clock genes such as Clock.
  • peptide (1) The difference between peptide (1) and peptide (2) is that the C-terminus of the peptide chain of the former is in the form of -OH, while the C-terminus of the peptide chain of the latter is in the form of amidation (-NH 2 ).
  • the peptides of the present invention have the same efficacy regardless of whether the C-terminus of the peptide chain is -NH 2 or -OH, and can be used for anti-aging and repair.
  • the peptides of the present invention can increase cell activity, promote cell proliferation, promote collagen production, increase skin elasticity, improve skin firmness, prevent or repair skin aging or photoaging, promote PER1 protein and Clock gene expression, restore circadian rhythm, resynchronize skin cell biological clocks, repair damaged cells, and delay skin aging. They can also be used to prepare PER1 protein activators or Clock protein activators, and have wide applications in the fields of cosmetics or medicine.
  • 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.

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Abstract

公开一种具有式（I）R1-Ser-Pro-X1-Gln-R2的肽、或其立体异构体、或其立体异构体的混合物、或其盐、或它们的组合物、以及它们在制备用于预防或修复皮肤老化或光老化，促进胶原蛋白生成，增加皮肤弹性和/或皮肤紧致度，促进PER1蛋白表达、调节皮肤昼夜节律和/或修复皮肤受损细胞，促进Clock基因表达和/或再同步皮肤细胞生物钟的组合物中的用途，亦可用于制备PER1蛋白激活剂或Clock蛋白激活剂。

Description

具有抗衰老作用的肽及其组合物和用途 技术领域
本发明涉及医药和化妆品技术领域，特别是一种具有抗衰老作用的肽及其组合物和用途。
背景技术
皮肤作为人体的第一道生理防线，在保护组织器官免受化学性、机械性、物理性以及病原微生物侵袭的同时，也时刻经受着外部环境的考验。近年来，由于UV辐射、环境污染等环境侵袭，使得皮肤疾病的患者逐年增多，也加速了皮肤的衰老。环境侵袭会对皮肤细胞的DNA造成破坏并影响其昼夜节律。
昼夜节律是指生命活动以24小时为周期的变动，又称生物钟，它调节我们的许多代谢、生理活动和行为。很多证据表明，昼夜节律的失调和紊乱会对人的健康产生很大的负面效应，包括时差反应、倒夜班引起的疲劳综合症、睡眠失调、皮肤疾病等。此外，很多严重威胁人类健康的疾病，比如心脑血管疾病和肿瘤也都与昼夜节律的紊乱有关。然而，随着现代文明的发展，越来越多的人背离了日出而作，日落而息的生活方式，导致昼夜节律的失调越来越普遍。
皮肤细胞的角质细胞、黑色素细胞、成纤维细胞中也存在独立生物钟系统。昼夜节律蛋白不仅在培养的人体细胞中表达，还在人体皮肤生物组织中表达，对皮肤功能产生影响。皮肤很容易暴露于光、温度、湿度、紫外线、病菌等的外部环境因素中，这些外部环境因素以一天为周期发生变化。皮肤为了适应这种环境，通过启动存在于皮肤内的生物钟来调节各种生理学功能。有实验表明，皮肤细胞增殖及分化、水分散失、皮脂的生成、温度、pH、皱纹的生成均以24小时为周期发生变化。例如，皮肤细胞的增殖及分化以一天24小时为周期，通过一系列的过程而发生。白天皮肤容易因紫外线暴露而损伤，所以皮肤细胞的分化主要发生于傍晚和凌晨之间。通过将这些一系列的过程时间性地分离，以从有害环境中保护皮肤。但是，当这一过程的节律被打乱时，就无法在适当时间产生所需的生理反应，如此一来，皮肤昼夜节律性被打乱，就会加速皮肤的损伤。
近年来研究发现，与自然身体昼夜节律相关的基因包括Clock基因和PER1基因，两者都编码调节昼夜节律的蛋白质(Clock蛋白和PER1蛋白)。PER1是调节皮肤昼夜节律周期的生物钟核心组分之一，是Period基因家族的成员，Period基因家族由PER1、PER2、PER3组成，这些基因参与机体的新陈代谢、运动活动和行为等，其中PER1发挥着核心作用。Clock 基因和PER1基因也存在于皮肤细胞中，PER1基因的表达诱导引发与在夜间进行的生物过程(例如修复)相关的细胞活动程序。表皮祖细胞/干细胞的DNA复制、DNA修复机制和细胞分裂表现出明显的昼夜节律特征。长期暴露于环境侵袭的皮肤细胞通常表现出降低的、不规律的或不同步的Clock基因或PER1基因表达，扰乱皮肤细胞中正常昼夜节律。久而久之正常细胞昼夜节律和同步性被扰乱，皮肤平衡性被破坏，从而加速皮肤的自然老化过程，造成皮肤损伤，出现皱纹、细纹、皮肤松弛、不均匀色素沉着、老年斑、色斑等问题。
通过对Clock基因和PER1基因进行分析，发现它们是调节皮肤昼夜节律周期的生物钟核心基因和负反馈基因，参与调节皮肤对环境压力的反应过程，而且Clock基因和PER1基因以大约24小时为周期，干涉各种生理学过程中的遗传基因及蛋白质的表达及活性调节，以此生物钟迎合外部环境，来调节生理学反应的活性度。根据昼夜节律对皮肤细胞的各项活动过程进行调控，保护皮肤免受UV辐射、温度、化学和物理伤害以及微生物感染等各种环境伤害。
因此，有必要从皮肤昼夜节律的机理出发，研究一种能够改善皮肤昼夜节律、激活皮肤昼夜节律调节基因及相关蛋白的活性物，将其应用于医药和化妆品领域，缓解皮肤衰老。
发明内容
本发明的目的在于提供一种稳定、高效、安全、对皮肤无刺激的活性物，通过激活皮肤昼夜节律周期调节基因及相关蛋白的表达，修复受损细胞，实现抗衰老效果，同时能够促进胶原蛋白生成，增加皮肤弹性和/或皮肤紧致度，预防皮肤松弛，使其在医药和化妆品领域具有广泛的应用。
鉴于此，本发明提供一种式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，
R1-Ser-Pro-X1-Gln-R2 (I)
式(I)中，
X1选自：-Gly-、-Asn-、-Ala-、-Ile-、-Val-、-Ser-、-Thr-或-Met-；
R1选自：H或R3-CO-，R3选自：取代的或未取代的烷基、取代的或未取代的烯基；
R2选自：-NR4R5或-OR4，其中R4和R5彼此独立地选自：H、取代的或未取代的烷基、取代的或未取代的烯基；
所述烷基是指具有1-24个碳原子(可选具有1-16个碳原子；可选具有1-14个碳原子；可选具有1-12个碳原子；可选具有1、2、3、4、5、或6个的碳原子)的饱和脂肪族直链 或支链的烷基；可选选自：甲基、乙基、异丙基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十二烷基、十四烷基、十六烷基、十八烷基、2-乙基己基、2-甲基丁基、或5-甲基己基；
所述烯基是指具有2-24个碳原子(可选具有2-16个碳原子；可选具有2-14个碳原子；可选具有2-12个碳原子；可选具有2、3、4、5、或6个碳原子)的直链或支链烯基；所述烯基具有一个或多个碳-碳双键，可选具有1、2或3个共轭或非共轭的碳-碳双键；所述烯基是通过一个单键而结合至分子的其余部分；可选选自：乙烯基、油烯基、或亚油烯基；
可选地，所述“取代的烷基”、“取代的烯基”中的取代基选自C1-C4烷基；羟基；C1-C4烷氧基；氨基；C1-C4氨基烷基；C1-C4羰氧基；C1-C4氧基羰基；卤素(如氟、氯、溴、以及碘)；氰基；硝基；叠氮化物；C1-C4烷基磺酰基；硫醇；C1-C4烷硫基；C6-C30芳氧基如苯氧基；-NRb(C＝NRb)NRbRc，其中Rb和Rc是独立地选自：H、C1-C4烷基、C2-C4烯基、C2-C4炔基、C3-C10环烷基、C6-C18芳基、C7-C17芳烷基、具有三至十元的杂环基、或氨基的保护基。
可选地，R1选自：H、乙酰基、叔-丁酰基、己酰基、2-甲基己酰基、辛酰基、癸酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基或亚油酰基；R4、R5彼此独立地选自：H、甲基、乙基、己基、十二烷基或十六烷基；
可选地，R1选自H、乙酰基、肉豆蔻酰基或棕榈酰基；R4是H并且R5选自：H、甲基、乙基、己基、十二烷基或十六烷基；
具体地，R1是H或乙酰基；R2是-OH或-NH2。
可选地，式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，选自下列肽(1)-(32)：
(1)H-Ser-Pro-Gly-Gln-OH；
(2)H-Ser-Pro-Gly-Gln-NH2；
(3)Ac-Ser-Pro-Gly-Gln-OH；
(4)Ac-Ser-Pro-Gly-Gln-NH2；
(5)H-Ser-Pro-Ala-Gln-OH；
(6)H-Ser-Pro-Ala-Gln-NH2；
(7)Ac-Ser-Pro-Ala-Gln-OH；
(8)Ac-Ser-Pro-Ala-Gln-NH2；
(9)H-Ser-Pro-Asn-Gln-NH2；
(10)H-Ser-Pro-Asn-Gln-OH；
(11)Ac-Ser-Pro-Asn-Gln-OH；
(12)Ac-Ser-Pro-Asn-Gln-NH2；
(13)H-Ser-Pro-Ile-Gln-OH；
(14)H-Ser-Pro-Ile-Gln-NH2；
(15)Ac-Ser-Pro-Ile-Gln-OH；
(16)Ac-Ser-Pro-Ile-Gln-NH2；
(17)H-Ser-Pro-Val-Gln-OH；
(18)H-Ser-Pro-Val-Gln-NH2；
(19)Ac-Ser-Pro-Val-Gln-OH；
(20)Ac-Ser-Pro-Val-Gln-NH2；
(21)H-Ser-Pro-Ser-Gln-OH；
(22)H-Ser-Pro-Ser-Gln-NH2；
(23)Ac-Ser-Pro-Ser-Gln-OH；
(24)Ac-Ser-Pro-Ser-Gln-NH2；
(25)H-Ser-Pro-Thr-Gln-OH；
(26)H-Ser-Pro-Thr-Gln-NH2；
(27)Ac-Ser-Pro-Thr-Gln-OH；
(28)Ac-Ser-Pro-Thr-Gln-NH2；
(29)H-Ser-Pro-Met-Gln-OH；
(30)H-Ser-Pro-Met-Gln-NH2；
(31)Ac-Ser-Pro-Met-Gln-OH；
(32)Ac-Ser-Pro-Met-Gln-NH2。
可选地，选自肽(1)、肽(2)、肽(3)、肽(4)、肽(9)、肽(10)、肽(11)、肽(12)；具体地，
(1)H-Ser-Pro-Gly-Gln-OH；
(2)H-Ser-Pro-Gly-Gln-NH2；
(3)Ac-Ser-Pro-Gly-Gln-OH；
(4)Ac-Ser-Pro-Gly-Gln-NH2；
(9)H-Ser-Pro-Asn-Gln-NH2；
(10)H-Ser-Pro-Asn-Gln-OH；
(11)Ac-Ser-Pro-Asn-Gln-OH；
(12)Ac-Ser-Pro-Asn-Gln-NH2。
本发明的式(I)所示的肽可以作为立体异构体或立体异构体的混合物存在；例如，其所包含的这些氨基酸可以具有L-、D-的构型、或彼此独立地是外消旋的。因此，有可能获得同分异构混合物以及外消旋混合物或非对映混合物、或纯的非对映异构体或对映异构体，这取决于不对称碳的数量和存在什么同分异构体或同分异构混合物。本发明的式(I)所示的肽的优选的结构是纯的同分异构体，即，对映异构体或非对映异构体。
例如，当本发明所述-Pro-时，应理解-Pro-选自-L-Pro-、-D-Pro-、或两者的混合物，是外消旋的或非外消旋的。在本文件中描述的制备方法使本领域的普通技术人员能够通过选择具有正确构型的氨基酸来获得本发明的肽的每种立体异构体。
本发明还包括式(I)所示的肽的所有合适的同位素变体。本发明的这些肽的同位素变体此处理解为是指这样的化合物：其中在本发明的肽内至少一个原子被替换为相同原子序数的另一个原子，但所述另一原子的原子质量不同于自然界中通常或主要存在的原子质量。可掺入本发明的肽中的同位素的实例是：氢、碳、氮、氧或硫的那些，例如2H(氘)、3H(氚)、13C、14C、15N、17O、18O、33S、34S、35S或36S。本发明的肽的特定的同位素变体(特别是其中已经掺入一种或多种放射性同位素的那些)可能有利于，例如检查在体内的作用机理或活性化合物的分布；由于相对简单的可制备性和可检测性，尤其是用3H或14C同位素标记的化合物适用于该目的。另外，由于化合物的更强的代谢稳定性，同位素(例如氘)的掺入可以产生特定的治疗益处，例如体内半衰期的延长或所需活性剂量的降低；因此，在某些情况下，本发明的肽的这种改性还可构成本发明的优选实施方案。通过本领域技术人员已知的方法，例如通过在下文中进一步描述的方法和在实施例中所述的方法，通过使用各自的试剂和/或起始物质的相应的同位素改性物，可制备本发明的肽的同位素变体。
此外，本发明还包括本发明的肽的前药。术语“前药”在本文中意指这样的化合物：其本身可以是生物学上有活性的或无活性的，但是在它们在身体内的停留时间期间，其反应(例如代谢或水解)生成本发明的肽。
术语“美容上可接受的盐或药学上可接受的盐”指被认可的在动物，并且更确切地说在人类中使用的一种盐，包括式(I)所示的肽的金属盐，所述金属包括，但不局限于：锂、钠、钾、钙、镁、锰、铜、锌或铝等；包括式(I)所示的肽与有机碱形成的盐，所述有机碱包括，但不局限于：乙胺、二乙胺、乙二胺、乙醇胺、二乙醇胺、精氨酸、赖氨酸、组氨 酸或哌嗪等；包括式(I)所示的肽与无机酸或有机酸形成的盐，所述有机酸包括，但不局限于：乙酸、柠檬酸、乳酸、丙二酸、马来酸、酒石酸、延胡索酸、苯甲酸、天冬氨酸、谷氨酸、琥珀酸、油酸、三氟乙酸、草酸、扑酸(pamoate)或葡萄糖酸等；可选地，所述无机酸包括：盐酸、硫酸、硼酸或碳酸。
本发明式(I)所示的肽、或其立体异构体或其美容上可接受的盐、或其药学上可接受的盐的合成可以根据现有技术中已知的常规方法来进行，例如固相合成法、液相合成法或固相与液相结合的方法，还可以通过以产生所希望的序列为目标的生物技术方法、或通过具有动物、真菌、或植物来源的蛋白质的控制水解来制备。
例如，一种获得式(I)所示的肽的方法包括以下步骤：
-将具有受保护的N-末端和自由的C-末端的氨基酸与具有自由的N-末端和受保护的或与固体载体结合的C-末端的氨基酸偶联；
-消除保护N-末端的基团；
-重复该偶联顺序和消除保护N-末端的基团，直到获得所希望的肽序列；
-消除保护C-末端的基团或从该固体载体裂解。
优选地，C-末端与一种固体载体结合并且该方法是在固相上进行，包括将具有受保护的N-末端和自由的C-末端的氨基酸与具有自由的N-末端和与一种聚合物载体结合的C-末端的氨基酸偶联；消除保护N-末端的基团；并且重复此顺序所需要的次数以便因此获得具有所希望的长度的肽，接着从最初的聚合物载体裂解所合成的肽。
在整个合成中这些氨基酸的侧链的官能团用临时或永久的保护基团保持充分地保护，并且可以与从该聚合物载体裂解肽的过程同时地或正交地脱保护。
可选地，固相合成可以通过将二肽或三肽偶联到聚合物支持体上或偶联到先前与聚合物支持体结合的二肽或氨基酸上的汇集策略(convergent strategy)来进行。
使用本领域已知的标准条件和方法对N-末端和C-末端脱保护和/或以非确定的次序从聚合物支持体裂解肽，随后可以修饰所述末端的官能团。可以对与聚合物支持体结合的式(I)的肽进行N-末端和C-末端的任选的修饰，或在肽已从聚合物支持体裂解后进行N-末端和C-末端的任选的修饰。
本发明的另一方面，提供一种美容或药用组合物，包括有效量的上述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，以及至少一种赋形剂和任选的美容上或药学上可接受的佐剂；
可选地，所述佐剂选自：胶原合成刺激剂、调节PGC-1α合成的剂、调节PPARγ的活性的剂、增加或减少脂肪细胞的甘油三酸酯含量的剂、刺激或延迟脂肪细胞分化的剂、脂解剂或刺激脂肪分解的剂、溶脂剂、生脂剂、乙酰胆碱受体聚集的抑制剂、抑制肌肉收缩的剂、抗胆碱能试剂、弹性蛋白酶抑制剂、基质金属蛋白酶抑制剂、黑色素合成刺激或抑制剂、增白剂或脱色剂、促色素沉着剂、自晒黑剂、抗老化剂、NO-合酶抑制剂、5α-还原酶抑制剂、赖氨酰羟化酶和/或脯氨酰羟化酶的抑制剂、抗氧化剂、自由基清除剂和/或抗大气污染的剂、活性羰基类物质清除剂、抗糖化剂、抗组胺剂、抗病毒剂、抗寄生虫剂、乳化剂、润肤剂、有机溶剂、液体推进剂、皮肤调理剂、保留水分的物质、α羟基酸、β羟基酸、增湿剂、表皮水解酶、维生素、氨基酸、蛋白质、色素、染料、生物聚合物、胶凝聚合物、增稠剂、表面活性剂、软化剂、粘合剂、防腐剂、抗皱剂、能够减少或治疗下眼袋的剂、去角质剂、抗微生物剂、灭菌剂、抑菌剂、刺激真皮或表皮大分子的合成和/或能够抑制或预防它们的降解的剂、刺激弹性蛋白合成的剂、刺激核心蛋白聚糖合成的剂、刺激层粘连蛋白合成的剂、刺激防御素合成的剂、刺激伴侣蛋白合成的剂、刺激cAMP合成的剂、刺激HSP70合成的剂、刺激热休克蛋白合成的剂、刺激透明质酸合成的剂、刺激纤连蛋白合成的剂、刺激去乙酰化酶合成的剂、刺激脂质和角质层组分的合成的剂、神经酰胺、脂肪酸、抑制胶原降解的剂、抑制弹性蛋白降解的剂、抑制丝氨酸蛋白酶的剂、刺激成纤维细胞增殖的剂、刺激角质形成细胞增殖的剂、刺激脂肪细胞增殖的剂、刺激黑色素细胞增殖的剂、刺激角质形成细胞分化的剂、抑制乙酰胆碱酯酶的剂、皮肤松弛剂、刺激糖胺聚糖合成的剂、抗角化过度剂、粉刺溶解剂、抗银屑病剂、抗湿疹剂、DNA修复剂、DNA防护剂、稳定剂、止痒剂、用于治疗和/或护理敏感性皮肤的剂、固化剂、紧致剂、重构剂、抗拉伸纹剂、调节皮脂产生的剂、止汗剂、刺激愈合的剂、协助愈合的剂、刺激再上皮化的剂、协助再上皮化的剂、细胞因子、镇静剂、抗炎剂、麻醉剂、作用于毛细血管循环和/或微循环的剂、刺激血管生成的剂、抑制血管渗透性的剂、静脉紧张剂、作用于细胞代谢的剂、用于改善真皮-表皮接合的剂、诱导毛发生长的剂、毛发生长抑制或延缓剂、香料、螯合剂、植物提取物、精油、海洋提取物、得自生物发酵过程的剂、无机盐、细胞提取物、防晒剂、以及有效抗A和/或B紫外线的有机或无机光防护剂或其混合物。
可选地，所述美容或药用组合物的制剂选自：霜剂、油、香膏、泡沫、洗剂、凝胶、擦剂、浆液、软膏、摩丝、粉末、杆剂、笔剂、喷雾剂、气溶胶、胶囊剂、片剂、颗粒剂、口香糖、溶液、混悬液、乳剂、酏剂、多糖薄膜、胶冻或明胶；
可选地，所述胶囊剂包括：软胶囊剂、硬胶囊剂，可选为明胶胶囊剂；
可选地，所述片剂包括：糖衣片剂。
本发明的肽根据它们的序列的性质或N-末端和/或C-末端中的任何可能的修饰，在水中具有可变的溶解度。因此本发明的肽可以通过水溶液掺入组合物中，且不溶于水的那些可溶解于美容上或药学上可接受的常规溶剂中，所述溶剂例如并且不限于乙醇、丙醇、异丙醇、丙二醇、甘油、丁二醇或聚乙二醇或其任何组合。
待施用的美容上或药学上有效量的本发明的肽以及它们的剂量将依赖于许多因素，包括年龄、患者的状态、病症或疾病的严重性、施用的途径和频率以及待使用的肽的具体性质。
“美容上或药学上有效量”意指无毒性的但足以提供希望的效果的本发明的一种或多种肽的量。在本发明的美容组合物或药物组合物中以获得希望的效果的美容上或药学上有效的浓度使用本发明的肽；在一个优选形式中，相对于组合物的总重量，在0.00000001％(按重量计)和20％(按重量计)之间，优选在0.000001％(按重量计)和15％(按重量计)之间、更优选在0.0001％(按重量计)和10％(按重量计)之间，并且甚至更优选在0.0001％(按重量计)和5％(按重量计)之间。
本发明的另一方面，提供一种美容上或药学上可接受的递送系统或缓释系统，以便实现有效成分的更好渗透和/或改进它的药物代谢动力学和药效动力学特性，其包含有效量的上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述的美容或药用组合物。
术语“递送系统”是指与本发明的肽一起施用的稀释剂、佐剂、赋形剂或载体，它们选自：水、油或表面活性剂、包括石油来源、动物来源、植物来源、或合成来源的那些，例如并且不限于花生油、大豆油、矿物油、芝麻油、蓖麻油、聚山梨醇酯、脱水山梨糖醇酯、醚硫酸酯、硫酸酯、甜菜碱、葡萄糖苷、麦芽糖苷、脂肪醇、壬苯醇醚、泊洛沙姆、聚氧乙烯、聚乙二醇、右旋糖、甘油、毛地黄皂苷和类似物。本领域的普通技术人员已知在可以给予本发明的肽的不同递送系统中可以使用的稀释剂。
术语“缓释”以常规含义使用，指提供化合物在一段时间内逐渐释放的化合物的递送系统，且优选地但不是必须地，在整个时间段内具有相对恒定的化合物释放水平。
递送系统或缓释系统的实例是脂质体、油质体、非离子型表面活性剂脂质体囊泡、醇质体、毫米胶囊、微米胶囊、纳米胶囊、纳米结构的脂质载体、海绵状物、环糊精、类脂囊泡、胶束、毫米球、微米球、纳米球、脂质球、微米乳液、纳米乳液、毫米粒子、微米粒子或纳 米粒子。优选的递送系统或缓释系统是脂质体和微米乳液，更优选具有反胶束的内部结构的油包水型微米乳液。
缓释系统可以通过现有技术中已知的方法来制备，并且可以例如通过以下方式来给予：通过局部或经皮给药，包括粘附贴剂、非粘附贴剂、封闭贴剂、以及微电子贴剂；或通过全身给药例如并且不局限于，口服或胃肠外途径，包括鼻、直肠、皮下植入或注射、或直接植入或注射至特定身体部位中，并且优选地应该释放相对恒定量的本发明的这些肽。在该缓释系统中包含的肽的量将取决于例如该组合物将被给予的部位、本发明的肽的释放动力学和持续时间、以及有待治疗和/或护理的病状、病症和/或疾病的性质。
本发明的另一方面，提供一种上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、或上述的美容上或药学上可接受的递送系统或缓释系统在制备用于治疗、预防或修复皮肤老化和/或光老化的美容组合物或药物组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、或上述的美容上或药学上可接受的递送系统或缓释系统在制备用于增加皮肤弹性和/或皮肤紧致度的美容组合物或药物组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、或上述的美容上或药学上可接受的递送系统或缓释系统在制备用于促进胶原蛋白生成的美容组合物或药物组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、或上述的美容上或药学上可接受的递送系统或缓释系统在制备用于促进PER1蛋白表达、调节皮肤昼夜节律和/或修复皮肤受损细胞的美容组合物或药物组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、或上述的美容上或药学上可接受的递送系统或缓释系统在制备PER1蛋白激活剂中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、 或上述的美容上或药学上可接受的递送系统或缓释系统在制备用于促进Clock基因表达和/或再同步皮肤细胞生物钟的组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、或上述的美容上或药学上可接受的递送系统或缓释系统在制备Clock蛋白激活剂中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、或上述的美容上或药学上可接受的递送系统或缓释系统在制备化妆品中的用途。
本发明的另一方面，提供一种化妆品，包含上述式(I)所示的肽，或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或上述美容或药用组合物、或上述的美容上或药学上可接受的递送系统或缓释系统；
可选地，所述化妆品的剂型包括膏霜、乳液、凝胶、粉剂、片剂、膜剂、气雾剂或喷雾剂。
为了便于理解本发明，对在本发明所使用的一些术语和表述的含义说明如下：
在本发明中，术语“皮肤”应理解为是构成它的多个层，从最上层或角质层至最下层或皮下组织，两个端点都包括在内。这些层由不同类型的细胞组成，如角质形成细胞、成纤维细胞、黑色素细胞、和/或脂肪细胞等。在本发明中，术语“皮肤”包括头皮。
术语“治疗”，指的是给予根据本发明的肽以减轻或消除一种疾病或病症、或减少或消除与这种疾病或病症相关的一种或多种症状。术语“治疗”还涵盖了减轻或消除该疾病或病症的生理后果的能力。
术语“预防”，指的是本发明的肽在一种疾病或病症出现前防止、延迟、或阻碍其出现或发展的能力。
术语“修复”，指的是本发明的肽在一种疾病或病症出现后改善、缓解或恢复其原状的能力。
术语“老化”指的是皮肤随着年龄的增长经历的变化(自然老化)，或通过暴露于阳光(光老化)或暴露于环境污染物，如化学污垢或污染物、烟草烟雾等而经历的变化，并且包括所有外在可见的和/或通过触摸可感知的变化，例如并且不局限于：皮肤上的不连续性的发展(如皱纹、细纹、表情纹、拉伸纹、条纹、沟纹、不平整或粗糙、毛孔尺寸增大、水分损失、弹性损失、紧致性损失、平滑性损失、变形恢复能力损失、回弹性损失)、皮肤下垂 (如脸颊下垂、眼睛下方出现眼袋、或出现双下巴等)、皮肤颜色的变化(如瘢痕、变红、眼袋、或出现色素过度沉着区域如老年斑或雀斑等)、异常分化、过度角质化、弹性组织变性、角化症、脱发、橘皮样皮肤、胶原结构损失，以及角质层、真皮、表皮、血管系统(例如出现蜘蛛静脉或毛细血管扩张症)或靠近皮肤的那些组织的其他组织学变化。
术语“光老化”指的是由于皮肤长期暴露于紫外线辐射而导致的皮肤过早老化，它呈现出与自然老化相同的生理特征，例如并且不局限于：松弛、下垂、颜色改变或色素沉着不规则、异常和/或过度角质化。
在本说明书中，用于氨基酸的缩写遵循IUPAC-IUB生化命名委员会(IUPAC-IUBCommission of Biochemical Nomenclature)在欧洲生物化学杂志(Eur.J.Biochem.1984,138:9-37)中所指定的规则。
因此，例如，Gly表示NH2-CH2-COOH，Gly-表示NH2-CH2-CO-，-Gly表示-NH-CH2-COOH，并且-Gly-表示-NH-CH2-CO-。因此，表示肽键的连字符消除了当位于该符号的右侧时的氨基酸(在此用常规非离子化形式来表示)1-羧基中的OH，并且消除了当位于该符号的左侧时的氨基酸2-氨基中的H；两种修饰可以应用于同一个符号(见表1)。
表1氨基酸残基的结构以及它们的单字母和三字母缩写符号

缩写“Ac-”在本发明中用来表示乙酰基(CH3-CO-)。
本发明相对于现有技术所取得的有益效果包括：
1、本发明所述的肽，合成方便，安全性高，能够增加皮肤成纤维细胞的活性，促进胶原蛋白生成，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛，治疗、预防和/或修复皮肤老化或光老化。
2、本发明的肽能够促进PER1蛋白表达，增加PER1蛋白含量，参与皮肤昼夜节律周期的调节，从而恢复昼夜节律及再同步皮肤细胞生物钟，可用于修复皮肤受损细胞，缓解皮肤衰老迹象；亦可用于制备PER1蛋白激活剂。
3、本发明的肽能够激活皮肤昼夜节律周期调节基因及Clock等相关蛋白的表达，调节皮肤昼夜节律，再同步皮肤细胞生物钟，增强细胞活力，修复受损细胞，缓解皮肤衰老迹象，在化妆品或医药领域具有广泛应用，亦可用于制备Clock蛋白激活剂。
附图说明
为了更清楚地说明本发明的技术方案，下面将对本发明的描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是肽(1)H-Ser-Pro-Gly-Gln-OH(分子式C15H25N5O7)质谱图，[M+H]+准分子离子峰的质荷比(m/z)为388.40，质谱测得的分子量为387.40。
图2是肽(2)H-Ser-Pro-Gly-Gln-NH2(分子式C15H26N6O6)质谱图，[M+H]+准分子离子峰的质荷比(m/z)为387.2045，质谱测得的分子量为386.20。
图3是肽(9)H-Ser-Pro-Asn-Gln-NH2(分子式C17H29N7O7)质谱图，[M+H]+准分子离 子峰的质荷比(m/z)为444.2271，质谱测得的分子量为443.23。
图4是测试样品对HaCaT细胞活性的影响结果图。
图5是测试样品对HSF细胞活性的影响结果图。
图6是测试样品对经UV处理后HSF细胞活性的影响结果图。
图7是测试样品对胶原蛋白含量的影响结果图。
图8是测试样品对PER1蛋白含量的影响结果图。
具体实施方式
为使本发明的所述目的、特征和优点能够更加明显易懂，下面结合附图和实施例对本发明作进一步详细的说明。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，在本发明中，用于氨基酸的缩写遵循IUPAC-IUB的生物化学命名委员会在Eur.J.Biochem.(1984)138:9-37和J.Chem.(1989)264:633-673中指定的规则。
Amide Resin：一种多肽合成用的起始树脂；WangResin：王树脂；Fmoc-Linker：4-[(2,4-二甲氧基苯基)(Fmoc-氨基)甲基]苯氧乙酸；DMF：N,N-二甲基甲酰胺；DCM：二氯甲烷；DMAP：4-二甲氨基吡啶；HOBt：1-羟基苯并三氮唑；DIC：二异丙基碳二亚胺；DIPEA：二异丙基乙胺；Ac2O：醋酸酐；piperidine：哌啶；TFA：三氟乙酸；TIS：三异丙基硅烷；Ser：丝氨酸；Pro：脯氨酸；Gly：甘氨酸；Asn：天冬酰胺；Gln：谷氨酰胺；Fmoc：9-芴基甲氧羰基；tBu：叔丁基；Trt：三苯甲基。
实施例1 H-Ser-Pro-Gly-Gln-OH的制备
1.1树脂的溶胀及反应
称取10g的WangResin于固相合成反应柱中，用DCM溶胀，洗涤树脂，抽走溶剂。
称取15g的Fmoc-Gln(Trt)-OH、6mL的DIC、4g的HOBt和1.5g的DMAP于干燥三角瓶中。用DMF溶解后加入溶胀后的树脂中反应2.5h，抽走反应液，洗涤树脂，抽走溶剂。
继续加入Ac2O、DIPEA和DMAP封端处理2h。洗涤树脂，抽走溶剂。
1.2脱Fmoc
Fmoc-Gln(Trt)-Wang Resin用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂7次，抽走溶剂。
1.3投料反应
称取8g的Fmoc-Gly-OH，3g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min。加5mL的DIC活化3min，避免水汽。将活化后的氨基酸加入脱保护后树脂中反应1.5h，抽走反应液。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在3g HOBt和5mL DIC的情况下，使用DMF作为溶剂，将活化后的7g的Fmoc-Pro-OH偶联至肽基树脂上，持续反应1h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在存在3g HOBt和5mL DIC的情况下，使用DMF作为溶剂，偶联9g的Fmoc-Ser(tBu)-OH至肽基树脂上，持续反应1.2h；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂，收缩干燥后得到14g的H-Ser(tBu)-Pro-Gly-Gln(Trt)-WangResin。
1.4裂解
量取80mL的TFA、2mL的TIS、2mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取14g的H-Ser(tBu)-Pro-Gly-Gln(Trt)-Wang Resin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2.5h。抽滤，收集滤液浓缩至不滴液后加入异丙醚搅拌离心洗涤6次，真空干燥，得到3.3g的H-Ser-Pro-Gly-Gln-OH粗肽。
1.5纯化
称取3.3g粗肽溶于100mL纯水中，超声直至目视无明显颗粒后，加入少量稀氨水调节pH至弱碱性，用孔径为0.22μm微孔滤膜过滤得到澄清透明溶液，通过反相HPLC纯化处理，纯化梯度如下表：
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度95.36％的肽(1)H-Ser-Pro-Gly-Gln-OH。
实施例2 H-Ser-Pro-Gly-Gln-NH2的制备
2.1 Fmoc-Linker-Amide Resin的制备
称取5g的Amide Resin树脂于固相合成反应柱中，用DMF溶胀，洗涤树脂，抽走溶剂。
称取7.1g的Fmoc-Linker、4.4g的HOBt于干燥三角瓶中。用DMF溶剂溶解后置于冰 水浴中冷却10min，加DIC活化10min，避免水汽。
将活化后的Fmoc-Linker加入溶胀后的树脂中反应2.5h，抽走反应液，洗涤树脂，抽走溶剂。
继续加入Ac2O与DIPEA封端处理1.5h。洗涤树脂，抽走溶剂。
2.2脱Fmoc
Fmoc-Linker-Amide Resin用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂7次，抽走溶剂。
2.3投料反应
称取11.7g的Fmoc-Gln(Trt)-OH，4.4g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min。加6.3mLDIC活化3min，避免水汽。将活化后的氨基酸加入脱保护后树脂中反应2h，抽走反应液。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在4.4g HOBt和6.3mL DIC的情况下，使用DMF作为溶剂，将活化后的5.7g的Fmoc-Gly-OH偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在4.4g HOBt和6.3mL DIC的情况下，使用DMF作为溶剂，顺序地偶联7.3g的Fmoc-Pro-OH以及随后8.3g的Fmoc-Ser(tBu)-OH；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂，收缩干燥后得到12.2g的Ser(tBu)-Pro-Gly-Gln(Trt)-Linker-Amide Resin。
2.4裂解
量取95mL的TFA、2.5mL的TIS和2.5mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取12.2g的Ser(tBu)-Pro-Gly-Gln(Trt)-Linker-Amide Resin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2h。抽滤，收集滤液浓缩到15mL后加入异丙醚搅拌离心洗涤6次，直至pH值为3-4，真空干燥，得到4.8g的H-Ser-Pro-Gly-Gln-NH2粗肽。
2.5纯化
称取4.8g粗肽溶于90mL甲醇：水(V：V＝1：2)中，用孔径为0.22μm微孔滤膜过滤得到澄清透明溶液，通过反相HPLC纯化处理，纯化梯度如下表：
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度98.726％的肽(2) H-Ser-Pro-Gly-Gln-NH2。
实施例3 H-Ser-Pro-Asn-Gln-NH2的制备
3.1 Fmoc-Linker-Amide Resin的制备
称取5g的Amide Resin树脂于固相合成反应柱中，用DMF溶胀，洗涤树脂，抽走溶剂。
称取7.1g的Fmoc-Linker、4.4g的HOBt于干燥三角瓶中。用DMF溶剂溶解后置于冰水浴中冷却10min，加DIC活化10min，避免水汽。
将活化后的Fmoc-Linker加入溶胀后的树脂中反应2.5h，抽走反应液，洗涤树脂，抽走溶剂。
继续加入Ac2O与DIPEA封端处理1.5h。洗涤树脂，抽走溶剂。
3.2脱Fmoc
Fmoc-Linker-Amide Resin用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂7次，抽走溶剂。
3.3投料反应
称取11.7g的Fmoc-Gln(Trt)-OH，4.4g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min。加6.3mLDIC活化3min，避免水汽。将活化后的氨基酸加入脱保护后树脂中反应2h，抽走反应液。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在4.4g HOBt和6.3mL DIC的情况下，使用DMF作为溶剂，将活化后的11.5g的Fmoc-Asn(Trt)-OH偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在4.4g HOBt和6.3mL DIC的情况下，使用DMF作为溶剂，顺序地偶联7.3g的Fmoc-Pro-OH以及随后8.3g的Fmoc-Ser(tBu)-OH；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂，收缩干燥后得到16.2g的Ser(tBu)-Pro-Asn(Trt)-Gln(Trt)-Linker-Amide Resin。
3.4裂解
量取95mL的TFA、2.5mL的TIS和2.5mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取16.2g的Ser(tBu)-Pro-Asn(Trt)-Gln(Trt)-Linker-Amide Resin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2h。抽滤，收集滤液浓缩到15mL后加入异丙醚搅拌离心洗涤6次，直至pH值为3-4，真空干燥，得到5.2g的H-Ser-Pro-Asn-Gln-NH2粗肽。
3.5纯化
称取5.2g粗肽溶于120mL甲醇：水(V：V＝1：2)中，用孔径为0.22μm微孔滤膜过 滤得到澄清透明溶液，通过反相HPLC纯化处理，纯化梯度如下表：
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度99.696％的肽(9)H-Ser-Pro-Asn-Gln-NH2。
实施例4
本发明式(I)中的其他肽可以通过类似的方法制备。
所获得的这些肽通过ESI-MS测定其分子量，部分肽的测试结果见下表2及图1-3：
表2质谱法测定分子量结果
实施例5细胞增殖实验
5.1试剂与材料
噻唑蓝(MTT)、二甲基亚砜(DMSO)、高糖培养基(DMEM)、胎牛血清、PBS。
5.2仪器
酶标仪、CO2培养箱、超净工作台。
5.3细胞株
人角质形成细胞(HaCaT)购买自中国科学院典型培养物保藏委员会昆明细胞库，人皮肤成纤维细胞(HSF)购买自中国科学院典型培养物保藏委员会上海细胞库。
5.4待测样品
给药组：肽(2)和肽(9)，测试浓度均为6.25ppm、12.5ppm、25ppm、50ppm、100ppm、200ppm。
空白对照组：PBS。
阳性对照组：2％DMSO。
5.5实验方法
分别取处于指数生长期状态良好的HaCaT角质形成细胞和HSF成纤维细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×105个/mL，制成细胞悬液。
取细胞悬液接种于96孔板上，200μL/孔，置恒温CO2培养箱中培养24h。
换液，分别加入给药组、空白对照组和阳性对照组样品，20μL/孔，置于37℃、5％CO2培养箱中孵育72h。
之后每孔加入20μL 5mg/ml MTT，继续于37℃、5％CO2培养箱中孵育4h。弃去原溶液，加入150μL/孔的DMSO，置于平板摇床上振摇5min后，使用酶标仪在波长570nm处测定每孔的OD值，并计算细胞活力。
细胞活力＝(给药孔OD-调零孔OD)/(空白对照孔OD-调零孔OD)×100％
5.6实验结果
MTT法是一种检测细胞存活和生长的方法，测得的OD值与细胞活性成正比。
测试样品对HaCaT细胞活性的影响结果见图4，结果显示，与空白对照组相比，阳性对照组的HaCaT细胞活性显著下降，表明2％的DMSO对HaCaT细胞具有毒性作用，给药组在200ppm范围内对HaCaT细胞没有毒性作用，而且本发明的肽(2)和肽(9)在低浓度6.25ppm时即能够明显地提高细胞活性，促进HaCaT细胞增殖，且随着浓度升高，促细胞增殖作用随之增强。
测试样品对HSF细胞活性的影响结果见图5，结果显示，与空白对照组相比，阳性对照组的HSF细胞活性显著下降，表明2％的DMSO对HSF细胞具有毒性作用，给药组在200ppm范围内对HSF成纤维细胞没有毒性作用，而且本发明的肽(2)和肽(9)在低浓度6.25ppm时即能够明显地提高细胞活性，促进HSF成纤维细胞增殖，且随着浓度升高，促细胞增殖作用随之增强。
由此可知，本发明的肽对HaCaT角质形成细胞和HSF成纤维细胞不仅没有毒性作用，而且还能提高细胞的活性，促进其增殖，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛。
实施例6光老化实验
6.1试剂与材料
胎牛血清、高糖培养基(DMEM)、青霉素、链霉素、噻唑蓝(MTT)。
6.2仪器
酶标仪、CO2培养箱、超净工作台。
6.3细胞株
人皮肤成纤维细胞(HSF)购买自中国科学院典型培养物保藏委员会上海细胞库。
6.4待测样品
给药组：
参比物1(Palm-Lys-Thr-Thr-Lys-Ser)，测试浓度分别为12.5ppm、25ppm、50ppm；
参比物2(Ser-Pro-Leu-Gln-NH2)，测试浓度分别为12.5ppm、25ppm、50ppm；
肽(2)，测试浓度分别为12.5ppm、25ppm、50ppm；
肽(9)，测试浓度分别为12.5ppm、25ppm、50ppm。
空白对照组：PBS。
UV组：UV辐射，加PBS。
6.5实验方法
取处于指数生长期状态良好的HSF成纤维细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×105个/mL，制成细胞悬液。
适当稀释取10000个/孔细胞悬液接种于96孔板上，待细胞长满至80％左右时，建立UV光老化模型。空白对照组加50μLPBS，补充培养基至200μL，不进行UV照射；UV组和给药组，加入适量PBS反复洗至无色后，加入50μL PBS，以80mJ/cm2UV灯下照射，灯源和培养瓶间距15cm，经照射后，弃去PBS，UV组加入PBS溶液和培养基至200μL，给药组加入培养液和倍比稀释药物至200μL。空白对照组、UV组、给药组继续于37℃、5％CO2培养箱中孵育24h。
之后每孔加入20μL 5mg/mL MTT，继续于37℃、5％CO2培养箱中孵育4h。弃去原溶液，加入150μL/孔的DMSO，置于平板摇床上振摇5min后，使用酶标仪读取490nm和630nm波长下的参比OD值。
6.6实验结果
皮肤衰老受到内源性因素和外源性因素的影响，如遗传、环境暴露、紫外线照射、激素变化等。这些因素的累积，特别是紫外线的照射，导致皮肤结构、功能和外观的改变。本实验选取80mJ/cm2UV能量进行辐射建立皮肤光老化模型。
实验结果如图6所示，经UV辐射后，HSF成纤维细胞活性显著下降，表明成功建立了 光老化模型；给药组在50ppm范围内均能够提高细胞活性，从而改善细胞老化，产生明显的抗光老化作用；与参比物1和参比物2相比，本发明的肽(2)和肽(9)能够更明显地提高细胞活性，显著促进HSF成纤维细胞增殖；其中，肽(2)在12.5ppm低浓度下即能够大为提高HSF成纤维细胞的活性，产生明显的抗光老化作用。由此可知，本发明的肽能够用于治疗、预防和/或修复皮肤老化或光老化，而且具有比现有技术更优的抗衰功效。
实施例7胶原蛋白含量测试
7.1试剂与材料
胎牛血清、DMEM培养基、磷酸盐缓冲液、胰蛋白酶、BCA蛋白试剂盒、胶原蛋白I ELISA试剂盒。
7.2仪器
酶标仪、CO2培养箱、超净工作台、恒温箱。
7.3细胞株
人皮肤成纤维细胞(HSF)购买自中国科学院典型培养物保藏委员会上海细胞库。
7.4待测样品
给药组：
参比物1(Palm-Lys-Thr-Thr-Lys-Ser)，测试浓度分别为12.5ppm、25ppm；
参比物2(Ser-Pro-Leu-Gln-NH2)，测试浓度分别为12.5ppm、25ppm；
肽(2)，测试浓度分别为12.5ppm、25ppm；
肽(9)，测试浓度分别为12.5ppm、25ppm。
空白对照组：PBS。
UV组：UV辐射，加PBS。
7.5实验方法
取处于指数生长期状态良好的HSF成纤维细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×106个/mL，制成细胞悬液。
适当稀释取100000个/孔细胞悬液接种于6孔板上，待细胞长满至80％左右时建模。空白对照组加200μLPBS，补充培养基至800μL，不进行UV照射；UV组和给药组，加入适量PBS反复洗至无色后，加入200μLPBS，置于80mJ/cm2UV灯下照射，灯源和培养瓶间距15cm。经照射后，弃去PBS，UV组加入PBS溶液和培养基至800μL，给药组加入培养 基和倍比稀释药物至800μL。空白对照组、UV组、给药组继续于37℃、5％CO2培养箱中孵育48h。
培养结束后，将第1孔细胞消化计数，并稀释至0.5×106个/mL，其余孔的细胞使用细胞刮刮下来，500μL重悬后，所有孔取50μL超声30s后使用BCA法测定总蛋白，根据第1孔蛋白浓度稀释其他孔，使所有细胞悬液浓度为0.5×106个/mL。将调整浓度后的细胞悬液超声破碎30s，1500xɡ离心15min，收集细胞上清获得样本液；按照胶原蛋白I ELISA试剂盒操作说明书进行操作。15min内用酶标仪在450nm处依序测量各孔的OD值。
7.6实验结果
皮肤的衰老与胶原蛋白密切相关，胶原蛋白是结缔组织中发现的最丰富的蛋白质，对于皮肤饱满和紧致具有重要作用。在紫外线过暴环境中，胶原蛋白的生成受到抑制。本实验采用测试样品处理经紫外线辐射后的细胞，检测相应细胞中的胶原蛋白I含量，以确定本发明的肽是否能够促进胶原蛋白生成。
测试样品对胶原蛋白含量的影响结果见图7。结果显示，与空白对照组相比，UV组的胶原蛋白含量显著降低；与UV组相比，参比物1和参比物2不能增加经紫外辐射后细胞中的胶原蛋白含量，而本发明的肽(2)和肽(9)在12.5-25ppm范围内即能够明显地提高胶原蛋白含量，促进胶原蛋白表达，具有优异的促胶原蛋白生成的作用。
由此可知，本发明的肽能够促进胶原蛋白生成，增加胶原蛋白含量，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛，治疗、预防和/或修复皮肤老化或光老化，可以在化妆品或医药领域用于抵抗皮肤衰老。
实施例8PER1蛋白含量测试
8.1试剂与材料
胎牛血清、DMEM培养基、磷酸盐缓冲液、胰蛋白酶、人节律昼夜蛋白1(PER1)ELISA试剂盒。
8.2仪器
酶标仪、CO2培养箱、超净工作台、恒温箱。
8.3细胞株
人皮肤成纤维细胞(HSF)购买自中国科学院典型培养物保藏委员会上海细胞库。
8.4待测样品
给药组：
参比物2(Ser-Pro-Leu-Gln-NH2)，测试浓度为25ppm；
肽(2)，测试浓度为25ppm。
空白对照组：PBS。
8.5实验方法
取处于指数生长期状态良好的HSF成纤维细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×106个/mL，制成细胞悬液。
适当稀释取100000个/孔细胞悬液接种于6孔板上，待细胞长满至80％左右时建模。空白对照组加200μL PBS，补充培养基至800μL；给药组加入适量PBS反复洗至无色后，加入培养基和药物至800μL，各组均置于37℃、5％CO2培养箱中孵育48h。
培养结束后使用细胞刮刮掉细胞，吹打混匀，将细胞悬液超声破碎30s，1500xɡ离心15min，收集细胞上清获得样本液；按照PER1蛋白ELISA试剂盒操作说明书进行操作。15min内用酶标仪在450nm处依序测量各孔的OD值。
8.6实验结果
节律昼夜蛋白1(PER1)是调节皮肤昼夜节律周期的生物钟核心组分之一，参与调节皮肤对环境压力的反应过程，保护皮肤免受UV辐射、温度、化学和物理伤害以及微生物感染等各种环境伤害，而且PER1蛋白的表达与衰老具有一定相关性，提高PER1蛋白的含量可以在一定程度上改善皮肤衰老。本实验通过检测相应细胞中PER1蛋白的含量，以确定本发明的肽是否能够促进PER1蛋白的表达。
测试样品对PER1蛋白含量的影响结果见图8。结果显示，与空白对照组相比，25ppm的肽(2)能够明显地提高PER1蛋白的含量，促进PER1蛋白的表达，而且与参比物2相比，本发明的肽(2)具有更优的促进PER1蛋白表达的效果。
由此可知，本发明的肽能够促进PER1蛋白表达，增加PER1蛋白含量，参与皮肤昼夜节律周期的调节，从而恢复昼夜节律及再同步皮肤细胞生物钟，可用于修复皮肤受损细胞，缓解皮肤衰老迹象；亦可用于制备PER1蛋白激活剂。
实施例9 Clock基因表达检测
9.1试剂与材料
磷酸盐缓冲液(PBS)(Gibco)、二甲基亚砜(DMSO)(Sigma)、高糖培养基(DMEM) (Gibco)、胎牛血清(Gibco)、RNA提取液(武汉赛维尔生物科技有限公司)、SweScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR(One-Step gDNA Remover)(武汉赛维尔生物科技有限公司)、2×SYBR Green qPCR Master Mix(None ROX)(武汉赛维尔生物科技有限公司)、引物(武汉赛维尔生物科技有限公司)。
9.2仪器
二氧化碳培养箱(上海一恒)、超净工作台(苏净)、生物倒置显微镜(重庆光电)、酶标仪(美国MD)、荧光正置显微镜(广州明美)、台式高速冷冻型微量离心机(DragonLab)、涡旋混匀仪(武汉赛维尔生物科技有限公司)、掌上离心机(武汉赛维尔生物科技有限公司)、荧光定量PCR仪(Bio-rad)、PCR仪(北京东胜创新生物科技有限公司)、干式恒温金属浴(武汉赛维尔生物科技有限公司)、超微量分光光度计(Thermo)、封膜仪(智能型)(武汉赛维尔生物科技有限公司)。
9.3细胞株
人皮肤成纤维细胞(HSF)。
9.4待测样品
多肽组：肽(1)、肽(2)，上述样品测试浓度均为25ppm。
对照组：PBS。
模型组：UVA照射。
9.5实验方法
9.5.1细胞给药及收集
(1)实验前处理：成纤维细胞复苏并将细胞培养至3cm培养皿中，直至细胞数量达到18皿。
(2)实验分组：将18皿细胞分为三组，分别为对照组，模型组和多肽组(n＝6×3)。
(3)同步化处理：使用100nM的地塞米松对成纤维细胞处理30min，使其具有与正常细胞相同的节律。
(4)建模：倒掉含有地塞米松的培养基，PBS清洗细胞后；加入适量PBS覆盖培养皿，对照组不照射，模型组及多肽组用UVA照射4h，使其偏离正常细胞的节律。随后倒掉PBS加入完全培养基于培养箱中培养6h。
(5)加样：加入相应的测试样品。
(6)收集细胞：分别在加样后的不同时间收集细胞。收集细胞时，倒掉培养基，加胰酶消化后，终止消化并离心得细胞沉淀。随后倒掉沉淀，每孔加500μL PBS洗涤细胞，离心，弃上清，并于-80℃冰箱保存。
9.5.2总RNA抽提(枪头和离心管均使用无菌无酶款)
(1)用微量移液器将培养瓶/板中培养液吸除干净，加入1mL 4℃预冷的PBS溶液，轻摇洗涤。用微量移液器将PBS吸除干净。
(2)加入1mL的RNA提取液，轻缓振荡或用枪头吹打，破碎细胞。
(3)将液体转移到灭过菌的1.5mL离心管内。
(4)加入400μL三氯甲烷，颠倒离心管15s，充分混匀，静置3min。
(5)4℃下，12000rpm离心10min。
(6)将400μL上清转移到一新的离心管中，加入550μL异丙醇，颠倒混匀。-20℃放置15min。
(7)4℃下，12000rpm离心10min，管底的白色沉淀即为RNA。
(8)吸除液体，加入75％乙醇1.5mL洗涤沉淀。
(9)4℃下，12000rpm离心10min。
(10)将液体吸除干净，将离心管置于超净台上吹3min。
(11)加入15μLWaterNuclease-Free溶解RNA。
(12)使用Nanodrop 2000检测RNA浓度及纯度：仪器空白调零后取2.5μL待测RNA溶液于检测基座上，放下样品臂，使用电脑上的软件开始吸光值检测。
(13)将浓度过高的RNA进行适当比例的稀释，使其终浓度为200ng/μL。
9.5.3反转录(枪头和PCR管均使用无菌无酶款)
(1)反转录反应体系配制(20μL反应体系，试剂盒货号G3337)
(2)轻轻混匀并离心
(3)反转录程序设置，于普通PCR仪上完成反转录。
9.5.4 PCR定量
(1)取0.1mL PCR反应板，配制如下反应体系，每个反转录产物配制3管。点完样用PCR封板膜配合封膜仪完成封膜。
(2)PCR扩增，于荧光定量PCR仪上完成扩增。
9.5.5结果处理
ΔΔCT法：
A＝CT(目的基因，待测样本)-CT(内标基因，待测样本)
B＝CT(目的基因，对照样本)-CT(内标基因，对照样本)
K＝A-B
表达倍数＝2-K
9.6实验结果
生物节律是指机体的活动随时间的变化所表现出来的规律，是生命体活动的重要特征之一。目前研究最多的生物节律是昼夜节律。Clock基因是研究昼夜节律相关动作的主要基因。人体内部的生物钟系统受到紫外线等外界因素干扰会导致昼夜节律紊乱，从而影响代谢活动，使修复功能变差，进而表现出衰老迹象，在基因层面表现为Clock等时钟基因表达异常。
表3测试样品对Clock基因表达的影响(16h的表达倍数)
测试样品对Clock基因表达的影响见表3。结果显示，本发明的肽(1)和肽(2)均能够明显提高经UVA辐射后的细胞中Clock基因的表达。由此可知，本发明的肽能够激活皮肤昼夜节律周期调节基因及Clock等相关蛋白的表达，调节皮肤昼夜节律，再同步皮肤细胞生物钟，增强细胞活力，修复受损细胞，缓解皮肤衰老迹象，在化妆品或医药领域具有广泛 应用，亦可用于制备Clock蛋白激活剂。
肽(1)和肽(2)的不同之处在于，前者的肽链C末端为-OH的形式，后者肽链C末端为酰胺化(-NH2)的形式。实验表明，本发明的肽，不管其肽链C末端是-NH2还是-OH，均具有相同的功效，可用于抗衰、修护。
综上所述，本发明的肽能够提高细胞活性、促进细胞增殖、促进胶原蛋白生成、增加皮肤弹性、提高皮肤紧致度、预防或修复皮肤老化或光老化、促进PER1蛋白和Clock基因表达、恢复昼夜节律、再同步皮肤细胞生物钟、修复受损细胞、延缓皮肤衰老，亦可用于制备PER1蛋白激活剂或Clock蛋白激活剂，在化妆品或医药领域具有广泛的应用。
实施例10含肽(1)的爽肤水的制备
将尿囊素、甘油用水溶解，加热至85℃，保温30分钟；将PEG-7甘油椰油酸酯、肽(1)用水溶解；上述溶液冷却后混合，搅拌均匀，得混合溶液；将丙二醇、防腐剂、香精依次加入上述混合溶液，加水搅拌均匀，即得。
实施例11含肽(2)的精华液的制备

将处方量的透明质酸钠加入水中，搅拌使混合均匀，然后加热到80～85℃，保温搅拌使其分散均匀。温度降到40℃以下，加入甘油、芦芭胶、肽(2)、维生素C、辛甘醇和1,2-己二醇，搅拌均匀。用15％三乙醇胺调溶液的pH值至5.5左右，即得。
实施例12含肽(9)的脂质体的制备
将二棕榈酰基磷脂酰胆碱称重且溶于氯仿。于真空下蒸发溶剂，直至得到磷脂薄层，将此层于55℃以所需浓度的肽水溶液处理而水化，得到多室脂质体。将多室脂质体通过高压均质处理，得到尺寸更加小而均一的单室脂质体。
以上内容是结合具体的优选实施方式对本发明所做的进一步详细的说明，但是不表示本发明的具体实施是局限于这些说明。对于本发明所属领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或是替换，都应视为属于本发明的保护范围。

Claims (17)

  1. 式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，
    R1-Ser-Pro-X1-Gln-R2   (I)
    式(I)中，
    X1选自：-Gly-、-Asn-、-Ala-、-Ile-、-Val-、-Ser-、-Thr-或-Met-；
    R1选自：H或R3-CO-，R3选自：取代的或未取代的烷基、取代的或未取代的烯基；
    R2选自：-NR4R5或-OR4，其中R4和R5彼此独立地选自：H、取代的或未取代的烷基、取代的或未取代的烯基；
    所述烷基是指具有1-24个碳原子(可选具有1-16个碳原子；可选具有1-14个碳原子；可选具有1-12个碳原子；可选具有1、2、3、4、5、或6个的碳原子)的饱和脂肪族直链或支链的烷基；可选选自：甲基、乙基、异丙基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十二烷基、十四烷基、十六烷基、十八烷基、2-乙基己基、2-甲基丁基、或5-甲基己基；
    所述烯基是指具有2-24个碳原子(可选具有2-16个碳原子；可选具有2-14个碳原子；可选具有2-12个碳原子；可选具有2、3、4、5、或6个碳原子)的直链或支链烯基；所述烯基具有一个或多个碳-碳双键，可选具有1、2或3个共轭或非共轭的碳-碳双键；所述烯基是通过一个单键而结合至分子的其余部分；可选选自：乙烯基、油烯基、或亚油烯基；
    可选地，所述“取代的烷基”、“取代的烯基”中的取代基选自C1-C4烷基；羟基；C1-C4烷氧基；氨基；C1-C4氨基烷基；C1-C4羰氧基；C1-C4氧基羰基；卤素(如氟、氯、溴、以及碘)；氰基；硝基；叠氮化物；C1-C4烷基磺酰基；硫醇；C1-C4烷硫基；C6-C30芳氧基如苯氧基；-NRb(C＝NRb)NRbRc，其中Rb和Rc是独立地选自：H、C1-C4烷基、C2-C4烯基、C2-C4炔基、C3-C10环烷基、C6-C18芳基、C7-C17芳烷基、具有三至十元的杂环基、或氨基的保护基。
  2. 根据权利要求1所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，R1选自：H、乙酰基、叔-丁酰基、己酰基、2-甲基己酰基、辛酰基、癸酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基或亚油酰基；R4、R5彼此独立地选自：H、甲基、乙基、己基、十二烷基或十六烷基；
    可选地，R1选自H、乙酰基、肉豆蔻酰基或棕榈酰基；R4是H并且R5选自：H、甲基、 乙基、己基、十二烷基或十六烷基；
    具体地，R1是H或乙酰基；R2是-OH或-NH2。
  3. 根据权利要求1所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，选自下列肽(1)-(32)：
    (1)H-Ser-Pro-Gly-Gln-OH；
    (2)H-Ser-Pro-Gly-Gln-NH2；
    (3)Ac-Ser-Pro-Gly-Gln-OH；
    (4)Ac-Ser-Pro-Gly-Gln-NH2；
    (5)H-Ser-Pro-Ala-Gln-OH；
    (6)H-Ser-Pro-Ala-Gln-NH2；
    (7)Ac-Ser-Pro-Ala-Gln-OH；
    (8)Ac-Ser-Pro-Ala-Gln-NH2；
    (9)H-Ser-Pro-Asn-Gln-NH2；
    (10)H-Ser-Pro-Asn-Gln-OH；
    (11)Ac-Ser-Pro-Asn-Gln-OH；
    (12)Ac-Ser-Pro-Asn-Gln-NH2；
    (13)H-Ser-Pro-Ile-Gln-OH；
    (14)H-Ser-Pro-Ile-Gln-NH2；
    (15)Ac-Ser-Pro-Ile-Gln-OH；
    (16)Ac-Ser-Pro-Ile-Gln-NH2；
    (17)H-Ser-Pro-Val-Gln-OH；
    (18)H-Ser-Pro-Val-Gln-NH2；
    (19)Ac-Ser-Pro-Val-Gln-OH；
    (20)Ac-Ser-Pro-Val-Gln-NH2；
    (21)H-Ser-Pro-Ser-Gln-OH；
    (22)H-Ser-Pro-Ser-Gln-NH2；
    (23)Ac-Ser-Pro-Ser-Gln-OH；
    (24)Ac-Ser-Pro-Ser-Gln-NH2；
    (25)H-Ser-Pro-Thr-Gln-OH；
    (26)H-Ser-Pro-Thr-Gln-NH2；
    (27)Ac-Ser-Pro-Thr-Gln-OH；
    (28)Ac-Ser-Pro-Thr-Gln-NH2；
    (29)H-Ser-Pro-Met-Gln-OH；
    (30)H-Ser-Pro-Met-Gln-NH2；
    (31)Ac-Ser-Pro-Met-Gln-OH；
    (32)Ac-Ser-Pro-Met-Gln-NH2。
  4. 根据权利要求3所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，选自肽(1)、肽(2)、肽(3)、肽(4)、肽(9)、肽(10)、肽(11)、肽(12)；具体地，
    (1)H-Ser-Pro-Gly-Gln-OH；
    (2)H-Ser-Pro-Gly-Gln-NH2；
    (3)Ac-Ser-Pro-Gly-Gln-OH；
    (4)Ac-Ser-Pro-Gly-Gln-NH2；
    (9)H-Ser-Pro-Asn-Gln-NH2；
    (10)H-Ser-Pro-Asn-Gln-OH；
    (11)Ac-Ser-Pro-Asn-Gln-OH；
    (12)Ac-Ser-Pro-Asn-Gln-NH2。
  5. 根据权利要求1-4任一项所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，
    所述美容上可接受的盐或药学上可接受的盐包括式(I)所示的肽的金属盐，所述金属包括：锂、钠、钾、钙、镁、锰、铜、锌或铝；
    可选地，所述美容上可接受的盐或药学上可接受的盐包括式(I)所示的肽与有机碱形成的盐，所述有机碱包括：乙胺、二乙胺、乙二胺、乙醇胺、二乙醇胺、精氨酸、赖氨酸、组氨酸或哌嗪；
    可选地，所述美容上可接受的盐或药学上可接受的盐包括式(I)所示的肽与无机酸或有机酸形成的盐，所述有机酸包括：乙酸、柠檬酸、乳酸、丙二酸、马来酸、酒石酸、延胡索酸、苯甲酸、天冬氨酸、谷氨酸、琥珀酸、油酸、三氟乙酸、草酸、扑酸或葡萄糖酸；
    可选地，所述无机酸包括：盐酸、硫酸、硼酸或碳酸。
  6. 一种美容或药用组合物，其特征在于，包括有效量的权利要求1-5任一项所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或 其药学上可接受的盐，以及至少一种赋形剂和任选的美容上或药学上可接受的佐剂；
    可选地，所述佐剂选自：胶原合成刺激剂、调节PGC-1α合成的剂、调节PPARγ的活性的剂、增加或减少脂肪细胞的甘油三酸酯含量的剂、刺激或延迟脂肪细胞分化的剂、脂解剂或刺激脂肪分解的剂、溶脂剂、生脂剂、乙酰胆碱受体聚集的抑制剂、抑制肌肉收缩的剂、抗胆碱能试剂、弹性蛋白酶抑制剂、基质金属蛋白酶抑制剂、黑色素合成刺激或抑制剂、增白剂或脱色剂、促色素沉着剂、自晒黑剂、抗老化剂、NO-合酶抑制剂、5α-还原酶抑制剂、赖氨酰羟化酶和/或脯氨酰羟化酶的抑制剂、抗氧化剂、自由基清除剂和/或抗大气污染的剂、活性羰基类物质清除剂、抗糖化剂、抗组胺剂、抗病毒剂、抗寄生虫剂、乳化剂、润肤剂、有机溶剂、液体推进剂、皮肤调理剂、保留水分的物质、α羟基酸、β羟基酸、增湿剂、表皮水解酶、维生素、氨基酸、蛋白质、色素、染料、生物聚合物、胶凝聚合物、增稠剂、表面活性剂、软化剂、粘合剂、防腐剂、抗皱剂、能够减少或治疗下眼袋的剂、去角质剂、抗微生物剂、灭菌剂、抑菌剂、刺激真皮或表皮大分子的合成和/或能够抑制或预防它们的降解的剂、刺激弹性蛋白合成的剂、刺激核心蛋白聚糖合成的剂、刺激层粘连蛋白合成的剂、刺激防御素合成的剂、刺激伴侣蛋白合成的剂、刺激cAMP合成的剂、刺激HSP70合成的剂、刺激热休克蛋白合成的剂、刺激透明质酸合成的剂、刺激纤连蛋白合成的剂、刺激去乙酰化酶合成的剂、刺激脂质和角质层组分的合成的剂、神经酰胺、脂肪酸、抑制胶原降解的剂、抑制弹性蛋白降解的剂、抑制丝氨酸蛋白酶的剂、刺激成纤维细胞增殖的剂、刺激角质形成细胞增殖的剂、刺激脂肪细胞增殖的剂、刺激黑色素细胞增殖的剂、刺激角质形成细胞分化的剂、抑制乙酰胆碱酯酶的剂、皮肤松弛剂、刺激糖胺聚糖合成的剂、抗角化过度剂、粉刺溶解剂、抗银屑病剂、抗湿疹剂、DNA修复剂、DNA防护剂、稳定剂、止痒剂、用于治疗和/或护理敏感性皮肤的剂、固化剂、紧致剂、重构剂、抗拉伸纹剂、调节皮脂产生的剂、止汗剂、刺激愈合的剂、协助愈合的剂、刺激再上皮化的剂、协助再上皮化的剂、细胞因子、镇静剂、抗炎剂、麻醉剂、作用于毛细血管循环和/或微循环的剂、刺激血管生成的剂、抑制血管渗透性的剂、静脉紧张剂、作用于细胞代谢的剂、用于改善真皮-表皮接合的剂、诱导毛发生长的剂、毛发生长抑制或延缓剂、香料、螯合剂、植物提取物、精油、海洋提取物、得自生物发酵过程的剂、无机盐、细胞提取物、防晒剂、以及有效抗A和/或B紫外线的有机或无机光防护剂或其混合物。
  7. 根据权利要求6所述的美容或药用组合物，其特征在于，所述美容或药用组合物的制剂选自：霜剂、油、香膏、泡沫、洗剂、凝胶、擦剂、浆液、软膏、摩丝、粉末、杆剂、 笔剂、喷雾剂、气溶胶、胶囊剂、片剂、颗粒剂、口香糖、溶液、混悬液、乳剂、酏剂、多糖薄膜、胶冻或明胶；
    可选地，所述胶囊剂包括：软胶囊剂、硬胶囊剂，可选为明胶胶囊剂；
    可选地，所述片剂包括：糖衣片剂。
  8. 一种美容上或药学上可接受的递送系统或缓释系统，其特征在于，包含有效量的权利要求1-5任一项所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述的美容或药用组合物；
    所述美容上或药学上可接受的递送系统或缓释系统选自：脂质体、油质体、非离子型表面活性剂脂质体囊泡、醇质体、毫米胶囊、微米胶囊、纳米胶囊、纳米结构的脂质载体、海绵状物、环糊精、类脂囊泡、胶束、毫米球、微米球、纳米球、脂质球、微米乳液、纳米乳液、毫米粒子、微米粒子或纳米粒子；可选为脂质体或微米乳液，可选具有反胶束的内部结构的油包水型微米乳液。
  9. 权利要求1-5任一项所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于治疗、预防或修复皮肤老化和/或光老化的美容组合物或药物组合物中的用途。
  10. 权利要求1-5任一项所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于增加皮肤弹性和/或皮肤紧致度的美容组合物或药物组合物中的用途。
  11. 权利要求1-5任一项所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于促进胶原蛋白生成的美容组合物或药物组合物中的用途。
  12. 权利要求1-5任一项所述的式(I)所示的肽、或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于促进PER1蛋白表达、调节皮肤昼夜节律和/或修复皮肤受损细胞的美容组合物或药物组合物 中的用途。
  13. 权利要求1-5任一项所述的式(I)所示的肽，或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备PER1蛋白激活剂中的用途。
  14. 权利要求1-5任一项所述的式(I)所示的肽，或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述的美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于促进Clock基因表达和/或再同步皮肤细胞生物钟的组合物中的用途。
  15. 权利要求1-5任一项所述的式(I)所示的肽，或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述的美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备Clock蛋白激活剂中的用途。
  16. 权利要求1-5任一项所述的式(I)所示的肽，或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述的美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备化妆品中的用途。
  17. 一种化妆品，其特征在于，包含有效量的权利要求1-5任一项所述的式(I)所示的肽，或其立体异构体、或其立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐、或权利要求6或7所述的美容或药用组合物、或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统；
    可选地，所述化妆品的剂型包括膏霜、乳液、凝胶、粉剂、片剂、膜剂、气雾剂或喷雾剂。
PCT/CN2023/133632 2022-11-25 2023-11-23 具有抗衰老作用的肽及其组合物和用途 WO2024109878A1 (zh)

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CN116284241B (zh) * 2023-04-20 2023-10-31 深圳市维琪科技股份有限公司 肽及其组合物和用途
CN117164663B (zh) * 2023-08-01 2024-05-10 青岛双元泰和药业有限公司 多肽化合物、其组合物及其用途
CN117088941B (zh) * 2023-10-20 2023-12-19 深圳市维琪科技股份有限公司 一种三肽衍生物及其组合物和用途
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CN117304265B (zh) * 2023-11-29 2024-01-30 深圳市维琪科技股份有限公司 一种皮肤护理多肽、组合物及其应用

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