CN115594735A - Peptide with anti-aging effect, and cosmetic composition or medicinal composition and application thereof - Google Patents

Peptide with anti-aging effect, and cosmetic composition or medicinal composition and application thereof Download PDF

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CN115594735A
CN115594735A CN202211490952.7A CN202211490952A CN115594735A CN 115594735 A CN115594735 A CN 115594735A CN 202211490952 A CN202211490952 A CN 202211490952A CN 115594735 A CN115594735 A CN 115594735A
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ser
pro
gln
peptide
agents
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CN115594735B (en
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丁文锋
肖玉
赵文豪
孙新林
陈雪
观富宜
彭晏
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SHENZHEN WINKEY MEDICAL RESEARCH DEVELOPMENT CO LTD
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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

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  • Peptides Or Proteins (AREA)
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Abstract

The invention provides a compound of formula (I) R 1 ‑Ser‑Pro‑X 1 ‑Gln‑R 2 Or a stereoisomer thereof, or a mixture of stereoisomers thereof, 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 the circadian rhythm of the skin and/or repairing damaged cells of the skinThe use of (1). The peptide of the formula (I) can regulate the skin circadian rhythm and resynchronize the skin cell biological clock by activating the expression of the skin circadian rhythm cycle regulating gene and related proteins, enhance the cell activity and repair damaged cells, thereby relieving the skin aging sign, can be used for resisting skin aging in the cosmetic or medical field, and can also be used for preparing a PER1 protein activator.

Description

Peptide with anti-aging effect, and cosmetic composition or medicinal composition and application thereof
Technical Field
The invention relates to the technical field of medicines and cosmetics, in particular to a peptide with an anti-aging effect, a cosmetic composition or a medicinal composition and application thereof.
Background
The skin, as the first physiological defense line of the human body, is constantly subjected to the external environment while protecting tissues and organs from chemical, mechanical, physical and pathogenic microorganisms. In recent years, the number of patients with skin diseases has increased year by year due to environmental attacks such as UV radiation and environmental pollution, and aging of the skin has also been accelerated. Environmental attacks can damage the DNA of skin cells and affect their circadian rhythm.
Circadian rhythm is the variation of vital activity in a 24-hour cycle, also called biological clock, which regulates many of our metabolic, physiological activities and behaviors. There is a lot of evidence that disorders and disturbances of circadian rhythms have a great negative effect on human health, including jet lag, fatigue syndrome caused by shifts of night, sleep disorders, skin diseases, etc. In addition, many diseases that seriously threaten human health, such as cardiovascular and cerebrovascular diseases and tumors, are also associated with disorders of circadian rhythms. However, with the development of modern civilization, more and more people have gone away from doing business and from the lifestyle of the sunset and rest, resulting in an increasingly common imbalance of circadian rhythms.
The independent biological clock system is also present in keratinocytes, melanocytes, fibroblasts of skin cells. 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, germs, etc., which vary in a one-day cycle. Skin to adapt to this environment, various physiological functions are regulated by activating the biological clock present in the skin. Experiments show that the proliferation and differentiation of skin cells, water loss, sebum generation, temperature, pH and wrinkle generation all change in a 24-hour period. For example, the proliferation and differentiation of skin cells occur through a series of processes in a 24-hour day cycle. Skin is easily damaged by uv exposure during the day, so that differentiation of skin cells mainly occurs between evening and early morning. By separating these series of processes temporally, the skin is protected from the harmful environment. However, when the rhythm of this process is disturbed, a desired physiological response cannot be produced at an appropriate time, and thus, the skin circadian rhythm is disturbed, which accelerates the skin damage.
In recent years, it has been found that genes associated with the circadian rhythm of the natural body include a Clock gene and a PER1 gene, both of which encode proteins (Clock protein and PER1 protein) that regulate the circadian rhythm. PER1 is one of the core components of biological clocks for regulating the circadian cycle of skin, and is a member of a Period gene family, the Period gene family is composed of PER1, PER2 and PER3, the genes are involved in the metabolism, the motor activity, the behavior and the like of the organism, and PER1 plays a core role. The Clock gene and the PER1 gene are also present in skin cells, and the expression of the PER1 gene induces a cellular activity program associated with biological processes (e.g., repair) that occur overnight. The DNA replication, DNA repair mechanisms and cell division of epidermal progenitor/stem cells exhibit pronounced circadian rhythm characteristics. Skin cells exposed to environmental attack for prolonged periods of time typically exhibit reduced, irregular or asynchronous expression of the Clock gene or the PER1 gene, disrupting the normal circadian rhythm in the skin cells. The circadian rhythm and synchronicity of normal cells are disturbed over time, and the skin balance is disrupted, thereby accelerating the natural aging process of the skin, causing skin damage, and causing problems of wrinkles, fine lines, skin laxity, uneven pigmentation, age spots, color spots, and the like.
Through analyzing the Clock gene and the PER1 gene, the Clock gene and the PER1 gene are found to be a biological Clock core gene and a negative feedback gene which regulate the circadian cycle of skin and participate in the reaction process of regulating the skin to the environmental pressure, and the Clock gene and the PER1 gene take about 24 hours as a period, interfere with the expression and activity regulation of genetic genes and proteins in various physiological processes, so that the biological Clock meets the external environment to regulate the activity degree of physiological reaction. Regulating and controlling various activity processes of skin cells according to circadian rhythm, protect the skin from various environmental insults such as UV radiation, temperature, chemical and physical insults, and microbial infections.
Therefore, it is necessary to study an active substance capable of improving the skin circadian rhythm and activating the skin circadian rhythm control gene and related proteins, based on the mechanism of the skin circadian rhythm, and to apply the active substance to the fields of medicine and cosmetics for the purpose of alleviating skin aging.
Disclosure of Invention
The invention aims to provide an active substance which is stable, efficient, safe and non-irritant to skin, repairs damaged cells by activating the expression of a skin circadian cycle regulatory gene and related proteins, achieves an anti-aging effect, can promote the generation of collagen, increases the skin elasticity and/or the skin firmness, prevents the skin from relaxing, and has wide application in the fields of medicines and cosmetics.
In view of this, 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）
in the formula (I), the compound is shown in the specification,
X 1 selected from: -Gly-, -Asn-, -Ala-, -Ile-, -Val-, -Ser-, -Thr-or-Met-;
R 1 selected from: h or R 3 -CO-，R 3 Selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
R 2 selected from: -NR 4 R 5 OR-OR 4 Wherein R is 4 And R 5 Independently of one another, selected from: H. substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
the alkyl group refers to a saturated aliphatic linear or branched alkyl group having 1 to 24 carbon atoms (optionally having 1 to 16 carbon atoms; optionally having 1 to 14 carbon atoms; optionally having 1 to 12 carbon atoms; optionally having 1,2, 3, 4, 5, or 6 carbon atoms); optionally 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 means a straight or branched chain alkenyl group having 2 to 24 carbon atoms (optionally having 2 to 16 carbon atoms; optionally having 2 to 14 carbon atoms; optionally having 2 to 12 carbon atoms; optionally having 2, 3, 4, 5, or 6 carbon atoms); said alkenyl having one or more carbon-carbon double bonds, optionally 1,2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bonded to the remainder of the molecule by a single bond; optionally selected from: vinyl, oleyl, or linoleyl;
alternatively, the substituents in the "substituted alkyl", "substituted alkenyl" are selected from C 1 -C 4 An alkyl group; a hydroxyl group; c 1 -C 4 An alkoxy group; an amino group; c 1 -C 4 An aminoalkyl group; c 1 -C 4 A carbonyloxy group; c 1 -C 4 An oxycarbonyl group; halogen (e.g., fluorine, chlorine, bromine, and iodine); a cyano group; a nitro group; an azide; c 1 -C 4 An alkylsulfonyl group; a thiol; c 1 -C 4 An alkylthio group; c 6 -C 30 Aryloxy groups such as phenoxy; -NR b (C=NR b )NR b R c Wherein R is b And R c Is independently selected from: H. c 1 -C 4 Alkyl radical, C 2 -C 4 Alkenyl radical, C 2 -C 4 Alkynyl, C 3 -C 10 Cycloalkyl, C 6 -C 18 Aryl radical, C 7 -C 17 An aralkyl group, a heterocyclic group having three to ten members, or a protecting group of an amino group.
Alternatively, R 1 Selected from: H. acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl or linoleoyl; r 4 、R 5 Independently of one another, selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;
alternatively, R 1 Selected from H, acetyl, myristoyl or palmitoyl; r 4 Is H and R 5 Selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;
specifically, R 1 Is H or acetyl; r 2 is-OH or-NH 2 。
Alternatively, 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, is selected from the following peptides (1) to (32):
（1）H-Ser-Pro-Gly-Gln-OH；
（2）H-Ser-Pro-Gly-Gln-NH 2 ；
（3）Ac-Ser-Pro-Gly-Gln-OH；
（4）Ac-Ser-Pro-Gly-Gln-NH 2 ；
（5）H-Ser-Pro-Ala-Gln-OH；
（6）H-Ser-Pro-Ala-Gln-NH 2 ；
（7）Ac-Ser-Pro-Ala-Gln-OH；
（8）Ac-Ser-Pro-Ala-Gln-NH 2 ；
（9）H-Ser-Pro-Asn-Gln-NH 2 ；
（10）H-Ser-Pro-Asn-Gln-OH；
（11）Ac-Ser-Pro-Asn-Gln-OH；
（12）Ac-Ser-Pro-Asn-Gln-NH 2 ；
（13）H-Ser-Pro-Ile-Gln-OH；
（14）H-Ser-Pro-Ile-Gln-NH 2 ；
（15）Ac-Ser-Pro-Ile-Gln-OH；
（16）Ac-Ser-Pro-Ile-Gln-NH 2 ；
（17）H-Ser-Pro-Val-Gln-OH；
（18）H-Ser-Pro-Val-Gln-NH 2 ；
（19）Ac-Ser-Pro-Val-Gln-OH；
（20）Ac-Ser-Pro-Val-Gln-NH 2 ；
（21）H-Ser-Pro-Ser-Gln-OH；
（22）H-Ser-Pro-Ser-Gln-NH 2 ；
（23）Ac-Ser-Pro-Ser-Gln-OH；
（24）Ac-Ser-Pro-Ser-Gln-NH 2 ；
（25）H-Ser-Pro-Thr-Gln-OH；
（26）H-Ser-Pro-Thr-Gln-NH 2 ；
（27）Ac-Ser-Pro-Thr-Gln-OH；
（28）Ac-Ser-Pro-Thr-Gln-NH 2 ；
（29）H-Ser-Pro-Met-Gln-OH；
（30）H-Ser-Pro-Met-Gln-NH 2 ；
（31）Ac-Ser-Pro-Met-Gln-OH；
（32）Ac-Ser-Pro-Met-Gln-NH 2 。
optionally, selected from peptide (1), peptide (2), peptide (3), peptide (4), peptide (9), peptide (10), peptide (11), peptide (12); in particular, the amount of the solvent to be used,
（1）H-Ser-Pro-Gly-Gln-OH；
（2）H-Ser-Pro-Gly-Gln-NH 2 ；
（3）Ac-Ser-Pro-Gly-Gln-OH；
（4）Ac-Ser-Pro-Gly-Gln-NH 2 ；
（9）H-Ser-Pro-Asn-Gln-NH 2 ；
（10）H-Ser-Pro-Asn-Gln-OH；
（11）Ac-Ser-Pro-Asn-Gln-OH；
（12）Ac-Ser-Pro-Asn-Gln-NH 2 。
the peptides of formula (I) of the invention may exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids comprised therein may have the L-, D-configuration or, independently of one another, be racemic. It is thus possible to obtain isomeric mixtures, as well as racemic or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and what isomer or isomeric mixture is present. Preferred structures of the peptides of formula (I) of the invention are pure isomers, i.e. enantiomers or diastereomers.
For example, when the present invention is said to be-Pro-then, it is understood that-Pro-is selected from-L-Pro-, -D-Pro-, or a mixture of both, is racemic or non-racemic. The preparation methods described in this document enable one of ordinary skill in the art to obtain each stereoisomer of the peptides of the invention by selecting amino acids with the correct configuration.
The invention also includes all suitable isotopic variants of the peptide of formula (I). Isotopic variants of these peptides of the invention are understood herein to mean compounds which: wherein at least one atom is replaced within a peptide of the invention by another atom of the same atomic number, but having an atomic mass different from the atomic mass usually or predominantly present in nature. Examples of isotopes that can be incorporated into the peptides of the invention are: of hydrogen, carbon, nitrogen, oxygen or sulfur, e.g. 2 H (deuterium), 3 H (tritium), 13 C、 14 C、 15 N、 17 O、 18 O、 33 S、 34 S、 35 S or 36 And S. Specific isotopic variations of the peptides of the invention (particularly those into which one or more radioisotopes have been incorporated) may be advantageous, for example, in examining the mechanism of action or the distribution of active compounds in vivo; due to the relatively simple manufacturability and detectability, in particular 3 H or 14 C-isotopically labelled compounds are suitable for this purpose. In addition, incorporation of isotopes (e.g., deuterium) may be due to greater metabolic stability of the compoundsTo produce specific therapeutic benefits, such as an increase in the in vivo half-life or a reduction in the required active dose; thus, in certain cases, such modifications of the peptides of the invention may also constitute preferred embodiments of the invention. Isotopic variants of the peptides of the invention can be prepared by methods known to those skilled in the art, for example by methods described further below and in the examples, by using the respective reagents and/or the corresponding isotopic modifications of the starting materials.
In addition, the present invention also includes prodrugs of the peptides of the present invention. The term "prodrug" means herein a compound that: which may be biologically active or inactive per se, but which react (e.g. metabolize or hydrolyze) during their residence time in the body to produce the peptides of the invention.
The term "cosmetically or pharmaceutically acceptable salt" refers to a salt that is recognized for use in animals, and more specifically humans, and includes metal salts of peptides represented by formula (I), including, but not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, aluminum, or the like; comprising a salt of a peptide of formula (I) with an organic base, including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, piperazine, or the like; comprising a salt of a peptide of formula (I) with an inorganic or organic acid, including, but not limited to: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid (pamoate), gluconic acid, or the like; optionally, the inorganic acid comprises: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.
The synthesis of the peptide of formula (I) of the present invention, or a stereoisomer thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, can be carried out according to conventional methods known in the art, such as solid phase synthesis methods, liquid phase synthesis methods, or methods combining solid phase and liquid, 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.
For example, a method of obtaining a peptide of formula (I) comprises the steps of:
-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 protected or solid support bound C-terminus;
-elimination of the group protecting the N-terminus;
-repeating the coupling sequence and eliminating the group protecting the N-terminus until the desired peptide sequence is obtained;
-elimination of the group protecting the C-terminus or cleavage from the solid support.
Preferably, the C-terminus is bound to a solid support and the method is carried out on a solid phase comprising coupling the amino acid having a protected N-terminus and a free C-terminus with the amino acid having a free N-terminus and a C-terminus bound to a polymeric support; elimination of the group protecting the N-terminus; and repeating this sequence as many times as necessary to thereby obtain a peptide having a desired length, followed by cleavage of the synthesized peptide from the original polymer support.
The functional groups of the side chains of these amino acids remain sufficiently protected with temporary or permanent protecting groups throughout the synthesis and may be deprotected simultaneously or orthogonally to the process of cleaving the peptide from the polymeric support.
Alternatively, solid phase synthesis may be performed by a pooling strategy (convergent strategy) of coupling di-or tripeptides to a polymeric support or to di-or amino acids previously bound to a polymeric support.
The functional groups at the N-terminus and C-terminus can be subsequently modified by deprotecting the termini and/or cleaving the peptide from the polymeric support in a non-defined order using standard conditions and methods known in the art. The peptide of formula (I) bound to the polymeric support may be optionally modified at the N-terminus and C-terminus, or after the peptide has been cleaved from the polymeric support.
In another aspect of the present invention, there is provided a cosmetic or pharmaceutical composition comprising an effective amount of a peptide of formula (I) as described above, or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically or pharmaceutically acceptable salt thereof, together with at least one excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant;
optionally, the adjuvant is selected from: stimulators of collagen synthesis, agents for modulating PGC-1 alpha synthesis, agents for modulating the activity of PPAR gamma, agents for increasing or decreasing the triglyceride content of adipocytes, agents for stimulating or delaying adipocyte differentiation, lipolytic or lipolysis agents, lipid solubilizing agents, lipid producing agents, inhibitors of acetylcholine receptor aggregation, agents for inhibiting muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulators or inhibitors, whitening or depigmenting agents, pro-pigmentation agents, self-tanning agents, anti-ageing agents, NO-synthase inhibitors, 5 alpha-reductase inhibitors, lysyl hydroxylase and/or prolyl hydroxylase inhibitors, antioxidants, free radical scavengers and/or anti-atmospheric pollution agents, active carbonyl scavenger, anti-glycation agents, anti-histamine agents, anti-viral agents antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioners, moisture retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, emollients, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating the lower eye pouch, exfoliating agents, antimicrobial agents, disinfectants, bacteriostats, agents that stimulate the synthesis of dermal or epidermal macromolecules and/or that inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate decorin synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, agents that stimulate chaperonin synthesis, agents that stimulate cAMP synthesis, agents that stimulate HSP70 synthesis, <xnotran> , , , , , , , , , , , , , , , , , , , , , , DNA , DNA , , , / , , , , , , , , , , , , , , , / , , , , , - , , , , , , , , , , , , A / B . </xnotran>
Optionally, the formulation of the cosmetic or pharmaceutical composition is selected from: creams, oils, balms, foams, lotions, gels, liniments, serums, ointments, mousses, powders, sticks, pencils, sprays, aerosols, capsules, tablets, granules, chewing gums, solutions, suspensions, emulsions, elixirs, polysaccharide films, jellies or gelatins;
optionally, the capsule comprises: soft capsule, hard capsule, optionally gelatin capsule;
optionally, the tablet comprises: sugar-coated tablets.
The peptides of the invention have variable solubility in water, depending on the nature of their sequence or any possible modification in the N-and/or C-terminus. The peptides of the invention may thus be incorporated into the composition by aqueous solution and those which are insoluble in water may be dissolved in conventional cosmetically or pharmaceutically acceptable solvents such as, and without limitation, 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 this invention to be administered and their dosage will depend on a number of factors, including age, the state of the patient, the severity of the condition or disease, the route and frequency of administration, and the specific nature of the peptide to be used.
By "cosmetically or pharmaceutically effective amount" is meant a non-toxic but sufficient amount of one or more peptides of the invention to provide the desired effect. Using the peptide of the invention in a cosmetically or pharmaceutically effective concentration to obtain the desired effect in the cosmetic or pharmaceutical composition of the invention; in a preferred form, it 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.
In another aspect of the present invention, there is provided a cosmetically or pharmaceutically acceptable delivery system or sustained release system for achieving better penetration of the active principle and/or for improving its pharmacokinetic and pharmacodynamic properties, comprising an effective amount of a peptide of formula (I) above, or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically or pharmaceutically acceptable salt thereof, or a cosmetically or pharmaceutically acceptable composition of the above.
The term "delivery system" refers to a diluent, adjuvant, excipient or carrier to be administered with the peptide of the invention, selected from: water, oil or surfactant, including those of petroleum, animal, vegetable, or synthetic origin, such as, and not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynol, poloxamers, polyoxyethylene, polyethylene glycols, dextrose, glycerol, digitonin, and the like. Diluents which can be used in different delivery systems in which the peptides of the invention can be administered are known to the person skilled in the art.
The term "sustained release" is used in the conventional sense to refer to a delivery system of a compound that provides for gradual release of the compound over a period of time, and preferably, but not necessarily, has a relatively constant level of compound release over the entire period of time.
Examples of delivery systems or sustained release systems are liposomes, oleosomes, non-ionic surfactant liposome vesicles, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, liposomes, micelles, nanospheres, microspheres, nanospheres, lipid globules, microemulsions, nanoemulsions, nanoparticles, microparticles or nanoparticles. Preferred delivery systems or sustained release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions with an internal structure of reverse micelles.
Sustained release systems can be prepared by methods known in the art and can be administered, for example, by: by topical or transdermal administration, including adhesive patches, non-adhesive patches, occlusive patches, and microelectronic patches; or by systemic administration such as, 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 relatively constant amounts of the peptides of the invention. The amount of peptide included in the sustained release system will depend on, for example, the site to which the composition is to be administered, the kinetics and duration of release of the peptide of the invention, and the nature of the condition, disorder and/or disease to be treated and/or cared for.
In another aspect of the present invention, there is provided a use 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 above, or the cosmetically or pharmaceutically acceptable delivery system or sustained release system above, in the manufacture of a cosmetic composition or pharmaceutical composition for the treatment, prevention or repair of skin aging and/or photoaging.
In another aspect of the present invention, there is provided a use of the peptide of 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 above, or the cosmetically or pharmaceutically acceptable delivery system or sustained release system above, in the preparation of a cosmetic or pharmaceutical composition for increasing skin elasticity and/or skin firmness.
In another aspect of the present invention, there is provided a use of the 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, or the cosmetic or pharmaceutical composition, or the cosmetically or pharmaceutically acceptable delivery system or sustained release system for preparing a cosmetic composition or a pharmaceutical composition for promoting collagen production.
In another aspect of the present invention, there is provided a use 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 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 the expression of PER1 protein, regulating the circadian rhythm of the skin, and/or repairing damaged cells of the skin.
In another aspect of the present invention, there is provided a use 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 above, or the cosmetically or pharmaceutically acceptable delivery system or sustained release system above, in the preparation of a PER1 protein activator.
To facilitate an understanding of the invention, some terms and expressions used in the invention are explained in the following meanings:
in the present invention, the term "skin" is understood to mean the layers that make up it, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both endpoints being inclusive. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and/or adipocytes, among others. In the present invention, the term "skin" includes the scalp.
The term "treatment" refers to the administration of a peptide according to the invention to reduce or eliminate a disease or disorder, or to reduce or eliminate one or more symptoms associated with such a disease or disorder. The term "treating" also encompasses the ability to reduce or eliminate the physiological consequences of the disease or disorder.
The term "prevention" refers to the ability of a peptide of the invention to prevent, delay, or hinder the appearance or progression of a disease or disorder prior to its appearance.
The term "repair" refers to the ability of a peptide of the invention to improve, alleviate or restore its original shape after the appearance of a disease or disorder.
The term "aging" refers to the changes that skin undergoes with age (natural aging), or by exposure to sunlight (photoaging) or to environmental contaminants, such as chemical soils or contaminants, tobacco smoke, and the like, and includes all externally visible and/or tactile changes, such as, and not limited to: the development of discontinuities in the skin (e.g., wrinkles, fine lines, expression lines, stretch lines, streaks, furrows, irregularities or roughness, increased pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of restitution, loss of resiliency), sagging of the skin (e.g., sagging of the cheeks, the appearance of bags under the eyes, or the appearance of double chin, etc.), changes in the color of the skin (e.g., scarring, reddening, bags under the eyes, or the appearance of hyperpigmented areas such as age spots or freckles, etc.), abnormal differentiation, hyperkeratinization, elastosis, keratosis, hair loss, cellulite-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 adjacent to the skin.
The term "photoaging" refers to the premature aging of skin due to prolonged exposure of the skin to ultraviolet radiation, which exhibits the same physiological characteristics as natural aging, such as, and not limited to: laxity, sagging, color change or pigmentation irregularities, abnormalities and/or hyperkeratinization.
In the present specification, the abbreviations used for amino acids follow the rules specified in the European journal of biochemistry (Eur.J.Biochem.1984, 138: 9-37) by the IUPAC-IUB Commission on Biochemical Nomenclature.
Thus, for example, gly represents NH 2 -CH 2 -COOH, gly-represents NH 2 -CH 2 -CO-, -Gly represents-NH-CH 2 -COOH, and-Gly-represents-NH-CH 2 -CO-. Thus, the hyphen representing a peptide bond eliminates the OH in the 1-carboxyl group of the amino acid (represented herein in the conventional non-ionized form) when located to the right of the symbol, and eliminates the H in the 2-amino group of the amino acid when located to the left of the symbol; both modifications can be applied to the same symbol (see table 1).
TABLE 1 structures of amino acid residues and their single-letter and three-letter abbreviations
Figure DEST_PATH_IMAGE001
The abbreviation "Ac-" is used herein to denote acetyl (CH) 3 -CO-）。
Compared with the prior art, the invention has the advantages that:
1. the peptide disclosed by the invention is convenient to synthesize, high in safety, and capable of increasing the activity of skin fibroblasts and promoting the generation of collagen, so that the skin elasticity and/or the skin firmness are increased, and the peptide can be used for preventing and even treating skin relaxation and treating, preventing and/or repairing skin aging or photoaging.
2. The peptide can promote the expression of PER1 protein, increase the content of PER1 protein and participate in the regulation of the skin circadian rhythm cycle, thereby recovering the circadian rhythm and resynchronizing the skin cell biological clock, and can be used for repairing damaged cells of the skin and relieving the skin aging signs; can also be used for preparing PER1 protein activator.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the description of the present invention will be briefly introduced below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without inventive labor.
FIG. 1 is the peptide (2) H-Ser-Pro-Gly-Gln-NH 2 (formula C) 15 H 26 N 6 O 6 ) Mass spectrum, [ M + H] + The mass-to-charge (m/z) ratio of the excimer ion peak was 387.2045, and the molecular weight was 386.20 as determined by mass spectrometry.
FIG. 2 is the peptide (9) H-Ser-Pro-Asn-Gln-NH 2 (formula C) 17 H 29 N 7 O 7 ) Mass spectrum, [ M + H] + The mass-to-charge (m/z) ratio of the excimer ion peak was 444.2271, and the molecular weight was 443.23 as determined by mass spectrometry.
FIG. 3 is a graph showing the effect of test samples on HaCaT cell activity.
FIG. 4 is a graph showing the effect of test samples on HSF cell activity.
FIG. 5 is a graph showing the effect of test samples on HSF cell activity after UV treatment.
FIG. 6 is a graph showing the results of the effect of the test samples on collagen content.
FIG. 7 is a graph showing the effect of the test samples on the PER1 protein content.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
It is to be noted that, in the present invention, the abbreviations used for the amino acids follow the rules specified by the Committee for Biochemical nomenclature of IUPAC-IUB in Eur.J. biochem. (1984) 138:9-37 and J.chem. (1989) 264: 633-673.
Amide Resin: a starting resin for polypeptide synthesis; fmoc-Linker:4- [ (2,4-dimethoxyphenyl) (Fmoc-amino) methyl]Phenoxyacetic acid; DMF: n, N-dimethylformamide; HOBt: 1-hydroxybenzotriazole; DIC: diisopropylcarbodiimide; DIPEA: diisopropylethylamine; ac of 2 O: acetic anhydride; piperidine: piperidine; TFA: trifluoroacetic acid; and (3) TIS: triisopropylsilane; ser: serine; pro: (ii) proline; gly: glycine; asn: asparagine; gln: (ii) glutamine; fmoc: 9-fluorenylmethoxycarbonyl; tBu: a tertiary butyl group; trt: a trityl group.
Example 1H-Ser-Pro-Gly-Gln-NH 2 Preparation of
1.1 Preparation of Fmoc-Linker-Amide Resin
Weighing 5g of Amide Resin in a solid phase synthesis reaction column, swelling with DMF, washing the Resin, and pumping away the solvent.
7.1g of Fmoc-Linker and 4.4g of HOBt were weighed into a dry flask. Dissolving with DMF solvent, cooling in ice water bath for 10min, and activating with DIC for 10min to avoid water vapor.
And adding the activated Fmoc-Linker into the swelled resin for reaction for 2.5h, pumping reaction liquid, washing the resin, and pumping the solvent.
Continuing to add Ac 2 And end-capping with DIPEA for 1.5h. The resin was washed and the solvent was removed.
1.2 Fmoc removal
Fmoc-Linker-Amide Resin was Fmoc-removed twice with 20% piperidine/DMF for 10min each time, sampled for K test, and developed dark blue. The resin was washed 7 times with DMF and the solvent was removed by suction.
1.3 Feeding reaction
11.7g of Fmoc-Gln (Trt) -OH and 4.4g of HOBt were weighed into a dry flask, dissolved by adding DMF, sealed and placed in a freezer at-18 ℃ for 30min. 6.3mL DIC was added for 3min to avoid water vapor. Adding the activated amino acid into the deprotected resin to react for 2h, and pumping the reaction solution. The K detection resin is colorless and transparent, which indicates that the reaction is complete.
The N-terminal Fmoc group was deprotected and 5.7g of activated Fmoc-Gly-OH was coupled to the peptidyl resin in the presence of 4.4g of HOBt and 6.3mL of DIC using DMF as solvent and the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. In each coupling, 7.3g of Fmoc-Pro-OH followed by 8.3g of Fmoc-Ser (tBu) -OH were coupled sequentially in the presence of 4.4g of HOBt and 6.3mL of DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was pumped away.
Deprotection of Fmoc group at N-terminal of peptidyl resin, fmoc removal twice with 20% piperidine/DMF for 10min each time, sampling for K detection, and deep blue color development. The Resin was washed 6 times with DMF, the solvent was removed, and 12.2g of Ser (tBu) -Pro-Gly-Gln (Trt) -Linker-Amide Resin was obtained after shrinkage and drying.
1.4 Cracking
Measuring 95mL of TFA, 2.5mL of TIS and 2.5mL of water, mixing and stirring uniformly to obtain a lysate, sealing, and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether was frozen in a freezer at-18 ℃ for further use.
Weighing 12.2g of Ser (tBu) -Pro-Gly-Gln (Trt) -Linker-Amide Resin, adding into a round-bottom flask, adding the frozen lysate, and stirring for reaction for 2h. Suction filtering, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging, washing for 6 times until pH is 3-4, and vacuum drying to obtain 4.8g of H-Ser-Pro-Gly-Gln-NH 2 Crude peptide.
1.5 Purification of
4.8g of the crude peptide are weighed out in 90mL of methanol: in water (V: V = 1:2), filtration through a 0.22 μm pore size microfiltration membrane afforded a clear, clear solution which was purified by reverse phase HPLC with the purification gradients as given in the following table:
time (min) Flow rate (mL/min) A% (acetonitrile) B% (0.1% acetic acid + pure water)
0 15 0 100
10 15 1 99
20 15 2 98
Purifying the filtered sample by sample introduction, collecting the fraction, concentrating and freeze-drying to obtain the peptide (2) H-Ser-Pro-Gly-Gln-NH with the purity of 98.726% 2 。
Example 2H-Ser-Pro-Asn-Gln-NH 2 Preparation of
2.1 Preparation of Fmoc-Linker-Amide Resin
Weighing 5g of Amide Resin in a solid phase synthesis reaction column, swelling with DMF, washing the Resin, and pumping away the solvent.
7.1g of Fmoc-Linker and 4.4g of HOBt were weighed into a dry flask. Dissolving with DMF solvent, cooling in ice water bath for 10min, and activating with DIC for 10min to avoid water vapor.
And adding the activated Fmoc-Linker into the swelled resin for reaction for 2.5h, pumping out reaction liquid, washing the resin, and pumping out the solvent.
Continuing to add Ac 2 And end-capping with DIPEA for 1.5h. The resin was washed and the solvent was removed.
2.2 Fmoc removal
Fmoc-Linker-Amide Resin was Fmoc-removed twice with 20% piperidine/DMF for 10min each time, sampled for K test, and developed dark blue. The resin was washed 7 times with DMF and the solvent was removed by suction.
2.3 Feeding reaction
11.7g of Fmoc-Gln (Trt) -OH and 4.4g of HOBt were weighed into a dry flask, dissolved by adding DMF, sealed and placed in a freezer at-18 ℃ for 30min. 6.3mL DIC was added for 3min to avoid water vapor. Adding the activated amino acid into the deprotected resin to react for 2h, and pumping the reaction solution. The K detection resin is colorless and transparent, which indicates that the reaction is complete.
The N-terminal Fmoc group was deprotected and 11.5g of Fmoc-Asn (Trt) -OH after activation were coupled to the peptidyl resin in the presence of 4.4g of HOBt and 6.3mL of DIC using DMF as solvent and the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. In each coupling, 7.3g of Fmoc-Pro-OH followed by 8.3g of Fmoc-Ser (tBu) -OH were coupled sequentially in the presence of 4.4g of HOBt and 6.3mL of DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was pumped away.
Deprotection of Fmoc group at N-terminal of peptidyl resin, fmoc removal twice with 20% piperidine/DMF for 10min each time, sampling for K detection, and deep blue color development. The Resin was washed 6 times with DMF, the solvent was removed, and 16.2g of Ser (tBu) -Pro-Asn (Trt) -Gln (Trt) -Linker-Amide Resin was obtained after shrinkage and drying.
2.4 Cracking
Measuring 95mL of TFA, 2.5mL of TIS and 2.5mL of water, mixing and stirring uniformly to obtain a lysate, sealing, and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether is placed in a refrigerator at-18 ℃ for freezing for standby.
Weighing 16.2g of Ser (tBu) -Pro-Asn (Trt) -Gln (Trt) -Linker-Amide Resin, adding into a round-bottom flask, adding the frozen lysate, and stirringStirring and reacting for 2h. Suction filtering, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging, washing for 6 times until pH is 3-4, and vacuum drying to obtain 5.2g of H-Ser-Pro-Asn-Gln-NH 2 Crude peptide.
2.5 Purification of
5.2g of crude peptide are weighed out in 120mL of methanol: in water (V: V = 1:2), filtration through a 0.22 μm pore size microfiltration membrane gave clear, clear solutions which were purified by reverse phase HPLC with the purification gradients given in the following table:
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
Purifying the filtered sample by sampling, and collectingThe fraction is concentrated and freeze-dried to obtain the peptide (9) H-Ser-Pro-Asn-Gln-NH with the purity of 99.696% 2 。
Example 3
Other peptides of formula (I) of the invention may be prepared by similar methods.
The molecular weight of the obtained peptides was determined by ESI-MS, and the results of the partial peptide test are shown in Table 2 below and FIGS. 1-2:
TABLE 2 Mass Spectrometry results
Numbering Sequence of Molecular weight mass spectrometry results
（2） H-Ser-Pro-Gly-Gln-NH 2 386.20
（9） H-Ser-Pro-Asn-Gln-NH 2 443.23
Example 4 cell proliferation assay
4.1 Reagents and materials
Thiazole blue (MTT), dimethyl sulfoxide (DMSO), a high-sugar medium (DMEM), fetal bovine serum, and PBS.
4.2 Instrument for measuring the position of a moving object
Microplate reader, CO 2 Incubator, superclean bench.
4.3 Cell line
Human keratinocytes (HaCaT) were purchased from kunming cell banks of the typical culture collection committee of the chinese academy of sciences, and Human Skin Fibroblasts (HSF) were purchased from shanghai cell banks of the typical culture collection committee of the chinese academy of sciences.
4.4 Sample to be tested
Administration group: peptide (2) and peptide (9) were tested at concentrations of 6.25ppm, 12.5ppm, 25ppm, 50ppm, 100ppm, 200ppm.
Blank control group: PBS.
Positive control group: 2% DMSO.
4.5 Experimental methods
Respectively taking HaCaT keratinocyte and HSF fibroblast in exponential growth phase, adding 0.25% trypsin digestive juice, digesting to make adherent cells fall off, counting 1-4 × 10 5 And (4) preparing cell suspension per mL.
Inoculating the cell suspension on a 96-well plate at a concentration of 200 μ L/well, and placing in constant temperature CO 2 Culturing in an incubator for 24h.
Changing the solution, adding the samples of the administration group, blank control group and positive control group, respectively, 20 μ L/well, standing at 37 deg.C and 5% CO 2 Incubate in incubator for 72h.
Thereafter, 20. Mu.L of 5mg/ml MTT was added to each well, and the reaction was continued at 37 ℃ with 5% CO 2 Incubate in incubator for 4h. The stock solution was discarded, 150. Mu.L/well of DMSO was added, the mixture was shaken on a shaker for 5min, and then the OD value of each well was measured at a wavelength of 570nm using a microplate reader, and the cell viability was calculated.
Cell viability = (dose well OD-zero OD)/(blank control well OD-zero OD) × 100%
4.6 Results of the experiment
The MTT method is a method for measuring the survival and growth of cells, and the OD value measured is proportional to the activity of the cells.
The results of the test samples on the activity of the HaCaT cells are shown in FIG. 3, and the results show that the activity of the HaCaT cells of the positive control group is remarkably reduced compared with that of the blank control group, which indicates that 2% DMSO has a toxic effect on the HaCaT cells, the administration group has no toxic effect on the HaCaT cells within the range of 200ppm, and the peptides (2) and (9) of the invention can obviously improve the cell activity and promote the proliferation of the HaCaT cells at low concentration of 6.25ppm, and the cell proliferation promoting effect is enhanced along with the increase of the concentration.
The results of the test samples on the activity of the HSF cells are shown in FIG. 4, and the results show that the activity of the HSF cells of the positive control group is remarkably reduced compared with that of the blank control group, which indicates that 2% DMSO has a toxic effect on the HSF cells, the administration group has no toxic effect on the HSF fibroblasts within the range of 200ppm, and the peptides (2) and (9) of the invention can obviously improve the cell activity and promote the proliferation of the HSF fibroblasts at a low concentration of 6.25ppm, and the cell proliferation promoting effect is enhanced along with the increase of the concentration.
Therefore, the peptide has no toxic effect on HaCaT keratinocytes and HSF fibroblasts, can improve the activity of cells and promote the proliferation of the cells, thereby increasing the skin elasticity and/or the skin firmness, and can be used for preventing and even treating skin relaxation.
EXAMPLE 5 photo aging test
5.1 Reagents and materials
Fetal bovine serum, high-sugar medium (DMEM), penicillin, streptomycin, and thiazole blue (MTT).
5.2 Instrument for measuring the position of a moving object
Microplate reader, CO 2 Incubator, superclean bench.
5.3 Cell line
Human Skin Fibroblasts (HSF) were purchased from the Shanghai cell Bank of the China academy of sciences type culture Collection.
5.4 samples to be tested
Administration group:
reference 1 (Palm-Lys-Thr-Thr-Lys-Ser) at concentrations tested 12.5ppm, 25ppm, 50ppm, respectively;
reference 2 (Ser-Pro-Leu-Gln-NH) 2 ) The test concentrations are respectively 12.5ppm, 25ppm and 50ppm;
peptide (2) at concentrations tested of 12.5ppm, 25ppm, 50ppm, respectively;
peptide (9) was tested at concentrations of 12.5ppm, 25ppm, 50ppm, respectively.
Blank control group: PBS.
UV group: UV irradiation, PBS was added.
5.5 Experimental method
Taking HSF fibroblasts in a good exponential growth phase, adding 0.25% trypsin digestion solution, digesting to make adherent cells fall off, counting by 1-4 multiplied by 10 5 And (4) preparing cell suspension per mL.
10000 cells/well of cell suspension are taken out and inoculated on a 96-well plate after being properly diluted, and when the cells grow to be about 80 percent, a UV light aging model is established. Adding 50 μ L PBS into blank control group, supplementing culture medium to 200 μ L, and not performing UV irradiation; adding appropriate amount of PBS to wash repeatedly to colorless in UV group and administration group, adding 50 μ L PBS at 80mJ/cm 2 Irradiating under UV lamp at a distance of 15cm between lamp source and culture bottle, discarding PBS after irradiation, adding PBS solution and culture medium to 200 μ L in UV group, and adding culture solution and diluted drug at multiple ratio to 200 μ L in administration group. Blank control group, UV group, and administration group were continued at 37 deg.C and 5% CO 2 Incubate in incubator for 24h.
Then 20. Mu.L of 5mg/mL MTT per well was added, continuing at 37 ℃ with 5% CO 2 Incubate in incubator for 4h. The original solution was discarded, 150. Mu.L/well of DMSO was added, the mixture was shaken on a shaker for 5min, and then the reference OD values at 490nm and 630nm were read using a microplate reader.
5.6 Results of the experiment
Skin aging is affected by endogenous and exogenous factors, such as genetics, environmental exposure, ultraviolet irradiation, hormonal changes, and the like. The accumulation of these factors, particularly the exposure to ultraviolet light, results in changes in skin structure, function and appearance. 80mJ/cm is selected for this experiment 2 The UV energy is radiated to establish a skin photoaging model.
The experimental result is shown in fig. 5, after UV irradiation, the activity of HSF fibroblasts is significantly reduced, indicating that the photoaging model is successfully established; the administration group can improve the cell activity within the range of 50ppm, thereby improving the cell aging and generating obvious anti-photoaging effect; compared with reference substances 1 and 2, the peptide (2) and the peptide (9) can obviously improve the cell activity and obviously promote the proliferation of HSF fibroblasts; wherein, the peptide (2) can greatly improve the activity of HSF fibroblasts under the low concentration of 12.5ppm and generate obvious anti-photoaging effect. Therefore, the peptide of the present invention can be used for treating, preventing and/or repairing skin aging or photoaging, and has superior anti-aging efficacy compared to the prior art.
Example 6 collagen content test
6.1 Reagents and materials
Fetal bovine serum, a DMEM culture medium, a phosphate buffer solution, trypsin, a BCA protein kit and a collagen I ELISA kit.
6.2 Instrument for measuring the position of a moving object
Microplate reader, CO 2 Incubator, superclean bench, thermostated container.
6.3 Cell line
Human Skin Fibroblasts (HSF) were purchased from the Shanghai cell Bank of the China academy of sciences type culture Collection.
6.4 samples to be tested
Administration group:
reference 1 (Palm-Lys-Thr-Thr-Lys-Ser) at concentrations tested 12.5ppm, 25ppm, respectively;
reference 2 (Ser-Pro-Leu-Gln-NH) 2 ) The test concentrations are respectively 12.5ppm and 25ppm;
peptide (2) at test concentrations of 12.5ppm, 25ppm, respectively;
peptide (9) was tested at 12.5ppm and 25ppm, respectively.
Blank control group: PBS.
UV group: UV irradiation, PBS was added.
6.5 Experimental method
Taking HSF fibroblasts in a good exponential growth phase, adding 0.25% trypsin digestion solution, digesting to make adherent cells fall off, counting by 1-4 multiplied by 10 6 And (4) preparing cell suspension per mL.
After 100000/hole cell suspensions are properly diluted and inoculated on a 6-hole plate, modeling is carried out when the cells grow to be about 80%. Adding 200 μ L PBS into blank control group, supplementing culture medium to 800 μ L, and not performing UV irradiation; adding appropriate amount of PBS to wash repeatedly to colorless in UV group and administration group, adding 200 μ L PBS, placing in 80mJ/cm 2 The UV lamp was used for irradiation, and the distance between the lamp source and the culture flask was 15cm. After irradiation, PBS was discarded, the PBS solution and medium were added to the UV group to 800. Mu.L, and the medium and the drug diluted in multiples to 800. Mu.L were added to the administration group. Blank control group, UV group, and administration group were continued at 37 deg.C and 5% CO 2 Incubate in incubator for 48h.
After the incubation, the 1 st well cells were counted and diluted to 0.5X 10 6 one/mL, cells from the remaining wells were scraped off using a cell scraper, 500. Mu.L was resuspended, 50. Mu.L of all wells were sonicated for 30s before total protein was measured using the BCA method, and the other wells were diluted to a concentration of 0.5X 10 of all cell suspensions based on the 1 st well protein concentration 6 one/mL. The cell suspension after adjusting the concentration was sonicated 30s,1500xɡCentrifuging for 15 min, and collecting cell supernatant to obtain sample solution; the procedures were performed according to the instructions for the collagen I ELISA kit. 15 The OD value of each well was measured sequentially at 450 nm with a microplate reader during min.
6.6 Results of the experiment
Skin aging is closely related to collagen, the most abundant protein found in connective tissue, and has an important role in skin plumpness and firmness. In the UV-overexposure environment, collagen production is inhibited. In the experiment, cells irradiated by ultraviolet rays are treated by using a test sample, and the content of collagen I in corresponding cells is detected to determine whether the peptide can promote the generation of collagen.
The results of the effect of the test samples on collagen content are shown in FIG. 6. The results show that the collagen content of the UV group is significantly reduced compared to the blank control group; compared with the UV group, the reference substances 1 and 2 can not increase the collagen content in the cells after ultraviolet radiation, and the peptides (2) and (9) can obviously improve the collagen content and promote the collagen expression within the range of 12.5-25ppm, thereby having excellent collagen generation promoting effect.
Therefore, the peptide can promote the generation of collagen, increase the content of the collagen, and further increase the elasticity and/or firmness of the skin, can be used for preventing and even treating the skin relaxation, treating, preventing and/or repairing the skin aging or photoaging, and can be used for resisting the skin aging in the fields of cosmetics or medicines.
Example 7 PER1 protein content assay
7.1 Reagents and materials
Fetal bovine serum, DMEM medium, phosphate buffer solution, trypsin and human circadian protein 1 (PER 1) ELISA kit.
7.2 Instrument for measuring the position of a moving object
Microplate reader, CO 2 Incubator, superclean bench, thermostated container.
7.3 Cell line
Human Skin Fibroblasts (HSF) were purchased from the Shanghai cell Bank of the China academy of sciences type culture Collection.
7.4 samples to be tested
Administration group:
reference 2 (Ser-Pro-Leu-Gln-NH) 2 ) The test concentration is 25ppm;
peptide (2) was tested at a concentration of 25ppm.
Blank control group: PBS.
7.5 Experimental method
Taking HSF fibroblasts in a good exponential growth phase, adding 0.25% trypsin digestion solution, digesting to make adherent cells fall off, counting by 1-4 multiplied by 10 6 And (4) preparing cell suspension per mL.
After 100000 cell suspensions are properly diluted and inoculated on a 6-well plate, modeling is carried out when the cells grow to about 80%. Adding 200 μ L PBS into blank control group, and supplementing culture medium to 800 μ L; adding appropriate amount of PBS into administration group, repeatedly washing to colorless, adding culture medium and medicine to 800 μ L, placing each group at 37 deg.C and 5% CO 2 Incubator 48h was incubated.
Scraping off cells after the culture is finished, blowing, beating and mixing uniformly, and carrying out ultrasonic disruption on the cell suspension for 30s,1500xɡCentrifuging for 15 min, and collecting cell supernatant to obtain sample solution; the procedures were performed according to the instructions of PER1 protein ELISA kit. 15 The OD of each well was measured sequentially at 450 nm with a microplate reader within min.
7.6 Results of the experiment
Circadian rhythm protein 1 (PER 1) is one of biological clock core components for regulating circadian rhythm cycle of skin, participates in the reaction process of regulating skin to environmental pressure, protects skin from UV radiation, temperature, chemical and physical damage and various environmental damages such as microbial infection, and expression of PER1 protein has certain correlation with aging, and the increase of PER1 protein content can improve skin aging to a certain extent. The experiment confirmed whether the peptide of the present invention can promote the expression of PER1 protein by detecting the content of PER1 protein in the corresponding cells.
The results of the effect of the test samples on the PER1 protein content are shown in FIG. 7. The results showed that 25ppm of peptide (2) significantly increased the content of PER1 protein and promoted the expression of PER1 protein compared to the blank control group, and that peptide (2) of the present invention had a superior effect of promoting the expression of PER1 protein compared to reference 2.
Therefore, the peptide can promote the expression of PER1 protein, increase the content of PER1 protein, participate in the regulation of the skin circadian rhythm cycle, restore the circadian rhythm and resynchronize the skin cell biological clock, and can be used for repairing damaged skin cells and relieving the skin aging signs; can also be used for preparing PER1 protein activator.
EXAMPLE 8 preparation of essence containing peptide (2)
INCI name By weight%
Peptide (2) 0.05
Vitamin C 1
Glycerol 5
Luba adhesive 5
Hyaluronic acid sodium salt 0.4
Octylene glycol 0.25
1,2 hexanediol 0.25
15% triethanolamine 0.04
Water (I) Proper amount to 100
Adding the sodium hyaluronate of the formula amount into water, stirring to mix uniformly, heating to 80-85 ℃, and keeping the temperature and stirring to disperse uniformly. Cooling to below 40 deg.C, adding glycerol, gemba gum, peptide (2), vitamin C, caprylyl glycol and 1,2-hexanediol, and stirring. Adjusting pH of the solution to about 5.5 with 15% triethanolamine.
EXAMPLE 9 preparation of liposome containing peptide (9)
Composition (I) By weight%
Dipalmitoyl phosphatidylcholine 4.0
Peptide (9) 0.2
Preservative 0.5
Water (W) Proper amount to 100
Dipalmitoylphosphatidylcholine was weighed and dissolved in chloroform. The solvent is evaporated under vacuum until a thin phospholipid layer is obtained, which is hydrated by treatment with an aqueous peptide solution of the desired concentration at 55 ℃ to obtain a multilamellar liposome. The multi-chamber liposome is subjected to high-pressure homogenization treatment to obtain the single-chamber liposome with smaller and uniform size.
The above description is further intended to describe the present invention in detail with reference to specific preferred embodiments, but it is not intended to limit the present invention to the specific embodiments. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (13)

1. A peptide represented by the 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）
in the formula (I), the compound is shown in the specification,
X 1 selected from the group consisting of: -Gly-, -Asn-, -Ala-, -Ile-, -Val-, -Ser-, -Thr-or-Met-;
R 1 selected from: h or R 3 -CO-，R 3 Selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstitutedAlkenyl of (a);
R 2 selected from the group consisting of: -NR 4 R 5 OR-OR 4 Wherein R is 4 And R 5 Independently of one another, selected from: H. substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
the alkyl group refers to a saturated aliphatic linear or branched alkyl group having 1 to 24 carbon atoms (optionally having 1 to 16 carbon atoms; optionally having 1 to 14 carbon atoms; optionally having 1 to 12 carbon atoms; optionally having 1,2, 3, 4, 5, or 6 carbon atoms); optionally 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 means a straight or branched chain alkenyl group having 2 to 24 carbon atoms (optionally having 2 to 16 carbon atoms; optionally having 2 to 14 carbon atoms; optionally having 2 to 12 carbon atoms; optionally having 2, 3, 4, 5, or 6 carbon atoms); said alkenyl having one or more carbon-carbon double bonds, optionally 1,2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bonded to the remainder of the molecule by a single bond; optionally selected from: vinyl, oleyl, or linoleyl;
alternatively, the substituents in the "substituted alkyl", "substituted alkenyl" are selected from C 1 -C 4 An alkyl group; a hydroxyl group; c 1 -C 4 An alkoxy group; an amino group; c 1 -C 4 An aminoalkyl group; c 1 -C 4 A carbonyloxy group; c 1 -C 4 An oxycarbonyl group; halogen (e.g., fluorine, chlorine, bromine, and iodine); a cyano group; a nitro group; an azide; c 1 -C 4 An alkylsulfonyl group; a thiol; c 1 -C 4 An alkylthio group; c 6 -C 30 Aryloxy groups such as phenoxy; -NR b (C=NR b )NR b R c Wherein R is b And R c Is independently selected from: H. c 1 -C 4 Alkyl radical, C 2 -C 4 Alkenyl radical, C 2 -C 4 Alkynyl, C 3 -C 10 Cycloalkyl radicals、C 6 -C 18 Aryl radical, C 7 -C 17 An aralkyl group, a heterocyclic group having three to ten members, or a protecting group of an amino group.
2. The peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein R is 1 Selected from: H. acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl or linoleoyl; r is 4 、R 5 Independently of one another, selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;
alternatively, R 1 Selected from H, acetyl, myristoyl or palmitoyl; r 4 Is H and R 5 Selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;
specifically, R 1 Is H or acetyl; r 2 is-OH or-NH 2 。
3. The peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to claim 1, selected from the following peptides (1) to (32):
（1）H-Ser-Pro-Gly-Gln-OH；
（2）H-Ser-Pro-Gly-Gln-NH 2 ；
（3）Ac-Ser-Pro-Gly-Gln-OH；
（4）Ac-Ser-Pro-Gly-Gln-NH 2 ；
（5）H-Ser-Pro-Ala-Gln-OH；
（6）H-Ser-Pro-Ala-Gln-NH 2 ；
（7）Ac-Ser-Pro-Ala-Gln-OH；
（8）Ac-Ser-Pro-Ala-Gln-NH 2 ；
（9）H-Ser-Pro-Asn-Gln-NH 2 ；
（10）H-Ser-Pro-Asn-Gln-OH；
（11）Ac-Ser-Pro-Asn-Gln-OH；
（12）Ac-Ser-Pro-Asn-Gln-NH 2 ；
（13）H-Ser-Pro-Ile-Gln-OH；
（14）H-Ser-Pro-Ile-Gln-NH 2 ；
（15）Ac-Ser-Pro-Ile-Gln-OH；
（16）Ac-Ser-Pro-Ile-Gln-NH 2 ；
（17）H-Ser-Pro-Val-Gln-OH；
（18）H-Ser-Pro-Val-Gln-NH 2 ；
（19）Ac-Ser-Pro-Val-Gln-OH；
（20）Ac-Ser-Pro-Val-Gln-NH 2 ；
（21）H-Ser-Pro-Ser-Gln-OH；
（22）H-Ser-Pro-Ser-Gln-NH 2 ；
（23）Ac-Ser-Pro-Ser-Gln-OH；
（24）Ac-Ser-Pro-Ser-Gln-NH 2 ；
（25）H-Ser-Pro-Thr-Gln-OH；
（26）H-Ser-Pro-Thr-Gln-NH 2 ；
（27）Ac-Ser-Pro-Thr-Gln-OH；
（28）Ac-Ser-Pro-Thr-Gln-NH 2 ；
（29）H-Ser-Pro-Met-Gln-OH；
（30）H-Ser-Pro-Met-Gln-NH 2 ；
（31）Ac-Ser-Pro-Met-Gln-OH；
（32）Ac-Ser-Pro-Met-Gln-NH 2 。
4. the peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to claim 3, selected from the group consisting of peptide (1), peptide (2), peptide (3), peptide (4), peptide (9), peptide (10), peptide (11), peptide (12); in particular, the amount of the solvent to be used,
（1）H-Ser-Pro-Gly-Gln-OH；
（2）H-Ser-Pro-Gly-Gln-NH 2 ；
（3）Ac-Ser-Pro-Gly-Gln-OH；
（4）Ac-Ser-Pro-Gly-Gln-NH 2 ；
（9）H-Ser-Pro-Asn-Gln-NH 2 ；
（10）H-Ser-Pro-Asn-Gln-OH；
（11）Ac-Ser-Pro-Asn-Gln-OH；
（12）Ac-Ser-Pro-Asn-Gln-NH 2 。
5. the peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 4,
the cosmetically or pharmaceutically acceptable salt comprises a metal salt of a peptide of formula (I), the metal comprising: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum;
optionally, the cosmetically or pharmaceutically acceptable salt comprises a salt of the peptide of formula (I) with an organic base comprising: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine;
optionally, the cosmetically or pharmaceutically acceptable salt comprises a salt of the peptide of formula (I) with an inorganic or organic acid, the organic acid comprising: acetic, citric, lactic, malonic, maleic, tartaric, fumaric, benzoic, aspartic, glutamic, succinic, oleic, trifluoroacetic, oxalic, pamoic or gluconic acids;
optionally, the inorganic acid comprises: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.
6. A cosmetic or pharmaceutical composition comprising an effective amount of a peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically or pharmaceutically acceptable salt thereof, according to any of claims 1 to 5, together with at least one excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant;
optionally, the adjuvant is selected from: stimulators of collagen synthesis, agents for modulating PGC-1 alpha synthesis, agents for modulating the activity of PPAR gamma, agents for increasing or decreasing the triglyceride content of adipocytes, agents for stimulating or delaying adipocyte differentiation, lipolytic or lipolysis agents, lipid solubilizing agents, lipid producing agents, inhibitors of acetylcholine receptor aggregation, agents for inhibiting muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulators or inhibitors, whitening or depigmenting agents, pro-pigmentation agents, self-tanning agents, anti-ageing agents, NO-synthase inhibitors, 5 alpha-reductase inhibitors, lysyl hydroxylase and/or prolyl hydroxylase inhibitors, antioxidants, free radical scavengers and/or anti-atmospheric pollution agents, active carbonyl scavenger, anti-glycation agents, anti-histamine agents, anti-viral agents antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioners, moisture retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, emollients, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating the lower eye pouch, exfoliating agents, antimicrobial agents, disinfectants, bacteriostats, agents that stimulate the synthesis of dermal or epidermal macromolecules and/or that inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate decorin synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, agents that stimulate chaperonin synthesis, agents that stimulate cAMP synthesis, agents that stimulate HSP70 synthesis, <xnotran> , , , , , , , , , , , , , , , , , , , , , , DNA , DNA , , , / , , , , , , , , , , , , , , , / , , , , , - , , , , , , , , , , , , A / B . </xnotran>
7. Cosmetic or pharmaceutical composition, according to claim 6, characterized in that the formulation of said cosmetic or pharmaceutical composition is selected from: creams, oils, balms, foams, lotions, gels, liniments, serums, ointments, mousses, powders, sticks, pencils, sprays, aerosols, capsules, tablets, granules, chewing gums, solutions, suspensions, emulsions, elixirs, polysaccharide films, jellies or gelatins;
optionally, the capsule comprises: soft capsule, hard capsule, optionally gelatin capsule;
optionally, the tablet comprises: sugar-coated tablets.
8. A cosmetically or pharmaceutically acceptable delivery system or sustained release system comprising an effective amount of a peptide of formula (I) according to any one of claims 1 to 5, or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically or pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to claim 6 or 7;
the cosmetically or pharmaceutically acceptable delivery system or sustained release system is selected from: liposomes, oleosomes, non-ionic surfactant liposome vesicles, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, liposomes, micelles, millispheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, nanoparticles, microparticles, or nanoparticles; optionally liposomes or microemulsions, optionally water-in-oil microemulsions having an internal structure of reverse micelles.
9. Use of a peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to any of claims 1 to 5, or a cosmetically or pharmaceutically acceptable composition according to claim 6 or 7, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system according to claim 8, for the preparation of a cosmetic or pharmaceutical composition for the treatment, prevention or repair of skin aging and/or photoaging.
10. Use of a peptide of formula (I), or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 5, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system according to claim 6 or 7, for the preparation of a cosmetic or pharmaceutical composition intended for increasing the elasticity and/or firmness of the skin.
11. Use of a peptide of formula (I), or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 5, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system according to claim 6 or 7, for the preparation of a cosmetic or pharmaceutical composition intended to promote collagen production.
12. Use of a peptide of formula (I) as defined in any one of claims 1 to 5, or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as defined in claim 6 or 7, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as defined in claim 8, for the preparation of a cosmetic or pharmaceutical composition for promoting the expression of PER1 protein, for modulating the circadian rhythm of the skin and/or for repairing damaged cells of the skin.
13. Use of a peptide of formula (I), or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to any of claims 1 to 5, or a cosmetically or pharmaceutically acceptable composition according to claim 6 or 7, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system according to claim 8, for the preparation of an activator of PER1 proteins.
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