CN115594735B - Peptides with anti-aging effect, compositions and uses thereof - Google Patents

Peptides with anti-aging effect, compositions and uses thereof Download PDF

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Publication number
CN115594735B
CN115594735B CN202211490952.7A CN202211490952A CN115594735B CN 115594735 B CN115594735 B CN 115594735B CN 202211490952 A CN202211490952 A CN 202211490952A CN 115594735 B CN115594735 B CN 115594735B
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agents
agent
acceptable salt
pharmaceutically acceptable
skin
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CN115594735A (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 Weiqi Technology Co ltd
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Shenzhen Weiqi Technology Co ltd
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Priority to CN202311070431.0A priority patent/CN116987140A/en
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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

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 manufacture of a cosmetic or pharmaceutical composition for treating, preventing or repairing skin aging or photoaging, promoting collagen production, increasing skin elasticity and/or skin firmness, promoting PER1 protein expression, regulating skin circadian rhythm, and/or repairing damaged cells of the skin. The peptide of formula (I) can regulate the circadian rhythm of skin and resynchronize the biological clock of skin cells, strengthen the cell activity and repair damaged cells by activating the expression of circadian rhythm regulation genes and related proteins of skin, thereby relieving the signs of skin aging, and can be used for resisting skin aging in the fields of cosmetics or medicines and also can be used for preparing PER1 protein activators.

Description

Peptides with anti-aging effect, compositions and uses thereof
Technical Field
The invention relates to the technical field of medicines and cosmetics, in particular to a peptide with an anti-aging effect, and a cosmetic composition or a medicinal composition and application thereof.
Background
Skin is used as the first physiological defense line of human body, and is also subjected to the test of external environment at any time while protecting tissue and organs from being affected by chemical, mechanical, physical and pathogenic microorganisms. In recent years, patients suffering from skin diseases are increasing year by year due to environmental attack such as UV radiation and environmental pollution, and aging of skin is also accelerated. Environmental attack can cause damage to the DNA of skin cells and affect its circadian rhythm.
Circadian rhythm refers to the variation of vital activities over a 24-hour period, also known as biological clock, which regulates our many metabolic, physiological activities and behaviors. Much evidence suggests that circadian rhythm disturbances and disorders can have significant negative effects on human health, including jet lag, night shift induced fatigue syndrome, sleep disorders, skin disorders, and the like. In addition, many diseases that severely threaten human health, such as cardiovascular and cerebrovascular diseases and tumors, are also associated with circadian rhythm disturbances. However, with the development of modern civilization, more and more people are working away from sunrise, sunset and rest lifestyle, resulting in an increasingly common imbalance of circadian rhythms.
An independent biological clock system is also present in keratinocytes, melanocytes and fibroblasts of skin cells. Circadian rhythm proteins are expressed not only in cultured human cells but also in human skin biological tissues, and exert influence on skin functions. The skin is easily exposed to external environmental factors such as light, temperature, humidity, ultraviolet rays, germs, etc., which vary with the period of one day. To adapt to this environment, the skin regulates various physiological functions by activating a biological clock that is present within the skin. Experiments show that the proliferation and differentiation of skin cells, water loss, sebum generation, temperature, pH and wrinkle generation all change in 24-hour period. For example, proliferation and differentiation of skin cells occur through a series of processes in a 24-hour period. Skin is easily damaged by uv exposure during the day, so differentiation of skin cells mainly occurs between the evening and early morning. By separating these series of processes temporally, the skin is protected from the harmful environment. However, when the rhythms of this process are disturbed, a desired physiological reaction cannot be generated at an appropriate time, and thus, the circadian rhythms of the skin are disturbed, and the damage of the skin is accelerated.
In recent years, it has been found that genes related to the circadian rhythm of the natural body include Clock gene and PER1 gene, both of which encode proteins regulating the circadian rhythm (Clock protein and PER1 protein). PER1 is one of the core components of biological clock for regulating the circadian cycle of skin, and is a member of Period gene family consisting of PER1, PER2, PER3, which are involved in metabolism, motor activity, behavior, etc. of the body, wherein PER1 plays a core role. The Clock gene and the PER1 gene are also present in skin cells, and expression of the PER1 gene induces cellular activity programs associated with biological processes (e.g., repair) that take place during the night. The DNA replication, DNA repair mechanism and cell division of epidermal progenitor/stem cells exhibit distinct circadian features. Skin cells exposed to environmental attack for a long period of time typically exhibit reduced, irregular or unsynchronized Clock gene or PER1 gene expression, disrupting the normal circadian rhythm in the skin cells. The circadian rhythm and synchronism of normal cells are disturbed over time, and the skin balance is destroyed, so that the natural aging process of the skin is accelerated, the skin is damaged, and the problems of wrinkles, fine lines, loose skin, uneven pigmentation, senile plaques, color spots and the like appear.
Analysis of Clock gene and PER1 gene shows that they are biological Clock core gene and negative feedback gene for regulating circadian cycle of skin, and participate in regulating reaction course of skin to environmental pressure, and Clock gene and PER1 gene interfere expression and activity regulation of genetic gene and protein in various physiological processes in about 24 hr period so that biological Clock caters to external environment to regulate activity of physiological reaction. Regulating and controlling various activity processes of skin cells according to circadian rhythm, and protecting skin from various environmental injuries such as UV radiation, temperature, chemical and physical injury, microbial infection and the like.
Therefore, it is necessary to develop an active substance capable of improving the circadian rhythm of the skin, activating the circadian rhythm regulating gene of the skin and related proteins from the viewpoint of the mechanism of the circadian rhythm of the skin, and apply the active substance to the fields of medicine and cosmetics to alleviate skin aging.
Disclosure of Invention
The invention aims to provide an active substance which is stable, efficient, safe and free from skin irritation, and can repair damaged cells by activating the expression of circadian rhythm regulation genes and related proteins of skin, thereby realizing anti-aging effect, promoting collagen production, increasing skin elasticity and/or skin compactness, preventing skin relaxation, and having wide application in the fields of medicine and cosmetics.
In view of this, the present invention provides a peptide represented by the formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, 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) of the present invention,
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 is 5 Independently of each other selected from: H. substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
alkyl 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 refers to a straight or branched 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); the alkenyl group has one or more carbon-carbon double bonds, optionally 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bound to the remainder of the molecule by a single bond; optionally selected from: vinyl, oleyl, or linoleyl;
Optionally, the substituents in the "substituted alkyl", "substituted alkenyl" are selected from C 1 -C 4 An alkyl group; a hydroxyl group; c (C) 1 -C 4 An alkoxy group; an amino group; c (C) 1 -C 4 An aminoalkyl group; c (C) 1 -C 4 A carbonyloxy group; c (C) 1 -C 4 An oxycarbonyl group; halogen (e.g., fluorine, chlorine, bromine, and iodine); cyano group; a nitro group; an azide; c (C) 1 -C 4 An alkylsulfonyl group; a mercaptan; c (C) 1 -C 4 Alkylthio; c (C) 6 -C 30 Aryloxy groups such as phenoxy; -NR b (C＝NR b )NR b R c Wherein R is b And R is c Is independently selected from: H. c (C) 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, C 3 -C 10 Cycloalkyl, C 6 -C 18 Aryl, C 7 -C 17 Aralkyl groups, heterocyclic groups having three to ten members, or protecting groups for amino groups.
Alternatively, R 1 Selected from: H. acetyl, t-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl or linoleoyl; r is R 4 、R 5 Independently of each other selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;
alternatively, R 1 Selected from H, acetyl, myristoyl or palmitoyl; r is R 4 Is H and R 5 Selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;
specifically, R 1 Is H or acetyl; r is R 2 is-OH or-NH 2 。
Alternatively, 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, is selected from the following peptides (1) - (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 the group consisting of peptide (1), peptide (2), peptide (3), peptide (4), peptide (9), peptide (10), peptide (11), peptide (12); in particular, the method comprises the steps of,
(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 peptide of formula (I) of the present invention may exist as stereoisomers or as mixtures of stereoisomers; for example, the amino acids they comprise may have the L-, D-configuration, or be racemic independently of each other. 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 the pure isomer, i.e., enantiomer or diastereomer.
For example, when the present invention is directed to-Pro-, it is understood that-Pro-is selected from-L-Pro-, -D-Pro-, or a mixture of both, 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 peptides of formula (I). The peptides of the invention are identical The potential variant is understood here to mean such compounds: wherein at least one atom is replaced by another atom of the same atomic number within the peptide of the invention, but the atomic mass of said other atom is different from the atomic mass normally or predominantly present in nature. Examples of isotopes that can be incorporated into the peptides of the invention are: those 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 S, S. Specific isotopic variations of the peptides of the present invention (particularly those into which one or more radioisotopes have been incorporated) may be advantageous, for example, for examining the mechanism of action or distribution of active compounds in vivo; due to relatively simple producibility and detectability, especially with 3 H or 14 C isotopically labeled compounds are suitable for this purpose. In addition, due to the greater metabolic stability of the compounds, the incorporation of isotopes (e.g., deuterium) may yield particular therapeutic benefits, such as increased in vivo half-life or reduced amounts of active agent required; thus, in some 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 further described below and in the examples, by using the respective reagents and/or corresponding isotopic modifications of the starting materials.
In addition, the invention also includes prodrugs of the peptides of the invention. The term "prodrug" means herein such compounds: which may be biologically active or inactive in itself, but which react (e.g., metabolize or hydrolyze) to form the peptides of the invention during their residence time in the body.
The term "cosmetically or pharmaceutically acceptable salt" refers to a salt that is approved for use in animals, and more specifically in humans, including metal salts of peptides of formula (I), including, but not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum, etc.; including salts of peptides of formula (I) with organic bases including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine and the like; including salts of peptides of formula (I) with inorganic or organic acids including, but not limited to: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoate (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, may be carried out according to conventional methods known in the art, such as solid phase synthesis, liquid phase synthesis or a combination of solid and liquid phases, and may also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins of animal, fungal or plant origin.
For example, a method for 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 carrier-bound C-terminus;
-elimination of the group protecting the N-terminal end;
-repeating the coupling sequence and elimination of the N-terminal protecting group until the desired peptide sequence is obtained;
-elimination of the C-terminal protecting group or cleavage from the solid support.
Preferably, the C-terminus is bound to a solid support and the method is performed 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 polymeric support; eliminating the group protecting the N-terminus; and repeating this sequence as many times as necessary to thereby obtain a peptide of the desired length, followed by cleavage of the synthesized peptide from the original polymer carrier.
The functional groups of the side chains of these amino acids remain fully 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 carrier.
Alternatively, solid phase synthesis may be performed by a pooling strategy (convergent strategy) of coupling a dipeptide or tripeptide to a polymeric support or to a dipeptide or amino acid previously bound to a polymeric support.
The functional groups of the ends may be subsequently modified using standard conditions and methods known in the art to deprotect the N-and C-termini and/or cleave the peptide from the polymeric support in a non-defined order. The peptide of formula (I) bound to the polymeric support may be optionally modified at the N-and C-termini, 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 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;
Optionally, the adjuvant is selected from: collagen synthesis stimulators, agents that modulate PGC-1 alpha synthesis, agents that modulate PPARgamma activity, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic or lipolysis-stimulating agents, lipolytic agents, adipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulating or inhibiting agent, whitening or depigmenting agent, pigmentation promoting agent, self-tanning agent, antiaging agent, NO-synthase inhibitor, 5α -reductase inhibitor, and pigment inhibitors of lysyl hydroxylase and/or prolyl hydroxylase, antioxidants, free radical scavengers and/or agents against atmospheric pollution, active carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioning agents, moisture retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermohydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating the lower eye bags, exfoliants, antimicrobial agents, bactericides, bacteriostats, agents capable of stimulating the synthesis of dermal or epidermal macromolecules and/or inhibiting or preventing 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, an agent that stimulates heat shock protein synthesis, an agent that stimulates hyaluronic acid synthesis, an agent that stimulates fibronectin synthesis, an agent that stimulates deacetylase synthesis, an agent that stimulates synthesis of lipids and stratum corneum components, ceramides, fatty acids, an agent that inhibits collagen degradation, an agent that inhibits elastin degradation, an agent that inhibits serine proteases, an agent that stimulates fibroblast proliferation, an agent that stimulates keratinocyte proliferation, an agent that stimulates adipocyte proliferation, an agent that stimulates melanocyte proliferation, an agent that stimulates keratinocyte differentiation, an agent that inhibits acetylcholinesterase, a skin relaxant, an agent that stimulates glycosaminoglycan synthesis, an anti-hyperkeratosis agent, an acne lytic agent, an anti-psoriasis agent, an anti-rash agent, a DNA repair agent, a DNA protectant, a stabilizer, an antipruritic agent, an agent for treating and/or caring for sensitive skin solidifying agents, tightening agents, restructuring agents, stretch-proofing agents, agents that modulate sebum production, antiperspirant agents, agents that stimulate healing, agents that assist healing, agents that stimulate re-epithelialization, agents that assist re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents that act on capillary circulation and/or microcirculation, agents that stimulate angiogenesis, agents that inhibit vascular permeability, intravenous tonicity agents, agents that act on cellular metabolism, agents for improving dermal-epidermal junction, agents that induce hair growth, hair growth inhibition or delay agents, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents derived from biofermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents that are effective against a and/or B uv light, or mixtures thereof.
Alternatively, the formulation of the cosmetic or pharmaceutical composition is selected from: cream, oil, balm, foam, lotion, gel, wipe, slurry, ointment, mousse, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, elixir, polysaccharide film, jelly, or gelatin;
optionally, the capsule comprises: soft capsule and 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 sequences or any possible modification in the N-terminal and/or C-terminal. The peptides of the invention may thus be incorporated into the composition by aqueous solutions, and those that are insoluble in water may 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, as well as their dosage, will depend on a number of factors including the age, the state of the patient, the severity of the condition or disease, the route and frequency of administration, and the particular nature of the peptide to be used.
By "cosmetically or pharmaceutically effective amount" is meant an amount of one or more peptides of the invention that is non-toxic but sufficient to provide the desired effect. The peptides of the invention are used in the cosmetic or pharmaceutical compositions of the invention in cosmetically or pharmaceutically effective concentrations to obtain the desired effect; in a preferred form, between 0.00000001% and 20% by weight, preferably between 0.000001% and 15% by weight, more preferably between 0.0001% and 10% by weight, and even more preferably between 0.0001% 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 comprising an effective amount of a peptide of formula (I) above, or a stereoisomer, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition thereof, in order to achieve better penetration of the active ingredient and/or to improve its pharmacokinetic and pharmacodynamic properties.
The term "delivery system" refers to a diluent, adjuvant, excipient or carrier with which the peptides of the invention are administered, selected from the group consisting of: water, oils or surfactants, including those of petroleum origin, animal origin, vegetable origin, 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 to which the peptides of the invention can be administered are known to those of ordinary skill in the art.
The term "sustained release" is used in a conventional sense to refer to a delivery system that provides a gradual release of a compound over a period of time, and preferably, but not necessarily, has a relatively constant level of release of the compound over the entire period of time.
Examples of delivery systems or slow release systems are liposomes, oleosomes, nonionic surfactant liposome vesicles, ethosomes, millimeter-capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, lipid vesicles, micelles, millimeter-spheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, millimeter-particles, microparticles or nanoparticles. Preferred delivery systems or slow release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions having 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 and without limitation, oral or parenteral routes, including nasal, rectal, subcutaneous implantation or injection, or direct implantation or injection into a particular body part, and preferably a relatively constant amount of these peptides of the invention should be released. The amount of peptide contained in the sustained release system will depend, for example, on the site where 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 the use of a peptide of formula (I) above, or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition as described above, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as described above, for the preparation of a cosmetic 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 the use of a peptide of formula (I) above, or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition as described above, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as described above, for 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 the use of a 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 a cosmetic or pharmaceutical composition as described above, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as described above, in the preparation of a cosmetic or pharmaceutical composition for promoting collagen production.
In another aspect of the present invention there is provided the use of a peptide of formula (I) above, or a stereoisomer thereof, or a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition as described above, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as described above, for the preparation of a cosmetic or pharmaceutical composition for promoting expression of PER1 protein, modulating circadian rhythm of skin and/or repairing damaged cells of skin.
In another aspect of the present invention, there is provided a use of a 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 a cosmetic or pharmaceutical composition as described above, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as described above, in the preparation of a PER1 protein activator.
In order to facilitate understanding of the invention, the meaning of some terms and expressions used in the present invention is described as follows:
in the present invention, the term "skin" is understood to mean the layers that make up it, from the uppermost or stratum corneum to the lowermost or subcutaneous tissue, both endpoints being included. 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 administration of a peptide according to the invention to reduce or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with such a disease or condition. The term "treating" also encompasses the ability to reduce or eliminate the physiological consequences of the disease or disorder.
The term "preventing" 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 condition.
The term "aging" refers to changes experienced by skin with age (natural aging), or by exposure to sunlight (photoaging) or to environmental pollutants such as chemical dirt or pollutants, tobacco smoke, etc., and includes all externally visible and/or perceivable changes by touch, such as, and not limited to: the development of discontinuities in the skin (e.g., wrinkles, fine lines, expression lines, stretch lines, streaks, grooves, irregularities or roughness, increased pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of deformability, loss of resilience), sagging skin (e.g., sagging cheek, under-eye appearance of bags, or appearance of double chin, etc.), changes in skin color (e.g., scarring, redness, bags, or appearance of hyperpigmented areas such as age spots or freckles, etc.), abnormal differentiation, hyperkeratinization, elastosis, keratosis, alopecia, orange-peel-like skin, loss of collagen structure, and other histological changes in the stratum corneum, dermis, epidermis, vascular system (e.g., appearance of spider veins or telangiectasia), or those tissues adjacent to the skin.
The term "photoaging" refers to premature aging of the skin due to prolonged exposure of the skin to ultraviolet radiation, which exhibits the same physiological characteristics as natural aging, for example and without limitation: relaxation, sagging, color changes or pigmentation irregularities, abnormalities and/or hyper-keratinization.
In the present specification, abbreviations for amino acids follow the rules specified in the European journal of biochemistry (Eur.J. biochem.1984, 138:9-37) by the IUPAC-IUB Biochemical nomenclature Commission (IUPAC-IUBCommission of 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 the peptide bond eliminates the OH in the 1-carboxyl group of the amino acid (represented here in 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 Structure of amino acid residues and their single and three letter abbreviations
The abbreviation "Ac-" is used in the present invention to denote acetyl (CH) 3 -CO-)。
The beneficial effects obtained by the invention relative to the prior art include:
1. The peptide disclosed by the invention is convenient to synthesize and high in safety, can increase the activity of skin fibroblasts and promote collagen generation, so that the skin elasticity and/or the skin compactness are increased, and can be used for preventing and even treating skin relaxation, and treating, preventing and/or repairing skin aging or photoaging.
2. The peptide of the invention can promote PER1 protein expression, increase PER1 protein content, participate in the regulation of skin circadian cycle, thereby recovering circadian cycle and resynchronizing skin cell biological clock, and can be used for repairing skin damaged cells and relieving skin aging signs; can also be used for preparing PER1 protein activator.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the drawings that are needed in the description of the present invention will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a 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 ratio (m/z) of the excimer ion peak was 387.2045 and the mass spectrum measured molecular weight was 386.20.
FIG. 2 is 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 ratio (m/z) of the excimer ion peak was 444.2271, mass spectrumThe molecular weight was measured to be 443.23.
FIG. 3 is a graph of the effect of test samples on HaCaT cell activity.
FIG. 4 is a graph of the effect of test samples on HSF cell activity.
FIG. 5 is a graph of the effect of test samples on HSF cell activity after UV treatment.
FIG. 6 is a graph showing the effect of the test sample on collagen content.
FIG. 7 is a graph of the effect of test samples on PER1 protein content.
Detailed Description
In order that the described objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
In the present invention, the abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Biochemical nomenclature Commission 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 (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: a tertiary butyl group; trt: trityl.
EXAMPLE 1H-Ser-Pro-Gly-Gln-NH 2 Is prepared from
1.1 preparation of Fmoc-Linker-Amide Resin
5g of Amide Resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed the Resin and the solvent was removed.
7.1g Fmoc-Linker, 4.4g HOBt were weighed into a drying flask. Dissolving with DMF solvent, cooling in ice water bath for 10min, and activating with DIC for 10min to avoid water vapor.
Adding the activated Fmoc-Linker into the swelled resin, reacting for 2.5 hours, pumping out the reaction liquid, washing the resin, and pumping out the solvent.
Continuing to add Ac 2 O and DIPEA were capped for 1.5h. The resin was washed and the solvent was pumped away.
1.2 Fmoc removal
Fmoc-Linker-Amide Resin was Fmoc-removed twice with 20% piperidine/DMF for 10min each time, sampled K for detection, and developed dark blue. The resin was washed 7 times with DMF and the solvent was removed.
1.3 feeding reaction
11.7g of Fmoc-Gln (Trt) -OH,4.4g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a refrigerator at-18℃for 30min. 6.3mL DIC was added to activate for 3min to avoid water vapor. And adding the activated amino acid into the deprotected resin for reaction for 2 hours, and pumping away the reaction solution. 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 HOBt and 6.3mLDIC using DMF as solvent and the reaction continued for 2h. These 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 using DMF as solvent in the presence of 4.4g of HOBt and 6.3mL of DIC; after the reaction was completed, the resin was washed and the solvent was removed.
The N-terminal Fmoc group of the peptidyl resin was deprotected and Fmoc was removed twice with 20% piperidine/DMF for 10min each time, sampling K and developing dark blue. The Resin was washed 6 times with DMF, the solvent was removed, and after shrinkage drying 12.2g of Ser (tBu) -Pro-Gly-Gln (Trt) -Linker-Amide Resin was obtained.
1.4 cleavage
Measuring 95mL of TFA, 2.5mL of TIS and 2.5mL of water, uniformly mixing and stirring to obtain a lysate, sealing and placing in a refrigerator at the temperature of minus 18 ℃ for later use; the isopropyl ether is placed in a refrigerator at the temperature of minus 18 ℃ for refrigeration for standby.
12.2g of Ser (tBu) -Pro-Gly-Gln (Trt) -Linker-Amide Resin was weighed, added to a round bottom flask, the frozen lysate was added and the reaction was stirred for 2h. Suction filtering, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging, washing for 6 times until pH value is 3-4, and vacuum drying to obtain 4.8g H-Ser-Pro-Gly-Gln-NH 2 Crude peptide.
1.5 purification
Weigh 4.8g of crude peptide in 90mL methanol: in water (V: v=1:2), a clear and transparent solution was obtained by filtration through a microporous filter membrane with a pore size of 0.22 μm, purification by reverse phase HPLC, the purification gradient being as follows:
purifying the filtered sample, 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 Is prepared from
2.1 preparation of Fmoc-Linker-Amide Resin
5g of Amide Resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed the Resin and the solvent was removed.
7.1g Fmoc-Linker, 4.4g HOBt were weighed into a drying flask. Dissolving with DMF solvent, cooling in ice water bath for 10min, and activating with DIC for 10min to avoid water vapor.
Adding the activated Fmoc-Linker into the swelled resin, reacting for 2.5 hours, pumping out the reaction liquid, washing the resin, and pumping out the solvent.
Continuing to add Ac 2 O and DIPEA were capped for 1.5h. The resin was washed and the solvent was pumped away.
2.2 Fmoc removal
Fmoc-Linker-Amide Resin was Fmoc-removed twice with 20% piperidine/DMF for 10min each time, sampled K for detection, and developed dark blue. The resin was washed 7 times with DMF and the solvent was removed.
2.3 feeding reaction
11.7g of Fmoc-Gln (Trt) -OH,4.4g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a refrigerator at-18℃for 30min. 6.3mL DIC was added to activate for 3min to avoid water vapor. And adding the activated amino acid into the deprotected resin for reaction for 2 hours, and pumping away the reaction solution. 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 activated Fmoc-Asn (Trt) -OH was coupled to the peptidyl resin using DMF as solvent in the presence of 4.4g HOBt and 6.3mLDIC, and the reaction was continued for 2h. These 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 using DMF as solvent in the presence of 4.4g of HOBt and 6.3mL of DIC; after the reaction was completed, the resin was washed and the solvent was removed.
The N-terminal Fmoc group of the peptidyl resin was deprotected and Fmoc was removed twice with 20% piperidine/DMF for 10min each time, sampling K and developing dark blue. 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 shrink drying.
2.4 cleavage
Measuring 95mL of TFA, 2.5mL of TIS and 2.5mL of water, uniformly mixing and stirring to obtain a lysate, sealing and placing in a refrigerator at the temperature of minus 18 ℃ for later use; the isopropyl ether is placed in a refrigerator at the temperature of minus 18 ℃ for refrigeration for standby.
16.2g of Ser (tBu) -Pro-Asn (Trt) -Gln (Trt) -Linker-Amide Resin was weighed, added to a round bottom flask, the frozen lysate was added and the reaction was stirred for 2h. Suction filtering, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging, washing for 6 times until pH value is 3-4, and vacuum drying to obtain 5.2g H-Ser-Pro-Asn-Gln-NH 2 Crude peptide.
2.5 purification
Weigh 5.2g of crude peptide in 120mL methanol: in water (V: v=1:2), a clear and transparent solution was obtained by filtration through a microporous filter membrane with a pore size of 0.22 μm, purification by reverse phase HPLC, the purification gradient being as follows:
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, collecting the fraction, concentrating and freeze-drying 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 present invention may be prepared by similar methods.
The molecular weight of these peptides obtained was determined by ESI-MS and the results of the partial peptide tests are shown in Table 2 below and FIGS. 1-2:
table 2 mass spectrometry determination of molecular weight results
Numbering device Sequence(s) Molecular weight mass spectrometry analysis 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), high sugar culture medium (DMEM), fetal bovine serum, and PBS.
4.2 instruments
Enzyme-labeled instrument, CO 2 Incubator, superclean bench.
4.3 cell lines
Human keratinocytes (HaCaT) were purchased from the institute of chinese academy of sciences 'typical culture preservation committee kunming cell bank, and Human Skin Fibroblasts (HSF) were purchased from the institute of chinese academy of sciences' typical culture preservation committee Shanghai cell bank.
4.4 sample to be tested
Drug administration group: the concentrations of peptide (2) and peptide (9) were 6.25ppm, 12.5ppm, 25ppm, 50ppm, 100ppm and 200ppm.
Blank control group: PBS.
Positive control group: 2% DMSO.
4.5 Experimental methods
Respectively taking HaCaT keratinocyte and HSF fibroblast in good exponential growth phase, adding 0.25% trypsin digestion solution, digesting to make adherent cells fall off, counting 1-4×10 5 Cell suspensions were prepared at each mL.
Inoculating cell suspension into 96-well plate at 200 μl/well, and standing at constant temperature with CO 2 Culturing in an incubator for 24 hours.
Changing liquid, adding the administration group, blank control group and positive control group sample respectively, 20 μl/well, placing at 37deg.C and 5% CO 2 Incubate in incubator for 72h.
Then 20. Mu.L of 5mg/ml MTT was added to each well, followed by 5% CO at 37℃ 2 Incubate in incubator for 4h. The original solution was discarded, 150. Mu.L/well of DMSO was added, and after shaking for 5min on a plate shaker, the OD value of each well was measured at a wavelength of 570nm using an ELISA reader, and the cell viability was calculated.
Cell viability= (dosing well OD-zeroing well OD)/(blank well OD-zeroing well OD) ×100%
4.6 experimental results
The MTT method is a method for detecting the survival and growth of cells, and the measured OD value is proportional to the activity of the cells.
The effect of the test sample on HaCaT cell activity is shown in fig. 3, and the result shows that compared with the blank control group, the positive control group has obviously reduced HaCaT cell activity, which indicates that 2% of DMSO has toxic effect on HaCaT cells, the administration group has no toxic effect on HaCaT cells within the range of 200ppm, and the peptide (2) and the peptide (9) can obviously improve cell activity at the low concentration of 6.25ppm, promote the proliferation of HaCaT cells, and the proliferation of the cells is enhanced along with the increase of the concentration.
The effect of the test sample on the activity of HSF cells is shown in FIG. 4, and the result shows that compared with the blank control group, the activity of HSF cells in the positive control group is obviously reduced, which shows that 2% of DMSO has toxic effect on HSF cells, the administration group has no toxic effect on HSF fibroblasts within the range of 200ppm, and the peptide (2) and the peptide (9) can obviously improve the activity of cells at the low concentration of 6.25ppm, promote the proliferation of HSF fibroblasts, and the proliferation of the cells is enhanced along with the increase of the concentration.
From this, it is clear that the peptide of the present invention has no toxic effect on HaCaT keratinocytes and HSF fibroblasts, and can also enhance the activity of the cells and promote the proliferation thereof, thereby increasing skin elasticity and/or skin firmness, and can be used for preventing or even treating skin laxity.
Example 5 photo aging test
5.1 reagents and materials
Fetal bovine serum, high sugar medium (DMEM), penicillin, streptomycin, thiazole blue (MTT).
5.2 instruments
Enzyme-labeled instrument, CO 2 Incubator, superclean bench.
5.3 cell lines
Human Skin Fibroblasts (HSF) were purchased from the national academy of sciences typical culture collection committee Shanghai cell bank.
5.4 sample to be tested
Drug administration group:
Reference 1 (Palm-Lys-Thr-Thr-Lys-Ser) at test concentrations of 12.5ppm, 25ppm, 50ppm, respectively;
reference 2 (Ser-Pro-Leu-Gln-NH) 2 ) The test concentrations were 12.5ppm, 25ppm, 50ppm, respectively;
peptide (2) at test concentrations of 12.5ppm, 25ppm, 50ppm, respectively;
peptide (9) at test concentrations of 12.5ppm, 25ppm, 50ppm, respectively.
Blank control group: PBS.
UV group: UV radiation, PBS was added.
5.5 Experimental methods
Taking HSF fibroblasts in an exponential growth phase, adding 0.25% trypsin digestion solution, and digesting to adhere the cellsFalling off and counting 1-4 x 10 5 Cell suspensions were prepared at each mL.
10000 cell suspensions per well are properly diluted and inoculated on a 96-well plate, and when the cells grow to about 80%, a UV photoaging model is established. The blank group was supplemented with 50 μl of LPBS, medium was supplemented to 200 μl, and UV irradiation was not performed; UV group and administration group, adding appropriate amount of PBS, repeatedly washing to colorless, adding 50 μLPBS, and mixing at 80mJ/cm 2 Irradiating under UV lamp, wherein the distance between the lamp source and the culture flask is 15cm, removing PBS after irradiation, adding PBS solution and culture medium to 200 μl in UV group, and adding culture solution and multiple dilution medicine to 200 μl in administration group. The blank, UV, and dosing groups continued at 37℃with 5% CO 2 Incubate in incubator for 24h.
Then 20. Mu.L of 5mg/mL MTT was added per well and the reaction was continued at 37℃with 5% CO 2 Incubate in incubator for 4h. The original solution was discarded, 150. Mu.L/well DMSO was added, and after shaking on a plate shaker for 5min, the reference OD values at 490nm and 630nm wavelengths were read using an ELISA reader.
5.6 experimental results
Skin aging is affected by endogenous and exogenous factors, such as genetics, environmental exposure, ultraviolet radiation, hormonal changes, and the like. The accumulation of these factors, particularly the irradiation of ultraviolet light, leads to alterations in the structure, function and appearance of the skin. The experiment selects 80mJ/cm 2 UV energy is radiated to build up a skin photoaging model.
The experimental results are shown in fig. 5, and after UV radiation, the activity of the HSF fibroblasts is significantly reduced, which indicates that the photoaging model is successfully established; the administration group can improve the cell activity within the range of 50ppm, so that the cell aging is improved, and obvious anti-photoaging effect is generated; compared with the reference substances 1 and 2, the peptides (2) and (9) can obviously improve the cell activity and obviously promote the proliferation of the HSF fibroblasts; wherein, the peptide (2) can greatly improve the activity of the HSF fibroblast at a low concentration of 12.5ppm, and has obvious anti-photoaging effect. From this, it is clear that 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 to the prior art.
EXAMPLE 6 collagen content test
6.1 reagents and materials
Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, BCA protein kit, collagen I ELISA kit.
6.2 instruments
Enzyme-labeled instrument, CO 2 Incubator, superclean bench, incubator.
6.3 cell lines
Human Skin Fibroblasts (HSF) were purchased from the national academy of sciences typical culture collection committee Shanghai cell bank.
6.4 sample to be tested
Drug administration group:
reference 1 (Palm-Lys-Thr-Thr-Lys-Ser) at test concentrations of 12.5ppm and 25ppm, respectively;
reference 2 (Ser-Pro-Leu-Gln-NH) 2 ) The test concentrations were 12.5ppm and 25ppm, respectively;
peptide (2) at test concentrations of 12.5ppm and 25ppm, respectively;
peptide (9) at test concentrations of 12.5ppm and 25ppm, respectively.
Blank control group: PBS.
UV group: UV radiation, PBS was added.
6.5 Experimental methods
Taking HSF fibroblasts in an exponential growth phase, adding 0.25% trypsin digestion solution, digesting to enable adherent cells to fall off, and counting 1-4×10 6 Cell suspensions were prepared at each mL.
The 100000 cell suspensions per well are properly diluted and inoculated on a 6-well plate, and the model is built when the cells grow to about 80 percent. 200. Mu.L of PBS was added to the blank, medium was supplemented to 800. Mu.L, and UV irradiation was not performed; UV group and administration group, adding appropriate amount of PBS, repeatedly washing to colorless, adding 200 μl PBS, and standing at 80mJ/cm 2 And irradiating under a UV lamp, wherein the distance between the lamp source and the culture flask is 15cm. After irradiation, PBS was discarded, the UV group was added to the PBS solution and medium to 800. Mu.L, and the dosing group was added to the medium and the drug was diluted to 800. Mu.L. The blank, UV, and dosing groups continued at 37℃with 5% CO 2 Incubate in incubator for 48h.
After the end of the culture, the 1 st well cells were digested and counted and diluted to 0.5X10 6 After cells in the remaining wells were scraped off with cell scrapes and resuspended in 500. Mu.L, 50. Mu.L of ultrasound was applied to all wells for 30s and the total protein was measured by BCA, and the other wells were diluted to a concentration of 0.5X10 s according to the 1 st well protein concentration 6 And each mL. Ultrasonically crushing the cell suspension with the adjusted concentration for 30s, centrifuging at 1500x N for 15min, and collecting cell supernatant to obtain a sample liquid; the operation was performed according to the collagen I ELISA kit protocol. The OD of each well was measured sequentially with an microplate reader at 450nm over 15 min.
6.6 experimental results
Aging of the skin is closely related to collagen, which is the most abundant protein found in connective tissue, and has an important role in skin plumping and tightening. In an ultraviolet overexposure environment, collagen production is inhibited. The experiment adopts a test sample to treat cells after ultraviolet radiation, and detects the content of collagen I in corresponding cells so as to determine whether the peptide of the invention can promote collagen generation.
The results of the effect of the test sample on collagen content are shown in fig. 6. The results show that the collagen content of the UV group is significantly reduced compared with that of the blank control group; compared with the UV group, the reference 1 and the reference 2 can not increase the collagen content in cells after ultraviolet irradiation, but the peptide (2) and the peptide (9) can obviously increase the collagen content within the range of 12.5-25ppm, promote the collagen expression and have excellent effect of promoting the collagen generation.
From the results, the peptide of the invention can promote collagen production, increase collagen content, thereby increasing skin elasticity and/or skin firmness, can be used for preventing and even treating skin relaxation, treating, preventing and/or repairing skin aging or photoaging, and can be used for resisting skin aging in the fields of cosmetics or medicines.
Example 7PER1 protein content test
7.1 reagents and materials
Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, human circadian protein 1 (PER 1) ELISA kit.
7.2 instruments
Enzyme-labeled instrument, CO 2 Incubator, superclean bench, incubator.
7.3 cell lines
Human Skin Fibroblasts (HSF) were purchased from the national academy of sciences typical culture collection committee Shanghai cell bank.
7.4 sample to be tested
Drug administration group:
reference 2 (Ser-Pro-Leu-Gln-NH) 2 ) The test concentration was 25ppm;
peptide (2) at a test concentration of 25ppm.
Blank control group: PBS.
7.5 Experimental methods
Taking HSF fibroblasts in an exponential growth phase, adding 0.25% trypsin digestion solution, digesting to enable adherent cells to fall off, and counting 1-4×10 6 Cell suspensions were prepared at each mL.
The 100000 cell suspensions per well are properly diluted and inoculated on a 6-well plate, and the model is built when the cells grow to about 80 percent. 200 mu L of PBS was added to the blank group, and the medium was supplemented to 800 mu L; adding appropriate amount of PBS, repeatedly washing to colorless, adding culture medium and medicine to 800 μl, placing each group at 37deg.C and 5% CO 2 Incubate in incubator for 48h.
Scraping off cells by using the cells after the culture is finished, blowing and uniformly mixing, ultrasonically crushing the cell suspension for 30s, centrifuging at 1500x for 15min, and collecting cell supernatant to obtain a sample liquid; the procedure was as PER1 protein ELISA kit protocol. The OD of each well was measured sequentially with an microplate reader at 450nm over 15 min.
7.6 experimental results
Circadian rhythm protein 1 (PER 1) is one of biological clock core components for regulating circadian rhythm period of skin, participates in regulating reaction process of skin to environmental pressure, protects skin from various environmental injuries such as UV radiation, temperature, chemical and physical injury, microbial infection and the like, has certain correlation with aging in expression of PER1 protein, and can improve skin aging to a certain extent by improving content of PER1 protein. The present experiment determines 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 effect of the test sample on PER1 protein content is shown in FIG. 7. The results show that the 25ppm peptide (2) can significantly increase the content of PER1 protein and promote the expression of PER1 protein compared with the blank group, and the peptide (2) of the invention has better effect of promoting the expression of PER1 protein compared with the reference 2.
From the results, the peptide of the invention can promote PER1 protein expression, increase PER1 protein content, participate in the regulation of the circadian cycle of skin, thereby recovering the circadian cycle and resynchronizing the biological clock of skin cells, and can be used for repairing damaged cells of skin and relieving the signs of skin aging; can also be used for preparing PER1 protein activator.
Example 8 preparation of essence containing peptide (2)
INCI name Weight percent
Peptide (2) 0.05
Vitamin C 1
Glycerol 5
Ardisia japonica glue 5
Transparent and transparentSodium salt of 0.4
Octyl glycol 0.25
1, 2-hexanediol 0.25
15% triethanolamine 0.04
Water and its preparation method Moderate to 100
Adding the sodium hyaluronate with the prescription amount into water, stirring to uniformly mix, heating to 80-85 ℃, and carrying out heat preservation and stirring to uniformly disperse. Cooling to below 40deg.C, adding glycerol, ardisia, peptide (2), vitamin C, octanediol and 1, 2-hexanediol, and stirring. Regulating the pH value of the solution to about 5.5 by using 15% triethanolamine.
EXAMPLE 9 preparation of liposomes containing peptide (9)
Composition of the components Weight percent
Dipalmitoyl phosphatidylcholine 4.0
Peptide (9) 0.2
Preservative agent 0.5
Water and its preparation method Moderate to 100
Dipalmitoyl phosphatidylcholine was weighed and dissolved in chloroform. Evaporating the solvent under vacuum until a thin layer of phospholipid is obtained, and hydrating the layer by treating it 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 single-chamber liposome with smaller and uniform size.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, but is not intended to limit the practice of the invention to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the invention, and the invention is not limited to the above-mentioned embodiments.

Claims (19)

1. A peptide of formula (I) 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) of the present invention,
X 1 is-Gly-;
R 1 is H;
R 2 is-OH or-NH 2 。
2. The peptide of formula (I) or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof according to claim 1, characterized by being selected from the following peptides (2):
(2)H-Ser-Pro-Gly-Gln-NH 2 。
3. The peptide of formula (I) or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof according to any one of claim 1 to 2,
the cosmetically or pharmaceutically acceptable salts include metal salts of peptides of formula (I), the metals including: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.
4. The peptide of formula (I) or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 2, wherein the cosmetically acceptable salt 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.
5. The peptide of formula (I) or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 2, wherein the cosmetically acceptable salt or pharmaceutically acceptable salt comprises a salt of the peptide of formula (I) with an inorganic or organic acid, the organic acid comprising: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.
6. A cosmetic or pharmaceutical composition comprising an effective amount of a peptide of formula (I) or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 5, together with at least one excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant.
7. The cosmetic or pharmaceutical composition according to claim 6, wherein the adjuvant is selected from the group consisting of: collagen synthesis stimulators, agents that modulate PGC-1 alpha synthesis, agents that modulate PPARgamma activity, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic or lipolysis-stimulating agents, lipolytic agents, adipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulating or inhibiting agent, whitening or depigmenting agent, pigmentation promoting agent, self-tanning agent, antiaging agent, NO-synthase inhibitor, 5α -reductase inhibitor, and pigment inhibitors of lysyl hydroxylase and/or prolyl hydroxylase, antioxidants, free radical scavengers and/or agents against atmospheric pollution, active carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioning agents, moisture retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermohydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating the lower eye bags, exfoliants, antimicrobial agents, bactericides, bacteriostats, agents capable of stimulating the synthesis of dermal or epidermal macromolecules and/or inhibiting or preventing 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, an agent that stimulates heat shock protein synthesis, an agent that stimulates hyaluronic acid synthesis, an agent that stimulates fibronectin synthesis, an agent that stimulates deacetylase synthesis, an agent that stimulates synthesis of lipids and stratum corneum components, ceramides, fatty acids, an agent that inhibits collagen degradation, an agent that inhibits elastin degradation, an agent that inhibits serine proteases, an agent that stimulates fibroblast proliferation, an agent that stimulates keratinocyte proliferation, an agent that stimulates adipocyte proliferation, an agent that stimulates melanocyte proliferation, an agent that stimulates keratinocyte differentiation, an agent that inhibits acetylcholinesterase, a skin relaxant, an agent that stimulates glycosaminoglycan synthesis, an anti-hyperkeratosis agent, an acne lytic agent, an anti-psoriasis agent, an anti-rash agent, a DNA repair agent, a DNA protectant, a stabilizer, an antipruritic agent, an agent for treating and/or caring for sensitive skin solidifying agents, tightening agents, restructuring agents, stretch-proofing agents, agents that modulate sebum production, antiperspirant agents, agents that stimulate healing, agents that assist healing, agents that stimulate re-epithelialization, agents that assist re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents that act on capillary circulation and/or microcirculation, agents that stimulate angiogenesis, agents that inhibit vascular permeability, intravenous tonicity agents, agents that act on cellular metabolism, agents for improving dermal-epidermal junction, agents that induce hair growth, hair growth inhibition or delay agents, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents derived from biofermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents that are effective against a and/or B uv light, or mixtures thereof.
8. Cosmetic or pharmaceutical composition according to claim 6 or 7, characterized in that the formulation of said cosmetic or pharmaceutical composition is selected from: cream, oil, balm, foam, lotion, gel, wipe, slurry, ointment, mousse, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, elixir, polysaccharide film, jelly, or gelatin.
9. The cosmetic or pharmaceutical composition according to claim 8, wherein the capsule comprises: soft capsule and hard capsule.
10. The cosmetic or pharmaceutical composition according to claim 9, wherein the capsule is a gelatin capsule.
11. The cosmetic or pharmaceutical composition according to claim 8, wherein the tablet comprises: sugar-coated tablets.
12. A cosmetically or pharmaceutically acceptable delivery system or slow release system comprising an effective amount of a peptide of formula (I) according to any one of claims 1 to 5 or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6 to 11;
The cosmetically or pharmaceutically acceptable delivery system or slow release system is selected from: liposomes, oleosomes, nonionic surfactant liposome vesicles, ethosomes, millimeter capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, lipid vesicles, micelles, millimeter spheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, millimeter particles, microparticles or nanoparticles.
13. The cosmetically or pharmaceutically acceptable delivery system or slow release system of claim 12 wherein the cosmetically or pharmaceutically acceptable delivery system or slow release system is a liposome or microemulsion.
14. The cosmetically or pharmaceutically acceptable delivery system or slow release system of claim 13 wherein the microemulsion is a water-in-oil microemulsion having an internal structure of reverse micelles.
15. Use of a peptide of formula (I) according to any one of claims 1 to 5 or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6 to 11, or a cosmetically or pharmaceutically acceptable delivery system or slow release system according to any one of claims 12 to 14, for the preparation of a cosmetic or pharmaceutical composition for the treatment, prevention or repair of skin ageing and/or photoaging.
16. Use of a peptide of formula (I) according to any one of claims 1 to 5 or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6 to 11, or a cosmetically or pharmaceutically acceptable delivery system or slow release system according to any one of claims 12 to 14, for the preparation of a cosmetic or pharmaceutical composition for increasing skin elasticity and/or skin firmness.
17. Use of a peptide of formula (I) according to any one of claims 1 to 5 or a cosmetically or pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6 to 11, or a cosmetically or pharmaceutically acceptable delivery system or slow release system according to any one of claims 12 to 14, for the preparation of a cosmetic or pharmaceutical composition for promoting collagen production.
18. Use of a peptide of formula (I) according to any one of claims 1 to 5 or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6 to 11, or a cosmetically or pharmaceutically acceptable delivery system or slow release system according to any one of claims 12 to 14, for the preparation of a cosmetic or pharmaceutical composition for promoting PER1 protein expression, regulating circadian rhythm of skin and/or repairing damaged cells of skin.
19. Use of a peptide of formula (I) according to any one of claims 1 to 5 or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6 to 11, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system according to any one of claims 12 to 14, for the preparation of a PER1 protein activator.
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