A hexapeptide compound, its composition and use

Abstract

Disclosed is a hexapeptide compound, its composition and use, relating to the technical field of polypeptides. The peptide or its salt has the structure shown in formula (I): R1-Val-Leu-Gln-Trp-Val-Lys-R2 (I). Specifically, it relates to the peptide or its salt, or their composition, and their use in the preparation of a composition for caring for or treating the skin or mucous membranes.

Description

Technical Field

[0001] The present disclosure relates to the field of polypeptide technology, and in particular to a hexapeptide compound, a composition thereof, and uses thereof. Background Art

[0002] Skin aging is a complex process, driven by both intrinsic aging and exogenous photoaging. It involves multiple factors, including dermal matrix degradation, elastic fiber rupture, and excessive muscle contraction. Muscle contraction is closely linked to the release of acetylcholine, for which calcium ions are essential. When neurons are excited, nerve impulses are transmitted to the axon terminal, triggering the opening of voltage-gated calcium channels and an influx of extracellular calcium ions. This influx of calcium ions prompts synaptic vesicles containing acetylcholine to migrate toward the presynaptic membrane. Under the influence of calcium ions, these vesicles fuse with the presynaptic membrane, releasing acetylcholine into the synaptic cleft. Acetylcholine acts on the postsynaptic membrane, leading to muscle contraction. Furthermore, acetylcholine binding to acetylcholine receptors on muscle cells triggers the opening of ion channels, increasing the permeability of the cell membrane to calcium ions and allowing a large influx of calcium ions, which also triggers muscle contraction. Research has shown that an imbalance in calcium homeostasis is a key factor in the sustained contraction of facial muscles. Excessive muscle contraction can lead to wrinkles. Therefore, inhibiting calcium influx can inhibit muscle contraction and thus improve wrinkles.

[0003] In terms of collagen metabolism, with aging or external stimuli, the synthesis rate of type I and type III collagen in the dermis decreases, while matrix metalloproteinase activity increases abnormally, leading to the gradual collapse of the skin's supporting structure and a loss of elasticity and firmness. Among external environmental factors, ultraviolet light is a significant contributor to collagen loss. Photoaging is a major cause of skin aging. Prolonged exposure to ultraviolet light and other rays can cause wrinkles, sagging, and other problems on the skin, prematurely entering a state of aging. Hyaluronidase, on the other hand, is an enzyme that hydrolyzes hyaluronic acid, reducing the viscosity of the intercellular matrix and increasing the permeability of fluids in tissues. However, overactivation of hyaluronidase accelerates the degradation of hyaluronic acid. As a key moisturizing factor in the skin, its degradation reduces the skin's ability to store water, leading to a decrease in the water content of the stratum corneum. This can cause the skin to exhibit a range of adverse conditions, such as roughness, dryness, and sensitivity, further exacerbating skin aging.

[0004] Skin aging involves many factors, and it is of great significance to develop an active substance that can exert multiple functions. Summary of the Invention

[0005] The present disclosure relates to a hexapeptide compound, a composition thereof, and uses thereof. The peptides and compositions containing the peptides have the effects of caring for or treating skin or mucous membranes.

[0006] In one aspect, the present disclosure provides a peptide represented by formula (I), or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof,

[0007] R1-Val-Leu-Gln-Trp-Val-Lys-R2(I)

[0008] In formula (I),

[0009] R1 is selected from: H or R3-CO-, wherein R3 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

[0010] R2 is selected from: -NR4R5 or -OR4, wherein each R4 and R5 are independently selected from: H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

[0011] The alkyl group refers to a saturated aliphatic linear or branched alkyl group having 1 to 24 carbon atoms (or 1 to 16 carbon atoms; or 1 to 14 carbon atoms; or 1 to 12 carbon atoms; or 1, 2, 3, 4, 5 or 6 carbon atoms); in some embodiments, the alkyl group is selected from: methyl, ethyl, isopropyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 2-ethylhexyl, 2-methylbutyl or 5-methylhexyl;

[0012] The alkenyl group refers to a straight or branched alkenyl group having 2 to 24 carbon atoms (or 2 to 16 carbon atoms; or 2 to 14 carbon atoms; or 2 to 12 carbon atoms; or 2, 3, 4, 5 or 6 carbon atoms); the alkenyl group has one or more carbon-carbon double bonds, and in some embodiments, the alkenyl group has 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bonded to the rest of the molecule through a single bond; in some embodiments, the alkenyl group is selected from: vinyl, oleyl or linoleyl;

[0013] In some embodiments, the substituents in the "substituted alkyl" and "substituted alkenyl" are selected from C1-C4 alkyl; hydroxy; C1-C4 alkoxy; amino; C1-C4 aminoalkyl; C1-C4 carbonyloxy; C1-C4 oxycarbonyl; halogen (such as fluorine, chlorine, bromine, and iodine); cyano; nitro; azide; C1-C4 alkylsulfonyl; thiol; C1-C4 alkylthio; C6-C 30 Aryloxy such as phenoxy; -NR b (C=NR b )NR b R c , where R b and R care independently selected from: H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C 10 Cycloalkyl, C6-C 18 Aryl, C7-C 17 an aralkyl group, a three- to ten-membered heterocyclic group, or a protecting group for an amino group.

[0014] In some embodiments, R1 is selected from: H, acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl or linoleoyl; R4 and R5 are independently selected from: H, methyl, ethyl, hexyl, dodecyl or hexadecyl;

[0015] In some embodiments, R1 is selected from H, acetyl, lauroyl, myristoyl, or palmitoyl; R4 is H and R5 is selected from H, methyl, ethyl, hexyl, dodecyl, or hexadecyl;

[0016] In some embodiments, R1 is H, acetyl, lauroyl, myristoyl, or palmitoyl; and R2 is -OH or -NH2.

[0017] In some embodiments, when R1 is H and R2 is -OH, the structure of the peptide is Cyclo-[Val-Leu-Gln-Trp-Val-Lys], and its structural formula is as follows:

[0018] .

[0019] The peptides of the present invention contain a large number of asymmetric carbon atoms. It is understood by those skilled in the art that the peptides of the present invention have stereoisomers and can exist as stereoisomers or mixtures of stereoisomers, and therefore it is possible to obtain isomeric mixtures and racemic mixtures or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and the isomers or isomeric mixtures present. In some embodiments, the peptides of the present invention are pure isomers, that is, enantiomers or diastereomers. In some embodiments, the structure of the peptides of the present invention is the L-isomer.

[0020] The present disclosure also includes all suitable isotopic variants of the above-mentioned peptides. Isotopic variants of the peptides of the present disclosure are understood herein to mean compounds in which at least one atom in the peptides of the present disclosure is replaced by another atom of the same atomic number, but the atomic mass of the other atom is different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be incorporated into the peptides of the present disclosure are those of hydrogen, carbon, nitrogen or oxygen, e.g. 2 H (deuterium), 3 H (tritium), 13 C.14 C. 15 N. 17 O or 18 O. Certain isotopic variants of the peptides disclosed herein (particularly those into which one or more radioactive isotopes have been incorporated) may be useful, for example, for examining the mechanism of action or distribution of the active compound in vivo; due to their relative ease of preparation and detectability, particularly with 3 H or 14 Compounds labeled with a C isotope are suitable for this purpose. In addition, due to the greater metabolic stability of the compound, the incorporation of an isotope (e.g., deuterium) can produce specific therapeutic benefits, such as an extension of the in vivo half-life or a reduction in the required active dose. Isotopic variants of the peptides of the present disclosure can be prepared by methods known to those skilled in the art, for example, by the methods further described below and in the examples, by using corresponding isotopic modifications of the respective reagents and / or starting materials.

[0021] The term "salt" refers to a salt approved for use in animals, and more specifically, humans, including metal salts of the peptides of the present disclosure, the metals including, but not limited to, lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum; including salts formed between the peptides of the present disclosure and organic bases, the organic bases including, but not limited to, ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine; including salts formed between the peptides of the present disclosure and inorganic or organic acids, the organic acids including, but not limited to, acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoate or gluconic acid; the inorganic acids including, but not limited to, hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0022] The nature of the salt is not critical and salts of the disclosed peptides may be obtained by conventional methods well known in the art.

[0023] The synthesis of the peptides of the present invention, or their stereoisomers, or mixtures of stereoisomers, or salts thereof can be carried out according to conventional methods known in the art, such as solid phase synthesis, liquid phase synthesis, or a combination of solid phase and liquid phase methods. They can also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins of animal, fungal, or plant origin.

[0024] For example, a method of obtaining the peptides of the present disclosure comprises the following steps:

[0025] - 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;

[0026] - Elimination of the group protecting the N-terminus;

[0027] - repeating this coupling sequence and eliminating the group protecting the N-terminus until the desired peptide sequence is obtained;

[0028] - Elimination of the group protecting the C-terminus or cleavage from the solid support.

[0029] In some embodiments, the C-terminus is bound to a solid support and the method is carried out on a solid phase, comprising coupling an amino acid having a protected N-terminus and a free C-terminus to an amino acid having a free N-terminus and a C-terminus bound to a polymer support; eliminating the group protecting the N-terminus; and repeating this sequence as many times as required to thereby obtain a peptide of the desired length, followed by cleavage of the synthesized peptide from the initial polymer support, and coupling and cyclizing the amino group at the N-terminus of the peptide chain to the carboxyl group at the C-terminus, followed by elimination of the group protecting the side chain.

[0030] The functional groups of the side chains of these amino acids remain adequately protected with temporary or permanent protecting groups throughout the synthesis.

[0031] In some embodiments, solid phase synthesis can be performed by a convergent strategy of coupling a dipeptide or tripeptide to a polymer support or to a dipeptide or amino acid previously bound to a polymer support.

[0032] Due to the application outside the body of a mammal, the peptides of the present disclosure can form part of various types of compositions. Therefore, another aspect of the present disclosure provides a composition comprising an effective amount of the above-mentioned peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, and at least one excipient and optionally an adjuvant. The composition can be prepared by conventional methods known to those skilled in the art.

[0033] In some embodiments, the adjuvant is selected from the group consisting of analgesics, agents that inhibit PAR-2 ​​activity, agents that regulate PGC-1α synthesis, agents that regulate PPARγ activity, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic agents or agents that stimulate lipolysis, lipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulators or inhibitors, whitening agents or depigmenting agents, pro-pigmentation agents, self-tanning agents, anti-aging agents, NO-synthase inhibitors, 5α-reductase inhibitors, inhibitors of lysyl hydroxylase and / or prolyl hydroxylase, antioxidants, free radical scavengers. and / or anti-atmospheric pollution agents, active carbonyl scavengers, anti-glycation agents, antihistamines, antivirals, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, moisture-retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating under-eye bags, keratolytic agents, antimicrobial agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and / or can 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 chaperone protein synthesis, agents that stimulate cAMP synthesis, agents that stimulate hyaluronic acid synthesis, agents that stimulate fibronectin synthesis, agents that stimulate deacetylase synthesis, agents that stimulate the synthesis of lipids and stratum corneum components, ceramides, fatty acids, agents that inhibit elastin degradation, agents that inhibit serine proteases, agents that stimulate fibroblast proliferation, agents that stimulate keratinocyte proliferation, agents that stimulate adipocyte proliferation, agents that stimulate melanocyte proliferation, agents that stimulate keratinocyte differentiation, agents that inhibit acetylcholinesterase, skin relaxants, agents that stimulate glycosaminoglycan synthesis, anti-hyperkeratosis agents, comedolytic agents, anti-psoriatic agents, anti-eczema agents, DNA repair agents, DNA protective agents, stabilizers, antipruritic agents, for the treatment and / or care of sensitive skin agents that act on the capillary circulation and / or microcirculation, agents that stimulate angiogenesis, agents that inhibit vascular permeability, agents that act on venous tension, agents that act on cellular metabolism, agents for improving the dermal-epidermal junction, agents that induce hair growth, agents that inhibit or delay hair growth, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents obtained from biological fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents effective against ultraviolet A and / or UVB rays, or mixtures thereof.

[0034] The effective amount of the disclosed peptides to be administered, and their dosage, will depend on a number of factors, including the age, condition of the user, severity of the condition, route and frequency of administration, and the specific nature of the peptide to be used.

[0035] "Effective amount" means a non-toxic amount of one or more peptides of the present disclosure that is sufficient to provide the desired effect. The peptides of the present disclosure are used in the compositions of the present disclosure at concentrations effective to achieve the desired effect. In some embodiments, the concentration is between 0.00000001% (by weight) and 20% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.000001% (by weight) and 15% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.0001% (by weight) and 10% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.0001% (by weight) and 5% (by weight) relative to the total weight of the composition.

[0036] Another aspect of the present disclosure provides a delivery system or sustained-release system for achieving better penetration of active ingredients, which comprises an effective amount of the above peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above composition.

[0037] The term "delivery system" refers to a diluent, adjuvant, excipient or carrier to be administered with the peptides of the present invention, selected from the group consisting of water, oil or surfactant, including those of petroleum origin, animal origin, plant origin, or synthetic origin, such as, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynol ether, poloxamers, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin and the like. Those of ordinary skill in the art are aware of the diluents, adjuvants, excipients or carriers that can be used in different delivery systems for administering the peptides of the present invention.

[0038] The term "sustained release" is used in its conventional sense to refer to a delivery system that provides for the gradual release of a compound over a period of time. In some embodiments, a sustained release system has a relatively constant level of compound release over a period of time.

[0039] Examples of delivery systems or sustained-release systems include, but are not limited to, liposomes, oleosomes, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion compounds, lipid vesicles, micelles, millispheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, milliparticles, microparticles, or nanoparticles.

[0040] Another aspect of the present disclosure provides a cosmetic comprising an effective amount of the above peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above composition, or the above delivery system or sustained-release system.

[0041] In some embodiments, the dosage form of the cosmetic comprises ointment, cream, emulsion, aqueous solution, oil, gel, powder, tablet, mud, patch, film, aerosol, spray, freeze-dried preparation or nano preparation.

[0042] Another aspect of the present disclosure provides a use of the above-mentioned peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in preparing a composition for caring for or treating skin or mucous membranes.

[0043] Another aspect of the present disclosure provides a use of the above-mentioned peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in the preparation of a composition for anti-aging, repairing, soothing or moisturizing.

[0044] Another aspect of the present disclosure provides a use of the above-mentioned peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in preparing a composition for inhibiting hyaluronidase activity; or in preparing a composition for promoting collagen production; or in preparing a composition for increasing skin elasticity and / or improving skin firmness; or in preparing a composition for promoting re-epithelialization or healing of skin or mucous membranes; or in preparing a composition for treating, preventing or repairing photoaging of the skin; or in preparing a composition for reducing, preventing or treating wrinkles; or in preparing a composition for inhibiting calcium ion influx.

[0045] Another aspect of the present disclosure provides a use of the above-mentioned peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in the preparation of a composition for treating or caring for conditions, disorders or diseases caused by muscle contraction.

[0046] In some embodiments, the condition, disorder, or disease is dystonia.

[0047] In some embodiments, the dystonia comprises focal dystonia, segmental dystonia, multifocal dystonia, or hemidystonia;

[0048] In some embodiments, the focal dystonia comprises hemifacial spasm, torsion dystonia, cervical dystonia or torticollis, laryngeal dystonia or spasmodic dysphonia, oromandibular dystonia, limb dystonia, nocturnal bruxism, hemifacial spasm, tics, or strabismus;

[0049] In some embodiments, the limb dystonia comprises writer's cramp, musician's cramp, or foot dystonia;

[0050] In some embodiments, the segmental dystonia comprises Meige's syndrome;

[0051] In some embodiments, the dystonia comprises dopamine-responsive dystonia.

[0052] Another aspect of the present disclosure provides use of the above-mentioned peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in preparing cosmetics.

[0053] In the present disclosure, the term "skin" is understood to include the multiple layers comprising it, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both ends inclusive. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes. In the present disclosure, the term "skin" includes the scalp.

[0054] The term "skin care" refers to the maintenance and care of the skin, improving the condition of the skin, making the skin delicate, smooth, tender and healthy.

[0055] The term "treating" refers to administering a peptide according to the present disclosure to alleviate or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with the disease or condition. The term "treating" also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or condition.

[0056] The term "prevent" refers to the ability of the disclosed peptides to prevent, delay, or hinder the onset or development of a disease or condition before it occurs.

[0057] The term "repair" refers to the ability of the disclosed peptides to improve, alleviate, or restore a disease or condition after it occurs.

[0058] The term "aging" refers to changes that the skin undergoes with age (natural aging) or through exposure to sunlight (photoaging) or to environmental pollutants, such as chemical dirt or pollutants, tobacco smoke, etc., and includes all changes that are outwardly visible and / or perceptible by touch, such as and not limited to: the development of discontinuities in the skin (such as wrinkles, fine lines, expression lines, stretch lines, striae, furrows, unevenness or roughness, increase in pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of ability to recover from deformation, loss of resilience), sagging of the skin (such as sagging cheeks, bags under the eyes, or the development of a double chin), changes in skin color (such as scarring, redness, bags under the eyes, or the development of areas of hyperpigmentation such as age spots or freckles), abnormal differentiation, hyperkeratinization, elastosis, keratosis, hair loss, orange peel skin, loss of collagen structure, and other histological changes in the stratum corneum, dermis, epidermis, vasculature (such as the development of spider veins or telangiectasias), or those tissues adjacent to the skin.

[0059] The term "photoaging" refers to the premature aging of the skin due to long-term exposure to ultraviolet radiation, which presents the same physiological characteristics as natural aging, such as but not limited to: loosening, sagging, color changes or irregular pigmentation, abnormalities and / or hyperkeratinization.

[0060] The present disclosure has the following advantages and effects:

[0061] The peptides disclosed herein can effectively inhibit calcium ion influx, thereby inhibiting muscle contraction, and can be used to reduce, prevent or treat wrinkles. By inhibiting calcium ion influx, the peptides disclosed herein can be used to treat or manage conditions, disorders or diseases caused by muscle contraction.

[0062] The peptides disclosed herein can effectively increase collagen content in cells exposed to ultraviolet radiation and can be used to treat, prevent, or repair skin photoaging. By promoting collagen production, the peptides disclosed herein can help increase skin elasticity, improve skin firmness, and promote re-epithelialization or healing of the skin or mucous membranes.

[0063] The peptide disclosed herein can effectively inhibit hyaluronidase activity, repair the skin barrier, and has the effects of repairing, soothing, and moisturizing.

[0064] The peptide disclosed herein has anti-aging, firming, anti-wrinkle, repairing, soothing, moisturizing and other effects, and can be used to care for or treat the skin or mucous membranes. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the technical solution of the present disclosure, the following briefly introduces the drawings required for use in the description of the present disclosure. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0066] Figure 1 The cyclic hexapeptide A (molecular formula C) in Example 1 of the present disclosure is 38 H 59 N9O7) mass spectrum.

[0067] Figure 2 It is the hexapeptide B (molecular formula C) in Example 2 of the present disclosure. 38 H 61 N9O8) mass spectrum. DETAILED DESCRIPTION

[0068] To make the objectives, features, and advantages of the present disclosure more readily apparent, the present disclosure is further described below in detail with reference to the accompanying drawings and examples. It should be understood that the described embodiments are only a portion of the embodiments of the present disclosure, and not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present disclosure without inventive effort are intended to fall within the scope of protection of the claims appended to the present disclosure.

[0069] In the present disclosure, abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Commission of Biochemical Nomenclature in Eur. J. Biochem. 1984, 138: 9-37.

[0070] Unless otherwise specified, the experimental reagents and materials used in this disclosure can be obtained commercially. The following are the abbreviations of some reagents and materials:

[0071] 2-CTC Resin: a starting resin for peptide synthesis (2-chlorotrityl chloride resin); DCM: dichloromethane; HOBt: 1-hydroxybenzotriazole; DMF: N,N-dimethylformamide; DIPEA: diisopropylethylamine; MeOH: methanol; piperidine: piperidine; DIC: diisopropylcarbodiimide; HBTU: O-benzotriazole-tetramethyluronium hexafluorophosphate; TFA: trifluoroacetic acid; Tis: triisopropylsilane; EDT: 1,2-ethanedithiol; Lys: lysine; Val: valine; Trp: tryptophan; Leu: leucine; Gln: glutamine; Fmoc: 9-fluorenylmethoxycarbonyl; Boc: tert-butyloxycarbonyl; Trt: trityl.

[0072] Example 1 Preparation of Cyclo-[Val-Leu-Gln-Trp-Val-Lys]

[0073] Cyclo-[Val-Leu-Gln-Trp-Val-Lys] was prepared by the following steps:

[0074] 1.1 Resin swelling

[0075] 100 g of 2-CTC Resin was weighed and placed in a solid phase synthesis reaction column, swollen with DCM, the resin was washed, and the solvent was removed.

[0076] 1.2 Feeding reaction

[0077] Weigh 111.1 g of Fmoc-Lys(Boc)-OH into a dry Erlenmeyer flask, dissolve it in DMF, and cool it in an ice-water bath for 10 minutes. Activate it with 85 mL of DIPEA for 10 minutes. Add the activated Fmoc-Lys(Boc)-OH to the swollen resin and allow to react for 3 hours. Aspirate the reaction solution, wash the resin, and remove the solvent. End-cap the resin by adding DCM, MeOH, and DIPEA for 15 minutes. Repeat this process once. Wash the resin and remove the solvent to obtain Fmoc-Lys(Boc)-2-CTC Resin.

[0078] Fmoc-Lys(Boc)-2-CTC Resin was deprotected twice with 20% piperidine / DMF, each for 10 minutes. Samples were taken for K-test, revealing a dark blue color. The resin was washed seven times with DMF, and the solvent was removed. 66.7 g of Fmoc-Val-OH and 32 g of HOBt were weighed and added to a dry Erlenmeyer flask. DMF was added to dissolve the mixture, and the mixture was sealed and placed in a -18°C refrigerator for 30 minutes. 46 mL of DIC was added for activation for 3 minutes to avoid moisture. The activated amino acid was added to the deprotected resin and reacted for 1 hour. The reaction solution was then removed. The colorless and transparent resin after K-test indicated that the reaction was complete, yielding Fmoc-Val-Lys(Boc)-2-CTC Resin.

[0079] The N-terminal Fmoc group was deprotected, and 103.4 g of activated Fmoc-Trp(Boc)-OH was coupled to the peptidyl resin in the presence of 32 g of HOBt and 46 mL of DIC using DMF as the solvent. The reaction was continued for 1 hour. The resin was then washed and the Fmoc group deprotection treatment was repeated to couple the next amino acid. In each coupling, 120.0 g of Fmoc-Gln(Trt)-OH, 70 g of Fmoc-Leu-OH, and then 66.7 g of Fmoc-Val-OH were sequentially coupled in the presence of 32 g of HOBt and 46 mL of DIC using DMF as the solvent. After the reaction was complete, the resin was washed and the solvent was removed to obtain Fmoc-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC resin.

[0080] The N-terminal Fmoc group of the peptidyl resin was deprotected using 20% ​​piperidine / DMF twice, each for 10 minutes. Samples were taken for K analysis, revealing a dark blue color. The resin was washed six times with DMF, and the solvent was removed by evacuation. After shrinkage and drying, 300 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin was obtained.

[0081] 1.3 Deresinization

[0082] Measure 23 mL of TFA and 2277 mL of DCM, mix and stir evenly to obtain a cleavage solution, seal it and place it in a -18°C refrigerator for later use; place isopropyl ether in a -18°C refrigerator for later use.

[0083] Weigh 230 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin into a round-bottom flask. Add the chilled lysate and stir for 0.5 h. Repeat once for a total of two lysates. Filter, collect, and concentrate the filtrate to yield 160 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-OH.

[0084] 1.4 Loop closure

[0085] 160 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-OH was added to a flask, 4 L of DCM was added with stirring, and 80 g of HBTU and 68.2 g of DIPEA were added. The reaction was allowed to react for 4 h. After completion of the reaction, post-treatment was performed to obtain 170 g of crude cyclic peptide Cyclo-[Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)] with a wet weight of 1.

[0086] 1.5 Cleavage (Removal of Protecting Groups)

[0087] 765 mL of TFA, 21.25 mL of Tis, 21.25 mL of EDT, and 21.25 mL of thioanisole were mixed, and 170 g of the above crude cyclic peptide was added. The mixture was cleaved for 2 h, precipitated with isopropyl ether, and then washed with isopropyl ether to obtain the crude Cyclo-[Val-Leu-Gln-Trp-Val-Lys] peptide.

[0088] 1.6 Purification

[0089] 26.5 g of crude Cyclo-[Val-Leu-Gln-Trp-Val-Lys] peptide was dissolved in methanol, diluted with acetic acid and purified water, and filtered through a 0.45 μm microporous filter membrane to obtain a clear and transparent solution. The solution was purified by reverse-phase HPLC using the following purification gradient:

[0090] Table 1

[0091] Time (min) Flow rate (mL / min) A% (acetonitrile) B% (0.1% acetic acid + pure water) 0 40 5 95 10 40 18 92 30 40 35 65 45 40 38 62 80 40 40 60

[0092] The filtered sample was injected for purification, and the fractions were collected, concentrated, and lyophilized to obtain a peptide Cyclo-[Val-Leu-Gln-Trp-Val-Lys] with a purity of >95%, which was designated as cyclohexapeptide A. The chemical structure is shown below:

[0093] .

[0094] The molecular weight of cyclohexapeptide A was determined by ESI-MS, and the mass spectrum is shown in FIG. Figure 1 The results showed that [M+H] + The mass-to-charge ratio (m / z) of the quasi-molecular ion peak is 754.4630, and the molecular weight measured by mass spectrometry is 753.46, which is consistent with the theoretical accurate molecular weight of cyclohexapeptide A. Its NMR data and analysis results are as follows:

[0095] 1H NMR (600 MHz, D2O) δ: 7.53 (d, J = 6 Hz, 1H); 7.32 (d, J = 6 Hz,1H); 7.08 (q, J = 6 Hz, 2H); 7.03 (d, J = 6 Hz, 1H); 4.64 (q, J = 6 Hz, 2H); 4.36 (dd, J = 12 Hz, 6 Hz, 1H); 4.24 (d, J = 6 Hz, 1H); 4.11 (m, 2H); 3.48(t, J = 6 Hz, 1H); 3.11 (dd, J = 12 Hz, 6 Hz, 1H); 2.85 (t, J = 6 Hz, 2H); 2.30 (m, 1H); 1.99 (m, 1H); 1.64 (m, 7H); 1.51 (m, 1H); 1.39 (m, 4H); 1.24(s, 1H); 0.83 (m, 9H); 0.75 (q, J = 6 Hz, 6H); 0.66 (d, J = 6 Hz, 3H).

[0096] Table 2 Proton spectrum analysis of the tested compounds

[0097] Serial number Chemical shift (ppm) Number of protons Peak shape (coupling constant) Structural attribution 1 7.53 1 d, J = 6 Hz The CH of the indole ring 2 7.32 1 d, J = 6 Hz The CH of the indole ring 3 7.08 2 q, J = 6 Hz The CH of the indole ring 4 7.03 1 d, J = 6 Hz The CH of the indole ring 5 4.64 2 q, J = 6 Hz α-CH connected to the amide bond 6 4.36 1 dd, J = 12 Hz, 6 Hz α-CH connected to the amide bond 7 4.24 1 d, J = 6 Hz α-CH connected to the amide bond 8 4.11 2 m α-CH connected to the amide bond 9 3.48 1 t, J = 6 Hz Hydrogen of saturated alkyl group (no obvious characteristics) 10 3.11 1 dd, J = 12 Hz, 6 Hz Hydrogen of saturated alkyl group (no obvious characteristics) 11 2.85 2 t, J = 6 Hz Hydrogen of saturated alkyl group (no obvious characteristics) 12 2.30 1 m Hydrogen of saturated alkyl group (no obvious characteristics) 13 1.99 1 m Hydrogen of saturated alkyl group (no obvious characteristics) 14 1.64 7 m Hydrogen of saturated alkyl group (no obvious characteristics) 15 1.51 1 m Hydrogen of saturated alkyl group (no obvious characteristics) 16 1.39 4 m Hydrogen of saturated alkyl group (no obvious characteristics) 17 1.24 1 s Hydrogen of saturated alkyl group (no obvious characteristics) 18 0.83 9 m Hydrogen of the methyl group of the valine side chain 19 0.75 6 q, J = 6 Hz Hydrogen of the methyl group of the leucine and valine side chains 20 0.66 3 d, J = 6 Hz Hydrogen of the methyl group of the leucine side chain

[0098] 13 C NMR (150 MHz, D2O) δ: 177.15, 175.13, 173.79, 173.57, 172.93,171.19, 136.20, 126.67, 124.57, 122.06, 119.45, 118.20, 112.14, 108.67,59.05, 58.27, 56.11, 54.73, 54.54, 51.03, 39.05, 30.79, 30.29, 29.36, 28.36,27.15, 26.48, 25.34, 24.47, 23.34, 22.58, 22.21, 19.78, 18.73, 18.19, 17.37,16.04.

[0099] Table 3 Carbon spectrum analysis of the tested compounds

[0100] Serial number Chemical shift (ppm) Number of carbon atoms Structural attribution 1 177.15 1 Carbon of a carbonyl (amide) group 2 175.13 1 Carbon of a carbonyl (amide) group 3 173.79 1 Carbon of a carbonyl (amide) group 4 173.57 2 Carbon of a carbonyl (amide) group 5 172.93 1 Carbon of a carbonyl (amide) group 6 171.19 1 Carbon of a carbonyl (amide) group 7 136.20 1 C on the indole ring 8 126.67 1 C on the indole ring 9 124.57 1 C on the indole ring 10 122.06 1 C on the indole ring 11 119.45 1 C on the indole ring 12 118.20 1 C on the indole ring 13 112.14 1 C on the indole ring 14 108.67 1 C on the indole ring 15 59.05 1 α-C connected to the amide bond 16 58.27 1 α-C connected to the amide bond 17 56.11 1 α-C connected to the amide bond 18 54.73 1 α-C connected to the amide bond 19 54.54 1 α-C connected to the amide bond 20 51.03 1 α-C connected to the amide bond 21 39.05 1 Carbon of a saturated alkyl group (no distinct characteristics) 22 30.79 1 Carbon of a saturated alkyl group (no distinct characteristics) 23 30.29 1 Carbon of a saturated alkyl group (no distinct characteristics) 24 29.36 1 Carbon of a saturated alkyl group (no distinct characteristics) 25 28.36 1 Carbon of a saturated alkyl group (no distinct characteristics) 26 27.15 1 Carbon of a saturated alkyl group (no distinct characteristics) 27 26.48 1 Carbon of a saturated alkyl group (no distinct characteristics) 28 25.34 1 Carbon of a saturated alkyl group (no distinct characteristics) 29 24.47 1 Carbon of a saturated alkyl group (no distinct characteristics) 30 23.34 1 Carbon of a saturated alkyl group (no distinct characteristics) 31 22.58 1 Carbon of a saturated alkyl group (no distinct characteristics) 32 22.21 1 The carbon of the methyl group of the leucine side chain 33 19.78 1 The carbon of the methyl group of the leucine side chain 34 18.73 1 The carbon of the methyl group of the valine side chain 35 18.19 1 The carbon of the methyl group of the valine side chain 36 17.37 1 The carbon of the methyl group of the valine side chain 37 16.04 1 The carbon of the methyl group of the valine side chain

[0101] Comprehensive analysis of the above nuclear magnetic resonance hydrogen spectrum and carbon spectrum showed that the structure of the tested compound was consistent with that of cyclohexapeptide A.

[0102] Example 2 Preparation of H-Val-Leu-Gln-Trp-Val-Lys-OH

[0103] 2.1 Resin swelling

[0104] 100 g of 2-CTC Resin was weighed and placed in a solid phase synthesis reaction column, swollen with DCM, the resin was washed, and the solvent was removed.

[0105] 2.2 Feeding reaction

[0106] Weigh 111.1 g of Fmoc-Lys(Boc)-OH into a dry Erlenmeyer flask, dissolve it in DMF, and cool it in an ice-water bath for 10 minutes. Activate it with 85 mL of DIPEA for 10 minutes, avoiding moisture. Add the activated Fmoc-Lys(Boc)-OH to the swollen resin and allow to react for 3 hours. Aspirate the reaction solution, wash the resin, and aspirate the solvent. Continue capping the resin with DCM, MeOH, and DIPEA for 0.5 hours. Wash the resin and aspirate the solvent to obtain Fmoc-Lys(Boc)-2-CTC Resin.

[0107] Fmoc-Lys(Boc)-2-CTC Resin was depolymerized twice with 20% piperidine / DMF for 10 min each. Samples were taken for K-test, and the color developed to be dark blue. The resin was washed seven times with DMF, and the solvent was removed.

[0108] Weigh 66.7 g of Fmoc-Val-OH and 32 g of HOBt into a dry Erlenmeyer flask. Dissolve in DMF, seal, and freeze at -18°C for 30 min. Activate with 46 mL of DIC for 3 min, avoiding moisture. Add the activated amino acid to the deprotected resin and react for 1 h. Aspirate the reaction solution. A colorless, transparent K-test indicates a complete reaction.

[0109] The N-terminal Fmoc group was deprotected, and 103.4 g of activated Fmoc-Trp(Boc)-OH was coupled to the peptidyl resin using DMF as a solvent in the presence of 32 g of HOBt and 46 mL of DIC. The reaction was continued for 1 hour. The resin was then washed and the Fmoc group deprotection treatment was repeated to couple the next amino acid. In each coupling, 120 g of Fmoc-Gln(Trt)-OH, 70 g of Fmoc-Leu-OH, and then 66.7 g of Fmoc-Val-OH were sequentially coupled in the presence of 32 g of HOBt and 46 mL of DIC using DMF as a solvent. After the reaction was complete, the resin was washed and the solvent was removed.

[0110] The N-terminal Fmoc group of the peptidyl resin was deprotected using 20% ​​piperidine / DMF for two 10-min cycles. Samples were taken for K-analysis, revealing a dark blue color. The resin was washed six times with DMF, and the solvent was removed to obtain H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin.

[0111] 2.3 Lysis

[0112] Measure 180 mL of TFA, 5 mL of EDT, 5 mL of TIS, 5 mL of thioanisole and 5 mL of water, mix and stir evenly to obtain a cleavage solution, seal the container and place it in a -18°C refrigerator for later use; place isopropyl ether in a -18°C refrigerator for later use.

[0113] Weigh 40 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin into a round-bottom flask. Add the frozen lysate and stir for 2 hours. Filter the mixture, collect the filtrate, and wash it three times with isopropyl ether, centrifuge, and stir. Vacuum dry to obtain H-Val-Leu-Gln-Trp-Val-Lys-OH.

[0114] 2.4 Purification

[0115] 17 g of H-Val-Leu-Gln-Trp-Val-Lys-OH was weighed and dissolved in 250 mL of purified water and 50 mL of acetic acid, sonicated, filtered through diatomaceous earth, and then filtered through a 0.45 μm microporous filter membrane to obtain a clear solution. The solution was purified by reverse-phase HPLC using the following purification gradient:

[0116] Table 4

[0117] Time (min) Flow rate (mL / min) A% (acetonitrile) B% (purified water) 0 40 2 98 5 40 5 95 30 40 15 85 45 40 20 80 80 40 40 60

[0118] The filtered sample was injected for purification, fractions were collected, concentrated and lyophilized to obtain peptide H-Val-Leu-Gln-Trp-Val-Lys-OH with a purity of >97%, which was recorded as hexapeptide B. The molecular weight of hexapeptide B was determined, and the mass spectrum was as shown in Figure 2 The results showed that [M+H] + The mass-to-charge ratio (m / z) of the quasi-molecular ion peak was 772.86, and the molecular weight measured by mass spectrometry was 771.86, which was consistent with the theoretical accurate molecular weight of hexapeptide B.

[0119] Other peptides disclosed herein, including but not limited to: H-Val-Leu-Gln-Trp-Val-Lys-NH2, Ac-Val-Leu-Gln-Trp-Val-Lys-OH, Ac-Val-Leu-Gln-Trp-Val-Lys-NH2, Myr-Val-Leu-Gln-Trp-Val-Lys-OH, Myr-Val-Leu-Gln-Trp-Val-Lys-NH2, Pal-Val-Leu-Gln-Trp-Val-Lys-OH, Pal-Val-Leu-Gln-Trp-Val-Lys-NH2, etc., can be prepared by similar polypeptide solid-phase synthesis methods.

[0120] Example 3 Calcium Ion Influx Test

[0121] 3.1 Reagents and Materials

[0122] 0.25% trypsin digestion solution, phosphate buffered saline (PBS), acetylcholine solution containing 1% fetal bovine serum, and Fluo-3AM calcium ion probe.

[0123] 3.2 Instruments

[0124] Constant temperature CO2 incubator, clean bench, fluorescence microscope.

[0125] 3.3 Cell lines

[0126] Mouse neuroblastoma cells (Neuro-2a cells).

[0127] 3.4 Samples to be tested and grouping

[0128] 3.4.1 Samples to be tested

[0129] Cyclic hexapeptide A, hexapeptide B, and reference peptide (dipeptide diaminobutyryl benzylamide diacetate) were all tested at a concentration of 50 ppm.

[0130] 3.4.2 Grouping

[0131] Experimental group: test sample, acetylcholine solution containing 1% fetal bovine serum.

[0132] Control group: PBS, acetylcholine solution containing 1% fetal bovine serum.

[0133] 3.5 Experimental methods

[0134] Take a bottle of Neuro-2a cells in good condition in the exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count (1-4)×10 5Cells were plated at 400 μg / mL to prepare a cell suspension. An appropriate amount of the cell suspension was inoculated onto a 12-well plate containing complete culture medium and cultured in a constant temperature CO2 incubator for 24 h. The complete culture medium in the wells was aspirated, and complete culture medium and the test sample were added to the experimental group, while complete culture medium and an equal volume of PBS were added to the control group, and cultured in a CO2 incubator for 24 h. The supernatant was discarded, the cells were washed twice with PBS, and Fluo-3AM working solution was added to the 12-well plate, and incubated at 37°C for 30 min. The Fluo-3AM working solution was discarded, and the test sample and acetylcholine solution containing 1% fetal bovine serum were added to the experimental group, while an equal volume of acetylcholine solution containing 1% fetal bovine serum was added to the control group, and incubated for 5 min. The supernatant was discarded, and the cells were washed twice with PBS. Observe and photograph under a fluorescence microscope, and analyze the relative fluorescence intensity using Image J software.

[0135] 3.6 Experimental Results

[0136] Calcium influx plays a key role in skin muscle contraction. Inhibiting calcium influx can reduce excessive muscle contraction, improve the appearance of wrinkles on the skin surface, and reduce their depth, thereby achieving a wrinkle-reducing effect. The calcium ion content in cells is directly proportional to the intensity of fluorescence staining; that is, the stronger the fluorescence intensity, the higher the calcium ion content in the cells. This experiment measured the relative fluorescence intensity of calcium ions in Neuro-2a cells to determine whether the disclosed peptides could inhibit calcium ion influx.

[0137] The results of the effects of the test samples on calcium influx in Neuro-2a cells are shown in Table 5.

[0138] Table 5 Relative fluorescence intensity of calcium ions in Neuro-2a cells

[0139] Group Relative fluorescence intensity (Mean±SD) control group 100.00%±1.60% Reference peptide group <![CDATA[52.43%±3.47% *** ]]> Cyclic hexapeptide group A <![CDATA[46.84%±2.61% *** ]]> Hexapeptide B group <![CDATA[55.91%±2.09% *** ]]>

[0140] Note: Compared with the control group, *** P <0.001.

[0141] The reference peptide (dipeptide diaminobutyryl benzylamide diacetate) is a commercially available anti-wrinkle polypeptide ingredient that reduces wrinkle formation by inhibiting muscle contraction and has excellent anti-wrinkle effects. The results in Table 5 show that the relative fluorescence intensity of the reference peptide group was significantly reduced compared to the control group, indicating that the experimental system is effective and reliable. Both cyclic hexapeptide A and hexapeptide B disclosed herein can significantly reduce the calcium ion fluorescence intensity of Neuro-2a cells and inhibit calcium ion influx. Among them, cyclic hexapeptide A has a better effect of inhibiting calcium ion influx than the reference peptide. It can be seen that the peptides disclosed herein can significantly inhibit calcium ion influx, thereby inhibiting muscle contraction, and can be used to reduce, prevent or treat wrinkles, exerting anti-aging and anti-wrinkle effects. By inhibiting calcium ion influx, the peptides disclosed herein can be used to treat or care for conditions, disorders or diseases caused by muscle contraction.

[0142] Example 4 Collagen I content test

[0143] 4.1 Reagents and Materials

[0144] Fetal bovine serum, DMEM medium, phosphate buffered saline (PBS), trypsin, RIPA lysis buffer, collagen I ELISA kit, BCA protein kit.

[0145] 4.2 Instruments

[0146] Microplate reader, CO2 incubator, clean bench, and gas bath constant temperature oscillator.

[0147] 4.3 Cell lines

[0148] Human skin fibroblasts (HSF).

[0149] 4.4 Samples to be tested and grouping

[0150] 4.4.1 Samples to be tested

[0151] The test concentrations of cyclic hexapeptide A and hexapeptide B were both 15.6 ppm.

[0152] 4.4.2 Grouping

[0153] Experimental group: UV radiation + test sample.

[0154] Blank control group: PBS.

[0155] UV group: UV radiation + PBS.

[0156] 4.5 Experimental Methods

[0157] Take a bottle of HSF fibroblasts in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count (1-4) × 10 5cells / mL to prepare a cell suspension.

[0158] The diluted cell suspension was inoculated into a 12-well plate and cultured in a CO2 incubator for 24 hours. The complete culture medium in the well was aspirated, and each group was repeatedly washed with an appropriate amount of PBS until colorless. The blank control group was added with 100 μL PBS and the complete culture medium was replenished to 1000 μL without UV irradiation. The UV group and the experimental group were added with 200 μL PBS and placed in 80 mJ / cm 2 Irradiate under UV light for 15 minutes. After irradiation, discard the PBS. Add 100 μL of PBS to the UV group and replenish the complete medium to 1000 μL. Add 100 μL of the test sample to the experimental group and replenish the complete medium to 1000 μL. Incubate the blank control, UV, and experimental groups in a 37°C, 5% CO2 incubator for 48 hours.

[0159] After the culture, the cells were collected and centrifuged to discard the supernatant. RIPA lysis buffer was added and the cells were shaken three times with a vortexer (30 s / time, 3 min interval). The cells were centrifuged at 12000 rpm for 10 min. The supernatant was aspirated and tested according to the instructions of the collagen I detection kit. The total protein concentration of the supernatant was detected using a BCA protein quantification kit.

[0160] 4.6 Experimental Results

[0161] Type I collagen is the most abundant collagen in the human body. It occurs in thick, tightly packed bundles and possesses strong tensile strength, providing a strong structural support and strength for the skin, giving it elasticity and toughness. Therefore, increasing type I collagen content is crucial for preventing aging and increasing skin elasticity and firmness. This experiment used test samples to treat cells exposed to ultraviolet radiation and measured the type I collagen content in the cells to determine whether the disclosed peptides could promote type I collagen production.

[0162] The results of the effects of the test samples on the collagen I content are shown in Table 6.

[0163] Table 6 Effects of test samples on collagen I content

[0164] Group Relative content of collagen I (Mean±SD) Blank control group 100.00%±8.46% UV Group <![CDATA[47.32%±6.31% ### ]]> Cyclic hexapeptide group A <![CDATA[72.98%±3.08% *** ]]> Hexapeptide B group <![CDATA[72.73%±4.65% ** ]]>

[0165] Note: Compared with the blank control group, ### P <0.001; compared with the UV group, ** P <0.01, *** P <0.001.

[0166] The results showed that compared with the blank control group, the collagen I content in the UV group was significantly reduced, indicating that the modeling was successful; compared with the UV group, both cyclic hexapeptide A and hexapeptide B in the experimental group were able to significantly increase the collagen I content and promote the production of type I collagen.

[0167] This suggests that the peptides disclosed herein can significantly increase collagen content in cells exposed to ultraviolet radiation and can be used to treat, prevent, or repair skin photoaging. By promoting collagen production, the peptides disclosed herein can be used to increase skin elasticity, improve skin firmness, and delay skin aging.

[0168] Example 5 Collagen III content test

[0169] 5.1 Reagents and Materials

[0170] Fetal bovine serum, DMEM medium, phosphate buffered saline (PBS), trypsin, RIPA lysis buffer, collagen III ELISA kit, BCA protein kit.

[0171] 5.2 Instruments

[0172] Microplate reader, CO2 incubator, clean bench, and gas bath constant temperature oscillator.

[0173] 5.3 Cell lines

[0174] Human skin fibroblasts (HSF).

[0175] 5.4 Samples to be tested and grouping

[0176] 5.4.1 Samples to be tested

[0177] The test concentrations of cyclic hexapeptide A and hexapeptide B were both 31.2 ppm.

[0178] 5.4.2 Grouping

[0179] Experimental group: UV radiation + test sample.

[0180] Blank control group: PBS.

[0181] UV group: UV radiation + PBS.

[0182] 5.5 Experimental Methods

[0183] Take a bottle of HSF fibroblasts in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count (1-4) × 10 5 cells / mL to prepare a cell suspension.

[0184] The diluted cell suspension was inoculated into a 12-well plate and cultured in a CO2 incubator for 24 hours. The complete culture medium in the well was aspirated, and each group was repeatedly washed with an appropriate amount of PBS until colorless. The blank control group was added with 100 μL PBS and the complete culture medium was replenished to 1000 μL without UV irradiation. The UV group and the experimental group were added with 200 μL PBS and placed in 80 mJ / cm 2 Irradiate under UV light for 15 minutes. After irradiation, discard the PBS. Add 100 μL of PBS to the UV group and replenish the complete medium to 1000 μL. Add 100 μL of the test sample to the experimental group and replenish the complete medium to 1000 μL. Incubate the blank control, UV, and experimental groups in a 37°C, 5% CO2 incubator for 48 hours.

[0185] After the culture, the cells were collected and centrifuged to discard the supernatant. RIPA lysis buffer was added and the cells were shaken three times with a vortexer (30 s / time, 3 min interval). The cells were centrifuged at 12000 rpm for 10 min. The supernatant was aspirated and tested according to the instructions of the collagen III detection kit. The total protein concentration of the supernatant was detected using a BCA protein quantification kit.

[0186] 5.6 Experimental Results

[0187] Type III collagen plays a crucial role in supporting and repairing the skin, providing elasticity and stress resistance. It also participates in skin regeneration and repair, accelerating wound healing and tissue regeneration. Therefore, increasing type III collagen levels is crucial for firming and repairing damaged skin. This experiment used test samples to treat cells exposed to ultraviolet radiation and measured the type III collagen content in the cells to determine whether the disclosed peptides could promote type III collagen production.

[0188] The results of the effects of the test samples on the collagen III content are shown in Table 7.

[0189] Table 7 Effects of test samples on collagen III content

[0190] Group Relative content of collagen III (Mean±SD) Blank control group 100.00%±12.37% UV Group <![CDATA[54.79%±4.17% ## ]]> Cyclic hexapeptide group A <![CDATA[99.95%±15.00% ** ]]> Hexapeptide B group <![CDATA[90.21%±5.42% *** ]]>

[0191] Note: Compared with the blank control group, ## P <0.01; compared with the UV group, ** P <0.01, *** P <0.001.

[0192] The results showed that compared with the blank control group, the collagen III content in the UV group was significantly reduced, indicating that the modeling was successful; compared with the UV group, both cyclic hexapeptide A and hexapeptide B in the experimental group were able to significantly increase the collagen III content and promote the production of type III collagen.

[0193] As can be seen from this, the peptides disclosed herein can effectively increase collagen content in cells exposed to ultraviolet radiation and can be used to treat, prevent, or repair skin photoaging. By promoting collagen production, they can increase skin elasticity, improve skin firmness, and promote re-epithelialization or healing of the skin or mucous membranes, thereby achieving anti-aging, firming, and repairing effects.

[0194] Example 6 Hyaluronidase inhibition experiment

[0195] 6.1 Reagents and Materials

[0196] Sodium acetate buffer (pH=5.6), hyaluronidase, calcium chloride, sodium hyaluronate, acetylacetone solution, anhydrous ethanol, sodium hydroxide solution, P-DAB color developer [prepared by uniformly mixing p-dimethylaminobenzaldehyde (0.8 g), concentrated hydrochloric acid (15 mL) and an equal amount of glacial acetic acid].

[0197] 6.2 Instruments

[0198] Microplate reader, electronic balance, and gas bath constant temperature oscillator.

[0199] 6.3 Samples to be tested and grouping

[0200] 6.3.1 Samples to be tested

[0201] The test concentration of cyclic hexapeptide A and hexapeptide B was 1000ppm.

[0202] 6.3.2 Grouping

[0203] Sample group: test sample, hyaluronidase, sodium hyaluronate;

[0204] Sample zero adjustment group: test sample, sodium acetate buffer;

[0205] Blank control group: distilled water, hyaluronidase, sodium hyaluronate;

[0206] Blank zero adjustment group: distilled water, sodium acetate buffer.

[0207] 6.4 Experimental Methods

[0208] Hyaluronidase and sodium hyaluronate were dissolved in sodium acetate buffer.

[0209] In a 96-well plate, 25 μL of the test sample and 25 μL of hyaluronidase (500 U / mL) were added to the sample group; 25 μL of the test sample and 25 μL of sodium acetate buffer were added to the sample zero adjustment group; 25 μL of distilled water and 25 μL of hyaluronidase (500 U / mL) were added to the blank control group; and 25 μL of distilled water and 25 μL of sodium acetate buffer were added to the blank zero adjustment group. After shaking at 37°C in a constant-temperature air bath for 20 minutes, 5 μL of calcium chloride solution (2.5 mol / L) was added to each well, and the plates were shaken at 37°C in a constant-temperature air bath for 20 minutes. 25 μL of sodium hyaluronate (1 mg / mL) was added to the sample and blank control groups, and 25 μL of sodium acetate buffer was added to the sample and blank zero adjustment groups, and the plates were shaken at 37°C in a constant-temperature air bath for 40 minutes. Then, 25 μL of distilled water, 5 μL of sodium hydroxide solution (5 mol / L), and 25 μL of acetylacetone solution were added to each well and placed in a boiling water bath for 15 minutes, followed by an ice bath for 10 minutes, and finally placed at room temperature for 10 minutes. 50 μL of P-DAB was added to each well, followed by 100 μL of anhydrous ethanol, and the cells were placed at room temperature for 30 minutes. The OD was measured at 570 nm. 570 value.

[0210] Hyaluronidase inhibition rate (%)

[0211] Where: A1 is the OD of the sample zeroing group 570 value, A2 is the sample group OD 570 A3 is the OD of the blank zero adjustment group. 570 A4 is the OD of the blank control group. 570 value.

[0212] 6.5 Experimental Results

[0213] Hyaluronidase activity is closely linked to type I allergic reactions. Inhibiting hyaluronidase activity can exert soothing and anti-allergic effects, and is therefore often used as an evaluation indicator for soothing and anti-allergic effects. Furthermore, hyaluronidase is a hydrolase that degrades hyaluronic acid. A reduction in hyaluronic acid can disrupt the skin's barrier function and lead to water loss. Inhibiting hyaluronidase activity can reduce hyaluronic acid degradation in cells and increase their content, thereby increasing hydration of the skin or mucous membranes, repairing the skin barrier, and exerting moisturizing, soothing, and repairing effects.

[0214] Sodium hyaluronate is a substrate for hyaluronidase. Under the catalytic action of hyaluronidase, sodium hyaluronate is degraded to produce glucuronic acid and N-acetylglucosamine. Under the action of P-DAB, it develops color and absorbs visible light at a wavelength of 570 nm. In this experiment, the test sample was treated with hyaluronidase, and the reaction amount of sodium hyaluronate was measured to determine whether the test sample disclosed herein could inhibit the activity of hyaluronidase.

[0215] The results of hyaluronidase activity inhibition rates of different test samples are shown in Table 8.

[0216] Table 8 Hyaluronidase activity inhibition rate of test samples (Mean±SD)

[0217] concentration Cyclohexapeptide A Hexapeptide B 1000ppm 54.43%±6.42% 21.48%±3.98%

[0218] The results showed that both cyclic hexapeptide A and hexapeptide B of the present disclosure could inhibit hyaluronidase activity, with the inhibition rate of cyclic hexapeptide A reaching 54.43%. This indicates that the peptides of the present disclosure have excellent hyaluronidase inhibition effects and can be used to repair the skin barrier, with repairing, soothing, and moisturizing effects.

[0219] Example 7

[0220] A cream is prepared by the following steps, and the specific formula is as shown in Table 9:

[0221] Table 9

[0222]

[0223] According to the recipe, heat Phase C in a suitable container to 55-60°C until completely dissolved and set aside. Add Phase A to a stirring pot, stir and heat to 80-85°C. Add Phase B to the oil phase pot, stir and heat to 75-80°C until completely dissolved and transparent. Pump Phase B into Phase A, turn on the vacuum, homogenize for 5 minutes, maintain stirring, and keep warm for 20 minutes. Start cooling to 60-65°C, add Phase C, and homogenize for 2 minutes. Cool to 35-40°C, add the pre-dissolved Phase D material, and stir for 10-15 minutes.

[0224] Example 8

[0225] An essence is prepared by the following steps, and the specific formula is shown in Table 10 below:

[0226] Table 10

[0227]

[0228] According to the formula and dosage, add the materials of Phase A to a stirring pot, stir and heat to 80-85°C; mix the materials of Phase B evenly until there are no powder particles, add to the stirring pot, and continue stirring for 10-15 minutes; start cooling, cool to 60-65°C, add the materials of Phase C; cool to 35-40°C, add the materials of Phase D and Phase E, and stir for 10-15 minutes.

[0229] In this disclosure, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or terminal device. In the absence of further restrictions, an element defined by the statement "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article, or terminal device comprising the element.

[0230] Although the specific embodiments of the present disclosure have been described for illustrative purposes, those skilled in the art may make various modifications or improvements without departing from the spirit and scope of the present disclosure. These modifications or improvements should fall within the scope of the appended claims of the present disclosure.

Claims

1. A peptide represented by formula (I) or a salt thereof, R1-Val-Leu-Gln-Trp-Val-Lys-R2(I) In formula (I), R1 is selected from H, acetyl, lauroyl, myristoyl or palmitoyl; R2 is -OH or -NH2.

2. The peptide or salt thereof according to claim 1, characterized in that When R1 is H and R2 is -OH, the structure of the peptide is Cyclo-[Val-Leu-Gln-Trp-Val-Lys].

3. The peptide or salt thereof according to claim 1 or 2, characterized in that The salts include metal salts of the peptide, wherein the metal includes lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum; Alternatively, the salt includes a salt of the peptide with an organic base, the organic base including ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine; Alternatively, the salt includes a salt formed by the peptide with an inorganic acid or an organic acid, wherein the organic acid includes: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; and the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

4. A composition, characterized in that The composition comprises an effective amount of the peptide or salt thereof according to any one of claims 1 to 3, at least one excipient and optionally an adjuvant.

5. A delivery system or sustained-release system, characterized in that Comprising an effective amount of the peptide or salt thereof according to any one of claims 1 to 3, or the composition according to claim 4.

6. The delivery system or sustained-release system according to claim 5, characterized in that The delivery system or sustained-release system includes: liposomes, oleosomes, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion compounds, lipid vesicles, micelles, millispheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, milliparticles, microparticles or nanoparticles.

7. A cosmetic, characterized in that: Comprising an effective amount of the peptide or salt thereof according to any one of claims 1 to 3, or the composition according to claim 4, or the delivery system or sustained-release system according to claim 5 or 6.

8. The cosmetic according to claim 7, characterized in that The dosage forms of the cosmetics include ointments, creams, emulsions, aqueous solutions, oils, gels, powders, tablets, muds, patches, films, aerosols, sprays, freeze-dried preparations or nano preparations.

9. Use of the peptide or salt thereof according to any one of claims 1 to 3, or the composition according to claim 4, or the delivery system or sustained-release system according to claim 5 or 6 in the preparation of a composition for anti-aging, repairing, soothing or moisturizing.

10. The use according to claim 9, characterized in that The anti-aging effect includes one or more of promoting collagen production or inhibiting calcium ion influx.

11. The use according to claim 9, characterized in that The soothing includes inhibiting hyaluronidase activity.

12. Use of the peptide or salt thereof according to any one of claims 1 to 3, or the composition according to claim 4, or the delivery system or sustained-release system according to claim 5 or 6 in the preparation of a composition for increasing skin elasticity and / or improving skin firmness; or in the preparation of a composition for promoting re-epithelialization or healing of skin or mucous membranes; or in the preparation of a composition for treating, preventing or repairing photoaging of the skin; or in the preparation of a composition for reducing, preventing or treating wrinkles.

13. Use of the peptide or salt thereof according to any one of claims 1 to 3, or the composition according to claim 4, or the delivery system or sustained-release system according to claim 5 or 6 in the preparation of cosmetics for anti-aging, firming, anti-wrinkle, repairing, soothing or moisturizing.

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