Hexapeptide compound as well as composition and application thereof

Abstract

Disclosed are a hexapeptide compound and a composition and use thereof, relating to the technical field of polypeptides, the peptide or a salt thereof having a structure as shown in formula (I): R1-Val-Leu-Gln-Trp-Val-Lys-R2 (I). In particular, the invention relates to the peptide or the salt thereof, or a composition of the peptide and the salt thereof, and application of the peptide and the salt in preparation of a composition for nursing or treating skin or mucous membranes.

Description

Technical Field

[0001] The present disclosure relates to the technical field of polypeptides, and particularly relates to a hexapeptide compound, its composition and uses. Background Art

[0002] Skin aging is a complex process, which is jointly affected by endogenous aging and exogenous photoaging, and involves multiple aspects such as degradation of the dermal matrix, rupture of elastic fibers, and excessive muscle contraction. Muscle contraction is closely related to the release of acetylcholine, and calcium ions are essential substances for the release of acetylcholine. When a neuron is excited, the nerve impulse is conducted to the axon terminal, triggering the opening of voltage-gated calcium channels, and extracellular calcium ions flow into the axon terminal. The influx of calcium ions promotes the movement of synaptic vesicles containing acetylcholine towards the presynaptic membrane, and under the action of calcium ions, they fuse with the presynaptic membrane, releasing acetylcholine into the synaptic cleft. Acetylcholine acts on the postsynaptic membrane, thereby causing muscle contraction. In addition, when acetylcholine binds to acetylcholine receptors on muscle cells, it will cause the opening of ion channels, leading to an increase in the permeability of the cell membrane to calcium ions, enabling a large amount of calcium ions to flow in, which will also cause muscle contraction. Research shows that the imbalance of calcium ion homeostasis is the key inducement for the persistent contraction of facial muscles. Excessive muscle contraction will lead to the appearance of wrinkles. Therefore, inhibiting the influx of calcium ions can inhibit muscle contraction, thereby improving wrinkles.

[0003] In terms of collagen metabolism, with age or external stimuli, the synthesis rate of type I and type III collagens in the dermis decreases, while the activity of matrix metalloproteinases increases abnormally, resulting in the gradual collapse of the skin's supporting structure and the loss of skin elasticity and firmness. Among external environmental factors, ultraviolet rays are an important factor causing collagen loss. Photoaging is the main cause of skin aging. Prolonged exposure to light such as ultraviolet rays will cause problems such as wrinkles and sagging of the skin, and the skin will enter the aging state prematurely. On the other hand, hyaluronidase is an enzyme that can hydrolyze hyaluronic acid and can reduce the viscosity of the extracellular matrix and improve the permeability of fluids in tissues. However, the over-activation of hyaluronidase will accelerate the degradation of hyaluronic acid, and as an important moisturizing factor in the skin, its degradation will reduce the skin's water storage capacity, leading to a decrease in the water content of the stratum corneum, resulting in a series of adverse states such as rough, dry, and sensitive skin, further exacerbating skin aging.

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

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

[0006] On the one hand, the present disclosure provides a peptide represented by formula (I), or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, R 1 -Val-Leu-Gln-Trp-Val-Lys-R 2 (I) In formula (I), R 1 is selected from: H or R 3 -CO-, where R 3 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl; R 2 is selected from: -NR 4 R 5 or -OR 4 , where each R 4 and R 5 are independently selected from: H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl; The alkyl refers to a saturated aliphatic straight-chain or branched-chain alkyl having 1-24 carbon atoms (or having 1-16 carbon atoms; or having 1-14 carbon atoms; or having 1-12 carbon atoms; or having 1, 2, 3, 4, 5 or 6 carbon atoms); In some embodiments, the alkyl 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; The alkenyl refers to a straight-chain or branched-chain alkenyl having 2-24 carbon atoms (or having 2-16 carbon atoms; or having 2-14 carbon atoms; or having 2-12 carbon atoms; or having 2, 3, 4, 5 or 6 carbon atoms); The alkenyl has one or more carbon-carbon double bonds, and in some embodiments, the alkenyl has 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; The alkenyl is bonded to the rest of the molecule through a single bond; In some embodiments, the alkenyl is selected from: vinyl, oleyl or linoleyl; In some embodiments, the substituents in the "substituted alkyl" and "substituted alkenyl" are selected from C 1 -C 4 alkyl; hydroxyl; C 1 -C 4 alkoxy; amino; C 1 -C 4 aminoalkyl; C 1 -C 4 carbonyloxy; C 1 -C 4Oxycarbonyl; halogen (such as fluorine, chlorine, bromine, and iodine); cyano; nitro; azide; C 1 -C 4 Alkylsulfonyl; thiol; C 1 -C 4 Alkylthio; C 6 -C 30 Aryloxy such as phenoxy; -NR b (C=NR b )NR b R c , wherein R b and R c are independently selected from: H, 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, a heterocyclic group having three to ten members or a protecting group for an amino group.

[0007] In some embodiments, R 1 is selected from: H, acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl or linoleoyl; R 4 , R 5 are independently selected from: H, methyl, ethyl, hexyl, dodecyl or hexadecyl; In some embodiments, R 1 is selected from H, acetyl, lauroyl, myristoyl or palmitoyl; R 4 is H and R 5 is selected from: H, methyl, ethyl, hexyl, dodecyl or hexadecyl; In some embodiments, R 1 is H, acetyl, lauroyl, myristoyl or palmitoyl; R 2 is -OH or -NH 2 .

[0008] In some embodiments, when R 1 is H and R 2 is -OH, the structure of the peptide is Cyclo-[Val-Leu-Gln-Trp-Val-Lys], and its structural formula is shown as follows: .

[0009] The peptides of the present disclosure contain a large number of asymmetric carbon atoms. Those skilled in the art understand that the peptides of the present disclosure have stereoisomers and can exist as stereoisomers or mixtures of stereoisomers, and thus it is possible to obtain isomeric mixtures as well as racemic mixtures or mixtures of diastereomers, 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 disclosure are pure isomers, i.e., enantiomers or diastereomers. In some embodiments, the structure of the peptides of the present disclosure is the L-isomer.

[0010] The present disclosure also includes all suitable isotopic variants of the above peptides. These isotopic variants of the peptides of the present disclosure are understood herein to mean compounds in which at least one atom within 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 that is normally or predominantly present in nature. Examples of isotopes that can be incorporated into the peptides of the present disclosure are those of hydrogen, carbon, nitrogen, or oxygen, such as 2 H (deuterium), 3 H (tritium), 13 C, 14 C, 15 N, 17 O or 18 O. Specific isotopic variants of the peptides of the present disclosure (especially those in which one or more radioactive isotopes have been incorporated) may be useful, for example, for examining the mechanism of action or the distribution of the active compound in the body; due to their relatively simple preparability and detectability, especially compounds labeled with 3 H or 14 C isotopes are suitable for this purpose. Additionally, due to the greater metabolic stability of the compound, the incorporation of an isotope (such as deuterium) can produce specific therapeutic benefits, such as an extended in vivo half-life or a reduced required active dose. The isotopic variants of the peptides of the present disclosure can be prepared by methods known to those skilled in the art, such as by the methods further described hereinafter and those described in the examples, by using the corresponding isotopically modified forms of the respective reagents and / or starting materials.

[0011] The term "salt" refers to a salt that is recognized for use in animals, and more particularly in humans, including metal salts of the peptides of the present disclosure, said metals including, but not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum, etc.; including salts formed by the peptides of the present disclosure with organic bases, said organic bases including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine, etc.; including salts formed by the peptides of the present disclosure with inorganic acids or organic acids, said 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, etc.; said inorganic acids including, but not limited to: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

[0012] The nature of the salt is not decisive, and the salts of the peptides of the present disclosure can be obtained by conventional methods well known in the art.

[0013] The synthesis of the peptides of the present disclosure, or their stereoisomers, or mixtures of their stereoisomers, or their salts can be carried out according to conventional methods known in the prior art, such as solid-phase synthesis, liquid-phase synthesis, or a method combining solid and liquid phases, and 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.

[0014] For example, a method for obtaining the peptides of the present disclosure includes the following steps: - Coupling an amino acid with a protected N-terminus and a free C-terminus to an amino acid with a free N-terminus and a protected or solid support-bound C-terminus; - Removing the group protecting the N-terminus; - Repeating the coupling sequence and removing the group protecting the N-terminus until the desired peptide sequence is obtained; - Removing the group protecting the C-terminus or cleaving from the solid support.

[0015] In some embodiments, the C-terminus is bound to a solid support and the method is carried out on a solid phase, including coupling an amino acid with a protected N-terminus and a free C-terminus to an amino acid with a free N-terminus and a C-terminus bound to a polymeric support; removing the group protecting the N-terminus; and repeating this sequence the required number of times so as to thereby obtain a peptide of the desired length, then cleaving the synthesized peptide from the initial polymeric support and cyclizing the coupling of the amino group at the N-terminus of the peptide chain with the carboxyl group at the C-terminus, and then removing the groups protecting the side chains.

[0016] Throughout the synthesis, the functional groups of the side chains of these amino acids are kept sufficiently protected with temporary or permanent protecting groups.

[0017] In some embodiments, solid-phase synthesis can be carried out by a convergent strategy of coupling a dipeptide or tripeptide to a polymer support or to a dipeptide or amino acid previously attached to the polymer support.

[0018] Due to applications outside the mammalian body, the peptides of the present disclosure can form part of various types of compositions. Thus, in another aspect of the present disclosure, there is provided a composition comprising an effective amount of the above-mentioned peptide, or its stereoisomer, or a mixture of its stereoisomers, or its salt, and at least one excipient and optionally an adjuvant. The composition can be prepared by conventional methods known to those skilled in the art.

[0019] In some embodiments, the adjuvant is selected from: analgesics, agents that inhibit PAR-2 activity, agents that regulate PGC-1α synthesis, agents that regulate the activity of PPARγ, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic agents or agents that stimulate lipolysis, lipolytic solvents, lipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, stimulants or inhibitors of melanin synthesis, whitening agents or depigmenting agents, pigmentation promoters, 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 agents against air pollution, reactive carbonyl species scavengers, anti-glycation agents, antihistamines, antiviral agents, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, moisture-retaining substances, α-hydroxy acids, β-hydroxy acids, humectants, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gum 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 are capable of inhibiting or preventing their degradation, agents that stimulate elastin synthesis, agents that stimulate the synthesis of decorin, agents that stimulate the synthesis of laminin, agents that stimulate the synthesis of defensins, agents that stimulate the synthesis of chaperone proteins, 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-hyperkeratotic agents, comedolytic agents, anti-psoriatic agents, anti-eczema agents, DNA repair agents, DNA protectants, stabilizers, antipruritic agents, agents for treating and / or caring for sensitive skin, curing agents, firming agents, restructuring agents, anti-stretch mark agents, agents that regulate sebum production, antiperspirants, agents that stimulate healing, agents that assist in healing, agents that stimulate re-epithelialization, agents that assist in 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, venotonic agents, agents that act on cell metabolism, agents for improving the dermal-epidermal junction, agents that induce hair growth, hair growth inhibitors or retardants, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents obtained from biological fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic light protectants that are effectively resistant to UVA and / or UVB or mixtures thereof.

[0020] The effective amount of the peptides of the present disclosure to be administered and their dosages will depend on many factors, including age, the condition of the user, the severity of the condition, the route and frequency of administration, and the specific nature of the peptide to be used.

[0021] "Effective amount" means an amount of one or more peptides of the present disclosure that is non-toxic but sufficient to provide the desired effect. The peptides of the present disclosure are used at an effective concentration in the compositions to obtain 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.

[0022] Another aspect of the present disclosure provides a delivery system or sustained-release system to achieve better penetration of the active ingredient, which comprises an effective amount of the above-mentioned peptide, or its stereoisomer, or a mixture of its stereoisomers, or its salt, or the above-mentioned composition.

[0023] The term "delivery system" refers to a diluent, adjuvant, excipient or carrier administered together with the peptides of the present disclosure, which are selected from: water, oil or surfactant, including those of petroleum origin, animal origin, plant origin, or synthetic origin, such as and not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitan ester, ether sulfate, sulfate, betaine, glucoside, maltoside, fatty alcohol, nonoxynol, poloxamer, polyethylene oxide, polyethylene glycol, dextran, glycerol, digitonin and the like. Diluents, adjuvants, excipients or carriers that can be used in different delivery systems in which the peptides of the present disclosure can be administered are known to those of ordinary skill in the art.

[0024] The term "sustained-release" is used in its conventional meaning and refers to a delivery system of a compound that provides for the gradual release of the compound over a period of time. In some embodiments, the sustained-release system has a relatively constant level of compound release over the entire time period.

[0025] Examples of the delivery system or sustained-release system include, but are not limited to: liposomes, oleosomes, ethosomes, millimeter capsules, micron capsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion complexes, niosomes, micelles, millimeter spheres, micron spheres, nanospheres, lipid spheres, micron emulsions, nanoemulsions, millimeter particles, micron particles or nanoparticles.

[0026] On the other hand, the present disclosure provides a cosmetic comprising an effective amount of the above peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or the above composition, or the above delivery system or sustained-release system.

[0027] In some embodiments, the dosage form of the cosmetic includes paste, cream, emulsion, aqueous solution, oil, gel, powder, tablet, mud, patch, film, aerosol, spray, freeze-dried preparation or nano-preparation.

[0028] On the other hand, the present disclosure provides the use of the above peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or the above composition, or the above delivery system or sustained-release system in the preparation of a composition for caring for or treating the skin or mucosa.

[0029] On the other hand, the present disclosure provides the use of the above peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or the above composition, or the above delivery system or sustained-release system in the preparation of a composition for anti-aging, repair, soothing or moisturizing.

[0030] On the other hand, the present disclosure provides the use of the above peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or the above composition, or the above delivery system or sustained-release system in the preparation of a composition for inhibiting hyaluronidase activity; or in the preparation of a composition for promoting collagen production; or 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 the skin or mucosa; or in the preparation of a composition for treating, preventing or repairing skin photoaging; or in the preparation of a composition for reducing, preventing or treating wrinkles; or in the preparation of a composition for inhibiting calcium ion influx.

[0031] On the other hand, the present disclosure provides the use of the above peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or the above composition, or the above delivery system or sustained-release system in the preparation of a composition for treating or caring for a condition, disorder or disease caused by muscle contraction.

[0032] In some embodiments, the condition, disorder or disease is dystonia.

[0033] In some embodiments, the dystonia includes focal dystonia, segmental dystonia, multifocal dystonia or hemidystonia; In some embodiments, the focal dystonia includes blepharospasm, dystonia musculorum deformans, cervical dystonia or torticollis, laryngeal dystonia or spasmodic dysphonia, oromandibular dystonia, limb dystonia, bruxism, hemifacial spasm, tic disorder or strabismus; In some embodiments, the limb dystonia includes writer's cramp, musician's cramp or foot dystonia; In some embodiments, the segmental dystonia includes Meige syndrome; In some embodiments, the dystonia includes dopamine-responsive dystonia.

[0034] Another aspect of the present disclosure provides the use of the above-mentioned peptide, or its stereoisomer, or a mixture of its stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in the preparation of cosmetics.

[0035] In the present disclosure, the term "skin" should be understood as the multiple layers that constitute it, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both endpoints being included. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes, etc. In the present disclosure, the term "skin" includes the scalp.

[0036] The term "caring for the skin" refers to maintaining and nourishing the skin, improving the state of the skin, making the skin delicate, smooth, tender and healthy.

[0037] The term "treatment" refers to administering the peptide according to the present disclosure to alleviate or eliminate a disease or disorder, or to reduce or eliminate one or more symptoms associated with such disease or disorder. The term "treatment" also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or disorder.

[0038] The term "prevention" refers to the ability of the peptide of the present disclosure to prevent, delay, or impede the occurrence or development of a disease or disorder before it appears.

[0039] The term "repair" refers to the ability of the peptide of the present disclosure to improve, relieve, or restore the original state after the occurrence of a disease or disorder.

[0040] The term "aging" refers to the changes that the skin undergoes with increasing age (natural aging), or those resulting from exposure to sunlight (photoaging) or environmental pollutants such as chemical dirt or contaminants, tobacco smoke, etc., and includes all externally visible and / or perceptible by touch changes, such as and not limited to: the development of discontinuities on the skin (such as wrinkles, fine lines, expression lines, stretch marks, striations, furrows, unevenness or roughness, increased pore size, water loss, loss of elasticity, loss of firmness, loss of smoothness, loss of the ability to recover from deformation, loss of resilience), skin sagging (such as sagging cheeks, the appearance of eye bags below the eyes, or the appearance of a double chin, etc.), changes in skin color (such as scars, redness, eye bags, or the appearance of hyperpigmented areas such as age spots or freckles, etc.), abnormal differentiation, hyperkeratosis, elastosis, keratosis, hair loss, cellulite, loss of collagen structure, and other histological changes in the stratum corneum, dermis, epidermis, vascular system (such as the appearance of spider veins or telangiectasia) or those tissues close to the skin.

[0041] The term "photoaging" refers to the premature aging of the skin caused by long-term exposure of the skin to ultraviolet radiation, which exhibits the same physiological characteristics as natural aging, such as and not limited to: laxity, sagging, color change or irregular pigmentation, abnormal and / or excessive keratinization.

[0042] The present disclosure has the following advantages and effects: The peptide of the present disclosure can effectively inhibit the influx of calcium ions, thereby inhibiting muscle contraction, and can be used to reduce, prevent or treat wrinkles. By inhibiting the influx of calcium ions, the peptide of the present disclosure can be used to treat or care for conditions, disorders or diseases caused by muscle contraction.

[0043] The peptide of the present disclosure can effectively increase the collagen content in cells after ultraviolet radiation, and can be used to treat, prevent or repair skin photoaging. By promoting collagen production, the peptide of the present disclosure is beneficial to increasing skin elasticity, improving skin firmness, and promoting re-epithelialization or healing of the skin or mucosa.

[0044] The peptide of the present disclosure can effectively inhibit the activity of hyaluronidase, repair the skin barrier, and has the effects of repair, soothe and moisturize.

[0045] The peptide of the present disclosure has the functions of anti-aging, firming, anti-wrinkle, repair, soothe and moisturize, and can be used to care for or treat the skin or mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] To more clearly illustrate the technical solutions of the present disclosure, the accompanying drawings required for the description of the present disclosure will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0047] Figure 1 is the mass spectrometry diagram of cyclohexapeptide A (molecular formula C 38 H 59 N 9 O 7 )in Example 1 of the present disclosure.

[0048] Figure 2 is the mass spectrometry diagram of hexapeptide B (molecular formula C 38 H 61 N 9 O 8 )in Example 2 of the present disclosure. Detailed implementation manners

[0049] To make the objectives, features, and advantages of the present disclosure more obvious and understandable, the present disclosure will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are some embodiments of the present disclosure, rather than all embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope protected by the appended claims of the present disclosure.

[0050] In the present disclosure, the abbreviations for amino acids follow the rules specified by the IUPAC-IUB Commission of Biochemical Nomenclature in the European Journal of Biochemistry (Eur. J. Biochem. 1984, 138: 9-37).

[0051] Unless otherwise specified, the experimental reagents and materials used in the present disclosure can be obtained commercially. The following are the abbreviations for some reagents and materials:

[0052] 2-CTC Resin: an initial resin for polypeptide synthesis (2-chlorotrityl chloride resin); DCM: dichloromethane; HOBt: 1-hydroxybenzotriazole; DMF: N,N-dimethylformamide; DIPEA: diisopropylethylamine; MeOH: methanol; piperidine: piperidine; DIC: diisopropylcarbodiimide; HBTU: O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate; TFA: trifluoroacetic acid; Tis: triisopropylsilane; EDT: 1,2-ethanedithiol; Lys: lysine; Val: valine; Trp: tryptophan; Leu: leucine; Gln: glutamine; Fmoc: 9-fluorenylmethyloxycarbonyl; Boc: tert-butoxycarbonyl; Trt: trityl.

[0053] Example 1 Preparation of Cyclo-[Val-Leu-Gln-Trp-Val-Lys]

[0054] Cyclo-[Val-Leu-Gln-Trp-Val-Lys] was prepared through the following steps:

[0055] 1.1 Swelling of the resin

[0056] Weigh 100 g of 2-CTC Resin into a solid-phase synthesis reaction column, swell it with DCM, wash the resin, and draw off the solvent.

[0057] 1.2 Feeding and reaction

[0058] Weigh 111.1 g of Fmoc-Lys(Boc)-OH into a dry Erlenmeyer flask, dissolve it with DMF solvent, cool it in an ice-water bath for 10 min, and add 85 mL of DIPEA to activate it for 10 min. Add the activated Fmoc-Lys(Boc)-OH to the swollen resin and react for 3 h, draw off the reaction solution, wash the resin, and draw off the solvent. Continue to add DCM, MeOH, and DIPEA for capping treatment for 15 min, and repeat once. Wash the resin and draw off the solvent to obtain Fmoc-Lys(Boc)-2-CTC Resin.

[0059] Deprotect the Fmoc group at the N-terminus twice with 20% piperidine / DMF for 10 minutes each time. Take a sample for Kaiser test, and the color shows dark blue. Wash the resin with DMF 7 times and pump out the solvent. Weigh 66.7 g of Fmoc-Val-OH and 32 g of HOBt and add them to a dry Erlenmeyer flask. Add DMF to dissolve them, seal it and place it in a -18°C refrigerator for 30 minutes. Add 46 mL of DIC to activate for 3 minutes, avoiding water vapor. Add the activated amino acid to the deprotected resin and react for 1 hour, then pump out the reaction solution. The Kaiser test shows that the resin is colorless and transparent, indicating that the reaction is complete, and Fmoc-Val-Lys(Boc)-2-CTC Resin is obtained.

[0060] Deprotect the Fmoc group at the N-terminus of the peptidyl resin. Using DMF as the solvent, couple 103.4 g of activated Fmoc-Trp(Boc)-OH to the peptidyl resin in the presence of 32 g of HOBt and 46 mL of DIC, and continue the reaction for 1 hour. Then wash these resins and repeat the deprotection process of the Fmoc group to couple the next amino acid. In each coupling, in the presence of 32 g of HOBt and 46 mL of DIC, using DMF as the solvent, sequentially couple 120.0 g of Fmoc-Gln(Trt)-OH, 70 g of Fmoc-Leu-OH, and then 66.7 g of Fmoc-Val-OH; after the reaction is complete, wash the resin and pump out the solvent to obtain Fmoc-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin.

[0061] Deprotect the Fmoc group at the N-terminus of the peptidyl resin twice with 20% piperidine / DMF for 10 minutes each time. Take a sample for Kaiser test, and the color shows dark blue. Wash the resin with DMF 6 times and pump out the solvent. After shrinkage drying, 300 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin is obtained.

[0062] 1.3 Deprotection of resin

[0063] 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 standby; place isopropyl ether in a -18°C refrigerator for freezing and standby.

[0064] Weigh 230 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin, add it to a round-bottom flask, add the above-prepared frozen cleavage solution, stir and react for 0.5 h, repeat once, and perform a total of 2 cleavages. Filter by suction, collect the filtrate and concentrate to obtain 160 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-OH.

[0065] 1.4 Cyclization

[0066] Add 160 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-OH to a flask, add 4 L of DCM and stir, then add 80 g of HBTU and 68.2 g of DIPEA, and react for 4 h. After the reaction is completed, perform post-treatment, and after treatment, obtain 170 g of wet weight of the crude cyclic peptide Cyclo-[Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)].

[0067] 1.5 Cleavage (deprotection of groups)

[0068] Mix 765 mL of TFA, 21.25 mL of Tis, 21.25 mL of EDT, and 21.25 mL of benzyl methyl sulfide, add 170 g of the above-mentioned crude cyclic peptide, cleave for 2 h, precipitate with isopropyl ether, and then wash with isopropyl ether to obtain the crude peptide Cyclo-[Val-Leu-Gln-Trp-Val-Lys].

[0069] 1.6 Purification

[0070] Dissolve 26.5 g of the crude peptide Cyclo-[Val-Leu-Gln-Trp-Val-Lys] in methanol, add acetic acid and purified water for dilution, filter through a microporous membrane with a pore size of 0.45 μm to obtain a clear and transparent solution, and purify it by reverse-phase HPLC. The purification gradient is shown in Table 1 below:

[0071] Table 1 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

[0072] Inject the filtered sample for purification, collect the fractions, concentrate and freeze-dry to obtain the peptide Cyclo-[Val-Leu-Gln-Trp-Val-Lys] with a purity > 95%, denoted as cyclic hexapeptide A, and its chemical structural formula is as follows: .

[0073] Determine the molecular weight of cyclic hexapeptide A by ESI-MS, and the mass spectrum is as Figure 1 shown. The results show 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 exact molecular weight of cyclohexapeptide A. The NMR data and analysis results are as follows:

[0074] 1 H NMR (600 MHz, D 2 O) 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).

[0075] Table 2 Hydrogen spectrum analysis table of the tested compounds Serial number Chemical shift (ppm) Number of protons Peak shape (coupling constant) Structural assignment 1 7.53 1 d, J = 6 Hz CH of indole ring 2 7.32 1 d, J = 6 Hz CH of indole ring 3 7.08 2 q, J = 6 Hz CH of indole ring 4 7.03 1 d, J = 6 Hz CH of indole ring 5 4.64 2 q, J = 6 Hz α-CH connected to amide bond 6 4.36 1 dd, J = 12 Hz, 6 Hz α-CH connected to amide bond 7 4.24 1 d, J = 6 Hz α-CH connected to amide bond 8 4.11 2 m α-CH connected to 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 valine side chain methyl 19 0.75 6 q, J = 6 Hz Hydrogen of leucine and valine side chain methyl 20 0.66 3 d, J = 6 Hz Hydrogen of leucine side chain methyl

[0076] 13 C NMR (150 MHz, D 2 O) 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.

[0077] Table 3 Carbon spectrum analysis table of the tested compound Serial number Chemical shift (ppm) Number of carbon atoms Structural assignment 1 177.15 1 Carbon of carbonyl (amide) 2 175.13 1 Carbon of carbonyl (amide) 3 173.79 1 Carbon of carbonyl (amide) 4 173.57 2 Carbon of carbonyl (amide) 5 172.93 1 Carbon of carbonyl (amide) 6 171.19 1 Carbon of carbonyl (amide) 7 136.20 1 C on indole ring 8 126.67 1 C on indole ring 9 124.57 1 C on indole ring 10 122.06 1 C on indole ring 11 119.45 1 C on indole ring 12 118.20 1 C on indole ring 13 112.14 1 C on indole ring 14 108.67 1 C on indole ring 15 59.05 1 α-C connected to amide bond 16 58.27 1 α-C connected to amide bond 17 56.11 1 α-C connected to amide bond 18 54.73 1 α-C connected to amide bond 19 54.54 1 α-C connected to amide bond 20 51.03 1 α-C connected to amide bond 21 39.05 1 Carbon of saturated alkyl group (no obvious characteristics) 22 30.79 1 Carbon of saturated alkyl group (no obvious characteristics) 23 30.29 1 Carbon of saturated alkyl group (no obvious characteristics) 24 29.36 1 Carbon of saturated alkyl group (no obvious characteristics) 25 28.36 1 Carbon of saturated alkyl group (no obvious characteristics) 26 27.15 1 Carbon of saturated alkyl group (no obvious characteristics) 27 26.48 1 Carbon of saturated alkyl group (no obvious characteristics) 28 25.34 1 Carbon of saturated alkyl group (no obvious characteristics) 29 24.47 1 Carbon of saturated alkyl group (no obvious characteristics) 30 23.34 1 Carbon of saturated alkyl group (no obvious characteristics) 31 22.58 1 Carbon of saturated alkyl group (no obvious characteristics) 32 22.21 1 Carbon of leucine side chain methyl 33 19.78 1 Carbon of leucine side chain methyl 34 18.73 1 Carbon of valine side chain methyl 35 18.19 1 Carbon of valine side chain methyl 36 17.37 1 Carbon of valine side chain methyl 37 16.04 1 Carbon of valine side chain methyl

[0078] Comprehensive analysis of the above nuclear magnetic resonance hydrogen spectrum and carbon spectrum shows that the structure of the tested compound is consistent with that of cyclohexapeptide A.

[0079] Example 2 Preparation of H-Val-Leu-Gln-Trp-Val-Lys-OH

[0080] 2.1 Swelling of resin

[0081] Weigh 100 g of 2-CTC Resin into a solid-phase synthesis reaction column, swell it with DCM, wash the resin, and suck out the solvent.

[0082] 2.2 Feeding reaction

[0083] Weigh 111.1 g of Fmoc-Lys(Boc)-OH into a dry Erlenmeyer flask, dissolve it with DMF solvent, cool it in an ice-water bath for 10 min, add 85 mL of DIPEA to activate it for 10 min, avoiding water vapor. Add the activated Fmoc-Lys(Boc)-OH to the swollen resin and react for 3 h, suck out the reaction solution, wash the resin, and suck out the solvent. Continue to add DCM, MeOH and DIPEA for capping treatment for 0.5 h. Wash the resin and suck out the solvent to obtain Fmoc-Lys(Boc)-2-CTC Resin.

[0084] Deprotect Fmoc from Fmoc-Lys(Boc)-2-CTC Resin twice with 20% piperidine / DMF, 10 min each time, sample for Kaiser test, and the color shows dark blue. Wash the resin 7 times with DMF and suck out the solvent.

[0085] Weigh 66.7 g of Fmoc-Val-OH and 32 g of HOBt into a dry Erlenmeyer flask, add DMF to dissolve it, seal it and place it in a -18 °C refrigerator for 30 min. Add 46 mL of DIC to activate it for 3 min, avoiding water vapor. Add the activated amino acid to the deprotected resin and react for 1 h, suck out the reaction solution. The Kaiser test shows that the resin is colorless and transparent, indicating that the reaction is complete.

[0086] The N-terminal Fmoc group was deprotected, and 103.4 g of activated Fmoc-Trp(Boc)-OH was coupled to the peptide resin in the presence of 32 g of HOBt and 46 mL of DIC using DMF as the solvent, and the reaction was continued for 1 h. Then the resins were washed and the deprotection process of the Fmoc group 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 the solvent; after the reaction was complete, the resin was washed and the solvent was removed by suction.

[0087] The N-terminal Fmoc group of the peptide resin was deprotected, and the Fmoc was removed twice with 20% piperidine / DMF for 10 min each time. Samples were taken for Kaiser test, and the color development was dark blue. The resin was washed 6 times with DMF, and the solvent was removed by suction to obtain H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin.

[0088] 2.3 Cleavage

[0089] 180 mL of TFA, 5 mL of EDT, 5 mL of TIS, 5 mL of benzyl methyl sulfide, and 5 mL of water were measured and mixed and stirred evenly to obtain a cleavage solution, which was sealed and placed in a refrigerator at -18 °C for standby; isopropyl ether was placed in a refrigerator at -18 °C for freezing and standby.

[0090] 40 g of H-Val-Leu-Gln(Trt)-Trp(Boc)-Val-Lys(Boc)-2-CTC Resin was weighed and added to a round-bottom flask, and the above-mentioned frozen cleavage solution was added, and the reaction was stirred for 2 h. Suction filtration was carried out, and after the filtrate was collected, isopropyl ether was added and stirred and centrifuged for washing 3 times, and then vacuum dried to obtain H-Val-Leu-Gln-Trp-Val-Lys-OH.

[0091] 2.4 Purification

[0092] 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 and sonicated. After filtration through diatomaceous earth, it was filtered through a microporous filter membrane with a pore size of 0.45 μm to obtain a clear solution, and was purified by reverse-phase HPLC. The purification gradient is shown in Table 4 below:

[0093] Table 4 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

[0094] The filtered sample was injected for purification, fractions were collected, concentrated and freeze-dried to obtain peptide H-Val-Leu-Gln-Trp-Val-Lys-OH with a purity > 97%, denoted as hexapeptide B. The molecular weight of hexapeptide B was measured, and the mass spectrum was as shown in Figure 2 shown. The results showed that the mass-to-charge ratio (m / z) of the [M+H] + quasi-molecular ion peak was 772.86, and the molecular weight measured by mass spectrometry was 771.86, which was consistent with the theoretical exact molecular weight of hexapeptide B.

[0095] Other peptides of the present disclosure include, but are not limited to: H-Val-Leu-Gln-Trp-Val-Lys-NH 2 2, Ac-Val-Leu-Gln-Trp-Val-Lys-OH, Ac-Val-Leu-Gln-Trp-Val-Lys-NH 2 2, Myr-Val-Leu-Gln-Trp-Val-Lys-OH, Myr-Val-Leu-Gln-Trp-Val-Lys-NH 2 2, Pal-Val-Leu-Gln-Trp-Val-Lys-OH, Pal-Val-Leu-Gln-Trp-Val-Lys-NH 2 2, etc., which can be prepared by similar solid-phase peptide synthesis methods.

[0096] Example 3 Calcium ion influx test

[0097] 3.1 Reagents and materials

[0098] 0.25% trypsin digestion solution, phosphate buffer (PBS), acetylcholine solution containing 1% fetal bovine serum, Fluo-3AM calcium ion probe.

[0099] 3.2 Instruments

[0100] Thermostatic CO 2 2 incubator, laminar flow hood, fluorescence microscope.

[0101] 3.3 Cell line

[0102] Mouse neuroblastoma cells (Neuro-2a cells).

[0103] 3.4 Test samples and grouping

[0104] 3.4.1 Test samples

[0105] Cyclohexapeptide A, hexapeptide B, reference peptide (dipetidyl diamino butyryl benzylamide diacetate), and the test concentrations were all 50 ppm.

[0106] 3.4.2 Grouping

[0107] Experimental group: Test sample, acetylcholine solution containing 1% fetal bovine serum.

[0108] Control group: PBS, acetylcholine solution containing 1% fetal bovine serum.

[0109] 3.5 Experimental method

[0110] 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 detached, count (1 - 4)×10 5 cells / mL to make a cell suspension. Take an appropriate amount of the cell suspension and inoculate it on a 12-well plate containing complete medium, and culture it in a constant temperature CO 2 incubator for 24 h. Aspirate the complete medium in the wells, add complete medium and the test sample to the experimental group, add complete medium and an equal volume of PBS to the control group, and culture in a CO 2 incubator for 24 h. Discard the supernatant, wash the cells 2 times with PBS, add Fluo-3AM working solution to the 12-well plate, and incubate at 37°C for 30 min. Discard the Fluo-3AM working solution, add the test sample and acetylcholine solution containing 1% fetal bovine serum to the experimental group, add an equal volume of acetylcholine solution containing 1% fetal bovine serum to the control group, and incubate for 5 min. Discard the supernatant, add PBS to wash the cells 2 times. Observe and take pictures under a fluorescence microscope, and perform relative fluorescence intensity analysis using Image J software.

[0111] 3.6 Experimental results

[0112] Calcium ion influx plays a key role in skin muscle contraction. By inhibiting calcium ion influx, excessive muscle contraction can be reduced, wrinkles on the skin surface can be improved, and the wrinkle depth can be reduced, thus achieving the effect of removing wrinkles. The calcium ion content in cells is proportional to the fluorescence staining intensity, that is, the stronger the fluorescence intensity, the higher the calcium ion content in the cells. In this experiment, the relative fluorescence intensity of calcium ions in Neuro-2a cells was detected to determine whether the peptide disclosed in the present invention can inhibit calcium ion influx.

[0113] The results of the influence of the test sample on calcium ion influx in Neuro-2a cells are shown in Table 5.

[0114] Table 5 Relative fluorescence intensity of calcium ions in Neuro-2a cells Group Relative fluorescence intensity (Mean±SD) Control group 100.00%±1.60% Reference peptide group <![CDATA[52.43%±3.47% *** > Cyclohexapeptide A group <![CDATA[46.84%±2.61% *** > Hexapeptide B group <![CDATA[55.91%±2.09% *** >

[0115] Note: Compared with the control group, *** P <0.001.

[0116] The reference peptide (dipetide 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 with that of the control group, indicating that the experimental system is effective and reliable. Both the cyclic hexapeptide A and hexapeptide B of the present disclosure 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 on inhibiting calcium ion influx than the reference peptide. It can be seen that the peptides of the present disclosure can significantly inhibit calcium ion influx, thereby inhibiting muscle contraction, and can be used to reduce, prevent or treat wrinkles and play anti-aging and anti-wrinkle roles. By inhibiting calcium ion influx, the peptides of the present disclosure can be used to treat or care for conditions, disorders or diseases caused by muscle contraction.

[0117] Example 4 Determination of Collagen I Content

[0118] 4.1 Reagents and Materials

[0119] Fetal bovine serum, DMEM medium, phosphate buffer solution (PBS), trypsin, RIPA lysis buffer, collagen I ELISA kit, BCA protein kit.

[0120] 4.2 Instruments

[0121] Microplate reader, CO 2 Incubator, laminar flow hood, air bath constant temperature shaker.

[0122] 4.3 Cell Lines

[0123] Human skin fibroblasts (HSF).

[0124] 4.4 Samples to be Tested and Grouping

[0125] 4.4.1 Samples to be Tested

[0126] Cyclic hexapeptide A and hexapeptide B, with test concentrations of 15.6 ppm.

[0127] 4.4.2 Grouping

[0128] Experimental group: UV radiation + sample to be tested.

[0129] Blank control group: PBS.

[0130] UV group: UV radiation + PBS.

[0131] 4.5 Experimental Method

[0132] Take a bottle of HSF fibroblasts in good condition in the exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells detached, and count (1 - 4) × 105 cells / mL to prepare a cell suspension.

[0133] The diluted cell suspension was inoculated into a 12-well plate and cultured in a CO 2 incubator for 24 h. The complete medium in the wells was aspirated, and in each group, an appropriate amount of PBS was added and repeatedly washed until colorless. Then, in the blank control group, 100 μL of PBS was added and the complete medium was supplemented to 1000 μL without UV irradiation; in the UV group and the experimental group, 200 μL of PBS was added, and the cells were irradiated under a UV lamp at 80 mJ / cm 2 for 15 min. After irradiation, the PBS was discarded. In the UV group, 100 μL of PBS solution was added and the complete medium was supplemented to 1000 μL, while in the experimental group, 100 μL of the test sample was added and the complete medium was supplemented to 1000 μL. The blank control group, the UV group, and the experimental group were continued to be incubated in an incubator at 37 °C and 5% CO 2 for 48 h.

[0134] After the culture, the cells were collected, the supernatant was discarded by centrifugation, RIPA lysis buffer was added, and the mixture was shaken evenly 3 times with a vortex mixer (30 s each time, with an interval of 3 min), then centrifuged at 12000 rpm for 10 min. The supernatant was aspirated, and the detection was carried out according to the operation instructions of the collagen type I detection kit, and the total protein concentration of the supernatant was detected with a BCA protein quantification kit.

[0135] 4.6 Experimental results

[0136] Collagen type I is the most abundant collagen in the human body, showing a thick and tightly arranged bundle structure. It has strong tensile strength, provides a strong supporting structure and support force for the skin, endows the skin with elasticity and toughness. Therefore, increasing the content of collagen type I is of great significance for anti-aging, increasing skin elasticity and firmness. In this experiment, cells irradiated with ultraviolet rays were treated with the test sample, and the content of collagen type I in the corresponding cells was detected to determine whether the peptide disclosed in this application can promote the production of collagen type I.

[0137] The results of the effect of the test sample on the content of collagen I are shown in Table 6.

[0138] Table 6 Effect of the test sample on the content of collagen I Group Relative content of collagen I (Mean±SD) Blank control group 100.00%±8.46% UV group <![CDATA[47.32%±6.31% ### > Cyclohexapeptide A group <![CDATA[72.98%±3.08% *** > Hexapeptide B group <![CDATA[72.73%±4.65% ** >

[0139] Note: Compared with the blank control group, ### P <0.001; compared with the UV group, ** P <0.01, *** P <0.001.

[0140] The results showed that, compared with the blank control group, the content of collagen I in the UV group was significantly decreased, indicating successful modeling; compared with the UV group, both cyclohexapeptide A and hexapeptide B in the experimental group could significantly increase the content of collagen I and promote the production of type I collagen.

[0141] It can be seen therefrom that the peptides of the present disclosure can significantly increase the collagen content in cells irradiated with ultraviolet rays and can be used for treating, preventing or repairing skin photoaging. By promoting collagen production, the peptides of the present disclosure can be used to increase skin elasticity, improve skin firmness and delay skin aging.

[0142] Example 5 Measurement of Collagen III Content

[0143] 5.1 Reagents and Materials

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

[0145] 5.2 Instruments

[0146] Microplate reader, CO 2 Incubator, laminar flow hood, air bath thermostatic shaker.

[0147] 5.3 Cell Line

[0148] Human skin fibroblasts (HSF).

[0149] 5.4 Test Samples and Grouping

[0150] 5.4.1 Test Samples

[0151] Cyclohexapeptide A, hexapeptide B, and the test concentration is 31.2 ppm for both.

[0152] 5.4.2 Grouping

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

[0154] Blank control group: PBS.

[0155] UV group: UV radiation + PBS.

[0156] 5.5 Experimental Method

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

[0158] Inoculate the diluted cell suspension into a 12-well plate and place it in a CO2 Cultivate for 24 h in an incubator. Aspirate the complete medium in the wells. After each group is washed repeatedly with an appropriate amount of PBS until colorless, the blank control group adds 100 μL of PBS and supplements the complete medium to 1000 μL without UV irradiation; the UV group and the experimental group add 200 μL of PBS and are placed under an 80 mJ / cm 2 UV lamp and irradiated for 15 min. After irradiation, discard the PBS. The UV group adds 100 μL of PBS solution and supplements the complete medium to 1000 μL. The experimental group adds 100 μL of the sample to be tested and supplements the complete medium to 1000 μL. The blank control group, the UV group, and the experimental group continue to incubate in an incubator at 37 °C and 5% CO 2 for 48 h.

[0159] After the culture is completed, collect the cells, centrifuge to discard the supernatant, add RIPA lysis buffer, and homogenize with a vortex mixer 3 times (30 s each time, with an interval of 3 min). Centrifuge at 12,000 rpm for 10 min, aspirate the supernatant, and detect according to the operation manual of the collagen III detection kit, and detect the total protein concentration of the supernatant with a BCA protein quantification kit.

[0160] 5.6 Experimental results

[0161] Collagen type III plays an important role in skin support and repair, can provide elasticity and stress resistance to the skin, and is also involved in the skin regeneration and repair process, which can accelerate wound healing and tissue regeneration. Therefore, increasing the content of collagen type III is of great significance for tightening the skin and repairing damaged skin. In this experiment, cells irradiated with ultraviolet rays were treated with the test sample, and the content of collagen type III in the corresponding cells was detected to determine whether the peptide disclosed in the present invention can promote the production of collagen type III.

[0162] The results of the influence of the test sample on the content of collagen III are shown in Table 7.

[0163] Table 7 Influence of the test sample on the content of collagen III Group Relative content of collagen III (Mean±SD) Blank control group 100.00%±12.37% UV group <![CDATA[54.79%±4.17% ## > Cyclohexapeptide A group <![CDATA[99.95%±15.00% ** > Hexapeptide B group <![CDATA[90.21%±5.42% *** >

[0164] Note: Compared with the blank control group, ## P <0.01; compared with the UV group, ** P <0.01, *** P <0.001.

[0165] The results showed that, compared with the blank control group, the content of collagen III in the UV group was significantly decreased, indicating successful modeling; compared with the UV group, both cyclohexapeptide A and hexapeptide B in the experimental group could significantly increase the content of collagen III and promote the production of type III collagen.

[0166] It can be seen therefrom that the peptides of the present disclosure can effectively increase the collagen content in cells irradiated with ultraviolet rays and can be used for treating, preventing or repairing skin photoaging. By promoting collagen production, it is beneficial to increase skin elasticity, improve skin firmness, and promote re-epithelialization or healing of the skin or mucosa, thereby achieving the effects of anti-aging, firming, and repairing.

[0167] Example 6 Hyaluronidase inhibition experiment

[0168] 6.1 Reagents and materials

[0169] Sodium acetate buffer solution (pH = 5.6), hyaluronidase, calcium chloride, sodium hyaluronate, acetylacetone solution, absolute ethanol, sodium hydroxide solution, P-DAB color reagent [prepared by uniformly mixing p-dimethylaminobenzaldehyde (0.8 g), concentrated hydrochloric acid (15 mL) and an equal amount of glacial acetic acid].

[0170] 6.2 Instruments

[0171] Microplate reader, electronic balance, air bath constant temperature shaker.

[0172] 6.3 Samples to be tested and grouping

[0173] 6.3.1 Samples to be tested

[0174] Cyclohexapeptide A and hexapeptide B, with test concentrations both being 1000 ppm.

[0175] 6.3.2 Grouping

[0176] Sample group: Samples to be tested, hyaluronidase, sodium hyaluronate;

[0177] Sample zero adjustment group: Samples to be tested, sodium acetate buffer solution;

[0178] Blank control group: Distilled water, hyaluronidase, sodium hyaluronate;

[0179] Blank zero adjustment group: Distilled water, sodium acetate buffer solution.

[0180] 6.4 Experimental method

[0181] Both hyaluronidase and sodium hyaluronate are dissolved with sodium acetate buffer solution.

[0182] Take a 96-well plate. Add 25 μL of the sample to be tested and 25 μL of hyaluronidase (500 U / mL) to the sample group; add 25 μL of the sample to be tested and 25 μL of sodium acetate buffer to the sample zero-adjustment group; add 25 μL of distilled water and 25 μL of hyaluronidase (500 U / mL) to the blank control group; add 25 μL of distilled water and 25 μL of sodium acetate buffer to the blank zero-adjustment group. After placing them in a 37°C constant-temperature air bath and shaking for 20 min, add 5 μL of calcium chloride solution (2.5 mol / L) to each well, and then place them in a 37°C constant-temperature air bath and shake for 20 min. Add 25 μL of sodium hyaluronate (1 mg / mL) to the sample group and the blank control group, add 25 μL of sodium acetate buffer to the sample zero-adjustment group and the blank zero-adjustment group, and place them in a 37°C constant-temperature air bath and shake for 40 min. Then add 25 μL of distilled water, 5 μL of sodium hydroxide solution (5 mol / L), and 25 μL of acetylacetone solution to each well, place them in a boiling water bath for 15 min, then in an ice bath for 10 min, and finally at room temperature for 10 min. Add 50 μL of P-DAB to each well, then add 100 μL of absolute ethanol, place them at room temperature for 30 min, and measure the OD at 570 nm. 570 value.

[0183] Hyaluronidase inhibition rate (%)

[0184] Where: A 1 is the OD value of the sample zero-adjustment group, A 570 is the OD value of the sample group, A 2 is the OD value of the blank zero-adjustment group, A 570 is the OD value of the blank control group, A 3 is the OD value of the blank zero-adjustment group, A 570 is the OD value of the blank control group, A 4 is the OD value of the blank control group. 570 value.

[0185] 6.5 Experimental results

[0186] The activity of hyaluronidase is closely related to type I allergic reactions. Inhibiting the activity of hyaluronidase can play a soothing and anti-allergic role. Therefore, the inhibition of hyaluronidase activity is often used as an evaluation index for studying the soothing and anti-allergic effects. In addition, hyaluronidase is a hydrolase that degrades hyaluronic acid. The reduction of hyaluronic acid will damage the skin barrier function and cause water loss inside the skin. Inhibiting the activity of hyaluronidase can reduce the degradation of hyaluronic acid in cells, increase the content of hyaluronic acid in cells, facilitate the hydration of the skin or mucosa, repair the skin barrier, and play the effects of moisturizing, soothing, and repairing.

[0187] Sodium hyaluronate is the substrate of hyaluronidase. Under the catalysis of hyaluronidase, sodium hyaluronate degrades to produce glucuronic acid and N-acetylglucosamine, which develops color under the action of P-DAB and absorbs visible light with a wavelength of 570 nm. In this experiment, test samples were used to treat hyaluronidase, and by detecting the reaction amount of sodium hyaluronate, it was determined whether the test samples to be disclosed could inhibit the activity of hyaluronidase.

[0188] The results of the inhibition rate of hyaluronidase activity of different test samples are shown in Table 8.

[0189] Table 8 Inhibition rate of hyaluronidase activity of test samples (Mean±SD) Concentration Cyclohexapeptide A Hexapeptide B 1000 ppm 54.43%±6.42% 21.48%±3.98%

[0190] The results showed that both cyclohexapeptide A and hexapeptide B to be disclosed could inhibit the activity of hyaluronidase, and the inhibition rate of cyclohexapeptide A was as high as 54.43%. Thus, it can be seen that the peptides to be disclosed have excellent effects of inhibiting the activity of hyaluronidase, can be used to repair the skin barrier, and have the effects of repair, soothing and moisturizing.

[0191] Example 7

[0192] A cream was prepared through the following steps, and the specific formula is shown in Table 9 below:

[0193] Table 9

[0194] According to the formula dosage, the materials in Phase C were heated to 55 - 60 °C in a suitable container and completely dissolved for standby. The materials in Phase A were added to the stirring pot and stirred and heated to 80 - 85 °C. The materials in Phase B were added to the oil phase pot and stirred and heated to 75 - 80 °C until completely dissolved and transparent. The materials in Phase B were pumped into Phase A, the vacuum was turned on, homogenized for 5 minutes, and stirring was maintained and kept warm for 20 minutes. Cooling was started, and when the temperature dropped to 60 - 65 °C, the materials in Phase C were added and homogenized for 2 minutes. When the temperature dropped to 35 - 40 °C, the pre-dissolved materials in Phase D were added and stirred for 10 - 15 minutes to obtain the product.

[0195] Example 8

[0196] An essence was prepared through the following steps, and the specific formula is shown in Table 10 below:

[0197] Table 10

[0198] According to the formula dosage, the materials in Phase A were added to the stirring pot and stirred and heated to 80 - 85 °C; the materials in Phase B were mixed evenly until there were no powder particles and then added to the stirring pot and stirred continuously for 10 - 15 minutes; cooling was started, and when the temperature dropped to 60 - 65 °C, the materials in Phase C were added; when the temperature dropped to 35 - 40 °C, the materials in Phase D and Phase E were added and stirred for 10 - 15 minutes to obtain the product.

[0199] In this disclosure, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variation thereof is 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 expressly listed, or elements inherent to such process, method, article or terminal device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or terminal device comprising the element.

[0200] Although specific embodiments of the present disclosure have been described for purposes of illustration, those skilled in the art can 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 peptides, wherein the metals include 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, and 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 caring for or treating skin or mucous membranes.

10. 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.

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

12. The use according to claim 10, characterized in that The soothing includes inhibiting hyaluronidase activity.

13. Use of a peptide or salt thereof according to any one of claims 1 to 3, or a composition according to claim 4, or a 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.

14. 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.

Images