Cyclic hexapeptide as well as composition and application thereof

Abstract

The invention discloses a cyclic hexapeptide as well as a composition and application thereof, and relates to the technical field of polypeptides, and the cyclic peptide has the following structure: Cyclo-[Glu-Glu-Met-Gln-Arg-Arg]. Specifically, the invention relates to the cyclic peptide or the salt thereof, or a composition of the cyclic peptide and the salt thereof, and application of the cyclic peptide and the salt in preparation of a composition for nursing or treating skin or mucosa.

Description

Technical Field

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

[0002] In recent years, with the continuous improvement of consumers' requirements for the efficacy and safety of cosmetics, the research and development of cosmetic raw materials have been increasingly emphasized. The Chinese cosmetic raw material industry is in a stage of rapid development. Active polypeptide raw materials have become the core components in the fields of anti-aging, repair, etc. due to their high biological activity and safety. At present, significant progress has been made in the research of polypeptide raw materials, and a variety of polypeptides with functions such as moisturizing, anti-aging, and whitening have been successfully developed and applied in cosmetics. In addition to the research and development of linear polypeptides, cyclic peptides, as a novel-structured polypeptide, have also received extensive attention and become a new hot spot in the research and development of polypeptide raw materials.

[0003] There are still few cyclic peptide products on the current market. Therefore, the development of cyclic peptide raw materials with novel structures, high efficiency and safety is of great significance for meeting the increasingly diverse market demands and promoting the development of the cosmetics industry. Summary of the Invention

[0004] The present disclosure relates to a cyclohexapeptide, its composition and uses. The cyclohexapeptide and the composition containing the cyclohexapeptide have the effects of nursing or treating the skin, mucous membrane, etc.

[0005] On the one hand, the present disclosure provides a cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or its salt. The structure of the cyclic peptide is Cyclo-[Glu-Glu-Met-Gln-Arg-Arg], and its structural formula is shown as follows:

[0006]

[0007] The cyclic peptide of the present disclosure contains a large number of asymmetric carbon atoms. Those skilled in the art understand that the cyclic peptide of the present disclosure has stereoisomers and can exist as stereoisomers or a mixture of stereoisomers, and thus it is possible to obtain a mixture of isomers and racemic mixtures or diastereoisomer mixtures, or pure diastereoisomers or enantiomers, depending on the number of asymmetric carbons and the isomers or isomer mixtures present. In some embodiments, the cyclic peptide described in the present disclosure is a pure isomer, that is, an enantiomer or a diastereoisomer.

[0008] The present disclosure also includes all suitable isotopic variants of the cyclic peptides. Isotopic variants of the cyclic peptides of the present disclosure are understood to mean compounds in which at least one atom within the cyclic 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 cyclic peptides of the present disclosure are those of hydrogen, carbon, nitrogen, oxygen, or sulfur, such as 2 H (deuterium), 3 H (tritium), 13 C, 14 C, 15 N, 17 O, 18 O, 33 S, 34 S, 35 S or 36 S. Specific isotopic variants of the cyclic peptides of the present disclosure (especially those in which one or more radioactive isotopes have been incorporated) may be advantageous, for example, for examining the mechanism of action or the distribution of active compounds in the body; due to their relatively simple preparability and detectability, especially cyclic peptides labeled with 3 H or 14 C isotopes are suitable for this purpose. Additionally, due to the greater metabolic stability of the cyclic peptides, the incorporation of isotopes (such as deuterium) can produce specific therapeutic benefits, such as an extended in vivo half-life or a reduced required active dose. Isotopic variants of the cyclic peptides of the present disclosure can be prepared by methods known to those skilled in the art, such as by the methods further described below and those described in the examples, by using the corresponding isotopically modified forms of the respective reagents and / or starting materials.

[0009] The term "salt" refers to an approved salt for use in animals, and more particularly in humans, and includes metal salts of the cyclic peptides, where the metals include, but are not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum, etc.; includes salts formed by the cyclic peptides with organic bases, where the organic bases include, but are not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine, etc.; includes salts formed by the cyclic peptides with inorganic acids or organic acids, where the organic acids include, but are not limited to: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoate, or gluconic acid, etc.; the inorganic acids include, but are not limited to: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

[0010] The nature of the salt is not decisive, and salts of the cyclic peptides can be obtained by conventional methods well known in the art.

[0011] The synthesis of the cyclic peptides, or their stereoisomers, or mixtures of their stereoisomers, or salts thereof according to the present disclosure 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 generating the desired sequence, or by controlled hydrolysis of proteins of animal, fungal, or plant origin.

[0012] For example, a method for obtaining the cyclic peptides according to the present disclosure includes the following steps:

[0013] - 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;

[0014] - Removing the group protecting the N-terminus;

[0015] - Repeating the coupling sequence and removing the group protecting the N-terminus until the desired peptide sequence is obtained;

[0016] - Removing the group protecting the C-terminus or cleaving from the solid support;

[0017] - Coupling and cyclizing the amino group at the N-terminus of the peptide chain with the carboxyl group at the C-terminus;

[0018] - Removing the groups protecting the side chains.

[0019] In some embodiments, the C-terminus is bound to a solid support and the method is carried out on the 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 original polymeric support, and coupling and cyclizing the amino group at the N-terminus of the peptide chain with the carboxyl group at the C-terminus.

[0020] During the entire synthesis, the functional groups of the side chains of these amino acids are kept sufficiently protected with temporary or permanent protecting groups.

[0021] In some embodiments, solid-phase synthesis can be carried out by a convergent strategy of coupling a dipeptide or tripeptide to a polymeric support or to a dipeptide or amino acid previously bound to a polymeric support.

[0022] Due to applications outside the body of mammals, the cyclic 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 cyclic 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.

[0023] In some embodiments,The adjuvant is selected from: an agent that activates Clock expression, an analgesic, an agent that inhibits PAR-2 activity, an agent that regulates PGC-1α synthesis, an agent that regulates the activity of PPARγ, an agent that increases or decreases the triglyceride content of adipocytes, an agent that stimulates or delays adipocyte differentiation, a lipolytic agent or an agent that stimulates lipolysis, a lipolytic agent, a lipogenic agent, an inhibitor of acetylcholine receptor aggregation, an agent that inhibits muscle contraction, an anticholinergic agent, an elastase inhibitor, a matrix metalloproteinase inhibitor, a stimulator or inhibitor of melanin synthesis, a whitening agent or a depigmenting agent, a pigmenting agent, a self-tanning agent, an anti-aging agent, a NO-synthase inhibitor, a 5α-reductase inhibitor, an inhibitor of lysyl hydroxylase and / or prolyl hydroxylase, an antioxidant, a free radical scavenger and / or an agent against air pollution, an advanced glycation end product scavenger, an anti-glycation agent, an antihistamine, an antiviral agent, an antiparasitic agent, an emulsifier, an emollient, an organic solvent, a liquid propellant, a water-retaining substance, an α-hydroxy acid, a β-hydroxy acid, a humectant, an epidermal hydrolase, a vitamin, an amino acid, a protein, a pigment, a dye, a biopolymer, a gum polymer, a thickening agent, a surfactant, a softening agent, an adhesive, a preservative, an anti-wrinkle agent, an agent capable of reducing or treating lower eye bags, a keratolytic agent, an antimicrobial agent, an agent that stimulates the synthesis of dermal or epidermal macromolecules and / or is capable of inhibiting or preventing their degradation, an agent that stimulates elastin synthesis, an agent that stimulates the synthesis of decorin, an agent that stimulates the synthesis of laminin, an agent that stimulates the synthesis of defensin, an agent that stimulates the synthesis of chaperone protein, an agent that stimulates cAMP synthesis, an agent that stimulates hyaluronic acid synthesis, an agent that stimulates fibronectin synthesis, an agent that stimulates deacetylase synthesis, an agent that stimulates the synthesis of lipids and stratum corneum components, a ceramide, a fatty acid, an agent that inhibits elastin degradation, an agent that inhibits serine protease, an agent that stimulates fibroblast proliferation, an agent that stimulates keratinocyte proliferation, an agent that stimulates adipocyte proliferation, an agent that stimulates melanocyte proliferation, an agent that stimulates keratinocyte differentiation, an agent that inhibits acetylcholinesterase, a skin relaxant, an agent that stimulates glycosaminoglycan synthesis, an anti-hyperkeratotic agent, an acne solvent, an anti-psoriatic agent, an anti-eczema agent, a DNA repair agent, a DNA protecting agent, a stabilizer, an antipruritic agent, an agent for treating and / or caring for sensitive skin, a curing agent, a firming agent, a reconstructing agent, an anti-stretch mark agent, an agent that regulates sebum production, an antiperspirant, an agent that stimulates healing, an agent that assists healing, an agent that stimulates re-epithelialization, an agent that assists re-epithelialization, a cytokine, a sedative, an anti-inflammatory agent, an anesthetic, an agent that acts on capillary circulation and / or microcirculation, an agent that stimulates angiogenesis, an agent that inhibits vascular permeability, a venotonic agent, an agent that acts on cell metabolism, an agent for improving the dermal-epidermal junction, an agent that induces hair growth, an agent that inhibits or delays hair growth, a fragrance, a chelating agent, a plant extract, an essential oil, a marine extract, an agent obtained from a biological fermentation process, an inorganic salt, a cell extract, a sunscreen, and an organic or inorganic light protection agent or a mixture thereof that is effectively resistant to ultraviolet A and / or B.

[0024] The effective amount of the cyclic 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 cyclic peptide to be used.

[0025] "Effective amount" means an amount of the cyclic peptides of the present disclosure that is non-toxic but sufficient to provide the desired effect. The cyclic 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.

[0026] Another aspect of the present disclosure provides a delivery system or a sustained-release system for better penetration of the active ingredient, which comprises an effective amount of the above-mentioned cyclic peptides, or their stereoisomers, or a mixture of their stereoisomers, or their salts, or the above-mentioned compositions.

[0027] The term "delivery system" refers to a diluent, adjuvant, excipient or carrier administered together with the cyclic peptides of the present disclosure, which are selected from: water, oils or surfactants, 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, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynols, poloxamers, polyethylene oxides, polyethylene glycols, dextrans, glycerols, digitonin and the like. Diluents that can be used in different delivery systems in which the cyclic peptides of the present disclosure can be administered are known to those of ordinary skill in the art.

[0028] 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 period of time.

[0029] Examples of the delivery system or the sustained-release system include, but are not limited to: liposomes, oleosomes, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion complexes, niosomes, micelles, milliballs, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, milliparticles, microparticles or nanoparticles.

[0030] Another aspect of the present disclosure provides a cosmetic comprising an effective amount of the above-mentioned cyclic 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.

[0031] 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.

[0032] Another aspect of the present disclosure provides the use of the above-mentioned cyclic 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 a composition for oil control, acne treatment or repair.

[0033] Another aspect of the present disclosure provides the use of the above-mentioned cyclic 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 a composition for reducing skin oil synthesis or secretion.

[0034] Another aspect of the present disclosure provides the use of the above-mentioned cyclic 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 a composition for promoting re-epithelialization or healing of the skin or mucosa.

[0035] Another aspect of the present disclosure provides the use of the above-mentioned cyclic 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.

[0036] In the present disclosure, the term "skin" should be understood to be the multiple layers that make it up, 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.

[0037] The term "caring for the skin" refers to maintaining and nourishing the skin, improving the state of the skin, and making the skin delicate, smooth, tender and healthy.

[0038] The present disclosure has the following advantages and effects:

[0039] 1. The cyclic peptide of the present disclosure can effectively promote cell migration. Promoting cell migration can promote re-epithelialization or wound healing of the skin or mucosa and repair the skin barrier. Therefore, the cyclic peptide of the present disclosure can be used to promote re-epithelialization or wound healing of the skin or mucosa, repair the damaged skin barrier, etc., so as to achieve the effect of repair.

[0040] 2. The cyclic peptides of the present disclosure can inhibit the synthesis or secretion of sebum by sebaceous gland cells, slow down sebum deposition, reduce the occurrence of acne or pimples, and also contribute to the recovery of the skin after the occurrence of acne or pimples, having the effects of controlling oil, removing acne, and repairing.

[0041] 3. The cyclic peptides of the present disclosure are obtained by coupling and cyclizing the N-terminus and C-terminus of linear EEMQRR. However, compared with linear polypeptides, the cyclic peptides of the present disclosure have better effects of promoting cell migration and inhibiting oil synthesis or secretion, thereby achieving excellent effects of controlling oil, removing acne, and repairing. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the present disclosure, the drawings required for the description of the present disclosure will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present disclosure. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0043] Figure 1 It is the mass spectrum of cyclohexapeptide A (molecular formula C 32 H 55 N 13 O 11 S) in Example 1 of the present disclosure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] To make the above objects, 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 drawings and embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present disclosure. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present disclosure without creative efforts fall within the scope of the claims of the present disclosure.

[0045] In the present disclosure, the abbreviations for amino acids follow the rules specified by the IUPAC-IUB Commission of Biochemical Nomenclature in Eur. J. Biochem. 1984, 138: 9-37.

[0046] 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:

[0047] 2-CTC Resin: An initial resin for polypeptide synthesis; DCM: Dichloromethane; HOBt: 1-Hydroxybenzotriazole; DMF: N,N-Dimethylformamide; DIPEA: Diisopropylethylamine; MeOH: Methanol; piperidine: Piperidine; DIC: Diisopropylcarbodiimide; TFA: Trifluoroacetic acid; Tis: Triisopropylsilane; EDT: 1,2-Ethanedithiol; Glu: Glutamic acid; Met: Methionine; Gln: Glutamine; Arg: Arginine; Fmoc: 9-Fluorenylmethyloxycarbonyl; OtBu: tert-Butoxy; Trt: Triphenylmethyl; Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-3-sulfonyl.

[0048] Example 1 Preparation of Cyclo-[Glu-Glu-Met-Gln-Arg-Arg]

[0049] Cyclo-[Glu-Glu-Met-Gln-Arg-Arg] is prepared through the following steps:

[0050] S1. Swelling of the resin

[0051] Weigh 8 g of 2-CTC Resin into a solid-phase synthesis reaction column, swell it with DCM, wash the resin, and draw off the solvent.

[0052] S2. Feeding and reaction

[0053] S2.1. Weigh 12.0 g of Fmoc-Arg(Pbf)-OH and 3.5 g of HOBt into a dry Erlenmeyer flask, dissolve them with DMF solvent, cool in an ice bath for 10 min, add 5 mL of DIC for activation for 10 min, avoiding water vapor. Add the activated Fmoc-Arg(Pbf)-OH to the swollen resin for reaction for 2.5 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 1.5 h. Wash the resin and draw off the solvent to obtain Fmoc-Arg(Pbf)-2-CTC Resin.

[0054] S2.2. Deprotect the Fmoc group of Fmoc-Arg(Pbf)-2-CTC Resin twice with 20% piperidine / DMF for 10 minutes each time. Take a sample for K test, and the color shows dark blue. Wash the resin 7 times with DMF and remove the solvent by suction. Weigh 14.0 g of Fmoc-Arg(Pbf)-OH and 3.6 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 5.0 mL of DIC to activate for 3 minutes, avoiding water vapor. Add the activated amino acid to the deprotected resin and react for 2 hours, then remove the reaction solution by suction. The colorless and transparent resin in the K test indicates that the reaction is complete, and Fmoc-Arg(Pbf)-Arg(Pbf)-2-CTC Resin is obtained.

[0055] S2.3. Repeat step S2.2 to deprotect the N-terminal Fmoc group, and in the presence of 3.6 g of HOBt and 5.0 mL of DIC, use DMF as the solvent to couple the activated amino acid to the peptide resin for 2 hours of continuous reaction. Then wash these resins and repeat the deprotection treatment of the Fmoc group to couple the next amino acid. In each coupling, in the presence of 3.6 g of HOBt and 5.0 mL of DIC, use DMF as the solvent to sequentially couple 13 g of Fmoc-Gln(Trt)-OH, 8 g of Fmoc-Met-OH, 9 g of Fmoc-Glu(OtBu)-OH, and then 9 g of Fmoc-Glu(OtBu)-OH; after the reaction is complete, wash the resin and remove the solvent by suction to obtain Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-2-CTC Resin.

[0056] S2.4. Deprotect the N-terminal Fmoc group of the peptide resin. Deprotect the Fmoc group twice with 20% piperidine / DMF for 10 minutes each time. Take a sample for K test, and the color shows dark blue. First, wash the resin 6 times with DMF and remove the solvent by suction. After shrinkage drying, H-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-2-CTC Resin is obtained.

[0057] S3. Deproteinize the resin

[0058] S3.1. Measure 1.8 mL of TFA and 178.2 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.

[0059] S3.2. Add H-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-2-CTCResin into a round-bottom flask, add the above-prepared frozen lysis solution, stir and react for 0.5 h, repeat once, for a total of 2 lysis times. Pour it into isopropyl ether to precipitate a white solid, filter to obtain H-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-OH.

[0060] S4. Cyclization

[0061] Add H-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-OH into a flask, add 1000 mL of DCM, then add 7 g of HBTU and 6 g of DIPEA, and react for 48 h. After the reaction is completed, perform post-treatment to obtain the solid crude cyclic peptide Cyclo-[Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)].

[0062] S5. Cleavage (deprotection of functional groups)

[0063] Mix 67.5 mL of TFA, 1.9 mL of Tis, 1.9 mL of purified water, 1.9 mL of EDT, and 1.9 mL of benzyl methyl sulfide, add 15.0 g of the crude cyclic peptide, cleave for 2 h, precipitate with isopropyl ether, and then wash with isopropyl ether to obtain the crude peptide Cyclo-[Glu-Glu-Met-Gln-Arg-Arg].

[0064] S6. Purification

[0065] Dissolve 6.5 g of the crude peptide Cyclo-[Glu-Glu-Met-Gln-Arg-Arg] in 120 mL of pure water, 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 as follows:

[0066] Time (min) Flow rate (mL / min) A% (acetonitrile) B% (0.1% acetic acid + pure water) 0 40 2 98 10 40 8 92 30 40 12 88 45 40 30 70

[0067] Inject the filtered sample for purification, collect the fractions, concentrate and freeze-dry to obtain the cyclic peptide Cyclo-[Glu-Glu-Met-Gln-Arg-Arg] with a purity > 95%, denoted as cyclic hexapeptide A, and its structural formula is as follows:

[0068]

[0069] Determine the molecular weight of cyclic hexapeptide A by ESI-MS, and the mass spectrum is asFigure 1 As shown. The results show that [M-H] - The mass-to-charge ratio (m / z) of the quasi-molecular ion peak is 828.3824, and the molecular weight measured by mass spectrometry is 829.38, which is consistent with the theoretical exact molecular weight of cyclohexapeptide A. The NMR data and analysis results are as follows:

[0070] 1 H NMR(600MHz,D2O)δ:4.22(m,4H),4.13(dd,J=12,6Hz,1H),4.08(t,J=6Hz,1H),3.12(m,4H),2.48(m,2H),2.25(m,2H),2.11(m,9H),2.00(s,3H),1.86(m,7H),1.52(m,4H).

[0071] Table 1 Hydrogen spectrum analysis table of the tested compound

[0072]

[0073]

[0074] 13 C NMR(150MHz,D2O)δ:181.39,181.15,177.72,173.49,173.43,173.14,172.88,172.80,156.70,156.66,54.83,54.63,53.91,53.83,53.61,40.47,40.44,33.84,33.32,31.17,29.55,29.39,27.99,27.77,27.38,26.94,26.25,24.64,24.60,14.20.

[0075] Table 2 Carbon spectrum analysis table of the tested compound

[0076]

[0077]

[0078] The results of the above nuclear magnetic resonance hydrogen spectrum and carbon spectrum both show that the structure of the tested compound is consistent with the structure of cyclohexapeptide A.

[0079] The linear peptide H-Glu-Glu-Met-Gln-Arg-Arg-OH (hexapeptide B) can be obtained by a similar preparation method.

[0080] Example 2 Oil content test

[0081] 2.1 Reagents and materials

[0082] 0.25% Trypsin Digestive Solution (prepared by dissolving 0.25 g of trypsin in 100 mL of water), PBS, FFA (obtained by mixing linoleic acid and palmitic acid at a molar ratio of 1:1).

[0083] 2.2 Instruments

[0084] Thermostatic CO2 Incubator, Laminar Flow Hood.

[0085] 2.3 Cell Line

[0086] Human Sebaceous Gland Cells SZ-95.

[0087] 2.4 Test Samples and Grouping

[0088] 2.4.1 Test Samples

[0089] Cyclic Hexapeptide A and Hexapeptide B, both dissolved in PBS, with a test concentration of 50 ppm;

[0090] Simvastatin, dissolved in PBS, with a test concentration of 0.05 μM.

[0091] 2.4.2 Grouping

[0092] Experimental Group: Test Samples, FFA;

[0093] Normal Group: PBS;

[0094] Model Group: PBS, FFA.

[0095] 2.5 Experimental Method

[0096] Take a bottle of SZ-95 cells in the exponential growth phase with good condition, add 0.25% trypsin digestive 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 thermostatic CO2 incubator for 24 h. Except for the normal group which adds PBS, add FFA to each of the other wells to a final concentration of 225 μmol / mL. While inducing the model, add the corresponding test samples to the experimental group respectively, and culture it in a thermostatic CO2 incubator for 48 h. Aspirate the culture medium and operate according to the instructions of the Oil Red O Staining Kit.

[0097] 2.6 Experimental Results

[0098] Oil Red O is a fat-soluble dye that can be highly dissolved in fat. Its staining principle is that Oil Red O can specifically adsorb neutral triglycerides, lipids, and lipoproteins in tissues and cells, thereby staining the fat. FFA is an inducer. Under the stimulation and induction of FFA, SZ-95 cells will secrete a large amount of oils that can be stained by Oil Red O. In this experiment, test samples were used to treat SZ-95 cells after being stimulated and induced by FFA. By detecting the amount of oil production in SZ-95 cells, it was determined whether the cyclohexapeptide A of the present disclosure could inhibit the oil secretion of human sebaceous gland cells.

[0099] The results of the effects of test samples on the oil secretion of SZ-95 cells are shown in Table 3.

[0100] Table 3 Relative oil secretion of SZ-95 cells

[0101]

[0102]

[0103] Note: Compared with the model group, **P < 0.01, ***P < 0.001.

[0104] The experimental results show that compared with the linear hexapeptide B, the cyclohexapeptide A obtained by cyclizing the head and tail of hexapeptide B has a significantly improved ability to inhibit oil synthesis and secretion. It can be seen that the cyclohexapeptide A of the present disclosure can not only inhibit the synthesis and secretion of oils by sebaceous gland cells, slow down the oil deposition, but also has a more superior inhibitory effect compared with the linear hexapeptide B and simvastatin. It can be used to improve problems such as excessive skin oiliness and skin water-oil imbalance, reduce the occurrence of acne or pimples, and also contribute to the recovery of the skin after the occurrence of acne or pimples, having the functions of oil control, acne removal, and skin repair.

[0105] Example 3 Cell Migration Test

[0106] 3.1 Reagents and Materials

[0107] 0.25% Trypsin Digest (prepared by dissolving 0.25 g of trypsin in 100 mL of water), complete medium, PBS.

[0108] 3.2 Instruments

[0109] Biological inverted microscope, constant temperature CO2 incubator, laminar flow hood.

[0110] 3.3 Cell Lines

[0111] Human keratinocytes (HaCaT).

[0112] 3.4 Test Samples and Grouping

[0113] 3.4.1 Samples to be tested

[0114] Cyclohexapeptide A and hexapeptide B were dissolved in PBS, and the test concentrations were both 200 ppm.

[0115] 3.4.2 Grouping

[0116] Sample group: Samples to be tested;

[0117] Blank control group: PBS.

[0118] 3.5 Experimental method

[0119] Take a bottle of cells in good condition in the exponential growth phase, add 0.25% trypsin digestion solution for digestion to make a cell suspension, inoculate it on a 12-well plate containing complete medium, and culture it in a constant temperature CO2 incubator for 24 h. Change the medium, draw two vertical lines in the middle of the well with a yellow pipette tip, wash it once with PBS, find the upper and lower positions marked by the horizontal line and take pictures. After taking pictures, add complete medium containing 2% FBS and the samples to be tested to the sample group; add complete medium containing 2% FBS and PBS to the blank control group. Then culture it in a constant temperature CO2 incubator for 24 h. After culturing for 24 h, take pictures and observe at the same position under an inverted biological microscope, and calculate the scratch area.

[0120] Scratch healing rate (%) = (Scratch area before applying the sample - Scratch area after applying the sample) / Scratch area before applying the sample × 100%.

[0121] 3.6 Experimental results

[0122] Cell migration is one of the basic functions of normal cells, a physiological process for the normal growth and development of the body, and also a common form of movement existing in living cells. The process of cell migration is similar to the skin wound healing process. Therefore, promoting cell migration has a positive effect on skin repair.

[0123] The results of the influence of the test samples on cell migration are shown in Table 4.

[0124] Table 4 Influence of test samples on cell migration

[0125] Group Scratch healing rate (Mean±SD) Blank control group 10.66％±2.52％ Cyclohexapeptide A group 48.57％±0.43％*** Hexapeptide B group 34.68％±0.04％***

[0126] Note: Compared with the blank control group, ***P < 0.001.

[0127] The results showed that the cyclic hexapeptide A of the present disclosure could significantly promote the migration of HaCaT cells, showing a significant effect of promoting the re-epithelialization of the skin or mucosa or wound healing. In addition, compared with the linear hexapeptide B, the cyclic hexapeptide A obtained by cyclizing the head and tail of the hexapeptide B had significantly improved technical effects. It can be seen that the cyclic hexapeptide A of the present disclosure has excellent effects in promoting cell migration and can be used to promote the re-epithelialization of the skin or mucosa or wound healing, repair damaged skin barriers, etc., to achieve the effect of repair.

[0128] Example 4

[0129] A cream is prepared by the following steps:

[0130]

[0131] According to the prescription dosage, heat the materials in phase C in a suitable container to 55 - 60 °C and dissolve them completely for standby. Add the materials in phase A to the stirring pot and stir and heat to 80 - 85 °C. Add the materials in phase B to the oil phase pot, stir and heat to 75 - 80 °C until completely dissolved and transparent. Pump the materials in phase B into the materials in phase A, turn on the vacuum, homogenize for 5 minutes, maintain stirring, and keep warm for 20 minutes. Start to cool down, cool down to 60 - 65 °C, add the materials in phase C, and homogenize for 2 minutes. Cool down to 35 - 40 °C, add the pre-dissolved materials in phase D, and stir for 10 - 15 minutes to obtain the product.

[0132] In the present 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 terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device comprising a series of elements not only includes those elements, but also includes 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 one..." does not exclude the existence of additional identical elements in the process, method, article or terminal device comprising the element.

[0133] Although specific embodiments of the present disclosure have been described for purposes of illustrative explanation, 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 cyclic peptide or a salt thereof, characterized in that The structure of the cyclic peptide is Cyclo-[Glu-Glu-Met-Gln-Arg-Arg].

2. A composition, characterized in that The invention comprises an effective amount of the cyclic peptide or a salt thereof according to claim 1, and at least one excipient and an optional adjuvant.

3. A delivery system or sustained-release system, characterized in that: Comprising an effective amount of the cyclic peptide or a salt thereof according to claim 1, or the composition according to claim 2.

4. The delivery system or sustained-release system according to claim 3, 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.

5. A cosmetic, characterized in that: Comprising an effective amount of the cyclic peptide or salt thereof according to claim 1, or the composition according to claim 2, or the delivery system or sustained-release system according to claim 3 or 4.

6. The cosmetic according to claim 5, 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.

7. Use of the cyclic peptide or salt thereof according to claim 1, or the composition according to claim 2, or the delivery system or sustained-release system according to claim 3 or 4 in the preparation of a composition for oil control, acne removal or repair.

8. Use of the cyclic peptide or salt thereof according to claim 1, or the composition according to claim 2, or the delivery system or sustained-release system according to claim 3 or 4 in the preparation of a composition for reducing skin oil synthesis or secretion.

9. Use of the cyclic peptide or salt thereof according to claim 1, or the composition according to claim 2, or the delivery system or sustained-release system according to claim 3 or 4 in the preparation of a composition for promoting re-epithelialization or healing of skin or mucous membranes.

10. Use of the cyclic peptide or salt thereof according to claim 1, or the composition according to claim 2, or the delivery system or sustained-release system according to claim 3 or 4 in the preparation of cosmetics.

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