Cyclic hexapeptide for skin care and composition and application thereof

Abstract

The invention discloses a cyclic hexapeptide for skin care and a composition and application thereof, and relates to the technical field of polypeptides, and the cyclic peptide has the following structure: Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu]. 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 in particular to a cyclohexapeptide for skin care, a composition thereof, and uses thereof. Background Art

[0002] Skin oil plays a vital role in the human body. It not only acts as a natural moisturizing barrier to lock in moisture and maintain skin softness and elasticity, but also effectively blocks the invasion of harmful substances and bacteria from the outside world and protects skin health. However, when skin oil secretion is too strong, it will bring a series of negative effects, such as clogging pores, causing skin problems such as acne and acne, and making the skin look greasy and not refreshing, affecting the overall appearance.

[0003] The male hormone secretion is relatively strong in the male group, and the skin oil secretion is also more vigorous. The skin areas such as the face and back are prone to a lot of oil. Therefore, some personal cleaning products such as facial cleansers, shower gels, and shampoos for men on the market currently focus on oil control. However, frequent use of personal cleaning products with too strong cleaning power or improper cleaning methods can easily remove the natural oil on the surface of the skin excessively, resulting in an imbalance of water and oil on the skin, which in turn causes damage to the skin barrier function. Once the skin barrier is damaged, the skin becomes sensitive and fragile. Not only does the skin's moisture evaporate faster, causing symptoms of dryness and cracking, but it is also easily stimulated by external factors, causing discomfort symptoms such as skin redness, swelling, and itching. In severe cases, it may also cause more complex skin inflammation. Maintaining the water-oil balance of the skin is an important part of protecting skin health. In order to address the problem of strong oil secretion and damaged skin barrier caused by excessive daily cleaning, it is necessary not only to continuously control oil, but also to repair the skin barrier and reduce water loss to maintain the water-oil balance of the skin. Summary of the invention

[0004] The present disclosure relates to a cyclohexapeptide and a composition and use thereof. The cyclohexapeptide and a composition containing the cyclohexapeptide have the effects of caring for or treating skin or mucous membranes.

[0005] In one aspect, the present disclosure provides a cyclic peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, wherein the structure of the cyclic peptide is Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu].

[0006] The cyclic peptides disclosed herein contain a large number of asymmetric carbon atoms, and those skilled in the art understand that the cyclic peptides disclosed herein have stereoisomers and may exist as stereoisomers or stereoisomer mixtures, and thus may be obtained as isomeric mixtures and racemic mixtures or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and the isomers or isomeric mixtures present. In some embodiments, the cyclic peptides disclosed herein are pure isomers, i.e., enantiomers or diastereomers.

[0007] 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 herein to refer to compounds in which at least one atom in 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 usually 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, e.g. 2 H (deuterium), 3 H (tritium), 13 C. 14 C. 15 N. 17 O. 18 O. 33 S. 34 S. 35 S or 36 Certain isotopic variants of the cyclic peptides disclosed herein (particularly those into which one or more radioactive isotopes have been incorporated) may be useful, for example, for examining the mechanism of action or distribution of the active compound in vivo; due to the relative ease of preparation and detectability, particularly with 3 H or 14 C isotope-labeled cyclic peptides are suitable for this purpose. In addition, due to the stronger metabolic stability of cyclic peptides, the incorporation of isotopes (e.g., deuterium) can produce specific therapeutic benefits, such as an extension of the half-life in vivo or a reduction in the required active dose. Isotopic variants of the cyclic peptides disclosed herein can be prepared by methods known to those skilled in the art, such as by the methods further described below and in the examples, by using the respective reagents and / or corresponding isotopic modifications of the starting materials.

[0008] The term "salt" refers to a salt approved for use in animals, and more specifically in humans, including metal salts of the cyclic peptide, the metal including, but not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, etc.; including salts formed by the cyclic peptide and organic bases, the organic bases including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine, etc.; including salts formed by the cyclic peptide and inorganic or organic acids, the organic acids including, but not limited to: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoate or gluconic acid, etc.; the inorganic acids including, but not limited to: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0009] The nature of the salt is not critical and salts of the cyclic peptide may be obtained by conventional methods known in the art.

[0010] The synthesis of the cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt disclosed in the present invention can be carried out according to conventional methods known in the prior art, such as solid phase synthesis, liquid phase synthesis, or a combination of solid phase and liquid phase. It can also be prepared by a biotechnological method aimed at producing a desired sequence, or by controlled hydrolysis of proteins having animal, fungal, or plant origin.

[0011] For example, a method for obtaining the cyclic peptide disclosed herein comprises the following steps:

[0012] - coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a C-terminus that is protected or bound to a solid support;

[0013] - Elimination of the group protecting the N-terminus;

[0014] - repeating this coupling sequence and eliminating the group protecting the N-terminus until the desired peptide sequence is obtained;

[0015] - elimination of the group protecting the C-terminus or cleavage from the solid support;

[0016] - Cycling the amino group at the N-terminus of the peptide chain and the carboxyl group at the C-terminus;

[0017] - Elimination of groups protecting side chains.

[0018] In some embodiments, the C-terminus is bound to a solid support and the method is carried out on a solid phase, comprising coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a C-terminus bound to a polymer support; eliminating the group protecting the N-terminus; and repeating this sequence as many times as required so as to thereby obtain a peptide of the desired length, followed by cleaving the synthesized peptide from the initial polymer support and coupling and cyclizing the amino group at the N-terminus of the peptide chain to the carboxyl group at the C-terminus.

[0019] The functional groups of the side chains of these amino acids remain adequately protected with temporary or permanent protecting groups throughout the synthesis.

[0020] In some embodiments, solid phase synthesis can be performed by a convergent strategy of coupling a dipeptide or tripeptide to a polymer support or to a dipeptide or amino acid previously bound to a polymer support.

[0021] Due to the application outside the body of a mammal, the cyclic peptide of the present disclosure can form a part of various types of compositions. Therefore, another aspect of the present disclosure provides a composition comprising an effective amount of the above-mentioned cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or a salt thereof, and at least one excipient and an optional adjuvant. The composition can be prepared by conventional methods known to those skilled in the art.

[0022] In some embodiments,The adjuvant is selected from the group consisting of agents that activate Clock expression, analgesics, agents that inhibit PAR-2 ​​activity, agents that regulate PGC-1α synthesis, agents that regulate PPARγ activity, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic agents or agents that stimulate lipolysis, lipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulators or inhibitors, whitening agents or depigmenting agents, pigmentation promoting agents, self-tanning agents, anti-aging agents, NO-synthase inhibitors, 5α-reductase inhibitors, inhibitors of lysyl hydroxylase and / or prolyl hydroxylase, antioxidants, free radicals. Agents for removing and / or preventing air pollution, agents for removing active carbonyl substances, anti-glycation agents, antihistamines, antivirals, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, substances for retaining moisture, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating bags under the eyes, keratolytic agents, antimicrobial agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and / or that can inhibit or prevent their degradation, agents that stimulate the synthesis of elastin, agents that stimulate the synthesis of decorin, agents that stimulate the synthesis of laminin, agents that stimulate the synthesis of defensins agents, agents that stimulate chaperone protein synthesis, agents that stimulate cAMP synthesis, agents that stimulate hyaluronic acid synthesis, agents that stimulate fibronectin synthesis, agents that stimulate deacetylase synthesis, agents that stimulate the synthesis of lipids and stratum corneum components, ceramides, fatty acids, agents that inhibit elastin degradation, agents that inhibit serine proteases, agents that stimulate fibroblast proliferation, agents that stimulate keratinocyte proliferation, agents that stimulate adipocyte proliferation, agents that stimulate melanocyte proliferation, agents that stimulate keratinocyte differentiation, agents that inhibit acetylcholinesterase, skin relaxants, agents that stimulate glycosaminoglycan synthesis, anti-hyperkeratosis agents, comedolytic agents, anti-psoriatic agents, anti-eczema agents, DNA repair agents, DNA protective agents, stabilizers, antipruritic agents, for the treatment and / or care of allergies Sensitive skin agents, firming agents, tightening agents, restructuring agents, stretch mark resistance agents, agents regulating sebum production, antiperspirants, agents stimulating healing, agents assisting healing, agents stimulating re-epithelialization, agents assisting re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents acting on capillary circulation and / or microcirculation, agents stimulating angiogenesis, agents inhibiting vascular permeability, agents of venous tension, agents acting on cell metabolism, agents for improving dermal-epidermal junction, agents inducing hair growth, agents inhibiting or retarding hair growth, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents obtained from biological fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents effective against A and / or B ultraviolet rays, or mixtures thereof. ,

[0023] The effective amount of the cyclic peptides of the present disclosure to be administered, as well as their dosage, will depend on a number of factors, including the age, state of the user, severity of the condition, route and frequency of administration, and the specific nature of the cyclic peptide to be used.

[0024] "Effective amount" means an amount of the cyclic peptide of the present disclosure that is non-toxic but sufficient to provide the desired effect. The cyclic peptide of the present disclosure is used in the composition of the present disclosure at an effective concentration 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.

[0025] 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 cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or its salt, or the above-mentioned combination.

[0026] The term "delivery system" refers to a diluent, adjuvant, excipient or carrier to be administered with the cyclic peptides of the present disclosure, 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, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynol ethers, poloxamers, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin and the like. Those of ordinary skill in the art are aware of the diluents that can be used in the different delivery systems in which the cyclic peptides of the present disclosure can be administered.

[0027] The term "sustained release" is used in a conventional sense to refer to a delivery system that provides for the gradual release of a compound over a period of time. In some embodiments, a sustained release system has a relatively constant level of compound release over a period of time.

[0028] Examples of delivery systems or sustained-release systems include, but are not limited to, liposomes, oleosomes, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion compounds, lipid vesicles, micelles, millispheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, milliparticles, microparticles or nanoparticles.

[0029] 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 a salt thereof, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system.

[0030] In some embodiments, the dosage form of the cosmetic comprises ointment, cream, emulsion, aqueous solution, oil, gel, powder, tablet, mud, patch, film, aerosol, spray, freeze-dried preparation or nano preparation.

[0031] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or a salt thereof, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in the preparation of a composition for caring for or treating skin or mucous membranes.

[0032] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or a salt thereof, 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 removal, repair or moisturizing.

[0033] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or a salt thereof, 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 a use of the above-mentioned cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or a salt thereof, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in preparing a composition for promoting the expression of filaggrin (FLG) and / or loricrin (LOR), or in preparing a composition for promoting re-epithelialization or healing of the skin or mucosa, or in preparing a composition for repairing the skin barrier.

[0035] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in the preparation of cosmetics.

[0036] In the present disclosure, the term "skin" is understood to refer to the multiple layers that make it up, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both ends are 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 "skin care" refers to the maintenance and care of the skin, improving the condition of the skin, making it delicate, smooth, tender and healthy.

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

[0039] 1. The cyclic peptide disclosed in the present invention can effectively promote the expression of FLG and LOR. FLG is an important factor involved in the skin barrier. In the process of participating in the formation of the keratin capsule of the outer layer of the epidermis, it promotes epidermal differentiation and forms a unique barrier structure of the epidermal stratum corneum, which plays an important role in maintaining the barrier function of the skin. LOR is the main component of the keratin capsule and plays an important role in the normal functioning of the epidermal barrier function. Promoting the expression of FLG and LOR can repair the skin barrier, improve the skin barrier function, help lock in skin moisture, and enhance the skin's moisturizing ability. Therefore, the cyclic peptide disclosed in the present invention has the effects of repairing and moisturizing.

[0040] 2. The cyclic peptide disclosed in the present invention can effectively promote cell migration. Promoting cell migration can promote re-epithelialization or wound healing of the skin or mucous membrane, and repair the skin barrier. Therefore, the cyclic peptide disclosed in the present invention can be used to promote re-epithelialization or wound healing of the skin or mucous membrane, repair damaged skin barriers, etc., thereby achieving the effect of repair.

[0041] 3. The cyclic peptide disclosed in the present invention can inhibit the synthesis or secretion of oil by sebaceous gland cells, slow down oil deposition, reduce the occurrence of blackheads or acne, and also help the recovery of the skin after the occurrence of blackheads or acne, and has the effects of oil control, acne removal and repair.

[0042] 4. The cyclic peptide disclosed in the present invention is obtained by coupling and cyclizing the N-terminus and C-terminus of the linear RRQ(d)MEE. However, compared with the linear polypeptide, the cyclic peptide disclosed in the present invention has better effects in promoting cell migration, promoting the expression of LOR and FLG, and inhibiting oil synthesis or secretion, thereby achieving excellent oil control, acne removal, repair and moisturizing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solution of the present disclosure, the drawings required for use in 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 ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 The cyclohexapeptide A (molecular formula C) in Example 1 of the present disclosure 32 H 55 N 13 O 11 S) mass spectrum. DETAILED DESCRIPTION

[0045] In order to make the objects, features and advantages of the present disclosure more obvious and understandable, the present disclosure is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the claims attached to the present disclosure.

[0046] In the present disclosure, the abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Commission of Biochemical Nomenclature in Eur. J. Biochem. 1984, 138: 9-37.

[0047] Unless otherwise specified, the experimental reagents and materials used in this disclosure can be obtained from commercial sources. The following are the abbreviations of some reagents and materials:

[0048] 2-CTC Resin: a starting resin for peptide synthesis; DCM: dichloromethane; HOBt: 1-hydroxybenzotriazole; DMF: N,N-dimethylformamide; DIPEA: diisopropylethylamine; MeOH: methanol; piperidine: piperidine; DIC: diisopropylcarbodiimide; TFA: trifluoroacetic acid; PE: petroleum ether; Tis: triisopropylsilane; EDT: 1,2-ethanedithiol; Glu: glutamic acid; D-Met: D-methionine; Gln: glutamine; Arg: arginine; Fmoc: 9-fluorenylmethoxycarbonyl; OtBu: tert-butyloxy; Trt: trityl; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-3-sulfonyl.

[0049] Example 1 Preparation of Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu]

[0050] Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu] was prepared by the following steps:

[0051] S1. Swelling of resin

[0052] Weigh 10 g of 2-CTC Resin into a solid phase synthesis reaction column, swell it with DCM, wash the resin, and remove the solvent.

[0053] S2, feeding reaction

[0054] S2.1. Weigh 10.3 g of Fmoc-Glu(OtBu)-OH into a dry conical flask, dissolve it with DMF solvent, and place it in an ice-water bath to cool for 10 min. Add 11.3 mL of DIPEA and stir for 3 min. Then add the above mixed solution to the swollen resin and react for 2.5 h. Draw off the reaction solution, wash the resin 4 times, and draw off the solvent. Continue to add DCM, MeOH, and DIPEA for 10 min and 2 times for end-capping. Wash the resin 4 times and draw off the solvent to obtain Fmoc-Glu(OtBu)-2-CTC Resin.

[0055] S2.2, Fmoc-Glu(OtBu)-2-CTC Resin was de-Fmoced twice with 20% piperidine / DMF: 8 min for the first time and 5 min for the second time. Sample K test, dark blue color. Wash the resin with DMF 7 times and remove the solvent. Weigh 10.5g of Fmoc-Glu(OtBu)-OH and 3.9g of HOBt into a dry conical flask, add DMF to dissolve it, seal it and place it in a -18℃ refrigerator for 30min. Add 5.6mL DIC to activate for 3min. Add the activated amino acid to the deprotected resin and react for 2h, and remove the reaction solution. The K test resin is colorless and transparent, indicating that the reaction is complete, and Fmoc-Glu(OtBu)-Glu(OtBu)-2-CTC Resin is obtained.

[0056] S2.3, repeat step S2.2, deprotect the N-terminal Fmoc group, and in the presence of 3.9g HOBt and 5.6mL DIC, use DMF as solvent to couple the activated amino acid to the peptidyl resin for 2h. Then wash the resin and repeat the deprotection of the Fmoc group to couple the next amino acid. In each coupling, 9.1 g of Fmoc-D-Met-OH, 14.9 g of Fmoc-Gln(Trt)-OH, 15.8 g of Fmoc-Arg(Pbf)-OH and then 15.8 g of Fmoc-Arg(Pbf)-OH were sequentially coupled in the presence of 3.9 g of HOBt and 5.6 mL of DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was removed to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-D-Met-Glu(OtBu)-Glu(OtBu)-2-CTC Resin.

[0057] S2.4. Deprotect the N-terminal Fmoc group of the peptide resin and use 20% piperidine / DMF to remove Fmoc twice: 8 minutes for the first time and 5 minutes for the second time. Take a sample for K inspection and the color is dark blue. Wash the resin 4 times with DMF, then rinse it 4 times with DCM, and finally wash it once with MeOH and remove the solvent. After shrinkage and drying, 28.7g H-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-D-Met-Glu(OtBu)-Glu(OtBu)-2-CTC Resin was obtained.

[0058] S3, Resin removal

[0059] S3.1. Take 4.5 mL of TFA and 445.5 mL of DCM, mix and stir evenly to obtain the cleavage solution, seal it and place it in a -18°C refrigerator for later use; place isopropyl ether in a -18°C refrigerator for later use.

[0060] S3.2. Weigh 28.7g H-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-D-Met-Glu(OtBu)-Glu (OtBu)-2-CTC Resin, added Add the frozen lysate to the bottom flask, stir and react for 0.5h, repeat once, and lyse twice in total. Filter by suction, collect the filtrate and concentrate to obtain an oily substance, add PE and stir to solidify to obtain 26.5g of H-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-D-Met-Glu(OtBu)-Glu(OtBu)-OH.

[0061] S4, cyclization

[0062] 26.5 g H-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-D-Met-Glu(OtBu)-Glu(OtBu)-OH was added to a flask, 500 mL DCM was added and stirred, and then 24.5 g HATU, 14 g DIPEA, and 300 mL DMF were added and reacted for 7 h. After the reaction was completed, post-treatment was performed to obtain 26.7 g of oily crude cyclic peptide Cyclo-[Arg(Pbf)-Arg(Pbf)-Gln(Trt)-D-Met-Glu(OtBu)-Glu(OtBu)].

[0063] S5, cleavage (deprotection group)

[0064] Take 144 mL of TFA, 4 mL of Tis, 4 ml of EDT, and 4 mL of thioanisole, mix well, add 26.7 g of crude cyclic peptide, cleave for 2.5 hours, precipitate with isopropyl ether, and then wash with isopropyl ether to obtain Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu] crude peptide.

[0065] S6. Purification

[0066] 16.0 g of Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu] crude peptide was dissolved in 200 mL of pure water, filtered with a microporous filter membrane with a pore size of 0.45 μm to obtain a clear and transparent solution, and purified by reverse phase HPLC. The purification gradient is as shown in the following table:

[0067] Time (min) Flow rate (mL / min) A% (acetonitrile) B% (0.1% acetic acid + pure water) 0 40 2 98 10 40 6 94 30 40 10 90 45 40 12 88 60 40 20 80

[0068] The filtered sample was injected for purification, and the fractions were collected, concentrated and freeze-dried to obtain a cyclic peptide Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu] with a purity of >95.3%, which was recorded as cyclohexapeptide A and has the following structural formula:

[0069]

[0070] The molecular weight of cyclohexapeptide A was determined by ESI-MS, and the mass spectrum is shown in Figure 1 The results show that [M+H] + The mass-to-charge ratio (m / z) of the quasi-molecular ion peak is 830.3440, and the molecular weight measured by mass spectrometry is 829.34, which is consistent with the theoretical precise molecular weight of cyclohexapeptide A. Its NMR data and analysis results are as follows:

[0071] 1 H NMR(600MHz,D2O)δ:4.41(dd,J=12,6Hz,1H),4.30(q,J=6Hz,2H),4.23(t,J=6Hz,1H),4.16(t,J=6Hz,1H),4.10(t,J=6Hz,1H),3 .09(dt,J=24,6Hz,4H),2.50(m,2H),2.22(m,5H),2.08(m,5H),2.00(s,3H),1.94(m,3H),1.82(m,3H),1.70(m,2H),1.48(m,4H).

[0072] Table 1 Hydrogen spectrum analysis of the tested compounds

[0073] Serial number Chemical shift (ppm) Number of protons (hydrogen atoms) Peak shape (coupling constant) Structural attribution 1 4.41 1 dd,J=12,6Hz α-CH connected to the amide bond 2 4.30 2 q,J=6Hz α-CH connected to the amide bond 3 4.23 1 t,J=6Hz α-CH connected to the amide bond 4 4.16 1 t,J=6Hz α-CH connected to the amide bond 5 4.10 1 t,J=6Hz α-CH connected to the amide bond 6 3.09 4 dt,J=24,6Hz Hydrogen of saturated alkanes 7 2.50 2 m Hydrogen of saturated alkanes 8 2.22 5 m Hydrogen of saturated alkanes 9 2.08 5 m Hydrogen of saturated alkanes 10 2.00 3 s Hydrogen of saturated alkanes 11 1.94 3 m Hydrogen of saturated alkanes 12 1.82 3 m Hydrogen of saturated alkanes 13 1.70 2 m Hydrogen of saturated alkanes 14 1.48 4 m Hydrogen of saturated alkanes

[0074] 13C NMR(150MHz,D2O)δ:181.23,181.18,177.41,174.07,173.51,173.21,172.74,172.55,156.71,54.96,54.36,53.86,53 .37,53.30,40.54,40.40,33.90,33.83,30.48,29.44,28.95,28.41,28.25,27.93,27.63,26.51,24.76,24.17,14.15.

[0075] Table 2 Carbon spectrum analysis of the tested compounds

[0076]

[0077]

[0078]

[0079] The results of the above-mentioned nuclear magnetic resonance hydrogen spectrum and carbon spectrum both showed that the structure of the tested compound was consistent with the structure of cyclohexapeptide A.

[0080] The linear peptides H-Arg-Arg-Gln-D-Met-Glu-Glu-OH (hexapeptide B) and Ac-Arg-Arg-Gln-D-Met-Glu-Glu-NH2 (acetyl hexapeptide C) can be obtained by similar preparation methods.

[0081] Example 2 Oil content test

[0082] 2.1 Reagents and Materials

[0083] 0.25% trypsin digestion solution (prepared with 0.25 g trypsin in 100 mL water), PBS, FFA (prepared with linoleic acid and palmitic acid mixed in a molar ratio of 1:1).

[0084] 2.2 Instruments

[0085] Constant temperature CO2 incubator, clean workbench.

[0086] 2.3 Cell lines

[0087] Human sebaceous gland cells SZ-95.

[0088] 2.4 Samples to be tested and grouping

[0089] 2.4.1 Samples to be tested

[0090] Cyclohexapeptide A and hexapeptide B were both dissolved in PBS, and the test concentration was 50 ppm;

[0091] Simvastatin was dissolved in PBS and tested at a concentration of 0.05 μM.

[0092] 2.4.2 Grouping

[0093] Experimental group: samples to be tested, FFA;

[0094] Normal group: PBS;

[0095] Model group: PBS, FFA.

[0096] 2.5 Experimental methods

[0097] Take a bottle of SZ-95 cells in good condition in the exponential growth phase, add 0.25% trypsin digestion solution, digest to make the attached cells fall off, and count (1-4)×10 5 / mL, and make a cell suspension. Take an appropriate amount of cell suspension and inoculate it on a 12-well plate containing complete culture medium, and culture it in a constant temperature CO2 incubator for 24 hours. Except for the normal group, PBS was added to each well of the remaining wells to make the final concentration of 225μmol / mL. While inducing modeling, the corresponding samples to be tested were added to the experimental groups respectively, and cultured in a constant temperature CO2 incubator for 48 hours. Aspirate the culture medium and operate according to the instructions of the Oil Red O staining kit.

[0098] 2.6 Experimental Results

[0099] Oil Red O is a fat-soluble dye that is highly soluble in fat. The principle of its staining is that Oil Red O can specifically adsorb neutral triglycerides, lipids, and lipoproteins in tissues and cells to stain fat. FFA is an inducer. Under the stimulation and induction of FFA, SZ-95 cells will secrete a large amount of oil that can be stained with Oil Red O. This experiment used a test sample to treat SZ-95 cells stimulated and induced by FFA, and detected the amount of oil produced by SZ-95 cells to determine whether the cyclohexapeptide A disclosed in the present invention can inhibit the secretion of oil by human sebaceous gland cells.

[0100] The results of the effects of the test samples on the lipid secretion of SZ-95 cells are shown in Table 3.

[0101] Table 3 Relative secretion of lipids in SZ-95 cells

[0102] Group Relative oil secretion (Mean±SD) Normal group 100.00％±2.72％ Model Group 222.71％±26.37％ Simvastatin group 107.52％±3.51％*** Cyclic hexapeptide group A 113.82％±2.79％*** Hexapeptide B group 138.17％±5.85％**

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

[0104] The experimental results show that the ability of cyclic hexapeptide A obtained by cyclizing hexapeptide B head-to-tail is significantly improved compared to linear hexapeptide B in inhibiting oil synthesis and secretion. It can be seen that the cyclic hexapeptide A disclosed in the present invention can not only inhibit the synthesis and secretion of oil by sebaceous gland cells and slow down oil deposition, but also has a more superior inhibitory effect than linear hexapeptide B, and can be used to improve problems such as excessive oil production of the skin and imbalance of water and oil in the skin, can reduce the occurrence of blackheads or acne, and also help the recovery of the skin after blackheads or acne occurs, and has the effects of oil control, acne removal, and repair.

[0105] Example 3 Cell migration test

[0106] 3.1 Reagents and Materials

[0107] 0.25% trypsin digestion solution (0.25 g trypsin in 100 mL water), complete culture medium, PBS.

[0108] 3.2 Instruments

[0109] Biological inverted microscope, constant temperature CO2 incubator, and clean workbench.

[0110] 3.3 Cell lines

[0111] Human keratinocytes (HaCaT).

[0112] 3.4 Samples to be tested and grouping

[0113] 3.4.1 Samples to be tested

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

[0115] 3.4.2 Grouping

[0116] Sample group: samples to be tested;

[0117] Blank control group: PBS.

[0118] 3.5 Experimental methods

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

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

[0121] 3.6 Experimental Results

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

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

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

[0125] Group Scratch healing rate (Mean±SD) Blank control group 10.66％±2.52％ Cyclic hexapeptide group A 47.80％±0.11％*** Hexapeptide B group 35.65％±0.01％***

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

[0127] The results show that the cyclic hexapeptide A disclosed in the present invention can significantly promote HaCaT cell migration, and exhibits a significant effect of promoting re-epithelialization or wound healing of the skin or mucous membrane. In addition, the cyclic hexapeptide A obtained by the head-to-tail cyclization of hexapeptide B has a significantly improved technical effect compared to the linear hexapeptide B. It can be seen that the cyclic hexapeptide A disclosed in the present invention has an excellent effect of promoting cell migration, and can be used to promote re-epithelialization or wound healing of the skin or mucous membrane, repair damaged skin barriers, etc., to achieve a repair effect.

[0128] Example 4 FLG content determination

[0129] 4.1 Reagents and Materials

[0130] 0.25% trypsin digestion solution (0.25g trypsin prepared with 100mL PBS), complete culture medium, PBS, RIPA lysis buffer, human silk polymer protein (FLG) ELISA detection kit (Human FLG Filaggrin ELISA Kit), BCA protein quantification kit.

[0131] 4.2 Instruments

[0132] UVB lamp, constant temperature CO2 incubator, clean workbench.

[0133] 4.3 Cell lines

[0134] Human keratinocytes (HaCaT).

[0135] 4.4 Samples to be tested and grouping

[0136] 4.4.1 Samples to be tested

[0137] Cyclohexapeptide A, hexapeptide B, and acetylhexapeptide C were dissolved in PBS, and the test concentration was 50 ppm.

[0138] 4.4.2 Grouping

[0139] Sample group: UVB + sample to be tested;

[0140] UV group: UVB+PBS;

[0141] Blank control group: PBS.

[0142] 4.5 Experimental methods

[0143] Take a bottle of human keratinocytes (HaCaT) in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count (1-4)×10 5 / mL, and make a cell suspension. Take an appropriate amount of cell suspension and inoculate it in a 12-well plate containing complete culture medium, and culture it in a CO2 incubator for 24h. Aspirate the complete culture medium in the well, add PBS, and expose it to UVB light for 15min; the blank control group does not need to be irradiated. After 15min of irradiation, aspirate the PBS solution, add complete culture medium and test samples to the sample group respectively; the blank control group and UV group add complete culture medium and PBS and culture for 48h. Collect the cells, centrifuge to obtain the cell pellet, add RIPA lysis buffer, shake it evenly with a vortex instrument 3 times (30s / time, 3min interval), and centrifuge it at 12000rpm*10min. Aspirate the supernatant and test it with an ELISA kit, and use a BCA protein quantification kit to detect the total protein concentration of the supernatant.

[0144] 4.6 Experimental Results

[0145] Filaggrin (FLG) is an important factor involved in the skin barrier. It promotes epidermal differentiation and forms a unique barrier structure of the epidermal stratum corneum in the process of participating in the formation of the keratin envelope of the outer layer of the epidermis. It plays an important role in maintaining the physical strength of the stratum corneum and reducing transepidermal water loss, which is beneficial to maintaining barrier integrity and moisturizing. Therefore, the expression of FLG can be used as an indicator to evaluate the effect of skin barrier repair. By testing the effect of a certain substance on the expression of FLG, the effect of the substance on skin barrier repair can be evaluated. Increased expression of FLG is beneficial to repair the skin barrier, improve skin barrier function, and enhance the skin's moisturizing ability. In this experiment, HaCaT cells were treated with test samples, and the content of FLG synthesized in HaCaT cells was detected to determine whether the cyclohexapeptide A disclosed in the present invention can promote the expression of FLG in HaCaT cells.

[0146] The results of the effects of the test samples on the expression of FLG in HaCaT cells are shown in Table 5.

[0147] Table 5 Effects of test samples on the expression of FLG in HaCaT cells

[0148] Group FLG relative content (Mean±SD) Blank control group 100.00％±12.39％ UV Groups 28.00％±9.28％ Cyclic hexapeptide group A 57.55％±14.57％** Hexapeptide B group 48.82％±0.74％*** Acetyl Hexapeptide Group C 35.90％±14.32％

[0149] Note: Compared with the UV group, **P<0.01, ***P<0.001.

[0150] The results showed that compared with the UV group, the cyclic hexapeptide A disclosed in the present invention can significantly promote the expression of FLG in HaCaT cells, but the results of acetyl hexapeptide C were not statistically different from those of the UV group. At the same time, the experimental results showed that the cyclic hexapeptide A obtained by cyclizing hexapeptide B head-to-tail has a significantly improved ability to promote FLG expression compared to linear hexapeptide B. It can be seen that the cyclic hexapeptide A disclosed in the present invention has an excellent effect of promoting FLG expression, can be used to repair the skin barrier, and has the effects of repairing and moisturizing.

[0151] Example 5 LOR content determination

[0152] 5.1 Reagents and Materials

[0153] 0.25% trypsin digestion solution (0.25g trypsin prepared with 100mL water), complete culture medium, PBS, RIPA lysis buffer, human loricrin (LOR) ELISA kit (Human LORLoricrin ELISA Kit), BCA protein quantification kit.

[0154] 5.2 Instruments

[0155] UVB lamp, constant temperature CO2 incubator, clean workbench.

[0156] 5.3 Cell lines

[0157] Human keratinocytes (HaCaT).

[0158] 5.4 Samples to be tested and grouping

[0159] 5.4.1 Samples to be tested

[0160] Cyclohexapeptide A and hexapeptide B were dissolved in PBS, and the test concentration was 200 ppm.

[0161] 5.4.2 Grouping

[0162] Sample group: UVB + sample to be tested;

[0163] UV group: UVB+PBS;

[0164] Blank control group: PBS.

[0165] 5.5 Experimental methods

[0166] Take a bottle of human keratinocytes (HaCaT) in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count (1-4)×10 5 / mL, and make a cell suspension. Take an appropriate amount of cell suspension and inoculate it in a 12-well plate containing complete culture medium, and culture it in a CO2 incubator for 24h. Aspirate the complete culture medium in the well, add PBS, and expose it to UVB light for 15min; the blank control group does not need to be irradiated. After 15min of irradiation, aspirate the PBS solution, add complete culture medium and test samples to the sample group respectively; the blank control group and UV group add complete culture medium and PBS and culture for 48h. Collect the cells, centrifuge to obtain the cell pellet, add RIPA lysis buffer, shake it evenly with a vortex instrument 3 times (30s / time, 3min interval), and centrifuge it at 12000rpm*10min. Aspirate the supernatant and test it with an ELISA kit, and use a BCA protein quantification kit to detect the total protein concentration of the supernatant.

[0167] 5.6 Experimental Results

[0168] Loricrin (LOR) is a major component of the keratin envelope and plays an important role in the normal function of the epidermal barrier. The expression of LOR plays an important role in the repair of the skin barrier, and increased expression of LOR is an important sign of skin barrier repair. In this experiment, the test sample was used to treat HaCaT cells, and the content of LOR synthesized in HaCaT cells was detected to determine whether the cyclohexapeptide A disclosed in the present invention can promote the expression of LOR in HaCaT cells.

[0169] The results of the effects of the test samples on the expression of LOR in HaCaT cells are shown in Table 6.

[0170] Table 6 Effects of test samples on HaCaT cell expression of LOR

[0171] Group LOR relative content (Mean±SD) Blank control group 100.00％±10.21％ UV Groups 51.57％±4.84％ Cyclic hexapeptide group A 87.05％±14.91％** Hexapeptide B group 68.81％±16.67％*

[0172] Note: Compared with the UV group, *P<0.05, **P<0.01.

[0173] The results show that the cyclic hexapeptide A disclosed in the present invention can significantly promote the expression of LOR in HaCaT cells. At the same time, the experimental results show that the cyclic hexapeptide A obtained by cyclizing hexapeptide B head-to-tail has a significantly improved ability to promote LOR expression compared to linear hexapeptide B. It can be seen that the cyclic hexapeptide A disclosed in the present invention has an excellent effect of promoting LOR expression, can be used to repair the skin barrier, and has the effects of repairing and moisturizing.

[0174] Example 6

[0175] A cream is prepared by the following steps:

[0176]

[0177] According to the prescribed dosage, heat the phase C material in a suitable container to 55-60℃, dissolve completely, and set aside. Add phase A to the stirring pot, stir and heat to 80-85℃. Add phase B to the oil phase pot, stir and heat to 75-80℃, and completely dissolve and become transparent. Pump phase B into phase A, turn on the vacuum, homogenize for 5 minutes, maintain stirring, and keep warm for 20 minutes. Start cooling, cool to 60-65℃, add phase C, and homogenize for 2 minutes. Cool to 35-40℃, add pre-dissolved phase D material, stir for 10-15 minutes, and you are done.

[0178] In the present disclosure, relational terms such as first and second, etc. are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the statement "comprise one..." do not exclude the presence of other identical elements in the process, method, article or terminal device including the elements.

[0179] Although the specific embodiments of the present disclosure are described for the purpose of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the present disclosure. These modifications or improvements should all 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-[Arg-Arg-Gln-D-Met-Glu-Glu].

2. The cyclic peptide or salt thereof according to claim 1, characterized in that The salt includes a metal salt of the cyclic peptide, and the metal includes: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum; Alternatively, the salt includes a salt formed by the cyclic peptide and an organic base, and the organic base includes: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine; Alternatively, the salt includes a salt formed by the cyclic peptide and 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.

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

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

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

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

7. The cosmetic according to claim 6, 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.

8. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3, or the delivery system or sustained-release system according to claim 4 or 5 in the preparation of a composition for caring for or treating skin or mucous membranes.

9. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3, or the delivery system or sustained-release system according to claim 4 or 5 in the preparation of a composition for oil control, acne removal, repair or moisturizing.

10. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3, or the delivery system or sustained-release system according to claim 4 or 5 in the preparation of a composition for reducing skin oil synthesis or secretion.

11. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3, or the delivery system or sustained-release system according to claim 4 or 5 in the preparation of a composition for promoting the expression of filaggrin and / or loricrin, or in the preparation of a composition for promoting re-epithelialization or healing of the skin or mucous membrane, or in the preparation of a composition for repairing the skin barrier.

12. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3, or the delivery system or sustained-release system according to claim 4 or 5 in the preparation of cosmetics.

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