Cyclohexapeptides for skin care, compositions and uses thereof

Abstract

Disclosed is a cyclohexapeptide for skin care and a composition and use thereof, relating to the technical field of polypeptides, the cyclopeptide having the following structure: Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu]. Specifically relates to the cyclopeptide or a salt thereof, or a composition thereof, and the use thereof in the preparation of a composition for caring for or treating skin or mucosa.

Description

Technical Field

[0001] This disclosure relates to the field of peptide technology, and in particular to a cyclic hexapeptide for skin care, its composition and uses. Background Technology

[0002] Skin sebum plays a vital role in the human body. It not only acts as a natural moisturizing barrier, locking in moisture and maintaining the skin's softness and elasticity, but also effectively blocks the invasion of harmful substances and bacteria from the outside world, protecting skin health. However, when sebum secretion is excessive, it can lead to a series of negative effects, such as clogged pores, causing skin problems like blackheads and acne, and making the skin look greasy and unclean, affecting its overall appearance.

[0003] Men generally have higher levels of testosterone, leading to more sebum production, particularly on the face and back. Consequently, many men's facial cleansers, shower gels, and shampoos on the market emphasize oil control. However, frequent use of overly strong cleansing products or improper cleansing methods can excessively remove the skin's natural oils, causing an imbalance of oil and water, and ultimately damaging the skin barrier. Once the skin barrier is damaged, the skin becomes sensitive and fragile. This not only leads to accelerated moisture evaporation causing dryness and cracking, but also makes it more susceptible to external stimuli, resulting in redness, itching, and other discomfort. In severe cases, it can even lead to more complex skin inflammations. Maintaining the skin's oil-water balance is crucial for healthy skin. Addressing the issues of excessive sebum production and damaged skin barriers caused by over-cleansing requires not only continuous oil control but also repairing the skin barrier and reducing moisture loss to maintain the skin's oil-water balance. Summary of the Invention

[0004] This disclosure relates to cyclic hexapeptides, compositions thereof, and uses, wherein the cyclic hexapeptides and compositions containing the cyclic hexapeptides have effects such as care or treatment of the skin or mucous membranes.

[0005] On one hand, this 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. Those skilled in the art will understand that the cyclic peptides of this disclosure have stereoisomers and can exist as stereoisomers or mixtures of stereoisomers. Therefore, it is possible to obtain mixtures of isomers, racemic mixtures, or diastereomer mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and the presence of isomers or mixtures of isomers. In some embodiments, the cyclic peptides of this disclosure are pure isomers, i.e., enantiomers or diastereomers.

[0007] This disclosure also includes all suitable isotopic variants of the cyclic peptides. Isotopic variants of the cyclic peptides of this disclosure are understood herein to refer to compounds in which at least one atom within the cyclic peptide of this disclosure is replaced by another atom of the same atomic number, but the atomic mass of said other atom differs from the atomic mass normally or predominantly found in nature. Examples of isotopes that can be incorporated into the cyclic peptides of this disclosure are those of hydrogen, carbon, nitrogen, oxygen, or sulfur, for example... 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 disclosed herein (especially those already incorporated with one or more radioisotopes) may be advantageous, for example, in examining mechanisms of action or distribution of active compounds in vivo; due to their relatively simple prepareability and detectability, especially with 3 H or 14 C-isotope-labeled cyclic peptides are suitable for this purpose. Furthermore, due to the enhanced metabolic stability of cyclic peptides, the incorporation of isotopes (e.g., deuterium) can produce specific therapeutic benefits, such as prolonged in vivo half-life or reduced required active dose. Isotope variants of the cyclic peptides of this disclosure can be prepared by methods known to those skilled in the art, such as those further described below and those described in the examples, using respective reagents and / or corresponding isotope modifiers of the starting materials.

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

[0009] The nature of the salt is not decisive, and the salt of the cyclic peptide can be obtained by conventional methods known in the art.

[0010] The synthesis of the cyclic peptides, or stereoisomers thereof, or mixtures thereof, or salts thereof described in this disclosure can be carried out using conventional methods known in the art, such as solid-phase synthesis, liquid-phase synthesis, or a combination of solid and liquid methods. It can also be prepared by biotechnological methods aimed at producing a desired sequence, or by controlled hydrolysis of proteins of animal, fungal, or plant origin.

[0011] For example, a method for obtaining the cyclic peptide described in this disclosure includes the following steps:

[0012] - Couple amino acids with a protected N-terminus and a free C-terminus to amino acids with a free N-terminus and a protected C-terminus or a C-terminus bound to a solid support.

[0013] - Eliminate the groups protecting the N-terminus;

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

[0015] - Eliminate the groups protecting the C-terminus or cleave them from the solid support;

[0016] - The amino group at the N-terminus of the peptide chain is coupled and cyclized with the carboxyl group at the C-terminus;

[0017] - Eliminate groups that protect the 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 to an amino acid having a free N-terminus and a C-terminus bound to a polymer support; removing the group protecting the N-terminus; and repeating this sequence a number of times as required to thus obtain a peptide of the desired length, followed by cleaving the synthesized peptide from the original polymer support and cyclizing the amino group at the N-terminus of the peptide chain to the carboxyl group at the C-terminus.

[0019] Throughout the synthesis, the functional groups of the side chains of these amino acids are adequately protected by temporary or permanent protecting groups.

[0020] In some embodiments, solid-phase synthesis can be carried out using a convergent strategy, which involves coupling dipeptides or tripeptides to a polymer support or to dipeptides or amino acids previously bound to a polymer support.

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

[0022] In some implementations...The adjuvants are selected from: 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 triglyceride content in adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic agents or agents that stimulate lipolysis, lipolytic agents, 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 bleaching agents, pigmentation promoters, self-tanning agents, anti-aging agents, NO-synthesizers, 5α-reductase inhibitors, lysyl hydroxylase and / or prolyl hydroxylase inhibitors, antioxidants, and free radical scavengers. Scavengers and / or anti-air pollution agents, active carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, water-retaining substances, alpha-hydroxy acids, beta-hydroxy acids, humectants, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents that reduce or treat under-eye bags, keratolytic agents, antimicrobial agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and / or inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate core proteoglycan synthesis, agents that stimulate laminin synthesis, and agents that stimulate defensin synthesis. Agents, agents stimulating chaperone protein synthesis, agents stimulating cAMP synthesis, agents stimulating hyaluronic acid synthesis, agents stimulating fibronectin synthesis, agents stimulating deacetylase synthesis, agents stimulating lipid and stratum corneum component synthesis, ceramides, fatty acids, agents inhibiting elastin degradation, agents inhibiting serine proteases, agents stimulating fibroblast proliferation, agents stimulating keratinocyte proliferation, agents stimulating adipocyte proliferation, agents stimulating melanocyte proliferation, agents stimulating keratinocyte differentiation, agents inhibiting acetylcholinesterase, skin relaxants, agents stimulating glycosaminoglycan synthesis, anti-hyperkeratosis agents, comedolytic agents, anti-psoriasis agents, anti-eczema agents, DNA repair agents, DNA protectants, stabilizers, antipruritic agents, for the treatment and / or care of sensitive skin. Agents for sensitive skin, including hardening agents, firming agents, reconstructing agents, anti-stretch mark agents, agents that regulate sebum production, antiperspirants, agents that stimulate healing, agents that assist healing, agents that stimulate re-epithelialization, agents that assist re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents acting on capillary circulation and / or microcirculation, agents that stimulate angiogenesis, agents that inhibit vascular permeability, venous tension agents, agents acting on cell metabolism, agents for improving dermal-epidermal junction, agents that induce hair growth, agents that inhibit or delay hair growth, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents derived from bio-fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents or mixtures thereof that effectively protect against UVA and / or UVB rays.

[0023] The effective amount of the cyclic peptides of this disclosure to be administered, and their dosage, will depend on many factors, including age, the user's condition, the severity of the condition, the 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 this disclosure that is non-toxic but sufficient to provide the desired effect. The effective concentration of the cyclic peptide of this disclosure is used in the compositions of this disclosure 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 this disclosure provides a delivery system or sustained-release system for better penetration of the active ingredient, comprising an effective amount of the aforementioned cyclic peptide, or a stereoisomer thereof, or a mixture thereof, a salt thereof, or a combination thereof.

[0026] The term "delivery system" refers to a diluent, adjuvant, excipient, or carrier applied with the cyclic peptides of this disclosure, selected from water, oil, or surfactants, including those of petroleum, animal, plant, or synthetic origin, such as and not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitol ester, ether sulfate, sulfate, betaine, glucosinolate, maltodextrin, fatty alcohol, nonyl alcohol ether, poloxamer, polyoxyethylene, polyethylene glycol, dextran, glycerol, digitalis saponins, and the like. Those skilled in the art are familiar with various diluents that can be used in different delivery systems for administering the cyclic peptides of this disclosure.

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

[0028] Examples of delivery systems or sustained-release systems include, but are not limited to: liposomes, oil bodies, alcohol bodies, millimeter capsules, micrometer capsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion complexes, lipid vesicles, micelles, millimeter spheres, micrometer spheres, nanospheres, lipid spheres, micrometer emulsions, nanoemulsions, millimeter particles, micrometer particles, or nanoparticles.

[0029] In another aspect of this disclosure, a cosmetic product is provided, comprising an effective amount of the above-described cyclic peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition thereof, or a delivery system or sustained-release system thereof.

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

[0031] Another aspect of this disclosure provides the use of the above-described cyclic peptide, or a stereoisomer thereof, or a mixture thereof, or a salt thereof, or a composition thereof, or a delivery system or sustained-release system thereof, in the preparation of a composition for the care or treatment of skin or mucous membranes.

[0032] Another aspect of this disclosure provides the use of the above-described cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or a salt thereof, or the above-described composition, or the above-described delivery system or sustained-release system in the preparation of compositions for oil control, acne treatment, repair, or moisturizing.

[0033] In another aspect of this disclosure, there is a use of the above-described cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or a salt thereof, or the above-described composition, or the above-described delivery system or sustained-release system in the preparation of a composition for reducing skin sebum synthesis or secretion.

[0034] In another aspect of this disclosure, there is provided the use of the above-described cyclic peptide, or its stereoisomer, or a mixture of its stereoisomers, or a salt thereof, or the above-described composition, or the above-described delivery system or sustained-release system in the preparation of a composition for promoting the expression of filaggrin (FLG) and / or lobelin (LOR), 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 repairing the skin barrier.

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

[0036] In this disclosure, the term "skin" should be understood as comprising its multiple layers, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, including both ends. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes. In this disclosure, the term "skin" includes the scalp.

[0037] The term "skin care" refers to the maintenance and care of the skin to improve its condition, making it delicate, smooth, soft, and healthy.

[0038] This disclosure has the following advantages and effects:

[0039] 1. The cyclic peptide disclosed herein can effectively promote the expression of FLG and LOR. FLG is an important factor involved in the skin barrier. In the process of forming the stratum corneum of the outer epidermis, it promotes epidermal differentiation, forming the unique barrier structure of the stratum corneum and playing a crucial role in maintaining the skin's barrier function. LOR is a major component of the stratum corneum and plays an important role in the normal functioning of the epidermal barrier. Promoting the expression of FLG and LOR can repair the skin barrier, improve its function, help retain skin moisture, and enhance the skin's moisturizing ability. Therefore, the cyclic peptide disclosed herein has repairing and moisturizing effects.

[0040] 2. The cyclic peptides disclosed herein can effectively promote cell migration. Promoting cell migration can facilitate re-epithelialization of the skin or mucous membranes, wound healing, and repair of the skin barrier. Therefore, the cyclic peptides disclosed herein can be used to promote re-epithelialization of the skin or mucous membranes, wound healing, and repair of damaged skin barriers, thereby achieving a repairing effect.

[0041] 3. The cyclic peptide disclosed herein can inhibit the synthesis or secretion of sebum by sebaceous gland cells, slow down sebum deposition, reduce the occurrence of pimples or acne, and also help the skin recover after pimples or acne occur. It has the effects of oil control, acne removal and repair.

[0042] 4. The cyclic peptide disclosed herein is obtained by cyclization of the N-terminus and C-terminus of linear RRQ(d)MEE. However, compared with linear polypeptides, the cyclic peptide disclosed herein has better effects on promoting cell migration, promoting LOR and FLG expression, and inhibiting sebum synthesis or secretion, thereby achieving excellent oil control, acne removal, repair and moisturizing effects. Attached Figure Description

[0043] To more clearly illustrate the technical solutions of this disclosure, the accompanying drawings used in the description of this disclosure will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is Example 1 of the present disclosure, Cyclohexapeptide A (molecular formula C). 32 H 55 N 13 O 11 The mass spectrum of S). Detailed Implementation

[0045] To make the objects, features, and advantages of this disclosure more apparent and understandable, the disclosure will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of the appended claims.

[0046] In this disclosure, the abbreviations used 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).

[0047] Unless otherwise specified, all experimental reagents and materials used in this disclosure are commercially available. The following are abbreviations for some reagents and materials:

[0048] 2-CTC Resin: A starting resin for polypeptide 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-Ethylenedithiol; Glu: Glutamic acid; D-Met: D-methionine; Gln: Glutamine; Arg: Arginine; Fmoc: 9-fluorenylmethoxycarbonyl; OtBu: Tert-butoxy; Trt: Triphenylmethyl; 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] is prepared through the following steps:

[0051] S1, Swelling of the resin

[0052] Weigh 10g 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 Erlenmeyer flask, dissolve it in DMF solvent, and cool it in an ice-water bath for 10 min. Add 11.3 mL of DIPEA and stir for 3 min. Then add the above mixture to the swollen resin and react for 2.5 h. Remove the reaction solution, wash the resin 4 times, and remove the solvent. Continue to add DCM, MeOH, and DIPEA for end-capping treatment for 10 min, and repeat twice. Wash the resin 4 times, remove the solvent, and obtain Fmoc-Glu(OtBu)-2-CTC Resin.

[0055] S2.2 Fmoc-Glu(OtBu)-2-CTC Resin was deprotected twice with 20% piperidine / DMF: the first time for 8 min, the second time for 5 min. A K test was performed on the sample; a deep blue color was observed. The resin was washed 7 times with DMF, and the solvent was removed. 10.5 g of Fmoc-Glu(OtBu)-OH and 3.9 g of HOBt were weighed and added to a dry Erlenmeyer flask. DMF was added to dissolve the flask, and the flask was sealed and placed in a -18°C refrigerator for 30 min. 5.6 mL of DIC was added for activation for 3 min. The activated amino acid was added to the deprotected resin and reacted for 2 h. The reaction solution was then removed. A colorless and transparent K test indicated that the reaction was complete, yielding Fmoc-Glu(OtBu)-Glu(OtBu)-2-CTC Resin.

[0056] S2.3. Repeat step S2.2 to deprotect the N-terminal Fmoc group, and in the presence of 3.9 g HOBt and 5.6 mL DIC, use DMF as a solvent to couple the activated amino acid to the peptide resin, continuing the reaction for 2 h. Then wash the resin and repeat the deprotection treatment of the Fmoc group to couple the next amino acid. In each coupling, 9.1 g Fmoc-D-Met-OH, 14.9 g Fmoc-Gln(Trt)-OH, 15.8 g Fmoc-Arg(Pbf)-OH, and subsequently 15.8 g Fmoc-Arg(Pbf)-OH were sequentially coupled using DMF as a solvent in the presence of 3.9 g HOBt and 5.6 mL DIC. 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. Deprotection of the N-terminal Fmoc group of the peptide resin was performed twice with 20% piperidine / DMF: the first time for 8 min, and the second time for 5 min. A sample was taken for K testing; the color was deep blue. The resin was first washed four times with DMF, then rinsed four times with DCM, and finally washed once with MeOH, after which the solvent was removed. After shrinkage and drying, 28.7 g of 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 Measure 4.5 mL of TFA and 445.5 mL of DCM, mix and stir evenly to obtain the lysis solution, seal and store at -18℃ for later use; store isopropyl ether at -18℃ 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, add circle Add the frozen lysis buffer to the bottom flask, stir and react for 0.5 h, repeat once, for a total of 2 lysis cycles. Filter, collect the filtrate and concentrate to obtain an oily substance, add PE and stir to solidify, yielding 26.5 g of H-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-D-Met-Glu(OtBu)-Glu(OtBu)-OH.

[0061] S4, cyclic

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

[0063] S5, cleavage (deprotection of groups)

[0064] Mix 144 mL TFA, 4 mL Tis, 4 mL EDT, and 4 mL anisole, add 26.7 g of crude cyclic peptide, lyse for 2.5 h, 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 and filtered through a 0.45 μm microporous membrane to obtain a clear and transparent solution. The solution was then purified by reversed-phase HPLC, and the purification gradient is shown in the table below:

[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 purified by injection, the fraction was collected, concentrated and lyophilized to obtain a cyclic peptide Cyclo-[Arg-Arg-Gln-D-Met-Glu-Glu] with a purity >95.3%, denoted as cyclic hexapeptide A, with the following structural formula:

[0069]

[0070] The molecular weight of cyclic hexapeptide A was determined by ESI-MS, and the mass spectrum is shown below. Figure 1 As shown. The results show that [M+H] + The mass-to-charge ratio (m / z) of the quasi-molecular ion peak was 830.3440, and the molecular weight determined by mass spectrometry was 829.34, which is consistent with the theoretical precise molecular weight of cyclic hexapeptide 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. Analysis of the proton NMR spectra 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 bonded to amide bond 2 4.30 2 q,J = 6Hz α-CH bonded to amide bond 3 4.23 1 t,J=6Hz α-CH bonded to amide bond 4 4.16 1 t,J=6Hz α-CH bonded to amide bond 5 4.10 1 t,J=6Hz α-CH bonded to amide bond 6 3.09 4 dt,J=24.6Hz Hydrogen in saturated alkanes 7 2.50 2 m Hydrogen in saturated alkanes 8 2.22 5 m Hydrogen in saturated alkanes 9 2.08 5 m Hydrogen in saturated alkanes 10 2.00 3 s Hydrogen in saturated alkanes 11 1.94 3 m Hydrogen in saturated alkanes 12 1.82 3 m Hydrogen in saturated alkanes 13 1.70 2 m Hydrogen in saturated alkanes 14 1.48 4 m Hydrogen in 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 proton and carbon NMR spectra both show that the structure of the tested compound is consistent with the structure of cyclic hexapeptide A.

[0080] 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 (0.25g trypsin was prepared with 100mL of water), PBS, and FFA (linoleic acid and palmitic acid were mixed in a molar ratio of 1:1).

[0084] 2.2 Instruments

[0085] Constant temperature CO2 incubator, ultra-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 Sample to be tested

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

[0091] Simvastatin was dissolved in PBS, and the test concentration was 0.05 μM.

[0092] 2.4.2 Grouping

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

[0094] Normal group: PBS;

[0095] Model groups: PBS, FFA.

[0096] 2.5 Experimental Methods

[0097] Take one flask of SZ-95 cells in good exponential growth phase, add 0.25% trypsin digestion solution, digest to detach the adherent cells, and count (1-4) × 10⁻⁶ cells. 5 Cells were prepared at a concentration of [number] cells / mL to form a cell suspension. An appropriate amount of the cell suspension was seeded into a 12-well plate containing complete culture medium and incubated in a CO2 incubator for 24 hours. Except for the normal group which received PBS, FFA was added to each well of the other groups to a final concentration of 225 μmol / mL for induction and modeling. Simultaneously, the corresponding test samples were added to the experimental groups, and the plates were incubated in a CO2 incubator for 48 hours. The culture medium was then discarded, and the Oil Red O staining kit was used according to the manufacturer's instructions.

[0098] 2.6 Experimental Results

[0099] Oil Red O is a fat-soluble dye that is highly soluble in fat. Its staining principle is that Oil Red O specifically adsorbs onto neutral triglycerides, lipids, and lipoproteins in tissues and cells, thus staining the fat. Free fatty acids (FFA) are an inducer; under FFA stimulation, SZ-95 cells secrete large amounts of oil that can be stained by Oil Red O. This experiment used test samples to treat SZ-95 cells induced by FFA stimulation. By detecting the amount of oil produced by SZ-95 cells, it was determined whether the cyclic hexapeptide A disclosed in this paper could inhibit the secretion of oil by human sebaceous gland cells.

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

[0101] Table 3. Relative lipid secretion in SZ-95 cells

[0102] Group Relative sebum secretion (Mean±SD) normal group 100.00％±2.72％ Model group 222.71％±26.37％ Simvastatin group 107.52％±3.51％*** Cyclic hexapeptide A group 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] Experimental results show that the cyclic hexapeptide A obtained by cycloaddition of hexapeptide B (head-to-tail cyclization) significantly enhances its ability to inhibit sebum synthesis and secretion compared to linear hexapeptide B. Therefore, the cyclic hexapeptide A disclosed herein not only inhibits sebum synthesis and secretion by sebaceous gland cells and slows sebum deposition, but its inhibitory effect is also superior to that of linear hexapeptide B. It can be used to improve problems such as excessive oil production and skin water-oil imbalance, reduce the occurrence of acne or pimples, and aid in skin recovery after acne or pimple formation, exhibiting oil-controlling, acne-removing, and repairing effects.

[0105] Example 3 Cell Migration Test

[0106] 3.1 Reagents and Materials

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

[0108] 3.2 Instruments

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

[0110] 3.3 Cell lines

[0111] Human keratinocytes (HaCaT).

[0112] 3.4 Samples to be tested and grouping

[0113] 3.4.1 Sample to be tested

[0114] Cyclic hexapeptide 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 Methods

[0119] One flask of cells in the exponential growth phase was digested with 0.25% trypsin solution to prepare a cell suspension. This suspension was then seeded into a 12-well plate containing complete culture medium and incubated in a CO2 incubator for 24 hours. The medium was changed, and two vertical lines were drawn in the center of each well using a yellow pipette tip. The cells were washed once with PBS, and photographs were taken at the positions marked above and below the horizontal lines. After photographing, complete culture medium containing 2% FBS and the test sample were added to the sample group; the blank control group was added to complete culture medium containing 2% FBS and PBS. The cells were then incubated in a CO2 incubator for 24 hours. After 24 hours of incubation, photographs were taken at the same locations under an inverted biological microscope, and the area of ​​the scratches was calculated.

[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, and a common form of movement among living cells. 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 effects of the test samples on cell migration are shown in Table 4.

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

[0125] Group Scratch healing rate (Mean±SD) Blank control group 10.66％±2.52％ Cyclic hexapeptide A group 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 showed that the cyclic hexapeptide A disclosed herein significantly promoted HaCaT cell migration, exhibiting a significant effect in promoting skin or mucous membrane re-epithelialization or wound healing. Furthermore, the cyclic hexapeptide A obtained by cyclically combining hexapeptide B head-to-tail showed a significantly improved technical effect compared to linear hexapeptide B. Therefore, the cyclic hexapeptide A disclosed herein possesses excellent cell migration-promoting effects and can be used to promote skin or mucous membrane re-epithelialization or wound healing, repair damaged skin barriers, and achieve restorative effects.

[0128] Example 4: Determination of FLG Content

[0129] 4.1 Reagents and Materials

[0130] 0.25% trypsin digestion solution (prepared by using 0.25g trypsin in 100mL PBS), complete culture medium, PBS, RIPA lysis buffer, human FLG Filaggrin ELISA kit, and BCA protein quantification kit.

[0131] 4.2 Instruments

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

[0133] 4.3 Cell lines

[0134] Human keratinocytes (HaCaT).

[0135] 4.4 Samples to be tested and grouping

[0136] 4.4.1 Sample to be tested

[0137] Cyclic hexapeptide A, hexapeptide B, and acetyl hexapeptide C were dissolved in PBS, and the test concentrations were all 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 one flask of human keratinocytes (HaCaT) in good exponential growth phase, add 0.25% trypsin digestion solution, digest to detach the adherent cells, and count (1-4) × 10⁻⁶ cells. 5 Cells were cultured at a density of [number] cells / mL to prepare a cell suspension. An appropriate amount of cell suspension was seeded into 12-well plates containing complete culture medium and incubated in a CO2 incubator for 24 h. The complete culture medium was aspirated from the wells, PBS was added, and the plates were exposed to UVB light for 15 min; the blank control group was not exposed. After 15 min of irradiation, the PBS solution was aspirated, and complete culture medium and the test sample were added to the sample groups, respectively; the blank control group and the UV group were added to complete culture medium and PBS, and cultured for 48 h. Cells were collected, the cell pellet was centrifuged, RIPA lysis buffer was added, and the cells were vortexed three times (30 s / time, 3 min interval), and centrifuged at 12000 rpm for 10 min. The supernatant was aspirated and analyzed using an ELISA kit, and the total protein concentration of the supernatant was determined using a BCA protein quantification kit.

[0144] 4.6 Experimental Results

[0145] Filamentin (FLG) is an important factor involved in the skin barrier. In the process of forming the stratum corneum of the outer epidermis, it promotes epidermal differentiation, forming the unique barrier structure of the stratum corneum. It plays a crucial role in maintaining the physical strength of the stratum corneum and reducing transepidermal water loss, thus contributing to barrier integrity and hydration. Therefore, FLG expression can serve as an indicator for evaluating the effectiveness of skin barrier repair. By testing the effect of a substance on FLG expression, the effect of that substance on skin barrier repair can be assessed. Increased FLG expression is beneficial for repairing the skin barrier, improving skin barrier function, and enhancing the skin's moisturizing ability. In this experiment, HaCaT cells were treated with test samples. By detecting the amount of FLG synthesized within HaCaT cells, the cyclic hexapeptide A disclosed herein could be used to promote FLG expression in HaCaT cells.

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

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

[0148] Group FLG relative content (Mean±SD) Blank control group 100.00％±12.39％ UV group 28.00％±9.28％ Cyclic hexapeptide A group 57.55％±14.57％** Hexapeptide B group 48.82％±0.74％*** Acetyl hexapeptide C group 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 herein significantly promoted FLG expression in HaCaT cells, but the results for acetyl hexapeptide C showed no statistically significant difference compared with the UV group. Simultaneously, these experimental results indicated that the cyclic hexapeptide A obtained by cyclically combining hexapeptide B end-to-end significantly enhanced its FLG expression-promoting ability compared to linear hexapeptide B. Therefore, the cyclic hexapeptide A disclosed herein possesses excellent FLG expression-promoting effects and can be used to repair the skin barrier, exhibiting repairing and moisturizing effects.

[0151] Example 5: Determination of LOR Content

[0152] 5.1 Reagents and Materials

[0153] 0.25% trypsin digestion solution (prepared by dissolving 0.25g trypsin in 100mL of water), complete culture medium, PBS, RIPA lysis buffer, Human LORLoricrin ELISA Kit, and BCA protein quantification kit.

[0154] 5.2 Instruments

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

[0156] 5.3 Cell lines

[0157] Human keratinocytes (HaCaT).

[0158] 5.4 Samples to be tested and grouping

[0159] 5.4.1 Sample to be tested

[0160] Cyclic hexapeptide A and hexapeptide B were dissolved in PBS, and the test concentrations were both 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 one flask of human keratinocytes (HaCaT) in good exponential growth phase, add 0.25% trypsin digestion solution, digest to detach the adherent cells, and count (1-4) × 10⁻⁶ cells. 5 Cells were cultured at a density of [number] cells / mL to prepare a cell suspension. An appropriate amount of cell suspension was seeded into 12-well plates containing complete culture medium and incubated in a CO2 incubator for 24 h. The complete culture medium was aspirated from the wells, PBS was added, and the plates were exposed to UVB light for 15 min; the blank control group was not exposed. After 15 min of irradiation, the PBS solution was aspirated, and complete culture medium and the test sample were added to the sample groups, respectively; the blank control group and the UV group were added to complete culture medium and PBS, and cultured for 48 h. Cells were collected, the cell pellet was centrifuged, RIPA lysis buffer was added, and the cells were vortexed three times (30 s / time, 3 min interval), and centrifuged at 12000 rpm for 10 min. The supernatant was aspirated and analyzed using an ELISA kit, and the total protein concentration of the supernatant was determined using a BCA protein quantification kit.

[0167] 5.6 Experimental Results

[0168] LOR (lobe-like proteins) are a major component of the keratin capsule and play a crucial role in the normal functioning of the epidermal barrier. LOR expression is vital in skin barrier repair, and increased LOR expression is an important marker of skin barrier repair. This experiment used test samples to treat HaCaT cells, and by detecting the level of LOR synthesized within HaCaT cells, determined whether the cyclic hexapeptide A disclosed herein could promote LOR expression in HaCaT cells.

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

[0170] Table 6. Effects of the test samples on LOR expression in HaCaT cells.

[0171] Group LOR relative content (Mean±SD) Blank control group 100.00％±10.21％ UV group 51.57％±4.84％ Cyclic hexapeptide A group 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 showed that the cyclic hexapeptide A disclosed herein significantly promoted LOR expression in HaCaT cells. Furthermore, these results indicated that the cyclic hexapeptide A obtained by cyclically cyclizing hexapeptide B (both ends) significantly enhanced its ability to promote LOR expression compared to linear hexapeptide B. Therefore, the cyclic hexapeptide A disclosed herein possesses excellent effects in promoting LOR expression and can be used to repair the skin barrier, exhibiting repairing and moisturizing effects.

[0174] Example 6

[0175] A type of frost is prepared through the following steps:

[0176]

[0177] According to the prescribed dosage, heat phase C in a suitable container to 55-60℃ until completely dissolved, then set aside. Add phase A to a mixing pot and heat to 80-85℃. Add phase B to the oil phase pot and heat to 75-80℃ until completely dissolved and transparent. Transfer phase B into phase A, apply vacuum, homogenize for 5 minutes, maintain stirring, and keep warm for 20 minutes. Begin cooling to 60-65℃, add phase C, and homogenize for 2 minutes. Cool to 35-40℃, add pre-dissolved phase D, and stir for 10-15 minutes to obtain the final product.

[0178] In this disclosure, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0179] While specific embodiments of this disclosure have been described for illustrative purposes, various modifications or alterations can be made by those skilled in the art without departing from the spirit and scope of this disclosure. All such modifications or alterations should fall within the scope of the appended claims.

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], and the cyclic peptide is shown in Formula I: Equation I.

2. The cyclic peptide or its salt 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 may comprise a salt formed by the cyclic peptide and an organic base, wherein the organic base may include: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine; Alternatively, the salt may comprise a salt formed by the cyclic peptide and an inorganic or 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, pyric acid, or gluconic acid; and the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

3. A composition, characterized in that, It includes an effective amount of the cyclic peptide or its salt as described in claim 1 or 2, as well as at least one excipient and optional adjuvant.

4. A delivery system or sustained-release system, characterized in that, Contains an effective amount of the cyclic peptide or its salt as described in claim 1 or 2, or the composition as described in 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, oil bodies, alcohol bodies, millimeter capsules, micrometer capsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion complexes, lipid vesicles, micelles, millimeter spheres, micrometer spheres, nanospheres, lipid spheres, micrometer emulsions, nanoemulsions, millimeter particles, micrometer particles, or nanoparticles.

6. A cosmetic product, characterized in that, Contains an effective amount of the cyclic peptide or its salt as described in claim 1 or 2, or the composition as described in claim 3, or the delivery system or sustained-release system as described in claim 4 or 5.

7. The cosmetic product according to claim 6, characterized in that, The dosage forms of the cosmetics include ointments, creams, emulsions, liquids, oils, gels, powders, tablets, muds, patches, films, aerosols, sprays, freeze-dried preparations, or nano-preparations.

8. Use of the cyclic peptide or its salt as claimed in claim 1 or 2, or the composition as claimed in claim 3, or the delivery system or sustained-release system as claimed in claim 4 or 5 in the preparation of compositions for oil control, acne treatment, repair or moisturizing.

9. The use according to claim 8, characterized in that, Oil control includes reducing the synthesis or secretion of sebum in the skin.

10. The use according to claim 8, characterized in that, The repair includes promoting the expression of filaggrin and / or lipogrin.

11. Use of the cyclic peptide of claim 1 or 2 or a salt thereof, or the composition of claim 3, or the delivery system or sustained-release system of claim 4 or 5 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 repairing the skin barrier.

12. Use of the cyclic peptide or its salt as claimed in claim 1 or 2, or the composition as claimed in claim 3, or the delivery system or sustained-release system as claimed in claim 4 or 5 in the preparation of cosmetics for oil control, acne treatment, repair, or moisturizing.

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