CN118240006A

CN118240006A - Cyclic tetrapeptides and compositions and uses thereof

Info

Publication number: CN118240006A
Application number: CN202410236462.7A
Authority: CN
Inventors: 丁文锋; 赵文豪; 观富宜; 孙新林; 肖玉; 彭晏
Original assignee: Shenzhen Weiqi Technology Co ltd
Current assignee: Shenzhen Weiqi Technology Co ltd
Filing date: 2024-03-01
Publication date: 2024-06-25

Abstract

Discloses a cyclic tetrapeptide, a composition and application thereof, and relates to the technical field of polypeptides, wherein the cyclic tetrapeptide has the following structure: cyclo- [ Pro-Lys-Glu-Lys ]. In particular to the cyclic peptides or salts thereof, or a composition thereof, and the use thereof in the preparation of a composition for caring for or treating skin or mucous membranes.

Description

Cyclic tetrapeptides and compositions and uses thereof

Technical Field

The present disclosure relates to the field of polypeptide technology, and in particular to cyclic tetrapeptides, compositions thereof, and uses thereof.

Background

Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase, is a key rate-limiting enzyme that is widely found in organisms and catalyzes the synthesis of melanin. During melanogenesis, tyrosinase catalyzes the oxidation of L-tyrosine to L-dopa, which is subsequently oxidized to L-dopa quinone, which is then converted to melanin by a series of non-enzymatic reactions. In humans, tyrosinase is overexpressed, which may lead to pigmentation disorders such as age spots, freckles, chloasma, and melanoma. Meanwhile, tyrosinase activity was found to be associated with parkinson's disease and other neurodegenerative diseases in the mammalian brain. The tyrosinase activity can be inhibited to reduce the generation of skin melanin, so that the tyrosinase inhibitor has wide application prospects in the fields of cosmetics, health-care products and medicines.

The conventional tyrosinase inhibitors mainly comprise kojic acid, ascorbic acid, arbutin and the like, however, the inventor realizes that the tyrosinase inhibitors have certain defects, such as skin cancer caused by long-term use of the kojic acid, easy oxidation reaction of the ascorbic acid, high photosensitivity of the arbutin and poor stability of the three. The polypeptide has special physiological activity and can better improve certain problems of skin, and is a research and development hot spot in the fields of cosmetology and medicine in recent years. There are few types of polypeptides currently available for inhibiting tyrosinase activity, and there is still a great need in the marketplace.

Disclosure of Invention

The present disclosure relates to cyclic tetrapeptides and compositions and uses thereof, which have the effect of caring or treating skin or mucous membrane, and the like.

In one aspect, the present disclosure provides a cyclic peptide, or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, having the structure Cyclo- [ Pro-Lys-Glu-Lys ].

The cyclic peptides of the present disclosure contain a large number of asymmetric carbon atoms, and those skilled in the art will appreciate that the cyclic peptides of the present disclosure have stereoisomers and may exist as stereoisomers or mixtures of stereoisomers and thus it is possible to obtain mixtures of isomers as well as racemic 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 described in the present disclosure are pure isomers, i.e., enantiomers or diastereomers. In some embodiments, the structure of a cyclic peptide of the present disclosure is an L-isomer.

The present disclosure also includes all suitable isotopic variants of the cyclic peptide. Isotopic variations of the cyclic peptides of the present disclosure are understood herein to mean such compounds: wherein at least one atom is replaced with another atom of the same atomic number within the cyclic peptide of the present disclosure, but the atomic mass of the other atom is different from the atomic mass normally or predominantly present in nature. Examples of isotopes that can be incorporated into the cyclic peptides of the present disclosure are: those of hydrogen, carbon, nitrogen or oxygen, such as ² H (deuterium), ³ H (tritium), ¹³C、¹⁴C、¹⁴N、¹⁵N、¹⁷ O or ¹⁸ O. Specific isotopic variants of the cyclic peptides of the present disclosure (particularly those into which one or more radioisotopes have been incorporated) may be advantageous, for example, for examining the mechanism of action or distribution of active compounds in vivo; cyclic peptides labeled with ³ H or ¹⁴ C isotopes are particularly suitable for this purpose due to their relatively simple producibility and detectability. In addition, due to the greater metabolic stability of cyclic peptides, the incorporation of isotopes (e.g., deuterium) may yield particular therapeutic benefits, such as increased in vivo half-life or reduced amounts of active agent required. Isotopic variants of the cyclic peptides of the present disclosure can be prepared by methods known to those skilled in the art, for example, by methods further described below and in the examples, by using the respective reagents and/or corresponding isotopic modifications of the starting materials.

The term "salt" refers to a salt that is approved for use in animals, and more specifically in humans, including metal salts of the cyclic peptides, including, but not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum, etc.; including salts of the cyclic peptides with organic bases including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine and the like; including salts of the cyclic peptides with inorganic or 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 (pamoate), gluconic acid, or the like; the inorganic acids include, but are not limited to: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

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

The synthesis of the cyclic peptides of the present disclosure, or stereoisomers thereof, or mixtures of stereoisomers thereof, or salts thereof, may be performed according to conventional methods known in the art, such as solid phase synthesis, liquid phase synthesis, or solid phase and liquid phase combination methods, and may also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins of animal, fungal, or plant origin.

For example, a method of obtaining a cyclic peptide of the present disclosure comprises the steps of:

-coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a protected or solid carrier-bound C-terminus;

-elimination of the group protecting the N-terminal end;

-repeating the coupling sequence and elimination of the N-terminal protecting group until the desired peptide sequence is obtained;

-elimination of the C-terminal protecting group or cleavage from the solid support;

-coupling and cyclizing the amino group at the N-terminus of the peptide chain with the carboxyl group at the C-terminus;

-elimination of the groups protecting the side chains.

In some embodiments, the C-terminus is bound to a solid support and the method is performed 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 polymeric support; eliminating the group protecting the N-terminus; and repeating this sequence as many times as necessary to thereby obtain a peptide of the desired length, followed by cleavage of the synthesized peptide from the original polymer carrier and coupling cyclization of the amino group at the N-terminus of the peptide chain with the carboxyl group at the C-terminus.

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

In some embodiments, solid phase synthesis can be performed by a pooling strategy (convergent strategy) of coupling a dipeptide or tripeptide to a polymeric support or to a dipeptide or amino acid previously bound to a polymeric support.

Due to application external to the body of a mammal, the cyclic peptides of the present disclosure may form part of various types of compositions. Thus in a further aspect of the present disclosure there is provided a composition comprising an effective amount of a cyclic peptide as described above, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, together with at least one excipient and optionally an adjuvant. The composition may be prepared by conventional methods known to those skilled in the art.

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 modulate PGC-1 alpha synthesis, agents that modulate PPARgamma activity, agents that increase or decrease triglyceride levels in adipocytes, agents that stimulate or retard adipocyte differentiation, lipolytic or lipolysis-stimulating agents, lipolysates, adipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis-stimulating or inhibiting agents, whitening or decolorizing agents, pigmentation-promoting agents, and, self-tanning agents, anti-aging agents, NO-synthase inhibitors, 5 alpha-reductase inhibitors, inhibitors of lysyl hydroxylase and/or prolyl hydroxylase, antioxidants, radical scavengers and/or anti-atmospheric agents, active carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, moisture retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermohydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gel polymers, thickeners, surfactants, softeners, adhesives, preservatives, Anti-wrinkle agents, agents capable of reducing or treating the lower pouch, keratolytic agents, antimicrobial agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and/or that inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate decorin synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, agents that stimulate chaperonin 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, acne solubilizers, anti-psoriasis agents, anti-eczema agents, DNA repair agents, DNA protectants, stabilizers, antipruritics, agents for treating and/or caring for sensitive skin, solidifying agents, tightening agents, restructuring agents, stretch mark agents, sebum production regulating agents, antiperspirant agents, healing stimulating agents, healing assisting agents, re-epithelialization stimulating agents, Agents that assist re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents that act on capillary circulation and/or microcirculation, agents that stimulate angiogenesis, agents that inhibit vascular permeability, venous tone agents, agents that act on cellular metabolism, agents that improve dermis-epidermis junction, agents that induce hair growth, hair growth inhibition or delay agents, fragrances, chelators, plant extracts, essential oils, marine extracts, agents derived from biological fermentation processes, inorganic salts, cellular extracts, sunscreens, and organic or inorganic photoprotective agents that are effective against a and/or B uv light, or mixtures thereof.

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, the state of the user, the severity of the condition, the route and frequency of administration, and the particular nature of the cyclic peptide to be used.

By "effective amount" is meant an amount of a cyclic peptide of the present disclosure that is non-toxic but sufficient to provide the desired effect. The cyclic peptides of the present disclosure are used in compositions of the present disclosure at concentrations effective to achieve the desired effect. In some embodiments, the concentration is between 0.00000001% (by weight) and 20% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.000001% (by weight) and 15% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.0001% (by weight) and 10% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.0001% (by weight) and 5% (by weight) relative to the total weight of the composition.

In another aspect of the present disclosure, a delivery system or a sustained release system is provided to achieve better penetration of an active ingredient comprising an effective amount of the cyclic peptide described above, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition of the above.

The term "delivery system" refers to a diluent, adjuvant, excipient, or carrier with which the cyclic peptides of the present disclosure are administered, selected from the group consisting of: water, oils or surfactants, including those of petroleum origin, animal origin, vegetable 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, betaines, glucosides, maltosides, fatty alcohols, nonoxynol, poloxamers, polyoxyethylene, polyethylene glycols, dextrose, glycerol, digitonin, and the like. Diluents that may be used in different delivery systems to which the cyclic peptides of the present disclosure may be administered are known to those of ordinary skill in the art.

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

Examples of delivery systems or sustained release systems include, but are not limited to: liposomes, oleosomes, ethosomes, millimeter capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, clathrates, lipid vesicles, micelles, millimeter spheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, millimeter particles, microparticles or nanoparticles.

In another aspect of the present disclosure, there is provided a cosmetic comprising an effective amount of the above cyclic peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or the above composition, or the above delivery system or sustained release system.

In some embodiments, the formulation of the cosmetic comprises a cream, an emulsion, a water, an oil, a gel, a powder, a tablet, a mud, a patch, a film, an aerosol, a spray, a lyophilized formulation, or a nano-formulation.

In another aspect of the present disclosure, there is provided a use of the above cyclic peptide, or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, or the above composition, or the above delivery system or sustained release system, for the preparation of a composition for treating or treating skin or mucous membrane.

In another aspect of the present disclosure, there is provided a use of the above cyclic peptide, or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, or the above composition, or the above delivery system or sustained release system, for the preparation of a composition for whitening, resolving macula, lightening skin color, or eliminating skin color non-uniformity.

In another aspect of the present disclosure, there is provided a use of the above cyclic peptide, or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, or the above composition, or the above delivery system or sustained release system, for the preparation of a composition for inhibiting tyrosinase activity or inhibiting melanogenesis.

In another aspect of the present disclosure, there is provided a use of the above cyclic peptide, or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, or the composition described above, or the delivery system or sustained release system described above, for the preparation of a composition for anti-aging, soothing, moisturizing or repairing.

In another aspect of the present disclosure, there is provided a use of the above cyclic peptide, or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, or the above composition, or the above delivery system or sustained release system, for the preparation of a composition for inhibiting elastase activity, or for the preparation of a composition for increasing skin elasticity and/or improving skin firmness, or for the preparation of a composition for inhibiting hyaluronidase activity, or for the preparation of a composition for repairing skin barrier.

In another aspect of the present disclosure, there is provided a use of the above cyclic peptide, or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, or the above composition, or the above delivery system or sustained release system, in the preparation of a cosmetic.

In this disclosure, the term "skin" is understood to be the layers that make up it, from the uppermost or stratum corneum to the lowermost or subcutaneous tissue, both endpoints being included. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and/or adipocytes, among others. In this disclosure, the term "skin" includes the scalp.

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

The present disclosure has the following advantages and effects:

1. The cyclic peptides of the present disclosure are effective in inhibiting tyrosinase activity. Inhibiting tyrosinase activity can reduce melanin synthesis of organism, thereby achieving whitening and speckle-reducing effects. Therefore, the cyclic peptide disclosed by the invention can reduce melanin synthesis and has the effects of whitening and removing freckles.

2. The cyclic peptides of the present disclosure are effective in inhibiting elastase activity. Inhibition of elastase activity inhibits elastin hydrolysis and increases elastin levels in skin tissue, while loss of elastin is a major cause of skin aging leading to sagging, sagging and fine wrinkles. Therefore, the cyclic peptide disclosed by the invention can inhibit the activity of elastase, thereby being beneficial to increasing skin elasticity, improving skin firmness and removing skin wrinkles.

3. The cyclic peptides of the present disclosure are effective in inhibiting hyaluronidase activity. Inhibiting hyaluronidase activity can reduce decomposition of hyaluronic acid in cells, increase hyaluronic acid content level in cells, promote hydration of skin or mucosa, repair skin barrier, and has effects of moistening, relieving, and repairing. Therefore, the cyclopeptide disclosed by the invention can improve the content level of hyaluronic acid in cells, and has the effects of moisturizing, relieving and repairing.

4. The cyclic peptide disclosed by the invention is prepared by coupling and cyclizing the N-terminal and the C-terminal of tetrapeptide-30, but compared with linear tetrapeptide-30, the cyclic peptide disclosed by the invention has higher stability and transdermal property, has better effects of inhibiting tyrosinase activity, elastase activity and hyaluronidase activity, and effectively improves the effects of whitening, lightening spots, tightening, removing wrinkles, moisturizing, soothing and repairing.

Drawings

In order to more clearly illustrate the technical solutions of the present disclosure, the drawings that are needed in the description of the present disclosure will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort to a person of ordinary skill in the art.

FIG. 1 is a mass spectrum of a cyclic tetrapeptide (formula C ₂₂H₃₈N₆O₆) of example 1 in the present disclosure.

FIG. 2 is a graph showing the results of tyrosinase activity assay experiments in example 2 of the present disclosure.

FIG. 3 is a graph showing the results of an elastase activity assay of example 3 of the present disclosure.

FIG. 4 is a graph showing the results of an experiment for measuring the hyaluronidase activity in example 4 of the present disclosure.

Detailed Description

In order that the manner in which the above recited objects, features and advantages of the present disclosure are obtained will become more readily apparent, a more particular description of the disclosure will be rendered by reference to the appended drawings and examples. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present disclosure. Based on the embodiments in this disclosure, all other embodiments that may be obtained by one of ordinary skill in the art without inventive effort are within the scope of the claims appended hereto.

In the present disclosure, the abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Biochemical nomenclature Commission (IUPAC-IUB Commission of Biochemical Nomenclature) in the European journal of biochemistry (Eur. J. Biochem.1984, 138:9-37).

Unless otherwise indicated, all reagents and materials used in the present disclosure are commercially available. The following are abbreviations for part of the reagents and materials:

2-CTC Resin: a starting resin for polypeptide synthesis; DMF: n, N-dimethylformamide; DCM: dichloromethane; DIPEA: diisopropylethylamine; meOH: methanol; piperidine: piperidine; HOBt: 1-hydroxybenzotriazole; DIC: diisopropylcarbodiimide; TFA: trifluoroacetic acid; PE: petroleum ether; HATU:2- (7-azabenzotriazol) -N, N' -tetramethylurea hexafluorophosphate; TIS: triisopropylsilane; lys: lysine; glu: glutamic acid; pro: proline; fmoc: 9-fluorenylmethoxycarbonyl; otBu: t-butoxy; boc: and tert-butoxycarbonyl group.

EXAMPLE 1 preparation of Cyclo- [ Pro-Lys-Glu-Lys ]

Cyclo- [ Pro-Lys-Glu-Lys ], prepared by the steps of:

S1 swelling of the resin

10G of 2-CTC Resin was weighed into a solid phase synthesis reaction column, swelled with DCM, the Resin washed, and the solvent was removed.

S2, feeding reaction

S2.1, 11.1g Fmoc-Lys (Boc) -OH, 9.7mL DIPEA were weighed into a dry flask. Dissolving with DMF solvent, adding into the swelled resin, reacting for 3h, pumping out the reaction solution, washing the resin, and pumping out the solvent. The capping treatments with DCM, meOH and DIPEA were continued for 30min. The resin was washed and the solvent was pumped away. Fmoc-Lys (Boc) -2-CTC Resin was obtained.

S2.2, fmoc-Lys (Boc) -2-CTC Resin was Fmoc-removed with 20% piperidine/DMF twice for 10min each time, sampled K for dark blue development. The resin was washed 7 times with DMF and the solvent was removed. 8.7g of Fmoc-Glu (OtBu) -OH and 3.5g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a-18℃refrigerator for 30min. Adding 4.7mLDIC to activate for 3min to avoid water vapor. And adding the activated amino acid into the deprotected resin to react for 1h, and pumping out the reaction solution. K detection resin is colorless and transparent, which indicates that the reaction is complete. Fmoc-Glu (OtBu) -Lys (Boc) -2-CTC Resin was obtained.

S2.3 deprotection of Fmoc group of Fmoc-Glu (OtBu) -Lys (Boc) -2-CTC Resin and coupling activated 9.6g Fmoc-Lys (Boc) -OH to the peptidyl Resin using DMF as solvent in the presence of 3.5g HOBt and 4.7mL DIC for 1h. These resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. The coupling was continued with 6.9g Fmoc-Pro-OH using DMF as solvent in the presence of 3.5g HOBt and 4.7mL DIC. After the reaction was completed, the resin was washed and the solvent was removed. Fmoc-Pro-Lys (Boc) -Glu (OtBu) -Lys (Boc) -2-CTC Resin was obtained.

S2.4, deprotection of Fmoc group of Fmoc-Pro-Lys (Boc) -Glu (OtBu) -Lys (Boc) -2-CTC Resin, removal of Fmoc with 20% piperidine/DMF was performed twice for 10min each time, sampling K and developing dark blue. The resin was washed 2 times with DMF and 5 times with DCM and the solvent was removed. After shrinkage drying, 18.0g of H-Pro-Lys (Boc) -Glu (OtBu) -Lys (Boc) -2-CTC Resin was obtained.

S3, resin removal

S3.1, measuring 144mL of 1% TFA/DCM, mixing and stirring uniformly to obtain a lysate for later use.

S3.2, taking 18g of H-Pro-Lys (Boc) -Glu (OtBu) -Lys (Boc) -2-CTC Resin, adding the Resin into a round bottom flask, adding the standby lysate, and carrying out cleavage reaction for 2 times each time for 0.5H. Suction filtration, collecting filtrate, concentrating to oily substance, adding PE, washing twice, soaking overnight, pouring out supernatant, and spin drying to obtain 11g oily H-Pro-Lys (Boc) -Glu (OtBu) -Lys (Boc) -OH total protective peptide.

S4, cyclization

S4.1, weighing 11g of H-Pro-Lys (Boc) -Glu (OtBu) -Lys (Boc) -OH full-protection peptide, 6.6g of HATU and 1.95g of HOBt, adding into a 250mL single-port bottle, adding 220mL of DMF, magnetically stirring, and adding 7.5g of DIPEA to make the reaction solution alkaline. The reaction was carried out overnight, and the reaction of the starting materials was complete by sampling HPLC.

S4.2, post-treatment: the reaction solution was centrifuged after ice-bath precipitation, washed with pure water for 4 times, and the solid portion was distilled with ethanol to obtain 5.9g of a crude cyclic peptide of Cyclo- [ Pro-Lys (Boc) -Glu (OtBu) -Lys (Boc) ] as a viscous solid.

S5 cleavage (deprotection group)

S5.1, measuring 33.63mL of TFA, 0.885mL of TIS and 0.885mL of water, mixing and stirring uniformly to obtain a lysate, sealing and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether is placed in a refrigerator at the temperature of minus 18 ℃ for refrigeration for standby.

S5.2, 5.9g of Cyclo- [ Pro-Lys (Boc) -Glu (OtBu) -Lys (Boc) ] crude cyclic peptide was added to a round bottom flask, the above frozen lysate was added, and the reaction was stirred at 25℃for 2h. Suction filtration, collecting filtrate, adding 100mL isopropyl ether, stirring, centrifuging and washing for 4 times to obtain 5.2g of crude Cyclo- [ Pro-Lys-Glu-Lys ] cyclopeptide.

S6, purifying

5.2G of crude Cyclo- [ Pro-Lys-Glu-Lys ] cyclic peptide is taken, added with 80mL of pure water, dissolved completely by ultrasonic, filtered by a microporous filter membrane with the pore diameter of 0.45 μm to obtain a clear and transparent solution, and purified by reversed phase HPLC, the purification gradient is shown in the following table:

Time (min)	Flow rate (mL/min)	Mobile phase a-acetonitrile (%)	Mobile phase B-pure water (%)
				0	40	0.5	99.5
30	40	3	97
				45	40	5	95
60	40	50	50

And (3) purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain the Cyclo- [ Pro-Lys-Glu-Lys ] with the purity of 98.905 percent, which is marked as the cyclic tetrapeptide A. The molecular weight was 482.88 as determined by ESI-MS and the mass spectrum was shown in FIG. 1.

The linear peptide Pro-Lys-Glu-Lys (tetrapeptide-30) can be obtained by a similar preparation method.

EXAMPLE 2 tyrosinase activity assay

2.1 Reagents and materials

Mushroom tyrosinase, L-DOPA.

2.2 Instruments

An enzyme-labeled instrument and an ultra-clean workbench.

2.3 Samples to be tested and groupings

2.3.1 Sample to be tested

The concentrations of cyclic tetrapeptide A, tetrapeptide-30 and kojic acid were all 10ppm.

2.3.2 Grouping

Sample group: sample to be tested, tyrosinase, PBS and L-DOPA;

sample zeroing group: sample to be measured, PBS, L-DOPA;

blank control group: PBS, tyrosinase, L-DOPA;

Blank zeroing group: PBS, L-DOPA.

2.4 Experimental methods

Taking a 96-well plate, adding 50 mu L of a sample to be detected and 50 mu L of tyrosinase (25U/mL) into a sample group; adding 50 mu L of sample to be detected and 50 mu L of PBS into the sample zeroing group; a blank was added with 50. Mu. LPBS, 50. Mu.L tyrosinase (25U/mL); blank zeroing groups were added with 100 μ LPBS. After incubation at 37℃for 5min, 50. Mu. LPBS and 50. Mu.LL-DOPA (0.5 mmol/L) were added to each well and incubated at 37℃for 10min. OD ₄₇₅ was measured at 475 nm.

Wherein: a ₁ is the OD ₄₇₅ value for the sample zeroed group, a ₂ is the OD ₄₇₅ value for the sample group, a ₃ is the OD ₄₇₅ value for the blank zeroed group, and a ₄ is the OD ₄₇₅ value for the blank control group.

2.5 Experimental results

L-DOPA is a substrate of tyrosinase, and is oxidized into black dopaquinone substances under the catalysis of tyrosinase, and absorbs visible light with a wavelength of 475 nm. The test samples are used for treating tyrosinase, and the reaction quantity of L-DOPA is detected to determine whether the cyclic tetrapeptide A can inhibit the activity of tyrosinase.

The effect of the test sample on tyrosinase activity inhibition is shown in fig. 2. The results show that the inhibition rate of the cyclic tetrapeptide A on the tyrosinase activity reaches 26.41%, and the inhibition rate of the tetrapeptide-30 on the tyrosinase activity is only 10.03%. The experimental result shows that the capability of inhibiting tyrosinase activity of the cyclic tetrapeptide A obtained by the head-tail cyclization of the tetrapeptide-30 is obviously improved compared with that of the linear tetrapeptide-30. From the results, the cyclic tetrapeptide A disclosed by the invention has excellent effect of inhibiting tyrosinase activity, can inhibit melanin generation, can be used for improving skin problems such as melanin precipitation and chloasma, and has the effects of whitening, removing freckles, brightening skin or eliminating uneven skin color.

Example 3 elastase Activity assay

3.1 Reagents and materials

PBS buffer, elastase solution, AAAPAN solution.

3.2 Instruments

An enzyme-labeled instrument and an electronic balance.

3.3 Samples to be tested and groupings

3.3.1 Sample to be tested

The concentrations of cyclic tetrapeptide A and tetrapeptide-30 were 200ppm.

3.3.2 Grouping

Sample group: sample to be tested, PBS, elastase, AAAPAN;

sample zeroing group: sample to be measured, PBS, AAAPAN;

blank control group: PBS, elastase, AAAPAN;

blank zeroing group: PBS, AAAPAN.

3.4 Experimental methods

Taking a 96-well plate, adding 85 mu L of PBS, 15 mu L of a sample to be tested and 25 mu L of elastase solution (2 mg/mL) into a sample group; adding 110 mu LPBS and 15 mu L of sample to be detected into the sample zeroing group; a blank group was added with 100. Mu. LPBS, 25. Mu.L elastase solution (2 mg/mL); blank zeroing groups were added with 125 μ LPBS. After incubation at 25℃for 15min, a further 25. Mu. LAAAPAN solution (1.015 mmol/L) was added to each well and incubated at 25℃for 15min. OD ₄₁₀ was measured at 410 nm.

Wherein: a ₁ is the OD ₄₁₀ value for the sample zeroed group, a ₂ is the OD ₄₁₀ value for the sample group, a ₃ is the OD ₄₁₀ value for the blank zeroed group, and a ₄ is the OD ₄₁₀ value for the blank control group.

3.5 Experimental results

AAAPAN (N-succinyl-alanine-p-nitroaniline) is a substrate for elastase, AAAPAN is decomposed under the catalysis of elastase, and the decomposition product absorbs visible light with a wavelength of 410 nm. The test samples were used to treat elastase in this experiment to determine whether the cyclic tetrapeptide a of the present disclosure inhibited elastase activity by detecting the amount of reaction AAAPAN.

The effect of the test sample on inhibition of elastase activity is shown in FIG. 3. The result shows that the cyclic tetrapeptide A can obviously inhibit the activity of elastase, the inhibition rate of the cyclic tetrapeptide A of the present disclosure to the activity of the elastase reaches 48.1%, and the inhibition rate of the tetrapeptide-30 to the activity of the elastase is only 23.5%. The experimental result shows that the capability of the cyclic tetrapeptide A obtained by the head-tail cyclization of the tetrapeptide-30 for inhibiting the elastase activity is remarkably improved compared with that of the linear tetrapeptide-30. From the results, the cyclic tetrapeptide A disclosed by the invention has excellent effect of inhibiting elastase activity, can be used for improving skin problems such as skin relaxation and skin wrinkles, effectively increasing skin elasticity and/or improving skin compactness, and realizes the effects of resisting aging, tightening and removing wrinkles.

EXAMPLE 4 hyaluronidase Activity assay

4.1 Reagents and materials

Sodium acetate buffer (ph=5.6), hyaluronidase, calcium chloride, sodium hyaluronate, acetylacetone, absolute ethanol, sodium carbonate, P-DAB (P-dimethylaminobenzaldehyde (0.8 g) and concentrated hydrochloric acid (15 mL) and an equal amount of glacial acetic acid were homogeneously mixed to obtain the final product.

4.2 Instruments

An enzyme-labeled instrument and an electronic balance.

4.3 Samples to be tested and groupings

4.3.1 Sample to be tested

And the test concentrations of the cyclic tetrapeptide A and the tetrapeptide-30 are 100ppm and 200ppm.

4.3.2 Grouping

Sample group: a sample to be tested, hyaluronidase and sodium hyaluronate;

sample zeroing group: sample to be measured, sodium acetate buffer;

blank control group: distilled water, hyaluronidase, sodium hyaluronate;

Blank zeroing group: distilled water and sodium acetate buffer.

4.4 Experimental methods

Hyaluronidase and sodium hyaluronate are dissolved in sodium acetate buffer.

Taking a 96-well plate, adding 25 mu L of a sample to be detected and 25 mu L of hyaluronidase (1000U/mL) into a sample group; adding 25 mu L of sample to be detected and 25 mu L of sodium acetate buffer solution into the sample zeroing group; 25. Mu.L distilled water and 25. Mu.L hyaluronidase (1000U/mL) were added to the blank; the blank zeroing group was added with 25. Mu.L distilled water and 25. Mu.L sodium acetate buffer. After shaking for 20min at 37℃in a constant temperature gas bath, 5. Mu.L of calcium chloride solution (2.5 mol/L) was added to each well, and the mixture was shaken for 20min at 37℃in a constant temperature gas bath. 25 mu L of sodium hyaluronate (1 mg/mL) is added into the sample group and the blank control group, 25 mu L of sodium acetate buffer solution is added into the sample zeroing group and the blank zeroing group, and the mixture is placed at the constant temperature of 37 ℃ for shaking for 40min, and then is placed at the room temperature for 10min. Each well was further charged with 25. Mu.L of distilled water, 5. Mu.L of sodium hydroxide solution (5 mol/L), and 25. Mu.L of acetylacetone solution, and the mixture was placed in an oven at 90℃for 15min, further ice-bath for 10min, and finally at room temperature for 10min. 50. Mu.L of DAB was added to each well, followed by 100. Mu.L of absolute ethanol, and the mixture was left at room temperature for 30min and at 570nm to determine OD ₅₇₀.

Wherein: a ₁ is the OD ₅₇₀ value for the sample zeroed group, a ₂ is the OD ₅₇₀ value for the sample group, a ₃ is the OD ₅₇₀ value for the blank zeroed group, and a ₄ is the OD ₅₇₀ value for the blank control group.

4.5 Experimental results

Sodium hyaluronate is a substrate of hyaluronidase, and is degraded under the catalysis of hyaluronidase to generate glucuronic acid and N-acetylglucosamine, and develops color under the action of P-DAB and absorbs visible light with 570nm wavelength. The present experiment employs a test sample to treat hyaluronidase, and the reaction amount of sodium hyaluronate is detected to determine whether the cyclic tetrapeptide A of the present disclosure can inhibit the activity of hyaluronidase.

The effect of the test sample on inhibition of hyaluronidase activity is shown in FIG. 4. The results show that the cyclic tetrapeptide a of the present disclosure can significantly inhibit the activity of hyaluronidase. At a concentration of 100ppm, the inhibition rate of the cyclic tetrapeptide A of the present disclosure on the hyaluronidase activity reaches 58.3%, while the inhibition rate of the tetrapeptide-30 on the hyaluronidase activity is only 37.7%. At a concentration of 200ppm, the inhibition rate of the cyclic tetrapeptide A of the present disclosure on the hyaluronidase activity reaches 62.6%, while the inhibition rate of the tetrapeptide-30 on the hyaluronidase activity is only 45.9%. The experimental result shows that the capability of the cyclic tetrapeptide A obtained by the head-tail cyclization of the tetrapeptide-30 for inhibiting the hyaluronidase activity is obviously improved compared with that of the linear tetrapeptide-30. From this, it can be seen that the cyclic tetrapeptide a of the present disclosure has an excellent effect of inhibiting hyaluronidase activity, can be used for repairing skin barrier, and has moisturizing, soothing and repairing effects.

Example 5

A whitening cream is prepared by the following steps:

Heating the D-phase material to 55-60 ℃ in a proper container according to the dosage of the prescription, and completely dissolving for standby; adding the phase A into a stirring pot, stirring and heating to 80-85 ℃; adding phase B into oil phase pot, stirring and heating to 75-80deg.C, completely dissolving and transparency; b phase is put into A phase, vacuum is started, homogenization is carried out for 5 minutes, stirring is maintained, and heat preservation is carried out for 20 minutes; cooling to 60-65deg.C, adding C phase and pre-dissolved D phase material, homogenizing for 2 min; cooling to 35-40deg.C, adding E phase material, and stirring for 10-15 min.

In this disclosure, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or terminal device that comprises the element.

Although specific embodiments of the disclosure have been described for illustrative purposes, various modifications or improvements can be made by those skilled in the art without departing from the spirit and scope of the disclosure. Such variations and modifications are intended to fall within the scope of the claims appended hereto.

Claims

1. A cyclic peptide or a salt thereof, wherein the cyclic peptide has a structure of Cyclo- [ Pro-Lys-Glu-Lys ].

2. The cyclic peptide or salt thereof according to claim 1, wherein the salt comprises a metal salt of the cyclic peptide, the metal comprising: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum;

or the salt comprises a salt of the cyclic peptide with an organic base comprising: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine;

Or the salt comprises a salt of the cyclic peptide with an inorganic acid or an organic acid comprising: 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; the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

3. A composition comprising an effective amount of a cyclic peptide or salt thereof according to claim 1 or 2, together with at least one excipient and optionally an adjuvant.

4. A composition according to claim 3, wherein the adjuvant is selected from the group consisting of: agents that activate Clock expression, analgesics, agents that inhibit PAR-2 activity, agents that modulate PGC-1 alpha synthesis, agents that modulate PPARgamma activity, agents that increase or decrease triglyceride levels in adipocytes, agents that stimulate or retard adipocyte differentiation, lipolytic or lipolysis-stimulating agents, lipolysates, adipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis-stimulating or inhibiting agents, whitening or decolorizing agents, Pigmentation-promoting agents, self-tanning agents, anti-ageing agents, NO-synthase inhibitors, 5 alpha-reductase inhibitors, inhibitors of lysyl hydroxylase and/or prolyl hydroxylase, antioxidants, free radical scavengers and/or agents against atmospheric pollution, active carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, moisture-retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermohydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, Adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating the lower pouch, keratolytic agents, antimicrobial agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and/or that inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate decorin synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, agents that stimulate chaperonin 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, acne solubilizers, anti-psoriasis agents, anti-rash agents, DNA repair agents, DNA protectants, stabilizers, antipruritics, agents for treating and/or caring for sensitive skin, solidifying agents, tightening agents, restructuring agents, anti-stretch marks agents, agents that regulate sebum production, antiperspirant agents, 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 that act on capillary circulation and/or microcirculation, agents that stimulate angiogenesis, agents that inhibit vascular permeability, venous tone agents, agents that act on cellular metabolism, agents that improve dermal-epidermal junction, agents that induce hair growth, hair growth inhibition or delay agents, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents derived from biological fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents that are effective against a and/or B ultraviolet light, or mixtures thereof.

5. A delivery system or sustained release system comprising an effective amount of the cyclic peptide or salt thereof of claim 1 or 2, or the composition of claim 3 or 4;

The delivery system or sustained release system comprises: liposomes, oleosomes, ethosomes, millimeter capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, clathrates, lipid vesicles, micelles, millimeter spheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, millimeter particles, microparticles or nanoparticles.

6. Cosmetic product, characterized in that it comprises an effective amount of a cyclic peptide or a salt thereof according to claim 1 or 2, or a composition according to claim 3 or 4, or a delivery system or a slow release system according to claim 5.

7. The cosmetic product according to claim 6, wherein the formulation of the cosmetic product comprises a cream, an emulsion, an aqueous solution, an oil, a gel, a powder, a tablet, a mud, a patch, a film, an aerosol, a spray, a lyophilized preparation or a nano-preparation.

8. Use of a cyclic peptide or a salt thereof according to claim 1 or 2, or a composition according to claim 3 or 4, or a delivery system or a slow release system according to claim 5, for the preparation of a composition for the care or treatment of skin or mucous membranes.

9. Use of a cyclic peptide or a salt thereof according to claim 1 or 2, or a composition according to claim 3 or 4, or a delivery system or a slow release system according to claim 5 for the preparation of a composition for whitening, freckle removing, skin lightening or skin colour non-uniformity eliminating.

10. Use of a cyclic peptide or a salt thereof according to claim 1 or 2, or a composition according to claim 3 or 4, or a delivery system or a slow release system according to claim 5 for the preparation of a composition for inhibiting tyrosinase activity or inhibiting melanogenesis.

11. Use of a cyclic peptide or a salt thereof according to claim 1 or 2, or a composition according to claim 3 or 4, or a delivery system or a slow release system according to claim 5, for the preparation of a composition for anti-ageing, soothing, moisturizing or repairing.

12. Use of a cyclic peptide or a salt thereof according to claim 1 or 2, or a composition according to claim 3 or 4, or a delivery system or a slow release system according to claim 5, for the preparation of a composition for inhibiting elastase activity, or for the preparation of a composition for increasing skin elasticity and/or improving skin firmness, or for the preparation of a composition for inhibiting hyaluronidase activity, or for the preparation of a composition for repairing skin barrier.

13. Use of a cyclic peptide or a salt thereof according to claim 1 or 2, or a composition according to claim 3 or 4, or a delivery system or a slow release system according to claim 5 for the preparation of a cosmetic.