CN115581633A - Novel use of peptide compounds in preparation of composition for skin aging repair - Google Patents

Abstract

Use of a compound of formula (I) or a salt thereof for the preparation of a composition for the rejuvenation of skin ageingRepair includes one or more of increasing fibroblast activity, promoting keratinocyte proliferation and migration, promoting re-epithelialization or healing of the skin or mucosa, repairing skin barriers, preventing or repairing photoaging damage, promoting collagen production, increasing skin elasticity, or increasing skin firmness.

Description

Novel use of peptide compounds in preparation of composition for skin aging repair

Technical Field

The invention belongs to the technical field of active polypeptide, and particularly relates to a new application of a peptide compound in preparing a composition for skin aging restoration.

Background

The skin, the largest and most important organ of the human body, covers the entire surface of the human body and is composed of the epidermis, dermis and subcutaneous tissue, where the epidermis and dermis layers play important roles, such as preventing water loss, forming a protective barrier and providing structural support. However, over time, under the influence of various factors such as age increase, internal and external environments and the like, the expression levels of the epidermis layer and the dermis layer of the skin can also change, for example, after long-term exposure or ultraviolet radiation, the proliferation speed and differentiation of epidermal layer keratinocytes are changed, the expression of molecules involved in basal layer cell adhesion is reduced, and the skin barrier function is weakened; the composition of the extracellular matrix of the dermis layer is also subjected to degenerative change along with the aging process, a large amount of collagen is lost, the activity of fibroblasts is weakened, the synthesized collagen is gradually reduced, the area of collagen fibers is also reduced, so that the reticular structure of the dermis layer of the skin is loosened, the moisture retention and the toughness of the skin tissue are reduced, and the skin is dried, rough, wrinkled, loosened, damaged and the like.

During the natural aging process of the skin, fibroblast function is reduced, collagen fibers and elastic fibers are damaged and broken, and the balance between the production and degradation of important biomolecules such as collagen in the skin is biased to the degradation process with age, so that the dermis is progressively thinned and damaged, the dermis is loosened, and wrinkles are formed subsequently; in addition, the secretion of matrix metalloproteinase is increased during skin photoaging, so that the degradation of collagen is accelerated, the loss of skin collagen is caused, the integrity of dermis is damaged, the strength and the elasticity of the skin are reduced, and even color spots appear; in addition, the decrease of collagen, elastin and fibronectin in skin aging causes the decrease of extracellular matrix, which is not favorable for skin repair, however, repair is a precise and complex dynamic process with time phases, and involves network regulation of various cells such as cell proliferation, migration, extracellular matrix degradation, angiogenesis and epithelial tissue remodeling. Therefore, the method has important significance in searching compounds with the functions of delaying senility and repairing the skin.

In recent years, more and more functional skin care products have been produced in order to repair skin and delay skin aging. However, most of the commercially available anti-aging repair skin care products improve the aging state of the skin by simply adding exogenous active substances (such as moisturizing agents, free radical scavengers, sunscreen agents and the like), and the real anti-aging effect is not satisfactory. In addition, the components of the skin care products can only be absorbed by the surface layer of the skin to a great extent, but cannot really enter the deep layer of the skin to achieve the purpose of durably renewing and supplementing collagen, so that the skin care products cannot well play the role of delaying senility and repairing. At present, several methods for repairing the skin aging have been found, for example, the method for repairing the skin aging by using retinol to promote the generation of collagen and increase the skin elasticity, and stimulate the cell renewal and regulate the metabolism of epidermis and horny layer, but the method is easy to have the side effects of dry skin mucous membrane, desquamation, pruritus, injury, photosensitivity to light and the like after a large amount of long-term use, and can also make the skin sensitive and weak, stimulate the secretion of skin melanocyte, and lead to the skin blackening. At present, compounds which can safely and efficiently play a role in delaying senescence and repairing are lacking in the market.

Therefore, there is still a need to develop more safe and effective compounds to promote cell proliferation, increase cell activity, promote synthesis and secretion of collagen, elastin and other components, improve skin mechanical properties such as flexibility, elasticity and firmness lost with aging or exposure to sunlight and/or environmental pollutants, and repair damaged skin to meet the increasing demands of consumers for treatment or care of skin.

Disclosure of Invention

The present inventors have completed the present invention by finding, through a large number of experimental studies, that a peptide compound has an efficacy of delaying aging and repairing the skin.

Accordingly, the present invention is directed to the use of a peptidic compound or a salt thereof in the preparation of a composition for skin aging repair including one or more of increasing fibroblast activity, promoting keratinocyte proliferation and migration, promoting re-epithelialization or healing of the skin or mucosa, repairing skin barriers, preventing or repairing photoaging damage, promoting collagen production, increasing skin elasticity, or increasing skin firmness.

The peptide compound or the salt thereof has the following general formula (I),

in the general formula (I), the compound has the following structure,

R ₁ selected from: h or R ₃ -CO-, wherein R ₃ Selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

R ₂ selected from the group consisting of: -OH, -NH ₂ 、-Leu-NH ₂ 、-Leu-OH、-Val-NH ₂ or-Val-OH;

the alkyl group refers to a saturated aliphatic straight or branched chain alkyl group having 1 to 24 carbon atoms (optionally having 1 to 16 carbon atoms; optionally having 1 to 14 carbon atoms; optionally having 1 to 12 carbon atoms; optionally having 1, 2, 3, 4, 5, or 6 carbon atoms); optionally selected from: methyl, ethyl, isopropyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 2-ethylhexyl, 2-methylbutyl, or 5-methylhexyl;

the alkenyl group means a straight or branched chain alkenyl group having 2 to 24 carbon atoms (optionally having 2 to 16 carbon atoms; optionally having 2 to 14 carbon atoms; optionally having 2 to 12 carbon atoms; optionally having 2, 3, 4, 5, or 6 carbon atoms); said alkenyl having one or more carbon-carbon double bonds, optionally 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bonded to the remainder of the molecule by a single bond; optionally selected from: vinyl, oleyl, or linoleyl;

alternatively, the substituents in the "substituted alkyl", "substituted alkenyl" are selected from C ₁ -C ₄ An alkyl group; a hydroxyl group; c ₁ -C ₄ An alkoxy group; an amino group; c ₁ -C ₄ An aminoalkyl group; c ₁ -C ₄ A carbonyloxy group; c ₁ -C ₄ An oxycarbonyl group; halogen (e.g., fluorine, chlorine, bromine, and iodine); a cyano group; a nitro group; an azide; c ₁ -C ₄ An alkylsulfonyl group; a thiol; c ₁ -C ₄ An alkylthio group; c ₆ -C ₃₀ Aryloxy groups such as phenoxy; -NR _b (C＝NR _b )NR _b R _c Wherein R is _b And R _c Is independently selected from: H. c ₁ -C ₄ Alkyl radical, C ₂ -C ₄ Alkenyl radical, C ₂ -C ₄ Alkynyl, C ₃ -C ₁₀ Cycloalkyl radical, C ₆ -C ₁₈ Aryl radical, C ₇ -C ₁₇ Aralkyl, heterocyclic group having three to ten members, or protective group of amino group.

Alternatively, R ₁ Selected from: H. acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl or phenylacetyl;

alternatively, R ₁ Is H, myristoyl or palmitoyl.

Alternatively, the compound is selected from (1) - (24):

(1)H-Tyr-Ser-OH；

(2)H-Tyr-Ser-NH ₂ ；

(3)Ac-Tyr-Ser-OH；

(4)Ac-Tyr-Ser-NH ₂ ；

(5)Palm-Tyr-Ser-OH；

(6)Palm-Tyr-Ser-NH ₂ ；

(7)Myr-Tyr-Ser-OH；

(8)Myr-Tyr-Ser-NH ₂ ；

(9)H-Tyr-Ser-Leu-OH；

(10)H-Tyr-Ser-Leu-NH ₂ ；

(11)Ac-Tyr-Ser-Leu-OH；

(12)Ac-Tyr-Ser-Leu-NH ₂ ；

(13)Palm-Tyr-Ser-Leu-OH；

(14)Palm-Tyr-Ser-Leu-NH ₂ ；

(15)Myr-Tyr-Ser-Leu-OH；

(16)Myr-Tyr-Ser-Leu-NH ₂ ；

(17)H-Tyr-Ser-Val-OH；

(18)H-Tyr-Ser-Val-NH ₂ ；

(19)Ac-Tyr-Ser-Val-OH；

(20)Ac-Tyr-Ser-Val-NH ₂ ；

(21)Palm-Tyr-Ser-Val-OH；

(22)Palm-Tyr-Ser-Val-NH ₂ ；

(23)Myr-Tyr-Ser-Val-OH；

(24)Myr-Tyr-Ser-Val-NH ₂ 。

alternatively, selected from the group consisting of compound (9), compound (13), compound (15), compound (17), compound (21), and compound (23); in particular, the amount of the solvent to be used,

(9)H-Tyr-Ser-Leu-OH；

(13)Palm-Tyr-Ser-Leu-OH；

(15)Myr-Tyr-Ser-Leu-OH；

(17)H-Tyr-Ser-Val-OH；

(21)Palm-Tyr-Ser-Val-OH；

(23)Myr-Tyr-Ser-Val-OH。

the peptidic compounds of the invention may exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids comprising them may have the L-, D-configuration, or be racemic independently of one another. It is thus possible to obtain isomeric mixtures, as well as racemic or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and what isomer or isomeric mixture is present. Preferred structures of these peptidic compounds of the invention are pure isomers, i.e., enantiomers or diastereomers.

For example, when-Ser-is mentioned in the present invention, it is understood that-Ser-is selected from-L-Ser-, -D-Ser-, or a mixture of both, is racemic or non-racemic. The preparation methods described in this document enable one of ordinary skill in the art to obtain each stereoisomer of the peptidic compounds of the invention by selecting amino acids with the correct configuration.

The invention also includes all suitable isotopic variations of the peptidic compounds of formula (I). Isotopic variations of the peptidic compounds of the invention are understood herein to mean compounds which: wherein at least one atom is replaced within the peptidic compounds of the invention by another atom of the same atomic number, but having an atomic mass different from the atomic mass usually or predominantly present in nature. Examples of isotopes that can be incorporated into the peptidic compounds of the invention are: of hydrogen, carbon, nitrogen or oxygen, e.g. ² H (deuterium) ³ H (tritium), ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁷ O or ¹⁸ And (O). Specific isotopic variations of the peptidic compounds of the invention (especially those into which one or more radioactive isotopes have been incorporated) may be advantageous, for example in order to examine the mechanism of action or the distribution of active compounds in vivo; due to the relatively simple manufacturability and detectability, in particular ³ H or ¹⁴ C-isotopically labelled compounds are suitable for this purpose. In addition, due to the greater metabolic stability of the compounds, the incorporation of isotopes (e.g., deuterium) can produce specific therapeutic benefits, such as increased in vivo half-life or decreased required active doses; thus, in certain cases, such modifications of the peptidic compounds of the invention may also constitute preferred embodiments of the invention. By methods known to the person skilled in the art, for example by the methods described further belowAnd the methods described in the examples, isotopic variations of the peptidic compounds of the invention can be prepared by using the respective reagents and/or the corresponding isotopic modifications of the starting materials.

In addition, the present invention also includes prodrugs of the peptidic compounds of the present invention. The term "prodrug" means herein a compound that: which may be biologically active or inactive per se, but which react (e.g. metabolize or hydrolyze) during their residence time in the body to form the compounds of the invention.

Optionally, the composition comprises 0.0001-5% of the compound shown in the general formula (I) or the salt thereof in percentage by mass;

optionally, the composition comprises the compound shown in the general formula (I) or the salt thereof with the mass percentage concentration of 0.0005-1%;

optionally, the composition comprises 0.001-0.1% by mass of a compound represented by the general formula (I) or a salt thereof;

optionally, the composition comprises 0.005-0.01 wt% of the compound shown in the general formula (I) or the salt thereof.

The compound represented by the general formula (I) can improve the activity of fibroblasts, promote the proliferation and migration of keratinocytes, promote the re-epithelization or healing of skin or mucosa, repair skin barriers, prevent or repair photoaging damage, promote the generation of collagen, increase the elasticity of skin or improve the firmness of skin.

Alternatively, the salt of the compound of formula (I) comprises a metal salt of a compound of formula (I), the metal comprising: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum;

alternatively, the salt of the compound of formula (I) comprises a salt of the compound of formula (I) with an organic base comprising: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine;

alternatively, the salt of the compound represented by the general formula (I) includes a salt of the compound represented by the general formula (I) with an inorganic acid or an organic acid, and the organic acid includes: acetic, citric, lactic, malonic, maleic, tartaric, fumaric, benzoic, aspartic, glutamic, succinic, oleic, trifluoroacetic, oxalic, pamoic or gluconic acids;

optionally, the inorganic acid comprises: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

The compounds of the general formula (I) or salts thereof according to the invention can be prepared by conventional methods known in the art, for example, by solid phase synthesis, liquid phase synthesis or a combination of solid phase and liquid phase methods, 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 for obtaining a compound of formula (I) 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 support bound C-terminus;

-elimination of the group protecting the N-terminus;

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

-elimination of the group protecting the C-terminus or cleavage from the solid support.

Preferably, the C-terminus is bound to a solid support and the method is carried out on a solid phase comprising coupling the amino acid having a protected N-terminus and a free C-terminus with the amino acid having a free N-terminus and a C-terminus bound to a polymeric support; elimination of the group protecting the N-terminus; and repeating this sequence as many times as necessary to thereby obtain a peptidic compound having a desired length, followed by cleavage of the synthesized peptidic compound from the original polymeric support.

The functional groups of the side chains of these amino acids remain sufficiently protected with temporary or permanent protecting groups throughout the synthesis and may be deprotected simultaneously or orthogonally to the process of cleaving the peptide from the polymeric support.

The method may comprise the further steps of: the functional groups at the N-terminus and C-terminus can be subsequently modified by deprotecting the N-terminus and C-terminus and/or cleaving the peptide from the polymeric support in a non-defined order using standard conditions and methods known in the art. The peptidic compounds of formula (I) bound to the polymeric support may be optionally modified at the N-and C-termini, or after the peptide has been cleaved from the polymeric support.

The compounds of formula (I) or salts thereof, as defined above, may be incorporated into cosmetically or pharmaceutically acceptable delivery systems or sustained release systems in order to achieve better penetration of the active ingredient and/or to improve its pharmacokinetic and pharmacodynamic properties, and may be applied to the skin and/or mucous membranes, or administered orally or parenterally as desired for the treatment and/or care of a condition, disorder and/or disease.

The frequency of administration or dosing can vary widely depending on the needs of each subject, with recommended administrations or dosing ranging from 1 to 10 times per month, preferably from 1 to 4 times per week, more preferably from 3 to 3 times per week, even more preferably 1 or 2 times per day.

The term "delivery system" refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered, selected from: water, oil or surfactant, including those of petroleum, animal, vegetable, or synthetic origin, such as, and not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitan esters, ether sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynol, poloxamers, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin, and the like. Diluents which may be used in the different delivery systems to which the compounds of the present invention may be administered are known to those of ordinary skill in the art.

The term "sustained release" is used in the conventional sense to refer to a delivery system of a compound that provides for gradual release of the compound over a period of time, and preferably, but not necessarily, has a relatively constant level of compound release over the entire period of time.

Examples of delivery systems or sustained release systems are liposomes, oleosomes, non-ionic surfactant liposome vesicles, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, liposomes, micelles, nanospheres, microspheres, nanospheres, lipid globules, microemulsions, nanoemulsions, nanoparticles, microparticles or nanoparticles. Preferred delivery systems or sustained release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions with an internal structure of reverse micelles.

Sustained release systems may be prepared by methods known in the art and may be administered, for example, by: by topical or transdermal administration, including adhesive patches, non-adhesive patches, occlusive patches, and microelectronic patches; or by systemic administration such as, but not limited to, oral or parenteral routes, including nasal, rectal, subcutaneous implantation or injection, or direct implantation or injection into a specific body site, and preferably should release relatively constant amounts of these compounds of the invention. The amount of compound included in the sustained release system will depend on, for example, the site to which the composition is to be administered, the kinetics and duration of release of the compounds of the invention, and the nature of the condition, disorder and/or disease to be treated and/or cared for.

The peptide derivative represented by the above general formula (I) or a salt thereof may be adsorbed on a solid organic polymer or a solid inorganic support, for example, but not limited to, talc, bentonite, silica, starch, maltodextrin, or the like.

Optionally, the composition is a cosmetic composition or a pharmaceutical composition.

The formulation of the composition is selected from: creams, oils, balms, foams, lotions, gels, liniments, serums, ointments, mousses, powders, sticks, pens, sprays, aerosols, capsules, tablets, granules, chewing gums, solutions, suspensions, emulsions, elixirs, polysaccharide films, jellies or gelatins.

Optionally, the composition further comprises at least one additional active agent for enhancing the skin's anti-aging repair effect described herein, said additional active agent being selected from one or more of peptides, natural plant ingredients, vitamin C and its derivatives, and retinoids.

The peptidic compounds of the invention have variable solubility in water, depending on the nature of their sequence or any possible modification in the N-and/or C-terminus. The peptidic compounds of the invention may thus be incorporated into the composition by aqueous solutions, and those that are insoluble in water may be dissolved in conventional cosmetically or pharmaceutically acceptable solvents such as, but not limited to, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol or polyethylene glycol or any combination thereof.

To facilitate an understanding of the invention, some terms and expressions used in the present invention are explained in the following:

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

The term "treating" refers to administering a peptidic compound according to the invention to alleviate or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with such a disease or condition. The term "treatment" also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or disorder.

The term "care" includes prevention of a disease and/or disorder.

The term "prevention" refers to the ability of a peptidic compound of the invention to prevent, delay, or retard the appearance or progression of a disease or condition before it occurs.

The term "repair" refers to the ability of a peptidic compound of the invention to improve, alleviate or restore its original state after the onset of a disease or condition.

The term "photoaging" refers to the premature aging of skin due to prolonged exposure of the skin to ultraviolet radiation, which exhibits the same physiological characteristics as natural aging, such as, and not limited to: laxity, sagging, color change or pigmentation irregularities, abnormalities and/or hyperkeratinization.

The amino acid abbreviations used in the present invention follow the rules specified by the IUPAC-IUB Commission on Biochemical nomenclature in the European journal of biochemistry (Eur.J.biochem.1984, 138: 9-37).

Thus, for example, val represents NH ₂ -CH(CH(CH ₃ ) ₂ ) -COOH, val-represents NH ₂ -CH(CH(CH ₃ ) ₂ ) -CO-, -Val represents-NH-CH (CH) ₃ ) ₂ ) -COOH, and-Val-represents-NH-CH (CH) ₃ ) ₂ ) -CO-. Thus, the hyphen representing the peptide bond eliminates the OH in the 1-carboxyl group of the amino acid (represented herein in the conventional non-ionized form) when located to the right of the symbol, and eliminates the H in the 2-amino group of the amino acid when located to the left of the symbol; both modifications can be applied to the same symbol (see table 1).

TABLE 1 structures of amino acid residues and their single-letter and three-letter abbreviations

The abbreviation "Ac-" is used herein to denote acetyl (CH) ₃ -CO-), the abbreviation "Palm-" is used herein to denote palmitoyl, and the abbreviation "Myr-" is used herein to denote myristoyl.

Compared with the prior art, the invention has the advantages that:

1. the compound is obtained by artificial design, is convenient to synthesize, is safe and non-irritant to human bodies, and can be applied to the field of cosmetics.

2. The compound can improve the activity of fibroblasts, promote the proliferation and migration of keratinocytes, promote the re-epithelization or healing of skin or mucosa, repair skin barriers, prevent or repair photoaging damage, promote the generation of collagen, increase the skin elasticity or improve the skin firmness, thereby having good skin aging repair effect and being applied to products for repairing skin aging.

Drawings

FIG. 1 shows that the compound (9) H-Tyr-Ser-Leu-OH (formula C) ₁₈ H ₂₇ N ₃ O ₆ ) Mass spectrum, [ M + H] ⁺ The mass-to-charge ratio (m/z) of the excimer ion peak was 382.2392, and the molecular weight was 381.24 as measured by mass spectrometry.

FIG. 2 shows that the compound (13) Palm-Tyr-Ser-Leu-OH (formula C) ₃₄ H ₅₇ N ₃ O ₇ ) Mass spectrum, [ M + Na ]] ⁺ The mass-to-charge ratio (m/z) of the added ion peak was 642.4174, and the molecular weight was 619.42 as measured by mass spectrometry.

FIG. 3 shows the compound (15) Myr-Tyr-Ser-Leu-OH (formula C) ₃₂ H ₅₃ N ₃ O ₇ ) Mass spectrum, [ M + Na ]] ⁺ The mass to charge ratio (m/z) of the added ion peak was 614.3734, and the molecular weight was 591.37 as measured by mass spectrometry.

FIG. 4 shows that compound (17) H-Tyr-Ser-Val-OH (formula C) ₁₇ H ₂₅ N ₃ O ₆ ) Mass spectrum, [ M + H] ⁺ The mass/charge ratio (m/z) of the excimer ion peak is 368.2215, and the molecular weight is 367.22 through mass spectrometry.

FIG. 5 is compound (23) Myr-Tyr-Ser-Val-OH (formula C) ₃₁ H ₅₁ N ₃ O ₇ ) Mass spectrum, [ M + Na ]] ⁺ The mass-to-charge ratio (m/z) of the added ion peak was 600.3671, and the molecular weight was 577.37 as measured by mass spectrometry.

FIG. 6 is a graph showing the effect of test samples on the proliferative activity of NIH3T3 cells.

FIG. 7 is a graph of the effect of test samples on the proliferative activity of HaCaT cells.

FIG. 8 is a graph of the effect of test samples on HaCaT cell adhesion.

FIG. 9 is a graph showing the effect of the test samples on the repair of photodamaged NIH3T3 cells.

FIG. 10 is a graph showing the results of a HaCaT cell scratch experiment observed under a 100-fold microscope.

FIG. 11 is a graph showing the effect of the test samples on collagen content.

FIG. 12 is a graph showing the results of HSF cell collagen fiber experiments observed under a 100-fold microscope.

Detailed Description

For a better understanding of the present invention, the following detailed description of the invention is given in conjunction with examples and figures, however, it should be understood that these examples and figures are for illustrative purposes only and are not intended to limit the scope of the present invention.

Abbreviations

Abbreviations for amino acids follow the committee on biochemical nomenclature of IUPAC-IUB in Eur j. Biochem. (1984) 138: chem (1989) 264: rules specified in 633-673.

Wang Resin: resin queen; DMF: n, N-dimethylformamide; DCM: dichloromethane; DIC: diisopropylcarbodiimide; ac of ₂ O: acetic anhydride; DIPEA: diisopropylethylamine; fmoc: 9-fluorenylmethoxycarbonyl; piperidine: piperidine; HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid; and (3) TIS: triisopropylsilane; palm-OH: palmitic acid; myr-OH: myristic acid; ac-: acetyl; palm-: a palmitoyl group; myr-: a myristoyl group; tyr: tyrosine; ser: serine; leu: leucine; val: valine; tBu: a tertiary butyl group.

Example 1 preparation of H-Tyr-Ser-Leu-OH

1.1 swelling of the resin

5g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, the resin was washed and the solvent was removed.

1.2 Charge reaction

6.1g of Fmoc-Leu-OH and 2.322g of HOBt are weighed and added into a drying triangular flask, DMF is added to dissolve the mixture, the mixture is sealed and placed in a refrigerator at the temperature of 18 ℃ below zero for 30min, and 2.7mL of DIC is added to activate the mixture for 3min, so that water vapor is avoided. Adding activated amino acid into the swelled resin, reacting for 3.5h, and pumpingMoving the reaction solution, and continuously adding Ac ₂ And end-capping with DIPEA for 1.5h. The resin was washed and the solvent was removed. The K detection resin is colorless and transparent, which indicates that the reaction is complete.

The N-terminal Fmoc group was deprotected and 2.6g of activated Fmoc-Ser (tBu) -OH was coupled to the peptidyl resin in the presence of 0.837g HOBt and 0.781mL DIC using DMF as solvent and the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Coupling 3.1g of Fmoc-Tyr (tBu) -OH after activation onto the peptidyl resin using DMF as solvent in the presence of 0.837g HOBt and 0.781mL DIC, and continuing the reaction for 2h; after the reaction was complete, the resin was washed and the solvent was pumped away.

Deprotection of the N-terminal Fmoc group of the peptidyl resin, fmoc removal twice with 20% piperidine/DMF for 10min each, sampling for K detection, development of dark blue. The Resin was washed 6 times with DMF, the solvent was removed, and the Resin was contracted and dried to give Tyr (tBu) -Ser (tBu) -Leu-Wang Resin.

1.3 cleavage

Weighing 14.25mL of TFA, 0.375mL of TIS and 0.375mL of water, mixing and stirring uniformly to obtain lysate, sealing and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether was frozen in a freezer at-18 ℃ for further use.

5g of Tyr (tBu) -Ser (tBu) -Leu-Wang Resin was weighed into a round-bottomed flask, and the frozen lysate was added thereto and reacted for 2.5 hours with stirring. And (3) carrying out suction filtration, collecting filtrate, concentrating to 1/4 of the volume, adding isopropyl ether, stirring, centrifuging, washing for 6 times until the pH value is 3-4, and carrying out vacuum drying to obtain 3.3g of H-Tyr-Ser-Leu-OH crude peptide.

1.4 purification

Weighing 3.3g of crude peptide, dissolving in 50mL of pure water, filtering with a 0.22 μm pore size microfiltration membrane to obtain a clear and transparent solution, pH 5, and purifying by reversed phase HPLC with the purification gradient as follows:

time (min)	Flow rate (mL/min)	A% (acetonitrile)	B% (0.1% acetic acid + pure water)
				0	40	20	80
5	40	20	80
				30	40	40	60
35	40	80	20
				45	40	80	20

And (3) sampling and purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain a compound (9) H-Tyr-Ser-Leu-OH with the purity of 98.35%.

Example 2 preparation of Palm-Tyr-Ser-Leu-OH

2.1 swelling of the resin

9g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed and the solvent was removed.

2.2 Charge reaction

Weighing 10.8g of Fmoc-Leu-OH and 4.95g of HOBt, adding the mixture into a drying triangular flask, adding DMF to dissolve the mixture, sealing and placing the flask in a refrigerator at the temperature of-18 ℃ for 30min, adding 6.24mL of DIC to activate the mixture for 3min, and avoiding water vapor. Adding the activated amino acid into the swelled resin to react for 3.5h, pumping out the reaction solution, and continuously adding Ac ₂ And end-capping with DIPEA for 1.5h. The resin was washed and the solvent was removed. The K detection resin is colorless and transparent, which indicates that the reaction is complete.

The N-terminal Fmoc group was deprotected and 5.3g of Fmoc-Ser (tBu) -OH after activation was coupled to the peptidyl resin in the presence of 2.24g of HOBt and 2.8mL of DIC using DMF as solvent and the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. 6.3g of Fmoc-Tyr (tBu) -OH were coupled in the presence of 2.24g of HOBt and 2.8mL of DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was pumped away.

Deprotection of the N-terminal Fmoc group of the peptidyl resin, fmoc removal twice with 20% piperidine/DMF for 10min each, sampling for K detection, development of dark blue. The resin was washed 6 times with DMF and the solvent was removed by suction.

In the presence of DIPEA, 5.6g of Palm-OH is coupled to the peptidyl Resin by using DMF as a solvent, the reaction is continued for 1.5h, the Resin is washed, the solvent is pumped out, and the Palm-Tyr (tBu) -Ser (tBu) -Leu-Wang Resin is obtained after contraction and drying.

2.3 cleavage

Weighing 70mL of TFA, 1.8mL of TIS and 1.8mL of water, mixing and stirring uniformly to obtain a lysate, sealing, and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether was frozen in a freezer at-18 ℃ for further use.

13.9g of Palm-Tyr (tBu) -Ser (tBu) -Leu-Wang Resin was weighed into a round-bottomed flask, and the frozen lysate was added and reacted for 2.5 hours with stirring. And (4) carrying out suction filtration, collecting filtrate, concentrating to 1/4 of the volume, adding isopropyl ether, stirring, centrifuging, washing for 6 times until the pH value is 3-4, and carrying out vacuum drying to obtain 4.5g of Palm-Tyr-Ser-Leu-OH crude peptide.

2.4 purification

Weighing 4.5g of crude peptide, dissolving in 30mL of acetic acid solution, adding 60mL of pure water to dissolve completely, filtering with a microporous membrane with a pore size of 0.22 μm to obtain a clear and transparent solution, and purifying by reversed-phase HPLC, wherein the purification gradient is as follows:

time (min)	Flow rate (mL/min)	A% (acetonitrile)	B% (0.1% acetic acid + pure water)
				0	15	0	100
10	15	0	100
				15	15	60	40
60	15	90	10
				78	15	100	0
80	15	100	0

And (3) sampling and purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain a compound (13) Palm-Tyr-Ser-Leu-OH with the purity of 95.37%.

Example 3 preparation of Myr-Tyr-Ser-Leu-OH

3.1 swelling of the resin

Weighing 8g of Wang resin in a solid phase synthesis reaction column, swelling the Wang resin with DMF, washing the resin, and pumping away the solvent.

3.2 Charge reaction

Weighing 8g of Fmoc-Leu-OH and 3.82g of HOBt, adding the mixture into a drying triangular flask, adding DMF to dissolve the mixture, sealing and placing the flask in a refrigerator at the temperature of 18 ℃ below zero for 30min, adding 4.8mL of DIC to activate the mixture for 3min, and avoiding water vapor. Adding the activated amino acid into the swelled resin to react for 3.5h, pumping out the reaction solution, and continuously adding Ac ₂ And end-capping with DIPEA for 1.5h. The resin was washed and the solvent was removed. The K detection resin is colorless and transparent, which indicates that the reaction is complete.

The N-terminal Fmoc group was deprotected and 4.9g of Fmoc-Ser (tBu) -OH after activation was coupled to the peptidyl resin in the presence of 2.07g of HOBt and 2.6mL DIC using DMF as solvent for 2h reaction. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. 5.9g of Fmoc-Tyr (tBu) -OH were coupled in the presence of 2.07g of HOBt and 2.6mL of DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was pumped away.

Deprotection of the N-terminal Fmoc group of the peptidyl resin, fmoc removal twice with 20% piperidine/DMF for 10min each, sampling for K detection, development of dark blue. The resin was washed 6 times with DMF and the solvent was removed by suction.

Coupling 4.7g of Myr-OH to peptidyl Resin in the presence of DIPEA using DMF as solvent, reacting for 1.5h, washing the Resin, pumping off the solvent, shrinking and drying to obtain Myr-Tyr (tBu) -Ser (tBu) -Leu-Wang Resin.

3.3 cleavage

Measuring 61.6mL of TFA, 1.6mL of TIS and 1.6mL of water, mixing and stirring uniformly to obtain a lysate, sealing and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether was frozen in a freezer at-18 ℃ for further use.

10.8g of Myr-Tyr (tBu) -Ser (tBu) -Leu-Wang Resin was weighed into a round-bottomed flask, and the above-mentioned frozen lysate was added and reacted for 2.5 hours with stirring. And (3) carrying out suction filtration, collecting filtrate, concentrating to 1/4 of the volume, adding isopropyl ether, stirring, centrifuging, washing for 6 times until the pH value is 3-4, and carrying out vacuum drying to obtain 1.5g of crude Myr-Tyr-Ser-Leu-OH peptide.

3.4 purification

1.5g of crude peptide was weighed and dissolved in acetic acid: to methanol (V: V = 1) was added 15mL of pure water to dissolve it completely, and the solution was filtered through a 0.22 μm pore size microporous membrane to obtain a clear and transparent solution, which was purified by reverse phase HPLC with the following purification gradient:

and (3) sampling and purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain a compound (15) Myr-Tyr-Ser-Leu-OH with the purity of 96.45%.

Example 4 preparation of H-Tyr-Ser-Val-OH

4.1 swelling of the resin

5g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, the resin was washed and the solvent was removed.

4.2 Charge reaction

5.9g of Fmoc-Val-OH and 2.322g of HOBt are weighed and added into a drying triangular flask, DMF is added to dissolve the Fmoc-Val-OH, the flask is sealed and placed in a refrigerator at the temperature of 18 ℃ below zero for 30min, and 2.7mL of DIC is added to activate the flask for 3min, so that water vapor is avoided. Adding the activated amino acid into the swelled resin to react for 3.5h, pumping out the reaction solution, and continuously adding Ac ₂ And end-capping with DIPEA for 1.5h. The resin was washed and the solvent was removed. The K detection resin is colorless and transparent, which indicates that the reaction is complete.

The N-terminal Fmoc group was deprotected and 2.9g of activated Fmoc-Ser (tBu) -OH was coupled onto the peptidyl resin in the presence of 0.837g HOBt and 0.781mL DIC using DMF as solvent and reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Coupling activated 3.4g of Fmoc-Tyr (tBu) -OH to the peptidyl resin in the presence of 0.837g of HOBt and 0.781mL of DIC using DMF as solvent, and reacting for 2h; after the reaction was complete, the resin was washed and the solvent was removed.

Deprotection of the N-terminal Fmoc group of the peptidyl resin, fmoc removal twice with 20% piperidine/DMF for 10min each time, sampling for K-test, development of dark blue. The Resin was washed 6 times with DMF, the solvent was removed, and 3.1g of Tyr (tBu) -Ser (tBu) -Val-Wang Resin was obtained after contraction and drying.

4.3 cleavage

Measuring 23.75mL of TFA, 0.625mL of TIS and 0.625mL of water, mixing and stirring uniformly to obtain a lysate, sealing and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether was frozen in a freezer at-18 ℃ for further use.

3.1g of Tyr (tBu) -Ser (tBu) -Val-Wang Resin is weighed and added into a round-bottom flask, the frozen lysate is added, and the reaction is stirred for 2.5h. And (3) carrying out suction filtration, collecting filtrate, concentrating to 1/4 of the volume, adding isopropyl ether, stirring, centrifuging, washing for 6 times until the pH value is 3-4, and carrying out vacuum drying to obtain 3.3g of crude H-Tyr-Ser-Val-OH peptide.

4.4 purification

Weighing 3.1g of crude peptide, dissolving in 50mL of pure water, filtering with a 0.22 μm pore size microfiltration membrane to obtain a clear and transparent solution, pH 5, and purifying by reversed phase HPLC with the purification gradient as follows:

time (min)	Flow rate (mL/min)	A% (acetonitrile)	B% (0.1% acetic acid + pure water)
				0	40	15	85
5	40	15	85
				30	40	35	65
31	40	70	30
				40	40	70	30

And (3) sampling and purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain a compound (17) H-Tyr-Ser-Val-OH with the purity of 98.37%.

Example 5 preparation of Palm-Tyr-Ser-Val-OH

5.1 swelling of the resin

8g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed and the solvent was removed.

5.2 Charge reaction

Weighing 8g of Fmoc-Val-OH and 3.82g of HOBt, adding the Fmoc-Val-OH and the HOBt into a drying triangular flask, adding DMF to dissolve the DMF, sealing and placing in a refrigerator at the temperature of 18 ℃ below zero for 30min, adding 4.8mL of DIC to activate for 3min, and avoiding water vapor. Adding the activated amino acid into the swelled resin to react for 3.5h, pumping out the reaction solution, and continuously adding Ac ₂ And end-capping with DIPEA for 1.5h. The resin was washed and the solvent was removed. The K detection resin is colorless and transparent, which indicates that the reaction is complete.

The N-terminal Fmoc group was deprotected and 4.9g of activated Fmoc-Ser (tBu) -OH was coupled to the peptidyl resin in the presence of 2.07g of HOBt and 2.6mL of DIC using DMF as solvent and the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. 5.9g of Fmoc-Tyr (tBu) -OH was coupled in the presence of 2.07g of HOBt and 2.6mL of DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was pumped away.

Deprotection of the N-terminal Fmoc group of the peptidyl resin, fmoc removal twice with 20% piperidine/DMF for 10min each, sampling for K detection, development of dark blue. The resin was washed 6 times with DMF and the solvent was removed by suction.

4.7g of Palm-OH was coupled to the peptidyl Resin using DMF as solvent in the presence of DIPEA, the reaction was continued for 1.5h, the Resin was washed, the solvent was pumped off, 10.8g of Palm-Tyr (tBu) -Ser (tBu) -Val-Wang Resin was obtained after drying by shrinkage.

5.3 cleavage

Measuring 61.6mL of TFA, 1.6mL of TIS and 1.6mL of water, mixing and stirring uniformly to obtain a lysate, sealing and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether was frozen in a freezer at-18 ℃ for further use.

10.8g of Palm-Tyr (tBu) -Ser (tBu) -Val-Wang Resin was weighed into a round-bottomed flask, and the above-mentioned frozen lysate was added and reacted for 2.5 hours with stirring. And (3) carrying out suction filtration, collecting filtrate, concentrating the filtrate to 1/4 of the volume, adding isopropyl ether, stirring, centrifuging, washing for 6 times until the pH value is 3-4, and carrying out vacuum drying to obtain 1.8g of Palm-Tyr-Ser-Val-OH crude peptide.

5.4 purification

1.8g of crude peptide was weighed out and dissolved in 115mL of acetic acid: methanol: to a solution of water (V: V =2 = 4) to be completely dissolved, filtered through a microporous membrane with a pore size of 0.22 μm to obtain a clear and transparent solution, and purified by reverse phase HPLC with the following purification gradient:

time (min)	Flow rate (mL/min)	A% (acetonitrile)	B% (0.1% acetic acid + pure water)
				0	15	0	100
10	15	0	100
				15	15	30	70
50	15	60	40
				55	15	100	0
60	15	100	0

And (3) sampling and purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain a compound (21) Palm-Tyr-Ser-Val-OH with the purity of 95.52%.

Example 6 preparation of Myr-Tyr-Ser-Val-OH

6.1 swelling of the resin

8g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed and the solvent was removed.

6.2 Charge reaction

Weighing 8g of Fmoc-Val-OH and 3.82g of HOBt, adding the Fmoc-Val-OH and the HOBt into a drying triangular flask, adding DMF to dissolve the mixture, sealing and placing the mixture in a refrigerator at the temperature of-18 ℃ for 30min, adding 4.8mL of DIC to activate the mixture for 3min, and avoiding water vapor. Adding the activated amino acid into the swelled resin to react for 3.5h, pumping out the reaction solution, and continuously adding Ac ₂ And end-capping with DIPEA for 1.5h. The resin was washed and the solvent was removed. The K detection resin is colorless and transparent, which indicates that the reaction is complete.

The N-terminal Fmoc group was deprotected and 4.9g of Fmoc-Ser (tBu) -OH after activation was coupled to the peptidyl resin in the presence of 2.07g of HOBt and 2.6mL DIC using DMF as solvent for 2h reaction. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. 5.9g of Fmoc-Tyr (tBu) -OH were coupled in the presence of 2.07g of HOBt and 2.6mL of DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was pumped away.

Deprotection of the N-terminal Fmoc group of the peptidyl resin, fmoc removal twice with 20% piperidine/DMF for 10min each, sampling for K detection, development of dark blue. The resin was washed 6 times with DMF and the solvent was removed by suction.

Coupling 4.7g of Myr-OH to peptidyl Resin using DMF as solvent in the presence of DIPEA, continuing the reaction for 1.5h, washing the Resin, drawing off the solvent, shrinking and drying to obtain 10.8g of Myr-Tyr (tBu) -Ser (tBu) -Val-Wang Resin.

6.3 cleavage

Measuring 61.6mL of TFA, 1.6mL of TIS and 1.6mL 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-18 ℃ for freezing for standby.

10.8g of Myr-Tyr (tBu) -Ser (tBu) -Leu-Wang Resin was weighed into a round-bottomed flask, and the above-mentioned frozen lysate was added and reacted for 2.5 hours with stirring. And (3) carrying out suction filtration, collecting filtrate, concentrating to 1/4 of the volume, adding isopropyl ether, stirring, centrifuging, washing for 6 times until the pH value is 3-4, and carrying out vacuum drying to obtain 1.5g of crude Myr-Tyr-Ser-Leu-OH peptide.

6.4 purification

1.5g of crude peptide was weighed and dissolved in acetic acid: methanol: in water (V: V = 2) to completely dissolve it, filtration through a 0.22 μm pore size microfiltration membrane gives a clear and transparent solution, which is purified by reverse phase HPLC with the following purification gradient:

time (min)	Flow rate (mL/min)	A% (acetonitrile)	B% (0.1% acetic acid + pure water)
				0	15	0	100
10	15	0	100
				15	15	30	70
50	15	60	40
				55	15	100	0
60	15	100	0

And (3) sampling and purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain a compound (23) Myr-Tyr-Ser-Val-OH with the purity of 96%.

Example 7

The other compounds of the general formula (I) according to the invention can be prepared in a similar manner.

The molecular weights of the obtained compounds were determined by ESI-MS, and the results of testing some of the compounds are shown in Table 2 below and FIGS. 1 to 5.

TABLE 2 determination of molecular weight by Mass Spectrometry

Numbering	Sequence of	Molecular weight Mass spectrometry results
			(9)	H-Tyr-Ser-Leu-OH	381.24
(13)	Palm-Tyr-Ser-Leu-OH	619.42
			(15)	Myr-Tyr-Ser-Leu-OH	591.37
(17)	H-Tyr-Ser-Val-OH	367.22
			(23)	Myr-Tyr-Ser-Val-OH	577.37

Example 8 cell proliferation assay

8.1 reagents and materials

Thiazole blue (MTT), dimethyl sulfoxide (DMSO), a high-sugar medium (DMEM), fetal bovine serum, and PBS.

8.2 instruments

Microplate reader, CO ₂ Incubator, superclean bench.

8.3 cell lines

Mouse skin fibroblasts (NIH 3T 3) were purchased from the shanghai cell bank of the typical culture collection committee of the chinese academy of sciences, and human keratinocytes (HaCaT) were purchased from the kunming cell bank of the typical culture collection committee of the chinese academy of sciences.

8.4 samples to be tested

Administration group: the concentrations of the compound (9), the compound (13), the compound (15), the compound (17), the compound (21) and the compound (23) in the samples were 12.5ppm, 25ppm, 50ppm and 100ppm, respectively.

Blank control group: PBS.

8.5 Experimental methods

Taking NIH3T3 cells and HaCaT keratinocytes in an exponential growth phase, adding 0.25% trypsin digestion solution, digesting to make adherent cells fall off, counting 1-4 multiplied by 10 ⁵ And (4) preparing cell suspension per mL.

Inoculating the cell suspension on a 96-well plate at a concentration of 200 μ L/well, and placing in constant temperature CO ₂ Culturing in an incubator for 24h.

Changing the solution, adding the samples of the administration group and the control group at 20 μ L/well, placing at 37 deg.C and 5% CO ₂ Incubate in incubator for 72h.

Then 20. Mu.L of 5mg/mL MTT per well was added, continuing at 37 ℃ with 5% CO ₂ Incubate in incubator for 4h. The stock solution was discarded, 150. Mu.L/well of DMSO was added, the mixture was shaken on a shaker for 5min, and then the OD value of each well was measured at a wavelength of 570nm using a microplate reader, and the cell viability was calculated.

Cell viability = (administration well OD-zero well OD)/(control well OD-zero well OD) × 100%

8.6 results

The MTT method is a method for detecting cell survival and growth, and the measured OD value is proportional to the cell activity.

The results of the effect of the test samples on the proliferation activity of the NIH3T3 cells are shown in FIG. 6, and the results show that compared with a blank control group, the administration group has no toxic effect on the NIH3T3 cells within the range of 100ppm, can also improve the cell activity of the administration group, and shows different effects of promoting the proliferation of the NIH3T3 fibroblasts at different concentrations; wherein, the compound (17) and the compound (21) can obviously improve the activity of the NIH3T3 fibroblast within the low concentration range of 12.5-50ppm, the compound (9) can improve the activity of the NIH3T3 fibroblast more preferably when the compound (9) is 100ppm, the compound (17) can improve the activity of the NIH3T3 fibroblast to the best degree when the compound (17) is 25ppm, and the proliferation of the NIH3T3 fibroblast is obviously promoted.

The effect results of the test samples on the proliferation activity of the HaCaT cells are shown in FIG. 7, and the results show that compared with a blank control group, the administration group has no toxic effect on the HaCaT cells within the range of 100ppm, can also improve the cell activity of the HaCaT cells, and shows different effects of promoting the proliferation of the HaCaT keratinocytes at different concentrations; wherein, the compound (9), the compound (17), the compound (21) and the compound (23) have better activity of improving HaCaT keratinocytes and obviously promote the proliferation of the HaCaT keratinocytes; furthermore, compound (17) and compound (21) at low concentrations of 12.5ppm were able to significantly promote HaCaT keratinocyte proliferation.

In conclusion, the compound provided by the invention can improve the activity of NIH3T3 fibroblasts and HaCaT keratinocytes and promote the proliferation of the cells. Wherein, the compound (9) and the compound (17) can obviously improve the activity of fibroblasts and promote the proliferation of keratinocytes; the new-structure compound (13), the compound (15), the compound (21) and the compound (23) obtained by respectively interconnecting palmitic acid and myristic acid with the N-terminal of the compound (9) and the compound (17) through covalent bonds can also obviously improve the cell activity under different concentrations, and the new-structure compound (21) obtained by interconnecting palmitic acid with the N-terminal of the compound (17) through covalent bonds can improve the cell activity and promote the cell proliferation under low concentration.

Therefore, the peptide compound has no toxic effect on fibroblasts and keratinocytes, and can promote cell proliferation, improve the activity of the fibroblasts and promote the proliferation of the keratinocytes, thereby indicating that the peptide compound has the skin repairing effect.

Example 9 cell adhesion experiments

9.1 reagents and materials

Fetal bovine serum, DMEM medium, penicillin, streptomycin and MTT.

9.2 instruments

Microplate reader, CO ₂ Incubator, superclean bench.

9.3 cell lines

Human keratinocytes (HaCaT) were purchased from kunming cell banks, typical culture collection committee of chinese academy of sciences.

9.4 samples to be tested

Administration group: the compound (9), the compound (13), the compound (15), the compound (17), the compound (21) and the compound (23) were dissolved in PBS, and the concentrations were 12.5ppm, 25ppm, 50ppm and 100ppm, respectively;

blank control group: PBS.

9.5 Experimental methods

Culturing frozen HaCaT human stratum corneum cells according to the ratio of 1: passage 2 to about 5, and selecting the cells with better growth as experimental objects.

The sample to be tested was added to a 96-well plate at 20. Mu.L/well and oven-dried overnight at 37 ℃. After digesting the better growing HaCaT cells the following day, plating at a density of 1 ten thousand HaCaT cells/well and supplementing the medium to 200. Mu.L, 5% CO at 37 ℃ ₂ Incubate in incubator for 3h. After the culture is finished, the culture plate is taken out, the culture medium is continuously supplemented until the liquid level just overflows, and the culture plate is sealed by a sealing film to ensure that no air bubbles exist. And turning clockwise for 20min. Discard original medium, add 90. Mu.L of fresh medium and 10. Mu.L of 5mg/mL MTT per well, and place at 37 ℃ in 5% CO ₂ Incubate in incubator for 3h. The solution was discarded and 150. Mu.L of DMSO was added. Reference OD values at 490nm and 630nm wavelengths were read using a microplate reader.

9.6 results of the experiment

HaCaT keratinocytes are selected, plated in a 96-well-coated drug-coated well-perforated plate, incubated, and after three-dimensional force action, cells with strong adhesion can be kept on the 96-well-perforated plate, MTT quantitative analysis is carried out on living cells on the plate, so that the adhesion effect of the cells can be reflected, and the influence of the drug on cell adhesion is evaluated, thereby determining whether the compound can improve the cell elasticity. The more viable cells that remain in the 96-well plate, the greater the measured OD value, indicating greater cell adhesion.

The results of the test samples on the adhesion influence on the HaCaT cells are shown in figure 8, the administration groups can improve the adhesion capacity of the HaCaT keratinocytes to different degrees at different concentrations, and compared with the blank control group, the compound (9), the compound (15), the compound (17) and the compound (23) can obviously improve the adhesion capacity of the HaCaT keratinocytes, so that the cell elasticity is improved. Wherein, the compound (15) can also obviously improve the cell adhesion capability under the low concentration of 25ppm, and the overall technical effect of the compound (9) and the compound (17) is better.

Therefore, the peptide compound can improve the cell adhesion capability, remarkably increase the cell adhesion and the cell-extracellular matrix adhesion, further increase the skin elasticity and improve the skin firmness, and can be used for preventing and even treating the skin relaxation.

Example 10 light Damage repair experiment

10.1 reagents and materials

Fetal bovine serum, high-sugar medium (DMEM), penicillin, streptomycin and MTT.

10.2 apparatus

Enzyme reader, CO ₂ Incubator, superclean bench.

10.3 cell lines

Mouse skin fibroblasts (NIH 3T 3) were purchased from Shanghai cell bank of the Committee for type culture Collection of Chinese academy of sciences.

10.4 samples to be tested

Administration group: the concentrations of compound (9), compound (13), compound (15), compound (17), compound (21) and compound (23) were 12.5ppm, 25ppm, 50ppm and 100ppm, respectively.

Control group: PBS.

UV group: PBS + UV.

10.5 Experimental methods

Taking NIH3T3 fibroblasts in an exponential growth phase with good state, adding 0.25% trypsin digestion solution, digesting to make adherent cells fall off, counting 1-4 multiplied by 10 ⁵ And (4) preparing cell suspension per mL.

10000 cells/well of cell suspension are taken out and inoculated on a 96-well plate after being properly diluted, and when the cells grow to be about 80 percent, a UV light aging model is established. Control group was supplemented with 50. Mu.L PBS to 200. Mu.L without UV irradiation; adding appropriate amount of PBS to wash repeatedly to colorless in UV group and administration group, adding 50 μ L PBS at 80mJ/cm ² Irradiating under UV lamp at a distance of 15cm between the lamp source and the culture flask, discarding PBS, adding PBS solution and culture medium to 200 μ L in UV group, and adding culture solution and diluted drug to 200 μ L in administration group. Control group, UV group, and administration group were continued at 37 deg.C and 5% CO ₂ Incubate in incubator for 24h.

Then 20. Mu.L of 5mg/mL MTT was added to each well, and the reaction was continued at 37 ℃ with 5% CO ₂ Incubate in incubator for 4h. The stock solution was discarded, 150. Mu.L/well of DMSO was added, the mixture was shaken on a shaker for 5min, and then the OD value of each well was measured at a wavelength of 570nm using a microplate reader, and the cell viability was calculated.

Cell viability = (administration well OD-zero well OD)/(control well OD-zero well OD) × 100%

10.6 results of the experiment

Skin aging is affected by endogenous and exogenous factors, such as genetics, environmental exposure, ultraviolet radiation, hormonal changes, and metabolism. The accumulation of these factors leads to changes in the structure, function and appearance of the skin, particularly the exposure to ultraviolet radiation. 80mJ/cm is selected in the experiment ² The UV energy is radiated to establish a skin photoaging model.

The experimental results are shown in fig. 9, compared with the blank control group, the activity of the fibroblasts is significantly reduced after UV irradiation, which indicates that the photoaging model is successfully established; the administration group can improve the activity of photodamaged NIH3T3 cells within the range of 100ppm to different degrees, thereby improving the cell aging and generating obvious anti-photoaging effect; compared with the UV group, the compound (9), the compound (13), the compound (17) and the compound (21) can obviously improve the cell activity within the range of 12.5-100ppm, the compound (13) can obviously improve the cell activity at a low concentration of 12.5ppm, and the compound (15) and the compound (23) can also obviously improve the cell activity at 50ppm. In general, the compound (9), the compound (13), the compound (15), the compound (17), the compound (21) and the compound (23) of the present invention all produced significant anti-photoaging effects, were able to repair photoaging damaged cells, and had good skin repair ability.

Therefore, the peptide compound can be used for preventing or repairing photoaging damage and has a skin repairing effect.

Example 11 cell scratch test

11.1 reagents and materials

Fetal bovine serum, DMEM medium, penicillin and streptomycin.

11.2 instruments

Optical microscope, CO ₂ An incubator.

11.3 cell lines

Human keratinocytes (HaCaT) were purchased from kunming cell banks, national academy of sciences type culture collection committee.

11.4 samples to be tested

Administration group: the concentrations of compound (9), compound (13), compound (15), compound (17), compound (21) and compound (23) to be tested were all 50ppm.

Blank control group: PBS.

Positive control group: 50U/mL EGF.

11.5 Experimental methods

Culturing frozen HaCaT human stratum corneum cells according to the ratio of 1: passage 2 to about 5 passages, and selecting cells with better growth as experimental objects. Inoculating the cells into a 12-hole plate at the density of 20 ten thousand per hole, wherein each hole is 2mL of cell suspension, and after the cells grow fully adherent to the wall, changing a culture medium containing 0.5-1% fetal calf serum to maintain the cells for 24h to synchronize the cells. Pipetting with sterilisationScratching the gun head, washing off dropped cells by PBS, adding a sample to be detected, and continuing to perform reaction at 37 ℃ and 5% CO ₂ Incubate in incubator for 24h, then place under light microscope to analyze wound healing area.

11.6 results of the experiment

A scratch test is carried out on the basis that HaCaT cells grow over a culture dish, the migration condition of the cells is observed under an optical microscope at 24h after administration, and the influence of the drug on the proliferation and migration of the cells is evaluated, so that whether the compound can promote the re-epithelialization or healing of skin or mucosa is determined.

The scratch migration experiment of the cells can reflect the proliferation and repair capacity of the cells, the experimental result is shown in figure 10, the scratch of the cells of the blank control group is still obvious, and the scratch distance is normal; compared with a blank control group, the EGF positive control group has obvious cell proliferation and migration, basically disappears the space and is connected into a whole; the scratch intervals of the compound (9), the compound (17) and the compound (21) groups are all shortened compared with the scratch intervals of cells of a blank control group, and a cell migration track appears; wherein, the scratch spacing of the compound (9) and the compound (17) groups is equivalent to the scratch spacing and the cell proliferation and migration effects of the EGF positive control group, which shows that the repair capacity of the compound (9) and the compound (17) on cells is equivalent to the effect of EGF.

Therefore, the peptide compound can promote the proliferation and migration of HaCaT keratinocytes, has a skin repair effect, can repair skin barriers, and can promote the re-epithelization or healing of skin or mucosa.

Example 12 collagen content test

12.1 reagents and materials

Fetal bovine serum, a DMEM culture medium, a phosphate buffer solution, trypsin, a collagen I ELISA kit and a BCA protein kit.

12.2 instruments

Microplate reader, CO ₂ Incubator, superclean bench, thermostated container.

12.3 cell lines

Human Skin Fibroblasts (HSF) were purchased from the shanghai cell bank of the committee for type culture collection of the chinese academy of sciences.

12.4 samples to be tested

Administration group: compound (9) and compound (17) at concentrations tested 12.5ppm, 50ppm, respectively;

blank control group: PBS;

UV group: UV irradiation, PBS was added.

12.5 Experimental methods

Taking HSF cells in logarithmic growth phase, performing enzymolysis and dilution, and performing enzymolysis and dilution according to the formula of 10 ⁵ And inoculating the cells/well on a 6-well plate, and establishing a UV light aging model until the cells grow to be about 80%. Adding 200 μ L PBS into blank control group, supplementing culture medium to 800 μ L, and not performing UV irradiation; adding appropriate amount of PBS to wash repeatedly to colorless in UV group and administration group, adding 200 μ L PBS, and placing at 80mJ/cm ² The culture flask was irradiated under a UV lamp with a lamp source spaced 15cm from the flask. After irradiation, PBS was discarded, the PBS solution and medium were added to the UV group to 800. Mu.L, and the medium and the drug diluted in multiples to 800. Mu.L were added to the administration group. The blank control group, UV group and administration group were continuously maintained at 37 deg.C and 5% CO ₂ And (5) incubating in an incubator for 48 hours.

After the incubation, the 1 st well cells were counted and diluted to 0.5X 10 ⁶ one/mL, cells from the remaining wells were scraped off using a cell scraper, 500. Mu.L was resuspended, 50. Mu.L of all wells were sonicated for 30s before total protein was measured using the BCA method, and the other wells were diluted to a concentration of 0.5X 10 of all cell suspensions based on the 1 st well protein concentration ⁶ one/mL. And (3) carrying out ultrasonication on the cell suspension with the adjusted concentration for 30s, centrifuging for 15min at 1500Xg, collecting cell supernatant to obtain a sample solution, and operating according to the collagen IELISA operation instruction. The OD of each well was measured sequentially at 450nm with a microplate reader within 15 min.

12.6 results of the experiment

Collagen is the most abundant protein found in connective tissue, and collagenase is synthesized and secreted by fibroblasts and is capable of degrading collagen in the skin to cause skin aging. Therefore, the expression of collagenase in cells is inhibited, and the increase of the collagen content plays an important role in preventing aging and increasing the skin plumpness and firmness. In the ultraviolet over-exposure environment, the activities of collagenase and elastase are greatly increased, and the hydrolysis of elastin and the synthesis of collagen are also inhibited. In the experiment, the cells irradiated by ultraviolet rays are treated by using a test sample, and the content of collagen I in the corresponding cells is detected to determine whether the peptide compound can promote the synthesis of collagen.

The results of the effect of the test samples on collagen content are shown in FIG. 11. The result shows that the collagen content of the UV group is greatly reduced compared with that of the blank control group, which indicates that the photoaging model is successfully established; compared with the UV group, the compound (9) and the compound (17) in the range of 12.5-50ppm can improve the collagen content and promote the collagen expression, and the compound (9) and the compound (17) can obviously improve the collagen content under the condition of low concentration of 12.5 ppm.

Therefore, the peptide compound can promote the generation of collagen, increase the content of the collagen, and further increase the skin elasticity and/or skin firmness, and can be used for preventing and even treating skin relaxation, treating, preventing or repairing skin aging or photoaging and resisting skin aging.

Example 13 collagen fiber experiment

13.1 reagents and materials

Fetal bovine serum, a DMEM medium, a phosphate buffer solution, trypsin and a biological sirius red staining kit.

13.2 Instrument

Optical microscope, CO ₂ An incubator.

13.3 cell lines

Human Skin Fibroblasts (HSF) were purchased from the Shanghai cell Bank of the China academy of sciences type culture Collection.

13.4 samples to be tested

Administration group: compound (9) and compound (17) at concentrations tested 12.5ppm, 50ppm, respectively;

blank control group: PBS;

UV group: UV irradiation, PBS was added.

13.5 methods of experiment

Taking HSF cells in logarithmic growth phase, performing enzymolysis and dilution, and performing enzymolysis and dilution according to the formula of 10 ⁵ And inoculating the cells/well on a 6-well plate, and establishing a UV light aging model until the cells grow to be about 80%. Adding 200 μ L PBS into blank control group, supplementing culture medium to 800 μ L, and not performing UV irradiation; adding appropriate amount of PBS to wash repeatedly to colorless in UV group and administration group, adding 200 μ L PBS, and placing at 80mJ/cm ² The culture flask was irradiated under a UV lamp with a lamp source spaced 15cm from the flask. After irradiation, PBS was discarded, and PBS solution and medium were added to 800. Mu.L in the UV group, and medium and drug were diluted to 800. Mu.L in the administration group. Blank control group, UV group, and administration group were continued at 37 deg.C and 5% CO ₂ Incubate in incubator for 48h.

After the culture, the cells were fixed with 4% paraformaldehyde for 15min, stained according to the instructions of the sirius red staining kit, and observed under an optical microscope.

13.6 results of the experiment

In the experiment, cells irradiated by ultraviolet rays are treated by using a test sample, the density of the corresponding cells and the area of collagen fibers in the same unit are detected, and the collagen fibers in the cells are qualitatively analyzed to determine whether the peptide compound can promote the expression of collagen.

The collagen fiber experiment of the cells can reflect the collagen expression capacity of the cells, and the experiment result is shown in figure 12, compared with a blank control group, the cell density of a UV group is reduced, and the area of the collagen fiber in the same unit is also reduced; compared with the UV group, the compounds (9) and (17) in the range of 12.5-50ppm can increase cell density, increase collagen fiber area in the same unit, promote collagen expression, and improve the symptoms of cell collagen content reduction caused by UV irradiation.

Therefore, the peptide compound can promote the expression of collagen, so as to increase the skin elasticity and/or the skin firmness, and can be used for preventing and even treating the skin relaxation, treating, preventing or repairing the skin aging or photoaging and resisting the skin aging.

EXAMPLE 14 preparation of liposome containing Compound (9)

The preparation method comprises the following steps: the phosphatidylcholine was weighed and dissolved in chloroform. The solvent is evaporated under vacuum until a thin phospholipid layer is obtained, which is hydrated by treatment with an aqueous solution of the compound at the desired concentration at 55 ℃ to obtain the multilamellar liposomes. The multi-chambered liposome is homogenized under high pressure, and the single-chambered liposome with smaller and uniform size is obtained.

EXAMPLE 15 microemulsion composition containing Compound (13)

The preparation method comprises the following steps: weighing the components of the B phase according to the dosage of the prescription, and adding the components into a container. Then, phase D is added to phase B and homogenized under continuous stirring. Phase a was then added to the mixture. And finally, adding the phase C, and uniformly stirring to obtain the composition.

Example 16 preparation of essence containing Compound (17)

The preparation method comprises the following steps: stirring purified water, heating to 85 deg.C, and maintaining the temperature for 30min; pre-dissolving sodium hyaluronate and xanthan gum in butanediol, adding into water, and stirring to dissolve completely; cooling to 35 deg.C under stirring, adding the rest components, and stirring.

EXAMPLE 17 preparation of toner containing Compound (21)

The preparation method comprises the following steps: dissolving allantoin and glycerol in water, heating to 85 deg.C, and maintaining the temperature for 30min; dissolving PEG-7 glyceryl cocoate and compound (21) in water; cooling the solution, mixing, and uniformly stirring to obtain a mixed solution; sequentially adding propylene glycol, antiseptic and essence into the above mixed solution, adding water, and stirring.

While preferred embodiments of the present invention have been described, additional variations and modifications of these embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the embodiments of the invention.

Finally, it should also be noted that, in this document, 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. Also, 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 phrases "comprising one of \ 8230; \8230;" does not exclude the presence of additional like elements in a process, method, article, or terminal device that comprises the element.

The present invention provides a novel use of peptide compounds for preparing compositions for skin aging repair, which is described in detail above, and the principle and embodiments of the present invention are illustrated herein by using specific examples, which are only used to help understanding the method of the present invention and the core concept thereof; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Claims (9)

1. Use of a compound of formula (I) or a salt thereof in the manufacture of a composition for skin rejuvenation including one or more of increasing fibroblast activity, promoting keratinocyte proliferation and migration, promoting re-epithelialization or healing of the skin or mucosa, repairing skin barriers, preventing or repairing photoaging damage, promoting collagen production, increasing skin elasticity, or increasing skin firmness;

in the general formula (I),

R ₁ selected from the group consisting of: h or R ₃ -CO-wherein R ₃ Selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

R ₂ selected from: -OH, -NH ₂ 、-Leu-NH ₂ 、-Leu-OH、-Val-NH ₂ or-Val-OH;

the alkyl group refers to a saturated aliphatic linear or branched alkyl group having 1 to 24 carbon atoms (optionally having 1 to 16 carbon atoms; optionally having 1 to 14 carbon atoms; optionally having 1 to 12 carbon atoms; optionally having 1, 2, 3, 4, 5, or 6 carbon atoms); optionally selected from: methyl, ethyl, isopropyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 2-ethylhexyl, 2-methylbutyl, or 5-methylhexyl;

the alkenyl group means a straight or branched chain alkenyl group having 2 to 24 carbon atoms (optionally having 2 to 16 carbon atoms; optionally having 2 to 14 carbon atoms; optionally having 2 to 12 carbon atoms; optionally having 2, 3, 4, 5, or 6 carbon atoms); said alkenyl having one or more carbon-carbon double bonds, optionally 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bonded to the remainder of the molecule by a single bond; optionally selected from: vinyl, oleyl, or linoleyl;

alternatively, in the "substituted alkyl", "substituted alkenyl" groupIs selected from C ₁ -C ₄ An alkyl group; a hydroxyl group; c ₁ -C ₄ An alkoxy group; an amino group; c ₁ -C ₄ An aminoalkyl group; c ₁ -C ₄ A carbonyloxy group; c ₁ -C ₄ An oxycarbonyl group; halogen (e.g., fluorine, chlorine, bromine, and iodine); a cyano group; a nitro group; an azide; c ₁ -C ₄ An alkylsulfonyl group; a thiol; c ₁ -C ₄ An alkylthio group; c ₆ -C ₃₀ Aryloxy groups such as phenoxy; -NR _b (C＝NR _b )NR _b R _c Wherein R is _b And R _c Is independently selected from: H. c ₁ -C ₄ Alkyl radical, C ₂ -C ₄ Alkenyl radical, C ₂ -C ₄ Alkynyl, C ₃ -C ₁₀ Cycloalkyl radical, C ₆ -C ₁₈ Aryl radical, C ₇ -C ₁₇ Aralkyl, heterocyclic group having three to ten members, or protective group of amino group.

2. Use according to claim 1, characterized in that R ₁ Selected from the group consisting of: H. acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl or phenylacetyl;

alternatively, R ₁ Is H, myristoyl or palmitoyl.

3. Use according to claim 1, characterized in that said compound is selected from (1) to (24):

(1)H-Tyr-Ser-OH；

(2)H-Tyr-Ser-NH ₂ ；

(3)Ac-Tyr-Ser-OH；

(4)Ac-Tyr-Ser-NH ₂ ；

(5)Palm-Tyr-Ser-OH；

(6)Palm-Tyr-Ser-NH ₂ ；

(7)Myr-Tyr-Ser-OH；

(8)Myr-Tyr-Ser-NH ₂ ；

(9)H-Tyr-Ser-Leu-OH；

(10)H-Tyr-Ser-Leu-NH ₂ ；

(11)Ac-Tyr-Ser-Leu-OH；

(12)Ac-Tyr-Ser-Leu-NH ₂ ；

(13)Palm-Tyr-Ser-Leu-OH；

(14)Palm-Tyr-Ser-Leu-NH ₂ ；

(15)Myr-Tyr-Ser-Leu-OH；

(16)Myr-Tyr-Ser-Leu-NH ₂ ；

(17)H-Tyr-Ser-Val-OH；

(18)H-Tyr-Ser-Val-NH ₂ ；

(19)Ac-Tyr-Ser-Val-OH；

(20)Ac-Tyr-Ser-Val-NH ₂ ；

(21)Palm-Tyr-Ser-Val-OH；

(22)Palm-Tyr-Ser-Val-NH ₂ ；

(23)Myr-Tyr-Ser-Val-OH；

(24)Myr-Tyr-Ser-Val-NH ₂ 。

4. use according to claim 3, characterized in that the compound is selected from compound (9), compound (13), compound (15), compound (17), compound (21) and compound (23); in particular, the amount of the solvent to be used,

(9)H-Tyr-Ser-Leu-OH；

(13)Palm-Tyr-Ser-Leu-OH；

(15)Myr-Tyr-Ser-Leu-OH；

(17)H-Tyr-Ser-Val-OH；

(21)Palm-Tyr-Ser-Val-OH；

(23)Myr-Tyr-Ser-Val-OH。

5. the use according to claim 1, wherein the composition comprises the compound of formula (I) or a salt thereof in a concentration of 0.0001% to 5% by mass;

optionally, the composition comprises the compound shown in the general formula (I) or the salt thereof with the mass percentage concentration of 0.0005-1%;

optionally, the composition comprises 0.001-0.1% by mass of a compound shown in the general formula (I) or a salt thereof;

optionally, the composition comprises the compound shown in the general formula (I) or the salt thereof, wherein the mass percentage concentration of the compound is 0.005% -0.01%.

6. Use according to claim 1, wherein the salt of the compound of formula (I) comprises a metal salt of a compound of formula (I), the metal comprising: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum;

alternatively, the salt of the compound of formula (I) comprises a salt of the compound of formula (I) with an organic base comprising: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine;

alternatively, the salt of the compound represented by the general formula (I) includes a salt of the compound represented by the general formula (I) with an inorganic acid or an organic acid, and the organic acid includes: acetic, citric, lactic, malonic, maleic, tartaric, fumaric, benzoic, aspartic, glutamic, succinic, oleic, trifluoroacetic, oxalic, pamoic or gluconic acids;

optionally, the inorganic acid comprises: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

7. Use according to claim 1, of a compound of general formula (I) or a salt thereof, incorporated in a cosmetically or pharmaceutically acceptable delivery system or sustained release system, or adsorbed onto a cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support;

the cosmetically or pharmaceutically acceptable delivery system or sustained release system is selected from: liposomes, oleosomes, nonionic surfactant liposome vesicles, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, liposomes, micelles, nanospheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, nanoparticles, microparticles, and nanoparticles; optionally liposome or microemulsion, optionally water-in-oil type microemulsion with reverse micelle internal structure;

the cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support is selected from: talc, bentonite, silica, starch or maltodextrin.

8. Use according to claim 1, characterized in that the composition is a cosmetic or pharmaceutical composition, the formulation of which is selected from: creams, oils, balms, foams, lotions, gels, liniments, serums, ointments, mousses, powders, sticks, pens, sprays, aerosols, capsules, tablets, granules, chewing gums, solutions, suspensions, emulsions, elixirs, polysaccharide films, jellies or gelatins.

9. Use according to claim 1, characterized in that said composition also comprises at least one other active agent for enhancing the skin's tonic repair action, said other active agent being selected from one or more of peptides, natural plant components, vitamin C and its derivatives or retinoids.

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