KR102056520B1 - Cosmetic composition containing a production method of peptide that promotes collagen synthesis in human fibroblasts and controls revitalization of collagen decomposing enzymes. - Google Patents

Abstract

As extensive research for the prevention and alleviation of wrinkle formation of the skin has been developed, important functions of collagen have been revealed in the skin, and by developing raw materials for promoting the synthesis of collagen in the skin, the present inventors have attempted to suppress skin wrinkles caused by aging. They have completed the present invention by confirming that the composition of the present invention containing the functional peptide represented by the following formula 1 to 4 has excellent wrinkle improvement function by inhibiting collagen synthesis enhancing effect and collagen degrading enzyme activation in human fibroblasts.
[Formula 1]
[Cys-Arg-X-Lys]
[Formula 2]
[X-Phe-Lys-Cys]
[Formula 3]
[Cys-Arg-X-Lys] ₂
[Formula 4]
[X-Phe-Lys-Cys] ₂
(In the formula 1 to 4, X is glutamic acid, aspartic acid, histidine, phenylalanine, alanine, cysteine, glycine, glutamine, asparagine, arginine, leucine, methionine, isoleucine, serine, tyrosine, threonine, lysine, tryptophan Selected from, proline and valine, and [] ₂ may represent a dimer of a thiol group (-SH), which is a cysteine residue, in a reduced form (-SS-).)

Description

Cosmetic composition containing a production method of peptide that promotes collagen synthesis in human fibroblasts and controls revitalization of collagen decomposing enzymes.

The present invention confirms the effect of a specific peptide on human fibroblasts, and more particularly, peptides that can be expected to prevent skin aging and wrinkle improvement through inducing collagen synthesis and inhibiting collagenase activation of fibroblasts. It is a technique regarding the utilized cosmetic composition.

The skin consists of the epidermis, the dermis and the subcutaneous tissue, the epidermis acting as a physical protective film that protects the body from thermoregulation and the external environment, and the dermis is a connective tissue between the epidermis and the subcutaneous fat layer. And structurally support the epidermis.

The dermal connective tissue in the dermal layer contains extracellular matrix protein, collagen, which is commonly known as about 30% of mammalian protein, and this collagen is mainly distributed in muscle, cartilage and bone. It consists of three amino acid residues: glycine, proline and hydroxyproline. To date, 19 types of collagen have been studied. In adult skin, there are type I, type III and the remaining type II, type IV, but most of the type I collagen is present in the dermal layer of the skin. It maintains, giving strength and tension to the skin, the newly synthesized procollagen is secreted into the extracellular space of skin cells through the enzymatic reaction to form collagen fibers.

Fibroblasts in the dermal layer are responsible for the expression of collagen.As age increases, the number and function of fibroblasts in the dermis decreases, thus reducing the synthesis of structural proteins such as collagen and degrading collagen. The amount of the enzyme, Matrix MetalloProteases (MMPs) increases, and water in the skin cells is lost (Fisher, G. J et al., 2002), which affects the stratum corneum and changes the skin structure. As age increases, the effect of ultraviolet light accumulates and collagen degradation reaches a point where collagen synthesis predominates, resulting in a decrease in the amount of collagen that acts as a supporting structure for skin (Talwar, JSet al., 1995).

Therefore, the discovery of safe materials that promote collagen synthesis and suppress collagenase action is important in skin aging and wrinkle improvement, and various studies on collagen change in skin are being conducted.

   Vitamin A, which is known to improve or inhibit skin photoaging by inhibiting the synthesis of MMPs and promoting collagen synthesis in the skin, has the same effect on naturally aged skin (Varani, J et. al., 2000)

The low molecular weight compound that promotes the production of collagen is currently widely used to improve skin wrinkles, blemishes, and pigmentation. (See US Patent Nos. 4,603,146 and 4,877,805). In addition to promoting collagen synthesis, new skin wrinkle treatments include ultrasonic treatment, laser dermabrasion, botulinum toxin injection, and restylene injection.

As extensive research for the prevention and alleviation of wrinkle formation has been developed, important functions of collagen have been revealed in the skin, and researches to suppress skin wrinkles caused by aging by developing raw materials for promoting collagen synthesis in the skin have been conducted. . The present inventors have completed the present invention by confirming that the composition of the present invention containing a functional peptide is excellent in anti-wrinkle function by enhancing collagen synthesis and inhibiting collagen degrading enzyme activation in human fibroblasts.

United States Patent No. 4,603,146 (registered July 29, 1986) U.S. Patent No. 4,720,353 (registered Jan. 19, 1988)

 'Mechanisms of photoaging and chronological skin aging.', Fisher, G. J. et al. , Arch. Dermatol., 2002, 138: 1462-1470.  'Reduced type I and type III procollagens in photodamaged adult human skin.' Talwar, J. S. et al., J. Invest. Dermatol., 1995, 105: 285-290.  'Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin' Varani, J. et al. , J. Invest. Dermatol, 2000, 114: 480-486. The present invention was completed by confirming the sound.

The present inventors treat peptides directly on fibroblasts present in the dermal layer to promote cell proliferation, induce the production of extracellular matrix such as collagen, and inhibit collagen degrading enzyme activation, thereby preventing skin aging and inhibiting wrinkle formation. I found it possible.

Therefore, an object of the present invention is to provide a cosmetic composition for inhibiting skin aging or skin wrinkle formation by containing the specific peptide.

[Formula 1]

[Cys-Arg-X-Lys]

[Formula 2]

[X-Phe-Lys-Cys]

[Formula 3]

[Cys-Arg-X-Lys] ₂

[Formula 4]

[X-Phe-Lys-Cys] ₂

In Formulas 1 to 4, X is glutamic acid, aspartic acid, histidine, phenylalanine, alanine, cysteine, glycine, glutamine, asparagine, arginine, leucine, methionine, isoleucine, serine, tyrosine, threonine, lysine, tryptophan, Selected from proline and valine,

[] 2 represents a dimer of a cysteine residue, a thiol group (-SH), in a reduced form (-S-S-).

Preferably, the peptide of Formula 1 to 4,

[Cys-Arg-Gly-Lys] (Compound 1)

[Cys-Arg-Pro-Lys] (Compound 2)

[Cys-Arg-Asn-Lys] (Compound 3)

[Cys-Arg-Lys-Lys] (Compound 4)

[Cys-Arg-Ser-Lys] (Compound 5)

[Cys-Arg-Tyr-Lys] (Compound 6)

[Cys-Arg-Asp-Lys] (Compound 7)

[Cys-Arg-Phe-Lys] (Compound 8)

[Cys-Arg-Leu-Lys] (Compound 9)

[Cys-Arg-Trp-Lys] (Compound 10)

Met-Phe-Lys-Cys (Compound 11)

Ser-Phe-Lys-Cys (Compound 12)

Lys-Phe-Lys-Cys (Compound 13)

His-Phe-Lys-Cys (Compound 14)

[Trp-Phe-Lys-Cys] (Compound 15)

[Thr-Phe-Lys-Cys] (Compound 16)

Val-Phe-Lys-Cys (Compound 17)

Gly-Phe-Lys-Cys (Compound 18)

Arg-Phe-Lys-Cys (Compound 19)

[Tyr-Phe-Lys-Cys] (Compound 20)

[Cys-Arg-Gly-Lys] ₂ (Compound 21)

[Cys-Arg-Pro-Lys] ₂ (Compound 22)

[Cys-Arg-Asn-Lys] ₂ (Compound 23)

[Cys-Arg-Lys-Lys] ₂ (Compound 24)

[Cys-Arg-Ser-Lys] ₂ (Compound 25)

[Cys-Arg-Tyr-Lys] ₂ (Compound 26)

[Cys-Arg-Asp-Lys] ₂ (Compound 27)

[Cys-Arg-Phe-Lys] ₂ (Compound 28)

[Cys-Arg-Leu-Lys] ₂ (Compound 29)

[Cys-Arg-Trp-Lys] ₂ (Compound 30)

Met-Phe-Lys-Cys ₂ (compound 31)

Ser-Phe-Lys-Cys ₂ (compound 32)

Lys-Phe-Lys-Cys ₂ (compound 33)

His-Phe-Lys-Cys ₂ (compound 34)

[Trp-Phe-Lys-Cys] ₂ (Compound 35)

[Thr-Phe-Lys-Cys] ₂ (Compound 36)

Val-Phe-Lys-Cys ₂ (compound 37)

Gly-Phe-Lys-Cys ₂ (compound 38)

Arg-Phe-Lys-Cys ₂ (compound 39)

[Tyr-Phe-Lys-Cys] ₂ (Compound 40)

In the compound, [] 2 represents a dimer of a cysteine residue, a thiol group (-SH), in a reduced form (-S-S-).

In another aspect, the present invention provides a composition for improving wrinkles containing at least one peptide selected from peptides of Formulas 1 to 4, or peptides of Compounds 1 to 40.

Thus, the present invention can provide a pharmaceutical composition for improving wrinkles containing at least one peptide selected from the peptides of Formulas 1 to 4, or the compounds 1 to 40. The peptide may be included in the pharmaceutical composition at 0.0001 to 1.0% by weight.

In still another aspect, the present invention provides a cosmetic composition for improving wrinkles containing at least one peptide selected from the peptides of Formulas 1 to 4, or the peptides of Compounds 1 to 40. The peptide may be included in the cosmetic composition in an amount of 0.0001 to 1.0% by weight. The cosmetics are lotion, emulsion, gel, cream, essence, pack, ampoule, lotion, cleansing

Ingredients, soaps, body products, soaps, oils, lipsticks and foundations.

Hereinafter, the present invention will be described in detail.

Among the peptides of the present invention, the peptides of the compounds 21 to 40 are obtained by modifying the thiol group (-SH), which is a cysteine (cysteine located at the terminal of the peptide) residues of the peptides of the compounds 1 to 20, respectively, into a reduced form (-SS-). It is a dimer. For example, a duplex in which a thiol group, which is a cysteine residue of Compound 1, is modified, is Compound 21, and a duplex, in which a thiol group, which is a cysteine residue of Compound 2, is reduced, is Compound 22. In this manner, a duplex of Compounds 1 to 20 can be prepared from Compounds 3 to 33, respectively.

Peptides of the invention can be prepared in such a way that one or more amino acids or suitably protected amino acids are continuously formed in the amino acid backbone, which are constantly bound to a solid phase, to form amide bonds. In addition, the peptide can be inserted, substituted, deleted other amino acids in a range that does not significantly reduce the stability, which also belongs to the scope of the present invention.

In addition, the cell permeable peptide (cell permeable peptide) for promoting intracellular migration of the peptide of the present invention may be further included by binding to the peptide C-terminal or N-terminal. For example, the cell permeable peptide includes TAT peptide (Arg-Lys-Lys -Arg-Arg-Tyr-Arg-Arg-Arg) and Tat-PTD peptide (Gly-Arg-Lys-Lys-Arg -Arg-Gln- Arg-Arg-Arg: Tat PTD), but the present invention is not limited thereto, and any cell-penetrating peptide known in the art may be used as long as it does not inhibit the activity of the peptide according to the present invention. Do.

On the other hand, the peptide of the present invention may exist in the form of a salt. Forms of salts usable in the present invention may be made during the final separation and purification of the compound or by reacting an amino group with a suitable acid. For example, as acid addition salts, acetates, adipates, alginates, citrate, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, camphorates, camphorsulfonates, digluconates, glycerophosphates, hemi Sulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroyodide, 2-hydroxyethane sulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, Naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate Trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, La-be-toluenesulfonate and undecanoate. However, the embodiment is not limited thereto. In addition, examples of acids that may be used to form acid addition salts may include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid. At this time, the peptide form containing the trifluoroacetate salt or the acetate salt is most preferred.

The amino groups or carboxyl groups of the amino acids used for preparing the peptides of the invention may be protected by suitable protecting groups. Protected amino acids can be reacted in solution by adding the following amino acids under conditions suitable to attach to a solid support or to form an amide bond. In addition, the protecting group can be removed completely before adding the amino acid protected with a suitable protecting group. After all amino acids have been linked as desired, the final desired peptide can be obtained by successive or simultaneous separation from the free residual protecting group and free solid support.

In the present invention, a condensation condensation obtained by synthesizing a desired hexapeptide by condensation of an appropriately protected tetrapeptide with another appropriately protected dipeptide to form an amide bond under the condition that the chiral center is not racemized. Peptides can be prepared using reaction techniques.

The most preferable synthetic method for preparing the peptide compound of the present invention may be a solid phase peptide synthesis method synthesized using a solid phase polymer retardation, wherein the α-amino group of the peptide prepared by the above method is acid or base sensitive. It can be protected by a functional group. The protecting group of the amino acid at this time must have a stable property in the conditions of peptide condensation reaction, and must be easily removable without breaking the extended peptide chain or without racemicization of any chiral center contained therein. Thus, suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyl-oxycarbonyl, t-amyloxycarbonyl , Isobornyloxycarbonyl, (α, α) -dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like, and such Other suitable protecting groups known in the art for the purpose are also usable within the scope of the present invention.

As the most preferable protecting group of the amino acid used in the peptide synthesis of the present invention, a 9-flu orenylmethyloxycarbonyl (Fmoc) protecting group can be used.

In particular, protecting groups for amino acid residues used in the peptide synthesis of the present invention are, for N-methyl glutamic acid, t-butyl (t-Bu); For lysine, t-butoxycarbonyl (Boc); For serine, 7t-butyl (t-Bu); For threonine and allothreonine, it is t-butyl (t-Bu); In the case of cysteine, it is preferable that it is trityl (Trt), but this invention is not limited to this.

In solid phase peptide synthesis methods, the C-terminal amino acid may be attached to a suitable solid support or resin. Suitable solid supports useful for such synthesis are preferably reagents of the staged condensation-deprotection reaction and materials which are inert to the reaction conditions and are insoluble in the medium used, for example rink amid or rink amide 4- Methylbenzylhydride

It may be a ricin amid MBHA resin.

In particular, the preferred solid support for the C-terminal amide peptide may be linkamide 4-methylbenzylhydrylamine resin commercially available from Novabiochem Cor poration.

C-terminal amide is a solvent such as dichloromethane, N-methylpyridone (NMP) or DMF at a temperature of 10 ℃ to 50 ℃, preferably at a temperature of 30 ℃ for 1 to 24 hours 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), benzotriazol-1-yloxy-tris (dimethylamino) phosphonium-hexafluorophosphate N, N'-dicyclohexylcarbodiimide (DCC), 'N, N'-diisoxo in the presence or absence of (BOP) or bis (2-oxo-3-oxazolidinyl) phosphine chloride (BOP CI) Propylcarbodiimide (DIC), [O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethylurojunium hexaflurophosphate] (HATU) or O-benzotriazole Activated carboxylic acid in -1-yl-N, N, N ', N'-tetra-methyluronium hexafluorophosphate (HBTU) to be condensed (bonded, coupled) to a resin or solid support via condensation There.

If the solid support is a link amide 4-methylbenzylhydrylamine resin, the preferred moiety as a protecting group is that the Fmoc functional group is excess of a secondary amine solution, preferably 20% piperidine DMF solution before condensing to the C-terminal amino acid. Use to cut. Preferred reagents used for condensation of the desired amithenoic acid in the deprotected '4- (2' ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxyacetamidoethyl resin are DMF for suitably protected amino acids. N-methylmorpholine (NMM), 1-hydroxybenzotriazole (H OBt) and O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium in a solvent Hexafluorophosphonate] (HATU), O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU), N, N'-dicyclohex Condensation reaction reagents such as silk carbodiimide (DCC) or N, N'-diisopropenylcarbodiimide (DIC).

Condensation of consecutive amino acids carried out in the present invention can be carried out manually or manually using an automated peptide synthesizer well known in the art. Preferred conditions for the synthesis reaction include deprotection of the α-amino acid protected with Fmoc group with a secondary amine solution, preferably piperidine, followed by washing with a sufficient excess of solvent and another protection desired for condensation. 3 to 7-fold molar excess of the amino acid can then be added, and the reaction can be preferably carried out in a DMF solvent.

In the final step of the synthesis of the peptide using the solid resin of the present invention, the peptide to be obtained from the resin can be removed in a continuous or single operation, and the protecting groups protecting the residues of the amino acids can be deprotected. Removal conditions of the peptide from the resin and deprotection conditions of the protecting groups present at the residues generally include cleavage reagent cocktails that cleave the bond between the resin-peptide, for example trifluoroacetic acid (TFA), triisopropylsilane. It can be obtained by treating dichloromethane mixed cocktail solution consisting of (TIS), thioanisol, water or ethanedithiol (EDT). The mixed solution thus obtained can produce a precipitate by excessively treating the refrigerated diethyl ether solvent. The precipitate obtained as described above was centrifuged to completely settle the precipitate, and the precipitate was solidified by first removing the excessive amounts of trifluoroacetic acid, triisopropylsilane, thioanizol, water and ethanedithiol, and repeating the above procedure two or more times. Can be obtained. At this time, the fully deprotected peptide salt can be separated and purified using a mixed solvent consisting of water and acetnitrile solvent and reversed phase high performance liquid chromatography (HPLC). The separated and purified peptide solution can be completely concentrated and dried using lyophilization to obtain a solid peptide.

Peptides of the present invention has a collagen synthesis effect.

According to a preferred embodiment of the present invention, the composition of the present invention may be provided as a pharmaceutical composition or cosmetic composition for wrinkle improvement.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, purified water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include conventionally used additives such as lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like. The pharmaceutical composition of the present invention is preferably parenteral administration, more preferably applied by topical administration by application.

Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be.

The dosage of the peptide, which is an active ingredient included in the pharmaceutical composition of the present invention, is 0.001 to 100 mg / kg, preferably 0.1 to 100 mg / kg, more preferably 1 to 50 mg / kg, based on an adult. Dosages may be administered once or several times a day. In addition, the peptide of the present invention may preferably contain 0.0001 to 1.0% by weight, more preferably 0.001 to 1.0% by weight, most preferably 0.001 to 0.01% by weight relative to the total weight of the pharmaceutical composition. It is not limited to this.

The cosmetic composition of the present invention includes not only peptides, which are effective ingredients thereof, but also components commonly used in cosmetic compositions, and include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, qubitatamines, pigments, and perfumes, and carriers It includes. In addition, as the carrier, purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butyrene glycol or propylene glycol), higher fatty acids (palmitylic acid or quilizonolenic acid), Oils and fats such as wheat germ oil, camellia oil, jojoba oil, olive oil, squalene, sunflower oil, bovine macadamia peanut oil, avocado oil, or fatty acid glycerides may be used, but are not limited thereto. If necessary, a surfactant, a humectant, a preservative, an antioxidant and the like can be added.

Surfactants that can be used in the cosmetic composition of the present invention, as anionic surfactant 알킬, alkylbenzene sulfonate, polyoxyalkylene alkyl sulfate ester salt, alkyl sulfate ester salt, olefin sulfonate, alkyl phosphate, polyoxyalkylene Alkyl ether phosphate, dialkyl sulfosuccinate, fatty acid salt, etc. are mentioned, As a nonionic surfactant, polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyhydric alcohol fatty acid partial ester, polyoxyethylene polyhydric alcohol fatty acid partial Ester, polyglycerol fatty acid ester, polyoxyethylene cured castor oil derivative, fatty acid diethanolamide, and the like. Examples of the cationic surfactant include tertiary aliphatic amine salts, alkyltrimethylammonium halides, and dialkyldimethylammonium halides. Examples of the amphoteric surfactants include amide betaine type, imidazolinium betaine type, and sulfobeta. Like dolls. Examples of the moisturizing agent include glycerin, propylene propylene glycol, 1,3-butylene glycol, dipropylene glycol, sorbitol, and the like. Examples of the preservative include benzoic acid, dehydroacetic acid, paraoxybenzoic acid esters (methyl paraoxybenzoate and butyl paraoxybenzoate), phenoxyethanol and the like. As the antioxidant, ascorbic acid, BHA, and the like can be cited. In addition, ultraviolet absorbers, anti-inflammatory agents, and refreshing agents can be added.

Peptide of the present invention may preferably contain 0.0001 to 1.0% by weight, more preferably 0.001 to 1.0% by weight, most preferably 0.001 to 0.01% by weight relative to the total weight of the cosmetic composition, but is not limited thereto. .

Moreover, the kind of cosmetics is not specifically limited, For example, skin care cosmetics, lipsticks, such as a lotion, an emulsion, a gel, cream, an essence, a pack, an ampoule, a lotion, a cleaning agent, a soap, body products, soap, oil, Makeup cosmetics, such as a foundation, etc. are mentioned, The formulation is not specifically limited.

The cosmetic composition of the present invention may be used daily and may also be used for an indefinite period of time. Preferably, the amount of use, frequency of use and duration may be adjusted according to the age, skin condition or skin type of the user, and the concentration of the peptide.

In accordance with the present invention, it has been found that certain peptides are activated at low concentrations to promote fibroblast proliferation, thereby promoting skin regeneration by the peptides. In addition, it was found that by acting on the fibroblasts of the dermal layer, it induces the production of extracellular matrix such as collagen and inhibits the formation of skin wrinkles by inhibiting collagenase activation. Therefore, the peptide may be usefully applied to the cosmetic composition for inhibiting skin aging or inhibiting skin wrinkle formation.

Figure 1 is a peptide of the present invention is a diagram of the cell survival rate of fibroblasts.
Figure 2 is a graphical representation of the effect of promoting peptide growth in fibroblasts peptides.
Figure 3 is a graphical representation of the effect of increasing the collagen synthesis of the compounds of the present invention.
Figure 4 is a graphical representation of the inhibitory effect of MMP-1 production of the compounds of the present invention.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the information introduced herein is intended to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

<Example 1-1. Synthesis of Compound 1>

Names and abbreviations of amino acids used in the present invention are indicated as follows.

Ala: Alanine / Cys: Cysteine / Gly: Glycine / Val: Valine / Pro: Proline / Phe: Phenylalanine / Met: Methionine / Trp: Tryptophan / Glu: Glutamine /

71.4 mg (0.10 mmol) of 2-chlorotrityl chloride resin (1.4 mmol loaded per gram) obtained from Novabiochem corporation was weighed into a reaction vessel. The resin was solvated with 3 ml of DMF and allowed to sweep for 5 minutes, then 3 ml of 20% (w / v) piperidine DMF solution was added and shaken for 20 minutes and the piperidine DMF solution After removal, the mixture was washed 5 times with 10 ml of DMF solvent (5 times with 10 ml each). * DMF: Dimethylformamide.

Fmoc-Gly (Trt) -OH (468.6 mg, 0.80 mmol), HOBt (108.1 mg, 0.80 mmol) and DIC (0.124 mL, 0.80 mmol) were completely dissolved in 2 mL of DMF solvent and then added to the resin. The reaction solution was shaken at room temperature for 8 hours, and then washed 5 times with 10 ml of DMF solvent. Add 3 ml of 20% (w / v) piperidine DMF solution, shake for 10 minutes, remove the piperidine solution, and then add 20% (w / v) piperidine DMF solution for 20 minutes. After the reaction, the Fmoc protecting group protected in the resin was completely removed and washed 5 times with 10 ml of DMF solvent (5 times with 10 ml each). The deprotection of the Fmoc protecting group at this stage was determined by Kaiser test [E. Kaiser et al. Anal. Biochem., 1970, 34 (2), 595-598.]. Fmoc: 9-fluorenylmethyloxycarbonyl / HOBt: 1-hydroxybenzotriazole / DIC: N, N'-diisopropylcarbodiimide.

Next, the peptides were condensed (coupled) continuously according to the same synthesis cycle as described below.

(1) 5 washes with DMF solvent (10 mL);

(2) deprotection twice for 10 min using 20% (w / v) piperidine DMF solution (3 mL);

(3) five washes with DMF solvent (10 mL);

(4) addition of Fmoc-amino acid;

(5) addition of a condensation reagent to activate amino acids and condensation for 2 hours;

(6) washing 5 times with DMF solvent (10 mL);

(1) to (6) were repeated again, and at this time, Fmoc-protected amino acid (0.80 mmol) after Fmoc-Cys (Trt) -OH was added to the resin reaction vessel and condensed in the order described below. .

(i) Fmoc-Lys-OH;

(ii) Fmoc-Gly-OH;

(iii) Fmoc-Arg-OH;

(iv) Fmoc-Cys (Trt) -OH;

After (7) after Fmoc-Cys (Trt) -OH condensation, finally, 20% piperidine DMF solution (3 mL) was treated.

Immediately after the end of the synthesis, a mixture of trifluoroacetic acid / thioanizol / ethanedithiol / triisopropylsilane / water (95: 5: 2.5: 2.5: 2.5) Using (10 mL), the peptide was cleaved from the resin. The precipitate was produced by treating 100 ml of the diethyl ether solvent refrigerated and stored in the thus obtained mixed solution. The precipitate obtained was centrifuged to completely settle and the first removal of trifluoroacetic acid, thioanisol and ethanedithiol was followed by the above procedure (100 ml of diethyl ether solvent was added to wash the precipitate and centrifugation-first removal). Trifluoroacetic acid, thioaniazole and ethanedithiol) were tried twice to obtain a precipitate which solidified. The precipitate (peptide) was purified by HPLC using a 5% to 100% acetonitrile / water gradient solvent system containing 0.01% trifluoroacetic acid over 50 minutes using a C-18 column. The pure purified fractions were lyophilized to give collagen synthesizing peptide H ₂ N- [Cys-Arg-Gly-Lys] -CO ₂ H (100 mg) as a white powder of trifluoroacetate salt.

Compound 1: H ₂ N- [Cys-Arg-Gly-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 462.62; (103 mg)

<Example 1-2. Synthesis of Compounds 2-20>

Using the same manufacturing process as in Example 1-1, but by changing the order of amino acids protected with Fmoc to prepare a peptide of the compound 2-20.

Compound 2: H ₂ N- [Cys-Arg-Pro-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 502.68; (105 mg)

Compound 3: H ₂ N- [Cys-Arg-Asn-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 519.67; (110 mg)

Compound 4: H ₂ N- [Cys-Arg-Lys-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 533.74; (105 mg)

Compound 5: H ₂ N- [Cys-Arg-Ser-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 492.64; (107 mg)

Compound 6: H ₂ N- [Cys-Arg-Tyr-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 568.74; (112 mg)

Compound 7: H ₂ N- [Cys-Arg-Asp-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 520.65; (109 mg)

Compound 8: H ₂ N- [Cys-Arg-Phe-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 552.74; (105 mg)

Compound 9: H ₂ N- [Cys-Arg-Leu-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 518.71; (117 mg)

Compound 10: H ₂ N- [Cys-Arg-Trp-Lys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 591.78; (102 mg)

Compound 11: H ₂ N- [Met-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 527.75; (100 mg)

Compound 12: H ₂ N- [Ser-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 483.63; (109 mg)

Compound 13: H ₂ N- [Lys-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 524.73; (114 mg)

Compound 14: H ₂ N- [His-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 533.7; (120 mg)

Compound 15: H ₂ N- [Trp-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 582.77; (104 mg)

Compound 16: H ₂ N- [Thr-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 497.66; (103 mg)

Compound 17: H ₂ N- [Val-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 495.69; (100 mg)

Compound 18: H ₂ N- [Gly-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 453.61; (107 mg)

Compound 19: H ₂ N- [Arg-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 552.74; (110 mg)

Compound 20: H ₂ N- [Tyr-Phe-Lys-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 559.73; (102 mg)

<Example 1-3. Synthesis of Compound 21>

The collagen synthesis promoter H ₂ N- [Cys-Arg-Gly-Lys] -CO ₂ H (50 mg) was dissolved in 2 ml of dimethyl sulfoxide (DMSO), and then 10 ml of water was added and stirred at room temperature for 3 days. To obtain a final collagen synthesizing peptide (50 mg) in the form of a dimer in which a thiol group (-SH), which is a cysteine residue, was converted into a reduced form (-SS-).

Compound 21: H ₂ N- [Cys-Arg-Gly-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 925.24; (53 mg)

<Example 1-4. Synthesis of Compounds 22-40>

Using the same production process as in Example 1-3, the peptide of the compounds 22 to 40 were prepared by changing the order of amino acids protected with Fmoc.

Compound 22: H ₂ N- [Cys-Arg-Pro-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1005.36; (52 mg)

Compound 23: H ₂ N- [Cys-Arg-Asn-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1039.34; (42 mg)

Compound 24: H ₂ N- [Cys-Arg-Lys-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1067.48; (46 mg)

Compound 25: H ₂ N- [Cys-Arg-Ser-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 985.28; (56 mg)

Compound 26: H ₂ N- [Cys-Arg-Tyr-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1137.48; (51 mg)

Compound 27: H ₂ N- [Cys-Arg-Asp-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1041.3; (48 mg)

Compound 28: H ₂ N- [Cys-Arg-Phe-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1105.48; (41 mg)

Compound 29: H ₂ N- [Cys-Arg-Leu-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1037.42; (51 mg)

Compound 30: H ₂ N- [Cys-Arg-Trp-Lys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1183.56; (55 mg)

Compound 31: H ₂ N- [Met-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1055.5; (48 mg)

Compound 32: H ₂ N- [Ser-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 967.26; (49 mg)

Compound 33: H ₂ N- [Lys-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1049.46; (56 mg)

Compound 34: H ₂ N- [His-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1067.4; (52 mg)

Compound 35: H ₂ N- [Trp-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1165.54; (47 mg)

Compound 36: H ₂ N- [Thr-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 995.32; (49 mg)

Compound 37: H ₂ N- [Val-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 991.38; (50 mg)

Compound 38: H ₂ N- [Gly-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 907.22; (54 mg)

Compound 39: H ₂ N- [Arg-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1105.48; (55 mg)

Compound 40: H ₂ N- [Tyr-Phe-Lys-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1119.46; (52 mg)

Through the experiments of collagen synthesis and mmp-1 synthesis inhibition of a total of 40 compounds of the above examples, the top three experiments with high collagen synthesis rate were carried out, it was selected in the same way as the monomer.

Experimental Example  1: Cell Viability Assay ( in vitro )

Human dermal fibroblasts (HDF: Human dermal fibroblast, manufactured by Thermo Co., Ltd.) were dispensed in 100-ul portions in 96-well plates at a concentration of 2.0 ³ cells / ml, and cultured daily at 37 ° C., 5% CO ₂ , and humidified conditions. . As the culture solution, 100 ul of each medium was used as a medium containing 5% of FBS (manufactured by Gibco) in DMEM medium (Dulbecco's Modified Eagle's Medium, manufactured by Gibco).

Subsequently, it was exchanged with DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Sigma) without Serum, and the compound of the present invention was incubated with 100 uM treatment. The same amount of PBS that did not dissolve the compound was added as a control.

After the test sample was added and cultured for 24 hours, MTT solution was added to the cultured cells at a concentration of 0.5 mg / ml and cultured at 37 ° C. and 5% CO 2 for 2 hours. After incubation, remove the medium from each well, place 100 μL of demethyl sulfoxide (DMSO; Sigma-Aldrich) in each well from which the medium was removed, and measure the absorbance at 595 nm using a microplate reader (Bio-Rad, USA). It was. The experiment was repeated three times and an average value was derived according to Equation 1. The results are shown in Table 1 and FIG.

[Equation 1]

% Cell viability = (Experimental group 540 nm absorbance / control group 540 nm absorbance) * 100

Cytotoxicity of Each Compound division Cell survival rate (%) division Cell survival rate (%) Control 100 Control 100 Compound 1 100.2 Compound 21 101.8 Compound 2 99.8 Compound 22 96.4 Compound 3 98.1 Compound 23 93.2 Compound 4 100.5 Compound 24 96.4 Compound 5 101.3 Compound 25 94.1 Compound 6 102.3 Compound 26 100.2 Compound 7 108.4 Compound 27 93.2 Compound 8 100.9 Compound 28 94.1 Compound 9 96.4 Compound 29 96.6 Compound 10 95.1 Compound 30 102.1 Compound 11 97.6 Compound 31 96.1 Compound 12 100.2 Compound 32 99.7 Compound 13 98.2 Compound 33 97.4 Compound 14 99.1 Compound 34 91.2 Compound 15 90.2 Compound 35 99.1 Compound 16 94.1 Compound 36 96.6 Compound 17 96.6 Compound 37 100.1 Compound 18 99.1 Compound 38 100.4 Compound 19 97.4 Compound 39 107.6 Compound 20 105.8 Compound 40 97.7

Referring to Table 1 and Figure 1, all compounds of the present invention was confirmed that all of the cell viability when compared to the control group, showed more than 90% cell viability. Through the results of this experiment, all compounds of the present invention was confirmed that there is no toxicity in animal cells including human skin fibroblasts, and confirmed that it is a safe raw material.

Experimental Example  2 : Peptide  Proliferation assay in fibroblasts (in vitro)

      [3H] -Thymidine uptake assay was performed to confirm the peptide effect on the proliferation of human dermal fibroblasts (HDF: Human dermal fibroblast, Thermo). The cells were incubated in fresh medium containing 1 μCi of [3H] -Thymidine (Amersham, Buckinghamshire, UK) and treated with fibroblasts in 12-well culture plates every 2 days for 6 days and incubated for a specified time. After that, the cells were washed twice with PBS and incubated with 0.1 N NaOH at room temperature. After lysis, the concentration of radioactivity was measured using a liquid scintillation counter as a measure of cell proliferation. The results are shown in Table 2 and FIG.

division [3H] -Thymidine
incorporation
(% ofcontrol) division [3H] -Thymidine incorporation
(% of control) Control 100 Control 100 Compound 1 95.2 Compound 21 110.5 Compound 2 102.1 Compound 22 128.5 Compound 3 96.3 Compound 23 100.7 Compound 4 101.2 Compound 24 99.8 Compound 5 102.3 Compound 25 108.3 Compound6 173.5 Compound26 140.8 Compound7 102.3 Compound27 108.3 Compound 8 169.4 Compound 28 150.8 Compound 9 100.3 Compound 29 105.3 Compound 10 170.9 Compound 30 180.7 Compound 11 90.5 Compound 31 109.8 Compound 12 100.8 Compound 32 108.6 Compound 13 105.6 Compound33 129.9 Compound 14 109.4 Compound 34 97.8 Compound 15 120.4 Compound 35 99.7 Compound 16 115.8 Compound36 99.6 Compound 17 100.7 Compound37 107.6 Compound 18 107.5 Compound38 108.7 Compound 19 109.6 Compound39 108.9 Compound 20 119.4 Compound 40 105.6

Referring to Table 2 and FIG. 2, most of the compounds of the present invention show an effect of increasing the proliferation of fibroblasts, which is confirmed to exhibit a sufficient effect on skin regeneration.

Experimental Example  3: Peptide  Type-1 Procollagen  Synthesis promoting effect measurement (in vitro)

Human dermal fibroblasts (HDF: Human dermal fibroblast, manufactured by Thermo) were dispensed in 1 wells in 12 well plates at a concentration of 6.0 ⁴ cells / ml, and cultured at 37 ° C., 5% CO ₂ , and humidified conditions for 2 days. . As the culture solution, 1 ml of each medium containing DBS medium (Dulbecco's Modified Eagle's Medium, manufactured by Gibco) at 5% FBS (manufactured by Gibco) was used.

Subsequently, it was exchanged with DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Sigma) without Serum, and the compound of the present invention was incubated with 100 uM treatment. The same amount of PBS that did not dissolve the compound was added as a control.

In the collagen production promotion test, after culturing for 2 days, the culture medium was collected, and the concentration of type I pro collagen secreted in the culture medium was measured by enzyme-linked immunoassay (ELSIA, Procollagen type I c-peptide EIA Kit; manufactured by R & D system). Quantification by

Based on the quantitative results, the amount of synthesized type I pro collagen (pg / ml) was measured, and the type I pro collagen production rate was calculated according to Equation 2 below, and the results are shown in Table 3 and FIG. 3.

[Equation 2]

Type I Pro Collagen Production Rate (%) = (Experiment Type I Pro Collagen Amount / Control Type I Pro Collagen Amount) * 100

division Skin Fibroblasts division Skin Fibroblasts Collagen Production (%) Collagen Production (%) Control 100 Control 100 Compound 1 102.3 Compound 21 107.3 Compound 2 101.3 Compound 22 132.3 Compound 3 102.3 Compound 23 105.6 Compound 4 122.3 Compound 24 105.3 Compound 5 126.3 Compound 25 102.3 Compound6 190.3 Compound26 155.1 Compound7 168.3 Compound27 100.6 Compound 8 175.4 Compound 28 185.6 Compound 9 99.4 Compound 29 111.1 Compound 10 160.4 Compound 30 187.1 Compound 11 110.2 Compound 31 137.5 Compound 12 110.3 Compound 32 133.8 Compound 13 113.2 Compound33 123.3 Compound 14 105.1 Compound 34 107.6 Compound 15 112.3 Compound 35 108.3 Compound 16 108.3 Compound36 113.3 Compound 17 99.8 Compound 37 108.6 Compound 18 105.3 Compound38 122.3 Compound 19 121.3 Compound39 102.3 Compound 20 112.3 Compound 40 107.3

Referring to Table 3 and Figure 3, all of the compounds 1 to 40 of the present invention show a collagen production promoting effect, it is confirmed that exhibits a sufficient effect on the prevention of skin wrinkles.

Experimental Example 4 Inhibitory Effects of Peptides on MMP-1 Production ( in vitro )

Human dermal fibroblasts (HDF: Human dermal fibroblast, manufactured by Thermo) were dispensed in 1 wells in 12 well plates at a concentration of 6.0 ⁴ cells / ml, and cultured at 37 ° C., 5% CO ₂ , and humidified conditions for 2 days. . As the culture medium, 1 ml of each medium containing DBS medium (Dulbecco's Modified Eagle's Medium, manufactured by Gibco) at 10% FBS (manufactured by Gibco) was used.

Subsequently, it was exchanged with DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Sigma) without Serum, and the compound of the present invention was incubated with 100 uM treatment. The same amount of PBS that did not dissolve the compound was added as a control.

In the MMP-1 production inhibition test, after culturing for 2 days, the culture medium was collected, and the concentration of MMP-1 secreted in the culture medium was quantified by enzyme-linked immunoassay (ELSIA, MMP-1 EIA Kit; manufactured by R & D system). It was.

Based on the quantitative results, the amount of synthesized MMP-1 (pg / ml) was measured, and MMP-1 production inhibition was calculated according to Equation 3 below, and the results are shown in Table 4 and FIG. 4.

[Equation 3]

% Inhibition of MMP-1 production = (experimental MMP-1 amount / control MMP-1 amount) * 100

division Skin fibroblasts
% Inhibition of MMP-1 production division Skin fibroblasts
% Inhibition of MMP-1 production Control 100 Control 100 Compound 1 70.3 Compound 21 87.2 Compound 2 80.2 Compound 22 64.5 Compound 3 82.3 Compound 23 88.1 Compound 4 94.3 Compound 24 80.1 Compound 5 87.6 Compound 25 77.1 Compound6 50.1 Compound26 49.5 Compound7 87.3 Compound27 66.5 Compound 8 60.7 Compound 28 44.4 Compound 9 87.9 Compound 29 87.2 Compound 10 40.1 Compound 30 35.1 Compound 11 87.9 Compound 31 87.6 Compound 12 97.5 Compound 32 92.2 Compound 13 87.9 Compound33 68.2 Compound 14 78.1 Compound 34 77.1 Compound 15 88.4 Compound 35 69.9 Compound 16 87.9 Compound36 78.1 Compound 17 91.0 Compound37 88.2 Compound 18 89.8 Compound38 90.3 Compound 19 87.5 Compound39 91.2 Compound 20 81.2 Compound 40 87.2

Referring to Table 4 and Figure 4, the compounds 1 to 40 of the present invention was confirmed that all of the excellent inhibitory effect of MMP-1 production, based on this it was confirmed that it has a sufficient effect on the skin wrinkle prevention and other peptides Compared with the MMP-1 production inhibitory effect was higher.

<110> Cellicon Lab Inc. <120> Cosmetic composition containing a production method of peptide          that promotes collagen synthesis in human fibroblasts and          controls revitalization of collagen decomposing enzymes. <130> 170324-5 <160> 40 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 1 Cys Arg Gly Lys   One <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 2 Cys Arg Pro Lys   One <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 3 Cys Arg Asn Lys   One <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 4 Cys Arg Lys Lys   One <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 5 Cys arg ser lys   One <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 6 Cys Arg Tyr Lys   One <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 7 Cys Arg Asp Lys   One <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 8 Cys arg phe lys   One <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 9 Cys arg leu lys   One <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 10 Cys Arg Trp Lys   One <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 11 Met phe lys cys   One <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 12 Ser Phe Lys Cys   One <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 13 Lys Phe Lys Cys   One <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 14 His Phe Lys Cys   One <210> 15 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 15 Trp Phe Lys Cys   One <210> 16 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 16 Thr Phe Lys Cys   One <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 17 Val Phe Lys Cys   One <210> 18 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 18 Gly Phe Lys Cys   One <210> 19 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 19 Arg Phe Lys Cys   One <210> 20 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 20 Tyr Phe Lys Cys   One <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 21 Lys Gly Arg Cys Cys Arg Gly Lys   1 5 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 22 Lys Pro Arg Cys Cys Arg Pro Lys   1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 23 Lys Asn Arg Cys Cys Arg Asn Lys   1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 24 Lys Lys Arg Cys Cys Arg Lys Lys   1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 25 Lys Ser Arg Cys Cys Arg Ser Lys   1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 26 Lys Tyr Arg Cys Cys Arg Tyr Lys   1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 27 Lys Asp Arg Cys Cys Arg Asp Lys   1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 28 Lys Phe Arg Cys Cys Arg Phe Lys   1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 29 Lys Leu Arg Cys Cys Arg Leu Lys   1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 30 Lys Trp Arg Cys Cys Arg Trp Lys   1 5 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 31 Met Phe Lys Cys Cys Lys Phe Met   1 5 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 32 Ser Phe Lys Cys Cys Lys Phe Ser   1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 33 Lys Phe Lys Cys Cys Lys Phe Lys   1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 34 His Phe Lys Cys Cys Lys Phe His   1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 35 Trp Phe Lys Cys Cys Lys Phe Trp   1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 36 Thr Phe Lys Cys Cys Lys Phe Thr   1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 37 Val Phe Lys Cys Cys Lys Phe Val   1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 38 Gly Phe Lys Cys Cys Lys Phe Gly   1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 39 Arg Phe Lys Cys Cys Lys Phe Arg   1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide that promotes collagen synthesis <400> 40 Tyr Phe Lys Cys Cys Lys Phe Tyr   1 5

Claims (7)

Collagen synthesis promoting peptides corresponding to the compounds 21 to 40 are

[Cys-Arg-Gly-Lys] ₂ (Compound 21),
Cys-Arg-Pro-Lys ₂ (compound 22),
[Cys-Arg-Asn-Lys] ₂ (compound 23),
[Cys-Arg-Lys-Lys] ₂ (compound 24),
Cys-Arg-Ser-Lys ₂ (compound 25),
Cys-Arg-Tyr-Lys ₂ (compound 26),
[Cys-Arg-Asp-Lys] ₂ (Compound 27),
Cys-Arg-Phe-Lys ₂ (compound 28),
Cys-Arg-Leu-Lys ₂ (compound 29),
Cys-Arg-Trp-Lys ₂ (compound 30),
Met-Phe-Lys-Cys ₂ (compound 31),
Ser-Phe-Lys-Cys ₂ (compound 32),
Lys-Phe-Lys-Cys ₂ (compound 33),
His-Phe-Lys-Cys ₂ (compound 34),
[Trp-Phe-Lys-Cys] ₂ (Compound 35),
Thr-Phe-Lys-Cys ₂ (compound 36),
Val-Phe-Lys-Cys ₂ (compound 37),
Gly-Phe-Lys-Cys ₂ (compound 38),
Arg-Phe-Lys-Cys ₂ (compound 39), and
[Tyr-Phe-Lys-Cys] ₂ (Compound 40)
And, the compounds 21 to 40 _{[]. 2} is a cysteine residue of a thiol collagen synthetic peptide promoting, characterized in that indicating the duplex modified with reduced material form (-SS-) to (-SH).
delete A pharmaceutical composition for preventing and treating skin wrinkles, comprising at least one peptide selected from the peptides according to claim 1.

delete The method of claim 3, wherein
The peptide is a pharmaceutical composition for preventing and treating skin wrinkles, characterized in that contained in 0.0001 ~ 1.0% by weight in the pharmaceutical composition.
Cosmetic composition for preventing and improving skin wrinkles, characterized in that it contains at least one peptide selected from the peptides according to claim 1.
The method of claim 6
The peptide is a cosmetic composition for improving wrinkles, characterized in that contained in the cosmetic composition 0.0001 ~ 1.0% by weight, lotion, milk, gel, cream, essence, pack, ampoule, lotion, cleaning agent, soap, body products, soap, Cosmetic composition for preventing and improving skin wrinkles, characterized in that selected from oils, lipsticks and foundations.

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