KR102007079B1 - Anti-aging peptide composition and cosmetic composition that prevents cells aging and improves wrinkles. - Google Patents

Abstract

The study on skin aging is to protect the skin cells by developing antioxidant and anti-aging substances in a manner that promotes the synthesis of collagen and inhibits biosynthesis or inhibits the activity of the matrix metalloproteinases (MMPs). To develop a peptide having a specific sequence according to the formula (1) to 4 to promote the proliferation of fibroblasts, induce the production of extracellular matrix such as collagen, skin by using this by confirming the wrinkle reduction in vivo as well as in vitro assay A cosmetic composition capable of inhibiting and preventing aging has been developed. In addition, 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.
[Formula 1]
[Cys-Val-Val-X-Gln]
[Formula 2]
Val-Val-X-Gln-Cys
[Formula 3]
[Cys-Val-Val-X-Gln] ₂
[Formula 4]
Val-Val-X-Gln-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

Anti-aging peptide composition and cosmetic composition for inhibiting cell aging and wrinkle improvement using peptides {Anti-aging peptide composition and cosmetic composition that prevents cells aging and improves wrinkles.}

The present invention relates to a cosmetic composition having a specific peptide component, and furthermore, to verify the effect of increasing the proliferation of fibroblasts and collagen synthesis and in vitro wrinkle improvement effect in addition to the in vitro assay to improve the skin wrinkles and anti-aging effect The present invention relates to a method for producing a peptide and a composition containing the same.

The present invention relates to a peptide for improving skin wrinkles and to cosmetics for improving skin wrinkles containing the same. More specifically, the peptide according to the present invention relates to matrix metalloproteinases related to collagen synthesis and collagen degradation related to skin wrinkle generation. MMP) inhibits secretion and has an effect of improving wrinkles.

Extracellular in the dermis in the skin due to wrinkles, sagging, pigmentation, etc., which are the main clinical signs of skin aging accompanying skin wrinkles caused by intrinsic and external factors, such as skin photoaging due to UV stimulation. A decrease in substrate protein. Type I collagen is the most abundant extracellular matrix protein in skin connective tissue, and elastin, fibronectin, integrin and fibrillin are also present. Collagen, which accounts for most of the dermis, protects the body from external physiological and chemical stimuli, and elastic elastin is involved in skin elasticity.

Recently, as living standards improve and interest in skin beauty increases, interest in skin aging is increasing. It is impossible to prevent skin aging, but various attempts have been made to suppress and slow skin aging and its representative symptoms, skin wrinkles. Therefore, recent studies on skin aging have been developed to protect the skin cells by developing antioxidant and anti-aging substances by promoting collagen synthesis and inhibiting biosynthesis or inhibiting the activity of matrix metalloproteinases (MMPs). Various studies have been conducted (Gilchrest BA., 1989, Bernstein EF. Et al., 1994).

As an active ingredient of functional cosmetics, it is possible to efficiently penetrate into skin cells, and even when absorbed by skin cells, there is a need to develop a material that can be stably existed for a long time without being degraded in vivo, and at the same time has no skin toxicity. Therefore, the present inventors have found that peptides having a specific sequence have excellent activity against skin aging inhibition and prevention by promoting the proliferation of fibroblasts and inducing the production of extracellular matrix such as collagen.

 ‘Aging and photoaging: an overview.’ Gilchrest BA, J Am Acad Dermatol., 1989; 21: 610-3.  'Elastin and fibrillin gene expression in chronically photodamaged skin.'Bernstein EF. et al., J Invest Dermatol., 1994; 103 (2): 182-6.

The inventors have found that peptides with specific sequences have excellent activity for wound healing and skin regeneration by promoting the proliferation of fibroblasts and inducing the production of extracellular matrix such as collagen. Accordingly, the present invention aims to provide certain peptides.

 The present invention, by using the composition utilizing the peptide, it is possible to provide a functional cosmetics for wrinkle improvement imparted functionality such as UV protection and anti-aging.

 The present inventors have described that it can be usefully used as a cosmetic composition for improving skin wrinkles containing a functional peptide.

[Formula 1]

[Cys-Val-Val-X-Gln]

[Formula 2]

Val-Val-X-Gln-Cys

[Formula 3]

[Cys-Val-Val-X-Gln] ₂

[Formula 4]

Val-Val-X-Gln-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 (-SS-).

Preferably, the peptide of Formula 1 to 4,

[Cys-Val-Val-Tyr-Gln] (Compound 1)

[Cys-Val-Val-Asp-Gln] (Compound 2)

[Cys-Val-Val-Phe-Gln] (Compound 3)

[Cys-Val-Val-Ser-Gln] (Compound 4)

[Cys-Val-Val-Leu-Gln] (Compound 5)

[Cys-Val-Val-Lys-Gln] (Compound 6)

[Cys-Val-Val-Asn-Gln] (Compound 7)

[Cys-Val-Val-Pro-Gln] (Compound 8)

[Cys-Val-Val-Gly-Gln] (Compound 9)

[Cys-Val-Val-Thr-Gln] (Compound 10)

Val-Val-Gln-Gln-Cys (Compound 11)

Val-Val-Trp-Gln-Cys (Compound 12)

Val-Val-Ser-Gln-Cys (Compound 13)

Val-Val-Glu-Gln-Cys (Compound 14)

Val-Val-Asn-Gln-Cys (Compound 15)

Val-Val-His-Gln-Cys (Compound 16)

Val-Val-Ala-Gln-Cys (Compound 17)

Val-Val-Ile-Gln-Cys (Compound 18)

Val-Val-Asp-Gln-Cys (Compound 19)

Val-Val-Thr-Gln-Cys (Compound 20)

Geological (Stearic, Palmitic, arachidic, etc.)

[Cys-Val-Val-Phe-Gln] (Compound 21)

[Cys-Val-Val-Pro-Gln] (Compound 22)

[Cys-Val-Val-Gly-Gln] (Compound 23)

[Cys-Val-Val-Thr-Gln] (Compound 24)

Val-Val-Thr-Gln-Cys (Compound 25)

[Cys-Val-Val-Tyr-Gln] ₂ (Compound 26)

[Cys-Val-Val-Asp-Gln] ₂ (Compound 27)

[Cys-Val-Val-Phe-Gln] ₂ (Compound 28)

[Cys-Val-Val-Ser-Gln] ₂ (Compound 29)

[Cys-Val-Val-Leu-Gln] ₂ (Compound 30)

[Cys-Val-Val-Lys-Gln] ₂ (Compound 31)

[Cys-Val-Val-Asn-Gln] ₂ (Compound 32)

[Cys-Val-Val-Pro-Gln] ₂ (Compound 33)

[Cys-Val-Val-Gly-Gln] ₂ (Compound 34)

[Cys-Val-Val-Thr-Gln] ₂ (Compound 35)

Val-Val-Gln-Gln-Cys ₂ (compound 36)

Val-Val-Trp-Gln-Cys ₂ (compound 37)

Val-Val-Ser-Gln-Cys ₂ (compound 38)

Val-Val-Glu-Gln-Cys ₂ (compound 39)

Val-Val-Asn-Gln-Cys ₂ (compound 40)

Val-Val-His-Gln-Cys ₂ (compound 41)

Val-Val-Ala-Gln-Cys ₂ (compound 42)

Val-Val-Ile-Gln-Cys ₂ (compound 43)

Val-Val-Asp-Gln-Cys ₂ (compound 44)

Val-Val-Thr-Gln-Cys ₂ (compound 45)

Geological (Stearic, Palmitic, arachidic, etc.)

[Cys-Val-Val-Phe-Gln] ₂ (Compound 46)

[Cys-Val-Val-Pro-Gln] ₂ (Compound 47)

[Cys-Val-Val-Gly-Gln] ₂ (Compound 48)

[Cys-Val-Val-Thr-Gln] ₂ (Compound 49)

Val-Val-Thr-Gln-Cys ₂ (compound 50)

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

The present invention also provides a 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 50.

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 peptides of Compounds 1 to 50. 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 compounds 1 to 50. The peptide may be included in the cosmetic composition in an amount of 0.0001 to 1.0% by weight. The cosmetics may be selected from lotion, milky lotion, gel, cream, essence, pack, ampoule, lotion, cleaning agent, soap, body products, soap, oil, lipstick and foundation.

Hereinafter, the present invention will be described in detail.

Among the peptides of the present invention, the peptides of Compounds 26 to 45 are each modified by reducing the thiol group (-SH), which is a cysteine (cysteine located at the terminal of the peptide) residues (-SS-) of the peptides of Compounds 1 to 20, respectively. 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 26, and a duplex, in which a thiol group, which is a cysteine residue of Compound 2, is reduced, is Compound 27. In this manner, the duplexes of Compounds 1 to 25 may be prepared using Compounds 3 to 28, 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- It may be a methyl benzyl hydryl amine resin (rink 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.

The inventors have found that peptides with specific sequences have excellent activity for wound healing and skin regeneration by promoting the proliferation of fibroblasts and inducing the production of extracellular matrix such as collagen. Accordingly, the present invention aims to provide certain peptides.

The present invention, by using the composition utilizing the peptide, it is possible to provide a functional cosmetics for wrinkle improvement imparted functionality such as UV protection and anti-aging.

 The present inventors have described that it can be usefully used as a cosmetic composition for improving skin wrinkles containing a functional peptide.

Figure 1 shows the cell viability of the peptide of the present invention.
Figure 2 is a graphical representation of the fibroblast proliferation promoting effect of the compounds of the present invention.
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.
Figure 5 is a graphical representation of the effect of improving the eye wrinkles of the composition containing a compound 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: Metheonine / Trp: Tryptophan / Glu: Glutamine /

71.4 mg (0.10 mmol) of 2-chlorotrityl chloride resin (1.4 mmol loaded per gram) purchased 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 repeatedly, at which 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-Gln-OH;

(ii) Fmoc-Tyr-OH;

(iii) Fmoc-Val-OH;

(iv) Fmoc-Val-OH;

(v) 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-Val-Val-Tyr-Gln] -CO ₂ H (100 mg) as a trifluoroacetate salt in white powder form.

Compound 1: H ₂ N- [Cys-Val-Val-Tyr-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 610.8; (103 mg)

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

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

Compound 2: H ₂ N- [Cys-Val-Val-Asp-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 562.71; (104 mg)

Compound 3: H ₂ N- [Cys-Val-Val-Phe-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 594.8; (109 mg)

Compound 4: H ₂ N- [Cys-Val-Val-Ser-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 534.7; (101mg)

Compound 5: H ₂ N- [Cys-Val-Val-Leu-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 560.77; (115 mg)

Compound 6: H ₂ N- [Cys-Val-Val-Lys-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 575.8; (113mg)

Compound 7: H ₂ N- [Cys-Val-Val-Asn-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 561.73; (108mg)

Compound 8: H ₂ N- [Cys-Val-Val-Pro-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 544.74; (119 mg)

Compound 9: H ₂ N- [Cys-Val-Val-Gly-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 504.68; (102 mg)

Compound 10: H ₂ N- [Cys-Val-Val-Thr-Gln] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 548.73; (107 mg)

Compound 11: H ₂ N- [Val-Val-Gln-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 575.76; (104 mg)

Compound 12: H ₂ N- [Val-Val-Trp-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 633.84; (110 mg)

Compound 13: H ₂ N- [Val-Val-Ser-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 534.7; (100 mg)

Compound 14: H ₂ N- [Val-Val-Glu-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 576.74; (102 mg)

Compound 15: H ₂ N- [Val-Val-Asn-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 561.73; (107 mg)

Compound 16: H ₂ N- [Val-Val-His-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 584.77; (103 mg)

Compound 17: H ₂ N- [Val-Val-Ala-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 518.7; (103 mg)

Compound 18: H ₂ N- [Val-Val-Ile-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 560.78; (107 mg)

Compound 19: H ₂ N- [Val-Val-Asp-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 562.71; (101mg)

Compound 20: H ₂ N- [Val-Val-Thr-Gln-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 548.73; (112 mg)

<Example 1-3. Synthesis of Compound 26>

The collagen synthesis promoting peptide H ₂ N- [Cys-Val-Val-Tyr-Gln] -CO ₂ H (50 mg) was dissolved in 2 ml of dimethyl sulfoxide (DMSO) and 10 ml of water was added thereto for 3 days. The mixture was stirred at 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 26: H ₂ N- [Cys-Val-Val-Tyr-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1221.6; (57 mg)

<Example 1-4. Synthesis of Compounds 26-45>

Using the same manufacturing process as in Example 1-3, but by changing the order of amino acids protected with Fmoc to prepare a peptide of the compounds 26 ~ 45.

Compound 27: H ₂ N- [Cys-Val-Val-Asp-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1125.42; (53 mg)

Compound 28: H ₂ N- [Cys-Val-Val-Phe-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1189.6; (52 mg)

Compound 29: H ₂ N- [Cys-Val-Val-Ser-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1069.4; (49 mg)

Compound 30: H ₂ N- [Cys-Val-Val-Leu-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1121.54; (44 mg)

Compound 31: H ₂ N- [Cys-Val-Val-Lys-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1151.6; (47 mg)

Compound 32: H ₂ N- [Cys-Val-Val-Asn-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1123.46; (59 mg)

Compound 33: H ₂ N- [Cys-Val-Val-Pro-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1089.48; (51 mg)

Compound 34: H ₂ N- [Cys-Val-Val-Gly-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1009.36; (44 mg)

Compound 35: H ₂ N- [Cys-Val-Val-Thr-Gln] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1097.46; (52 mg)

Compound 36: H ₂ N- [Val-Val-Gln-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1151.52; (49 mg)

Compound 37: H ₂ N- [Val-Val-Trp-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1267.68; (45 mg)

Compound 38: H ₂ N- [Val-Val-Ser-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1069.4; (56 mg)

Compound 39: H ₂ N- [Val-Val-Glu-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1153.48; (51 mg)

Compound 40: H ₂ N- [Val-Val-Asn-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1123.46; (48 mg)

Compound 41: H ₂ N- [Val-Val-His-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1169.54; (42 mg)

Compound 42: H ₂ N- [Val-Val-Ala-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1037.4; (52 mg)

Compound 43: H ₂ N- [Val-Val-Ile-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1121.56; (47 mg)

Compound 44: H ₂ N- [Val-Val-Asp-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1125.42; (42 mg)

Compound 45: H ₂ N- [Val-Val-Thr-Gln-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1097.46; (44 mg)

<Example 1-5. Synthesis of Compound 21>

Of the 5 peptides selected from the 20 peptides, DMF (4ML), 1.06 mL (10 eq) and DIPEA 1.96 mL (10 eq) were added to the compound 3 peptide before the peptide was cleaved from the resin. 5 washes with 10 mL of DMF solvent (5 washes with 10 mL each) * DMF: Dimethylformamide reaction was confirmed by Kaiser test, and then trifluoroacetic acid / thio Peptides were cleaved from the resin using a mixture of anisol / ethanedithiol / triisopropylsilane / water (95: 5: 2.5: 2.5: 2.5) (10 mL). 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 the collagen synthesis promoting peptide Stearyl- [Cys-Val-Pal-Phe-Gln] -CO ₂ H (102 mg) as a trifluoroacetate salt in white powder form. Compounds 21 to 25 were synthesized in the same manner.

* Type of Geology

Ste) Stearic acid

Ii) Palmitic acid

Arachidic acid

O) Oleic acid

Erucic acid

Lin) Linoleic acid

Iii) Linolenic acid

Through experiments on collagen synthesis and mmp-1 synthesis of 20 compounds of Examples 1-1 and 1-2 as described above, experiments such as skin permeability and stability were selectively attached to the top five high collagen synthesis rates. Proceeded.

Compound 21: Stearyl- [Cys-Val-Val-Phe-Gln] -CO ₂ H

Compound 22: Palmitoyl- [Cys-Val-Val-Pro-Gln] -CO ₂ H

Compound 23: Linoleyl- [Cys-Val-Val-Gly-Gln] -CO ₂ H

Compound 24: Linolenyl- [Cys-Val-Val-Thr-Gln] -CO ₂ H

Compound 25: Erucyl- [Val-Val-Thr-Gln-Cys] -CO ₂ H

<Example 1-6. Synthesis of Compound 41>

The collagen synthetase-promoting peptide Stearyl- [Cys-Val-Pal-Phe-Gln] -CO ₂ H (53 mg) methyl sulfoxide (DMSO) obtained in Example 1-5 was dissolved in 10 ml of water. After stirring for 3 days at room temperature, the final collagen synthesis promoting peptide Stearyl- [Cys-Val-Val in the form of a dimer in which the thiol group (-SH), which is a cysteine residue, is transformed into a reduced form (-S-S-). -Phe-Gln] ₂ -CO ₂ H (50 mg). Compounds 46 to 50 were synthesized in the same manner.

Compound 46: Stearyl- [Cys-Val-Val-Phe-Gln] ₂ -CO ₂ H

Compound 47: Palmitoyl- [Cys-Val-Val-Pro-Gln] ₂ -CO ₂ H

Compound 48: Linoleyl- [Cys-Val-Val-Gly-Gln] ₂ -CO ₂ H

Compound 49: Linolenyl- [Cys-Val-Val-Thr-Gln] ₂ -CO ₂ H

Compound 50: Erucyl- [Val-Val-Thr-Gln-Cys] ₂ -CO ₂ H

Examples 1-3,1-4 As described above, through experiments on collagen synthesis and mmp-1 synthesis inhibition of 20 compounds, the top five high collagen synthesis rates were selectively attached to lipids to conduct skin permeability and stability. It was selected in the same manner 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, a medium containing 5% of FBS (manufactured by Gibco) in 5% DMEM medium (Dulbecco's Modified Eagle's Medium, manufactured by Gibco) was used for each well.

Subsequently, DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Sigma) without Serum was exchanged, 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 1 hour. 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 below. 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 96.7 Compound 26 102.2 Compound 2 90.1 Compound 27 103.2 Compound 3 100.8 Compound 28 99.3 Compound 4 97.1 Compound 29 107.4 Compound 5 98.3 Compound 30 103.2 Compound 6 99.7 Compound 31 100.9 Compound 7 100.3 Compound 32 90.4 Compound 8 100.7 Compound 33 91.2 Compound 9 96.8 Compound 34 100.9 Compound 10 105.6 Compound 35 107.2 Compound 11 107.2 Compound 36 108.3 Compound 12 98.3 Compound 37 99.6 Compound 13 97.3 Compound 38 94.2 Compound 14 100.3 Compound 39 91.2 Compound 15 100.7 Compound 40 93.8 Compound 16 98.2 Compound 41 90.2 Compound 17 97.3 Compound 42 99.7 Compound 18 94.2 Compound 43 98.4 Compound 19 105.3 Compound 44 96.5 Compound 20 103.2 Compound 45 102.3 Compound 21 105.4 Compound 46 101.2 Compound 22 99.7 Compound 47 107.5 Compound 23 91.2 Compound 48 103.3 Compound 24 98.3 Compound 49 99.6 Compound 25 108.2 Compound 50 94.2

Referring to Table 1 and Figure 1, all the 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 promoting effect in fibroblasts (in vitro)

[3H] -Thymidine uptake assay was performed to confirm the peptide effect on the proliferation of human dermal fibroblast (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 12 days for a total of 48 hours 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 1 101.2 Compound 26 107.2 Compound 2 102.5 Compound 27 150.3 Compound 3 165.3 Compound 28 166.8 Compound 4 105.4 Compound 29 105.7 Compound 5 101.2 Compound 30 118.3 Compound 6 101.3 Compound 31 107.4 Compound 7 132.2 Compound 32 138.3 Compound 8 178.2 Compound 33 186.4 Compound 9 172.1 Compound 34 170.2 Compound 10 190.2 Compound 35 189.7 Compound 11 106.3 Compound 36 111.1 Compound 12 112.3 Compound 37 122.4 Compound 13 132.2 Compound 38 132.5 Compound 14 134.2 Compound 39 142.1 Compound 15 120.4 Compound 40 143.8 Compound 16 119.8 Compound 41 111.6 Compound 17 102.7 Compound 42 117.5 Compound 18 103.6 Compound 43 120.3 Compound 19 112.3 Compound 44 140.5 Compound 20 198.9 Compound 45 200.2 Compound 21 185.8 Compound 46 111.9 Compound 22 171.2 Compound 47 108.4 Compound 23 183.2 Compound 48 105.2 Compound 24 198.9 Compound 49 117.4 Compound 25 237.2 Compound 50 145.5

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

Human dermal fibroblasts (HDF: Human dermal fibroblast, manufactured by Thermo Co., Ltd.) were dispensed in a 12 well plate at a concentration of 6.0 ⁴ cells / ml in 1 ml portions, 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) with 5% FBS (manufactured by Gibco) was used.

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

In the collagen production promotion test, after 48 hours of incubation, 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
Collagen Production (%) division Skin fibroblasts
Collagen Production (%) Control 100 Control 100 Compound 3 165.3 Compound 28 182.2 Compound 8 175.4 Compound 33 205.1 Compound 9 197.7 Compound 34 225.1 Compound 10 200.3 Compound 35 186.3 Compound 20 204.2 Compound 45 178.3 Compound 21 208.9 Compound 46 180.6 Compound 22 196.2 Compound 47 203.3 Compound 23 189.3 Compound 48 211.3 Compound 24 197.5 Compound 49 202.3 Compound 25 200.3 Compound 50 210.3

Referring to Table 3 and Figure 3, all of the compounds of the present invention showed a collagen production promoting effect, it was confirmed to exhibit a sufficient effect on the prevention of skin wrinkles.

Experimental Example  4 : Peptide MMP -1 suppression effect ( in vitro )

Human dermal fibroblasts (HDF: Human dermal fibroblast, manufactured by Thermo Co., Ltd.) were dispensed in a 12 well plate at a concentration of 6.0 ⁴ cells / ml in 1 ml portions, 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) with 10% FBS (manufactured by Gibco) was used.

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

In the MMP-1 production inhibition rate 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 3 70.2 Compound 28 37.5 Compound 8 75.4 Compound 33 45.6 Compound 9 67.7 Compound 34 56.3 Compound 10 55.3 Compound 35 36.2 Compound 20 80.2 Compound 45 50.3 Compound 21 38.9 Compound 46 44.5 Compound 22 50.2 Compound 47 46.3 Compound 23 29.2 Compound 48 50.1 Compound 24 30.5 Compound 49 36.2 Compound 25 40.4 Compound 50 40.2

Referring to Table 4 and Figure 4, all of the compounds of the present invention was confirmed that the effect of inhibiting the production of MMP-1 was excellent, based on this it was confirmed that it has a sufficient effect on the prevention of skin wrinkles, among the plurality of peptide compounds, lipid The peptides had lower inhibitory effect on MMP-1 production than other peptides.

Experimental Example  5: eye wrinkle improvement effect human application test ( PRIMOS  High Resolution

In order to confirm the wrinkle improvement effect of the cosmetic composition according to the present invention, 22 Korean women aged 35 to 50 years were evaluated in the same manner as the daily use of the cream for each 4 weeks. The evaluation was performed by using PRIMOS High Resolution (Phaseshift Rapid In vivo Measurement Of Skin high resolution, manufactured by GFMesstechinik GmbH, Germany). The value of the eye wrinkle variable through roughness analysis is as follows.

Ra (Average roughness): The average of the absolute values of the lengths from the centerline to the cross-sectional curve of the surface when the centerline is drawn at the roughness height of the cross section.

Root mean square roughness (Rq): The root mean square root of the length from the center line to the cross-sectional curve of the surface when the center line is drawn at the roughness height of the cross section.

Rmax (Maximum roughness depth): The difference between the highest peak and the lowest valley within a single measurement range.

The smaller the value of the variable analyzed by the roughness measurement, the better the wrinkles. The decrease rate of the roughness parameter due to PRIMOS High Resolution was calculated according to Equation 4. The calculated values for each variable are shown in Table 5 and FIG.

[Equation 4]

% Reduction = (Measurement before application-Measurement after application / Measurement before application) * 100

Roughness Variable 2 weeks after application 4 weeks after application Ra 3.264 8.165 Rq 2.121 5.778 Rmax 1.651 3.588

As a result of analyzing the measured values of the roughness parameters of the wrinkles around the eye area using PRIMOS High Resolution, the Ra value after the use of Experiment 1 cream was applied at a statistically significant level (p <0.05) after 2 weeks of application. After 4 weeks, the decrease was 3.264% and 8.165%, respectively. The Rq value was also decreased after 2 weeks and after 4 weeks of application to a statistically significant level (p <0.05). In addition, the Rmax value decreased after 4 weeks of application to a statistically significant level (p <0.05) compared to before sample application. Therefore, the cream of Experimental Example 1 was confirmed to help improve eye wrinkles after two weeks of application, four weeks after application.

Experimental Example  6: Evaluation of the wrinkle improvement effect around the eyes (visual evaluation)

In order to confirm the wrinkle improvement effect of the cosmetic composition according to the present invention, 20 Korean women aged 35 to 50 years were evaluated in the same manner as the daily use of cream for 8 weeks.

The cosmetic preparations prepared according to Experimental Example 1 and Comparative Example 1 were used separately on each side of the face, and the wrinkle improvement effect of the subject was evaluated through visual observation by an experienced doctor.

division Wrinkle improvement effect Great slightly none Effectiveness ( % ) Experimental Example 1 13 5 2 90 Comparative Example 1 2 3 15 10

Referring to Table 6, it can be confirmed that the cosmetic preparations including collagen synthesis promoting peptides as in Experimental Example 1 was significantly improved wrinkle improvement effect than the cosmetics prepared without the above ingredients as in Comparative Example 1.

As mentioned above, although preferred embodiments of the present invention have been described, those skilled in the art can understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. There will be. Therefore, it should be understood that one embodiment described above is illustrative in all respects and not restrictive.

Claims (7)

Collagen synthesis promoting peptides corresponding to the following compounds 26 to 45

[Cys-Val-Val-Tyr-Gln] ₂ (Compound 26)
[Cys-Val-Val-Asp-Gln] ₂ (Compound 27)
[Cys-Val-Val-Phe-Gln] ₂ (Compound 28)
[Cys-Val-Val-Ser-Gln] ₂ (Compound 29)
[Cys-Val-Val-Leu-Gln] ₂ (Compound 30)
[Cys-Val-Val-Lys-Gln] ₂ (Compound 31)
[Cys-Val-Val-Asn-Gln] ₂ (Compound 32)
[Cys-Val-Val-Pro-Gln] ₂ (Compound 33)
[Cys-Val-Val-Gly-Gln] ₂ (Compound 34)
[Cys-Val-Val-Thr-Gln] ₂ (Compound 35)
Val-Val-Gln-Gln-Cys ₂ (compound 36)
Val-Val-Trp-Gln-Cys ₂ (compound 37)
Val-Val-Ser-Gln-Cys ₂ (compound 38)
Val-Val-Glu-Gln-Cys ₂ (compound 39)
Val-Val-Asn-Gln-Cys ₂ (compound 40)
Val-Val-His-Gln-Cys ₂ (compound 41)
Val-Val-Ala-Gln-Cys ₂ (compound 42)
Val-Val-Ile-Gln-Cys ₂ (compound 43)
Val-Val-Asp-Gln-Cys ₂ (compound 44)
Val-Val-Thr-Gln-Cys ₂ (compound 45)
And Compounds 26 to 45 _{[]. 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 Composition for preventing and improving skin wrinkles, characterized in that it contains at least one peptide selected from the peptides according to claim 1.

delete The method of claim 3, wherein
The peptide is a composition for preventing and improving skin wrinkles, characterized in that it comprises 0.0001 to 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.

Images