KR102007077B1 - Cosmetic composition containing anti-aging and wrinkle preventing pentapeptide and pentapeptide dimer production method - Google Patents

Abstract

The present invention relates to a method for preparing a novel penta peptide and penta peptide dimer represented by the following Chemical Formulas 1 to 4 and to a cosmetic composition for preventing skin aging and preventing skin wrinkles containing the same as an active ingredient. Skin aging or skin with excellent effect of preventing skin aging or preventing skin wrinkles by increasing the amount of collagen type I and type II reduced collagen production in aged skin by including peptide and pentapeptide dimer as active ingredients It relates to a cosmetic composition for preventing and improving wrinkles. In particular, the pentapeptide and pentapeptide dimer for skin aging or wrinkle prevention and improvement can be used in addition to various basic cosmetic compositions such as skin, lotion, cream, essence, gel, pack or soap form, without skin irritation and skin Stability also has excellent characteristics.
[Formula 1]
[Cys-Gly-Gln-X-Arg]
[Formula 2]
Gly-X-Pro-Arg-Cys
[Formula 3]
[Cys-Gly-Gln-X-Arg] ₂
[Formula 4]
Gly-X-Pro-Arg-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 cysteine residue, a thiol group (-SH), in a reduced form (-SS-).)

Description

Preparation method of pentapeptide and pentapeptide dimer for preventing skin aging and skin wrinkles and cosmetic composition comprising the same {Cosmetic composition containing anti-aging and wrinkle preventing pentapeptide and pentapeptide dimer production method}

The present invention relates to an anti-aging and anti-wrinkle cosmetic composition containing pentapeptide and pentapeptide dimer for preventing skin aging and preventing skin wrinkles as an active ingredient, and is particularly important in cosmetics for preventing skin aging and improving skin wrinkles. The present invention relates to a method for preparing a novel pentapeptide and pentapeptide dimer that promotes the synthesis and expression of phosphorus collagen, and a cosmetic and pharmaceutical composition containing the same.

The skin of the human body is a membrane covering the outside of the body, which protects the body from harmful environmental elements and plays an external aesthetic function. As the skin ages, the skin immunity is lowered as the aging progresses, and the skin is accompanied with a change in skin elasticity, skin color change, and wrinkle formation. The skin aging process is classified into endogenous aging and exogenous aging. Endogenous aging is a natural aging that occurs naturally with age, and is an aging phenomenon that appears as a genetic factor. On the other hand, in the case of exogenous aging, aging is influenced by environmental factors such as ultraviolet rays, which can be prevented through artificial control. Therefore, research on the discovery of functional materials for the prevention of skin aging and the prevention of skin wrinkles has been actively conducted mainly on exogenous aging.

Environmental factors that cause exogenous aging include UV exposure, air pollution, and stress, and continuous exposure to these harmful environments increases oxygen free radicals in the body and collagen, elastin, and hyaluronic acid, which are the connective tissues of the dermis. The back is broken to form wrinkles. In particular, the highest proportion of the various factors causing exogenous aging is repeated UV exposure. UV (UV) is classified into ultraviolet A (UVA) having a wavelength of 320 to 400 nm, ultraviolet B (UVB) having a wavelength of 290 to 320 nm, and ultraviolet C (UVC) having a wavelength of 200 to 290 nm. ) Has been reported to be the main cause of exogenous aging that affects the skin most and causes photoaging.

Collagen is a major component of the dermis of the skin, of which type I collagen accounts for 80% of the total. When the skin is exposed to ultraviolet rays for a long time, a large amount of free radicals are generated in the skin, which causes collagen synthesis to degrade or degrade, resulting in decreased skin elasticity and wrinkles. Therefore, in discovering new materials for preventing skin aging and preventing and treating skin wrinkles, finding a substance that promotes collagen synthesis may be a good strategy.

Retinoic acid, which is currently used as an anti-aging and therapeutic agent for skin aging, has low skin absorption ability due to fat-soluble properties. Therefore, it is difficult to expect the effect of enhancing collagen synthesis in the dermis, and it is a skin irritant and a fast enzyme in vivo. Safety and stability issues due to degradation are emerging. Therefore, it is required to develop a new skin anti-aging and prevention material having excellent absorption from the skin, maximizing skin anti-aging and skin wrinkle prevention in vivo, and improving skin safety.

Meanwhile, in recent years, peptides have attracted attention as a functional ingredient for preventing skin aging and preventing skin wrinkles. Peptides are attracting attention in various research fields such as cosmetics, foodstuffs, and pharmaceuticals, and are polymers in which about 2 to 50 of the 20 essential amino acids are linked through amide bonds. Peptides are simpler in size and structure than proteins, but are known to play an important role in metabolism in vivo because they interact with proteins in vivo. Therefore, the peptide itself is not only a substance present in vivo, but also decomposed into amino acids after being inactivated for a certain period of time even when introduced into the body, and thus is more safe than other conventional materials, and also helps skin regeneration activity as a component of protein. State is the material.

Domestic Publication No. 10-2012-0012904

 Katayama et al. (1993) J. Biol. Chem., 268, 9941-9944  Shuster S., (1975) British Journal of Dermatology, 93, 639-643 The present invention was completed by confirming the sound.

Against this background, the present inventors established an in vitro assay using a human dermal fibroblast cell line to confirm the anti-aging and anti-wrinkle effects of the compound pentapeptide and pentapeptide dimer, thereby synthesizing the biosynthesis of two procollagens. The present invention was completed by confirming the ability of the compound pentapeptide and pentapeptide dimer to have anti-aging and anti-wrinkle effects.

Accordingly, an object of the present invention is to provide a method for preparing pentapeptide and pentapeptide dimer, which is harmless to human body and has excellent skin permeability or safety, and a pharmaceutical composition and a cosmetic composition exhibiting anti-aging and anti-wrinkle effect containing the same.

[Formula 1]

[Cys-Gly-Gln-X-Arg]

[Formula 2]

Gly-X-Pro-Arg-Cys

[Formula 3]

[Cys-Gly-Gln-X-Arg] ₂

[Formula 4]

Gly-X-Pro-Arg-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-Gly-Gln-Gly-Arg] (Compound 1)

[Cys-Gly-Gln-Pro-Arg] (Compound 2)

[Cys-Gly-Gln-Asn-Arg] (Compound 3)

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

[Cys-Gly-Gln-Asp-Arg] (Compound 5)

Cys-Gly-Gln-Tyr-Arg (Compound 6)

[Cys-Gly-Gln-Ser-Arg] (Compound 7)

[Cys-Gly-Gln-Phe-Arg] (Compound 8)

[Cys-Gly-Gln-Leu-Arg] (Compound 9)

Gly-Gln-Ile-Arg-Cys (Compound 10)

Gly-Met-Pro-Arg-Cys (Compound 11)

Gly-Thr-Pro-Arg-Cys (Compound 12)

Gly-Arg-Pro-Arg-Cys (Compound 13)

Gly-Leu-Pro-Arg-Cys (Compound 14)

Gly-Phe-Pro-Arg-Cys (Compound 15)

Gly-Ser-Pro-Arg-Cys (Compound 16)

Gly-Asp-Pro-Arg-Cys (Compound 17)

Gly-Asn-Pro-Arg-Cys (Compound 18)

Gly-Lys-Pro-Arg-Cys (Compound 19)

Gly-Glu-Pro-Arg-Cys (Compound 20)

Geological (Stearic, Palmitic, arachidic, etc.)

[Cys-Gly-Gln-Gly-Arg] (Compound 21)

Cys-Gly-Gln-Pro-Arg (Compound 22)

[Cys-Gly-Gln-Leu-Arg] (Compound 23)

Gly-Asn-Pro-Arg-Cys (Compound 24)

Gly-Glu-Pro-Arg-Cys (Compound 25)

Cys-Gly-Gln-Gly-Arg ₂ (Compound 26)

[Cys-Gly-Gln-Pro-Arg] ₂ (Compound 27)

[Cys-Gly-Gln-Asn-Arg] ₂ (Compound 28)

[Cys-Gly-Gln-Lys-Arg] ₂ (Compound 29)

Cys-Gly-Gln-Asp-Arg ₂ (Compound 30)

[Cys-Gly-Gln-Tyr-Arg] ₂ (Compound 31)

[Cys-Gly-Gln-Ser-Arg] ₂ (Compound 32)

[Cys-Gly-Gln-Phe-Arg] ₂ (Compound 33)

[Cys-Gly-Gln-Leu-Arg] ₂ (Compound 34)

Gly-Gln-Ile-Arg-Cys ₂ (Compound 35)

Gly-Met-Pro-Arg-Cys ₂ (Compound 36)

Gly-Thr-Pro-Arg-Cys ₂ (Compound 37)

Gly-Arg-Pro-Arg-Cys ₂ (compound 38)

Gly-Leu-Pro-Arg-Cys ₂ (Compound 39)

Gly-Phe-Pro-Arg-Cys ₂ (compound 40)

Gly-Ser-Pro-Arg-Cys ₂ (Compound 41)

Gly-Asp-Pro-Arg-Cys ₂ (Compound 42)

Gly-Asn-Pro-Arg-Cys ₂ (Compound 43)

Gly-Lys-Pro-Arg-Cys ₂ (compound 44)

Gly-Glu-Pro-Arg-Cys ₂ (compound 45)

Geological (Stearic, Palmitic, arachidic, etc.)

[Cys-Gly-Gln-Gly-Arg] ₂ (Compound 46)

[Cys-Gly-Gln-Pro-Arg] ₂ (Compound 47)

[Cys-Gly-Gln-Leu-Arg] ₂ (Compound 48)

Gly-Asn-Pro-Arg-Cys ₂ (Compound 49)

Gly-Glu-Pro-Arg-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 50 are each modified by reducing the thiol group (-SH), which is a cysteine (cysteine located at the terminal of the peptide) residues of the peptides of Compounds 1 to 25, 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 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- 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.

According to the present invention, there is no skin irritation, excellent skin stability, and a collagen biosynthesis enhancing effect, a method for producing penta peptide and penta peptide dimer and an excellent effect on showing skin aging prevention and skin wrinkle prevention effect containing the same as an active ingredient Dermal pharmaceutical compositions and cosmetic compositions are provided.

1 graphically depicts the effect of increasing the synthesis of procollagen type I of the compounds of the present invention.
Figure 2 graphically depicts the effect of increasing the collagen type II synthesis of the compounds of the present invention.
Figure 3 shows the cell viability 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: 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-Arg-OH;

(ii) Fmoc-Gly-OH;

(iii) Fmoc-Gln-OH;

(iv) Fmoc-Glu-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-Gly-Gln-Gly-Arg] -CO ₂ H (100 mg) as a trifluoroacetate salt in white powder form.

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

MS (ESI) m / e, [M + H] ⁺ = 416.49; (100 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-Gly-Gln-Pro-Arg] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 456.55; (109 mg)

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

MS (ESI) m / e, [M + H] ⁺ = 473.54; (101mg)

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

MS (ESI) m / e, [M + H] ⁺ = 487.61; (107 mg)

Compound 5: H ₂ N- [Cys-Gly-Gln-Asp-Arg] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 474.52; (105 mg)

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

MS (ESI) m / e, [M + H] ⁺ = 522.61; (110 mg)

Compound 7: H ₂ N- [Cys-Gly-Gln-Ser-Arg] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 446.51; (104 mg)

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

MS (ESI) m / e, [M + H] ⁺ = 506.61; (120 mg)

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

MS (ESI) m / e, [M + H] ⁺ = 472.59; (101mg)

Compound 10: H ₂ N- [Gly-Gln-Ile-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 472.59; (109 mg)

Compound 11: H ₂ N- [Gly-Met-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 459.61; (102 mg)

Compound 12: H ₂ N- [Gly-Thr-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 542.69; (105 mg)

Compound 13: H ₂ N- [Gly-Arg-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 429.52; (109 mg)

Compound 14: H ₂ N- [Gly-Leu-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 441.56; (110 mg)

Compound 15: H ₂ N- [Gly-Phe-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 576.71; (100 mg)

Compound 16: H ₂ N- [Gly-Ser-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 516.61; (102 mg)

Compound 17: H ₂ N- [Gly-Asp-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 475.59; (103 mg)

Compound 18: H ₂ N- [Gly-Asn-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 442.52; (103 mg)

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

MS (ESI) m / e, [M + H] ⁺ = 456.59; (107 mg)

Compound 20: H ₂ N- [Gly-Glu-Pro-Arg-Cys] -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 457.53; (109 mg)

<Example 1-3. Synthesis of Compound 26>

The collagen synthesizing peptide H ₂ N- [Cys-Glu-Gln-Gly-Arg] -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-Glu-Gln-Gly-Arg] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 832.98; (50 mg)

<Example 1-4. Synthesis of Compounds 27-45>

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 27: H ₂ N- [Cys-Glu-Gln-Pro-Arg] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 913.1; (52 mg)

Compound 28: H ₂ N- [Cys-Glu-Gln-Asn-Arg] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 947.08; (48 mg)

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

MS (ESI) m / e, [M + H] ⁺ = 957.22; (44 mg)

Compound 30: H ₂ N- [Cys-Glu-Gln-Asp-Arg] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 949.04; (49 mg)

Compound 31: H ₂ N- [Cys-Glu-Gln-Tyr-Arg] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1045.22; (48 mg)

Compound 32: H ₂ N- [Cys-Glu-Gln-Ser-Arg] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 893.02; (54 mg)

Compound 33: H ₂ N- [Cys-Glu-Gln-Phe-Arg] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1013.22; (50 mg)

Compound 34: H ₂ N- [Cys-Glu-Gln-Leu-Arg] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 945.16; (48 mg)

Compound 35: H ₂ N- [Gly-Gln-Ile-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 945.18; (52 mg)

Compound 36: H ₂ N- [Gly-Met-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 919.22; (52 mg)

Compound 37: H ₂ N- [Gly-Thr-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1085.38; (48 mg)

Compound 38: H ₂ N- [Gly-Arg-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 859.04; (56 mg)

Compound 39: H ₂ N- [Gly-Leu-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 883.12; (59 mg)

Compound 40: H ₂ N- [Gly-Phe-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1153.42; (52 mg)

Compound 41: H ₂ N- [Gly-Ser-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 1033.22; (42 mg)

Compound 42: H ₂ N- [Gly-Asp-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 951.18; (52 mg)

Compound 43: H ₂ N- [Gly-Asn-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 885.04; (48 mg)

Compound 44: H ₂ N- [Gly-Lys-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 913.18; (45 mg)

Compound 45: H ₂ N- [Gly-Glu-Pro-Arg-Cys] ₂ -CO ₂ H

MS (ESI) m / e, [M + H] ⁺ = 915.06; (46 mg)

<Example 1-5. Synthesis of Compound 21>

Of the five 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 1 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-Gly-Gln-Gly-Arg] -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 of collagen synthesis and mmp-1 synthesis of 20 compounds of Examples 1-1 and 1-2 as described above, an experiment was performed by selectively attaching the top five high collagen synthesis rates.

Compound 21: Stearyl- [Cys-Gly-Gln-Gly-Arg] -CO ₂ H

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

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

Compound 24: Linolenyl- [Gly-Asn-Pro-Arg-Cys] -CO ₂ H

Compound 25: Erucyl- [Gly-Glu-Pro-Arg-Cys] -CO ₂ H

<Example 1-6. Synthesis of Compound 46>

The collagen synthetase-promoting peptide Stearyl- [Cys-Gly-Gln-Gly-Arg] -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-Gly-Gln 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-). -Gly-Arg] ₂ -CO ₂ H (50 mg) was obtained. Compounds 41 to 45 were synthesized in the same manner.

Compound 46: Stearyl- [Cys-Gly-Gln-Gly-Arg] ₂ -CO ₂ H

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

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

Compound 49: Linolenyl- [Gly-Asn-Pro-Arg-Cys] ₂ -CO ₂ H

Compound 50: Erucyl- [Gly-Glu-Pro-Arg-Cys] ₂ -CO ₂ H

Example 1-3, 1-4 as described above through the experiment of collagen synthesis ability and mmp-1 synthesis inhibition of 20 compounds, the experiment was carried out by selectively attaching the top five high collagen synthesis rate to the lipid, the same method as the monomer Was selected.

Experimental Example  One : Peptide Procollagen  Ⅰ Synthesis Promotion Measure in vitro )

Human dermal fibroblasts (HDF: Human dermal fibroblast) (HDF) were dispensed in a 12 well plate at a concentration of 0.5 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) with 5% FBS (manufactured by Gibco) was used.

Subsequently, the cell culture solution was washed with PBS, exchanged with DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Sigma) without FBS (Gibco), and cultured at 37 ° C., 5% CO ₂ , and humidified conditions for one day. Proceeded. After incubation, the cells were washed with 1 ml of PBS (Phosphate Buffered Saline, manufactured by Sigma), and then treated with 50 uM of each compound of the present invention. 100 μl of PBS that did not dissolve the compound was used as a control.

In the procollagen I production promotion test, after adding the compound of the present invention, the cells were cultured for 3 days, and then 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).

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 1 below, and the results are shown in Table 1 and FIG. 1.

[Equation 1]

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

Comparison of Procollagen I Production Rate of Each Compound division Skin fibroblasts
Procollagen I production rate (%) division Skin fibroblasts
Procollagen I production rate (%) Compound 1 150.1 Compound 26 167.9 Compound 2 152.7 Compound 27 168.1 Compound 3 142.0 Compound 28 159.9 Compound 4 144.4 Compound 29 155.2 Compound 5 146.6 Compound 30 154.1 Compound 6 148.9 Compound 31 154.3 Compound 7 147.9 Compound 32 166.0 Compound 8 148.0 Compound 33 166.0 Compound 9 149.5 Compound 34 166.6 Compound 10 144.5 Compound 35 164.2 Compound 11 137.2 Compound 36 163.5 Compound 12 135.0 Compound 37 161.7 Compound 13 134.4 Compound 38 162.4 Compound 14 135.4 Compound 39 159.8 Compound 15 134.3 Compound 40 159.9 Compound 16 134.1 Compound 41 166.3 Compound 17 132.8 Compound 42 166.0 Compound 18 152.9 Compound 43 170.2 Compound 19 149.0 Compound 44 165.9 Compound 20 154.3 Compound 45 171.3 Compound 21 158.6 Compound 46 178.8 Compound 22 155.7 Compound 47 176.1 Compound 23 154.9 Compound 48 175.0 Compound 24 157.2 Compound 49 171.4 Compound 25 160.1 Compound 50 170.9

Referring to Table 1 and FIG. 1, the compounds 1 to 50 of the present invention all show the effect of promoting the production of procollagen I and are confirmed to exhibit sufficient effects on the prevention of skin aging and the prevention of skin wrinkles.

On the other hand, since the peptide is degraded by various hydrolases very quickly when penetrated into the body, its capacity decreases rapidly, so it is very important to slow down the degradation rate of the peptide. In general, various hydrolytic enzymes decompose the amide bond (-C (= O) NH-) portion of the peptide structure with carboxylic acid (-CO2H) and amine (-NH2). The dimer structure has a (-SS-) backbone that functions to lower or disrupt the degradation of common hydrolytic enzymes. That is, among the peptide compounds, compounds 26 to 50 in the duplex form are slower to be degraded by hydrolase in vivo than compounds 1 to 25 in the monomer form. Therefore, in the case of the compounds 26-50 which have a cysteine dimer among the peptide compounds of this invention, it is judged that the body stability is very excellent. Furthermore, lipidized peptides 21-25 and 46-50 had similar or higher levels of collagen production promotion than peptides that did not.

Experimental Example  2 : Peptide Procollagen  Measurement of Synthesis Promoting Effect) in vitro )

Human dermal fibroblasts (HDF: Human dermal fibroblast) (HDF) were dispensed in a 12 well plate at a concentration of 0.5 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) with 5% FBS (manufactured by Gibco) was used.

Subsequently, the cell culture solution was washed with PBS, exchanged with DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Sigma) without FBS (Gibco), and cultured at 37 ° C., 5% CO ₂ , and humidified conditions for one day. Proceeded. After incubation, the cells were washed with 1 ml of PBS (Phosphate Buffered Saline, manufactured by Sigma), and then treated with 50 uM of each compound of the present invention. 100 μl of PBS that did not dissolve the compound was used as a control.

In the procollagen II production promotion test, three days after the addition of the compound of the present invention, the culture medium was collected, and the concentration of type II pro collagen secreted in the culture medium was measured by enzyme-linked immunoassay (ELSIA, Human Procollagen type II ELISA Kit). (Prepared by R & D system).

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

[Equation 2]

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

Comparison of Procollagen II Production Rate of Each Compound division Skin fibroblasts
Procollagen II production rate (%) division Skin fibroblasts
Procollagen II production rate (%) Compound 1 179.1 Compound 26 192.0 Compound 2 173.2 Compound 27 180.4 Compound 3 168.8 Compound 28 174.4 Compound 4 169.0 Compound 29 176.0 Compound 5 169.9 Compound 30 173.9 Compound 6 171.4 Compound 31 174.4 Compound 7 170.5 Compound 32 176.5 Compound 8 170.2 Compound 33 176.6 Compound 9 178.5 Compound 34 184.6 Compound 10 167.3 Compound 35 173.3 Compound 11 163.0 Compound 36 170.4 Compound 12 163.9 Compound 37 170.1 Compound 13 164.5 Compound 38 174.2 Compound 14 164.5 Compound 39 177.5 Compound 15 164.2 Compound 40 177.5 Compound 16 165.1 Compound 41 177.6 Compound 17 170.4 Compound 42 179.0 Compound 18 177.7 Compound 43 186.1 Compound 19 172.8 Compound 44 176.3 Compound 20 172.6 Compound 45 179.7 Compound 21 180.1 Compound 46 195.5 Compound 22 180.0 Compound 47 193.7 Compound 23 179.3 Compound 48 194.1 Compound 24 178.6 Compound 49 187.7 Compound 25 179.7 Compound 50 188.8

Referring to Table 2 and FIG. 2, the compounds 1 to 50 of the present invention all show a procollagen II production promoting effect, and have been shown to exhibit sufficient effects on skin aging prevention and skin wrinkle prevention. Among the many peptide compounds, lipidized peptides 21-25 and 46-50 had a similar or higher degree of promoting procollagen II production than peptides that did not.

Experimental Example  3: Pentapeptide  And Pentapeptide Dimer  Safety check ( in vitro )

Human dermal fibroblasts (HDF: Human dermal fibroblast, manufactured by Thermo) were dispensed in 100 ul portions in 96 well plates at a concentration of 0.5 ³ cells / ml, and cultured for 2 days under 37 ° C, 5% C0 ₂ , and humidification conditions. . As the culture solution, a medium containing 10% of FBS (manufactured by Gibco) in 10% DME medium (Dulbecco's Modified Eagle's Medium, manufactured by Gibco) was used for each well.

Subsequently, it was exchanged with DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Sigma) without FBS (Gibco), washed again with 100 ul of PBS (Phosphate Buffered Saline, manufactured by Sigma), and then the compound of the present invention was 200 uM. Treated and incubated. 100 μl of PBS that did not dissolve the compound was used as a control.

The test sample was added and cultured for 48 hours, then treated with MTT and incubated for 2 hours. Thereafter, the culture solution was removed, DMSO was added thereto, and the resulting formazan was sufficiently dissolved, and the absorbance was measured at 540 nm. Based on the measured values, cell viability was calculated according to Equation 3, and the results are shown in Table 3 and FIG. 3.

[Equation 3]

% 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 1 102.2 Compound 26 100.6 Compound 2 101.8 Compound 27 97.2 Compound 3 100.3 Compound 28 98.1 Compound 4 99.9 Compound 29 97.9 Compound 5 99.8 Compound 30 99.2 Compound 6 98.7 Compound 31 100.0 Compound 7 98.8 Compound 32 101.4 Compound 8 96.1 Compound 33 101.2 Compound 9 99.0 Compound 34 98.7 Compound 10 98.6 Compound 35 98.3 Compound 11 98.5 Compound 36 99.4 Compound 12 102.1 Compound 37 99.5 Compound 13 101.9 Compound 38 99.0 Compound 14 100.2 Compound 39 99.0 Compound 15 100.4 Compound 40 99.1 Compound 16 99.7 Compound 41 98.9 Compound 17 99.8 Compound 42 100.7 Compound 18 98.7 Compound 43 100.4 Compound 19 98.5 Compound 44 101.2 Compound 20 103.2 Compound 45 99.9 Compound 21 95.7 Compound 46 97.1 Compound 22 95.8 Compound 47 96.0 Compound 23 96.1 Compound 48 96.6 Compound 24 95.9 Compound 49 95.7 Compound 25 96.0 Compound 50 95.5

Referring to Table 3 and Figure 3, all the compounds of the present invention was confirmed that all of the cell viability compared to the control, showing a cell viability of 95% or more. Through the results of this experiment, all compounds of the present invention was confirmed that the safety (Safety) in animal cells including human skin fibroblasts (Safety) is a safe raw material.

Experimental Example  4: Preparation of Cosmetic Formulations

In order to evaluate the effect of the cosmetic composition including the novel heptapeptide monosaccharide and dimer, the collagen synthesis promoting peptide, a cosmetic formulation in the form of a cream was prepared by combining the components shown in Table 4 below.

Cream Formulation to Assess Wrinkle Improvement ingredient Experimental Example 1 (% by weight) Comparative Example 1 (% by weight) Heptapeptide Monosaccharide 0.01 - Heptapeptide dimer 0.01 - Moisturizing ingredients 10 10 Fiji-100 Stearate 0.5 0.5 Glyceryl Stearate 0.3 0.3 Polysorbate 60 0.2 0.2 Cetyl alcohol 0.1 0.1 Squalane 5 5 Tocopheryl Acetate 0.2 0.2 Fiji-5 Rape Seed Sterol 0.05 0.05 glycerin 2 2 Setes-3 0.02 0.02 Disodium ID 0.02 0.02 1,2-hexanediol 0.1 0.1 Octyldodecanol 0.25 0.25 Dipropylene glycol 0.23 0.23 Thickener Quantity Quantity Incense, Preservative Quantity Quantity Purified water Remaining amount Remaining amount Sum 100.0 100.0

Experimental Example  5: 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 30 to 55 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 inspector.

Eye wrinkle improvement effect evaluation result division Wrinkle improvement effect Great slightly none Effectiveness% Experimental Example 1 12 6 2 75% Comparative Example 1 One 8 11 25%

Referring to Table 5, the cosmetic preparations containing penta peptides and penta peptide dimers having collagen synthesis promoting effect as in Experimental Example 1 than the cosmetics prepared without containing the components as in Comparative Example 1 wrinkle improvement effect It was confirmed that the markedly improved.

As mentioned above, although preferred embodiments and experimental examples of the present invention have been described, those skilled in the art to which the present invention pertains may realize the present invention in other specific forms without changing the technical spirit or essential features thereof. I can understand that. Therefore, it should be understood that the embodiments and the experimental examples described above are exemplary in all respects and not limiting.

<110> Celliconlab <120> cosmetic composition containing anti-aging and wrinkle preventing          pentapeptide and pentapeptide dimer production method <130> cil-181116 <160> 40 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 1 Cys Gly Gln Gly Arg   1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 2 Cys Gly Gln Pro Arg   1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 3 Cys Gln Gln Asn Arg   1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 4 Cys Gly Gln Lys Arg   1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 5 Cys Gly Gln Asp Arg   1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 6 Cys Gly Gln Tyr Arg   1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 7 Cys Gly Gln Ser Arg   1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 8 Cys Gly Gln Phe Arg   1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 9 Cys Gly Gln Leu Arg   1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 10 Gly Gln Ile Arg Cys   1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 11 Gly Met Pro Arg Cys   1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 12 Gly Thr Pro Arg Cys   1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 13 Gly Arg Pro Arg Cys   1 5 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 14 Gly Leu Pro Arg Cys   1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 15 Gly Phe Pro Arg Cys   1 5 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 16 Gly Ser Pro Arg Cys   1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 17 Gly Asp Pro Arg Cys   1 5 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 18 Gly Asn Pro Arg Cys   1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 19 Gly Lys Pro Arg Cys   1 5 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cosmetic peptide <400> 20 Gly Glu Pro Arg Cys   1 5 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 21 Cys Gly Gln Gly Arg Cys Gly Gln Gly Arg   1 5 10 <210> 22 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 22 Cys Gly Gln Pro Arg Cys Gly Gln Pro Arg   1 5 10 <210> 23 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 23 Cys Gly Gln Asn Arg Cys Gly Gln Asn Arg   1 5 10 <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 24 Cys Gly Gln Lys Arg Cys Gly Gln Lys Arg   1 5 10 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 25 Cys Gly Gln Asp Arg Cys Gly Gln Asp Arg   1 5 10 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 26 Cys Gly Gln Tyr Arg Cys Gly Gln Tyr Arg   1 5 10 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 27 Cys Gly Gln Ser Arg Cys Gly Gln Ser Arg   1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 28 Cys Gly Gln Phe Arg Cys Gly Gln Phe Arg   1 5 10 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 29 Cys Gly Gln Leu Arg Cys Gly Gln Leu Arg   1 5 10 <210> 30 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 30 Gly Gln Ile Arg Cys Gly Gln Ile Arg Cys   1 5 10 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 31 Gly Met Pro Arg Cys Gly Met Pro Arg Cys   1 5 10 <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 32 Gly Thr Pro Arg Cys Gly Thr Pro Arg Cys   1 5 10 <210> 33 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 33 Gly Arg Pro Arg Cys Gly Arg Pro Arg Cys   1 5 10 <210> 34 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 34 Gly Leu Pro Arg Cys Gly Leu Pro Arg Cys   1 5 10 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 35 Gly Phe Pro Arg Cys Gly Phe Pro Arg Cys   1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 36 Gly Ser Pro Arg Cys Gly Ser Pro Arg Cys   1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 37 Gly Asp Pro Arg Cys Gly Asp Pro Arg Cys   1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 38 Gly Asn Pro Arg Cys Gly Asn Pro Arg Cys   1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 39 Gly Lys Pro Arg Cys Gly Lys Pro Arg Cys   1 5 10 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cosmetic peptide <400> 40 Gly Glu Pro Arg Cys Gly Glu Pro Arg Cys   1 5 10

Claims (7)

Peptide for promoting collagen synthesis containing any one peptide selected from the group consisting of SEQ ID NO: 1 to 50 as an active ingredient.

delete A pharmaceutical composition for preventing and treating skin wrinkles containing any one peptide selected from the group consisting of SEQ ID NOs: 1 to 50 as an active ingredient.

delete The method of claim 3, wherein
The peptide is a pharmaceutical composition for preventing and treating 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 containing any one peptide selected from the group consisting of SEQ ID NOs: 1 to 50 as an active ingredient
The method of claim 6,
The peptide is contained in the cosmetic composition 0.0001 to 1.0% by weight, lotion, milk, gel, cream, essence, pack, ampoule, lotion, cleaning agent, soap, body products, soap, oil, lipstick and foundation Cosmetic composition for preventing and improving skin wrinkles, characterized in that formulated in any one selected from the group consisting of

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