KR20150029884A - Peptides for promoting synthesis of collagen, method for preparing thereof, and, composition comprising thereof - Google Patents

Abstract

The present invention relates to peptides for promoting collagen synthesis having enhanced collagen synthesis capacity which are represented by chemical formulas 1-4, to a method for preparing the same, and to an anti-wrinkle composition utilizing the same. The peptides of the present invention have enhanced collagen synthesis promoting capacity, thereby being easily used as a composition for mitigating skin aging and improving wrinkles. Especially, the peptides of the present invention which are prepared in a dimer form have higher use safety and stability than conventional peptide compositions.

Description

TECHNICAL FIELD The present invention relates to a collagen synthesis promoting peptide, a process for producing the collagen synthesis peptide, a composition for promoting collagen synthesis,

The present invention relates to a collagen synthesis promoting peptide having excellent collagen synthesis ability, a method for producing the collagen synthesis promoting peptide, and a composition for improving wrinkles using the peptide.

Skin is the largest organ of the human body composed of the epidermis, dermis, and subcutaneous tissue. It is an important body that plays various roles such as protection function, barrier function, temperature control function, excretion function and respiration function. However, due to various external environments, the thickness of epidermis, dermis, and subcutaneous tissue becomes thinner, and as the composition and content of the lipid barrier, which functions as a skin barrier, is changed, its function is rapidly deteriorated and aging occurs. Molecular biologically, skin aging is known to be the main cause of the reduction of collagen forming matrix in skin dermis. Collagen is a substance that accounts for 70 to 80% of the skin's dermis and is a typical substance involved in maintaining the elasticity of the skin. According to the research results of the Korean Society of Cosmetic Scientists Volume 38, Issue 4, Vol. 87, collagen peptides are known to have skin regeneration and skin regeneration effects. Collagen is degraded by aging and its degradation is accelerated by the external environment such as ultraviolet rays, and wrinkles of the skin are increased, and skin aging is promoted.

In order to solve such problems as skin aging and wrinkle improvement, various studies on the change of collagen are under way. The low molecular weight compounds that promote the production of collagen are now widely reported as retinoids widely used for the improvement of skin wrinkles, stains and pigmentation, among which clinical results of removing wrinkles on retinol (vitamin A) have been reported. A cosmetic composition containing retinol has been reported to effectively improve skin wrinkles, skin sagging, and elasticity reduction caused by sunlight (see U.S. Patent No. 4,603,146, No. 4,877,805). It has also been reported that a cosmetic composition containing L-ascorbic acid (vitamin C), which is essential for the collagen synthesis process, is effective for improving skin wrinkles, ultraviolet light blocking, pigmentation such as spots / freckles / black spots 4,938,969, U.S. Patent No. 4,772,591 and European Patent No. 533,667). In recent years, studies for stabilizing the above-described active ingredients have been conducted.

As another composition for improving wrinkles, US Pat. No. 4,720,353 discloses that it can be stabilized by using BHA (butylated hydroxy anisole), vitamin C, propyl gallate and tocole ferulol linoleate as antioxidants , European Patent No. 440,398 discloses that retinol can be stabilized with a water-in-oil (W / O) type emulsion composition containing one or more water-soluble antioxidants and an oil-soluble antioxidant and a chelating agent over one term. A variety of liposome and capsule formulations for stabilizing retinol have been studied and used as cosmetic products. Other than retinol, TGF-beta (transforming growth factor), betulinic acid, wild yam extract (Korean Patent Publication No. 2009-0055079) and collagen peptide derived from soy protein (Japanese Patent No. 4030067) ≪ / RTI > However, the above-described skin-improving effective ingredients have unstable properties such that they are easily oxidized to light, moisture, air, heat, and the like, and thus they have a problem of being used as raw materials for cosmetics. In particular, when used for dry skin, retinol peels off the skin's skin, causing irritation to the skin, which can lead to redness or dryness of the skin. In addition, the cost of the recombinant protein raw material is high and the reproducibility of the natural extract is low.

Accordingly, the inventors of the present invention have completed the present invention by confirming that the composition of the present invention containing a functional peptide has an effect of synthesizing collagen and is excellent in the function of improving wrinkles.

Korean Patent No. 1135444, Korean Patent No. 1107312, and Korean Patent No. 753472 disclose a cosmetic composition for improving wrinkles containing various functional peptides. However, these peptides are not limited to those contained in the composition of the present invention As a substance completely different from a peptide, the prior arts can be said to have a different composition from the present invention.

U.S. Patent No. 4,603,146 (Methods for retarding the effects of aging of the skin, Registered on July 29, 1986) United States Patent No. 4,720,353 (Stable pharmaceutical w / o emulsion composition, registered on Jan. 19, 1988) U.S. Patent No. 4,772,591 (Method for accelerated wound healing, registered on September 20, 1988) U.S. Patent No. 4,877,805 (Registered on October 31, 1989, Methods for treatment of sundamaged human skin with retinoids) US Patent No. 4,938,969 (Registered on July 3, 1990) (Method for treatment of aging or photo-damaged skin) European Patent No. 533,667 (Registered on June 28, 1995, METHOD FOR THE TREATMENT OF AGING OR PHOTO-DAMAGED SKIN) Korean Patent Publication No. 2009-0055079 (cosmetic composition for preventing skin aging containing wild yam extract, published on Jun. 2, 2009) Japanese Patent No. 4030067 (new peptide, registered October 26, 2007) Korean Patent No. 1135444 (cosmetic composition for improving wrinkles containing palmitoyl oligopeptide and mulberry extract, registered April 04, 2012) Korea Patent No. 1107312 (Cosmetic composition for skin improvement and protection including peptides, registered on Jan. 11, 2012) Korean Patent No. 753472 (Fused Peptide in which TAT Peptide is Bound to Peptide Derived from C-Terminal of Human Collagen Type I, Method for Producing the Same, and Cosmetic Composition for Improving Skin Wrinkle, Registered on Aug. 23, 2007)

E. Kaiser et al. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides., Anal. Biochem., 1970, 34 (2), 595-598. Kim, In-Su; et al., Effects of oral low-molecular collagen peptide on skin recovery after fractionated photothermolysis treatment, Journal of the Korean Society of Cosmetic Scientists, Vol.38 No.47 (2012. 12), 321-326.

It is an object of the present invention to provide a collagen synthesis promoting peptide having excellent collagen synthesis ability, a process for producing the same, and a composition for improving wrinkles using the same.

The present invention relates to a collagen synthesis promoting peptide represented by the following Chemical Formulas (1) to (4).

[Chemical Formula 1]

[Lys-Thr- X- Lys-Ser-Cys]

(2)

[Cys-Lys-Thr- X- Lys-Ser]

(3)

[Lys-Thr- X- Lys-Ser-Cys] ₂

[Chemical Formula 4]

[Cys-Lys-Thr- X- Lys-Ser] ₂

In the above Chemical Formulas 1 to 4, X represents an amino acid selected from the group consisting of glutamic acid, aspartic acid, histidine, phenylalanine, alanine, cysteine, glycine, glutamine, asparagine, arginine, leucine, methionine, isoleucine, serine, tyrosine, threonine, Selected from proline and valine,

[] ₂ represents a dimer obtained by modifying a cysteine residue, thiol group (-SH), to a reduced form (-SS-).

Preferably, the peptides of the above formulas (1) to (4)

[Lys-Thr-Lys-Lys-Ser-Cys] (Compound 1),

[Lys-Thr-Glu-Lys-Ser-Cys] (Compound 2),

[Lys-Thr-Phe-Lys-Ser-Cys] (Compound 3),

[Lys-Thr-Thr-Lys-Ser-Cys] (Compound 4),

[Lys-Thr-Ala-Lys-Ser-Cys] (Compound 5),

[Cys-Lys-Thr-Lys-Lys-Ser] (Compound 6),

[Cys-Lys-Thr-Glu-Lys-Ser] (Compound 7),

[Cys-Lys-Thr-Phe-Lys-Ser] (Compound 8),

[Cys-Lys-Thr-Thr-Lys-Ser] (Compound 9),

[Cys-Lys-Thr-Ala-Lys-Ser] (Compound 10),

[Lys-Thr-Lys-Lys-Ser-Cys] ₂ (Compound 11),

[Lys-Thr-Glu-Lys-Ser-Cys] ₂ (Compound 12)

[Lys-Thr-Phe-Lys-Ser-Cys] ₂ (Compound 13)

[Lys-Thr-Thr-Lys-Ser-Cys] ₂ (Compound 14)

[Lys-Thr-Ala-Lys-Ser-Cys] ₂ (Compound 15),

[Cys-Lys-Thr-Lys-Lys-Ser] ₂ (Compound 16),

[Cys-Lys-Thr-Glu-Lys-Ser] ₂ (Compound 17),

[Cys-Lys-Thr-Phe-Lys-Ser] ₂ (Compound 18),

[Cys-Lys-Thr-Thr-Lys-Ser] ₂ (Compound 19)

[Cys-Lys-Thr-Ala-Lys-Ser] ₂ (Compound 20)

[ ₂ ] represents a dimer in which a thiol group (-SH), which is a cysteine residue, is modified to a reduced form (-SS-).

The present invention also relates to a peptide of the above formulas (1) to (4) or at least one peptide selected from the peptides of the above compounds 1 to 20 There is provided a composition for improving wrinkles containing a peptide.

Accordingly, the present invention provides a peptide of the above formulas (1) to (4), or a peptide of A pharmaceutical composition for improving wrinkles containing a peptide can be provided. The peptide may be included in the pharmaceutical composition in an amount of 0.0001 to 1.0% by weight.

In another aspect, the present invention provides a cosmetic composition for improving wrinkles containing peptides of the above formulas (1) to (4) or peptides selected from at least one of the peptides of the above compounds 1 to 20. The peptide may be contained in the cosmetic composition in an amount of 0.0001 to 1.0% by weight. The cosmetics may be selected from lotions, lotions, gels, creams, essences, packs, ampoules, lotions, cleansing agents, soaps, body products, soaps, oils, lipsticks and foundations.

Hereinafter, the present invention will be described in detail.

Among the peptides of the present invention, the peptides of the compounds 11 to 20 are obtained by modifying the thiol group (-SH), which is a residue of cysteine (the cysteine located at the terminal of the peptide) of the peptide of each of the compounds 1 to 10, It is a dimer. For example, Compound 11 is a compound obtained by modifying the thiol group, which is a cysteine residue of Compound 1, into a reducing form, Compound 11 is a compound obtained by modifying the thiol group, which is a cysteine residue of Compound 2, into a reducing form. In the same manner, a compound of Compound 13 to Compound 20 can be prepared from Compound 3 to Compound 10, respectively.

The peptide of the present invention can be produced by, but not limited to, forming one or more amino acids or a suitably protected amino acid continuously in an amino acid skeleton constantly bonded to a solid phase. In addition, the peptide can be inserted, substituted or deleted with other amino acids within a range that does not significantly deteriorate the stability, and this also falls within the scope of the present invention.

Also, a cell permeable peptide which promotes intracellular movement of the peptide of the present invention is bound to the C-terminal or N-terminal of the peptide . For example, the cell permeable peptide includes a TAT peptide (Arg-Lys-Lys-Arg-Arg-Arg-Arg-Arg) Arg-Arg-Arg: Tat PTD). However, the present invention is not limited thereto. Any cell permeable 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.

Meanwhile, the peptide of the present invention may exist in the form of a salt. The salt forms which can be used in the present invention may be those which are made during the final isolation and purification of the compound or by reacting the amino group with an appropriate acid. For example, an acid addition salt can be prepared by reacting an acid addition salt such as acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, Sulfates, heptanoates, hexanoates, formates, fumarates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, mesitylenesulfonates, methanesulfonates, Naphthalene sulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, Trichloroate, trifluoroacetate, phosphate, glutamate, bicarbonate, para -Toluenesulfonate and undecanoate, but are not limited thereto. In addition, examples of acids that can be used to form acid addition salts 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, a peptide form containing a triploloacetate salt or an acetate salt is most preferable.

The amino group or the carboxyl group of the amino acid used for producing the peptide of the present invention can be protected by a suitable protecting group. The protected amino acid can be reacted in solution by adding the following amino acid under conditions suitable for attachment to a solid support or formation of an amide bond. In addition, the protecting group may be completely removed prior to addition of the protected amino acid with a suitable protecting group. After all of the amino acids are linked as desired, the final desired peptide can be obtained by sequential or simultaneous separation from the free residual protecting group and the free solid support.

In the present invention, it is preferable that the chiral center is condensed with another appropriately protected dipeptide under a condition that the chiral center is not racemized to form an amide bond and deprotection to synthesize the desired hexapeptide, Technology to produce peptides.

As the most preferable synthesis method for producing the peptide compound of the present invention, there can be used a method of synthesizing a solid phase peptide synthesized by using a solid polymer conjugate. The α-amino group of the peptide prepared by the above method has an acid or base- Lt; / RTI > The protecting group of the amino acid at this time should have a stable property in the peptide condensation reaction condition and should have a property that it can be easily removed without destroying the extended peptide chain or without any racemization of any chiral center contained therein. Accordingly, suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyl- oxycarbonyl, t- N-butyl, isobornyloxycarbonyl, (α, α) -dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitrophenylsulphenyl, 2-cyano-t-butyloxycarbonyl, Other suitable protecting groups known in the art for this purpose are also within the scope of the present invention.

The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group can be used as the most preferable protecting group of the amino acid used in the peptide synthesis of the present invention.

In particular, the protecting group of the amino acid residue used in the peptide synthesis of the present invention is t-butyl (t-Bu) in the case of N-methylglutamic acid; For lysine, t-butoxycarbonyl (Boc); For serine, 7t-butyl (t-Bu); For threonine and allotreonine, t-butyl (t-Bu); In the case of cysteine, trityl (Trt) is preferable, but the present invention is not limited thereto.

In the solid phase peptide synthesis method, the C-terminal amino acid can be attached to a suitable solid support or resin. Suitable solid supports useful for this synthesis include reagents of the staged condensation-deprotection reaction and those which are inert to the reaction conditions and which are insoluble in the medium used, for example rink amid or link amide 4- Methylbenzylhydryl amine resin (rink amid MBHA resin).

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

The C-terminal amide is reacted in a solvent such as dichloromethane, N-methylpyridone (NMP) or DMF at a temperature of 10 ° C to 50 ° C, preferably at a temperature of 30 ° C, for 4 to 24 hours -Dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), benzotriazol-1-yloxy-tris (dimethylamino) phosphonium-hexafluorophosphate N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DCC) in the presence or absence of boron trifluoromethanesulfonate (BOP) or bis (DIC), [O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU) or O-benzotriazole- (Bonded, coupled) to the resin or solid support via condensation by activating the carboxylic acid on N, N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU).

When the solid support is a linkamide 4-methylbenzylhydrylamine resin, the Fmoc functional group, as a preferred protecting group, is used in excess of a solution of a secondary amine, preferably 20% piperidine DMF, before condensation with the C-terminal amino acid Cut it. Preferred reagents for use in condensing the desired amino acid in the deprotected 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxyacetamidoethyl resin include, but are not limited to, (NMM), 1-hydroxybenzotriazole (HOBt) and O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluoro (HATU), O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU), N, N'-dicyclohexylcarbodiimide (DCC) or N, N'-diisopropylcarbodiimide (DIC).

Condensation of consecutive amino acids carried out in the present invention can be carried out directly using an automatic peptide synthesizer, which is well known in the related art, or manually. Preferred synthetic reaction conditions include deprotection of the alpha -amino acid protected with the Fmoc group by treatment with a secondary amine solution, preferably piperidine, followed by washing with a sufficient excess of the solvent, The amino acid can then be added in an excess amount of 3 to 7 times by mole, preferably in a DMF solvent.

In the final step of synthesizing the peptide using the solid phase resin of the present invention, the peptides to be obtained from the resin by continuous or one-time operation of the peptides can be removed and the protective groups protecting the amino acid residues respectively can be deprotected. The removal of the peptide from the resin and the deprotection conditions of the protecting groups present in the residue generally include a cleavage reagent cocktail that cleaves the bond between the resin and the peptide, for example, trifluoroacetic acid (TFA), triisopropylsilane (TIS) , Thioanisole, water or ethanedithiol (EDT), and the like. The resulting mixed solution can be precipitated by treating excess refrigerated diethyl ether solvent. The precipitate thus obtained was centrifuged to complete precipitation, excess trifluoroacetic acid, triisopropylsilane, thioanisole, water and ethanedithiol were firstly removed, and the above procedure was repeated twice or more to solidify the precipitate Can be obtained. At this time, the completely deprotected peptide salt can be separated and purified using a mixed solvent composed of water and an acetonitrile solvent and reverse 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.

The peptides of the present invention have collagen synthesis effect.

According to a preferred embodiment of the present invention, the composition of the present invention can be provided as a pharmaceutical composition for improving wrinkles or a cosmetic composition.

When the composition of the present invention is prepared from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are those conventionally used in the field of the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, purified water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. The pharmaceutical composition of the present invention may further include additives commonly used such as lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives and the like in addition to the above components. The pharmaceutical composition of the present invention is preferably parenterally administered, and more preferably, is applied by a local administration method by application.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, .

The dose of the peptide as an active ingredient contained 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 on an adult basis, The dose may be administered once a day or divided into several times. The peptide of the present invention may be contained in an amount of preferably 0.0001 to 1.0% by weight, more preferably 0.001 to 1.0% by weight, and most preferably 0.001 to 0.01% by weight, based on the total weight of the pharmaceutical composition, It is not.

The cosmetic composition of the present invention contains not only peptides as active ingredients thereof, but also ingredients conventionally used in cosmetic compositions, such as conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, . The carrier may further contain at least one selected from the group consisting of purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butylene glycol or propylene glycol), higher fatty acids (palmitic acid or linolenic acid) But are not limited to, wheat germ oil, camellia oil, jojoba oil, olive oil, squalane, sunflower oil, macadamia peanut oil, avocado oil or fatty acid glyceride). If necessary, surfactants, moisturizers, preservatives, antioxidants and the like may be added.

Examples of the surfactant that can be used in the cosmetic composition of the present invention include anionic surfactants such as alkylbenzenesulfonates, polyoxyalkylene alkylsulfate ester salts, alkylsulfate ester salts, olefin sulfonates, alkyl phosphates, polyoxyalkylene Alkyl ether phosphates, dialkyl sulfosuccinates, fatty acid salts, and the like. Nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyhydric alcohol fatty acid partial esters, polyoxyethylene polyhydric alcohol fatty acid moieties Esters, polyglycerin fatty acid esters, polyoxyethylene hydrogenated castor oil derivatives, and fatty acid diethanolamides. Examples of the cationic surfactant include tertiary aliphatic amine salts, alkyltrimethylammonium halides and dialkyldimethylammonium halides. Examples of the amphoteric surfactants include amide betaine, imidazolinium betaine, sulfobetaine, And the like. Examples of the moisturizing agent include glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, and sorbitol. Examples of the preservative include benzoic acid, dehydroacetic acid, paraoxybenzoic acid esters (such as methyl parahydroxybenzoate, butyl parahydroxybenzoate), and phenoxyethanol. Examples of the antioxidant include ascorbic acid and BHA. In addition, an ultraviolet absorber, an anti-inflammatory agent, and a refreshing agent may be added.

The peptide of the present invention may be contained in an amount of preferably 0.0001 to 1.0% by weight, more preferably 0.001 to 1.0% by weight, and most preferably 0.001 to 0.01% by weight, based on the total weight of the cosmetic composition, but is not limited thereto .

The kind of the cosmetic material is not particularly limited, and examples thereof include skin care cosmetic materials such as lotion, milky lotion, gel, cream, essence, pack, ampoule, lotion, cleanser, soap, body products, soap, oil, Makeup cosmetics such as foundation, and the like, and the formulations thereof are not particularly limited.

The cosmetic composition of the present invention can be used everyday or can be used for an unspecified period. Preferably, the amount of usage, the number of times of use, and the period of time can be adjusted according to the user's age, skin condition or skin type, and the concentration of the peptide.

The present invention relates to a collagen synthesis promoting peptide having excellent collagen synthesis ability, a method for producing the collagen synthesis promoting peptide, and a composition for improving wrinkles using the same, wherein the peptide of the present invention exhibits excellent collagen synthesis promoting function, It is easy to use. In particular, the peptide of the present invention, which is produced in the form of a dimer, has higher safety and stability in use than existing peptide compositions.

1 is a graphical representation of the effect of the compounds of the present invention on increasing collagen synthesis.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Synthesis of peptide >

Nomenclature and abbreviations of amino acids used in the present invention are expressed as follows.

Ala: alanine / Cys: cysteine / Glu: glutamic acid / Phe: phenylalanine / Lys: lysine / Ser: serine / Thr: threonine.

Example 1-1. Synthesis of compounds 1 and 11

71.4 mg (0.10 mmol) of 2-chlorotrityl chloride resin (resin loaded with 1.4 mmol per gram) purchased from Novabiochem corporation was weighed into a reaction vessel. The resin was solubilized with 3 ml of DMF and sufficiently swelled for 5 minutes. Then, 3 ml of a 20% (w / v) piperidine DMF solution was added, shaking for 10 minutes, and a piperidine DMF solution After removing, it was washed 5 times with 10 ml of DMF solvent (washing 5 times in 10 ml). * DMF: Dimethylformamide.

Fmoc-Cys (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. 3 ml of a 20% (w / v) piperidine DMF solution was added and shaken for 10 minutes to remove the piperidine solution. Then, 20% (w / v) piperidine DMF solution was added thereto, After the reaction, the Fmoc protecting group protected by the resin was completely removed and washed 5 times with 10 ml of DMF solvent (5 times in 10 ml portions). At this stage, the deprotection of the Fmoc protecting group was carried out using the 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 continuously condensed (coupled) according to the same synthesis cycle as described below.

(1) washing 5 times with DMF solvent (10 ml);

(2) deprotection twice with 10% (w / v) piperidine DMF solution (3 ml) for 10 minutes;

(3) washing 5 times with DMF solvent (10 ml);

(4) Fmoc-amino acid addition;

(5) Amino acid activation and 2 hour condensation by addition of condensation reagent;

(6) washed 5 times with DMF solvent (10 ml);

The above steps (1) to (6) were repeated, wherein the Fmoc-protected amino acid (0.80 mmol) after Fmoc-Cys (Trt) -OH was added to the resin reaction vessel in the sequence described below and condensed .

(i) Fmoc-Ser (tBu) -OH;

(ii) Fmoc-Lys (Boc) -OH;

(iii) Fmoc-Lys (Boc) -OH;

(iv) Fmoc-Thr (tBu) -OH;

(v) Fmoc-Lys (Boc) -OH;

After (6) after Fmoc-Lys (Boc) -OH condensation, finally, 20% piperidine DMF solution (3 ml) was treated.

Immediately after such synthesis termination, the peptide condensed resin was treated with a mixture of trifluoroacetic acid / thioanisole / ethanediol / triisopropylsilane / water (95: 5: 2.5: 2.5: 2.5) (10 ml), and the peptide was cleaved from the resin. The thus obtained mixed solution was treated with 100 ml of refrigerated diethyl ether solvent to produce a precipitate. The obtained precipitate was centrifuged to completely precipitate, and then trifluoroacetic acid, thioanisole and ethanedithiol were firstly removed, and 100 ml of a diethyl ether solvent was added to wash the precipitate, followed by centrifugation, , A process for removing trifluoroacetic acid, thioanisole and ethanedithiol) was repeated twice to obtain a solidified precipitate. 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. By freeze-drying the pure fractions were purified as acetate, trifluoroacetate type of a white powder compound _{1: H 2 N- [Lys-} Thr-Lys-Lys-Ser-Cys] -CO 2 H (70㎎) was obtained.

The compound 1: H ₂ N- [Lys-Thr-Lys-Lys-Ser-Cys] -CO ₂ H (20 mg) was dissolved in 2 ml of dimethylsulfoxide (DMSO), 10 ml of water was added, And the mixture was stirred at room temperature to obtain the following compound 11 (18 mg) in the form of a dimer in which the thiol group (-SH) as the cysteine residue was modified to the reduced form (-SS-).

Compound 1 : H ₂ N- [Lys-Thr-Lys-Lys-Ser-Cys] ₂ -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 694.94; (70 mg)

Compound 11 : H ₂ N- [Lys-Thr-Lys-Lys-Ser-Cys] ₂ -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 1386.88; (18 mg)

Examples 1-2. Synthesis of compounds 2 to 10 and 12 to 20

Peptides of the following compounds 2 to 10 and 12 to 20 were prepared in the same manner as in Example 1-1 except that the order of amino acids protected by Fmoc was changed.

Compound 2 : H ₂ N- [Lys-Thr-Glu-Lys-Ser-Cys] -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 695.88; (79 mg)

Compound 12 : H ₂ N- [Lys-Thr-Glu-Lys-Ser-Cys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1388.76; (24 mg)

Compound 3 : H ₂ N- [Lys-Thr-Phe-Lys-Ser-Cys] -CO ₂ H; MS (ESI) m / e, [M + H] < ⁺ > = 713.94; (73 mg)

Compound 13 : H ₂ N- [Lys-Thr-Phe-Lys-Ser-Cys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1424.88; (19 mg)

Compound 4 : H ₂ N- [Lys-Thr-Thr-Lys-Ser-Cys] -CO ₂ H; MS (ESI) m / e, [M + H] < ⁺ > = 669.87; (67 mg)

Compound 14 : H ₂ N- [Lys-Thr-Thr-Lys-Ser-Cys] ₂ -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 1335.74

Compound 5 : H ₂ N- [Lys-Thr-Ala-Lys-Ser-Cys] -CO ₂ H; MS (ESI) m / e, [M + H] < ⁺ > = 638.34; (78 mg)

Compound 15 : H ₂ N- [Lys-Thr-Ala-Lys-Ser-Cys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1272.68; (19 mg)

Compound 6 : H ₂ N- [Cys-Lys-Thr-Lys-Lys-Ser] -CO ₂ H; MS (ESI) m / e, [M + H] < ⁺ > = 695.44; (72 mg)

Compound 16 : H ₂ N- [Cys-Lys-Thr-Lys-Lys-Ser] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1386.88; (17 mg)

Compound 7 : H ₂ N- [Cys-Lys-Thr-Glu-Lys-Ser] -CO ₂ H ; MS (ESI) m / e, [M + H] < ⁺ > = 696.38; (66 mg)

Compound 17 : H ₂ N- [Cys-Lys-Thr-Glu-Lys-Ser] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1388.76; (16 mg)

Compound 8 : H ₂ N- [Cys-Lys-Thr-Phe-Lys-Ser] -CO ₂ H; MS (ESI) m / e, [M + H] < ⁺ > = 714.44; (80 mg)

Compound 18 : H ₂ N- [Cys-Lys-Thr-Phe-Lys-Ser] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1424.88; (21 mg)

Compound 9 : H ₂ N- [Cys-Lys-Thr-Thr-Lys-Ser] -CO ₂ H; MS (ESI) m / e, [M + H] < ⁺ > = 669.87; (76 mg)

Compound 19 : H ₂ N- [Cys-Lys-Thr-Thr-Lys-Ser] ₂ -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 1335.74; (17 mg)

Compound 10 : H ₂ N- [Cys-Lys-Thr-Ala-Lys-Ser] -CO ₂ H ; MS (ESI) m / e, [M + H] < ⁺ > = 638.34; (74 mg)

Compound 20 : H ₂ N- [Cys-Lys-Thr-Ala-Lys-Ser] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1272.68; (19 mg)

<Example 2> Measuring effect of promoting collagen synthesis of peptide in vitro )>

Normal human skin fibroblast (NHDF-NB: Kurashiki bonded key manufactured manufacture, Ltd.) a, 1.0 × 10 ⁴ cells / ㎖ at a concentration and dispensed in a 96 well plate by 100㎕, 37 ℃, 5% C0 2, the humidification conditions Lt; / RTI &gt; for 3 days. The culture medium used was medium containing 10% by weight of LSGS (Low Serum Growth Supplement, manufactured by Kurashiki Boshiki Co., Ltd.) in Medium 106S (Kurashiki Boshiki Co., Ltd.) in an amount of 100 쨉 l per well. , And exchanged with DMEM (Dulbecco's Modified Eagle's Medium, Sigma) and treated with 100 μl of PBS (Phosphate Buffered Saline, manufactured by Takara Bio Industries Co., Ltd.) and cultured (final concentration: 10 μM). In the collagen production promotion test, after culturing for 7 days, the culture broth was collected, and the concentration of type I collagen secreted in the culture broth was measured by enzyme-linked immunosorbent assay (Procollagen type I c-peptide EIA Kit, Takara Bio Inc.). Based on the quantitative results, a control culture medium The amount of collagen in each test sample culture was calculated and calculated based on the amount of type I collagen in 100%. The results are shown in Table 1 and FIG.

Referring to Table 1 and FIG. 1, all of the compounds 1 to 20 of the present invention show an effect of stimulating collagen production, and showing sufficient effect to prevent skin wrinkles.

On the other hand, in general, it is very important to slow down the degradation rate of the peptide because the peptide is rapidly degraded by various hydrolytic enzymes when it enters the living body and its ability is rapidly reduced. In general, various hydrolytic enzymes decompose an amide bond (-C (= O) NH-) in a peptide structure into a carboxylic acid (-CO ₂ H) and an amine (-NH ₂ ) The cysteine dimer structure has a (-SS-) skeleton, which functions to lower or disturb the decomposition ability of common hydrolytic enzymes. That is, among the above peptide compounds, dimer-type compounds 11 to 20 are less decomposed by hydrolytic enzymes in vivo than compounds 1 to 10 in monomer form. Therefore, among the peptide compounds of the present invention, the compound 11 to 20 having cysteine dimer is considered to have excellent stability in the body.

PREPARATION EXAMPLE 1 Cosmetic Containing Peptide

A cosmetic containing the peptide of the present invention was prepared using the following components.

Production Example 1-1. cream

Compound 11 of the present invention 0.01 g

Paraben ................................ 0.2 g

Allantoin ............................. 2.0 g

Betaine ............................... 1.0 g

Sodium hyaluronate .................... 0.13 g

Tocopheryl acetate .......................... 10 g

Shea butter ............................... 10 g

Jojoba oil ............................ 1.7 g

Preservative ................................ 0.4 g

Incense ..................................... 0.2 g

Distilled water .............................. 74.36 g

Production Example 1-2. essence

Compound 11 of the present invention 0.01 g

Glycerin ............................. 10.0 g

Betaine ............................... 5.0 g

PEG 1500 ... 2.0 g

Allantoin ............................. 0.1 g

DL-Panthenol ............................ 0.3 g

EDTA-2Na ... 0.02 g

Benzophenone-9 .......................... 0.04 g

Hydroxyethyl cellulose 0.1 g

Sodium hyaluronate .................... 8.0 g

Carboxyvinyl polymer 0.2 g

Triethanolamine ...................... 0.18 g

Octyldodecanol .......................... 0.3 g

Octyldodeces-16 ....................... 0.4 g

Ethanol ............................... 6.0 g

Preservative ................................ 0.4 g

Incense .................................... 0.2 g

Distilled water .............................. 66.75 g

Production Example 1-3. Mask Pack

Compound 11 of the present invention ... 0.01 g

Polyvinyl alcohol ......................... 15.0 g

Cellulose sword ........................ 0.15 g

Glycerin .............................. 3.0 g

PEG-1500 ... 2.0 g

Cyclodextrin ...................... 0.15 g

DL-Panthenol .............................. 0.4 g

Allantoin .............................. 0.1 g

Monoammonium glycyrrhizin acid 0.3 g

Nicotinamide ........................... 0.5 g

Ethanol ................................. 6.0 g

Preservative ................................. 0.4 g

Incense ..................................... 0.2 g

Distilled water ............................... 71.79 g

Claims (10)

A peptide for promoting collagen synthesis, which is represented by the following formulas (1) and (2).
[Chemical Formula 1]
[Lys-Thr- X- Lys-Ser-Cys]
(2)
[Cys-Lys-Thr- X- Lys-Ser]
Wherein X is selected from the group consisting of glutamic acid, aspartic acid, histidine, phenylalanine, alanine, cysteine, glycine, glutamine, asparagine, arginine, leucine, methionine, isoleucine, serine, tyrosine, threonine, lysine, tryptophan, Selected from prolin and valine. A peptide for promoting collagen synthesis, which is represented by the following formulas (3) and (4).
(3)
[Lys-Thr- X- Lys-Ser-Cys] ₂
[Chemical Formula 4]
[Cys-Lys-Thr- X- Lys-Ser] ₂
In the above formulas (3) and (4), X represents an amino acid selected from the group consisting of glutamic acid, aspartic acid, histidine, phenylalanine, alanine, cysteine, glycine, glutamine, asparagine, arginine, leucine, methionine, isoleucine, serine, tyrosine, threonine, Selected from proline and valine,
[] ₂ represents a dimer obtained by modifying a cysteine residue, thiol group (-SH), to a reduced form (-SS-). The method according to claim 1,
The peptides of Formulas (1) and (2)
[Lys-Thr-Lys-Lys-Ser-Cys] (Compound 1),
[Lys-Thr-Glu-Lys-Ser-Cys] (Compound 2),
[Lys-Thr-Phe-Lys-Ser-Cys] (Compound 3),
[Lys-Thr-Thr-Lys-Ser-Cys] (Compound 4),
[Lys-Thr-Ala-Lys-Ser-Cys] (Compound 5),
[Cys-Lys-Thr-Lys-Lys-Ser] (Compound 6),
[Cys-Lys-Thr-Glu-Lys-Ser] (Compound 7),
[Cys-Lys-Thr-Phe-Lys-Ser] (Compound 8),
[Cys-Lys-Thr-Thr-Lys-Ser] (Compound 9)
[Cys-Lys-Thr-Ala-Lys-Ser] (Compound 10),
Wherein the peptide is selected from the group consisting of: 3. The method of claim 2,
The peptides of formulas (3) and (4)
[Lys-Thr-Lys-Lys-Ser-Cys] ₂ (Compound 11),
[Lys-Thr-Glu-Lys-Ser-Cys] ₂ (Compound 12)
[Lys-Thr-Phe-Lys-Ser-Cys] ₂ (Compound 13)
[Lys-Thr-Thr-Lys-Ser-Cys] ₂ (Compound 14)
[Lys-Thr-Ala-Lys-Ser-Cys] ₂ (Compound 15),
[Cys-Lys-Thr-Lys-Lys-Ser] ₂ (Compound 16),
[Cys-Lys-Thr-Glu-Lys-Ser] ₂ (Compound 17),
[Cys-Lys-Thr-Phe-Lys-Ser] ₂ (Compound 18),
[Cys-Lys-Thr-Thr-Lys-Ser] ₂ (Compound 19)
[Cys-Lys-Thr-Ala-Lys-Ser] ₂ (Compound 20)
, Wherein [] ₂ in the above compound represents a dimer obtained by modifying a thiol group (-SH), which is a cysteine residue, to a reduced form (-SS-). A wrinkle-improving composition characterized by containing at least one peptide selected from the peptides according to any one of claims 1 to 4. A pharmaceutical composition for improving wrinkles, characterized by containing at least one peptide selected from the peptides according to any one of claims 1 to 4. The method according to claim 6,
Wherein the peptide is contained in the pharmaceutical composition in an amount of 0.0001 to 1.0% by weight. A cosmetic composition for wrinkle improvement, characterized by containing at least one peptide selected from the peptides according to any one of claims 1 to 4. 9. The method of claim 8,
Wherein the peptide is contained in the cosmetic composition in an amount of 0.0001 to 1.0% by weight. 9. The method of claim 8,
Wherein the cosmetic composition is selected from cosmetics, lotions, gels, creams, essences, packs, ampoules, lotions, cleansing agents, soaps, body products, soaps, oils, lipsticks and foundations.

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