KR101870898B1 - Galloyl-peptide derivatives and Anti-aginig Skin External Composition Comprising the Same - Google Patents

Abstract

The present invention relates to a peptide derivative to which galactic acid is conjugated, and a composition for external application for skin which is excellent in antioxidant and antioxidative efficacy including the peptide derivative.
The galloyl-peptide derivative conjugated with gallic acid according to the present invention and the composition for external application for skin containing the same have excellent antioxidant ability and at the same time, they are excellent in improvement of skin wrinkle and elasticity by promoting collagen synthesis, Widely available.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a peptide derivative having gallic acid binding thereto and an anti-aging skin composition containing the same,

The present invention relates to a peptide derivative to which galactic acid is conjugated, and a composition for external application for skin which is excellent in antioxidant and antioxidative efficacy including the peptide derivative.

As the industry develops, environmental pollution becomes serious, and there is a growing interest in skin aging prevention and whitening. In particular, human skin constantly undergoes changes, the most representative of which is degradation of the skin caused by aging and a reduction in visual beauty. The final appearance of skin appearance as a result of skin aging is wrinkle formation, skin elasticity degradation, senile spots formation, and wrinkle formation is considered to be the most typical phenomenon.

In addition, as the environmental pollution becomes serious, more and more people are experiencing discomfort due to skin inflammation. Therefore, researches on new substances for skin inflammation mitigation and treatment are actively carried out. In addition, skin whitening, And to develop functional cosmetic products for skin conditions such as astringency, antioxidation, skin wrinkle improvement, and skin irritation alleviation (Patent Document 1).

In particular, there has been a desperate need for the development of new materials that will be used as functional cosmetic materials having both antioxidant ability and ability to enhance collagen aggregation for improving skin wrinkles.

Patent Document 1: Korean Patent Laid-Open No. 10-2008-080253

The present inventors have made efforts to produce a composition for external application for skin having a skin improving ability, and confirmed that the peptide derivative having galactanic acid-bonded peptides has excellent antioxidative and anti-aging properties including antioxidative ability and wrinkle-reducing ability.

Accordingly, an object of the present invention is to provide a peptide derivative having excellent antioxidant ability and anti-aging ability, and an external preparation for skin containing the same.

In order to achieve the above object, the present invention provides a galloyl-peptide derivative represented by the following formula (1)

[Chemical Formula 1]

In this formula,

wherein n is 1 or 2 and L is selected from the group consisting of lucyl-valyl-histidine (LVH), isoleucyl-valyl-histidine (IVH), lysyl-threonyl-threonyl-seryl-serine (KTTKS), alanyl- (AHK), glycyl-histidyl-lysine (GHK), glutamyl-glutamyl-methionyl-glutaminyl-arginyl-arginine (EEMQRR), cysteinyl- Glycyl-glycyl-glycine (PFG), glycyl-valyl-glycyl-valyl-proline (GVGVP), tyrosyl-glycyl-glycyl- phenylalanylmethionine (YGGFM), and tyrosine- Ribyl-aryvinyl-lysyl-tyrosyl-proline (YGGFLRKYP) amino acid sequence.

The present invention also provides an antioxidant or skin external composition for skin improvement comprising the above-mentioned galloyl-peptide derivative as an active ingredient.

In one aspect, the present invention relates to a galloyl-peptide derivative having excellent antioxidant and anti-aging effect.

In the present invention, the galloyl-peptide derivative means a peptide derivative to which gallic acid is bound.

In the present invention, a gallyl group may be bonded to the N-terminus of the peptide (L).

The structure of the galloyl-peptide derivative of the present invention can be represented by the following Formula 1:

[Chemical Formula 1]

Wherein L is selected from the group consisting of lucyl-valyl-histidine (SEQ ID NO: 1: LVH), isoyl-valyl-histidine (SEQ ID NO: 2: IVH), lysyl-tryonyl- Glutamyl-methionyl-glutamate (SEQ ID NO: 3), alanyl-histidyl-lysine (SEQ ID NO: (SEQ ID NO. 6: EEMQRR), cysteinyl-alanyl-serine (SEQ ID NO: 7: CAS), prolyl-phenylalanyl-glycine (SEQ ID NO: 8: PFG), glycyl-valyl Glycyl-phenylalanyl-methionine (SEQ ID NO: 10: YGGFM), and tyrosine-glycyl-glycyl-phenylalanyl-proline (SEQ ID NO: 9: GVGVP), tyrosyl- (SEQ ID NO: 11: YGGFLRKYP) amino acid sequence shown in SEQ ID NO: 11.

In the present invention, the Galyl group is bound to the N-terminus of the peptide (L).

[Reaction Scheme 1]

Thus, the resulting galloyl-peptide derivative is represented by the following formula (2) when n is 1:

The peptide derivative according to the present invention can be prepared by synthesizing a desired peptide and then reacting with a galactic acid derivative. Such a desired peptide can be obtained by extracting a protein in vivo and then treating it with a protease to lower the molecular weight, And may be produced using a recombinant protein and a protein expression system, preferably by a chemical synthesis method using a peptide synthesizer or the like.

The peptide derivative according to the present invention is a peptide derivative,

(1) obtaining an NH2-protected peptide-resin by a conventional solid phase peptide synthesis (SPPS);

(2) reacting the obtained NH2-protected peptide-resin with a benzoic acid derivative; And

(3) removing the resin.

When a functional group is present in the side chain of the amino acid residue constituting the desired peptide, a peptide can be synthesized using the amino acid in which the functional group is protected in the step (1) Lt; / RTI >

One embodiment of the process for preparing a galloyl-peptide derivative according to the present invention using a functional group-protected amino acid is shown schematically in Scheme 2 below:

[Reaction Scheme 2]

(1) Peptide synthesis and purification

The peptide (L) according to the present invention can be used in combination with derivatives (such as lucyl-valyl-histidine (LVH), isoylsilyl-valyl-histidine (IVH), lysyltreonyl-threonyl- Glutamyl-methionyl-glutaminyl-arginyl-arginine (EEMQRR), cysteinyl-alanyl-serine (CAS) (YGGFM), and tyrosine-proline (GVGVP), prolyl-phenylalanyl-glycine (PFG), glycyl-valyl-glycyl- valyl- Lysyl-glycyl-phenylalanyl-lucyl-arginyl-lysyl-tyrosyl-proline (YGGFLRKYP)) can be prepared using Solid Phase Peptide Synthesis using Fmoc.

(2) using each phytochemical-peptide solid phase peptide synthesis method

Solid-phase peptide synthesis is a repetition of coupling-deprotection. The C-terminal carboxy group of the next amino acid in which the N-terminal is protected is reacted to the unprotected N-terminal amide group of the peptide bound to the solid support. Thereafter, the N-terminal is exposed so that the amino acid can then be deprotected and reacted.

Fmoc and tert-butoxycarbonyl (Boc) are two strategies for solid phase peptide synthesis. Unlike ribosomal protein synthesis, solid phase peptide synthesis is synthesized from the C-terminal to the N-terminal. The amino acid N-terminal is protected by these protecting groups and is composed of Fmoc-amino acid and Boc-amino acid. Among them, the Fmoc strategy protects the side chain at the N-terminal end of the amino acid with the Fmoc protecting group, which is easily removed from the base, and the tertiary-butyl (tBu) protecting group, which is easily removed from the acid. After binding the first Fmoc-amino acid to the linker of the resin, Fmoc is removed using Piperidine. The Kaiser test confirms that the Fmoc protecting group has been removed and then activates the carboxy group of the Fmoc-amino acid to give a second reaction. The free amine is checked on Resin by Kaiser test to confirm completion of reaction. The synthesis is synthesized according to the peptide sequence with the removal of the Fmoc protecting group and the subsequent reaction of Fmoc-amino acid in one cycle. The synthesized peptide is removed from the resion by treatment with TFA and the protection of the side chain is removed at this time.

Another embodiment of the process for producing a galloyl-peptide derivative according to the present invention using a functional group-protected amino acid is schematically shown in Reaction Scheme 3 below.

[Reaction Scheme 3]

As described above, the peptide (L) according to the present invention is capable of binding one or more than two or more galyl groups.

In another aspect, the present invention relates to an antioxidant or skin external composition for skin improvement comprising a peptide derivative according to the present invention as an active ingredient.

In the present invention, facilitation of skin improvement includes, for example, skin protection and skin condition improvement, skin whitening, prevention or improvement of skin aging and wrinkles, skin protection and alleviation of skin inflammation reaction, immunological disease improvement, Skin irritation mitigation, skin cell proliferation and regeneration ability, antioxidant ability, and collagen synthesis promoting ability.

In the present invention, the expression "comprising as an active ingredient" means a composition for external application for skin, which shows a skin improving effect as an external composition for skin, such as collagen synthesis, elasticity improvement or antioxidative activity associated with wrinkle- ≪ RTI ID = 0.0 > and / or < / RTI >

Particularly, the composition for external application for skin according to the present invention has an antioxidative ability according to the capability of free radical scavenging activity, and in particular, it can increase collagen biosynthesis. Collagen degradation inhibitory ability, and can be applied for skin aging prevention, skin wrinkle improvement / prevention, and skin elasticity enhancement / improvement.

In the present invention, the composition for external application for skin may be a cosmetic composition or a pharmaceutical composition.

The cosmetic composition of the present invention may contain an acceptable carrier in cosmetic preparations. Herein, "an acceptable carrier for a cosmetic preparation" is a known or used compound or composition that can be contained in a cosmetic preparation, or a compound or composition to be developed in the future, which is toxic, instable or irritating It says nothing.

The carrier may be included in the skin topical composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 90% by weight to about 99.99% by weight of the composition, based on the total weight thereof. However, since the ratio varies depending on the formulations described below in which the composition for external application for skin of the present invention is prepared, and its specific application site (face, neck, etc.) or its preferable application amount and the like, And should not be construed as limiting the scope of the invention.

Examples of the carrier include an alcohol, an oil, a surfactant, a fatty acid, a silicone oil, a wetting agent, a moisturizer, a viscous modifier, an emulsifier, a stabilizer, an ultraviolet scattering agent, an ultraviolet absorber, a coloring agent and a perfume. The compounds / compositions which can be used as the above-mentioned alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscous modifier, emulsifier, stabilizer, ultraviolet scattering agent, ultraviolet absorber, The person skilled in the art can select and use the appropriate substance / composition.

As an embodiment of the present invention, the composition for external application for skin according to the present invention may contain glycerin, butylene glycol, propylene glycol, polyoxyethylene hardened castor oil, ethanol, triethanolamine and the like in addition to the above- , Antiseptic, antioxidant, coloring agent, purified water and the like may be included as needed.

The composition for external application for skin according to the present invention can be prepared in various forms such as lotion, essence, gel, emulsion, lotion, cream (underwater type, water type, multiphase), solution, suspension ), Capsules (soft capsules, hard capsules) having a coating such as anhydrous products (oil and glycol), gel, mask, pack, powder or gelatin.

The skin of the present invention includes not only the face but also the scalp and whole body. The composition for external application for skin which can be applied to such scalp includes shampoo, rinse, treatment, hair removal agent, etc., and a body cleanser And the like can be manufactured in various forms.

The method for preparing a skin external composition containing a peptide derivative according to the present invention is not limited to the above-described manufacturing method, and any person skilled in the art may make modifications The composition for external application for skin containing the galloyl peptide derivative can be prepared.

In particular, the composition for external application for skin may be prepared in the form of a general emulsified formulation and a solubilized formulation, by a known manufacturing method, in addition to the manufacturing method specifically disclosed in the present invention.

When the cosmetic composition is manufactured from a cosmetic composition, the cosmetic composition of the emulsified formulation includes nutritional lotion, cream, essence and the like. It can also be prepared in the form of adjuvants which can be applied topically or systemically, which are conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base.

In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing an oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) May be provided in the form of a follicular dispersing agent of the type, cream, skin, lotion, powder, ointment, spray or conical stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

In addition, the composition for external application for skin of the present invention may further comprise at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, As used herein means any emulsion or emulsion which is commonly used in emulsifying or emulsifying agents, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics Adjuvants commonly used in the cosmetics or dermatological fields, such as other ingredients. And, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

The composition for external application for skin according to the present invention includes functional cosmetics capable of preventing skin aging, improving wrinkles, promoting skin elasticity, and improving skin elasticity due to antioxidant ability, collagen synthesis promoting ability.

In the present invention, in the case of the pharmaceutical composition, in addition to the above-mentioned galloyl peptide derivative, the carrier may include a "pharmaceutically acceptable carrier" including a diluent, a lubricant, a binder, a disintegrant, a sweetener, ≪ / RTI > The pharmaceutical composition may further comprise an additive. The additives may include flavoring agents, vitamins, and antioxidants. Examples of the diluent include lactose, dextrin, tapioca starch, corn starch, soybean oil, microcrystalline cellulose or mannitol as a carrier, magnesium stearate or talc as a lubricant, , And the binding agent may be polyvinylpyrrolidone or hydroxypropylcellulose. Examples of the disintegrant include carboxymethylcellulose calcium, sodium starch glycolate, polacrilin potassium, or crospovidone; examples of sweeteners include white sugar, fructose, sorbitol, or aspartame; stabilizers include sodium carboxymethylcellulose, Or xanthan gum, and the preservative may be methyl paraoxybenzoate, propyl p-hydroxybenzoate, or potassium sorbate.

The pharmaceutical composition may be formulated into conventional pharmaceutical formulations known in the art. The pharmaceutical composition may be formulated and administered in the form of an oral administration preparation, an injection preparation, a suppository, a transdermal preparation, and a dosage form. For example, the formulations may be formulated for oral administration, such as solutions, suspensions, powders, granules, tablets, capsules, pills, or expanses.

The galloyl-peptide derivative conjugated with gallic acid according to the present invention and the composition for external application for skin containing the same have excellent antioxidant ability and at the same time, they are excellent in improvement of skin wrinkle and elasticity by promoting collagen synthesis, Widely available.

FIG. 1 is a graph showing the results of measurement of Ra of the eye wrinkle region as a result of confirming the effect of improving the wrinkles of the eye by applying the peptide derivative conjugated with gallic acid according to the present invention to a human body.
FIG. 2 is a graph showing the Rmax measurement result of the eye wrinkle region as a result of confirming the effect of improving the wrinkles of the eye by applying the peptide derivative conjugated with gallic acid according to the present invention to human body.
FIG. 3 is a 3D photograph of the eye wrinkle using PRIMOS High Resolution of the eye wrinkle region as a result of confirming the wrinkle improving effect of the human eye by applying the peptide derivative conjugated with gallic acid according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

One. Galois (3,4,5- Trihydroxybenzoyl ) -Tripeptide ( Lucil - valyl-histidine)

1-1. Tripeptide ( Lucil - valyl-histidine, LVH ) Synthesis of

The tripeptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group, and N-hydroxybenzotriazole Amino acid residues were extended using N-hydroxybenzotriazole (HOBt) and N, N'-diisopropylcarbodiimide (DIC) as activators (Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of His (Trt) resin (Nova Biochem, Inc.) protected with Fmoc in the glass reactor was bubbled with 3 ml of N, N-Dimethylformamide (DMF) After swelling, the solvent was removed and Fmoc was removed by treatment with 2 ml of 20% piperidine (3 ml). The Fmoc-removed His resin was treated 3 times with dichloromethane (DCM), 3 times with DMF and reacted with HOBt-DIC-activated Fmoc-Val-OH (valine) solution at room temperature for about 2 hours. The NH2-protected peptide (lucyl-valyl-histidine) resin obtained by successive reaction of leucine with this process was washed three times with DCM and three times with DMF.

1-2. 3,4,5- Trihydroxybenzoyl ( Galois ) -Tripeptide ( Lucil - valyl-histidine, LVH ) Synthesis of

The NH2-protected peptide (lucyl-valyl-histidine) -resin synthesized above was washed with DMF and DCM, and 5 equivalents of 3,4,5-trihydroxybenzoic acid and 5 equivalents of HOBt (5 eq.) And DIC (5 eq.) Were mixed and reacted at room temperature overnight. After the reaction was completed, the reaction mixture was washed three times with DMF and DCM, respectively, and then dried.

The dried 3,4,5-trihydroxybenzoyl (galloyl) -trepeptide (lucyl-valyl-histidine) resin was dissolved in trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: : 5: 2.5 (v / v)) at room temperature for 2 to 3 hours to remove the Boc group, which is a protecting group of the functional group present in the side chain of the amino acid residue constituting the peptide, -Trihydroxybenzoyl (galloyl) -treopeptide (lucyl-valyl-histidine) was separated and the peptide was precipitated with cold diethyl ether. The obtained dried 3,4,5-trihydroxybenzoyl (galloyl) -treopeptide (lucyl-valyl-histidine) was dissolved in water containing 0.1% trifluoroacetic acid and acetonitrile as a solvent by reverse phase high performance liquid chromatography (reverse phase high performance liquid chromatography; column: Gemini, C18 110A 25021.2 mm).

The thus obtained peptide was purified using high performance liquid chromatography (HPLC) and then lyophilized to obtain LVH having a purity of 95% or more.

2. Galois (3,4,5- Trihydroxybenzoyl ) -Tripeptide ( Isoleucyl - valyl-histidine)

2-1. Synthesis of tripeptide ( isoyl -valyl-histidine, IVH ); The tripeptide was obtained by using the same method as in the above method 1, except that only the desired tripeptide sequence was different.

2-2. Synthesis of 3,4,5 -trihydroxybenzoyl ( galloyl ) -treopeptide ( isoyl -valyl-histidine, IVH ); A tripeptide derivative was obtained by using the same method as in the above method 1 except that only the desired tripeptide sequence was different.

3. Galois (3,4,5- Trihydroxybenzoyl ) -Pentapeptide ( Rysil - Threonyl - Threonyl - Rysil - Synthesis of serine)

3-1. Penta peptide (florisil-TRE O'Neill-TRE O'Neill-florisil-serine, KTTKS) synthesis; The pentapeptide was obtained by using the same method as in the above method 1 except that only the desired pentapeptide sequence was different.

3-2. Synthesis of 3,4,5-trihydroxy benzoyl (galloyl) - penta peptide (serine, KTTKS florisil-TRE O'Neill-TRE O'Neill-florisil); A pentapeptide derivative was obtained by using the same method as in the above method 1 except that only the desired pentapeptide sequence was different.

4. Galois (3,4,5- Trihydroxybenzoyl ) -Tripeptide ( Alany - Histidyl - lysine) Synthesis of

4-1. Synthesis of (lysine, AHK alanyl-peptidyl Heath) tripeptide; The tripeptide was obtained by using the same method as in the above method 1, except that only the desired tripeptide sequence was different.

4-2. Synthesis of 3,4,5 -trihydroxybenzoyl ( galloyl ) -treopeptide ( alanyl-histidyl-lysine , AHK ); A tripeptide derivative was obtained by using the same method as in the above method 1 except that only the desired tripeptide sequence was different.

5. Galois (3,4,5- Trihydroxybenzoyl ) -Tripeptide (glycyl- Histidyl - lysine) Synthesis of

5-1. Synthesis of tripeptide (glycyl- histidyl -lysine, GHK ); The tripeptide was obtained by using the same method as in the above method 1, except that only the desired tripeptide sequence was different.

5-2. Synthesis of 3,4,5 -trihydroxybenzoyl ( galloyl ) -treopeptide (glycyl- histidyl -lysine, GHK ); A tripeptide derivative was obtained by using the same method as in the above method 1 except that only the desired tripeptide sequence was different.

6. Galois (3,4,5- Trihydroxybenzoyl ) -Hexapeptide ( Glutamyl - Glutamyl - Methionyl - Glutaminyl - Argigny - arginine) Synthesis of

6-1. Synthesis of hexapeptide ( glutamyl - glutamyl - methionyl - glutaminyl - arginyl -arginine, EEMQRR); A hexapeptide was obtained by using the same method as in the above method 1 except that only the desired hexapeptide sequence was different.

6-2. Synthesis of 3,4,5 -trihydroxybenzoyl ( galloyl ) -hexapeptide ( glutamyl-glutamyl-methionyl-glutaminyl-arginyl-arginine , EEMQRR ); A hexapeptide derivative was obtained by using the same method as in the above method 1 except that only the desired hexapeptide sequence was different.

7. Galois (3,4,5- Trihydroxybenzoyl ) -Tripeptide ( Systeigny - Alany - Synthesis of serine)

7-1. Synthesis of tripeptide ( cysteinyl - alanyl -serine, CAS ); The tripeptide was obtained by using the same method as in the above method 1, except that only the desired tripeptide sequence was different.

7-2. Synthesis of (serine, CAS System table carbonyl-alanyl) - 3,4,5-trihydroxy benzoyl (galloyl) tripeptide; A tripeptide derivative was obtained by using the same method as in the above method 1 except that only the desired tripeptide sequence was different.

8. Galois (3,4,5- Trihydroxybenzoyl ) -Tripeptide ( Ploll - Phenylalanyl - glycine) Synthesis of

8-1. Synthesis of tripeptide ( prolyl - phenylalanyl -glycine, PFG ); The tripeptide was obtained by using the same method as in the above method 1, except that only the desired tripeptide sequence was different.

8-2. Synthesis of 3,4,5 -trihydroxybenzoyl ( galloyl ) -treopeptide ( prolyl - phenylalanyl -glycine, PFG ); A tripeptide derivative was obtained by using the same method as in the above method 1 except that only the desired tripeptide sequence was different.

9. Galois (3,4,5- Trihydroxybenzoyl ) -Pentapeptide (glycyl-valyl-glycyl-valyl-proline)

9-1. Synthesis of pentapeptide (glycyl-valyl-glycyl-valyl-proline, GVGVP ); The pentapeptide was obtained by using the same method as in the above method 1 except that only the desired pentapeptide sequence was different.

9-2. Synthesis of 3,4,5-trihydroxy benzoyl (galloyl) - penta peptide (proline, GVGVP glycyl-balril-glycyl-balril); A pentapeptide derivative was obtained by using the same method as in the above method 1 except that only the desired pentapeptide sequence was different.

10. Galois (3,4,5- Trihydroxybenzoyl ) -Pentapeptide ( Tyrosine - glycyl-glycyl- Phenylalanyl - methionine)

10-1. Synthesis of pentapeptide ( tyrosyl -glycyl-glycyl- phenylalanyl -methionine, PFG ); The pentapeptide was obtained by using the same method as in the above method 1 except that only the desired pentapeptide sequence was different.

10-2. Synthesis of 3,4,5 -trihydroxybenzoyl ( galloyl ) -pentapeptide ( tyrosyl -glycyl-glycyl- phenylalanyl -methionine, YGGFM); A pentapeptide derivative was obtained by using the same method as in the above method 1 except that only the desired pentapeptide sequence was different.

11. Galois (3,4,5- Trihydroxybenzoyl ) -Octapeptide (tyrosine-glycyl-glycyl- Phenylalanyl - Lucil - Argigny - Rysil - Tyrosine - proline) Synthesis of

11-1. Synthesis of (proline, tyrosine YGGFLRKYP-glycyl-glycyl-phenyl alanyl-Lucille - are ginil-florisil-tee room) octa-peptides; The octapeptide was obtained by differentiating only the desired octapeptide sequence using the same method as the one described above.

11-2. Synthesis of (proline, tyrosine YGGFLRKYP-glycyl-glycyl-phenyl alanyl-Lucille - are ginil-florisil-tee room) - 3,4,5-trihydroxy benzoyl (galloyl) octa-peptides; The octapeptide was obtained by using the same method as in the above method 1 except that only the desired octapeptide sequence was different.

Experimental Example  1: cytotoxicity ( Cell viability ) Evaluation test

HaCaT cell (Keratinocyte) is divided into 1 × ¹⁰ 5/24 well and cultured. After 24 hours, the medium was replaced with a medium containing no FBS, starvation was performed for 24 hours, and the sample of the gallo-peptide derivative prepared in Example 1 at a concentration of 5 ppm was treated. After 24 hours of treatment, the medium is removed and the cells are treated with 5 mg / ml MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyl Tetrazolium Bromide) reagent at 40ul / well and incubated for 4 hours. After 4 hours, remove the medium, add 1 ml of DMSO (Amresco, 0231-500ML) and shake for 10 minutes. Take 200ul at 96 well and measure absorbance at 540nm with Spectrophotometer (Thermo). The degree of cytotoxicity was expressed as a percentage based on the absorbance intensity of the solvent in which the substance was dissolved in the control.

[Equation 1]

Cell viability (%) = (absorbance of test group / absorbance of control group) x 100

sample Cell survival rate
Cell viability (%) One Galloyl-LVH 110 2 Galloyl-IVH 105 3 Galloyl-KTTKS 100 4 Galloyl-AHK 103 5 Galloyl-GHK 104 6 Galloyl-EEMQRR 102 7 Galloyl-CAS 100 8 Galloyl-PFG 106 9 Galloyl-GVGVP 104 10 Galloyl-YGGFM 100 11 GalloylYGGFLRKYP 102

As a result, it was confirmed that the galloyl-peptide derivatives did not significantly affect cytotoxicity

Experimental Example  2: DPPH  freedom Radical The ability to eliminate  Antioxidant test

Compound 1, 1-diphenyl-2-picryl hydrazyl (DPPH, Sigma D9132-1G, USA) generates free radicals in ethanol, and is produced by mixing with the peptide derivative prepared in Example 1 at a certain ratio (Free radical scavenging activity test) to determine the amount of free radicals to be reduced. The free radical scavenging activity was modified by the method of Kim et al. (Kor. J. Pharmacogn., 24 (4), 299-303 (1993)) and the stable free radical DPPH (1,1-diphenyl- picryl hydrazyl, Sigma D9132-1G, USA) reagent was used.

150 μl of a 0.2 mM DPPH solution (ethanol in the case of blank) was diluted to a concentration of 5 ppm of a peptide derivative and galactic acid (a positive control), and 150 μl of each of them was added and mixed. After incubation at room temperature for 30 minutes, And the control group was set to use purified water. After the absorbance of the test group and the control group was measured, the degree of antioxidant activity was expressed as a percentage based on the absorbance of the control group using pure water. The free radical scavenging activity was measured using the following formula. In the positive control group, 5 ppm of gallic acid was used at the same concentration.

&Quot; (2) "

Free radical activity inhibition rate (%) = 100- (Reaction absorbance of each sample liquid / Reaction absorbance of the blank) X100

sample Free radical activity inhibition (%) Control group 0 Gallic acid 46 One Galloyl-LVH 56 2 Galloyl-IVH 50 3 Galloyl-KTTKS 44 4 Galloyl-AHK 52 5 Galloyl-GHK 56 6 Galloyl-EEMQRR 76 7 Galloyl-CAS 66 8 Galloyl-PFG 55 9 Galloyl-GVGVP 51 10 Galloyl-YGGFM 45 11 Galloyl YGGFLRKYP 34

As a result, it was confirmed that the galloyl-peptide derivative had much higher DPPH free radical scavenging activity than gallic acid, and thus showed excellent antioxidant ability.

Experimental Example  3: Evaluation of anti-aging

3.1 Procollagen Type I C- Peptides EIA  ( Procollagen type  I C- peptide EIA )

Human dermal fibroblast was dispensed into 1 x 10 ⁵ cells / ml on a 48 well plate, cultured for 1 day until reaching a confluency of about 60%, and further cultured for 1 day in FBS-free medium . The medium was removed prior to the treatment with the galloyl derivative peptide of Example 1, washed with PBS (phosphate buffered saline), diluted to 5 ppm of the galloyl derivative peptide of Example 1 in a DMEM medium to which no FBS was added, CO ₂ incubator for 48 hours. The negative control (NC) was prepared by culturing the cells in the same medium without the addition of the test substance. TGF- (10 ng / ml) was treated as a positive control (PC). In order to evaluate the expression of Procollagen, the culture was cultured by the above culture method and quantitatively analyzed using Procollagen Type I C-Peptide (PIP) EIA Kit (Cat #. MK101, Takara, Japan). The supernatant was applied to the coated 96-well plate and the primary antibody, anti-collagen antibody, was treated at 37 ° C for 90 minutes and then washed three times with TPBS (0.1% Tween 20 in PBS). Secondary antibody, anti-mouse IgG (whole mouse, alkaline phosphatase conjugated) was reacted for 90 minutes and then washed three times with TPBS. After incubation at room temperature for 30 minutes with 1 mg / mL p-nitrophenyl phosphate in a solution of alkaline phosphatease substrate solution, absorbance was measured at 405 nm with a microplate reader.

Procollagen Type C-Peptide EIA kit was used to test the effect of collagen synthesis on collagen synthesis. Galactic acid did not show collagen synthesis ability, and it was confirmed that it promoted Type I collagen synthesis in galloyl derivative peptide.

sample PIP (ng / ml) Control group 100 Gallic acid 98 One Galloyl-LVH 111 2 Galloyl-IVH 132 3 Galloyl-KTTKS 137 4 Galloyl-AHK 156 5 Galloyl-GHK 168 6 Galloyl-EEMQRR 189 7 Galloyl-CAS 132 8 Galloyl-PFG 124 9 Galloyl-GVGVP 180 10 Galloyl-YGGFM 177 11 Galloyl YGGFLRKYP 206

3.2. PCOLCE , MMP9  Expression test

(1) Cell culture

Human fibroblasts (CCD986sk, fibroblast; PCOLCE, MMP1) / human keratinocytes (HaCaT, keratinocyte; for MMP9) were cultured in Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) Were cultured in a 100 mm / 60.1 cm culture dish with antimycotic (GIBCO, Cat. # 15240-062) at 37 ° C and 5% CO ₂ . When human keratinocytes are confluent at a concentration of 80% or more, they are seeded at 5 × 10 ⁴ cells / well in a 96-well plate, cultured until confluence reaches 80% or more in cell culture conditions, Next, 50mJ of UVB was irradiated to induce inflammation, and then the test material was treated with DMEM medium (without FBS) after exchange. After incubation for 4 hours, the cells were incubated at the mRNA level to determine the effect of each substance on the expression of COX-2, an inflammation-related gene induced by UV irradiation. The test method is Real-time PCR, and the test procedure is as follows.

 (2) RNA Isolation  & Reverse transcription

RNA isolation and cDNA synthesis were performed using SuperPrep cell lysis & RT Kit for qPCR (TOYOBO Cat. # SCQ-101). The cells were washed once with DPBS, and incubated at room temperature for 5 minutes with 50 μl cell lysis mixture (gDAN remover added to lysis buffer). Stop buffer was added to stop the reaction and stored in ice. Add 8 ㎕ of extracted mRNA to 32 ㎕ of RT reaction mixture and perform PCR to synthesize cDNA. The PCR conditions are 37 캜 for 15 min, 50 캜 for 5 min, and 98 캜 for 5 min.

(3) Real - time PCR  analysis

In order to comparatively analyze the expression of the gene, the synthesized cDNA was used as a template and subjected to a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of Thunderbird SYBR qPCR Mix (TOYOBO Cat. # QPS-201). The primers used in the experiments were Qiagen QuantiTectprimerassays (PCOLCE, Cat. # QT01005725). The relative incidence was normalized by Housekeeping gene (GAPDH, Cat. # QT01192646). Real-time PCR conditions are as follows.

<Real-time cycler conditions> Step Time Temperature Reverse transcription 30 min 50 PCR initial activation step 15 min 95 3-step cycling

Denaturation 15 s 94 Annealing 30 s 60 Extension 30 s 72 Number of cycles 45

sample PCOLCE
Expression MMP 9
Suppression of expression Control group One One 0.4% Adenosine 1.4 0.49 Gallic acid 0.99 1.24 One Galloyl-LVH 2.9 0.67 2 Galloyl-IVH 3.24 0.66 3 Galloyl-KTTKS 2.2 0.36 4 Galloyl-AHK 2.1 0.35 5 Galloyl-GHK 2.2 0.42 6 Galloyl-EEMQRR 2.3 0.51 7 Galloyl-CAS 1.54 0.59 8 Galloyl-PFG 1.62 0.64 9 Galloyl-GVGVP 1.89 0.32 10 Galloyl-YGGFM 1.98 0.54 11 Galloyl YGGFLRKYP 2.4 0.41

&Quot; (3) &quot;

Expression (fold) = (Expression rate of sample treated group) / (Expression rate of control group)

In order to examine the effect on procollagen synthesis, the amount of mRNA expression of PCOLCE (gene related to collagen synthesis) was compared. As a result, it was confirmed that the expression of PCOLCE was increased when 11 kinds of Galloyl-derivative peptide were treated. In particular, in the case of Galloyl-LVH, Galloyl-IVH, Galloyl-KTTKS, Galloyl-AHK, Galloyl-GHK and Galloyl-EEMQRR, the expression of collagen synthesis gene was confirmed twice or more as compared with the control, Enhancement, and elasticity.

In addition, the expression of MMP9, which is known as Fibroblast collagenase, was also confirmed, and it was confirmed that the expression of MMP9 was decreased when treated with Galloyl-derivative peptide. It is known that when having MMP-9 inhibitory activity, it protects collagen and prevents elasticity and stretching of skin. Thus, these results suggest that Galloyl-derivative peptides inhibit collagen degradation and may be effective in improving wrinkles.

Experimental Example  4: Galois  Derivative eye wrinkle improvement effect Human body application test

: Serum and essence were prepared based on the composition shown in Example 2, containing Galloyl-YGGFLRKYP, Galloyl-YGGFM, and Galloyl-IVH.

4.1. Test evaluation method

(1) Study subjects: 21 (30 ~ 55 years old, female)

30s 40s 50s Sum 4.8% 28.6% 66.7% 100%

(2) Test period: 4 weeks (before use, after 2 weeks of use, after 4 weeks of use)

(3) After 2 weeks and 4 weeks after the use, the evaluation method was as follows:

- Evaluation of eye wrinkles using PRIMOS High Resolution

- Subjective questionnaire evaluation by subjects

- Assessment of adverse reactions by dermatologists and subjects

(4) Sample Usage: Twice a day (morning and evening) After cleansing, trim the skin with the usual foundation product and apply it to the eye area in the order of serum and essence containing the galloyl-peptide derivative.

(5) Sample information:

 - Sample A (test group): Galoyl-peptide derivatives Galloyl-YGGFLRKYP, Galloyl-YGGFM, Serum and Essence containing Galloyl-IVH

 Sample B (Control): Galoyl-peptide derivative Galloyl-YGGFLRKYP, Galloyl-YGGFM, Serum and essence not containing Galloyl-IVH

- The test subject is prohibited to use the foundation product and makeup from 12 hours before the visit.

- The human body test is conducted under constant temperature and humidity (20 ~ 24 ℃, 40 ~ 60% RH) without air movement and no direct sunlight

- After cleansing, take the stabilization under constant temperature and humidity condition for 30 minutes and conduct the test

4.2 Test results

1. Results of eye wrinkle measurement using PRIMOS High Resolution

The measurement results, reduction rate, and statistical analysis results of the Roughness parameters of the eye wrinkles using PRIMOS High Resolution are as follows.

As a result of analyzing the measured value of Roughness variable of the eye wrinkle area using PRIMOS High Resolution, the Ra value of the sample A applied region was statistically significant ( p <0.05 ) ( P <0.05 ), respectively. The Rmax values were 3.78% and 5.93% after 2 weeks and 4 weeks after use, respectively. In addition, the site of application of Sample A showed a statistically significant decrease ( p <0.05 ) after 4 weeks of use compared with the site of application of Sample B. Thus, Sample A was found to exert an excellent effect in improving eye wrinkles after 4 weeks of use. (Figs. 1 to 3).

Example 2: Preparation of cosmetic composition

Cosmetics of emulsified form such as nutritional lotion, cream, essence, etc. and cosmetics of solubilized form such as softened longevity were prepared with the cosmetic product containing the galloyl derivative peptide of the present invention as an active ingredient.

Production Example 2-1: Lotion

According to the following formulation, it was prepared according to the conventional lotion preparation method.

Raw material name Weight% (w / w) glycerin 5.0 Dipropylene glycol 3.0 Hyaluronic acid 0.5 Polyoxyethylene hardened castor oil 0.1 Polyethylene oleyl ethyl 0.1 ethanol 5.0 antiseptic 0.15 Beauty Suitable amount flash Suitable amount The galloyl peptide derivative of Example 1 2 Purified water to 100

Production Example 2-2: Essence

According to the following prescription, it was prepared according to a conventional method for producing essence.

Raw material name Weight% (w / w) glycerin 6.00 Butylene glycol 4.00 Cetearyl hexanoate 3.00 Microcrystalline wax 2.80 Diisostearyl malate 2.50 Beta-glucan 2.00 Glyceryl monostearate 1.50 Cyclomethicone 1.50 Glyceryl oleate 1.40 Polysorbate 60 1.20 Stearic acid 0.70 Dimethicone 0.60 Sorbitan stearate 0.30 Clofenine 0.12 Allantoin 0.10 Caffeine 0.10 Panthenol 0.10 The galloyl peptide derivative of Example 1 0.001 Spices Suitable amount flash Suitable amount Purified water to 100

Manufacturing example  2-3: Lotion

According to the following formulation, it was prepared according to a conventional lotion preparation method.

Raw material name Weight% (w / w) Cetostearyl alcohol 0.8 Self emulsifying monostearic acid 1.0 Wax 0.5 Stearic acid 0.5 Liquid paraffin 7.0 Squalane 5.0 Macadamia oil 3.0 Isocetyl octanoate 2.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 1.2 glycerin 4.0 Propylene glycol 4.0 Betaine 4.0 Carboxy polymer 0.12 Triethanolamine 0.15 antiseptic 0.25 Spices Suitable amount flash Suitable amount The galloyl peptide derivative of Example 1 5.0 Purified water to 100

Manufacturing example  2-4: Cream

According to the following formulation, it was prepared according to a conventional cream production method.

Raw material name Weight% (w / w) Cetostearyl alcohol 3.0 Self emulsifying monostearic acid 1.5 Chin type monostearate 1.5 Wax 0.5 Liquid paraffin 8.0 Squalane 7.0 Isocetyl octanoate 4.0 Refined jojoba oil 4.0 Dimethylsiloxane 0.3 Sorbitan monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 Propylene glycol 4.0 Betaine 4.0 Xanthan gum 0.06 Triethanolamine 0.10 antiseptic 0.25 Spices Suitable amount flash Suitable amount The galloyl peptide derivative of Example 1 7.0 Purified water to 100

Manufacturing example  2-5: Gel

According to the following prescription, it was prepared according to a conventional gel preparation method.

Raw material name Weight% (w / w) glycerin 4.0 Propylene glycol 4.0 ethanol 10 Polyoxyethylene hardened castor oil 0.1 Carboxy polymer 0.30 Triethanolamine 0.30 antiseptic Suitable amount Spices Suitable amount flash Suitable amount The galloyl peptide derivative of Example 1 1.0 Purified water to 100

Manufacturing example  2-6: Serum

Raw material name Weight% (w / w) Purified water to 100 Butylene glycol 3 glycerin 2 ethanol 2 Hydroxypropyl gua 1.2 Sodium hyaluronate 0.5 Betaine 0.5 PIGGY-60 Hydrogenated Castor Oil 0.3 1,2-hexanediol 0.24 Caprylic hydroxamic acid 0.07 Hydrolyzed corn starch 0.03 Beta-glucan 0.03 Disodium iodide 0.02 Spices 0.02 Sucroose 0.01 Polyglutamic acid 0.01 The galloyl peptide derivative of Example 1 0.001

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> BIO FD & C <120> Galloyl-peptide derivatives and Anti-aging Skin External          Composition Comprising the Same <130> P2015-0430 <160> 11 <170> KoPatentin 3.0 <210> 1 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 1 Leu Val His   One <210> 2 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 2 Ile Val His   One <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 3 Lys Thr Thr Lys Ser   1 5 <210> 4 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 4 Ala His Lys   One <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 5 Gly His Lys   One <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 6 Glu Glu Met Gln Arg Arg   1 5 <210> 7 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 7 Cys Ala Ser   One <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 8 Pro Phe Gly   One <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 9 Gly Val Gly Val Pro   1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 10 Tyr Gly Gly Phe Met   1 5 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 11 Leu Val His   One

Claims (5)

A galloyl-peptide derivative represented by the following formula (1): &lt; EMI ID =
[Chemical Formula 1]

Wherein n is 1 or 2 and L is a peptide consisting of a ribonyl-tryonyl-threonyl-lysyl-serine (KTTKS) amino acid sequence,
The galloyl-peptide derivative is characterized in that the galloyl-peptide derivative has antioxidative activity and collagen production promoting activity. The galloyl-peptide derivative according to claim 1, wherein a galloyl group is bonded to the N-terminus of the peptide (L).
delete A cosmetic composition for improving skin wrinkles or skin elasticity comprising a galloyl-peptide derivative according to claim 1. delete

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