KR20150011726A - Novel Peptide derivatives from Jellyfish Extract and Composition for Skin External Application Comprising the Same - Google Patents

Abstract

The present invention relates to a peptide derivative derived from jellyfish having anti-aging function, and to a composition containing the same for external application to skin and, more specifically, to a novel tripeptide derivative derived from jellyfish and to an anti-aging composition containing the same for external application to skin. According to the present invention, the composition for external application to skin reduces synthesis of melanin, is effective in whitening, has increased collagen synthesis ability, is effective against wrinkles, has inflammatory inhibitory effect by inhibiting iNOS activity and excellent antioxidant ability, and can prevent aging of skin.

Description

[0001] The present invention relates to a peptide derivative derived from a jellyfish having an anti-aging function and a composition for external application containing the peptide. [0002]

More particularly, the present invention relates to a novel tripeptide derivative derived from jellyfish and an anti-aging dermatological composition containing the same. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide derivative derived from a jellyfish having an anti-

The skin protects all the organs in the body from changes in temperature and humidity and from external stimuli such as ultraviolet rays.

However, constant physical and chemical stimuli received from the outside deteriorate the skin function and accelerate aging of the skin. Such aging of the skin accompanies the deterioration of the skin constituents as a whole, resulting in the skin being easily split. In addition, decreased fibroblasts and impaired activity of these cells are also an important part of the skin aging process.

In order to prevent such skin aging phenomenon and to maintain healthier and more beautiful skin, cosmetics containing physiologically active substances obtained from various plants, animals and microorganisms have been conventionally used to maintain the inherent functions of the skin, However, conventional cosmetic raw materials are still insufficient to satisfy satisfactory effects and efficacies, and have a problem of causing adverse skin reactions.

In addition, inflammation is one of the defense mechanisms that try to minimize or minimize the reaction when cells or tissues are damaged by causes such as stress or ultraviolet rays, causing cell reactions with nerves, blood vessels, tissues, blood, etc. Overexposure can result in itching, fever, redness, pain, skin irritation such as tissue damage, and skin trouble.

Such skin inflammation and skin troubles can regulate stimulation and inflammation by regulating cytokines and inhibiting the expression of inflammatory mediators to normalize skin. Removal of inflammation to eliminate inflammation, the action of reducing vital signs and symptoms is called anti-inflammatory. Until now, the substances that are used for anti-inflammation are nonsteroidal system such as flufenamicacid, ibuprofen,

(benzydamine), indomethacin (indomethacin), and the steroid system,

Prednisolone, and dexamethasone. In addition, neuropeptide antagonists, bradykinin antagonists, COX inhibitors, and the like have been proposed. However, most of them have problems in terms of safety to skin and stability in cosmetic preparation And its use is limited (Patent Document 10-2008-0020853).

Therefore, it is urgently required to develop new materials that are effective for activating skin cells without skin toxicity while eliminating these problems, and for improving whitening and wrinkle improvement.

Korean Patent Publication No. 10-2008-0020853

The present inventors newly synthesized a tripeptide having an amino acid sequence of LVH and a tripeptide derivative in which any one of benzyl, kojic acid, benzoic acid derivative and cinnamic acid derivative substituted with a dihydroxyl group was bonded, Wrinkle improvement, anti-inflammation, and antioxidation, and completed the present invention.

Therefore, an object of the present invention is to provide a peptide derivative in which a tripeptide having an amino acid sequence of LVH alone or a tripeptide is bonded to any one of benzyl, kojic acid, benzoic acid derivative and cinnamic acid derivative substituted with dihydroxyl groups, To provide a composition for external application.

The present invention relates to tripeptide derivatives comprising the following general formula 1:

[Formula 1]

A-B

Wherein A is a functional group selected from the group consisting of benzyl, kojic acid, benzoic acid derivatives and cinnamic acid derivatives absent or substituted with dihydroxyl groups, and B is a tripeptide having the amino acid sequence of LVH.

The present invention also provides a whitening dermatological external preparation composition comprising the tripeptide derivative.

The present invention also provides a composition for external application for skin for improving wrinkles comprising the tripeptide derivative.

The present invention also provides an anti-inflammatory dermatological composition comprising the tripeptide derivative.

The present invention also provides an antioxidant composition for external application for skin comprising the tripeptide derivative.

The composition for external application for skin according to the present invention is effective for whitening by reducing melanin synthesis and has an increased collagen combining ability and is excellent for wrinkle improvement. It has an anti-inflammatory effect and an excellent antioxidative effect by inhibition of iNOS activity, It has the effect of preventing aging.

Hereinafter, the present invention will be described in detail.

The present invention relates to a tripeptide having a LVH amino acid sequence or a tripeptide derivative in which any one of benzyl, kojic acid, benzoic acid and cinnamic acid derivatives substituted with a dihydroxyl group is bonded, 1 < / RTI >: < RTI ID = 0.0 >

 [Formula 1]

A-B

Wherein A is a functional group selected from the group consisting of benzyl, kojic acid, benzoic acid derivatives and cinnamic acid derivatives that are absent or substituted with dihydroxyl groups, and B is a tripeptide having the amino acid sequence of LVH.

Here, LVH is a tripeptide composed of lucyl-valyl-histidine.

In the present invention, the process of obtaining LVH is as follows.

The LVH peptide was derived from the full moon water jellyfish (Aurelia aurita). It is collected by collecting water jellyfish of full moon, removing salinity through several washing processes, lyophilizing and pulverizing and separating and purifying the powder from ethanol extract, ultrasonic and hot water extract. Full moon Water jellyfish ( Aurelia aurita ) is a species that lives in the coastal waters of Korea during the year, and it is a species that abundant in the summer and fill the intake of the power plant. The diameter of the umbrella is 10-20cm, and four gonads are arranged in a transparent circular umbrella, forming a flower leaf shape.

As an embodiment of the present invention, it may be a tripeptide derivative composed of lucyl-valyl-histidine without A.

Specifically, the tripeptide derivative can be obtained by the following process:

Is synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group, and N-hydroxybenzotriazole (N- hydroxybenzotriazole (HOBt) and N, N'-dicyclohexylcarbodiimide (DCC) are used as an activator to extend the amino acid residues. [Reference: Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

 Specifically, 0.04 g of histidine resin (Nova Biochem, Inc) in which the amino group was protected with Fmoc in a glass reactor was treated with 3 ml of N-methyl-2-pyrrolidone (NMP) After swelling, the solvent is removed and the Fmoc is removed by treating with 2 ml of 20% piperidine (3 ml). The histidine resin from which Fmoc was removed was treated twice with dichloromethane (DCM), twice with NMP, and reacted with the valine solution activated with HOBt-DCC at room temperature for about 2 hours. The NH2-protected peptide (lucyl-valyl-histidine) resin obtained by continuously reacting leucine in this manner is washed twice with DCM, twice with NMP, twice with DCM, and then completely dried.

In one embodiment of the present invention, A may be benzyl substituted with a dihydroxyl group.

Where the substitution position of the hydroxyl groups may be 2, 3 or 2, and in one embodiment may be benzyl substituted with a 2, 4 hydroxyl group. The structure of the tripeptide derivative to which a benzyl derivative having a hydroxy group is bonded is represented by the following formula (1). Although a dihydroxy group is represented by the following formula, a benzyl derivative having a hydroxy group is also included in the present invention.

 [Chemical Formula 1]

R 1, R 2 and R 3 are independently -H, -OH, a C 1 -C 6 alkyl group, a C 1 -C 6 alkoxy group, a cyano, a halo or an amino group, preferably a hydrogen or a hydroxyl group. The peptide is a tripeptide consisting of lucyl-valyl-histidine.

Scheme 1 is a schematic representation of a process for preparing a tripeptide derivative having a benzyl group substituted with the 2,4-dihydroxyl group, and a specific synthesis process is based on the description of Example 2.

[Reaction Scheme 1]

In another embodiment of the present invention, A may be kojic acid. Wherein the kojic acid may be substituted or unsubstituted koic acid and may be substituted with -OH, a C1-C6 alkyl group, a C1-C6 alkoxy group, a cyano, a halo or an amino group when substituted. (3-hydroxy-4-oxo - 4H-pyran-2-yl) methyloxycarbonyl-tripeptide (lucyl-valyl-histidine) .

(2)

In the above formula (2), the peptide is a tripeptide consisting of lucyl-valyl-histidine.

Scheme 2 is a schematic representation of the preparation of tripeptide derivative conjugated with kojic acid, and the specific synthesis procedure is based on Example 3.

[Reaction Scheme 2]

In another embodiment of the present invention, A may be a benzoic acid derivative. The tripeptide having a benzoic acid derivative having a substituent bonded thereto has the following structure (3).

 (3)

In the above formula, R4-R8 are each independently -H, -OH, a C1-C6 alkyl group, a C1-C6 alkoxy group, a cyano, a halo or an amino group, preferably a hydrogen or a hydroxy group. In the above formula, the peptide is a tripeptide consisting of lucyl-valyl-histidine.

In the present invention, the tripeptide derivative to which the benzoic acid derivative is bound includes the following examples:

(1) salicyloyl-tripeptide (lucyl-valyl-histidine) (Salicyloyl-LVH)

(2) 2,4-Dihydroxybenzoyl-tripeptide (2,4-dihydroxybenzoyl-tripeptide)

(3) 3,4,5-trihydroxybenzoyl (galloyl) tripeptide

Scheme 3 schematically shows a process for preparing the tripeptide derivative to which the benzoic acid derivative is bonded, and a specific synthesis process is based on the description of Example 4. [

[Reaction Scheme 3]

In another embodiment of the present invention, A may be a cinnamic acid derivative. The tripeptide to which a cinnamic acid derivative having a substituent is bonded has the following structure (4).

 [Chemical Formula 4]

R9-R13 are independently -H, -OH, a C1-C6 alkyl group, a C1-C6 alkoxy group, a cyano, a halo or an amino group, preferably a hydrogen or a hydroxyl group. In the above formula, the peptide is a tripeptide consisting of lucyl-valyl-histidine.

Scheme 4 schematically shows a process for preparing a tripeptide derivative to which the cinnamic acid derivative is bound, and a specific synthesis process is based on the description of Example 5.

[Reaction Scheme 4]

In another aspect, the present invention relates to a composition for external application for skin having whitening, wrinkle, anti-inflammation and antioxidative ability, which comprises the tripeptide derivative as an active ingredient.

In the present invention, the term "comprising as an active ingredient" means an external preparation for skin which exhibits reduced skin melanin synthesis and exhibits a whitening effect, exhibits collagen synthesis or anti-inflammation, ≪ RTI ID = 0.0 > and / or < / RTI > The content of the tripeptide derivative in the composition is in the range of 0.1 to 10% by weight based on the total composition.

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. In addition, conventionally known organic / inorganic UV blocking agents and natural products known to have ultraviolet shielding function may be additionally included.

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, etc., in addition to the tripeptide derivative, , Antioxidants, coloring agents, 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 method for preparing a skin external composition containing a tripeptide derivative according to the present invention is not limited to the method disclosed in the Examples, and any person skilled in the art may make modifications The composition for external application for skin containing pro-centramenimal minimal culture or extract according to the present invention 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, A lipid vesicle or a cosmetically active agent, a lipid vesicle or a cosmetically active agent, a lipid vesicle or a cosmetically active agent, a stabilizer, a stabilizer, Such as other ingredients of the cosmetic or dermatological science. 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 due to whitening, anti-inflammation, antioxidant, antioxidant ability, and ability to promote collagen synthesis.

Such a pharmaceutical composition may contain, in addition to the active ingredient pro-centramine minimal culture or its extract, a "pharmaceutically acceptable carrier ", which may be a diluent, a lubricant, a binder, a disintegrant, a sweetener, Preservatives, < / 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.

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.

In one embodiment of the present invention, the following tripeptides and derivatives thereof were prepared.

Functional group binding to tripeptide The When the dihydroxyl group is  Substituted benzyl N- (2,4-Dihydroxybenzyl) -LVH Kojic acid (5-Hydroxy-4-oxo -4 H -pyran-2-yl) -methoxy carbonyl-LVH Benzoic acid derivative 2-hydroxybenzoyl (Salicyloyl) -LVH 2,4-Dihydroxybenzoyl-LVH 3,4,5-Trihydroxybenzoyl (Galloyl) -LVH Shinnam-san  derivative 3,4-Dihydroxycinnamoyl (Caffeoyl) -LVH

Tripeptide ( Lucil - valyl-histidine) synthesis

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of an amino acid, and N-hydroxybenzotriazole N-hydroxybenzotriazole (HOBt) and N, N'-dicyclohexylcarbodiimide (DCC) were used as activating agents to extend the amino acid residues (Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of histidine resin (Nova Biochem, Inc) in which the amino group was protected with Fmoc in a glass reactor was treated with 3 ml of N-methyl-2-pyrrolidone (NMP) After swelling, the solvent was removed and Fmoc was removed by treatment with 2 ml of 20% piperidine (3 ml). The histidine resin from which Fmoc was removed was treated twice with dichloromethane (DCM), twice with NMP, and reacted with the valine solution activated with HOBt-DCC at room temperature for about 2 hours. The NH2-protected peptide (lucyl-valyl-histidine) resin obtained by successive reaction of leucine was washed twice with DCM, twice with NMP, twice with DCM, and completely dried.

N- (2,4- Dihydroxybenzyl ) -Tripeptide ( Lucil - valyl-histidine)

2.1 NH2 - Protected Peptides - Synthesis of Resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of an amino acid, and N-hydroxybenzotriazole N-hydroxybenzotriazole (HOBt) and N, N'-dicyclohexylcarbodiimide (DCC) were used as activating agents to extend the amino acid residues (Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of histidine resin (Nova Biochem, Inc) in which the amino group was protected with Fmoc in a glass reactor was treated with 3 ml of N-methyl-2-pyrrolidone (NMP) After swelling, the solvent was removed and Fmoc was removed by treatment with 2 ml of 20% piperidine (3 ml). The histidine resin from which Fmoc was removed was treated twice with dichloromethane (DCM), twice with NMP, and reacted with the valine solution activated with HOBt-DCC at room temperature for about 2 hours. The NH2-protected peptide (lucyl-valyl-histidine) resin obtained by successive reaction of leucine was washed twice with DCM, twice with NMP, twice with DCM, and completely dried.

2.2: N- (2,4- Dihydroxybenzyl ) -Tripeptide ( Lucil - valyl-histidine)

The 20% piperidine / NMP solution was added to the synthesized NH2-protected peptide (lucyl-valyl-histidine) -resin to remove the Fmoc bound to the amino group, washed with NMP and DCM, 2 equivalents of hydroxybenzaldehyde, NaBH (OAc) are added, and solvent DCE (Dichloroethane) is added and stirred. Further, 1-2 equivalents of acetic acid was added, followed by a coupling reaction at room temperature overnight. After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

(82.5: 5: 5: 5) of trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol was added to the dried N- (2,4-dihydroxybenzyl) - tripeptide (lucyl- valyl- 5: 2.5 (v / v)) at room temperature for 2 to 3 hours to obtain an amino acid

(2,4-dihydroxybenzyl) -treopeptide (lucyl-valyl-histidine) was isolated from the resin by removing the triphenylmethyl (trityl group) which is a protecting group of the functional group present in the side chain of the residue , And the peptide was precipitated with cold diethyl ether. The obtained dried N- (2,4-dihydroxybenzyl) -treopeptide (lucyl-valyl-histidine) was dissolved in acetonitrile and water containing 0.1% trifluoroacetic acid as a solvent by reverse phase high performance liquid chromatography phase high performance liquid chromatography; column: Gemini, C18 110A 250 x 21.2 mm).

Yield: 62%

(5- Hydroxy -4-oxo-4 H- Pyran-2-yl) Methyloxycarbonyl - tripeptide ( Lucil - valyl-histidine)

3.1 Protected Peptides - Synthesis of Resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of an amino acid, and N-hydroxybenzotriazole N-hydroxybenzotriazole (HOBt) and N, N'-dicyclohexylcarbodiimide (DCC) were used as activating agents to extend the amino acid residues (Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of histidine resin (Nova Biochem, Inc) in which the amino group was protected with Fmoc in a glass reactor was treated with 3 ml of N-methyl-2-pyrrolidone (NMP) After swelling, the solvent was removed and Fmoc was removed by treatment with 2 ml of 20% piperidine (3 ml). The histidine resin from which Fmoc was removed was treated twice with dichloromethane (DCM), twice with NMP, and reacted with the valine solution activated with HOBt-DCC at room temperature for about 2 hours. The NH2-protected peptide (lucyl-valyl-histidine) resin obtained by successive reaction of leucine was washed twice with DCM, twice with NMP, twice with DCM, and completely dried.

3.2 (5- Hydroxy -4-oxo-4 H -Pyran-2-yl) Methyloxycarbonyl - tripeptide synthesis

The 20% piperidine / NMP solution was added to the NH2-protected peptide (lucyl-valyl-histidine) -resin synthesized above to remove Fmoc bound to the amino group and washed with NMP and DCM.

 Kojic acid (5 g, 35 mmol) was added to a round bottom flask with anhydrous THF (90 ml) and DMF (10 ml) solvent and stirred until the solution became clear. Carbonyldiimidazole (CDI, 5.1 g, 0.9 equiv.), A precursor of carbamate, was dissolved in THF, and the solution was titrated to the solution. The solution was stirred at room temperature for 24 hours. The white solid started to precipitate after 3 hours of reaction. After stirring for 24 hours, the solution was filtered and evaporated under reduced pressure to obtain the product in a yield of 70%.

The activated kojic acid synthesized above was added to the washed resin under a THF solvent, HOBt was added as an additive, and the reaction was completed by stirring at room temperature for 24 hours.

The dried (5-hydroxy-4-oxo- 4H -pyran-2-yl) methyloxycarbonyl-tripeptide-resin was reacted with trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: 2.5 (v / v)) at room temperature for 2 to 3 hours to give triphenylmethyl (trityl group), which is a protecting group of the functional group present in the side chain of the amino acid residue constituting the peptide Hydroxy-4-oxo- 4H -pyran-2-yl) methyloxycarbonyl-tripeptide was isolated from the resin and the peptide was precipitated with cold diethyl ether. The resulting dried 5-hydroxy-4-oxo- 4H -pyran-2-yl) methyloxycarbonyl-tripeptide (lucyl-valyl-histidine) was dissolved in water containing 0.1% trifluoroacetic acid and acetonitrile Was purified by reverse phase high performance liquid chromatography (Gemini, C18 110A 250 x 21.2 mm) using a solvent.

Yield: 58%

4.1 NH2 - Protected Peptides - Synthesis of Resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of an amino acid, and N-hydroxybenzotriazole N-hydroxybenzotriazole (HOBt) and N, N'-dicyclohexylcarbodiimide (DCC) were used as activating agents to extend the amino acid residues (Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of histidine resin (Nova Biochem, Inc) in which the amino group was protected with Fmoc in a glass reactor was treated with 3 ml of N-methyl-2-pyrrolidone (NMP) After swelling, the solvent was removed and Fmoc was removed by treatment with 2 ml of 20% piperidine (3 ml). The histidine resin from which Fmoc was removed was treated twice with dichloromethane (DCM), twice with NMP, and reacted with the valine solution activated with HOBt-DCC at room temperature for about 2 hours. The NH2-protected peptide (lucyl-valyl-histidine) resin obtained by successive reaction of leucine was washed twice with DCM, twice with NMP, twice with DCM, and completely dried.

4.2 Salicyloyl - tripeptide ( Lucil - valyl-histidine) ( Salicyloyl - LVH ) Synthesis of

The 20% piperidine / NMP solution was added to the NH2-protected peptide (lucyl-valyl-histidine) -resin synthesized above to remove Fmoc bound to the amino group and washed with NMP and DCM.

 A 20% piperidine / NMP solution was added to the NH2-protected peptide (glutamyl-cystanyl-glycine) -resin synthesized by the above method to remove Fmoc bound to the amino group, After washing with N-methyl-2-pyrrolidone (NMP) and dichloromethane (DCM), 5 equivalents of salicylic acid, 5 equivalents of HOBt and 5 equivalents of DIC were mixed at room temperature overnight, Lt; / RTI > After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

The dried salicyloyl-tripeptide-resin was dissolved in a mixed solution of trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: 5: 2.5 (v / For 3 hours to remove the triphenylmethyl (trityl group) which is a protecting group of the functional group present in the side chain of the amino acid residues constituting the peptide, separating the salicyloyl-tripeptide from the resin, The peptide was precipitated. The obtained dried salicyloyl-tripeptide was reacted with water containing 0.1% trifluoroacetic acid and acetonitrile as a solvent by reverse phase high performance liquid chromatography (Gemini, C18 110A 250 x 21.2 mm ).

Yield: 65%

4.3 2,4- Dihydroxybenzoyl  Tripeptide (2,4- Dihydroxybenzoyl - tripeptide ) Synthesis of

The 20% piperidine / NMP solution was added to the NH2-protected peptide (lucyl-valyl-histidine) -resin synthesized above to remove Fmoc bound to the amino group and washed with NMP and DCM.

 A 20% piperidine / NMP solution was added to the NH2-protected peptide (glutamyl-cystanyl-glycine) -resin synthesized by the above method to remove Fmoc bound to the amino group, After washing with N-methyl-2-pyrrolidone (NMP) and dichloromethane (DCM), 5 equivalents of 2,4-dihydroxybenzoic acid and 5 equivalents of HOBt, DIC (5 eq.) Were mixed and reacted overnight at room temperature for coupling reaction. After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

The dried 2,4-dihydroxybenzoyl-tripeptide-resin was mixed with a mixture of trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: 5: 2.5 (v / v) Solution at room temperature for 2 to 3 hours to remove the triphenylmethyl (trityl group), which is a protecting group of the functional group present in the side chain of the amino acid residue constituting the peptide, and react with 2,4-dihydroxybenzoyl-tripeptide , And then the peptide was precipitated with cold diethyl ether. The obtained dried 2,4-dihydroxybenzoyl-tripeptide was dissolved in water containing 0.1% trifluoroacetic acid and acetonitrile as a solvent by reverse phase high performance liquid chromatography (column: Gemini, C18 110 A 250 x 21.2 mm).

Yield: 71%

4.4 3,4,5- Trihydroxybenzoyl ( Galois ) Synthesis of (3,4,5-Trihydroxybenzoyl (galloyl) -Tripeptide) Tripeptide

The 20% piperidine / NMP solution was added to the NH2-protected peptide (lucyl-valyl-histidine) -resin synthesized above to remove Fmoc bound to the amino group and washed with NMP and DCM.

 A 20% piperidine / NMP solution was added to the NH2-protected peptide (glutamyl-cystanyl-glycine) -resin synthesized by the above method to remove Fmoc bound to the amino group, After washing with N-methyl-2-pyrrolidone (NMP) and dichloromethane (DCM), 5 equivalents of 2,4-dihydroxybenzoic acid and 5 equivalents of HOBt, DIC (5 eq.) Were mixed and reacted overnight at room temperature for coupling reaction. After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

The dried 3,4,5-trihydroxybenzoyl (galloyl) -treopeptide-resin was dissolved in trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: / v)) at room temperature for 2 to 3 hours to remove triphenylmethyl (trityl group), which is a protecting group of a functional group present in the side chain of the amino acid residue constituting the peptide, -Trihydroxybenzoyl (galloyl) -tripeptide was isolated and the peptide was precipitated with cold diethyl ether. The obtained dried 3,4,5-trihydroxybenzoyl (galloyl) -treopeptide was dissolved in acetonitrile and water containing 0.1% trifluoroacetic acid as a solvent in a reverse phase high performance liquid chromatography ; column: Gemini, C18 110 A 250 x 21.2 mm).

Yield: 69%

3,4- Dihydroxycinnamoyl (Cafe oil) -tripeptide synthesis

5.1. NH2 - Protected Peptides - Synthesis of Resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of an amino acid, and N-hydroxybenzotriazole N-hydroxybenzotriazole (HOBt) and N, N'-dicyclohexylcarbodiimide (DCC) were used as activating agents to extend the amino acid residues (Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of histidine resin (Nova Biochem, Inc) in which the amino group was protected with Fmoc in a glass reactor was treated with 3 ml of N-methyl-2-pyrrolidone (NMP) After swelling, the solvent was removed and Fmoc was removed by treatment with 2 ml of 20% piperidine (3 ml). The histidine resin from which Fmoc was removed was treated twice with dichloromethane (DCM), twice with NMP, and reacted with the valine solution activated with HOBt-DCC at room temperature for about 2 hours. The NH2-protected peptide (lucyl-valyl-histidine) resin obtained by successive reaction of leucine was washed twice with DCM, twice with NMP, twice with DCM, and completely dried.

5.2. 3,4- Dihydroxycinnamoyl (Cafe oil) -tripeptide synthesis

The 20% piperidine / NMP solution was added to the NH2-protected peptide (lucyl-valyl-histidine) -resin synthesized above to remove Fmoc bound to the amino group and washed with NMP and DCM.

 A 20% piperidine / NMP solution was added to the NH2-protected peptide (glutamyl-cystanyl-glycine) -resin synthesized by the above method to remove Fmoc bound to the amino group, After washing with N-methyl-2-pyrrolidone (NMP) and dichloromethane (DCM)

5 equivalents of 3,4-dihydroxycinnamic acid (caffeic acid), 5 equivalents of HOBt (5 equivalents) and DIC (5 equivalents) were mixed and allowed to react overnight at room temperature. After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

The dried 3,4-dihydroxycinnamoyl-tripeptide (caffeoyl-tripeptide) resin was dissolved in trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: (v / v)) at room temperature for 2 to 3 hours to remove triphenylmethyl (trityl group), which is a protecting group of the functional group present in the side chain of the amino acid residue constituting the peptide, -Dihydroxycinnamoyl-tripeptide (caffeoyl-tripeptide) was separated, and the peptide was precipitated with cold diethyl ether. The resulting dried 3,4-dihydroxycinnamoyl-tripeptide (caffeoyl-tripeptide) was dissolved in acetonitrile and water containing 0.1% trifluoroacetic acid in a reverse phase high performance liquid chromatography column: Gemini, C18 110A 250 x 21.2 mm).

Yield: 65%

Experimental Example  1: Jellyfish derived from the present invention Peptides  Skin cell viability of derivative compositions Cell Viability ) On the test

To examine the proliferative activity of the peptide derivatives of the present invention, human keratinocyte cells (HaCaT) were cultured in an ATCC (American Type Culture Collection). Each cell was inoculated into a 24-well culture plate at a concentration of 1 × 10 ⁴ cells / ml. The medium was DMEM (Dubelco's Modified Eagle Medium, BRL, USA) containing 10% FBS. After culturing in DMEM containing 10% FBS for 48 hours and culturing by 25 to 30% of the surface area of the culture container, FBS-free DMEM containing each of the tripeptide derivatives prepared in Examples 1 to 5 was replaced with DMEM for 24 hours Lt; / RTI > 50 μl of a solution of 2.5 mg / ml of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma M5655, USA) (DMSO, Sigma D2650, USA) was added to each well, and the formazan crystals formed were dissolved by stirring for 20 minutes. Then, 100 μl of each of the formazan crystals was dissolved in 96 wells And the absorbance at 570 nm was measured by Enzyme-Linked Immunosorbent Assay (ELISA). The degree of proliferation activity of skin cells was calculated by the following formula based on the absorbance intensity of the control group using pure water and expressed as a percentage, and the results are shown in the table below.

[Equation 1]

Cell viability effect (%) = [(absorbance of experiment group - absorbance of control group) / absorbance of control group] × 100

The skin cell survival rate of the jellyfish-derived peptide derivative of the present invention Cell viability (% of control) 0.1% One % 2 % Control group 100 LVH 110 122 135 When the dihydroxyl group is  Substituted benzyl N- (2,4-Dihydroxybenzyl) -LVH 101 110 120 Kojic acid (5-Hydroxy-4-oxo -4 H -pyran-2-yl) -methoxy carbonyl-LVH 105 115 125 Benzoic acid derivative 2-hydroxybenzoyl (Salicyloyl) -LVH 110 120 130 2,4-Dihydroxybenzoyl-LVH 112 118 135 3,4,5-Trihydroxybenzoyl (Galloyl) -LVH 109 109 121 Shinnam-san  derivative 3,4-Dihydroxycinnamoyl (Caffeoyl) -LVH 98 101 103

Effects on skin cell toxicity As shown in Table 2, no cytotoxicity was observed with 1%, 2% and 5% of jellyfish-derived peptide derivatives. That is, it was confirmed that the cell survival rate was not affected, and the jellyfish derived peptide derivative according to the present invention had an effect on skin cell growth and proliferation activation.

Experimental Example  2: B16F10 malanoma cell Using melanogenesis  Inhibition measurement

B16F10, a melanin-producing cell, was purchased from Korean Cell Line Bank (KCLB) and cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics at 37 ° C, 5%, and CO ₂ . B16F10 cells were suspended in DMEM medium containing 10% FBS at a concentration of 1.5 × 10 ³ cells / well in order to confirm melanin production by jellyfish-derived peptide derivatives. Suspension cells (5 ml) are placed in a tissue culture flask and allowed to attach for 1 day. After the attachment, the samples of the peptide derivative derived from the jellyfish derived in Examples 1 to 5 were added at different concentrations and then cultured at 37 ° C for 5 days under 5% CO ₂ . The cultured B16F10 cells were washed with phosphate buffered saline (PBS) and trypsinized. Cells were collected in a corning tube, and 1 ml of 1N NaOH solution was added per 1 × 10 ⁶ cells / ml to dissolve the cells. Melanogenesis inhibition rates were determined by measuring absorbance at 490 nm using a microplate reader.

&Quot; (2) "

Inhibition of melanogenesis (%) = [1 - (Absample / Abcontrol)] 100

Melanin Contents
(% of control) 0.1% One % 2 % Control group 100 LVH 100 101 99 When the dihydroxyl group is  Substituted benzyl N- (2,4-Dihydroxybenzyl) -LVH 95 93 90 Kojic acid (5-Hydroxy-4-oxo -4 H -pyran-2-yl) -methoxy carbonyl-LVH 90 75 50 Benzoic acid derivative 2-hydroxybenzoyl (Salicyloyl) -LVH 100 101 100 2,4-Dihydroxybenzoyl-LVH 96 92 87 3,4,5-Trihydroxybenzoyl (Galloyl) -LVH 95 91 90 Shinnam-san  derivative 3,4-Dihydroxycinnamoyl-LVH 90 85 82

In order to examine the effect of the peptide derivative derived from jellyfish derived peptides on the melanin synthesis of B16 / F10 melanoma cells, total amount of melanin was measured after 3 days of treatment with the peptide derivative derived from jellyfish at different concentrations. As a result, it was confirmed that melanin synthesis was reduced compared with the control group except for LVH and 2-hydroxybenzoyl (Salicyloyl) -LVH, which is a benzoic acid derivative.

Experimental Example  3: Procollagen  Synthetic enhancement test

Human fibroblasts (Human Skin Fibroblast) for 37 ℃, 5% in a CO ₂ incubator of 10% in the cell culture medium to maintain a constant humidity FBS, Penicillin (50U / ml) , Streptomycin DMEM (Dulbecco's containing (50 / ml) (Gibco, USA). Cells were seeded at a density of 1 × 10 ⁵ cells / ml in a 24-well plate and cultured for 24 hours. The medium containing the peptide derivatives of the jellyfish derived from Examples 1 to 5 at concentrations of 0.1%, 1% and 2% was added to the control (DMEM medium) and cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours. After 48 hours, the amount of newly synthesized collagen was measured by measuring 20 μl of each supernatant of the culture medium and measuring it using Procollagen Type I C-Peptide EIA Kit (PICP, Takara, Cat No. MK101). The amount of PICP was converted into ng / 1 × 10 ⁵ cells and calculated according to the following formula, and the results are shown in Table 4.

&Quot; (3) "

Increase in collagen biosynthesis
(% of control) 0.1% One % 2 % Control group 100 LVH 105 122 140 When the dihydroxyl group is  Substituted benzyl N- (2,4-Dihydroxybenzyl) -LVH 101 108 120 Kojic acid (5-Hydroxy-4-oxo -4 H -pyran-2-yl) -methoxy carbonyl-LVH 105 118 125 Benzoic acid derivative 2-hydroxybenzoyl (Salicyloyl) -LVH 106 120 130 2,4-Dihydroxybenzoyl-LVH 103 118 135 3,4,5-Trihydroxybenzoyl (Galloyl) -LVH 107 109 121 Shinnam-san  derivative 3,4-Dihydroxycinnamoyl-LVH 100 105 109

As the concentration of 0.1%, 1%, and 2% jellyfish-derived peptide derivatives was increased, the amount of procollagen biosynthesis increased, indicating that the collagen synthesis enhancing effect was excellent.

Experimental Example  4: iNOS  Inactive inhibition Admission  Anti-inflammatory effect experiment

The amount of nitric oxide (NO) produced from the Raw 264.7 cell line was determined using the Griess reagent as the NO ₂ ^- form present in the cell culture medium. In order to measure inhibition of NO production in Raw 264.7 cells activated with 1 μg LPS, the experimental group treated with the 1% jellyfish derived peptide derivative samples prepared in Examples 1 to 5 and the control group were cultured for 24 hours, and then the Griess reagent was cultured in a cell culture 100 μl of the supernatant and 100 μl of Griess reagent (1% sulfanilamide in 5% phosphoric acid, 1% α-naphthylamide in H ₂ O) were mixed and reacted on 96-well plates for 10 minutes and absorbance was measured at 540 nm. The concentration of NO ₂ ^- was obtained by measuring the absorbance by diluting sodium nitrate.

LPS
Whether or not to process sample Cell Viability
(%) NO Production
(μM) - Control group 100 1.3


+ Control group 60 3.8 LVH 122 2.5 N- (2,4-Dihydroxybenzyl) -LVH 110 2.8 (5-Hydroxy-4-oxo -4 H -pyran-2-yl) -methoxy carbonyl-LVH 115 2.2 2-hydroxybenzoyl (Salicyloyl) -LVH 120 3.1 2,4-Dihydroxybenzoyl-LVH 118 2.8 3,4,5-Trihydroxybenzoyl (Galloyl) -LVH 109 2.1 3,4-Dihydroxycinnamoyl-LVH 101 3

The activity of iNOS was inhibited by 1% concentration of jellyfish-derived peptide derivative compared to the control group,

Experimental Example  5: Antioxidant effect ABTS Radical Scavenging Activity )

The antioxidant activity of ABTS radicals was measured by the method of van den Berg et al., Which is a method using ABTS free radicals produced by the reaction with potassium persulfate, Respectively.

1.0 mM AAPH (2,2'-azo-bis 2-amidinopropanediol hydrochloride) was mixed with 2.5 mM ABTS (2,2'-azino-bis-3-ethylbenzenothiazolin-6-sulfonic acid) dissolved in 100 mM PBS buffer. Lt; 0 > C for 12 minutes. 0.1%, 1% and 2% concentration The peptide derivatives of Examples 1 to 5 20 μl and 980 μl of ABTS solution were incubated in a 37 ° C water bath for 10 min. The absorbance at 734 nm was measured using a Trolox 0.1% positive control. The ABTS radical scavenging activity was calculated according to the following equation.

&Quot; (4) "

ABTS radical scavenging activity
(% of control) 0.1% One % 2 % Control group 0 0.1% Trolox (positive control) 21 45 84 LVH 2 5 6 When the dihydroxyl group is  Substituted benzyl N- (2,4-Dihydroxybenzyl) -LVH 5 15 40 Kojic acid (5-Hydroxy-4-oxo -4 H -pyran-2-yl) -methoxy carbonyl-LVH 15 35 50 Benzoic acid derivative 2-hydroxybenzoyl (Salicyloyl) -LVH 5 8 15 2,4-Dihydroxybenzoyl-LVH 7 21 39 3,4,5-Trihydroxybenzoyl (Galloyl) -LVH 8 24 56 Shinnam-san  derivative 3,4-Dihydroxycinnamoyl-LVH 10 30 60

It was found that the antioxidant ability was increased with the content of the peptide derivative derived from the jellyfish derived from the composition of the present invention, and the antioxidant ability was similar to that of the positive control substance.

Preparation of cosmetic composition

Cosmetic products of an emulsified form such as nutritional lotion, cream, essence, etc., and cosmetics of a solubilized form such as softening water were prepared by using the tripeptide derivatives of Examples 1 to 5 of the present invention as an active ingredient.

Manufacturing example  6-1: 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 tripeptide derivatives of Examples 1 to 5 2.0 to 7.0 Purified water to 100

Manufacturing example  6-2: Essence

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

Raw material name Weight% (w / w) Cetostearyl alcohol 1.0 Self emulsifying monostearic acid 1.0 Wax 0.5 Squalane 5.0 Isocetyl octanoate 3.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 Propylene glycol 0.2 Carboxy polymer 0.22 Triethanolamine 0.25 antiseptic Suitable amount Spices Suitable amount flash Suitable amount The tripeptide derivatives of Examples 1 to 5 2.0 to 7.0 Purified water to 100

Manufacturing example  6-3: 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 tripeptide derivatives of Examples 1 to 5 2.0 to 5.0 Purified water to 100

Manufacturing example  6-4: 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 tripeptide derivatives of Examples 1 to 5 2.0 to 7.0 Purified water to 100

Manufacturing example  6-5: 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 tripeptide derivatives of Examples 1 to 5 1.0 to 7.0 Purified water to 100

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.

Claims (5)

A tripeptide derivative comprising the following general formula 1:
[Formula 1]
AB
Wherein A is a functional group selected from the group consisting of benzyl, kojic acid, benzoic acid derivatives and cinnamic acid derivatives that are absent or substituted with dihydroxyl groups, and B is a tripeptide having the amino acid sequence of LVH.
A skin whitening external preparation for whitening comprising the peptide derivative according to claim 1.
A composition for external application for skin for improving wrinkles comprising the peptide derivative according to claim 1.
An anti-inflammatory dermatological composition comprising the peptide derivative according to claim 1.
An antioxidant composition for external application for skin comprising the peptide derivative according to claim 1.