KR102506020B1 - The Peptide having Excellent Antioxidant Activity and Tyrosinase Inhibition Activity and Composition Containing the same - Google Patents

Abstract

본 발명의 신규한 펩타이드는 항산화 활성 및 티로시나제 저해활성이 우수하여 피부 미백 효과가 뛰어나므로, 인체 적용시 본래의 기능을 효과적으로 발휘할 수 있어, 의약, 화장품, 기능성 식품 분야에서 유용하게 이용될 수 있다.The present invention relates to a peptide having antioxidant activity and tyrosinase inhibitory activity and a composition containing the same.
The peptide according to the present invention has the structure of Formula I below.
[Formula I]

Since the novel peptide of the present invention has excellent antioxidant activity and tyrosinase inhibitory activity and excellent skin whitening effect, it can effectively exert its original function when applied to the human body, and can be usefully used in the fields of medicine, cosmetics, and functional foods.

Description

Peptide having antioxidant activity and tyrosinase inhibitory activity and composition containing the same {The Peptide Having Excellent Antioxidant Activity and Tyrosinase Inhibition Activity and Composition Containing the same}

The present invention relates to a peptide having antioxidant activity and tyrosinase inhibitory activity and a composition containing the same.

Peptide is a core material of bio and is the smallest functional unit of protein, and consists of 2 to 50 amino acids. It shows excellent efficacy in small amounts and is non-toxic, so it is widely used as a major raw material for medicines, foods, and cosmetics. In particular, the use of peptide cosmetic materials that are safe and effective for the human body is increasing day by day, and the development of new materials is very important in the industry, such as import substitution effect.

The efficacy of peptides used as cosmetic raw materials is mainly composed of materials that have effects such as collagen proliferation, whitening, and anti-inflammatory, and are relatively expensive, so there is a limit to the amount used. If various effects such as antioxidant, anti-inflammatory, collagen proliferation, and whitening can be expressed through one peptide, and it is composed of a relatively short sequence and can be commercialized through mass production, it can be used in various fields.

As shown in FIG. 1, there are two types of melanin: eumelanin, which is purple to black, and pheomelanin, which is yellow to red. Transitions to reveal skin color.

Changes in skin color are determined by various factors, such as changes in the number of melanocytes, abnormal production or structure of melanocytes, abnormal melanization of melanocytes, degree of migration of keratinocytes, and degree of loss of melanin. It is highly influenced by endocrine nutrition, inflammation, infection, tumor, physical and chemical factors.

Inhibiting melanin production is largely composed of three directions: suppression of melanin production in melanocytes, control of melanocyte stimulating substances, and promotion of melanin excretion.

Inhibiting melanin production controls copper (Cu), which is an essential element of the active site of tyrosinase, an enzyme involved in melanin production, or Tyrosinase related protein I (TRP-I). ) and tyrosinase related protein II (TRP-II: Tyrosinase related protein II) by controlling iron ion (Fe) to inactivate enzyme activity or inhibit the production of melanin production intermediates. And, by inhibiting the production of melanin, the effect of skin whitening can be seen. Kojic acid, arbutin, vitamin C derivatives, and rucinol are known representative whitening agents.

Whitening cosmetics are products developed for the purpose of alleviating spots and freckles caused by ultraviolet rays and suppressing the production of melanin pigment. As representative new substances, mulberrin extract, mulberry extract, banha extract, etc., which inhibit melanin production by inhibiting the activity of tyrosinase, an enzyme that catalyzes the oxidation of tyrosine, have been developed and used in whitening products. Currently, raw materials for whitening cosmetics notified by the Ministry of Food and Drug Safety include mulberry extract, arbutin, ethyl ascorbyl ether, oil-soluble licorice extract, etc., and extracts are mainly used.

In addition, Korean Patent Publication No. 2020-0125517 discloses a composition for skin whitening containing a callus extract derived from Artemisia aster and its culture medium as an active ingredient, and Korean Patent Publication No. 2020-0034675 discloses an extract of Adlay rice bran or a fermented product thereof. A composition for skin whitening was disclosed.

On the other hand, Korean Patent Registration No. 2094220 as a peptide whitening composition discloses a cyclic tripeptide for skin whitening, but it is extracted from bacterial symbionts and is not suitable for mass production, and does not have antioxidant and tyrosinase inhibitory effects at the same time, There is an urgent need to develop peptides having multiple physiological activity effects.

Accordingly, the present inventors identified a peptide sequence having a whitening effect by inhibiting the activity of the tyrosinase enzyme, and as a result, found that it had excellent antioxidant and tyrosinase activity inhibitory properties, thereby completing the present invention.

An object of the present invention is to provide a novel peptide having excellent antioxidant activity and tyrosinase inhibitory activity and its use.

Another object of the present invention is to provide a composition for skin whitening.

As mentioned above, the inventors of the present invention completed the present invention by designing and preparing a novel peptide and confirming its efficacy, confirming that it has excellent antioxidant and tyrosinase enzyme activity inhibitory abilities.

An object of the present invention is to provide a peptide represented by the following formula (I) having antioxidant activity and tyrosinase inhibitory activity.

[Formula I]

The peptide of Formula I is a novel peptide having a sequence of H-γ-Glu-Cys-β-Ala-γ-Glu-Cys-Gly-His-Lys-OH.

In the present invention, the novel peptide of Formula I can be prepared using techniques known in the art. Representative synthesis methods are described below, but the present invention is not limited to these examples, and those skilled in the art may appropriately modify and use them within the scope of technology known in the art.

In order to achieve the above object, the present invention is (a) Fmoc-His (Trt) -Lys (Boc) represented by the formula I-a by binding Fmoc-His (Trt) -OH to H-Lys (Boc) -Wang resin - Obtaining Wang resin; (b) sequentially adding amino acids (Fmoc-Gly-OH, Fmoc-β-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-γ-Glu-OtBu, etc.) to the resin obtained in step (a) Obtaining a peptide bonded to a resin having a protecting group represented by Formula I-b; (c) obtaining a deprotected crude peptide represented by Formula I-c through a deprotection process from the resin to which the protected peptide obtained in step (b) is bound; and (d) purifying the crude peptide obtained in step (c) with high performance liquid chromatography (HPLC) to obtain the peptide represented by Formula I.

[Formula I-a]

[Formula I-b]

[Formula I-c]

[Formula I]

In the present invention, the peptide represented by Formula I is synthesized by solid phase synthesis.

In Formulas Ia and Ib, R1 may use hydrogen or an amine protecting group commonly used in the art. The amine protecting group is an acetaminomethyl group, a benzyloxycarbonyl group, a tert -butyloxycarbonyl group, an acetyl group, a benzoyl group, para-nitrobenzoyl ( para -Nitrobenzoyl) group, para-methoxybenzoyl group ( para -Methoxybenzoyl), etc. can be exemplified, and benzyloxycarbonyl group and tert -butyloxycarbonyl group are preferable, and tert - It is more preferable to use a butyloxycarbonyl ( tert -Butyloxycarbonyl) group, but is not limited thereto.

In Formula Ib, R2 may use hydrogen or an aromatic amine protecting group commonly used in the art. The amine protecting group is a methoxymethyl group, a benzyloxymethyl group, a triphenylmethyl group, a tert -butyldimethylsilyl group, a triphenylsily group, a triiso Propylsilyl group, para -methoxybenzyl group, tetrahydropyran group, tetrahydrofuran group, tert -butyl group, diphenylmethyl ) group, 2-chlorotrityl group, benzyl group, 4-methoxybenzyl group, allyl group, tert-butyloxycarbonyl ( tert -Butyloxycarbonyl) group, acetyl group, benzoyl group, etc. can be exemplified, and methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, tert-butyldimethylsilyl ( It is preferable to use a tert -Butyldimethylsilyl) group, a triphenylsilyl group or a triisopropylsilyl group, and it is more preferable to use a 2-chlorotrityl group or a triphenylmethyl group And, it is most preferable to use a triphenylmethyl group as the amide protecting group, but is not limited thereto.

In Formula Ib, R3 may use a thiol protecting group commonly used in the art. The thiol protecting group is a methoxymethyl group, a benzyloxymethyl group, a triphenylmethyl group, a tert -butyldimethylsilyl group, a triphenylsily group, a triiso Propylsilyl group, para -methoxybenzyl group, tetrahydropyran group, tetrahydrofuran group, tert -butyl group, diphenylmethyl ) group, 2-chlorotrityl group, benzyl group, 4-methoxybenzyl group, allyl group, tert-butyloxycarbonyl ( tert -Butyloxycarbonyl) group, acetyl group, benzoyl group, etc. can be exemplified, and methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, tert-butyldimethylsilyl ( It is preferable to use a tert -Butyldimethylsilyl) group, a triphenylsilyl group or a triisopropylsilyl group, and it is more preferable to use a 2-chlorotrityl group or a triphenylmethyl group And, it is most preferable to use a triphenylmethyl group as the amide protecting group, but is not limited thereto.

In Formula Ib, R₄ may use a hydroxyl protecting group of a carboxylic acid commonly used in the art. The thiol protecting group is a methoxymethyl group, a benzyloxymethyl group, a triphenylmethyl group, a tert -butyldimethylsilyl group, a triphenylsily group, a triiso Propylsilyl group, para -methoxybenzyl group, tetrahydropyran group, tetrahydrofuran group, tert -butyl group, diphenylmethyl ) group, 2-chlorotrityl group, benzyl group, 4-methoxybenzyl group, allyl group, tert-butyloxycarbonyl ( tert -Butyloxycarbonyl) group, acetyl group, benzoyl group, etc. can be exemplified, and methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, tert-butyldimethylsilyl ( It is preferable to use a tert -Butyldimethylsilyl group, a triphenylsilyl group or a triisopropylsilyl group, and more preferably a tert -butyl group or a triphenylmethyl group, and a thiol It is most preferable to use a tert-butyl ( tert -Butyl) group as the protecting group, but is not limited thereto.

Hereinafter, the synthesis method will be described in more detail step by step.

The solid-phase peptide synthesis method;

1) Swelling the lysine-linked resin and attaching the second protected amino acid to the resin;

2) removing the amino acid protecting group (N-terminal amino group) by treatment with a base (piperidine) and washing the resin;

3) reacting the protected amino acid with the reagent for binding to the washed resin to bind;

4) repeating steps 2) and 3) to synthesize in sequence;

5) simultaneously removing the protecting groups of the resin and constituent amino acids;

6) solidifying the peptide with a solvent and then purifying it; and

7) concentrating and lyophilizing the purified peptide.

In step 1), resins that can be used include 2-Chlorotritylchloride Resin, Wang Resin, and DHPP Resin, 4-(1',1'- At least one selected from the group consisting of dimethyl-1'-hydroxypropyl)phenoxyacetyl alanyl aminomethyl polystyrene), PDDM Resin (Diphenyldiazomethane Resin), and SASRIN Resin (2-Methoxy-4-alkoxybenzyl alcohol Resin) can be heard

In step 1), the solvent used is dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl Pyrrolidone (N-Methylpyrrolidone), Chloroform, 1,2-Dichloroethane, Tetrahydrofurane, 1,4-Dioxane, Aceto At least one selected from the group consisting of Acetonitrile, Methanol, Ethanol, Isopropanol, Ethylene glycol, Methyl acetate, Ethyl acetate, etc. can be heard

In step 2), the base used to remove the protecting group is piperidine, pyrrolidine, piperazine, DBU (1,8-diazabicyclo[5.4.0]undec-7). -ene), 4-methylpiperidine, morpholine, 1-methyl-3-butylimidazolium tetrafluoroborane (1-methyl-3-butyl imidazolium BF4), ethanol Ethanolamine, Cyclohexylamine, Tris(2-aminoethyl)amine, 1,3-Dicyclohexanebis-(methylamine) (1,3-Dicyclohexanebis- (methylamine)), 1,4-bis-(3-aminopropyl)piperazine (1,4-Bis-(3-aminopropyl)piperazine), diethylamine, 4-dimethylaminopyridine ) and inorganic bases such as lithium hydroxide, sodium hydroxide, calcium hydroxide, and potassium hydroxide.

In step 3), reagents used when the protected amino acid binds include DCC (N,N-Dicyclohexylcarbodiimide), DIC (N,N-Diisopropylcarbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-( dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), PyBrOP (Bromo-tripyrrolidino-phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate), PyOxim (Ethyl cyanophosphate) (hydooxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), HCTU (2-(6-Chloro-1H- benzotriazol-1-yl)-N,N,N',N',-tetramethylaminium hexafluorophosphate), HDMC (N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uronium hexafluorophosphate N- oxide), TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3, 3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3 -tetramethyluroniumtetrafluoroboratemethanaminium), CDI (carbonyldiimidazole), COMU (1-[1-(Cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate), TOTT (2-(1-Oxy-pyridin- 2-yl)-1,1,3,3-tetramethyl-isothiouronium tetrafluoroborate), EDCㅇHCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), TFFH (Tetramethylfluoroformamidinium hexafluorophosphate), EEDQ (N-Ethoxycarbony- 2-ethoxy-1,2-dihydro-quinoline), T3P (2-propanephosphonic acid anhydride), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate ), HOBt (1-Hydroxybenzotriazole), HOOBt (HODhbt, Hydroxy-3,4-dihydro-4-ox-1,2,3-benzo-triazine), BTC (bis-Trichloromethylcarbonate or Triphosgene), CDI (1,1 '-Carbonyldiimidazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole), HOSu (N-Hydroxysuccinimide), and the like.

In step 5), the process of simultaneously removing the protecting groups of the resin and the amino acid is performed in the presence of an acidic solution. The acidic solution is TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5), TFA/phenol/water/thioanisole/ 1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA-DODT-TIS-anisole-water (183:5:2:5: 5, v/v), TFA/phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/water (95/5), TFA/DCM/indole (70/28/2), and TFA/TIPS/water (95/2.5/2.5) solutions.

In step 6), the solvent used to purify the crude peptide is a mixed solution of methanol, ethanol, isopropanol, acetonitrile, and purified water. It can be purified by using trifluoroacetic acid, acetic acid, and formic acid within 0.1% to 5%.

Protecting groups for these functional groups are described in detail in “Protecting Groups in Organic Synthesis (Greene and Wuts, John Wiley & Sons, 1991)”.

As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by a peptide bond.

The novel peptide according to the present invention had strong antioxidant activity (FIG. 3) and also excellent tyrosinase inhibitory activity (FIG. 4).

Therefore, as another aspect, the present invention relates to a composition for skin whitening comprising the novel peptide. A skin whitening composition containing the novel peptide of the present invention as an active ingredient can be used for pharmaceutical, cosmetic, or health functional foods.

The pharmaceutical composition containing the peptide is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. may be used, but is not limited thereto.

Carriers, excipients and diluents that may be included in the pharmaceutical composition containing the peptide of the present invention include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. However, it is not limited thereto. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.

The cosmetic composition containing the peptide can be used for skin, etc., and can be used for a skin whitening effect.

The content of the peptide included in the cosmetic composition of the present invention can be appropriately adjusted according to the use, application form, purpose of use and desired effect, and considering the effect compared to the content, for example, within 0.0001 to 99.9% by weight based on the total weight of the composition can be used in

The composition for skin whitening of the present invention can be administered parenterally, and can be applied in a topical application method by transdermal application.

The cosmetic composition is applied percutaneously to the skin and scalp, and refers to a composition that can be used in the manufacture of all cosmetic products, including basic cosmetics, chest and buttocks creams, makeup cosmetics, body products, shaving products, hair products, etc. As such, it may be formulated in the form of oral preparations, sprays, suspensions, emulsions, creams, gels, foams, etc., but the form is not particularly limited.

In addition, the cosmetic composition is softening lotion, astringent lotion, nutrient lotion, nutrient cream, massage cream, eye cream, eye essence, essence, cleansing cream, cleansing lotion, cleansing foam, cleansing water, pack, powder, body lotion, body creams, body essences, body washes, sunscreen creams, hair dyes, shampoos, rinses, toothpastes, mouthwashes, lotions, ointments, gels, creams, patches, and sprays.

The functional food composition containing the peptide can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like.

The health functional food of the present invention refers to a food manufactured and processed using raw materials or ingredients having useful functionalities for the human body according to Act No. 6727 on Health Functional Food, which provides nutrients for the structure and function of the human body. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulation or physiological action.

The health functional food of the present invention may contain ordinary food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards.

Items listed in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; It includes, but is not limited to, mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.

In addition, the skin whitening composition of the present invention includes all kinds of ingredients that can be used for commercialization and formulation in addition to the above active ingredients, such as fragrances, pigments, bactericides, antioxidants, preservatives, moisturizers, thickeners, excipients, diluents, and inorganic substances. It may additionally include salts and synthetic polymer materials, and the type and content thereof may be appropriately adjusted depending on the use and purpose of the final product.

Since the novel peptide of the present invention has excellent antioxidant activity and tyrosinase inhibitory activity and excellent skin whitening effect, it can effectively exert its original function when applied to the human body, and can be usefully used in the fields of medicine, cosmetics, and functional foods.

1 is an explanatory view showing a melanin production pathway.
Figure 2 is a process chart for synthesizing a peptide having antioxidant activity and tyrosinase inhibitory activity according to an embodiment of the present invention.
Figure 3 is the result of evaluating the antioxidant activity of the novel peptide according to the present invention.
Figure 4 is the result of evaluating the tyrosinase inhibitory activity of the novel peptide according to the present invention.

Unless otherwise indicated herein, the abbreviations used for the designation of amino acids and protecting groups are based on terms recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB ( Biochemistry, 11:1726-1732(1972)). ( Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984).

Abbreviations for protecting groups and amino acids used herein are as follows:

β-Ala: β-Alanine

AcOH: Acetic acid

Cys: Cysteine

DCM: Dichloromethane

DMF: Dimethylformamide (N,N-Dimethylformamide)

DMSO: Dimethylsulfoxide

DTT: Dithiolthreitol

EDT: 1,2-Ethanedithiol

Gly: Glycine

γ-Glu: gamma-glutamic acid (γ-Glutamic acid)

His: Histidine

HPLC: High Performance Liquid Chromatography

Lys: Lysine

Boc: tert-Butyloxycarbonyl group ( tert -Butyloxycarbonyl)

OtBu: O-tert-butyl (O- tert -Butyl)

t-Bu: tert-butyl ( tert -Butyl)

Fmoc: 9-Fluorenyloxycarbonyl

Trt: Triphenylmethyl or Trityl

Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl)

PMC: 2,2,5,7,8-pentamethylchroman-6-sulfonyl (2,2,5,7,8-Pentamethylchroman-6-sulfonyl)

Mtr: 4-methoxy-2,3,6-trimethylphenyl-sulfonyl (4-Methoxy-2,3,6-trimethylphenyl-sulfonyl)

TIS: Triisopropylsilane

Tos: para-Toluenesulfonyl

TES: Triethylsilane

TFA: Trifluoroacetic acid

TIPS: Triisopropylsilane

Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

embodiment. Preparation of Peptide Represented by Formula I

Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (weight/volume) % for solid/solid, and liquid /liquid is (volume/volume) %.

Example 1: Preparation of H-His(Trt)-Lys(Boc)-Wang resin

[Formula 2]

Fmoc-His(Trt)-OH (molecular weight = 619.7 g/mol, 93.0 g, 150 mmole) and Oxyma (Ethyl cyanohydroxyiminoacetate , molecular weight = 142.11 g/mol, 23.45 g, 165 mmole) was dissolved in 500 ml of THF and then added. DIC (molecular weight = 126.2 g/mol, 20.82 g, 165 mmole) was slowly added dropwise to the mixed reaction solution, followed by gentle stirring at room temperature for 4 hours. After washing the resin twice with 500 ml of THF, 500 ml of a 20% piperidine/THF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 500 ml of THF, H-His(Trt)-Lys(Boc)-Wang resin represented by Chemical Formula 2 was obtained (yield >99%).

Example 2: Preparation of H-Gly-His(Trt)-Lys(Boc)-Wang resin

[Formula 3]

Fmoc-Gly-OH (molecular weight = 297.3 g/mol, 44.6 g, 150 mmole) and Oxyma (Ethyl cyanohydroxyiminoacetate, molecular weight = 142.11 g/mol, 23.45 g, 165 mmole) was dissolved in 500 ml of THF and then added. DIC (molecular weight = 126.2 g/mol, 20.82 g, 165 mmole) was slowly added dropwise to the mixed reaction solution, followed by gentle stirring at room temperature for 4 hours. After washing the resin twice with 500 ml of THF, 500 ml of a 20% piperidine/THF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 500 ml of THF, H-Gly-His(Trt)-Lys(Boc)-Wang resin represented by Chemical Formula 3 was obtained (yield >99%).

Example 3: Preparation of H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin

[Formula 4]

In a reactor containing H-Gly-His(Trt)-Lys(Boc)-Wang resin (50 mmole), Fmoc-Cys(Trt)-OH (molecular weight = 585.7 g/mol, 87.9 g, 150 mmole) and Oxyma ( Ethyl cyanohydroxyiminoacetate, molecular weight = 142.11 g/mol, 23.45 g, 165 mmole) was dissolved in 500 ml of THF and then added. DIC (molecular weight = 126.2 g/mol, 20.82 g, 165 mmole) was slowly added dropwise to the mixed reaction solution, followed by gentle stirring at room temperature for 4 hours. After washing the resin twice with 500 ml of THF, 500 ml of a 20% piperidine/THF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin 6 times with 500 ml of THF, H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin represented by Chemical Formula 4 was obtained (yield >98%).

Example 4: Preparation of H-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin

[Formula 5]

Fmoc-γ-Glu-(OtBu) (molecular weight = 425.5 g/mol, 63.83 g, 150 mmole) and Oxyma (Ethyl cyanohydroxyiminoacetate, molecular weight = 142.11 g/mol, 23.45 g, 165 mmole) were dissolved in 500 ml of THF and added thereto. DIC (molecular weight = 126.2 g/mol, 20.82 g, 165 mmole) was slowly added dropwise to the mixed reaction solution, followed by gentle stirring at room temperature for 4 hours. After washing the resin twice with 500 ml of THF, 500 ml of a 20% piperidine/THF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 500 ml of THF, an H-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin represented by Formula 5 was obtained ( Yield >99%).

Example 5: Preparation of H-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin

[Formula 6]

Fmoc-β-Ala-OH (molecular weight = 311.3 g /mol, 46.7 g, 150 mmole) and Oxyma (Ethyl cyanohydroxyiminoacetate, molecular weight = 142.11 g/mol, 23.45 g, 165 mmole) were dissolved in 500 ml of THF and added thereto. DIC (molecular weight = 126.2 g/mol, 20.82 g, 165 mmole) was slowly added dropwise to the mixed reaction solution, followed by gentle stirring at room temperature for 4 hours. After washing the resin twice with 500 ml of THF, 500 ml of a 20% piperidine/THF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 500 ml of THF, H-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin represented by Formula 6 was obtained (yield >99%).

Example 6: Preparation of H-Cys(Trt)-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang Resin

[Formula 7]

In a reactor containing H-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin (50 mmole), Fmoc-Cys(Trt)-OH (molecular weight = 585.7 g/mol, 87.9 g, 150 mmole) and Oxyma (Ethyl cyanohydroxyiminoacetate, molecular weight = 142.11 g/mol, 23.45 g, 165 mmole) were dissolved in 500 ml of THF and added thereto. DIC (molecular weight = 126.2 g/mol, 20.82 g, 165 mmole) was slowly added dropwise to the mixed reaction solution, followed by gentle stirring at room temperature for 4 hours. After washing the resin twice with 500 ml of THF, 500 ml of a 20% piperidine/THF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 500 ml of THF, H-Cys(Trt)-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys ( Boc)-Wang resin was obtained (yield >99%).

Example 7: H -γ- Glu-(OtBu)-Cys(Trt)-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang Resin manufacture of

[Formula 8]

Fmoc-Glu in a reactor containing H-Cys(Trt)-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin (50 mmole) -(OtBu) (molecular weight = 425.5 g/mol, 63.83 g, 150 mmole) and Oxyma (Ethyl cyanohydroxyiminoacetate, molecular weight = 142.11 g/mol, 23.45 g, 165 mmole) were dissolved in 500 ml of THF and added thereto. DIC (molecular weight = 126.2 g/mol, 20.82 g, 165 mmole) was slowly added dropwise to the mixed reaction solution, followed by gentle stirring at room temperature for 4 hours. After washing the resin twice with 500 ml of THF, 500 ml of a 20% piperidine/THF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 500 ml of THF, H-γ-Glu-(OtBu)-Cys(Trt)-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly represented by Formula 8 -His(Trt)-Lys(Boc)-Wang resin was obtained (yield >99%).

Example 8: Preparation of Crude H-γ-Glu-Cys-β-Ala-γ-Glu-Cys-Gly-His-Lys-OH

Add H-γ-Glu-(OtBu)-Cys(Trt)-β-Ala-γ-Glu-(OtBu)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Wang resin to the reactor 2 L of a cooled TFA/EDT/TIS/water (92.5/2.5/2.5/2.5) solution was slowly poured into the mixture, followed by stirring at room temperature for 3 hours. After receiving the reaction solution, the reaction solution was slowly added dropwise to 8 L of cooled diethyl ether to precipitate the peptide. After stirring at room temperature for 30 minutes, the peptide was recovered by filtration. After removing the solvent under reduced pressure as much as possible, it was dried in a vacuum dryer for 5 hours. 42.27 g of almost white crude peptide was obtained. (Molecular weight 875.97g/mol, crude peptide purity 78.5%, yield 96.4%)

Example 9: Preparation of H-γ-Glu-Cys-β-Ala-γ-Glu-Cys-Gly-His-Lys-OH

[Formula I]

42.27 g of the crude peptide obtained in Example 8 was dissolved in purified water and then filtered through a 0.46 μm membrane. The filtrate was repeatedly injected by industrial HPLC (230 nm, 500 ml/min, 10 micron C18 column, the initial concentration of acetonitrile in 0.1% TFA increased from 5% to 65% in 20 minutes), followed by separation and purification, represented by Formula I A peptide, 19.28 g (yield: 45.6%, purity: 98.5%) was obtained.

Experimental Example 1: Evaluation of novel peptide antioxidant activity (ABTS radical scavenging activity evaluation)

In order to evaluate the antioxidant activity of the peptide material obtained in Example 9, ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) was reacted with potassium persulfate to oxidize ABTS to a radical state. , which formed a deep blue color.

Afterwards, the absorbance was measured on a well plate reader to measure the antioxidant power using the principle of losing color and becoming transparent when reacted with an antioxidant material. First, ABTS and potassium persulfate were dissolved in distilled water, prepared at concentrations of 14 mM and 4.9 mM, respectively, mixed 1:1, and reacted at room temperature for 12 hours while blocking light with aluminum foil.

Prepare an ABTS radical solution by diluting the solution approximately 50 times with 1X PBS so that the absorbance at 734 nm is 0.7 (±0.02) when measured with an absorbance photometer. and 190 μL of an ABTS radical solution were placed, shielded with aluminum foil, left at room temperature for 5 minutes, and then absorbance at 734 nm was measured. The results are shown in FIG. 3 .

As shown in Figure 3, it was found that the peptide according to the present invention has excellent antioxidant activity at a concentration of 100 μM or more.

Experimental Example 2: Tyrosinase inhibition assay

Dissolve the sample in purified water, set a concentration range that can confirm the inhibition of tyrosinase activity, and dilute it so that it is at least 5 concentrations, and the concentration of the test sample is specifically specified. 220 μL of 0.1 M phosphate buffer (pH 6.5), 20 μL of sample solution, and 20 μL of mushroom tyrosinase solution (1500 U/mL to 2000 U/mL) were put into the test tube in order. 40 μL of 1.5 mM tyrosine solution was added to this solution, followed by reaction at 37° C. for 10 to 15 minutes, and then absorbance was measured at 490 nm. After calculating the sample concentration (IC ₅₀ ) when the activity inhibition rate was 50% using an appropriate program, it was corrected by using a solvent in which the sample was dissolved instead of the sample solution as a blank sample solution. Arbutin was used as a positive control group to compare the results, and the results are shown in FIG. 4 .

Tyrosinase activity inhibition rate (%) = 100 - [(b-b') / (a-a')] × 100

a: Absorbance after reaction of blank sample solution

b: Absorbance after reaction of sample solution

a', b': Absorbance measured by replacing tyrosinase with buffer

As shown in Figure 4, it was found that the peptide according to the present invention has excellent tyrosinase inhibitory activity depending on the concentration.

Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and thereby the scope of the present invention is not limited thereto. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

Peptide consisting of the structure of formula (I);
[Formula I]

The peptide according to claim 1, wherein the peptide has antioxidant activity and tyrosinase inhibitory activity.
A cosmetic composition for skin whitening comprising the peptide of claim 1.
The cosmetic composition for skin whitening according to claim 3, wherein the composition has antioxidant activity and tyrosinase inhibitory activity.
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