KR102101767B1 - C-terminal amidated peptides with melanogenesis-inhibiting activity and compositions thereof - Google Patents

Abstract

The present invention relates to a peptide having a carboxy terminal having a melanin production inhibitory activity and amidated carboxy terminal, and more specifically, a carboxy terminal amidated (-NH ₂ ) SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: It is for a pharmaceutical composition, a cosmetic composition, and a food composition for treating pigmentation diseases comprising a peptide composed of one amino acid sequence selected from the group consisting of 9 to 18 and an active ingredient thereof.
The peptide according to the present invention can be effectively used to develop pharmaceutical compositions for pigmentation treatment, cosmetics, and functional foods because it has an effect of inhibiting melanin production very effectively without compromising cell viability.

Description

A carboxyl terminal amidated peptide having a melanin production inhibitory activity and a composition comprising the same {C-terminal amidated peptides with melanogenesis-inhibiting activity and compositions thereof}

The present invention relates to a peptide having a carboxy terminal having a melanin production inhibitory activity and amidated carboxy terminal, and more specifically, a carboxy terminal amidated (-NH ₂ ) SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: Peptides consisting of one amino acid sequence selected from the group consisting of 9 to 18, and pharmaceutical compositions and cosmetic compositions for the treatment of pigmentation diseases comprising the same, and for food compositions.

Melanin is a brown or black pigment that is present in tissues where skin, hair, eyes, and other pigments are deposited. It is a major part of a person's appearance and is also important for maintaining skin homeostasis. Melanin biosynthesis of the skin is influenced by various factors such as hormonal changes and nutritional status, and when melanin is not normally produced and the biosynthesis process is disturbed, pigmentation defects classified as reduced pigmentation or excessive pigmentation occur. Defects in pigmentation can be genetic or acquired, temporary or permanent, and can also occur part or all of the body. Preventing the accumulation of undesired melanin is a very high concern in the cosmetic industry because melanin accumulates or distributes abnormally, which may cause tingling, freckles, and senile sunspots, which may give an unfavorable appearance.

Melanocytes (melanin-forming cells, melanocytes) interact not only with the endocrine system in the body, but also with external factors such as ultraviolet light and drugs. In the skin, one melanocyte is surrounded by about 30-40 keratinocytes, and melanin production is regulated by a closed paracrine system. Melanin is synthesized by a chain enzymatic reaction from L-tyrosine in the melanosome, a cell organelle in melanocytes. The keratinocytes secrete signaling substances that stimulate melanocytes to mature melanosomes and promote melanin biosynthesis. The mature melanosome that accumulates melanin is secreted from the dendrite of melanocytes and transferred to the cytoplasm of adjacent keratinocytes.

α-melanocyte-stimulating hormone (α-MSH) is very important for melanin production. α-MSH is composed of the amino acid sequence of Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH ₂ , melanocortin 1 receptor, MC1-R). When the agonist binds to MC1-R, MC1-R activates adenylate cyclase, cAMP increases to activate PKA, and PKA cAMP-responsive element binding protein transcription factor) to finally phosphorylate to activate microphthalmia-associated transcription factor (MITF). MITF is also activated by Wnt, GSK3β, and MAPK signaling systems, in response to various stimuli, tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and dopachrome tauto It regulates the expression level of enzymes involved in melanin biosynthesis, such as merase (dopachrome tautomerase, DCT; also called tyrosinase-related protein 2, TYRP2). Aguti signaling protein (Agouti) is known as an antagonist of MC1-R that competes with α-MSH and inhibits melanin production.

Peptides have attracted attention as an active ingredient in cosmetics and cosmoceuticals due to bioactivity suitable for skin care, and various peptides have already been included as cosmetic ingredients. The amino acid sequence of the peptide is very diverse, and various modes of action are possible. Furthermore, peptides are broken down into natural amino acids that are not particularly toxic. However, the shortcomings of peptides are the high cost of peptide biosynthesis, and there is also a drawback that skin penetration is inefficient due to ionicity. Palmitoyl pentapeptide-4 (KTTKS), glycine-histidine-lysine-Cu (glycyl-histidyl-lysine (GHK) -Cu) and acetyl hexapeptide (Argireline®) Eases certain aspects of. Peptides having melanin production inhibitory effects have been reported, such as disulfarnyl peptide and Angio-S (SFKLRY-NH ₂ ).

The present inventors apply a positional scanning of synthetic peptide combinatorial library (PS-SPCL) method of a synthetic peptide combination library that has been effectively used to screen various bioactive peptides to develop a peptide inhibitor that inhibits cell melanin production. Did. Using the PS-SPCL and α-MSH-stimulated rodent melanoma B16-F10 cell line, peptides expected to have melanin production inhibitory activity were identified and their effects were confirmed.

Accordingly, the present inventors completed the present invention by screening a peptide composed of 1 to 4 amino acid sequences based on the positional scanning of synthetic peptide combinatorial library (PS-SPCL), and identifying a novel peptide having an inhibitory activity for melanin production in cells. Did.

Accordingly, an object of the present invention is to provide a peptide composed of one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 9 to 18, wherein the carboxy terminus is amidated (-NH ₂ ).

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of pigmentation diseases comprising the peptide as an active ingredient.

Another object of the present invention is to provide a cosmetic composition comprising the peptide.

Another object of the present invention is to provide a food composition comprising the peptide.

In order to achieve the above object, the present invention

Provided is a peptide consisting of one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 9 to 18 wherein the carboxy terminus is amidated (-NH ₂ ).

In order to achieve another object of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of pigmentation diseases comprising the peptide as an active ingredient.

In order to achieve another object of the present invention, provides a cosmetic composition comprising the peptide.

In order to achieve another object of the present invention, there is provided a composition for food containing the peptide.

Hereinafter, the present invention will be described in detail.

In the present invention, the carboxy terminal is amidated (-NH ₂ ) SEQ ID NO: 3, SEQ ID NO: 5, provides a peptide consisting of one amino acid sequence selected from the group consisting of SEQ ID NO: 9 to 18.

The term "polypeptide" as used herein is used interchangeably with "protein" or "peptide, peptide," and refers to a polymer of amino acid residues, such as is commonly found in proteins in nature. The peptide provided by the present invention is composed of 1 to 4 amino acid sequences, and is also referred to herein as dipeptide, tripeptide, and tetrapeptide.

As used herein, one letter (three letters) of an amino acid refers to the following amino acid in accordance with standard abbreviation rules in the field of biochemistry:

A (Ala): Alanine; C (Cys): cysteine; D (Asp): aspartic acid; E (Glu): glutamic acid; F (Phe): phenylalanine; G (Gly): glycine; H (His): histidine; I (IIe): isoleucine; K (Lys): lysine; L (Leu): leucine; M (Met): methionine; N (Asn): asparagine; O (Ply): Yuppyrrolicin; P (Pro): proline; Q (Gln): glutamine; R (Arg): arginine; S (Ser): serine; T (Thr): Threonine; V (Val): valine; W (Trp): tryptophan; Y (Tyr): tyrosine.

In addition, the amino acid sequence of all peptides in the present specification is described in the direction of the amino terminal (N-terminal, amino terminal or N-terminal) to the carboxy terminal (C-terminal, carboxy terminal or C-terminal) according to standard regulations in the field of biochemistry. It is done. When the amino functional group (-NH ₂ ) is indicated at the carboxy terminus of the peptide described in the present specification, this does not mean the amino terminus of the amino acid sequence, but the amino terminus at the carboxy terminus by carboxy term amidation, which is a chemical synthesis method of the peptide. It shows that a functional group was added.

The peptide according to the present invention is specifically SEQ ID NO: 1 (Arg-Phe-Cys-Gly-NH ₂ ; D1 peptide) amidated carboxy terminus, SEQ ID NO: 2 (Arg-Phe-Cys-Arg-NH ₂ ; D2 peptide), SEQ ID NO: 3 (Arg-Phe-Trp-Gly-NH ₂ ; D3 peptide), SEQ ID NO: 4 (Arg-Phe -Trp-Arg-NH ₂ ; D4 peptide), SEQ ID NO: 5 (Arg-Leu- Trp-Gly-NH ₂ ; D5 peptide), SEQ ID NO: 6 (Arg-Leu-Trp-Arg-NH ₂ , D6 peptide), SEQ ID NO: 7 (Arg-Leu-Cys-Gly-NH ₂ ; D7 peptide), sequence No. 8 (Arg-Leu-Cys-Arg-NH ₂ ; D8 peptide), SEQ ID NO: 9 (Phe-Arg-Trp -Gly; D9 peptide) SEQ ID NO: 10 (Arg-Phe-Trp-NH ₂ ; E1 peptide), SEQ ID NO: 11 (Arg-Phe-Gly-NH ₂ ; E2 peptide), SEQ ID NO: 12 (Arg-Leu-Gly-NH ₂ ; E3 peptide), SEQ ID NO: 13 (Arg-Leu-Trp-NH ₂ ; E4 peptide) , SEQ ID NO: 14 (Phe-Trp-Gly-NH ₂ ; E5 peptide), SEQ ID NO: 15 (Leu-Trp-Gly-NH ₂ , E6 peptide), SEQ ID NO: 16 (Arg-Trp-Gly-NH ₂ ; E7 peptide ), SEQ ID NO: 17 (Trp-Gly-NH ₂ ; F1 peptide), SEQ ID NO: 18 (Gly-NH ₂ ; G1 peptide) It may be made of an amino acid sequence represented, preferably, may be made of an amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9 to 18. More preferably, it may be made of an amino acid sequence represented by SEQ ID NO: 14 to 18.

According to the inventors confirmed in the Examples, it was confirmed that all of the peptides have an inhibitory effect on melanin production and specifically reduce the cell melanin content. In addition, as confirmed by the present inventors, the peptide is not limited to inhibiting melanin production when stimulating B16-F10 cells with α-MSH, but has an effect of inhibiting melanin production regardless of melanin cell types or melanin-producing substances. It was confirmed to have. In other words, human epidermal melanocytes also inhibit the production of melanin caused by various stimuli such as foskolin, theophylline, cAMP, stem cell factor, and ultraviolet rays. Therefore, the peptide of the present invention is characterized by having melanin production inhibitory activity.

The peptide according to the present invention may be derived from nature, and may be synthesized using a known polypeptide synthesis method (genetic engineering method, chemical synthesis). Production of a peptide by genetic engineering methods can be carried out, for example, by preparing a nucleic acid encoding the polypeptide or a functional equivalent thereof according to a conventional method. The nucleic acid can be prepared by amplifying by PCR using an appropriate primer. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer. The produced nucleic acid is operatively linked thereto and inserted into a vector containing one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate expression of the nucleic acid, thereby producing a recombinant expression vector. After transformation into a host cell, cultivation is performed under appropriate medium and conditions to recover a substantially pure polypeptide expressed from the nucleic acid from the culture. The recovery can be performed using methods known in the art (eg, chromatography). In the above, "substally pure polypeptide" means that the polypeptide according to the present invention does not substantially contain any other protein derived from a host cell. The genetic engineering method for the synthesis of the polypeptide of the present invention can refer to the following documents: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second (1998) and Third (2000) Editions; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080, 1990.

In addition, the peptides according to the present invention can be easily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989). The present inventors have used chemically synthesized peptides using a C-terminal amidation reaction.

In addition, the peptide of the present invention is not only a polypeptide having a natural amino acid sequence, but also an amino acid sequence variant thereof having an effect of inhibiting melanin production is included in the scope of the present invention. Variants of the polypeptide of the present invention are peptides having different sequences by deletion, insertion, non-conservative or conservative substitution, substitution of amino acid analogs, or a combination of one or more amino acid residues in the amino acid sequence of the present invention, inhibiting melanin production It means maintaining the effect of. Amino acid exchanges that do not alter the activity of the molecule as a whole are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979).

In addition, if necessary, the peptides of the present invention are phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation. ) And the like, an appropriate functional group may be added.

The present invention provides a pharmaceutical composition for the prevention or treatment of pigmentation diseases comprising the peptide according to the present invention as an active ingredient.

As described above, the peptide according to the present invention has a very low cytotoxicity and excellent melanin production inhibitory effect, and thus provides a pharmaceutical composition for the prevention or treatment of pigmentation disease, including it as an active ingredient. The pharmaceutical composition according to the present invention is pigmented due to the pathological condition of excessive melanin pigmentation, for example, aging / photoaging, rapid hormonal changes such as pregnancy, skin damage and regeneration due to wounds, inflammation, burns, etc., It can be used to improve and alleviate freckles, freckles, blemishes, spots, spots, otamoban, blotch, senile surplus, melanin dermatosis, etc.

Therefore, the pharmaceutical composition comprising the peptide of the present invention may preferably be a pharmaceutical composition comprising the peptide of the present invention as an active ingredient, and has a function of helping to lighten the color of melanin pigment deposited on the skin. It may have a function of helping skin whitening by preventing the occurrence of skin pigmentation disease by preventing excessive melanin pigmentation on the skin.

The skin pigmentation disease may include blemishes, freckles, dark circles, black spots, blotch, melanocyte nevus, milk coffee spots, otamo birthmarks, blue birthmarks, hyperpigmentation spots, nipple pigmentation, noise net pigmentation and blemishes, etc. However, it is not limited thereto.

In the pharmaceutical composition according to the present invention, a peptide according to the present invention may be selected and included alone, or two or more peptides may be selected and mixed.

The peptides according to the invention can be used by themselves or in the form of pharmaceutically acceptable salts. "Pharmaceutically acceptable" in the present invention is physiologically acceptable, does not inhibit the action of the active ingredient when administered to a human, and usually refers to an allergic reaction such as gastrointestinal disorders, dizziness, or similar reactions. . The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid can be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid is not limited thereto, and includes hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid, and may preferably be hydrochloric acid.

The pharmaceutical composition comprising the peptide having the activity of inhibiting melanin production according to the present invention as an active ingredient varies according to the route of administration by a method known in the art with a pharmaceutically acceptable carrier for the effect of inhibiting or whitening the biosynthesis of melanin. Can be formulated. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

The pharmaceutical composition according to the present invention may be administered to a patient in an amount that exhibits an inhibitory or whitening effect on melanin production. For example, a typical daily dosage may be administered in the range of about 0.01 to 10000 mg / kg, and preferably, in the range of about 1 to 100 mg / kg. The pharmaceutical composition of the present invention may be administered once or several times within a preferred dosage range. In addition, the dosage of the pharmaceutical composition according to the present invention can be appropriately selected by a person skilled in the art according to the route of administration, the subject of administration, the age, the sex weight, the individual difference and the disease state.

The route of administration may be administered orally or parenterally. The parenteral administration method is not limited to this, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration. You can. Since the part where the production of melanin mainly occurs is skin, the pharmaceutical composition according to the present invention will be the main route of administration, but is not limited thereto.

When the pharmaceutical composition of the present invention is administered orally, powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, according to methods known in the art, with a suitable carrier for oral administration It can be formulated in the form of a back. Examples of suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including cellulose starch, wheat starch, rice starch and potato starch, cellulose, and the like. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like may be included, including methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropyl methyl cellulose. In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In addition, when administered parenterally, the pharmaceutical composition of the present invention may be formulated according to methods known in the art in the form of injections, transdermal administrations, and nasal inhalants together with suitable parenteral carriers. The injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media comprising water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, it may further include various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In addition, the injection may additionally include an isotonic agent such as sugar or sodium chloride in most cases.

In the case of transdermal administration agents, ointments, creams, lotions, gels, external solutions, pasta, linen agents, air rolls, and the like are included. In the above, "dermal administration" means that the pharmaceutical composition is topically administered to the skin to deliver an effective amount of the active ingredient contained in the pharmaceutical composition into the skin. For example, the pharmaceutical composition of the present invention may be administered by a method of preparing the injection-type formulation and lightly pricking the skin with a 30-gauge thin injection needle or directly applying it to the skin. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, a recipe generally known in pharmaceutical chemistry.

In the case of inhalation dosing agents, the compounds used in accordance with the present invention may be pressurized packs or, using suitable propellants, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in inhalers or insufflators can be formulated to contain a powder mixture of a compound and a suitable powder base such as lactose or starch.

As other pharmaceutically acceptable carriers, reference may be made to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

In addition, pharmaceutical compositions according to the present invention may include one or more buffers (e.g. saline or PBS), carbohydrates (e.g. glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (Eg, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and / or preservatives.

In addition, the pharmaceutical compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

In addition, the pharmaceutical composition of the present invention may be administered alone or in combination with a known substance (eg, a compound) having an effect of inhibiting or whitening melanin production.

The present invention provides a cosmetic composition comprising at least one peptide according to the present invention.

The peptide according to the present invention has an excellent effect of inhibiting melanin production and has very low cytotoxicity, and thus provides a cosmetic composition comprising at least one peptide according to the present invention.

In the cosmetic composition according to the present invention, the peptide according to the present invention may be selected and included alone, or two or more may be selected and mixed.

The cosmetic composition according to the present invention is preferably a cosmetic composition comprising the peptide according to the present invention as one or more active ingredients,

Skin whitening can be used without limitation on desired areas, but for example, it can be used on areas where pigmentation occurs, such as the face, nipples, areola, vulva, abdominal wall midline, navel, armpits, elbows, and knees.

Particularly, the distribution of melanin pigment in the nipple or vulva differs depending on the woman, and changes from pink to black according to the amount of melanin distribution, thereby using the peptide having the activity of inhibiting melanin production according to the present invention to reduce stress by the site color. It can be used for receiving women.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, and topical application commonly used in the field of dermatology by containing a dermatologically acceptable medium or base in addition to the peptide according to the present invention, or It can be prepared in the form of an adjuvant that can be applied systemically.

In addition, the cosmetic composition of the present invention, in addition to the peptide according to the present invention, in addition to fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, interfaces Active agents, water, ionic or nonionic emulsifiers, fillers, metal ion blockers and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants commonly used in the cosmetic or dermatological field, such as any other ingredients used as. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

Suitable cosmetic composition formulations include, for example, solutions, gels, solid or dough anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionic It can be provided in the form of a vesicle dispersant, cream, skin, lotion, powder, ointment, spray or conceal stick. In addition, it may be prepared in the form of a foam or aerosol composition further containing a compressed propellant. The products to which the cosmetic composition of the present invention can be added are not limited to, but are not limited to, skin lotion, skin softener, skin toner, convergence makeup, softening makeup, nutrition makeup, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, body Cream, massage cream, nutrition cream, moisturizing cream, hand cream, essence, nutrition essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, treatment, essence, latex, press powder, Formulations such as rust powder, eye shadow, and the like.

The content of the peptide of the present invention contained in the cosmetic composition of the present invention may be contained in the range of 0.00001 to 100% by weight, preferably 0.001 to 10% by weight, based on the total weight of the cosmetic composition, which is the desired effect of whitening, Factors such as the degree of application, the type of formulation, and stability of the peptide in the cosmetic composition may be appropriately determined by a person skilled in the art.

The present invention provides a food composition comprising at least one peptide according to the present invention.

The composition for food containing at least one peptide having a melanin production inhibitory activity according to the present invention includes all forms of functional food, nutritional supplement, health functional food and food additives, etc. do. These types can be prepared in various forms according to conventional methods known in the art. In order to use the food composition of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.

Meanwhile, the food composition according to the present invention may include the peptides according to the present invention alone, or may include two or more of them by mixing. The food composition according to the present invention is preferably a food composition comprising the peptide according to the present invention as one or more active ingredients.

In addition, the food composition according to the present invention may be a food composition for whitening.

The above-mentioned "health functional food" refers to food manufactured and processed using ingredients or ingredients having useful functions for the human body according to Act 627 on the Health Functional Food, and "functional" means the structure of the human body. And it means to ingest for the purpose of obtaining useful effects for health purposes such as controlling nutrients for function or physiological action.

For example, as a health food, the food composition itself of the present invention may be prepared in the form of tea, juice, and drink to be consumed or granulated, encapsulated, and powdered. In addition, the food composition of the present invention can be prepared in the form of a composition by mixing with a known substance or active ingredient known to have an effect of inhibiting or whitening melanin production.

The food composition of the present invention may include a conventional food additive, and whether or not it is suitable as the "food additive" according to the general rules and general test methods of food additives approved by the Food and Drug Administration unless otherwise specified. It is judged according to the standards and standards for the item.

Items listed in the "Food Additives Revolution" include, for example, chemical additives such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; And mixed preparations such as L-sodium glutamate, noodle-added alkalis, preservatives, and tar colorants.

In addition, the present invention is a food additive containing a peptide, such as preservatives, fungicides, antioxidants, spices, seasonings, sweeteners, flavoring agents, expanding agents, strengthening agents, improvers, emulsifiers, various nutrients, synthetic flavors and natural flavors, etc. Flavoring agents, coloring agents, coloring agents, neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, antifoaming agents, solvents, mold release agents, preservatives, quality improvers , Glycerin, alcohol, carbonic acid used in carbonated beverages, or other food additives or food additives (supplementary ingredients), characterized in that it is used as an essential raw material. At this time, the food additive may be added to the food by immersing, spraying or mixing the food.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruits, canned foods, jams, marmalades, etc.), fish, meat, and processed foods (e.g. ham, sausage corn beef, etc.) ), Breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible plant oil, margarine, vegetable protein, Retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the food composition of the present invention.

The preferred content of the food composition of the peptide according to the present invention is not limited to this, but is preferably 0.00001 to 100% by weight, preferably 0.001 to 10% by weight, of the final food.

Accordingly, the present invention provides a novel peptide having a melanin production inhibitory activity and a pharmaceutical, cosmetic or food composition comprising the same as an active ingredient. The peptide according to the present invention has an effect of inhibiting melanin production very effectively without compromising cell viability.

1A is a location scanning (PS-SPCL) result of a synthetic peptide combination library for intracellular melanin production. The rodent melanoma cell line B16-F10 cells were stimulated with a vehicle control group or a corresponding peptide pool and melanin content was measured. Each panel shows the result obtained from the tetrapeptide pool where the amino acid sequence was confirmed, and the X and O sequences at the top of the panel indicate the positional properties of the amino acids in the tetrapeptide pool. The O position is each determined by one of the 20 L-amino acids, and the remaining five Xs consist of a mixture of 20 L-amino acids. Figure 1b shows the effect on the viability of B16-F10 cells, as a test result of MTT assay for each peptide combination. [Figure 1a *: p <0.05 compared to cells stimulated only with α-MSH, Figure 1b *: p <0.05 compared to the control group]
2A is an experimental result showing the effect of each tetrapeptide on cellular melanin biosynthesis. In the cell-based assay of FIG. 2A, B16-F10 cells were treated with a vehicle or a peptide at a specific concentration, stimulated with α-MSH of 100 nM, and absorbance was measured at 475 nm after 72 hours to measure melanin content. All of the amino acid sequences described in FIG. 2A are described from the amino terminal to the carboxy terminal. The amino functional group (-NH ₂ ) indicated at the peptide end indicates that it was added by carboxy terminal amidation used for peptide synthesis. 2B shows the results of the MTT assay test of each tetrapeptide, and shows the effect of each tetrapeptide on B16-F10 cell viability. [Figure 2a *: p <0.05 compared to cells stimulated only with α-MSH, Figure 2b *: p <0.05 compared to the control group]
Figure 3a is an experimental result showing the effect of D3, D5, D9 tetrapeptide on the melanin content of B16-F10 cells. Figure 3a shows the melanin content of cells treated with peptides at a concentration of 10-30 μM and stimulated with 100 nM α-MSH for 72 hours. The melanin content was normalized to the total protein content. 3B shows the results of MTT assay test of D3, D5, and D9 tetrapeptides, and shows the effect of each tetrapeptide on B16-F10 cell viability. Figure 3 was used as a positive control coumaric acid and arbutin. [Figure 3a *: p <0.05 compared to cells stimulated only with α-MSH, Figure 3b *: p <0.05 compared to the control group]
Figure 4a shows the effect of peptides with 2 or 3 amino acid sequences defined. In the cell-based assay of FIG. 4A, B16-F10 cells were treated with a vehicle or a peptide at a specific concentration, stimulated with α-MSH of 100 nM, and absorbance was measured at 475 nm after 72 hours to measure melanin content. Figure 4b shows the effect of each peptide on the B16-F10 cell viability, as a result of the MTT assay test of peptides in which amino acid 2 or 3 sequences are defined. [Figure 4a *: p <0.05 compared to cells stimulated only with α-MSH, Figure 4b *: p <0.05 compared to the control group]
Figure 5a is an experimental result showing the effect of glycine derivatives on the melanin content of B16-F10 cells. 5A shows the melanin content of cells treated with various concentrations of glycine derivatives and stimulated with 100 nM α-MSH for 72 hours. The melanin content was normalized to the total protein content. Figure 5b shows the effect of each peptide on the B16-F10 cell viability as a result of the MTT assay test of peptides with defined sequences of glycine derivatives. Figure 5 was used as a positive control coumaric acid and arbutin. [Figure 5a *: p <0.05 compared to cells stimulated only with α-MSH, Figure 5b *: p <0.05 compared to the control group]
6A is an experimental result showing the effect of tetrapeptide D3, tripeptide E5, dipeptide F1, and monopeptide G1 on melanin content in the extracellular and intracellular HEMs stimulated with α-MSH. Figure 6b shows the effect of each peptide on the HEMs cell viability, as a result of the MTT assay test for the HEMs cell line. [Figure 6a *: p <0.05 compared to cells stimulated only with α-MSH, Figure 6b *: p <0.05 compared to the control group]
Figure 7a is the result of the MTT assay of glycineamide-hydrochloride (Glycinamide-HCl), showing the effect on B16-F10 cell viability. 7B is an experimental result showing the effect of glycine amide-hydrochloride (Glycinamide-HCl) on melanin content of B16-F10 cells. [Figure 7a *: p <0.05 compared to the control, Figure 7b *: p <0.05 compared to cells stimulated only with α-MSH]
Figure 8a shows the activity of tyrosinase (TYR) against the B16-F10 cell line stimulated by glycineamide-HCl with α-MSH Figure 8b shows TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (dopachrometautomerase) and β-actin protein expression levels are shown [*: p <0.05 compared to cells stimulated only with α-MSH]
FIG. 9 shows cAMP-responsive element binding protein (CREB) and extracellular signaling regulatory kinase (ERK) for glycinamide-hydrochloride (B16-F10 cell line stimulated with α-MSH). Signal-regulated kinase (phosphorylation kinase) level, and shows the effect on the protein level of the microphthalmia-associated transcription factor (MITF) [Figure 9a *: p <0.05 compared to the group, Figure 9b *: α- P <0.05 compared to cells stimulated with MSH only
10 shows a peptide sequence having a melanin production inhibitory activity identified in the present invention.

Hereinafter, the present invention will be described in detail.

However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

<Experiment method>

Peptide synthesis

The synthetic tetrapeptide combinatorial library package was purchased from Peptron (Daejeon, Korea). The library consists of four positional sub-libraries: OXXX-NH ₂ , XOXX-NH ₂ , XXOX-NH ₂ , and XXXO-NH ₂ . In the above, the O position is each one of 20 L-amino acids, and the X position is composed of an equimolar mixture of 20 L-amino acids. The peptides of the library were synthesized by C-terminal amidation reaction.

Additionally, the peptide pool and each peptide were prepared using Peptron Co. (Daejeon, Korea) 's peptide ordering service. In the examples, peptides chemically synthesized using a carboxy terminal amidation reaction were used. The type or purity of the peptide was confirmed by mass spectrometry (MS) and high performance liquid chromatography (HPLC).

Cell culture

Cells were cultured in a humidified incubator at 37 ° C and 5% CO ₂ conditions. Murine melanoma B16-F10 cell line purchased from the American Type Culture Collection (Manassas, VA, USA) with 10% fetal bovine serum and antibiotics (100 U / mL) Incubated with Dulbecco 'Modified Eagle Medium containing penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericinB). Human epidermal melanocytes (HEMs) derived from foreskin of pigmented human newborns are purchased from Cascade Biologics (Portland, OR, USA) and human melanocyte growth supplements (Cascade Biologics). And medium 254 containing antibiotics.

Cell viability

Cell viability was measured using a 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. The B16-F10 cell line was treated with test peptides of various concentrations for 72 hours. Cells were washed with PBS and incubated for 3 hours with 100 μl of a culture solution to which 1 mg / mL MTT (Amresco, Solon, OH, USA) was added. Then, the culture medium was removed, and formazan accumulated in the cells was extracted with 100 μl of Dimethyl sulfoxide (DMSO). The absorbance of the extracted solution was measured at 595 nm using a SPECTROstar Nano microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).

Melanin production inhibitory activity

The inhibitory effect of melanin formation of the peptide to be analyzed was confirmed using the B16-F10 cell line and HEM. B16-F10 cells were treated with various concentrations of test peptide and stimulated with 100 nM α-MSH for 72 hours. Hexapeptide B6 (Phe-Ser-His-His-Leu-Gly-NH ₂ ), p-Coumaric acid and arbutin were used as controls. Extracellular melanin levels were measured using conditioned medium. Intracellular melanin was extracted with 1.0 M NaOH at 60 ° C. for 60 minutes. The melanin content was measured spectroscopically by measuring absorbance at 475 nm, and the derived values were normalized to the total protein content of the cells using a Bio-Rad DC assay (normalization).

TYR activity assay

B16-F10 cells were treated with glycineamide hydrochloride for 60 minutes, and stimulated with 100 nM α-MSH for 24 hours. Cold 10 mM Tris-HCl buffer with 120 mM sodium chloride, 25 mM potassium chloride, 2.0 mM ethylene glycol tetraacetic acid, 1.0 mM ethylenediamine tetraacetic acid, 0.5% Triton X-100, Protease inhibitor cocktail (Roche, Mannheim, Germany) Cells were lysed at (pH 7.4). The cell lysate was centrifuged at 13,000 x g for 15 minutes at 4 ° C to obtain a cell-free extract. TYR activity was measured using L-tyrosine and L-3,4-dihydroxyphenylalanine. The reaction mixture (200 μL) consisted of 100 mM sodium phosphate (pH 6.8), 1.0 mM L-tyrosine, 42 μM L-3,4-dihydroxy phenylalanine and cell-free extracts (40 μg protein) cultured at 37 ° C. The change in absorbance was measured at 475 nm using a Spectrostar Nano microplate reader.

Western blotting

Western blot is described in Study on phenolic compounds and novel peptides inhibiting UVB- and PM10-induced MMP1 expression and melanogenesis in skin cells. PhD Thesis 2017. Kyungpook National University, Korea.]. B16-F10 cells were treated with 150 mM NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% deoxycholate, 1 mM phenylmethylsulfonyl fluoride and Protease inhibitor cocktail (Roche). It was dissolved in cold 10 mM Tris-HCl buffer (pH 7.2). A portion of the cell lysate (30 μg of protein) was mixed with Laemmli sample buffer, denatured by heating at 95 ° C. for 5 minutes, and then electrophoresed on 10% SDS-polyacrylamide gel. After electrophoresis, the protein was transferred from the gel to a polyvinylidene difluoride membrane (Amersham Pharmacia, Little Chalfont, UK). Primary antibodies to TYR, TYRP1, DCT, MITF and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against CREB, phospho-CREB (Ser133), extracellular signal-regulated kinase (ERK), and phospho-ERK (Thr202 / Tyr204) were purchased from Cell Signaling (Danvers, Massachusetts, USA). The membrane was incubated with primary antibody overnight at 4 ° C., followed by incubation with secondary antibody (Cell Signaling) bound to horseradish peroxidase for 1 hour at room temperature. Protein bands were visualized using the picoEPD Western Reagent kit (Elpis-Biotech, Daejeon, Korea), and image density was analyzed using the National Institutes of Health ImageJ program.

Statistical analysis

Data are expressed as the mean ± standard error (mean ± n = 3) of the results of three or more independent experiments. Statistical significance was determined based on p <0.05 using Student's t- test, and marked with * when there was a statistically significant difference from the control group.

Example 1: Identification of peptides having melanin production inhibitory activity using PS-SPCL

Screening using PS-SPCL was performed on B16-F10 melanoma cells stimulated with α-MSH in all peptide pools of the synthetic tetrapeptide combinatorial library to observe the effect on melanin production. It was carried out ( Fig. 1a ).

α-MSH increased the melanin content of B16F10 cells as expected, which was attenuated to varying degrees by various tetrapeptide pools at a concentration of 1.0 mM, among which tetrapeptide pools with excellent inhibitory effects on melanin production were identified. According to the experimental results and the amino acid sequence of the identified peptide pool, the peptide pool having an arginine residue at the first position of the amino acid residue significantly inhibited melanin production in cells stimulated by α-MSH. Similarly, those with phenylalanine or leucine at the second position, cysteine or tryptophan at the third position, and glycine, arginine or cysteine at the fourth position showed significant inhibitory effects on melanin synthesis. Therefore, according to the location scanning results, the sequence of the tetrapeptide having anti-melanin production effect was predicted as follows: (Arg)-(Phe / Leu)-(Cys / Trp)-(Gly / Arg / Cys) -NH ₂ .

Example 2: Melanin production inhibitory effect of individual tetrapeptides

According to the PS-SPCL results of the previous example, 8 individual tetrapeptides with specific amino acids at each position of the amino acid sequence were synthesized ( FIGS. 2A and 3A ). These tetrapeptides are Arg at the first position from the amino terminus; Phe or Leu in the second position; Cys or Trp in the third position, Gly or Arg in the fourth position, named as D1 to D8 (D1 peptide, RFCG-NH ₂ , SEQ ID NO: 1; D2 peptide, RFCR-NH ₂ , SEQ ID NO: 2; D3 peptide, RFWG-NH ₂ , SEQ ID NO: 3; D4 peptide, RFWR-NH ₂ , SEQ ID NO: 4; D5 peptide, RLWG-NH ₂ , SEQ ID NO: 5; D6 peptide, RLWR-NH ₂ , SEQ ID NO: 6; D7 peptide, RLCG-NH ₂ , SEQ ID NO: 7; D8 peptide, RLCR-NH ₂ , SEQ ID NO: 8). These individual peptides evaluated melanin inhibitory effects at 30 μM and 100 μM concentrations. Hexapeptide B6 (Phe-Ser-His-His-Leu-Gly-NH ₂ ) was referred to as a control. (Seok JK, Lee SW, Choi J, Kim YM, Boo YC. Identification of novel antimelanogenic hexapeptides via positional scanning of a synthetic peptide combinatorial library.Experimental Dermatology. 2017 Aug; 26 (8): 742-744).

As shown in Figure 2a, tetrapeptides D3 (Arg-Phe-Trp-Gly-NH ₂ ) and D5 (Arg-Leu-Trp-Gly-NH ₂ ) produce melanin in cells stimulated by α-MSH. It was suppressed excellently. Hexa-peptide B6 showed melanin production inhibitory effect only at 100 μM.

We have tetrapeptide D3 (Arg-Phe-Trp-Gly-NH ₂ ) and D5 (Arg-Leu-Trp-Gly-NH ₂ ) sequences of α-MSH, i.e. Ac-SYSMEHFRWGKPV-NH ₂ , In other words It was found to be similar to FRWG and thus tetrapeptide D9 (FRWG-NH ₂ , SEQ ID NO: 9) was included in the next experiment. The present inventors further confirmed the anti-melanin production activity in the same manner using the positive control coumaric acid and arbutin for the D3, D5, and D9 peptides. It was confirmed that tetrapeptide D3, D5 as well as tetrapeptide D9 have an activity of inhibiting melanin production at concentrations lower than p-Coumaric acid and arbutin when compared on a molar concentration basis ( 3a)

Example 3: Inhibitory effect of short peptides on melanin production

In the previous example, it was confirmed that peptides having amino acid sequences of D3, D5, and D9 have the strongest inhibitory effect on melanin production. Additionally, the effect of inhibiting melanin production and the possibility of utilization of a peptide having a shorter sequence than that of the tetrapeptide, which are expected to be more advantageous in terms of convenience and economical efficiency of peptide synthesis, were investigated (FIG. 4A).

For this, E1 to E8 peptides were additionally synthesized: E1 (RFW-NH _2, SEQ ID NO: 10); E2 (RFG-NH ₂ , SEQ ID NO: 11); E3 (RLG-NH ₂ , SEQ ID NO: 12); E4 (RLW-NH ₂ , SEQ ID NO: 13); E5 (FWG-NH ₂ , SEQ ID NO: 14); E6 (LWG-NH ₂ , SEQ ID NO: 15); E7 (RWG-NH ₂ , SEQ ID NO: 16); F1 (WG-NH _2, SEQ ID NO: 17). The E1 to F1 peptides were amidated at the carboxy terminus according to the chemical synthesis method, and the effect on melanin production was evaluated at a concentration of 30 μM to 100 μM.

As shown in Figure 4a, all of the peptides to be further analyzed showed excellent melanin inhibitory effect. E5 (Phe-Trp-Gly- NH 2), E6 (Leu-Trp-Gly-NH 2) and E7 (Arg-Trp-Gly- NH 2) containing a Trp-Gly-NH ₂ of the test tripeptide is It showed higher melanin production inhibitory activity than other tripeptides. In addition, it was confirmed that dipeptide F1 (Trp-Gly-NH ₂ , tryptophanyl glycinamide) also has an effect of inhibiting melanin production.

Example 4: Inhibitory effect of glycine derivatives on melanin production

The present inventors further confirmed that glycineamide (Gly-NH ₂ , glycinamide, SEQ ID NO: 18, G1 peptide) maintains anti-melanin production activity, and free glycine and acetyl glycinamide have no activity. .

As shown in FIG. 5A, when compared on the basis of molar concentration, the inhibitory effect of glycineamide on melanin production was similar to p-Coumaric acid and was estimated to be significantly superior to arbutin.

Example 5: Melanin production inhibitory effect on HEM cell line

Further analysis was performed using the HEM cell line (human epithelial melanocyte cell) to more clearly confirm the results of the B16-F10 cell experiment.

As shown in FIG. 6A, the intracellular and extracellular melanin content of HEMs was increased by α-MSH treatment, and the melanin content increase was attenuated by tetrapeptide D3, tripeptide E5, dipeptide F1, and monopeptide G1.

HEMs cell viability was not affected by peptide treatment (FIG. 6B).

Therefore, it was confirmed that the peptide of the present invention is a melanin production inhibitor that can be safely used without cytotoxicity.

Example 6: Efficacy test of glycineamide hydrochloride

Example 6-1: Cytotoxicity and inhibitory activity of melanin production of glycineamide hydrochloride

While showing the effect of glycine amide, the cytotoxicity and inhibitory activity of melanin production of glycineamide were more specifically confirmed by using glycinamide hydrochloride, which is an ingestible salt form.

As shown in Figure 7, glycinamide hydrochloride did not cause any cytotoxicity up to 300 μM in B16-F10 cells. Glycineamide hydrochloride was found to lower melanocyte levels outside and within cells in cells stimulated with α-MSH at 25-100 μM.

Example 6-2: Measurement of TYR activity and related protein levels of glycineamide hydrochloride

8A, α-MSH increased TYR activity, and it was confirmed that the change was significantly weakened by glycine amide hydrochloride. In addition, α-MSH increased the protein levels of tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1), but did not increase the expression level of dopachrometautomerase (DCT) or β-actin protein. However, treatment with glycinamide hydrochloride (Glycinamide ·) significantly reduced protein levels of TYR, TYRP1 and DCT in both the absence and presence of α-MSH.

 Glycine amide hydrochloride is also called tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT), tyrosinase-related protein 2, TYRP2 ) It was confirmed that the melanin production was effectively suppressed by controlling the expression level of enzymes related to melanin biosynthesis.

Example 6-3: Effect of glycineamide hydrochloride on CREB, ERK phosphorylation and MITF protein expression levels

Since the α-MSH-MC1R pathway is known to induce TYR expression by activating CREB and MITF, it was investigated whether glycineamide hydrochloride influences this signal transduction pathway.

As shown in FIG. 9A, phosphorylation of CREB (Ser) increased significantly at 20 minutes after α-MSH stimulation, and reached a maximum level after 60 minutes as measured by Western blot. In the meantime, the total CREB figure has not changed. α-MSH also increased phosphorylation of ERK (Thr and Tyr), but no significant change was observed at the total ERK level. Glycinamide hydrochloride inhibited phosphorylation of CREB stimulated by α-MSH, but did not affect phosphorylation of ERK.

In addition, as shown in FIG. 9B, α-MSH increased MITF expression level, but was attenuated by glycineamide hydrochloride.

Therefore, it was confirmed that glycine amide phosphate effectively inhibits melanin production by inhibiting phosphorylation of CREB and ERK, thereby reducing MITF protein expression.

Example 7: Cell viability measurement

After treating the peptides of Examples 1 to 4 with B16-F10 cells and HEM in the same manner, cell viability was measured and the results were respectively shown in FIGS. 1B, 2B, 3B, 4B, 5B, 6B, and 6B. Summarized in 7a.

When the peptide pool of Example 1 was treated at a concentration of 1.0 mM, the cell viability was sometimes decreased depending on the peptide pool, but it was determined that the melanin production inhibitory effect of the selected peptide pool was not due to the decrease in cell viability. ( Figure 1b)

Among the individual peptides of Examples 2 to 4, some individual peptides sometimes decreased the cell survival rate, but it was also determined that the melanin production inhibitory effect of the selected peptides was not due to the decrease in cell survival rate. (Figures 2b to 5b)

In addition, it was confirmed that the cytotoxicity of the D3, D5, F1, and G1 peptides was little in the HEM cell line that was additionally tested.

Therefore, it was confirmed that the peptides according to the present invention have low cytotoxicity and can be used safely and usefully.

As described above, by using the peptide having the activity of inhibiting the production of melanin identified in the present invention, it can be usefully used to develop a safer and more effective pigmentation disease prevention or treatment drug, cosmetic or functional food.

<110> ruby crown Co. Ltd. <120> C-terminal amidated peptides with melanogenesis-inhibiting          activity and compositions thereof <130> NP18-0036P <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D1 peptide <400> 1 Arg Phe Cys Gly   One <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D2 peptide <400> 2 Arg Phe Cys Arg   One <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D3 peptide <400> 3 Arg Phe Trp Gly   One <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D4 peptide <400> 4 Arg Phe Trp Arg   One <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D5 peptide <400> 5 Arg Leu Trp Gly   One <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D6 peptide <400> 6 Arg Leu Trp Arg   One <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D7 peptide <400> 7 Arg Leu Cys Gly   One <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D8 peptide <400> 8 Arg Leu Cys Arg   One <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> D9 peptide <400> 9 Phe Arg Trp Gly   One <210> 10 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> E1 peptide <400> 10 Arg Phe Trp   One <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> E2 peptide <400> 11 Arg Phe Gly   One <210> 12 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> E3 peptide <400> 12 Arg Leu Gly   One <210> 13 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> E4 peptide <400> 13 Arg Leu Trp   One <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> E5 peptide <400> 14 Phe Trp Gly   One <210> 15 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> E6 peptide <400> 15 Leu Trp Gly   One <210> 16 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> E7 peptide <400> 16 Arg Trp Gly   One <210> 17 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> F1 peptide <400> 17 Trp Gly 167 <210> 18 <211> 1 <212> PRT <213> Artificial Sequence <220> <223> G1 peptide <400> 18 Gly 117

Claims (9)

delete delete delete A pharmaceutical composition for the prevention or treatment of pigmentation disease, comprising the peptide of SEQ ID NO: 18 with the carboxy terminal amidated (-NH ₂ ) as an active ingredient.
A cosmetic composition comprising the peptide of SEQ ID NO: 18 wherein the carboxy terminus is amidated (-NH ₂ ).
A food composition comprising the peptide of SEQ ID NO: 18 wherein the carboxy terminus is amidated (-NH ₂ ).
According to claim 4, wherein the pigmentation disorders are blemishes, freckles, dark circles, black spots, blotch, melanocyte nevus, milk coffee spots, otamo birthmarks, blue birthmarks, hyperpigmentation spots, papillary pigmentation, noise net pigmentation and Pharmaceutical composition, characterized in that at least one selected from the group consisting of blemishes.
The composition of claim 5, wherein the composition is for whitening.
7. The composition of claim 6, wherein the composition is in the form of one selected from the group consisting of functional foods, nutritional supplements, dietary supplements and food additives.

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