Peptides that stimulate tyrosinase activity and uses thereof

Abstract

The composition for promoting tyrosinase activity according to the present invention comprises a peptide having an amino acid sequence represented by the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q), R2 is arginine (R) or glutamine (Q), R3, R4 and R5 are arginine (R) or lysine (K), R6 is asparagine (N) or serine (S), and R7 and R8 are lysine (K) or tyrosine (Y). The composition according to the present invention can prevent, improve or treat hypomelanosis by promoting melanin production in melanocytes.

Description

[Technical Field]

[0001] The present invention relates to a peptide that stimulates tyrosinase activity and its use, and more particularly to a peptide that stimulates tyrosinase activity in melanocytes for preventing or treating melanin hypopigmentation and its use. [Background technology]

[0002] Melanocytes exist in the epidermal layer of the skin. Melanin is a phenolic polymer that is widely distributed in nature and is a complex of black pigment and protein. In the process of melanogenesis, melanin is synthesized in melanosomes, organelles within melanocytes.

[0003] Melanocytes grow in the shape of dendrites and are surrounded by keratinocytes. Melanocytes form melanosomes and transmit them to keratinocytes. Melanosomes containing accumulated melanin rise through the dendrites of melanocytes and are placed around the keratinocytes that make up skin cells, thereby protecting the keratinocyte DNA from being damaged by ultraviolet rays and other factors.

[0004] Melanin is synthesized in melanocytes starting from the amino acid tyrosine to L-DOPA (3,4-dihydrocyphenylalanin), which is then further oxidized to produce dopaquinone. Dopaquinone is then converted to cysteinyldopa to form pheomelanin. However, in cysteine ​​deficiency, dopaquinone is converted to dopachrome by a non-enzymatic process. Dopachrome is converted to eumelanin by two processes: an enzymatic process involving TRP1 and TRP2, and a non-enzymatic process. The melanin thus produced is transported by melanocyte dendrites to surrounding keratinocytes in the form of endoplasmic reticulum called melanosomes, which then distribute throughout the epidermis.

[0005] On the other hand, vitiligo is a skin disease that develops later in life, in which a lack of melanocytes causes skin color loss, resulting in white-appearing depigmented patches on the skin. The clinical hallmark of vitiligo is the development of white patches. This is due to damage to melanocytes, but the exact cause of melanocyte damage is unknown. Various hypotheses exist, including genetic factors, autoimmunity, oxidative stress, or toxic chemicals.

[0006] Vitiligo commonly appears on the face and extremities. Because the skin discoloration caused by vitiligo affects appearance, it can be a source of stress in interpersonal relationships. It is also known that the stress caused by vitiligo significantly increases the risk of mental illness. The global prevalence of vitiligo is approximately 0.5% to 1%, with the prevalence rates in men and women being roughly equal. However, gender differences do exist, with women reportedly developing the condition at a slightly earlier age than men.

[0007] Because melanin accumulation is also promoted by light, such as ultraviolet light, phototherapy, which utilizes this principle, has been used to treat patients with vitiligo since the 1800s. Several studies have shown that phototherapy promotes the migration and proliferation of melanocytes in the epidermal layer of the skin, providing a favorable environment for melanocyte growth and suppressing autoimmunity. Phototherapy, which uses ultraviolet (UVB) light, has side effects such as itching, burning, erythema, temporary hyperpigmentation, blisters, and dry skin. Both types of phototherapy are known to induce premature photoaging, making them difficult to use as long-term treatments.

[0008] Vitiligo is known to be treated with corticosteroid therapy. Corticosteroids are a term that refers to all steroids with anti-inflammatory properties. When used in conjunction with the aforementioned phototherapy, better results are seen. Corticosteroid therapy induces pigmentation and halts the progression of the disease. However, side effects such as epidermal atrophy, steroid folliculitis, and telangiectasia can occur, and when used as systemic therapy, side effects such as insomnia, acne, menstrual disorders, and weight gain can occur. Due to concerns about these side effects, corticosteroid therapy requires periodic rest periods.

[0009] Other treatments, such as surgical epidermal transplantation and cell transplantation, have been attempted, but they are not yet widely used, cannot treat large areas, and have side effects such as scarring, infection, and graft failure.If vitiligo recurs even after the transplanted cells, these therapies may further worsen the condition. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a peptide that promotes the formation of melanosomes by stimulating the activity of tyrosinase in melanocytes, and uses thereof.

[0011] Another object of the present invention is to provide a peptide capable of regulating the activity of tyrosinase in melanocytes and preventing hypomelanosis, which is the formation of depigmented spots on the skin, and uses thereof.

[0012] Yet another object of the present invention is to provide a peptide capable of improving or treating hypomelanosis by promoting melanin production in melanocytes, and uses thereof.

[0013] Another object of the present invention is to provide a peptide that promotes the oxidation of L-dopa to dopaquinone in the process of melanin synthesis in melanocytes, and its use.

[0014] The objects of the present invention are not limited to those mentioned above, and other objects not mentioned will be clearly understood by those having ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0015] According to one aspect of the present invention for solving the above technical problems, there is provided a composition for promoting tyrosinase activity, comprising a peptide having an amino acid sequence represented by the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q), R3, R4, and R5 are arginine (R) or lysine (K), respectively; R6 is asparagine (N) or serine (S); R7 and R8 are lysine (K) or tyrosine (Y).

[0016] According to another aspect of the present invention, there is provided a composition for regulating melanin synthesis, comprising a peptide consisting of the amino acid sequence of general formula 1.

[0017] According to yet another aspect of the present invention, there is provided a composition for preventing, ameliorating or treating melanin hypopigmentation, comprising a peptide consisting of the amino acid sequence of general formula 1.

[0018] As long as they can exert the effect of promoting the activity of tyrosinase, mutant peptides having a sequence that differs by one or more amino acid residues from the amino acid sequence that constitutes them are also included in the category of peptides provided by the present invention.

[0019] In general, amino acid exchanges in proteins and polypeptides that do not change the overall activity of the molecule are known in the art. The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. Furthermore, mutations or modifications in the amino acid sequence may increase the structural stability of the peptide against heat, pH, etc., or may increase the ability to promote melanosome synthesis.

[0020] For example, the acidic amino acid glutamine at position 3 of the peptide of SEQ ID NO: 1 provided by the present invention can be substituted with the basic amino acid lysine or arginine and still exhibit the same effects; the basic amino acid arginine at position 4 or 5 of the peptide of SEQ ID NO: 1 can be substituted with the acidic amino acid glutamine or the basic amino acid lysine and still exhibit the same effects; the basic amino acid lysine at position 6, 7, or 9 of the peptide of SEQ ID NO: 1 can be substituted with the basic amino acid arginine or the aromatic amino acid tyrosine and still exhibit the same effects; the acidic amino acid asparagine at position 8 of the peptide of SEQ ID NO: 1 can be substituted with the neutral amino acid serine and still exhibit the same effects; and the aromatic amino acid tyrosine at position 10 of the peptide of SEQ ID NO: 1 can be substituted with the basic amino acid lysine and still exhibit the same effects.

[0021] Thus, even if the acidic amino acids, basic amino acids, or aromatic amino acids constituting the peptides of the present invention are substituted with different acidic amino acids, basic amino acids, neutral amino acids, or aromatic amino acids, the effects of the peptides provided by the present invention can be exhibited as they are. Therefore, it is obvious that mutant peptides having a sequence that differs by one or more amino acid residues from the amino acid sequence constituting the peptides of the present invention are also included in the category of peptides provided by the present invention.

[0022] Furthermore, the peptides of the present invention can exhibit the same effects as the peptides provided by the present invention even if they have any amino acids added to their N- or C-terminus. For example, the peptide may have 1 to 300 amino acids added to its N- or C-terminus, or 1 to 100 amino acids added to its N- or C-terminus, or 1 to 24 amino acids added to its N- or C-terminus.

[0023] In another aspect, the present invention provides a polynucleotide encoding the peptide.

[0024] The polynucleotide may be mutated by substitution, deletion, insertion, or a combination thereof of one or more bases. When the nucleotide sequence is produced by chemical synthesis, a synthesis method known in the art, such as the method described in the literature (Engels and Uhlmann, Angew Chem Int Ed Engl., 37:73-127, 1988), can be used, such as the phosphotriester, phosphite, phosphoramidite, and H-phosphate method, PCR and other autoprimer methods, oligonucleotide synthesis on a solid support, etc. For example, a polynucleotide encoding the peptide of the present invention may comprise the nucleotide sequence of SEQ ID NO: 4.

[0025] In another aspect, the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for producing the peptide using the transformant.

[0026] The term "expression vector" as used herein refers to a recombinant vector capable of expressing a target peptide in a target host cell, comprising essential regulatory elements operably linked to a gene insert for expression. The expression vector includes expression regulatory elements such as an initiation codon, a termination codon, a promoter, and an operator. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must be functional in an individual when the gene product is administered, and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

[0027] As used herein, the term "operably linked" refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked so as to perform their common functions. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the coding sequence. Operable linkage with an expression vector can be produced using recombinant gene technology known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes commonly known in the art.

[0028] Additionally, the expression vector may contain a signal sequence for peptide excretion to facilitate separation of the peptide from the cell culture medium. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, exogenous translational control signals, which may include the ATG initiation codon, must be provided. These exogenous translational control signals and initiation codons may be of various natural and synthetic origins. Expression efficiency may be increased by the introduction of appropriate transcriptional or translational enhancing factors.

[0029] In addition, the expression vector may further comprise a protein tag that can optionally be removed using an endopeptidase to facilitate detection of the peptide.

[0030] The term "tag" as used herein refers to a molecule that exhibits a quantifiable activity or property, and may be a fluorescent molecule, including a chemical fluorescein, a polypeptide fluorescein, such as green fluorescent protein (GFP) or related proteins, or an epitope tag, such as a Myc tag, Flag tag, histidine tag, leucine tag, IgG tag, or streptavidin tag. In particular, when an epitope tag is used, a peptide tag consisting of preferably 6 or more amino acid residues, more preferably 8 to 50 amino acid residues, may be used.

[0031] In the present invention, the expression vector may contain a nucleotide sequence encoding a peptide that provides the effect of promoting the activity of tyrosinase in the present invention. The vector used in this case is not particularly limited as long as it is capable of producing the peptide. Preferably, the vector is a plasmid DNA, a phage DNA, or the like. More preferably, commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus, etc.), and the like. The expression vectors may be vectors of various types, such as insect virus, insect virus vectors (e.g., baculovirus), etc. Since the expression level and modification of proteins vary depending on the host cell, it is desirable to select and use the host cell that is most suitable for the purpose.

[0032] The transformant provided by the present invention can be prepared by transforming a host with the expression vector provided by the present invention, and can be used to produce the peptide by expressing the polynucleotide contained in the expression vector. The transformation can be carried out by various methods, including, but not limited to, CaCl precipitation, the Hanahan method, which is a CaCl precipitation method with increased efficiency by using a reducing agent such as DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation. Furthermore, the host used to prepare the transformant is not particularly limited as long as it is capable of producing the peptide, and may be bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, etc.; fungal cells such as Pichia pastoris, insect cells such as Drosophila and Spodoptera frugiperda Sf9 cells, etc.; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells, or plant cells.

[0033] The transformant can also be used in a method for producing a peptide of the present invention that has the effect of promoting the activity of tyrosinase. Specifically, the method for producing a peptide of the present invention that has the effect of promoting the activity of tyrosinase can include the steps of (a) culturing the transformant to obtain a culture, and (b) recovering the peptide of the present invention from the culture.

[0034] The term "cultivation" as used herein refers to a method of growing a microorganism under artificially controlled environmental conditions. In the present invention, the method of culturing the transformant can be performed by a method known in the art. Specifically, the culture is not particularly limited as long as it can express and produce the peptide of the present invention that has the effect of promoting the activity of tyrosinase, and may be continuous culture by batch, fed batch, or repeated fed batch process.

[0035] The culture medium used for cultivation must meet the requirements of the specific strain by adjusting the temperature, pH, etc. under aerobic conditions in a standard medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. Carbon sources that can be used include a mixed sugar of glucose and xylose as the main carbon source, as well as sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used alone or in combination. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, as well as amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its hydrolyzed products, and defatted soybean cake or its hydrolyzed products. These nitrogen sources can be used alone or in combination. The medium can contain potassium monophosphate, potassium diphosphate, and the corresponding sodium-containing salts as phosphorus sources. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts, can be used as phosphorus sources. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above substances.

[0036] In addition, suitable precursors may be used in the culture medium. The raw materials may be added to the culture by an appropriate method during the culture process, such as batch, fed-batch, or continuous, but are not limited to these. The pH of the culture may be adjusted by an appropriate method using basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acidic compounds such as phosphoric acid or sulfuric acid.

[0037] Additionally, foam formation can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 35°C. The culture is continued until the maximum amount of the peptide is produced. For this purpose, the culture is usually continued for 10 to 100 hours.

[0038] Furthermore, the step of recovering the peptide from the culture can be carried out by a method known in the art. Specifically, the recovery method is not particularly limited as long as it can be used to recover the produced peptide, but preferably includes centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion), etc.

[0039] The term "prevention" as used herein means any action of inhibiting or delaying the occurrence of hypomelanin by administering a pharmaceutical composition for preventing or treating hypomelanin, which comprises the peptide of the present invention.

[0040] The term "treatment" as used herein means any action of administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual seeking treatment for hypomelanin pigmentation, thereby promoting melanosome synthesis and thereby treating hypomelanin pigmentation.

[0041] The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for treating melanin hypopigmentation, further comprising the peptide and a suitable carrier (natural or non-natural carrier), excipient, or diluent commonly used in preparing pharmaceutical compositions. Specifically, the pharmaceutical composition may be formulated into a sterile injectable solution that can be administered to the site of induced melanin hypopigmentation by conventional methods. Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and collagen. When formulated, the composition may be prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In particular, these may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, ointments (e.g., pulp liner, etc.), etc. Examples of non-aqueous solvents and suspensions that may be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases that may be used include witepsol (registered trademark), macrogol (registered trademark), Tween (registered trademark) 61, cocoa butter, laurin butter, glycerogelatin, etc.

[0042] The content of the peptide in the pharmaceutical composition of the present invention is not particularly limited, but may be 0.0001 to 50 wt %, more preferably 0.01 to 20 wt %, based on the total weight of the final composition.

[0043] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, and sex, the patient's sensitivity to the drug, the administration time, route of administration, and excretion rate of the composition of the present invention used, the duration of treatment, drugs used in combination with or concomitantly with the composition of the present invention, and other factors known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a known pharmaceutical composition for treating melanin hypopigmentation. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum efficacy at the minimum dose without side effects.

[0044] The dosage of the pharmaceutical composition of the present invention can be determined by one skilled in the art, taking into consideration the purpose of use, severity of the disease, the patient's age, weight, sex, medical history, and the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng / kg to about 100 mg / kg, preferably 1 ng / kg to about 10 mg / kg, per adult. The administration frequency of the composition of the present invention is not particularly limited, but it can be administered once a day or in divided doses administered several times a day. The above dosages do not limit the scope of the present invention in any way.

[0045] In another aspect, the present invention provides a method for treating melanin hypopigmentation, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to an individual suffering from melanin hypopigmentation.

[0046] The term "vitiligo" as used herein refers to an acquired depigmentation disease in which white spots of various sizes and shapes appear on the skin due to the destruction of pigment cells.

[0047] As used herein, the term "poliosis" refers to a depigmentation disorder in which melanin (or pigment) is reduced or disappears from hair, eyebrows, and eyelashes.

[0048] The term "individual" as used herein may include, without limitation, mammals, including rats, livestock, etc., for which treatment of hypomelanosis is desired, but excludes humans from the individuals affected by the disease.

[0049] The pharmaceutical composition for treating melanin hypopigmentation of the present invention may be administered via any common route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be provided in any dosage form suitable for topical application depending on the purpose, including, but not limited to, oral, transdermal, intravenous, intramuscular, or subcutaneous administration. The pharmaceutical composition may be in the form of an injection, a solution for topical application, a suspension, an emulsion, a gel, a patch, or a spray, but is not limited to these. The dosage form can be easily prepared by conventional methods in the art, and surfactants, excipients, hydrating agents, emulsifiers, suspending agents, salts or buffers for osmotic adjustment, colorants, spices, stabilizers, antiseptics, preservatives, or other commonly used adjuvants may be used as appropriate.

[0050] In another aspect, the present invention provides a quasi-drug composition for preventing or ameliorating melanin hypopigmentation, comprising the peptide.

[0051] The term "amelioration" according to the present invention refers to any action that at least reduces a parameter related to the condition being treated, such as the severity of the symptoms.

[0052] In the present invention, the improvement can be interpreted as meaning any action of administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual seeking treatment for hypomelanin pigmentation, thereby promoting melanosome synthesis and thereby improving or benefiting the symptoms of hypomelanin pigmentation.

[0053] The term "quasi-drug" as used herein means an article used for the purpose of diagnosing, curing, improving, mitigating, treating, or preventing a disease in humans or animals, which has a milder effect than a pharmaceutical product. For example, according to the Pharmaceutical Affairs Law, quasi-drugs exclude articles used for pharmaceutical purposes, and include fiber and rubber products used to treat or prevent diseases in humans and animals, items that have a mild effect on the human body or do not have a direct effect on the human body and are not tools or machines, or items similar to them, and disinfectants and insecticides used to prevent infectious diseases.

[0054] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, and examples thereof include disinfectants and cleansers for skin or hair, skin or hair cleaning products, soaps, shampoos, and skin ointments.

[0055] In another aspect, the present invention provides a health functional food composition for preventing or improving melanin hypopigmentation, comprising the peptide.

[0056] The term "food" as used herein includes all foods in the usual sense, such as meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, health supplements, alcoholic beverages, vitamin complexes, functional health foods, and health foods.

[0057] The term "functional food" refers to a food with high medical and therapeutic value that is processed to efficiently exert bioregulatory functions in addition to providing nutrients. The term "functional" refers to the ability to regulate nutrients or achieve beneficial health effects, such as physiological effects, on the structure and function of the human body. The food of the present invention can be prepared by methods commonly used in the art, and may be prepared by adding raw materials and ingredients commonly used in the art. The food may also be prepared in any dosage form that is recognized as a food. The food composition of the present invention can be prepared in various dosage forms. Unlike general pharmaceuticals, the food composition is made from food ingredients, which has the advantage of being free of side effects that can occur with long-term use of pharmaceuticals. It is also highly portable, and the food of the present invention can be taken as an adjuvant to enhance the effects of preventing or improving melanin hypopigmentation.

[0058] The term "health food" refers to food that has more active health maintenance and promotion effects than general foods, and "health supplement food" refers to food for the purpose of health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement" may be used interchangeably.

[0059] Specifically, the health functional foods are foods in which the peptides of the present invention are added to food ingredients such as beverages, teas, spices, gums, and confectioneries, or are manufactured as capsules, powders, suspensions, etc., and when ingested, they bring about specific health benefits. However, unlike general medicines, they have the advantage of being made from food ingredients and therefore free from side effects that can occur when taking medicines for a long period of time.

[0060] The food composition of the present invention can be taken on a daily basis and is therefore expected to be highly effective in preventing or improving melanin hypopigmentation, making it highly useful.

[0061] The food composition may further contain a physiologically acceptable carrier, but the type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0062] The food composition may also contain additional ingredients commonly used in food compositions to improve smell, taste, visual appearance, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. Minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr) may also be included. Amino acids such as lysine, tryptophan, cysteine, and valine may also be included.

[0063] The food compositions may further contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG), etc.), artificial sweeteners (dulcin, cyclamic acid, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum (potassium aluminum sulfate), potassium D-bitartrate, etc.), fortifiers, emulsifiers, thickeners (thickening agents), coating agents, gum bases, antifoaming agents, solvents, and improvers. The additives may be selected according to the type of food and used in appropriate amounts.

[0064] The peptides of the present invention can be added directly or in combination with other foods or food ingredients, and can be used appropriately in a conventional manner. The amount of active ingredient to be added can be determined appropriately depending on the intended use (prevention, health, or therapeutic treatment). Generally, when producing a food or beverage, the food composition of the present invention can be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, per 100 parts by weight of the food or beverage composition. However, when taking the food or beverage for long-term health and hygiene purposes, the active ingredient can be added in an amount below the above range, and there is no problem from the standpoint of safety, so the active ingredient can be used in an amount above the above range.

[0065] One example of the food composition of the present invention may be used as a health drink composition. In this case, similar to conventional beverages, various flavorings or natural carbohydrates may be added as additional ingredients. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners that may be used include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates per 100 mL of the health drink composition of the present invention may typically be about 0.01 g to 0.04 g, specifically about 0.02 g to 0.03 g.

[0066] In addition to the above, the health drink composition may contain various nutrients, vitamins, electrolytes, flavorings, coloring agents, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. Fruit pulp may also be included for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used individually or in combination. The proportion of these additives is not critical, but is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health drink composition of the present invention.

[0067] The food composition of the present invention may contain the peptide of the present invention in various weight percentages as long as it can exhibit the effect of preventing or improving melanin hypopigmentation. Specifically, the food composition may contain the peptide of the present invention in an amount of 0.00001 to 100 weight % or 0.01 to 80 weight % relative to the total weight of the food composition, but is not limited thereto. [Effects of the Invention]

[0068] The peptides according to embodiments of the present invention or compositions containing same may stimulate the activity of tyrosinase, which is involved in the formation of melanosomes in melanocytes.

[0069] The peptide according to the embodiments of the present invention or a composition containing the same can regulate the activity of tyrosinase in melanocytes and prevent hypomelanosis, which is the formation of depigmented spots on the skin.

[0070] The peptide according to the embodiments of the present invention or a composition comprising the same can improve or treat hypomelanosis by promoting melanin production in melanocytes.

[0071] The peptides according to the embodiments of the present invention or compositions containing the same can promote the process of oxidizing L-dopa to dopaquinone in the process of melanin synthesis in melanocytes.

[0072] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those having ordinary skill in the art to which the present invention pertains from the following description. [Brief explanation of the drawings]

[0073] [Figure 1]Figure 1 shows the results of B16F10 cells, which were dispensed into a 6-well plate at 230,000 cells / well and cultured for 24 hours in a 37°C, 5% CO2 incubator, treated with peptide KH001 (sequence number 96) at different concentrations for 72 hours to prepare cells, and then the cell pellet was dissolved in a strong base solution and measured at 405 nm absorbance. (a) The melanin content (%) at different peptide treatment concentrations and (b) the degree of melanin production at different peptide treatment concentrations are compared. [Figure 2] Figure 2 shows B16F10 cells dispensed into a 6-well plate at 230,000 cells / well and cultured for 24 hours in a 37°C, 5% CO2 incubator. Afterwards, the cells were treated with KH001 peptide at concentrations of 50 μM, 100 μM, and 200 μM and cultured for 72 hours. The absorbance of the media in each well was measured at 405 nm to quantify the secreted melanin. [Figure 3] Figure 3 shows the tyrosinase activity of B16F10 cells, which were dispensed into a 6-well plate at 230,000 cells / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. The cells were then treated with peptide KH001 (sequence number 96) at various concentrations for 72 hours. The cells were then fixed and reacted with L-DOPA to produce a product, which was then observed under an optical microscope. [Figure 4] FIG. 4 shows the results of measuring cell viability by MTT assay 72 hours after treating B16F10 cells with KH001 peptide at concentrations of 50 μM, 100 μM, and 200 μM. [Figure 5]Figure 5 shows B16F10 cells plated at 230,000 cells / well in a 6-well plate. They were incubated at 37°C in a 5% CO2 incubator for 24 hours, then treated with KH001 peptide at concentrations of 50 μM, 100 μM, or 200 μM and incubated for 72 hours. The cells were washed with PBS and lysed in 100 μL of 0.1 M potassium phosphate buffer (pH 6.8) containing 1% Triton X. The cells were then placed in a 1.5 ml tube and centrifuged at 12,000 rpm for 20 minutes. 40 μL of the supernatant was incubated in 200 μL of 2 mg / ml L-DOPA solution (0.1 M potassium phosphate, pH 7.0) at 37°C for 30 minutes. The absorbance at 475 nm was measured to determine tyrosinase activity (corrected for the protein concentration of the cell lysate). DETAILED DESCRIPTION OF THE INVENTION

[0074] The objects and advantages of the present invention, as well as the technical configurations for achieving them, will become apparent from the following detailed description of the embodiments together with the accompanying drawings. In describing the present invention, if it is determined that a detailed description of a known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted. The terms used below are defined for describing the embodiments of the present invention, and may vary depending on the intentions or practices of users and operators.

[0075] However, the present invention is not limited to the embodiments disclosed below and can be realized in various different forms. The embodiments are provided solely to ensure complete disclosure of the present invention and to fully inform those skilled in the art of the invention. The present invention is defined only by the claims. Therefore, the definition should be based on the entire content of this specification.

[0076] Examples of the present invention will be specifically described below. Example 1: Synthesis of peptides The present inventors synthesized a peptide (SEQ ID NO: 1) that exhibits the effect of promoting tyrosinase activity using 9-fluorenylmethyloxycarbonyl (Fmoc), and then substituted the amino acids of the synthesized peptide to synthesize peptides of each group (Tables 1 to 12). N-KYQRRKKNKY-C (SEQ ID NO: 1) First, the peptides of Group 1 were synthesized by substituting the peptide of SEQ ID NO: 1 or the fifth to seventh amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine (Table 1).

[0077] [Table 1]

[0078] Next, peptides in Group 2 were synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine, and substituting the 8th amino acid with serine (Table 2).

[0079] [Table 2]

[0080] Next, peptides in group 3 were synthesized by substituting the 5th to 7th amino acids of the peptide of sequence number 1 with lysine or arginine, the 9th amino acid with tyrosine, and the 10th amino acid with lysine (Table 3).

[0081] [Table 3]

[0082] Next, peptides in group 4 were synthesized by substituting the 5th to 7th amino acids of the peptide of sequence number 1 with lysine or arginine, the 8th amino acid with serine, the 9th amino acid with tyrosine, and the 10th amino acid with lysine (Table 4).

[0083] [Table 4]

[0084] Next, peptides in group 5 were synthesized by substituting the third amino acid of the peptide of sequence number 1 with arginine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 5).

[0085] [Table 5]

[0086] Next, the peptides of group 6 were synthesized by substituting the third amino acid of the peptide of sequence number 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, and the eighth amino acid with serine (Table 6).

[0087] [Table 6]

[0088] Next, the peptides of group 7 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 7).

[0089] [Table 7]

[0090] Next, the peptides of group 8 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 8).

[0091] [Table 8]

[0092] Next, the peptides of group 9 were synthesized by substituting the third amino acid of the peptide of sequence number 1 with lysine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 9).

[0093] [Table 9]

[0094] Next, the peptides in Group 10 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, and the eighth amino acid with serine (Table 10).

[0095] [Table 10]

[0096] Next, the peptides of Group 11 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 11).

[0097] [Table 11]

[0098] Finally, the peptides in Group 12 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth through seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 12).

[0099] [Table 12]

[0100] Example 2: Materials and Methods (Example 2-1: Cultivation of melanocytes) Melanocyte (B16F10) cell line was cultured in DMEM medium containing 10% FBS and 1% PS (penicillin / streptomycin) in an incubator at 37°C and 5% CO2.

[0101] (Example 2-2: MTT assay) The MTT reagent is used after diluting it in DMEM to a concentration of 0.5 mg / ml. B16F10 cells were plated at 230,000 cells / well into 6-well plates and cultured for 24 hours in a 37°C, 5% CO2 incubator. Cells were then treated with peptide KH001 (SEQ ID NO: 96) at various concentrations for 72 hours. MTT reagent was then added and incubated in a 37°C incubator for 1 hour in the dark. After removing the media, the formazan produced was dissolved in DMSO and measured at 560 nm absorbance.

[0102] Example 2-3: Quantification of melanin contents B16F10 cells were dispensed into 6-well plates at 230,000 cells / well and cultured for 24 hours in a 37°C, 5% CO2 incubator. The cells were then treated with KH001 peptide at concentrations of 50 μM, 100 μM, and 200 μM and cultured for 72 hours. After washing with PBS, the cells were lysed in a solution of 1N NaOH and 10% DMSO. The absorbance of the lysate was measured at 405 nm to determine the amount of melanin, which was then corrected for the protein concentration of the lysate. Melanin content (%) = (absorbance of peptide-treated group / absorbance of PBS-treated group) × 100

[0103] (Example 2-4: Quantification of secreted melanin) B16F10 cells were plated onto a 6-well plate at 230,000 cells / well and cultured for 24 hours in a 37°C, 5% CO2 incubator. The cells were then treated with KH001 peptide at concentrations of 50μM, 100μM, and 200μM and cultured for 72 hours. The absorbance of the media in each well was measured at 405nm to quantify the amount of melanin secreted. Secreted melanin (%) = (absorbance of peptide-treated group / absorbance of PBS-treated group) × 100

[0104] (Example 2-5: Measurement of tyrosinase activity) B16F10 cells were plated at 230,000 cells / well into 6-well plates and cultured for 24 hours in a 37°C, 5% CO2 incubator. They were then treated with KH001 peptide at concentrations of 50 μM, 100 μM, and 200 μM and cultured for 72 hours. The cells were then washed with PBS and lysed in 100 μL of 0.1 M potassium phosphate buffer (pH 6.8) containing 1% Triton X. The cells were then placed in a 1.5 ml tube and centrifuged at 12,000 rpm for 20 minutes. A 40 μL aliquot of the supernatant was then incubated with 200 μL of 2 mg / ml L-DOPA solution (0.1 M potassium phosphate, pH 7.0) at 37°C for 30 minutes. Absorbance at 475 nm was then measured to determine tyrosine activity. This value was corrected for the protein concentration of the cell lysate. Tyrosine activity (%) = (absorbance of peptide-treated group / absorbance of PBS-treated group) × 100

[0105] (Example 2-6: Measurement of tyrosinase activity 2) B16F10 cells were dispensed into 6-well plates at 230,000 cells / well and cultured for 24 hours in a 37°C, 5% CO2 incubator. They were then treated with KH001 peptide at concentrations of 50 μM, 100 μM, and 200 μM and cultured for 72 hours. After washing the cells with PBS, they were fixed with 2% paraformaldehyde for 20 minutes. Next, the cells were washed three times with PBS, and 0.1% L-DOPA (0.1 M potassium phosphate, pH 7.0) was dispensed into each well. The cells were then incubated for 3 hours in a 37°C incubator and photographed under an optical microscope.

[0106] Example 3: Results Example 3-1: Effect of peptide KH001 on melanin production in B16F10 melanocytes B16F10 cells were treated with KH001 peptide at various concentrations. 72 hours after peptide treatment, the cell pellet was dissolved in strong base, and the absorbance at 405 nm was measured to quantify the amount of intracellular melanin. The amount was then normalized to the protein value using a BSA assay. As shown in Figure 1, treatment with KH001 at concentrations of 50 μM, 100 μM, and 200 μM resulted in a 38%, 81%, and 176% increase in melanin production compared to the control group. This indicates that the KH001 peptide induces melanin synthesis in a concentration-dependent manner.

[0107] Example 3-2: Effect of peptide KH001 on tyrosinase activity in B16F10 melanocytes 1 The melanin synthesis process requires the oxidation of L-DOPA to dopaquinone, a process that requires the activity of tyrosinase. Therefore, in this study, tyrosinase activity was measured by reacting lysate from KH001 peptide-treated B16F10 cells with L-DOPA and quantifying the amount of dopaquinone produced. As shown in Figure 5, treatment with KH001 peptide resulted in tyrosinase activity that increased by 101%, 443%, and 433% compared to the control group at KH001 concentrations of 50 μM, 100 μM, and 200 μM, respectively. This indicates that KH001 peptide induces melanin synthesis by increasing tyrosinase activity.

[0108] Example 3-3: Effect of peptide KH001 on tyrosinase activity in B16F10 melanocytes 2 B16F10 cells were treated with KH001 peptide at various concentrations for 72 hours, then fixed and reacted with L-DOPA reagent. Intracellular tyrosinase reacted with L-DOPA and turned black, with the darker the color, the higher the tyrosinase activity. As shown in Figure 3, the experimental results showed that tyrosinase activity was significantly increased in the KH001 peptide-treated group compared to the control group.

[0109] Example 3-4: Effect of peptide KH001 on melanin secretion in B16F10 melanocytes B16F10 cells were treated with KH001 peptide at various concentrations, and the amount of melanin secreted into the culture media 72 hours after treatment was measured by absorbance at 405 nm to quantify the amount of melanin secreted into the media. As shown in Figure 2, treatment with KH001 peptide resulted in a concentration-dependent increase in melanin production of 17%, 31%, and 37% compared to the control group. This indicates that the effect of KH001 peptide causes B16F10 cells to secrete more melanin in a concentration-dependent manner.

[0110] (Example 3-5: Toxicity evaluation of peptide KH001 in B16F10 melanocytes) B16F10 cells were treated with KH001 peptide at various concentrations. Cell viability was measured by MTT assay 72 hours after treatment. As shown in Figure 4, treatment with KH001 at concentrations of 50 μM, 100 μM, and 200 μM did not result in significant differences in cell viability compared to the control group.

[0111] Therefore, it was confirmed that the KH001 peptide was not toxic within the set concentrations.

[0112] In the present specification and drawings, preferred embodiments of the present invention are disclosed, and although specific terms are used, these are used in a general sense merely to clearly explain the technical contents of the present invention and to aid in understanding the invention, and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art to which the present invention pertains that, in addition to the embodiments disclosed herein, other modifications based on the technical concept of the present invention can be implemented.

Claims

1. A composition for promoting tyrosinase activity, comprising a peptide having an amino acid sequence represented by the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); R7 and R8 are lysine (K) or tyrosine (Y).

2. A composition for regulating melanin synthesis, comprising a peptide having an amino acid sequence represented by the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); R7 and R8 are lysine (K) or tyrosine (Y).

3. A composition for preventing, improving or treating melanin hypopigmentation, comprising a peptide having an amino acid sequence represented by the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); R7 and R8 are lysine (K) or tyrosine (Y).

4. A quasi-drug composition for preventing or improving melanin hypopigmentation, comprising a peptide having an amino acid sequence represented by the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); R7 and R8 are lysine (K) or tyrosine (Y).

5. The composition according to claim 1, wherein the peptide has an amino acid sequence of any one of SEQ ID NOs: 1 to 96.

6. A polynucleotide encoding the peptide of claim 1.

7. An expression vector comprising the polynucleotide of claim 6.

8. The composition according to claim 1, characterized in that the composition comprises a polypeptide in which the peptide is repeatedly linked.

9. The composition according to claim 3, wherein the melanin hypopigmentation is vitiligo or albinism.

10. The composition of claim 3, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

11. A method for preventing, ameliorating, or treating melanin hypopigmentation, comprising administering the composition of claim 3 to an individual.

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