Peptides promoting tyrosinase activity and uses thereof

Abstract

A composition for promoting tyrosinase activity according to the present invention comprises a peptide consisting of an amino acid sequence of the following general formula 1: wherein K-Y-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) in which R1 is arginine (R), lysine (K) or glutamine (Q); r2 is arginine (R) or glutamine (Q); r3, R4 and R5 are respectively 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, ameliorate or treat melanogenesis by promoting melanin production in melanin cells.

Description

Technical Field

[0001] The present invention relates to a peptide for promoting the activity of tyrosinase and a use thereof, and more particularly, to a peptide for promoting the activity of tyrosinase and a use thereof, wherein the tyrosinase is tyrosinase in melanocytes for preventing or treating hypomelaninosis. Background Art

[0002] Melanocytes are located in the epidermis of the skin. Melanin is a phenolic high molecular substance widely distributed in nature, and is a complex of black pigment and protein. When observing the melanogenesis process, melanin is synthesized in melanosomes, which are organelles in melanocytes.

[0003] Melanocytes extend in dendrites, and keratinocytes surround them. Melanocytes form melanosomes and pass them to keratinocytes. Melanosomes with accumulated melanin rise through the dendrites of melanocytes and are arranged in a way that wraps around keratinocytes that form skin cells, thereby preventing the DNA of keratinocytes from being damaged by ultraviolet rays and the like.

[0004] Melanin is synthesized in the following steps, that is, starting from an amino acid called tyrosine, L-DOPA (3,4-dihydroxyphenylalanin) is generated in melanocytes, and L-DOPA is oxidized again to generate dopaquinone (DOPA-quinone). Dopaquinone becomes pheomelanin through cysteinylDOPA. However, in the absence of cysteine, dopaquinone becomes dopachrome (DOPAchrome) by non-enzymatic function. Dopachrome is formed into eumelanin by two methods, one is an enzymatic process in which TRP1 and TRP2 perform functions, and the other is a non-enzymatic process. The melanin thus generated is transferred to the surrounding keratinocytes in the form of endoplasmic reticulum called melanosomes through the dendrites of melanocytes, thereby evenly spreading to the epidermis.

[0005] Vitiligo is an acquired disease that causes white spots to appear on the skin due to a lack of melanocytes in the skin. The clinical feature of vitiligo is the occurrence of white spots. This is caused by damage to melanocytes, but the exact cause of damage to melanocytes is still unclear. There are various hypotheses about the cause, but it is speculated that it is caused by genetic factors, autoimmunity, oxidative stress, or harmful chemicals.

[0006] Vitiligo usually appears on the face and extremities of the body. Since the skin depigmentation caused by vitiligo affects appearance, it may become a cause of stress in interpersonal relationships, etc. It is reported that the stress caused by vitiligo can actually greatly increase the risk of mental illness. The global prevalence of vitiligo is about 0.5-1%. It is reported that the prevalence of men and women is almost the same. There are differences according to gender. Compared with men, women develop the disease earlier.

[0007] Light such as ultraviolet rays also promotes the accumulation of melanin, and phototherapy based on this principle has been used to treat vitiligo patients since the 1800s. Some studies have shown that phototherapy promotes the migration and proliferation of melanocytes in the epidermis of the skin, provides an environment conducive to the growth of melanocytes, and inhibits autoimmunity. Phototherapy using ultraviolet rays (UVB) has side effects such as itching, burning, erythema, temporary pigmentation, blisters, and xerosis. It is reported that all phototherapy may induce premature photoaging, so it is difficult to use as a long-term treatment method.

[0008] Vitiligo is widely known through corticosteroid therapy. Corticosteroid is a term for all steroids with anti-inflammatory effects. Better results are shown when used together with the aforementioned light therapy. Corticosteroid therapy induces pigmentation and interrupts the progression of the disease. However, it can cause side effects such as epidermal atrophy, steroid folliculitis, and telangiectasia. When used as a systemic therapy, side effects such as insomnia, acne, menstrual disorders, and weight gain may occur. Due to concerns about these side effects, corticosteroid therapy requires regular rest periods.

[0009] Another treatment method is surgical treatment. Although epidermal transplantation and cell transplantation have been tried, they are not yet widely used and cannot be used for large-scale treatment. They have side effects such as scarring, infection, and transplantation failure. This treatment may worsen vitiligo when vitiligo reappears in the transplanted cells. Summary of the invention

[0010] Technical issues

[0011] The object of the present invention is to provide a peptide and its use, wherein the peptide promotes melanosome formation by promoting the activity of tyrosinase in melanocytes.

[0012] Another object of the present invention is to provide a peptide and its use, wherein the peptide can prevent hypomelanin disease that forms depigmented spots on the skin by regulating the activity of tyrosinase in melanocytes.

[0013] Another object of the present invention is to provide a peptide and use thereof, wherein the peptide can improve or treat hypomelaninism by promoting melanin production in melanocytes.

[0014] Another object of the present invention is to provide a peptide and its use, wherein the peptide promotes the oxidation of L-DOPA to dopaquinone during the synthesis of melanin in melanocytes.

[0015] The objects of the present invention are not limited to the above-mentioned contents, and a person having ordinary knowledge in the technical field to which the present invention belongs can clearly understand other objects not mentioned from the following description.

[0016] Solution to the problem

[0017] According to one embodiment of the present invention for solving the above technical problems, a composition for promoting tyrosinase activity is provided, which comprises a peptide consisting of an amino acid sequence of the following general formula 1,

[0018] KY-R1-R2-R3-R4-R5-R6-R7-R8 (Formula 1)

[0019] In the general formula 1,

[0020] R1 is arginine (R), lysine (K) or glutamine (Q);

[0021] R2 is arginine (R) or glutamine (Q);

[0022] R3, R4 and R5 are arginine (R) or lysine (K), respectively;

[0023] R6 is asparagine (N) or serine (S); and

[0024] R7 and R8 are lysine (K) or tyrosine (Y).

[0025] According to another embodiment of the present invention, a composition for regulating melanin synthesis is provided, which comprises a peptide consisting of an amino acid sequence of Formula 1.

[0026] According to another embodiment of the present invention, there is provided a composition for preventing, improving or treating hypomelaninosis, comprising a peptide consisting of an amino acid sequence of Formula 1.

[0027] As long as they can show an effect of promoting tyrosinase activity, even mutant peptides having one or more amino acid residues different from the amino acid sequence constituting the peptide are included in the category of the peptides provided by the present invention.

[0028] Generally, the exchange of amino acids in proteins and polypeptides without changing the activity of the molecule as a whole is well known in the art to which the present invention belongs. The most common exchange is the exchange between amino acid residues and 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, Asp / Gly. In addition, the peptides may include peptides whose heat, pH, etc. related structural stability or melanosome synthesis promoting ability is increased by mutation or modification in the amino acid sequence.

[0029] For example, even if glutamine, an acidic amino acid, located at position 3 of the peptide of sequence number 1 provided in the present invention is replaced by lysine or arginine, which are basic amino acids, the effect of the peptide provided in the present invention can be directly shown; even if arginine, a basic amino acid, located at position 4 or 5 of the peptide of sequence number 1 is replaced by glutamine, an acidic amino acid or lysine, which are basic amino acids, the effect of the peptide provided in the present invention can be directly shown; even if lysine, a basic amino acid, located at position 6, 7 or 9 of the peptide of sequence number 1 is replaced by arginine, a basic amino acid or tyrosine, which are aromatic amino acids, the effect of the peptide provided in the present invention can be directly shown; even if asparagine, an acidic amino acid, located at position 8 of the peptide of sequence number 1 is replaced by serine, which is a neutral amino acid, the effect of the peptide provided in the present invention can be directly shown; even if tyrosine, an aromatic amino acid, located at position 10 of the peptide of sequence number 1 is replaced by lysine, which are basic amino acids, the effect of the peptide provided in the present invention can be directly shown.

[0030] As described above, the acidic amino acids, basic amino acids or aromatic amino acids constituting the peptide of the present invention can directly exhibit the effects of the peptide provided in the present invention even if they are replaced by different acidic amino acids, basic amino acids, neutral amino acids or aromatic amino acids. Therefore, it is obvious that mutant peptides having a sequence different from that of one or more amino acid residues constituting the amino acid sequence of the peptide of the present invention also fall within the scope of the peptides provided in the present invention.

[0031] Furthermore, even if the peptide of the present invention has a form in which any amino acid is added to its N-terminus or C-terminus, the effect of the peptide provided in the present invention can be directly shown, and therefore it is included in the category of the peptide provided in the present invention. As one example, it can be a form in which 1 to 300 amino acids are added to the N-terminus or C-terminus of the peptide, as another example, it can be a form in which 1 to 100 amino acids are added to the N-terminus or C-terminus of the peptide, and as yet another example, it can be a form in which 1 to 24 amino acids are added to the N-terminus or C-terminus of the peptide.

[0032] As another embodiment of the present invention, the present invention provides a polynucleotide encoding the peptide.

[0033] For the polynucleotide, more than one base can be mutated by substitution, knockout, insertion or a combination thereof. In the case of preparing by chemically synthesizing a nucleotide sequence, a synthesis method known in the art to which the present invention belongs can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem Int Ed Engl., 37: 73-127, 1988), and can be synthesized using triester, phosphite, phosphoramidite and H-phosphate methods, polymerase chain reaction (PCR) and other automatic primer methods, oligonucleotide synthesis on a solid support, etc. For example, the polynucleotide encoding the peptide of the present invention may include the base sequence of sequence number 4.

[0034] As another embodiment, the present invention provides an expression vector comprising the polynucleotide, a transformant comprising the expression vector, and a method for producing the peptide using the transformant.

[0035] The term "expression vector" of the present invention refers to a recombinant vector that can express a target peptide in a target host cell, and means a gene construct that contains necessary regulatory elements that are operably linked to express a gene insert. The expression vector contains expression regulatory elements such as a start codon, a stop codon, a promoter, and an operator gene. The start codon and the stop codon are usually considered to be part of the nucleotide sequence encoding the polypeptide, and must exhibit an effect in an individual when the gene construct is administered, and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

[0036] The term "operably linked" of the present invention refers to a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked in order to perform a common function. For example, operably linking a promoter and a nucleic acid sequence encoding a protein or RNA can affect the expression of the coding sequence. Operable linkage with an expression vector can be prepared using gene recombination techniques known in the art to which the present invention belongs, and site-specific DNA cleavage and ligation can use enzymes commonly known in the art to which the present invention belongs, etc.

[0037] In addition, in order to promote the separation of peptides from the cell culture fluid, the expression vector may include a signal sequence for discharging the peptides. Specific initiation signals may also be required when performing effective translation of the inserted nucleotide sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, an exogenous translation regulation signal that may include the ATG initiation codon may be provided. These exogenous translation regulation signals and initiation codons may be a variety of natural and synthetic sources. Expression efficiency may be increased by introducing appropriate transcription or translation enhancers.

[0038] Meanwhile, in order to facilitate the detection of the peptide, the expression vector may further comprise a protein tag which can be optionally removed using an endopeptidase.

[0039] The term "tag" of the present invention refers to a molecule that exhibits an activity or characteristic that can be quantified, and may be a fluorescent molecule including a chemical fluorescent substance (fluoracer) (such as fluorescein) and a polypeptide fluorescent substance (such as fluorescent protein (GFP) or related proteins); or an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, a streptavidin tag, etc. In particular, when an epitope tag is used, a peptide tag consisting of 6 or more amino acid residues may be preferably used, and a peptide tag consisting of 8 to 50 amino acid residues may be more preferably used.

[0040] In the present invention, the expression vector may contain a nucleotide sequence encoding the peptide providing the effect of promoting the activity of tyrosinase of the present invention. The vector used in this case may preferably be a plasmid DNA, a phage DNA, etc., and more preferably may be a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an Escherichia coli-derived plasmid (pYG601 BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, adenovirus, vaccinia virus, etc.), virus, etc.), insect virus vectors (baculovirus, etc.), but are not particularly limited thereto as long as the peptide can be produced. The expression vector presents different protein expression amounts and modifications depending on the host cell, so it is preferred to select and use the host cell that best suits the purpose.

[0041] The expression vector provided in the present invention can be introduced into the host for transformation, so that the transformant provided in the present invention can be prepared, and the polynucleotide contained in the expression vector is expressed to be used for the production of the peptide. The transformation can be carried out by a variety of methods, such as the CaCl2 precipitation method, the Hanahan method in which a reducing substance called dimethyl sulfoxide (DMSO (dimethyl sulfoxide)) is used to improve the efficiency in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation, protoplast fusion, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation method, transformation method using polyethylene glycol (PEG), dextran sulfate, liposome and drying / inhibition-mediated transformation method, etc., but as long as the peptide can be produced, it is not particularly limited thereto. Furthermore, the host used in preparing the transformant is not particularly limited as long as it can produce the peptide, but may be bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc.; fungal cells such as Pichia pastoris, etc.; insect cells such as Drosophila, Spodoptera Sf9 cells, etc.; animal cells such as CHO cells, COS cells, NSO cells, 293 cells, human melanoma cells, etc.; or plant cells.

[0042] The transformant can also be used for the method of producing the peptide of the present invention that provides the effect of promoting the activity of tyrosinase. Specifically, the method of producing the peptide of the present invention that provides the effect of promoting the activity of tyrosinase may include: step (a), obtaining a culture by culturing the transformant; and step (b), recovering the peptide of the present invention from the culture.

[0043] The term "culture" of the present invention means a method of growing and developing a microorganism under an appropriately artificially adjusted environmental condition. In the present invention, the method of culturing the transformant can be carried out using a method widely known in the technical field to which the present invention belongs. Specifically, with respect to the culture, continuous culture can be carried out in a batch process or a fed-batch or repeated fed-batch process, but as long as it can be produced by expressing the peptide of the present invention that provides the effect of promoting the activity of tyrosinase, it is not particularly limited thereto.

[0044] The culture medium used for the culture needs to be a conventional culture medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc., under aerobic conditions, and the temperature, pH, etc., and the conditions of the specific strain need to be adjusted in an appropriate manner. As a usable carbon source, a mixed sugar of glucose and xylose is used as the main carbon source, in addition to: sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose; and oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc.; and fatty acids such as palmitic acid, stearic acid, linoleic acid; and alcohols such as glycerol, ethanol; and organic acids such as acetic acid. These substances can be used individually or as a mixture. As usable nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used; amino acids such as glutamic acid, methionine, glutamine; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used. These nitrogen sources can be used alone or in combination. In the culture medium, as a phosphorus source, monopotassium phosphate, dipotassium phosphate and corresponding sodium salts can be included. As usable phosphorus sources, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium salts are included. And as inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate and calcium carbonate can be used. Finally, growth essential substances such as amino acids and vitamins can be added to the substance for use.

[0045] In addition, appropriate precursors may be used in the culture medium. The raw materials may be added to the culture in a batch, fed-batch or continuous manner during the culture process, but are not particularly limited thereto. The pH of the culture may be adjusted by using an alkaline compound such as sodium hydroxide, potassium hydroxide, ammonia, or the like; an acidic compound such as phosphoric acid or sulfuric acid, or the like, in an appropriate manner.

[0046] Furthermore, the generation of bubbles can be suppressed by using defoamers such as fatty acid polyethylene glycol esters. In order to maintain a good aerobic state, oxygen or 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. And the culture is continued until the production amount of the peptide reaches the maximum. For this purpose, it can usually be achieved within 10 hours to 100 hours.

[0047] Furthermore, the step of recovering the peptide from the culture may be performed by a method known in the art to which the present invention belongs. Specifically, the recovery method is not particularly limited as long as it is applicable to the recovery of the produced peptide, and preferably, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) and the like can be used.

[0048] The term "prevention" of the present invention means all actions to inhibit or delay the occurrence of hypomelaninism by administering a pharmaceutical composition for preventing or treating hypomelaninism comprising the peptide of the present invention.

[0049] The term "treatment" of the present invention means any action of treating hypomelaninism by promoting melanosome synthesis by administering a pharmaceutical composition comprising the peptide of the present invention as an active ingredient to an individual in need of treatment for hypomelaninism.

[0050] The pharmaceutical composition of the present invention can be prepared into a form of a pharmaceutical composition for treating hypomelaninosis, in which the peptide further includes an appropriate carrier (natural or non-natural carrier), excipient or diluent commonly used in the preparation of the pharmaceutical composition. Specifically, the pharmaceutical composition can be formulated into a sterile injection that can be administered to the site of hypomelaninosis according to the usual method. In the present invention, the carrier, excipient and diluent that can be included in the pharmaceutical composition include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, etc. When preparing the formulation, commonly used diluents or excipients such as fillers, brighteners, binders, wetting agents, disintegrants, surfactants, etc. can be used for preparation. In particular, it can include sterile aqueous solutions, non-aqueous solvents, suspending agents, emulsions, freeze-dried agents, suppositories, ointments (for example, endodontic transplant materials, etc.), etc. As non-aqueous solvents and suspending agents, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, etc. can be used; injectable esters such as ethyl oleate, etc. can be used. As the base of the suppository, synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, lauric acid glyceride fat, glycerin gelatin, etc. can be used.

[0051] The content of the peptide contained in the pharmaceutical composition of the present invention may be 0.0001 wt % to 50 wt %, more preferably 0.01 to 20 wt %, based on the total weight of the final composition, but is not limited thereto.

[0052] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention refers to a sufficient amount for the treatment or prevention of a disease at a reasonable benefit / risk ratio that can be applied to medical treatment or prevention. The effective dosage level can be determined based on the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and the excretion rate treatment time, the elements of the drugs used in combination with or containing the composition of the present invention used, and other elements known in the medical field. The pharmaceutical composition of the present invention can be administered independently or in combination with a known pharmaceutical composition for the treatment of melanin deficiency. It is important to take all the above factors into consideration and administer the drug in an amount that has no side effects and can achieve the maximum effect with the least amount.

[0053] A person skilled in the art of the present invention may determine the dosage of the pharmaceutical composition of the present invention by considering the purpose of use, the degree of disease poisoning, the age, weight, sex, previous illness of the patient, or the type of substance used as an active ingredient. For example, each adult may be administered with about 0.1 ng to about 100 mg / kg of the pharmaceutical composition of the present invention, preferably, it can be administered with 1 ng to about 10 mg / kg, and the administration frequency of the composition of the present invention may be once a day or divided into doses for multiple administrations, but is not particularly limited thereto. The dosage does not limit the scope of the present invention in any aspect.

[0054] As another embodiment, the present invention provides a method for treating hypomelaninosis, comprising the step of administering a pharmaceutically effective amount of the pharmaceutical composition to an individual suffering from hypomelaninosis.

[0055] The term "vitiligo" of the present invention 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.

[0056] The term "poliosis" of the present invention refers to a depigmentation disease in which the melanin (or pigment) in the hair, eyebrows, and eyelashes is reduced or disappeared.

[0057] The term "subject" of the present invention is intended to include without limitation mammals in need of treatment for hypomelaninosis, including mice, livestock, etc., but humans may be excluded among subjects suffering from the disease.

[0058] The administration route of the pharmaceutical composition for treating hypomelanosis of the present invention can also be administered by any common route as long as the target tissue can be reached. The pharmaceutical composition of the present invention can provide all dosage forms suitable for local application according to the purpose, but is not particularly limited thereto. For example, it can be administered by oral, transdermal, intravenous, intramuscular, or subcutaneous injection. The pharmaceutical composition can be an injection, a skin external solution, a suspension, an emulsion, a gel, a patch or a spray, but is not limited thereto. The dosage form can be easily prepared by the usual methods in the art, and surfactants, excipients, wettable powders, emulsifying agents, suspending agents, salts or buffers for controlling osmotic pressure, colorants, spices, stabilizers, preservatives, preservatives or other commonly used adjuvants can be appropriately used.

[0059] As another embodiment, the present invention provides a quasi-pharmaceutical composition for preventing or improving hypomelaninosis, comprising the peptide.

[0060] The term "improvement" according to the present invention is intended to refer to a parameter related to the state being treated, for example, all actions that at least reduce the degree of symptoms.

[0061] In the present invention, the improvement can be explained as follows: a pharmaceutical composition comprising the peptide of the present invention as an effective ingredient is administered to an individual in need of treatment for hypomelanosis to promote the synthesis of melanosomes, thereby improving the symptoms of hypomelanosis or obtaining favorable changes in all behaviors.

[0062] The term "quasi-drugs" in the present invention means items that are used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases in humans or animals and have a milder effect than drugs. For example, according to the Pharmaceutical Affairs Law, quasi-drugs refer to items other than items used as drugs, including fiber / rubber products used to treat or prevent human / animal diseases, items that are not instruments or machines and similar items that have a mild or no direct effect on the human body, sterilizers / insecticides used to prevent infectious diseases, etc.

[0063] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, but as an example, it can be a disinfectant cleanser for skin or hair, a skin or hair cleansing product, a soap, a shampoo, a skin ointment, etc.

[0064] As another embodiment, the present invention provides a health functional food composition for preventing or improving hypomelaninism, comprising the peptide.

[0065] The term "food" of the present invention includes meat, sausage, bread, chocolate, candy, fast food, biscuits, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, rehydration, alcoholic beverages, vitamin complexes, health functional foods and health foods, etc., including all foods in the conventional sense.

[0066] The functional food is a term identical to food for special health use (FoSHU), and refers to a food with high medical and medical effects that can be processed to effectively present the regulating function of the organism in addition to providing nutrition. Among them, the so-called "function" means to regulate nutrients (to the structure and function of the human body) or to achieve favorable effects in health care purposes (such as physiological effects, etc.). The food of the present invention can be prepared by the method commonly used in the technical field to which the present invention belongs, and the raw materials and ingredients that are usually added in the technical field can be added to prepare it during the preparation. In addition, as for the dosage form of the food, as long as it is a dosage form that can be identified as food, it can be prepared without restriction. The food composition of the present invention has the following advantages: it can be prepared into a variety of dosage forms, and unlike ordinary medicines, food can be used as a raw material, so there are no side effects that may occur when taking medicines for a long time, and it is excellent in portability, so the food of the present invention can be taken as an adjuvant for enhancing the effect of preventing or improving melanin deficiency.

[0067] The health food refers to a food that has a positive effect of maintaining or improving health compared to ordinary food, and the health supplement food refers to a food for health supplement purposes. Depending on the situation, the terms health functional food, health food, and health supplement food can be used interchangeably.

[0068] Specifically, the health functional food refers to a food prepared by adding the peptide of the present invention to food materials such as beverages, teas, spices, chewing gums, biscuits, etc., or preparing it as a capsule, powder, suspension, etc. When the food is ingested, it brings specific effects on health. However, unlike ordinary medicines, since food is used as a raw material, there is no advantage of side effects that may occur when taking medicines for a long time.

[0069] The food composition of the present invention can be ingested daily, and therefore can be expected to have a good effect in preventing or improving hypomelaninosis, and thus can be used very effectively.

[0070] The food composition may further include a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the technical field to which the present invention belongs may be used.

[0071] Furthermore, the food composition may include additives that can be commonly used in food compositions to improve smell, taste, vision, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folic acid, pantothenic acid, etc. may be included. Furthermore, minerals such as zinc (Zn), iron (Fe), calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. may be included. Furthermore, amino acids such as lysine, tryptophan, cysteine, valine, etc. may be included.

[0072] Moreover, the food composition may contain food additives, such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high-efficiency bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar pigments, etc.), color developers (sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (sweetness, cyclamates, saccharin, sodium, etc.), spices (vanillin, lactones, etc.), expanders (alum, D-potassium hydrogen tartrate, etc.), enhancers, emulsifiers, thickeners (paste), film coating agents, glue bases, foam inhibitors, solvents, improvers, etc. The additives may be selected and used in appropriate amounts according to the type of food.

[0073] Peptide of the present invention can be directly added or used together with other foods or food ingredients, and can be suitably used according to conventional methods.Can suitably determine the mixed amount of active ingredient according to its purpose of use (prevention, health or therapeutic treatment).Usually, when preparing food or beverage, relative to food or beverage, food composition of the present invention can add below 50 weight portions, specifically, can add the amount below 20 weight portions.But, in the case of long-term intake for the purpose of health and hygiene, can comprise the amount below the described scope, owing to there is no problem aspect safety, can also use the amount above the described scope as active ingredient.

[0074] As an example of the food composition of the present invention, it can be used as a health drink composition. In the case, it can contain a variety of flavoring agents or natural carbohydrates as added ingredients, just like general beverages. The natural carbohydrates can be monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; polysaccharides, such as dextrin and cyclodextrin; sugar alcohols, such as xylitol, sorbitol, erythritol, etc. Sweeteners can use natural sweeteners, such as thaumatin and stevia extract; synthetic sweeteners, such as saccharin and aspartame, etc. The ratio of the natural carbohydrates in each 100 mL of the health drink composition of the present invention can generally be about 0.01g to 0.04g, specifically about 0.02g to 0.03g.

[0075] In addition to the above, the health drink composition can include multiple nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid, pectinate, alginic acid, alginate, organic acid, protective colloid thickener, pH adjusting agent, stabilizer, preservative, glycerine, ethanol or carbonating agent etc. In addition, the pulp used to prepare natural fruit juice, fruit juice beverage or vegetable beverage can be included. This composition can be used independently or in combination. Although the ratio of this additive is not particularly important, the health drink composition of the present invention of every 100 weight portions is selected usually in the range of 0.01 to 0.1 weight portion.

[0076] As for the food composition of the present invention, if it can show the effect of preventing or improving hypomelaninism, the peptide of the present invention can be contained in various weight %, specifically, based on the total weight of the food composition, it can contain 0.00001 to 100 weight % or 0.01 to 80 weight % of the peptide of the present invention, but it is not limited to this.

[0077] Effects of the Invention

[0078] The peptides according to the embodiments of the present invention or the composition comprising the same can promote the activity of tyrosinase associated with the formation of melanosomes in melanocytes.

[0079] The peptides or compositions comprising the same according to the embodiments of the present invention can prevent hypomelanin disease that forms depigmented spots on the skin by regulating the activity of tyrosinase in melanocytes.

[0080] The peptides or compositions comprising the same according to the embodiments of the present invention can improve or treat hypomelaninism by promoting melanin production in melanocytes.

[0081] The peptides according to the embodiments of the present invention or the composition comprising the same can promote the process of oxidation of L-dopa to dopaquinone during the melanin synthesis process in melanocytes.

[0082] The effects of the present invention are not limited to the above-mentioned contents, and a person having ordinary knowledge in the technical field to which the present invention belongs can clearly understand other effects not mentioned from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The figure shows that B16F10 cells were divided at 230,000 cells / well in a 6-well plate and cultured in a 5% CO2 incubator at 37 degrees for 24 hours, and then treated with peptide KH001 (sequence number 96) at different concentrations for 72 hours to prepare cells. After the cell pellets were dissolved in a strong alkaline solution, the absorbance was measured at 405 nm, and (a) the melanin content (%) according to different peptide treatment concentrations and (b) the degree of melanin production according to different peptide treatment concentrations were compared.

[0084] Figure 2 The tyrosinase activity is shown by observing the product under an optical microscope. The product is prepared by dividing B16F10 cells at 230,000 cells / well in a 6-well plate, culturing them in a 5% CO2 incubator at 37 degrees for 24 hours, then treating the peptide KH001 (SEQ ID NO: 96) at different concentrations for 72 hours, fixing the cells, and reacting them with levodopa (L-DOPA).

[0085] Figure 3 B16F10 cells were divided at 230,000 cells / well in a 6-well plate and cultured in a 5% CO2 incubator at 37 degrees for 24 hours. KH001 peptide was treated at different concentrations of 50uM, 100uM, and 200uM and cultured for 72 hours. The cells were washed with PBS, and completely dissolved with 100μl of 0.1M potassium phosphate buffer (pH 6.8) containing 1% triton X. The cells were placed in a 1.5ml tube and centrifuged at 12000rpm for 20 minutes. 40μl of the supernatant was taken out and reacted with 200μl of 2mg / ml L-DOPA solution (0.1M potassium phosphate pH7.0) at 37 degrees for 30 minutes. The absorbance was then measured at 475nm to measure the tyrosinase activity. activity) (corrected by the protein concentration of cell lysate).

[0086] Figure 4The method is to divide B16F10 cells at 230,000 cells / well in a 6-well plate and culture them in a 5% CO2 incubator at 37 degrees for 24 hours. Then, the cells are treated with KH001 peptide at different concentrations of 50uM, 100uM, and 200uM and cultured for 72 hours. The absorbance of the media in each well is measured at 405nm to quantify the secreted melanin.

[0087] Figure 5 The results are shown in Figure 2. After B16F10 cells were treated with KH001 peptide at different concentrations of 50uM, 100uM, and 200uM, an MTT assay was performed at 72 hours to measure cell viability. DETAILED DESCRIPTION

[0088] If you refer to the following and Figure 1 The purpose and effect of the present invention and the technical structure used to achieve the same will be clear from the detailed description of the embodiments. In describing the present invention, when it is judged that the detailed description of the known function or structure may unnecessarily confuse the gist of the present invention, the detailed description will be omitted. Moreover, the terms described below, as terms defined in consideration of the functions in the present invention, may vary according to the intention or convention of the user or operator.

[0089] However, the present invention is not limited to the embodiments disclosed below, and can be embodied in various forms that are different from each other. However, this embodiment is provided to make the disclosure of the present invention more complete and to fully explain the scope of the invention to people with ordinary knowledge in the technical field to which the present invention belongs, and the present invention is only defined by the scope of the claims. Therefore, this definition should be made based on the entire content of this specification.

[0090] The following is a detailed description of the embodiments of the present invention.

[0091] Example 1: Synthesis of peptides

[0092] The present inventors synthesized a peptide (SEQ ID NO. 1) showing a tyrosinase activity-promoting effect by the 9-fluorenylmethyloxycarbonyl (Fmoc) method, and synthesized peptides of various groups (Tables 1 to 12) by substituting amino acids of the synthesized peptide.

[0093] N-KYQRRKKNKY-C (SEQ ID NO. 1)

[0094] First, the peptide of Group 1 is the peptide of Sequence No. 1, or is synthesized by substituting lysine or arginine for amino acids 5 to 7 of the peptide of Sequence No. 1 (Table 1).

[0095]

Table 1

[0096]

[0097] Then, the amino acids at positions 5 to 7 of the peptide of SEQ ID NO: 1 were substituted with lysine or arginine, and the amino acid at position 8 was substituted with serine, thereby synthesizing peptides of Group 2 (Table 2).

[0098]

Table 2

[0099]

[0100] Then, the amino acids at positions 5 to 7 of the peptide of SEQ ID NO: 1 were substituted with lysine or arginine, and the amino acid at position 9 was substituted with tyrosine, thereby synthesizing peptides of Group 3 (Table 3).

[0101]

Table 3

[0102]

[0103]

[0104] Then, the amino acids at positions 5 to 7 of the peptide of sequence number 1 were substituted with lysine or arginine, the amino acid at position 8 was substituted with serine, the amino acid at position 9 was substituted with tyrosine, and the amino acid at position 10 was substituted with lysine, thereby synthesizing peptides of group 4 (Table 4).

[0105]

Table 4

[0106]

[0107] Then, the amino acid at position 3 of the peptide of sequence number 1 was substituted with arginine, the amino acid at position 4 was substituted with glutamine, and the amino acids at positions 5 to 7 were substituted with lysine or arginine, thereby synthesizing peptides of group 5 (Table 5).

[0108]

Table 5

[0109]

[0110]

[0111] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced with arginine, the amino acid at position 4 was replaced with glutamine, the amino acids at positions 5 to 7 were replaced with lysine or arginine, and the amino acid at position 8 was replaced with serine, thereby synthesizing peptides of group 6 (Table 6).

[0112]

Table 6

[0113] Group 6 peptides

[0114]

[0115] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by arginine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 7 (Table 7).

[0116]

Table 7

[0117]

[0118]

[0119] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by arginine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 8 was replaced by serine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 8 (Table 8).

[0120]

Table 8

[0121]

[0122] Then, the amino acid at position 3 of the peptide of sequence number 1 was substituted with lysine, the amino acid at position 4 was substituted with glutamine, and the amino acids at positions 5 to 7 were substituted with lysine or arginine, thereby synthesizing peptides of group 9 (Table 9).

[0123]

Table 9

[0124]

[0125]

[0126] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, and the amino acid at position 8 was replaced by serine, thereby synthesizing peptides of group 10 (Table 10).

[0127]

Table 10

[0128]

[0129] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 11 (Table 11).

[0130]

Table 11

[0131]

[0132]

[0133] Finally, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 8 was replaced by serine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 12 (Table 12).

[0134]

Table 12

[0135]

[0136] Example 2: Materials and Methods

[0137] Example 2-1 Cultivation of melanocytes

[0138] Melanocyte (Melanocyte B16F10) cell line was cultured in a DMEM medium containing 10% FBS and 1% PS (penicillin / streptomycin) in an incubator at 37°C and 5% CO 2 .

[0139] Example 2-2 MTT Assay

[0140] MTT reagent was diluted in DMEM at a concentration of 0.5 mg / ml and used.

[0141] B16F10 cells were seeded at 230,000 cells / well in a 6-well plate and cultured in a 5% CO2 incubator at 37 degrees for 24 hours. Then, peptide KH001 (SEQ ID NO: 96) was treated at different concentrations for 72 hours to prepare cells, and MTT reagent was injected and reacted for 1 hour in a light-shielded, 37-degree incubator. Formazan generated after all substrates were removed was dissolved in DMSO, and absorbance was measured at 560 nm.

[0142] Example 2-3 Quantification of melanin contents

[0143] B16F10 cells were divided at 230,000 cells / well in a 6-well plate and cultured in a 5% CO2 incubator at 37 degrees for 24 hours. KH001 peptide was treated at different concentrations of 50uM, 100uM, and 200uM and cultured for 72 hours. After washing the cells with PBS, the cells were lysed with a solution of 10% DMSO added to 1N NaOH. The amount of melanin was measured by measuring the absorbance at 405nm for the lysate, and the protein concentration value of the lysate was used for correction.

[0144] Melanin content (%) = (absorbance of peptide-treated group / absorbance of PBS-treated group) x 100

[0145] Example 2-4 Quantification of secreted melanin

[0146] B16F10 cells were seeded at 230,000 cells / well in a 6-well plate and cultured in a 5% CO2 incubator at 37 degrees for 24 hours. KH001 peptide was treated at different concentrations of 50uM, 100uM, and 200uM and cultured for 72 hours. At this time, the absorbance of the matrix in each well was measured at 405nm to quantify secreted melanin.

[0147] Secreted melanin (%) = (absorbance of peptide-treated group / absorbance of PBS-treated group) x 100

[0148] Example 2-5 Measurement of tyrosinase activity

[0149] B16F10 cells were divided at 230,000 cells / well in a 6-well plate and cultured in a 5% CO2 incubator at 37 degrees for 24 hours. For it, KH001 peptide was treated with different concentrations of 50uM, 100uM, and 200uM and cultured for 72 hours. Then the cells were washed with PBS, and after all the cells were dissolved with 100μl of 0.1M potassium phosphate buffer (pH 6.8) containing 1% Triton X, they were placed in a 1.5ml tube and centrifuged at 12000rpm for 20 minutes. Among them, 40μl of the supernatant was taken out and reacted with 200μl of 2mg / ml levodopa solution (0.1M potassium phosphate pH7.0) at 37 degrees for 30 minutes. Then the absorbance was measured at 475nm to measure the tyrosinase activity. This value was corrected with the protein concentration of the cell lysate.

[0150] Tyrosinase activity (%) = (absorbance of peptide-treated group / absorbance of PBS-treated group) x 100

[0151] Example 2-6 Measurement of tyrosinase activity 2

[0152] B16F10 cells were divided at 230,000 cells / well in a 6-well plate and cultured in a 5% CO2 incubator at 37 degrees for 24 hours. For them, KH001 peptide was treated at different concentrations of 50uM, 100uM, and 200uM and cultured for 72 hours. After washing the cells with PBS, they were fixed with 2% paraformaldehyde for 20 minutes. Then, after washing the cells three times with PBS, 0.1% L-DOPA (0.1M potassium phosphate pH7.0) was injected into each well. Then, the cells were reacted in an incubator at 37 degrees for 3 hours and photos were taken with an optical microscope.

[0153] Example 3: Results

[0154] Example 3-1. Effect of peptide KH001 on melanin production in B16F10 melanocytes

[0155] B16F10 cells were treated with KH001 peptide at different concentrations. At 72 hours after peptide treatment, the cell pellet was dissolved in a strong alkaline solution and the absorbance was measured at 405 nm to quantify the amount of melanin in the cells. The protein value was corrected by BSA assay. Figure 1 The results of treating KH001 at concentrations of 50uM, 100uM, and 200uM showed that melanin production increased by 38%, 81%, and 176% compared to the control group. Therefore, it can be seen that KH001 peptide can induce melanin synthesis in a concentration-dependent manner.

[0156] Example 3-2. Effect of peptide KH001 on tyrosinase activity of B16F10 melanocytes 1

[0157] In the synthesis of melanin, the oxidation of L-DOPA to DOPA quinone is required, and this process requires the activity of tyrosinase. Therefore, in this study, the lysate of B16F10 cells treated with KH001 peptide was reacted with L-DOPA to quantify the generated DOPA quinone, thereby measuring the tyrosinase activity. Figure 5(Tyrosinase activity), the results of KH001 peptide treatment showed that compared with the control group, the tyrosinase activity increased by 101%, 443%, and 433% at different concentrations of KH001 treatment concentrations of 50uM, 100uM, and 200Um, respectively. Therefore, it can be seen that KH001 peptide induces melanin synthesis by increasing tyrosinase activity.

[0158] Example 3-3. Effect of peptide KH001 on tyrosinase activity of B16F10 melanocytes 2

[0159] B16F10 cells were treated with KH001 peptide at different concentrations for 72 hours, and the cells were fixed and reacted with L-DOPA reagent. The tyrosinase in the cells reacts with L-DOPA and turns black. The darker it turns, the higher the tyrosinase activity. Figure 2 According to the experimental results, the tyrosinase activity in the KH001 peptide-treated group increased significantly compared with the control group.

[0160] Example 3-4. Effect of peptide KH001 on melanin secretion of B16F10 melanocytes

[0161] After B16F10 cells were treated with KH001 peptide at different concentrations, the absorbance of melanin secreted into the culture media was measured at 405 nm 72 hours after the treatment with KH001 peptide, thereby quantifying the amount of melanin secreted into the matrix. Figure 2 (Secreted melanin) As a result of KH001 peptide treatment, melanin production increased by 17%, 31%, and 37% respectively compared to the control group in a concentration-dependent manner. Therefore, it can be seen that due to the effect of KH001 peptide, B16F10 cells can secrete more melanin in a concentration-dependent manner.

[0162] Example 3-5. Toxicity evaluation of peptide KH001 in B16F10 melanocytes

[0163] B16F10 cells were treated with KH001 peptide at different concentrations. MTT assay was performed to measure cell viability 72 hours after treatment with KH001 peptide. Figure 4 (Survival rate) As a result of treating KH001 at concentrations of 50 uM, 100 uM, and 200 uM, there was no significant difference in cell survival rate compared to the control group.

[0164] Therefore, it was confirmed that the KH001 peptide had no toxicity within the specified concentration range.

[0165] The preferred embodiments of the present invention are disclosed in this specification and the accompanying drawings. Although specific terms are used, they are used in a general sense only to easily explain the technical content of the present invention and to help understand the invention, and are not used to limit the scope of the present invention. In addition to the embodiments disclosed herein, other variations based on the technical ideas of the present invention may also be implemented, which is obvious to a person skilled in the art of the present invention.

Claims

1. A composition for promoting tyrosinase activity, comprising a peptide consisting of an amino acid sequence of the following general formula 1, wherein: KY-R1-R2-R3-R4-R5-R6-R7-R8 (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); and R7 and R8 are lysine (K) or tyrosine (Y).

2. A composition for regulating melanin synthesis, comprising a peptide consisting of an amino acid sequence of the following general formula 1, wherein: KY-R1-R2-R3-R4-R5-R6-R7-R8 (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); and R7 and R8 are lysine (K) or tyrosine (Y).

3. A composition for preventing, improving or treating melanin deficiency, comprising a peptide consisting of an amino acid sequence of the following general formula 1, wherein: KY-R1-R2-R3-R4-R5-R6-R7-R8 (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); and R7 and R8 are lysine (K) or tyrosine (Y).

4. A quasi-medical composition for preventing or improving melanin deficiency, comprising a peptide consisting of an amino acid sequence of the following general formula 1, wherein: KY-R1-R2-R3-R4-R5-R6-R7-R8 (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); and R7 and R8 are lysine (K) or tyrosine (Y).

5. The composition according to claim 1, characterized in that The peptide is any one of the amino acid sequences from sequence number 1 to sequence number 96.

6. A polynucleotide encoding the peptide according to 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 formed by repeatedly linking the peptides.

9. The composition according to claim 3, characterized in that The hypomelaninosis is vitiligo or leucoderma.

10. The composition according to claim 3, characterized in that The composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

11. A method for preventing, improving or treating hypomelaninosis by administering the composition of claim 3 to an individual.

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