Peptide with melanogenesis inhibiting activity and use thereof

Abstract

The present invention relates to a peptide composed of an amino acid sequence of FLG having melanin production inhibitory activity, a cosmetic composition for skin whitening containing the same, a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease, and a health functional food composition for the prevention or improvement of melanin hyperpigmentation.

Description

Technology Field

[0001] The present invention relates to a peptide having melanin production inhibitory activity and the use thereof, and more specifically, to a peptide composed of an amino acid sequence of FLG having melanin production inhibitory activity, a cosmetic composition for skin whitening containing the same, a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease, and a health functional food composition for the prevention or improvement of melanin hyperpigmentation.

[0002] This result was researched with funding from the Ministry of Agriculture, Food and Rural Affairs and supported by the Private Sector-led R&D Commercialization Support Project of the Korea Institute of Planning and Evaluation for Agricultural and Food Technology (RS-2024-00402482). Background Technology

[0003] The skin is the largest organ possessed by humans. It consists of the epidermis and the dermis. The cells of the epidermis continuously divide to form various layers. During this process, the outermost cells die and harden, serving as one of the most excellent defense organs that protects the body from external stress, and bacteria cannot penetrate this skin either.

[0004] Since the skin has sebaceous glands and sweat glands, the sebaceous glands secrete sebum and the sweat glands secrete moisture, allowing humans to create a protective barrier and shield the stratum corneum. Because of this, human skin is always moist, shiny, and maintains a smooth state.

[0005] In humans, the most ideal skin condition, where beauty is at its peak, is when the moisture content of the stratum corneum reaches 20%. However, the skin undergoes various changes and aging due to the influence of the external environment, and in particular, exposure to sunlight or various external rays can cause skin diseases.

[0006] In particular, when skin is exposed to ultraviolet rays, its color darkens, and the degree of darkening varies from person to person.

[0007] Most people have a desire to cultivate clear and transparent skin, and there is a distinct tendency to prefer white skin.

[0008] Although cosmetics are intended to protect, adorn, and cleanse the skin from external factors, a closer look at their composition reveals that ingredients essential for product formation are included—contrary to their original intent—which can actually irritate the skin. Examples of this include emulsifiers, fragrances, and preservatives. Since most of these are produced through chemical synthesis, individuals with sensitive skin are at an increased risk of inflammatory reactions, allergies, and redness.

[0009] As cosmetic compositions used for skin care or whitening, there are traditionally cosmetics applied to the skin, and inner beauty products (edible cosmetics) that produce beauty effects when consumed as food are also being developed.

[0010] Regarding prior art for skin whitening compositions, various studies are being conducted, such as a skin whitening composition that inhibits excessive melanin pigment deposition through mechanisms like the inhibition of melanin-forming cell activity (Published Patent Application No. 10-2019-0050058), but the current situation is still insufficient. Prior art literature

[0011] Published Patent Application No. 10-2019-0050058 The problem to be solved

[0012] The inventors of the present invention have completed the present invention by continuously conducting research to develop a formulation that inhibits melanin production and has a skin whitening effect, developing a novel tripeptide composed of the amino acid sequence of FLG according to the present invention, and identifying that the tripeptide has an excellent effect of inhibiting melanin production without exhibiting cytotoxicity.

[0013] Therefore, the objective of the present invention is to provide a peptide composed of the amino acid sequence of FLG.

[0014] Another objective of the present invention is to provide a peptide composed of the amino acid sequence of the FLG for use as a cosmetic, pharmaceutical, or health functional food. means of solving the problem

[0015] To solve the above problem, the present invention provides a peptide composed of the amino acid sequence of FLG.

[0016] According to a preferred embodiment of the present invention, the peptide may have melanin production inhibitory activity.

[0017] To solve the above problem, the present invention provides a cosmetic composition for skin whitening comprising a peptide consisting of the amino acid sequence of the FLG as an active ingredient.

[0018] To solve the above problem, the present invention provides a pharmaceutical composition for the prevention or treatment of hypermelanin pigmentation disease comprising a peptide composed of the amino acid sequence of the FLG as an active ingredient.

[0019] According to a preferred embodiment of the present invention, the melanin hyperpigmentation disorder in the pharmaceutical composition may be melasma, freckles, senile pigment spots, or solar lentigo.

[0020] To solve the above problem, the present invention provides a health functional food composition for preventing or improving excessive melanin pigmentation, comprising a peptide consisting of the amino acid sequence of the FLG as an active ingredient.

[0021] According to a preferred embodiment of the present invention, melasma, freckles, senile pigment spots, or solar lentigo may be formed in the health functional food composition due to excessive deposition of melanin pigment. Effects of the invention

[0022] Since the peptide composed of the amino acid sequence of the FLG according to the present invention has excellent melanin production inhibition effect and pigment deposition inhibition effect, it can be utilized as an active ingredient in cosmetic compositions for skin whitening, inner beauty compositions, pharmaceutical compositions for the prevention or treatment of melanin hyperpigmentation disease, and health functional food compositions for the prevention or improvement of melanin hyperpigmentation. Brief explanation of the drawing

[0023] Figure 1 shows the experimental results of the whitening effect of a peptide composed of the amino acid sequence of FLG according to the present invention on a melanocyte culture medium. Figure 2 shows a graph comparing the whitening effect test results of Figure 1 with the control group. Specific details for implementing the invention

[0024] The description of the present invention is merely an example for structural or functional explanation, and therefore the scope of the present invention should not be interpreted as being limited by the examples described in the text. That is, since the examples may be modified in various ways and may take various forms, the scope of the present invention should be understood to include equivalents capable of realizing the technical concept.

[0025] Furthermore, the purposes or effects presented in this invention do not imply that specific embodiments must include all of them or only such effects; therefore, the scope of the rights of this invention should not be understood as being limited by them.

[0026] Unless otherwise defined, all terms used herein have the same meaning as generally understood by those skilled in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having meanings consistent with the context of the relevant technology and should not be interpreted as having an ideal or overly formal meaning unless explicitly defined in this application.

[0027] The present invention will be described in more detail below as one embodiment.

[0028] According to one aspect of the present invention, the present invention provides a peptide composed of the amino acid sequence of FLG.

[0029] The amino acid of the FLG of the present invention is a tripeptide composed of phenylalanine (F), leucine (L), and glycine (G). In one embodiment, the peptide containing the amino acid sequence of the present invention has no cytotoxicity and inhibits the production of melanin, thereby exhibiting a skin whitening effect or an effect that inhibits pigmentation.

[0030] In another embodiment of the present invention, the peptide comprising the amino acid sequence of the present invention exhibits an inhibitory effect on the DOPA oxidation reaction.

[0031] As used herein, the term "peptide" means a linear molecule formed by amino acid residues being joined together by peptide bonds. The peptide of the present invention may be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54(1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111(1984)) or liquid-phase synthesis techniques (US Patent No. 5,516,891).

[0032] The peptide of the present invention may select a portion of the amino acid sequence and induce modifications at the N-terminus or C-terminus to increase its activity. Through such modifications, the polypeptide of the present invention may have a high half-life with an increased half-life upon administration in vivo.

[0033] In another embodiment of the present invention, the C-terminus of the peptide of the present invention may be modified to a hydroxyl group (-OH), an amino group (-NH2), an azide group (-NHNH2), etc., and the N-terminus of the peptide may be bonded to a protecting group selected from the group consisting of an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG).

[0034] The modification of the amino acid described above serves to significantly improve the stability of the peptide according to the present invention. In this specification, the term "stability" refers not only to in vivo stability but also to storage stability (e.g., room temperature storage stability). The protecting group serves to protect the peptide of the present invention from attack by protein-cleaving enzymes in vivo.

[0035] According to another aspect of the present invention, the present invention provides a cosmetic composition for skin whitening comprising, as an active ingredient, a polypeptide consisting of the amino acid sequence of FLG or comprising said FLG amino acid sequence.

[0036] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of hypermelanosis of pigmentation disease comprising a peptide consisting of the amino acid sequence of the FLG as an active ingredient.

[0037] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of hypermelanosis of pigmentation disease comprising a peptide consisting of the amino acid sequence of the FLG as an active ingredient.

[0038] According to another aspect of the present invention, the present invention provides a health functional food composition for the prevention or improvement of melanin hyperpigmentation disease comprising a peptide consisting of the amino acid sequence of the FLG as an active ingredient.

[0039] In one embodiment of the present invention, the cosmetic composition, pharmaceutical composition and health functional food composition may comprise, in addition to the peptide comprising the amino acid sequence of the FLG described above, at least one pharmaceutically and / or cosmetically acceptable excipient, carrier, or diluent selected in relation to the intended route of administration and standard pharmaceutical or cosmetic application.

[0040] The peptide of the present invention can be freeze-dried for storage and reconstituted into a suitable carrier before use.

[0041] The term "pharmaceuticalally acceptable" in the present invention refers to a formulation that is sterile and free of pyrogens. Suitable pharmaceutical carriers are well known in the field of pharmaceuticals. The carrier must be "acceptable" in the sense that it does not adversely affect the efficacy of the active ingredient of the present invention and is not harmful to its recipient. Typically, the carrier may be sterile and pyrogen-free water or saline solution, but is not limited thereto.

[0042] Accordingly, "pharmaceutically acceptable carriers" and "pharmaceutically acceptable excipients" comprise compound(s) used to form part of a formulation, intended simply to act as carriers, that is, not intended to have biological activity of their own. Pharmaceutically acceptable carriers or excipients are generally safe and non-toxic. Pharmaceutically acceptable carriers and / or excipients such as those used herein may comprise all of one or more carriers and / or excipients.

[0043] Additionally, the term "cosmetically acceptable" is used to denote formulations suitable for use as cosmetics. Suitable cosmetic carriers are well known in the art, such as those commonly used in shampoos, lotions, creams, sprays, and other such products.

[0044] Excipients may be one or more carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrin, which are added to the composition, for example, to enable freeze-drying. Examples of polymers include starch, cellulose ether, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate of different degrees of hydrolysis, and polyvinylpyrrolidone of all different molecular weights, which are added to the composition, for example, to control viscosity, to achieve adhesion, or to protect lipids from chemical and proteolytic degradation.

[0045] Examples of lipids include fatty acids, phospholipids, phosphates, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids of all different acyl chain lengths and saturation, egg lecithin, soy lecithin, hydrogenated egg lecithin, and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals include talc, magnesium oxide, zinc oxide, and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or favorable pigment properties.

[0046] The term “diluent” is intended to mean an aqueous or non-aqueous solution intended to dilute a peptide in pharmaceutical manufacturing. The diluent may be, but is not limited to, one or more saline, water, polyethylene glycol, propylene glycol, ethanol, or vegetable oils (safflower oil, corn oil, peanut oil, cottonseed oil, sesame oil, etc.).

[0047] Diluents can also function as buffers. The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture intended to stabilize pH. Examples of buffers include, but are not limited to, Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartarate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, TES, etc.

[0048] The polypeptide of the present invention may be formulated at various concentrations depending on the efficacy or toxicity of the active ingredient used. Preferably, the composition may contain the polypeptide at a concentration of 1 nM to 1 M, for example, 0.1 μM (micromole) to 1 mM, 1 μM to 100 μM, 5 μM to 50 μM, 10 μM to 50 μM, 20 μM to 40 μM, and optionally about 30 μM, but is not limited thereto.

[0049] The compositions of the present invention may be administered via various routes, e.g., topically, subcutaneously, parenterally, or orally. The compositions of the present invention may preferably be administered topically or intracutaneously. Thus, the compositions of the present invention may be administered topically to the skin (e.g., scalp).

[0050] Meanwhile, the composition of the present invention may be administered parenterally, for example, intradermally. Such composition is best used in the form of a sterile aqueous solution containing sufficient salt or glucose to form an isotonic solution with other substances, for example, blood. If necessary, said aqueous solution should be appropriately buffered (preferably to a pH of 3 to 9). The preparation of a suitable parenteral formulation under sterile conditions is easily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0051] A composition containing the peptide of the present invention is administered to a subject in an effective amount. As used herein, "therapeutically effective amount," "effective amount," or "therapeutically effective" refers to an amount that provides a skin whitening effect or a preventive, alleviating, inhibiting, or therapeutic effect of pigmentation. This is a predetermined amount of an active substance calculated to produce a desired therapeutic effect.

[0052] The above-described health functional food composition of the present invention may additionally contain auxiliary components or additives, such as nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages, in addition to the active ingredients.

[0053] In addition, the carrier, excipient, or diluent may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline solution, but is not limited thereto.

[0054] Health functional foods that can be added to the above-mentioned health functional food composition of the present invention include, for example, various types of food, beverages, gum, candy, tea, vitamin complexes, functional foods, etc.

[0055] As is recognized by those skilled in the art, the amount of the active ingredient may vary depending on its specific activity. An appropriate dose may contain a predetermined amount of the active substance calculated to produce the desired therapeutic effect together with the required diluent.

[0056] In the method and use for preparing the composition of the present invention, a therapeutically effective amount of the active ingredient is provided. As is well known in the art, the therapeutically effective amount may be determined by a practitioner in the medical or veterinary field based on patient characteristics such as age, weight, gender, condition, complications, other diseases, etc.

[0057] In one embodiment of the present invention, "melanin hyperpigmentation" is the result of melanin synthesis, proliferation of melanocytes, and apoptosis of melanocytes. The melanin synthesis, proliferation of melanocytes, and apoptosis of melanocytes may be the result of post-inflammatory hyperpigmentation (PIH). Post-inflammatory hyperpigmentation is induced by acne, atopic dermatitis, contact allergic dermatitis, hypopigmentation, lichen planus, chronic lupus erythematosus, scleroderma, mechanical trauma, ionizing or non-ionizing radiation, burns, laser or drug therapy, skin infections, or combinations thereof. For example, post-inflammatory hyperpigmentation (PIH) is induced in acne.

[0058] Meanwhile, the above "excessive melanin pigmentation" may result in the formation of melasma, freckles, senile pigment spots, or solar lentigo, but is not limited thereto.

[0059] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention should not be interpreted as being limited to the following embodiments. The embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0061] <Example 1> Evaluation of the whitening effect of FLG peptide using melanoma cells

[0062] Experiments were conducted using the B16F1 melanoma cell line (Murine melanoma cell line). B16F1 melanoma cells grown attached to plates using 0.05% trypsin-EDTA (GIBCO BRL) were floated, and cell counting was performed to determine the number of cells in a 24-well plate: 2 × 10⁶ 4The cells were dispensed into wells and cultured for 24 hours.

[0063] After replacing the cultured cell line with New-10% FBS / DMEM medium, samples diluted to different concentrations were pretreated in the replaced medium for 1 hour, and then 100 nM of alpha-melanocyte stimulating hormone (α-Melanocyte stimulating hormone, α-MSH) was added.

[0064] After treating with a peptide composed of FLG (FLG-NH2) as a sample, the cells were incubated in carbon dioxide (CO2 incubation, Thermo) for 72 hours. 100 µl of the cell culture medium was taken and transferred to a 96-well plate, and the amount of secreted melanin was measured by measuring the absorbance at 405 nm.

[0065] After culture, cells were treated with 0.05% trypsin-EDTA (GIBCO BRL) to recover the cells, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed to compare the degree of inhibition of melanin production by the treated samples, as shown in Figure 1.

[0066] After culture, cells were harvested by treatment with 0.05% trypsin-EDTA (GIBCO BRL), centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. To the remaining residue, 100 µl of 1N sodium hydroxide containing 10% dimethylformamide (DMSO) was added, incubated at 60°C for 30 minutes, and then stirred to mix. Melanin was quantified by measuring the absorbance at 405 nm. The measured OD 405 It was converted into a percentage, and the degree to which melanin production was inhibited by the treated samples was compared based on the amount of melanin produced by melanoma cells in the α-MSH treatment group as 100%, as shown in Figure 2.

[0067] As shown in Figures 1 and 2, the results of the experiment on the melanocyte culture medium showed that the sample exhibited a superior melanin inhibition effect compared to the α-MSH treatment group in the range of 100 to 200 μg / ml.

[0068] From these experimental results, it can be confirmed that the skin whitening effect of the peptide composed of FLG according to the present invention is significantly superior.

[0070] <Example 2> Cytotoxicity Test

[0071] The MTT Assay method is a method for measuring cell viability using a dehydrogenase found in the mitochondria of living cells, and the toxicity of the FLG-based peptide (FLG-NH2) according to the present invention was evaluated.

[0072] B16F1 cells were used, and DMEM (Dubelcco's Modified Eagle Medium, Gibco) containing 10% FBS and 100 UL S / P was used as the animal cell culture medium. 4 x 10⁶ cells were placed in each well of a 96-well plate. 3 Cells were placed in wells and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0073] Subsequently, a peptide composed of FLG according to the present invention (FLG-NH2) was added to the cultured medium plate (experimental group) and cultured for an additional 72 hours. 0.5 mg / ml MTT solution was added to all wells and reacted for 4 hours; after removing the supernatant, 100 µl of DMSO was added to dissolve all the formazan crystals. Cell viability was compared by measuring absorbance at 520 nm using a plate reader.

[0074] The above measurement results are shown in comparison to Table 1 below.

[0075] division Relative Viability (%) control group 100% experimental group 101.1%

[0076] As can be seen in Table 1 above, it can be confirmed that the peptide composed of amino acids of FLG according to the present invention is not cytotoxic.

[0078] <Example 3> Skin irritation test

[0079] A human patch test was performed to evaluate the degree of skin irritation. The human patch test was conducted on 30 normal individuals aged 20 years or older. The sample (FLG-NH2) was prepared as aqueous solutions at concentrations of 0.001%, 0.01%, 0.1%, 1%, and 10%, and each was applied to the inner upper arm using a Finn chamber for 48 hours. After removal, the first reading was taken, and a second reading was performed after 72 hours to determine the skin reaction results. As a result of the evaluation, the colostrum hydrolysis product of the example was assessed as having no skin irritation.

[0081] <Preparation Example> Preparation of a cosmetic (mask pack composition)

[0082] A composition for a mask pack was prepared by dispersing and dissolving the aqueous phase and the oil phase separately with the composition and content shown in Table 2 below, mixing, saponifying, and cooling to room temperature. A mask pack was prepared using this.

[0083] ingredient Content (weight%) stearic acid 6.5 myristinic acid 28.0 Self-emulsifying monostearic acid glycerin 3.0 FLG-NH2 12.0 Propylene glycol 5.0 Concentrated glycerin 10.0 Sodium hydroxide 7.0 Sodium ethylenediaminetetraacetate 0.1 Pulsatilla extract 0.5. spices 0.01 purified water Remaining amount

[0085] As specific parts of the content of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention.

[0086] Accordingly, the substantial scope of the present invention shall be defined by the appended claims and their equivalents. Simple variations or modifications of the present invention may be readily utilized by those skilled in the art, and all such variations or modifications shall be deemed to be included within the scope of the present invention.

Claims

Claim 1 delete Claim 2 delete Claim 3 A cosmetic composition for skin whitening comprising, as an active ingredient, a peptide composed of the amino acid sequence of FLG having melanin production inhibitory activity, wherein the concentration of the peptide composed of the amino acid sequence of FLG is 100 to 200 μg / ml. Claim 4 A pharmaceutical composition for the prevention or treatment of a melanin hyperpigmentation disease comprising, as an active ingredient, a peptide composed of the amino acid sequence of FLG having melanin production inhibitory activity, wherein the concentration of the peptide composed of the amino acid sequence of FLG is 100 to 200 μg / ml. Claim 5 A pharmaceutical composition for the prevention or treatment of a melanin hyperpigmentation disease according to claim 4, characterized in that the melanin hyperpigmentation disease is melasma, freckles, senile pigment spots, or solar lentigo. Claim 6 A health functional food composition for the prevention or improvement of melanin hyperpigmentation, comprising as an active ingredient a peptide composed of the amino acid sequence of FLG having melanin production inhibitory activity, wherein the concentration of the peptide composed of the amino acid sequence of FLG is 100 to 200 μg / ml. Claim 7 A health functional food composition for preventing or improving excessive melanin pigmentation, characterized in that, in claim 6, melasma, freckles, age spots, or solar lentigo are formed due to the excessive melanin pigmentation.

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