Peptides with anti-inflammatory activity and their uses

Abstract

The present invention relates to a novel peptide having anti-inflammatory activity and the use of said peptide for preventing, treating or improving inflammatory diseases by its inflammation-suppressing effect. Specifically, the novel peptide according to the present invention has an advantage that it can be usefully used as an active ingredient of a pharmaceutical composition for preventing or treating inflammatory diseases induced by or accompanied by an inflammatory response, or as an active ingredient of a health functional food or cosmetic composition for the effect of preventing or improving inflammatory diseases, by inhibiting the expression or secretion of genes or proteins that promote inflammatory responses.

Description

[Technical Field]

[0001] The present invention relates to novel peptides having anti-inflammatory activity and inflammation suppressing effects, and uses thereof. [Background technology]

[0002] Inflammation is a defense mechanism of living tissues that occurs when tissues or cells are damaged or injured, or when they are infected by external sources of infection (viruses, bacteria, fungi, allergens, etc.). It refers to a complex condition caused by immune cells involved in various immune responses that gather around the damaged or infected area and the inflammatory factors they secrete. It is a lesion that induces three types of symptoms: tissue degeneration, circulatory disorders and exudation, and tissue proliferation. In general, inflammation is initiated and maintained by the chemotaxis of neutrophils, a type of white blood cell. Major factors involved in the inflammatory response include histamine secreted by mast cells, interferon gamma (IFNγ) secreted by T cells and NK cells, various interleukins secreted by macrophages, nitric oxide (NO), and prostaglandins.

[0003] Inflammatory responses are essentially defense responses of the body, working to restore damaged tissue or remove external sources of infection, restoring damaged body functions. However, if external sources of infection are not completely removed or if persistent and excessive inflammatory responses occur due to internal substances, such abnormal inflammatory responses can lead to a variety of human diseases, including autoimmune diseases and cancer. Representative examples include acute inflammation originating in the joints, diseases such as rheumatoid arthritis, skin diseases such as psoriasis, and allergic inflammatory diseases such as bronchial asthma.

[0004] It is possible to devise a method for treating or ameliorating inflammatory diseases by interrupting the inflammatory response by suppressing the activity of cells involved in inducing or maintaining the inflammatory response, or by suppressing the production of inflammation-inducing substances, enzymes, etc. Cytokines that induce inflammatory diseases have been understood at the molecular level thanks to the development of molecular biology, and research into inflammation-related factors has been progressing, with efforts being made to develop therapeutic agents by attempting to suppress inflammation by inhibiting the expression or activity of the cytokines.

[0005] Currently known therapeutic agents for inflammatory diseases include dexamethasone and cortisone, which contain adrenal cortical hormone components. However, although these are active therapeutic agents, they have the drawback of being highly toxic and causing side effects such as edema. Furthermore, since they do not selectively act on the cause of inflammation, they have been reported to cause severe immunosuppression, which can lead to problems. As described above, the use of drugs containing steroid components to treat inflammatory diseases is accompanied by side effects and problems. Therefore, there is an urgent need to develop therapeutic agents for inflammatory diseases that use drugs containing non-steroid components and that have no side effects and can provide stable anti-inflammatory therapeutic effects. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent Publication No. 10-2020-0043476 [Patent Document 2] Korean Patent Publication No. 10-2017-0124472 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a peptide that has anti-inflammatory activity, suppresses the expression of inflammatory cytokines, and suppresses the expression of inflammatory marker proteins such as Cox2 and iNos, and can be used to suppress inflammation.

[0008] Another object of the present invention is to provide a pharmaceutical composition containing the above peptide as an active ingredient, which can prevent or treat inflammatory diseases.

[0009] Another object of the present invention is to provide a health functional food that contains the above peptide as an active ingredient and can prevent or improve inflammatory diseases.

[0010] Another object of the present invention is to provide a cosmetic composition that contains the above peptide as an active ingredient and is capable of preventing or ameliorating inflammatory diseases. [Means for solving the problem]

[0011] To achieve the above object, one aspect of the present invention provides a peptide having anti-inflammatory activity, which comprises the amino acid sequence of SEQ ID NO:1.

[0012] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0013] Another aspect of the present invention provides a health functional food for preventing or ameliorating inflammatory diseases, which contains a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0014] Another aspect of the present invention provides a cosmetic composition for preventing or ameliorating inflammatory diseases, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0015] The present invention will be described in detail below.

[0016] 1. Peptides with anti-inflammatory activity One aspect of the present invention provides novel peptides that have anti-inflammatory activity.

[0017] The peptide refers to a polymer consisting of two or more amino acids linked by peptide bonds. However, peptides have drawbacks in that they are too large to be effectively delivered to target tissues or cells, or have a short half-life and are eliminated in the body within a short period of time. Therefore, the peptide of the present invention has anti-inflammatory activity and consists of 20 or less, for example, 15 or less, or 10 or less amino acids.

[0018] The peptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 1, or may be an amino acid variant or fragment having a different sequence due to deletion, insertion, substitution, or a combination thereof of amino acid residues, provided that the anti-inflammatory activity is not affected. Amino acid replacement at the peptide level without overall alteration of the anti-inflammatory activity of the peptide is known in the art. In some cases, modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. may be performed. Therefore, the present invention includes peptides comprising substantially the same amino acid sequence as a peptide comprising the amino acid sequence of SEQ ID NO: 1, as well as variants or active fragments thereof. The term "substantially identical protein" refers to an amino acid sequence having at least 75% sequence identity, e.g., at least 80%, at least 90%, or at least 95%, respectively, with the amino acid sequence of SEQ ID NO: 1. The peptide may further comprise a targeting sequence, a tag, a labeled residue, or an amino acid sequence specifically engineered to increase half-life or peptide stability.

[0019] The peptides of the present invention can be obtained by various methods well known in the art, such as polynucleotide recombination and protein expression systems, in vitro synthesis through chemical synthesis such as peptide synthesis, and cell-free protein synthesis.

[0020] Furthermore, a protecting group may be attached to the N- or C-terminus of the peptide to achieve better chemical stability, enhanced pharmacological properties (e.g., half-life, absorption, potency, efficacy), altered specificity (e.g., a broader spectrum of biological activity), or reduced antigenicity. For example, the protecting group may be an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or a polyethylene glycol (PEG). However, any component that can enhance peptide modification, particularly peptide stability, may be included without limitation. The term "stability" refers not only to in vivo stability, which protects the peptide of the present invention from attack by in vivo protease enzymes, but also to storage stability (e.g., storage stability at room temperature).

[0021] The anti-inflammatory activity of the present invention refers to the inhibition of inflammation, a type of defense response of biological tissues against a certain stimulus, and refers to a pathological state of abscess formed when tissues or cells are damaged or infected with various infectious agents, such as bacteria, fungi, viruses, and allergens, from the outside. The inflammatory response is a complex physiological response that occurs when inflammatory mediators and immune cells interact with local blood vessels and body fluids, resulting in enzyme activation, secretion of inflammatory mediators, fluid infiltration, cell migration, and tissue destruction, as well as external symptoms such as erythema, edema, fever, and pain. Therefore, the peptide of the present invention has the activity of inhibiting inflammation, and is therefore effective in reducing and ameliorating a series of pathological conditions and symptoms.

[0022] Furthermore, the peptide of the present invention can suppress the expression of inflammatory cytokines. When an inflammatory response occurs, such as when a wound is created or an external infectious agent penetrates the wound site and enters the body, leukocytes responsible for the initial immune response gather around the wound site or the infectious agent, and express and secrete inflammation-related cytokines, inducing an inflammatory response. Therefore, by suppressing the expression of inflammatory cytokines, the peptide of the present invention can exhibit anti-inflammatory activity. Furthermore, the anti-inflammatory activity and inflammation-suppressing effects of the peptide of the present invention can be confirmed by checking the expression level of the inflammatory cytokines.

[0023] The pro-inflammatory cytokine may be one or more selected from the group consisting of TNFα, IL-6, IL-17, IL-1β, and IFNγ. TNFα, an abbreviation for "tumor necrosis factor α," is a cytokine produced and secreted by macrophages and various cells activated during immune responses to bacterial infections and tumor diseases. It is known as a major mediator of inflammatory responses and plays an important role in inflammatory diseases such as rheumatoid arthritis (RA), psoriatic arthritis, Crohn's disease, psoriasis, and ankylosing spondylitis (AS). IL-6 (interleukin 6) is a cytokine produced by macrophages and various lymphocytes, and is known to promote inflammatory responses and, if produced in excess, to induce inflammatory diseases. IL-17 (interleukin 17) is also a pro-inflammatory cytokine, produced by Th17 cells and responsible for inducing or mediating inflammatory responses. IFNγ (interferon γ) can be produced by T lymphocytes and macrophages, is secreted in response to external viral or bacterial infection, and is known to play a role in autoimmune or autoinflammatory diseases. Therefore, the peptide of the present invention has the effect of suppressing inflammation through its activity of suppressing the expression of such inflammatory cytokines and suppressing the secretion of the expressed cytokines.

[0024] The peptides of the present invention can suppress the expression of Cox2. Cox2 (cyclooxygenase 2) is an enzyme involved in stimulating the process of prostaglandin biosynthesis, and its expression can be regulated by NF-κB, regulating inflammatory responses. Cox2 is a protein that is barely expressed under normal conditions but is rapidly expressed in response to stimuli such as cytokines, inflammatory factors, and endotoxins. Therefore, the anti-inflammatory activity of the peptides of the present invention can be confirmed by measuring the expression level of the Cox2 gene and the amount of Cox2 protein. The peptides of the present invention have the effect of suppressing inflammation by suppressing Cox2 expression.

[0025] Furthermore, the peptides of the present invention can inhibit the expression of iNos. iNos (inducible nitric oxide synthase) is an enzyme that catalyzes the production of nitric oxide (NO), and in particular, the secreted form of iNos is known to be involved in immune responses. Excessive production of nitric oxide by iNos in immune cells can induce cell damage, and the expression of iNos can be induced by stimulation of macrophages with LPS. Since iNos, like Cox2, is a protein involved in regulating inflammatory responses, the anti-inflammatory activity of the peptides of the present invention can be confirmed by measuring the expression level and protein amount of iNos. The peptides of the present invention can suppress and improve inflammatory responses by inhibiting iNos expression.

[0026] In order to confirm the anti-inflammatory effect of the peptide of the present invention, in a specific embodiment of the present invention, human keratinocytes (HaCaT) were treated with the inflammatory cytokine TNFα together with a peptide comprising the amino acid sequence of SEQ ID NO: 1, or with TGFβ, IL-23 (a Th17 type inflammatory cytokine), and the inflammatory antigen LPS, and the amounts of Cox2 and iNos proteins were measured. As a result, it was confirmed that although the inflammatory response of the cells was induced by the inflammatory factors treated, the amounts of Cox2 and iNos proteins were reduced by treatment with the peptide of the present invention (see Figures 1 and 2).

[0027] Furthermore, to confirm the effect of reducing the expression levels of inflammation-related genes by treatment with the peptide of the present invention, in a specific example of the present invention, mouse splenocytes were treated with LPS, an inflammation-inducing antigen, or TNFα, an inflammation-inducing cytokine, together with a peptide comprising the amino acid sequence of SEQ ID NO: 1, and the mRNA levels of TNFα, IL-6, Cox2, and IL-1β genes were examined. As a result, it was confirmed that, although the inflammatory response of the cells was induced by the treated inflammation-inducing factor, the expression levels of the inflammation-inducing cytokines and related enzyme genes were reduced by treatment with the peptide of the present invention (see Figures 3 and 4).

[0028] Furthermore, to confirm the effect of treatment with the peptide of the present invention on reducing the secretion of inflammation-related cytokines, in a specific embodiment of the present invention, mouse splenocytes were treated with an inflammation-inducing antigen, LPS, together with a peptide comprising the amino acid sequence of SEQ ID NO: 1, or with inflammation-inducing cytokines, TNFα, TGFβ, and IL-23, and the secretion levels of IL-17, IL-1β, and IFNγ were measured using ELISA. As a result, it was confirmed that, although the inflammatory response of the cells was induced by the treated inflammation-inducing factors, the secretion of such pro-inflammatory cytokines was reduced by treatment with the peptide of the present invention (see Figures 5 to 7).

[0029] Therefore, it is clear that the peptide of the present invention has anti-inflammatory activity that can reduce and ameliorate inflammatory responses by reducing the expression and secretion of inflammatory cytokines such as TNFα, IL-6, IL-17, IL-1β, and IFNγ, which can promote inflammatory responses, and by suppressing the expression of inflammation-related factors such as Cox2 and iNos.Therefore, the peptide of the present invention may be useful as an active ingredient of a composition for preventing, treating, or ameliorating inflammatory diseases induced by inflammation or accompanied by inflammatory responses.

[0030] 2. Pharmaceutical composition for preventing or treating inflammatory diseases Another aspect of the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0031] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the above section "1. Peptides with anti-inflammatory activity." Therefore, the specific description will be referred to the above section "1. Peptides with anti-inflammatory activity." Hereinafter, only the components specific to the pharmaceutical composition for the prevention or treatment of inflammatory diseases will be described.

[0032] The peptide of the present invention has the effect of inhibiting the expression or secretion of inflammation-related cytokines and inflammation-related factors, and therefore, a pharmaceutical composition containing the peptide as an active ingredient can suppress the expression of TNFα, IL-6, IL-17, IL-1β, IFNγ, Cox2, and iNos, and can therefore be used to prevent or treat inflammatory diseases.

[0033] The inflammatory disease refers to a pathological condition that causes inflammation induced by neutrophil chemotaxis among leukocytes, and may include any disease caused by or accompanied by an inflammatory response. For example, the inflammatory disease may include rhinitis, bronchitis, periodontitis, pancreatitis, gastritis, gastric ulcer, inflammatory skin disease, atopic dermatitis, encephilitis, sepsis, inflammatory bowel disease, chronic obstructive pulmonary disease, pulmonary hemoptysis, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic inflammatory diseases caused by chronic viral or bacterial infection, colitis, inflammatory bowel disease, type 1 diabetes, rheumatoid arthritis, reactive arthritis, and the like. The disease may be, but is not limited to, inflammatory bowel disease (IGN), osteoarthritis, psoriasis, scleroderma, osteoporosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, borreliosis, neuro-borreliosis, tuberculosis, sarcoidosis, lupus, discoid lupus, chilblain lupus, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Crohn's disease, Sjogren's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immune deficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, or multiple sclerosis.

[0034] The pharmaceutical composition of the present invention may be used to prevent the onset of inflammatory diseases by suppressing the expression or secretion of factors that induce inflammatory responses, or to treat diseases by suppressing further inflammatory responses that occur in damaged or injured cells in patients with the diseases and thereby inhibiting the progression of the disease.

[0035] Meanwhile, pharmaceutical compositions containing the peptides of the present invention as active ingredients may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients according to a method easily performed by a person skilled in the art to which the present invention pertains. In this case, the dosage form may be a solution, suspension, or emulsion in an oil or aqueous medium, or an extract, powder, granules, tablets, capsules, or gel (e.g., hydrogel), and may further contain a dispersant or stabilizer.

[0036] The peptides contained in the pharmaceutical compositions may be delivered in pharmaceutically acceptable carriers such as colloidal suspensions, powders, saline solutions, lipids, liposomes, microspheres, or nanospheres, which may be complexed or associated with delivery vehicles and delivered in vivo using delivery systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers, or fatty acids.

[0037] Other pharmaceutically acceptable carriers may include, but are not limited to, commonly used ingredients in pharmaceutical formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the formulation may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0038] The pharmaceutical compositions of the present invention can be administered orally or parenterally during clinical administration and may be used in the form of common pharmaceutical formulations. That is, the pharmaceutical compositions of the present invention can be administered in various oral and parenteral dosage forms during clinical administration. When formulated, they are formulated using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid formulations are formulated by mixing herbal extracts or fermented herbal products with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, glycerol, gelatin, and the like.

[0039] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level may be determined based on factors including the type and severity of the patient's disease, the activity of the drug, its sensitivity, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents for inflammatory diseases. The pharmaceutical composition may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum efficacy with the minimum amount without side effects, which can be easily determined by one skilled in the art.

[0040] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, and weight, the degree of absorption of the active ingredient into the body, the inactivation rate, the excretion rate, the type of disease, and concomitant drugs, and may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc. For example, the peptide of the present invention may be administered in an amount of about 0.0001 μg to 500 mg, e.g., 0.01 μg to 100 mg, per kg of patient body weight per day. Furthermore, the peptide may be administered in divided doses several times a day at regular intervals, for example, two to three times a day, at the discretion of a physician or pharmacist.

[0041] 3. Health functional foods for preventing or improving inflammatory diseases Another aspect of the present invention provides a health functional food for preventing or ameliorating inflammatory diseases, which contains a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0042] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the above section "1. Peptides with anti-inflammatory activity," so for specific explanations, refer to the above section "1. Peptides with anti-inflammatory activity," and below, only the unique composition of the health functional food for preventing or improving inflammatory diseases will be described.

[0043] Similar to the pharmaceutical composition, inflammatory diseases can be prevented or ameliorated by suppressing the inflammatory response, and functional health foods containing as an active ingredient the peptide of the present invention, which suppresses inflammation by inhibiting the expression or secretion of inflammatory cytokines (e.g., TNFα, IL-6, IL-17, IL-1β, IFNγ, etc.) and inflammatory response-related proteins (e.g., Cox2, iNos, etc.), can be useful for preventing or ameliorating inflammatory diseases.

[0044] The health functional food may be used before or after the onset of a disease to prevent or improve the disease, either simultaneously with or separately from a therapeutic drug.

[0045] In the health functional food of the present invention, the active ingredient may be added directly to the food or may be used together with other foods or food ingredients, and may be used appropriately by conventional methods. The amount of the active ingredient to be mixed may be determined appropriately depending on the intended use (prevention or improvement). Generally, when producing a food or beverage, the composition of the present invention may be added in an amount of preferably 15% by weight or less, and more preferably 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purposes of health and hygiene or health regulation, the amount may be less than the above range.

[0046] In addition to the active ingredient, the health functional food of the present invention may contain other essential ingredients without any particular limitation. For example, various flavorings or natural carbohydrates, like common beverages, may be included as additional ingredients. Examples of the natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Other flavorings that can be advantageously used include natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.). The proportion of the natural carbohydrates may be appropriately determined by the skilled artisan.

[0047] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0048] 4. Cosmetic composition for preventing or improving inflammatory diseases Another aspect of the present invention provides a cosmetic composition for preventing or ameliorating inflammatory diseases, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0049] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the above section "1. Peptides with anti-inflammatory activity," so for specific explanations, refer to the above section "1. Peptides with anti-inflammatory activity," and below, only the unique components of the cosmetic composition for preventing or improving inflammatory diseases will be described.

[0050] Similar to the pharmaceutical composition, inflammatory diseases can be prevented or ameliorated by suppressing the inflammatory response. A cosmetic composition containing as an active ingredient the peptide of the present invention, which suppresses inflammation by inhibiting the expression or secretion of inflammatory cytokines (e.g., TNFα, IL-6, IL-17, IL-1β, IFNγ, etc.) or inflammatory response-related proteins (e.g., Cox2, iNos, etc.), may be useful for preventing or ameliorating inflammatory diseases. In particular, the cosmetic composition of the present invention may be useful for preventing or ameliorating inflammatory diseases that occur in the skin, such as atopic dermatitis.

[0051] The peptide may be contained in an amount of 0.001 to 30% by weight, for example, 0.1 to 20% by weight, 0.1 to 10% by weight, 1 to 10% by weight, or 2 to 5% by weight, out of a total of 100% by weight of the cosmetic composition, but is not limited thereto.

[0052] Cosmetic compositions containing the peptide of the present invention as an active ingredient may further contain other ingredients that have a synergistic effect on the activity of the peptide, provided that the ingredients do not affect the anti-inflammatory activity of the peptide. For example, adjuvants commonly used in the cosmetic and dermatological fields, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, fragrances, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in cosmetics, may be included in amounts commonly used in the cosmetic and dermatological fields.

[0053] The cosmetic composition of the present invention may be prepared in any dosage form commonly prepared in the art, for example, a solution, a suspension, an emulsion, a gel, a lotion, an essence, a cream, a powder, a soap, a shampoo, conditionerThe cosmetic composition may be formulated into a cosmetic product such as a face mask, a surfactant-containing cleanser, a cleansing foam, a cleansing water, an oil, a liquid foundation, a cream foundation, or a spray.

[0054] When the dosage form is a solution or emulsion, the carrier component may be a solvent, solubilizer, or emulsifier, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, or sorbitan fatty acid esters.When the dosage form is a suspension, the carrier component may be a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters, and polyoxyethylene sorbitan esters, aluminum metahydroxide, microcrystalline cellulose, bentonite, agar, or tragacanth.When the dosage form is a cream or gel, the carrier component may be wax, paraffin, tragacanth, animal oil, starch, cellulose derivatives, silicone, bentonite, polyethylene glycol, silica, zinc oxide, or talc. When the formulation is a powder or spray, the carrier component may include a propellant such as silica, talc, aluminum hydroxyl group, lactose, calcium silicate, chlorofluorohydrocarbon, propane / butane, or dimethyl ether.When the formulation is a surfactant-containing cleanser, the carrier component may include fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, imidazolinium derivative, isethionate, methyl taurine, sarcosinate, fatty acid amide ether sulfate, fatty alcohol, alkylamidobetaine, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

[0055] In another aspect of the present invention, there is provided a method for treating an inflammatory disease, comprising administering a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject in need of such treatment.

[0056] In another aspect of the present invention, there is provided a method for ameliorating an inflammatory disease, comprising administering a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject in need of amelioration of the inflammatory disease.

[0057] In another aspect of the present invention, there is provided a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the treatment of an inflammatory disease.

[0058] In another aspect of the present invention, there is provided the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the manufacture of a medicament for treating or preventing an inflammatory disease. [Effects of the Invention]

[0059] The peptides provided by the present invention have the effect of suppressing inflammatory responses, thereby suppressing the expression and secretion of inflammatory cytokines such as TNFα, IL-6, IL-17, IL-1β, and IFNγ, and suppressing the expression of inflammation-related proteins such as Cox2 and iNos. Therefore, the peptides can be usefully used as an active ingredient in pharmaceutical compositions for preventing or treating inflammatory diseases accompanied by inflammatory responses or caused by inflammation, and can also be usefully used as an active ingredient in health functional foods or cosmetic compositions for preventing or ameliorating the inflammatory diseases.

[0060] However, the effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. [Brief explanation of the drawings]

[0061] [Figure 1]This figure shows the results of Western blotting to measure the amount of Cox2 protein in HaCaT cells treated with TNFα to induce an inflammatory response and then treated with 5 or 50 μM of the peptide of the present invention. NON indicates the results for the group not treated with TNFα, and NC indicates the results for the group treated with TNFα but not the peptide of the present invention. [Figure 2] 1 shows the results of Western blotting in which HaCaT cells were treated with LPS, TGFβ, and IL-23 to induce an inflammatory response, and then treated with 5 or 50 μM of the peptide of the present invention to measure the levels of iNos and Cox2 proteins. NON indicates the results for a group not treated with LPS, TGFβ, or IL-23, and NC indicates the results for a group treated with LPS, TGFβ, and IL-23 but not the peptide of the present invention. [Figure 3] 1 shows the results of RT-PCR in which mouse splenocytes were treated with LPS to induce an inflammatory response, and then treated with 5 or 50 μM of the peptide of the present invention to measure the mRNA levels of TNFα and IL-6 genes. NON means the result of a group not treated with LPS, and NC means the result of a group treated with LPS but not with the peptide of the present invention. [Figure 4] 1 shows the results of RT-PCR in which mouse splenocytes were treated with TNFα to induce an inflammatory response, and then treated with 5 or 50 μM of the peptide of the present invention to measure the mRNA levels of TNFα, Cox2, and IL-1β genes. NON means the result of a group not treated with TNFα, and NC means the result of a group treated with TNFα but not the peptide of the present invention. [Figure 5] 1 is a graph showing the results of ELISA in which mouse splenocytes were treated with LPS to induce an inflammatory response, and then treated with 5 or 50 μM of the peptide of the present invention to measure the secretion levels of TNFα, IL-1β, and IFNγ. NON means the result of a group not treated with LPS, and NC means the result of a group treated with LPS but not with the peptide of the present invention. [Figure 6]1 is a graph showing the results of ELISA in which mouse splenocytes were treated with TNFα to induce an inflammatory response, and then treated with 5 or 50 μM of the peptide of the present invention to measure the secretion levels of IL-17, IL-1β, and IFNγ. NON represents the results for a group not treated with TNFα, and NC represents the results for a group treated with TNFα but not with the peptide of the present invention. [Figure 7] 1 is a graph showing the results of ELISA in which mouse splenocytes were treated with LPS, TGFβ, and IL-23 to induce an inflammatory response, and then treated with 5 or 50 μM of the peptide of the present invention to measure the secretion levels of IL-17, IL-1β, and IFNγ. NON represents the results for a group not treated with LPS, TGFβ, or IL-23, and NC represents the results for a group treated with LPS, TGFβ, and IL-23 but not the peptide of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0063] Example [Production example] Peptide production The peptides having the amino acid sequence of SEQ ID NO: 1 listed in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were purified and separated using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA) on an ACQUITY UPLC BEH300 C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Co., USA).

[0064] [Table 1]

[0065] [Experimental Example 1] Confirmation of decrease in Cox2 protein expression level by peptide treatment To confirm the anti-inflammatory effect of treatment with the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1, inflammation was induced in human keratinocytes (HaCaT cells) and the expression level of Cox2 protein was measured. To induce inflammation, the inflammatory cytokine TNFα was treated, and Cox2 is an enzyme involved in the synthesis of prostaglandins and corresponds to an inflammation-related protein. Therefore, by measuring the expression level of Cox2, it was possible to confirm whether the inflammatory response was suppressed.

[0066] HaCaT cells were plated in a 24-well plate at 3 × 10 5 The cells were seeded at 1000 cells / well and cultured until the next day. After 4 hours in serum-free medium, cells were treated with TNFα and the peptides of the present invention and cultured for another 24 hours. Inflammatory responses were induced by treatment with 20 nM TNFα, and the peptides were treated at 5 and 50 μM. HaCaT cells were incubated as described above for a group without TNFα treatment (NON), a negative control group (NC) treated with TNFα but no peptide, and a group treated with the peptides of the present invention at the above concentrations. Lysates were collected after incubation as described above. Western blotting was performed to measure Cox2 protein levels using an anti-Cox2 antibody (Cell Signaling Technology, USA). To compare Cox2 protein levels, β-actin levels were also measured.

[0067] As a result, it was confirmed that when an inflammatory response was induced by treatment with TNFα, the expression level of Cox2 protein increased, and was more than two-fold higher than that of a group not treated with TNFα. In contrast, when treated with the peptide of the present invention, the level of Cox2 protein did not increase despite the treatment with TNFα, and in fact, the expression level of Cox2 decreased compared to a group not treated with TNFα (see Figure 1).

[0068] [Experimental Example 2] Confirmation of decrease in iNOS / Cox2 protein expression level by peptide treatment To further confirm the anti-inflammatory effect of treatment with the peptide of the present invention in addition to the results of Experimental Example 1, HaCaT cells were treated with Th17 (type 17) inflammation inducers and the expression levels of inflammation induction markers iNos and Cox2 were measured. The inflammation inducers were LPS, TGFβ, and IL-23. iNos is an enzyme involved in the production of nitric oxide (NO) and is a protein related to the inflammatory response induced by LPS, etc. Therefore, the suppression of the inflammatory response can be confirmed by measuring the expression levels of iNos and Cox2.

[0069] HaCaT cells were plated in a 24-well plate at 3 × 10 5 The cells were seeded at 1000 cells / well and cultured until the next day. After 4 hours in serum-free medium, the cells were treated with Th17-type inflammation-inducing cytokines TGFβ, IL-23, and LPS. Then, the peptides of the present invention were added and cultured for another 24 hours. Inflammatory responses were induced by treatment with 20 ng / ml TGFβ, 20 ng / ml IL-23, and 2 μg / ml LPS, and the peptides were treated at 5 and 50 μM, respectively. HaCaT cells were incubated as described above and collected, and lysates were collected. Western blotting was performed using anti-iNos and anti-Cox2 antibodies (Cell Signaling Technology, USA) to measure the levels of iNos and Cox2 proteins. To compare the amounts of iNos and Cox2 proteins, the amounts of β-actin were also confirmed.

[0070] As a result, when an inflammatory response was induced by treatment with TGFβ, IL-23, and LPS, the expression levels of iNos and Cox2 proteins all increased, resulting in darker bands, compared to a group not treated with TGFβ, IL-23, or LPS. In contrast, when treated with the peptide of the present invention, the levels of iNos and Cox2 proteins did not increase despite treatment with TGFβ, IL-23, and LPS, and the expression levels were reduced compared to a group not treated with the peptide (see Figure 2).

[0071] [Experimental Example 3] Confirmation of reduction in expression of pro-inflammatory cytokine genes by peptide treatment <3-1> Confirmation of inflammatory cytokine mRNA expression levels in cells induced by LPS inflammation Inflammation was induced in mouse splenocytes, and the expression levels of the inflammatory cytokines TNFα and IL-6 genes were measured. To induce inflammation, the cells were treated with LPS, an inflammation-inducing antigen. TNFα is a signaling protein involved in the inflammatory response, and IL-6 is a cytokine that promotes the inflammatory response. Therefore, the level of the inflammatory response can be confirmed by measuring the expression levels of TNFα and IL-6 genes.

[0072] Mouse splenocytes were plated in a 24-well plate at 1 × 10 7Cells were seeded at a concentration of 10 ... To compare the amount of mRNA, the amount of GADPH mRNA was also confirmed.

[0073] [Table 2]

[0074] As a result, it was confirmed that when an inflammatory response was induced by treatment with LPS, the levels of TNFα and IL-6 mRNA increased compared to the group without LPS treatment. In contrast, when treated with the peptide of the present invention, the levels of TNFα and IL-6 mRNA did not increase despite the LPS treatment, and the expression levels were reduced compared to the group without peptide treatment (see Figure 3).

[0075] <3-2> Confirmation of inflammatory cytokine mRNA expression levels in cells induced by TNFα In addition to the results of Experimental Example <3-1>, to further confirm the anti-inflammatory effect of treatment with the peptide of the present invention, mouse splenocytes were treated with TNFα to induce inflammation, and the expression levels of inflammatory cytokines TNFα and IL-1β, and the inflammation-inducing marker Cox2 gene were measured. The cytokine TNFα was treated to induce inflammation, and TNFα is a signaling protein involved in inflammatory responses, IL-1β is a cytokine that promotes inflammatory responses, and Cox2 is an inflammation-related protein. Therefore, the level of inflammatory responses can be confirmed by measuring the expression levels of TNFα, Cox2, and IL-1β genes.

[0076] Mouse splenocytes were plated in a 24-well plate at 1 × 10 7 Only cells / well were cultured and cultured until the next day. After changing to serum-free medium, 3 hours later TNF-alpha The cells were treated with the peptide of the present invention and then cultured for another 3 hours. An inflammatory response was induced by treatment with 20 nM TNFα, and the peptides were treated at 5 and 50 μM, respectively. Mouse splenocytes from a group without TNFα treatment (NON), a negative control group (NC) treated with TNFα but no peptide, and a group treated with the peptide of the present invention at the above concentrations together with TNFα were incubated as described above, harvested, and RNA was isolated. The amount of isolated RNA was quantified, and cDNA was synthesized using a cDNA synthesis kit (Intron, Korea). PCR was performed using PCR premix (Intron, Korea) and primers for TNFα, Cox2, and IL-1β. The primer sequences for TNFα were listed in Table 2, and the primer sequences for Cox2 and IL-1β were listed in Table 3. After PCR, each sample was loaded onto a 5% agarose gel to measure mRNA expression levels. To compare the amount of mRNA, the amount of GADPH mRNA was also confirmed.

[0077] [Table 3]

[0078] As a result, it was confirmed that when an inflammatory response was induced by treatment with TNFα, the levels of TNFα, Cox2, and IL-1β mRNA increased compared to a group that was not treated with TNFα. In contrast, when treated with the peptide of the present invention, the levels of TNFα, Cox2, and IL-1β mRNA did not increase despite the treatment with TNFα, and the expression levels were reduced compared to a group that was not treated with the peptide (see Figure 4).

[0079] [Experimental Example 4] Confirmation of reduction in secretion of pro-inflammatory cytokines by peptide treatment <4-1> Confirmation of the amount of inflammatory cytokines secreted in cells induced by LPS inflammation Inflammation was induced in mouse splenocytes, and the secretion levels of the inflammatory cytokines TNFα, IL-1β, and IFNγ were measured. To induce inflammation, the splenocytes were treated with the inflammation-inducing antigen LPS. TNFα is a signaling protein involved in the inflammatory response, IL-1β is a cytokine that promotes the inflammatory response, and IFNγ is also a cytokine involved in the inflammatory response. Therefore, the level of the inflammatory response can be determined by measuring the secretion levels of TNFα, IL-1β, and IFNγ.

[0080] Mouse splenocytes were plated in a 24-well plate at 1 × 10 7Cells were seeded at a rate of 1000 cells / well and cultured until the next day. After 4 hours in serum-free medium, the cells were treated with LPS and the peptides of the present invention and cultured for an additional 24 hours. An inflammatory response was induced by treatment with 2 μg / ml LPS, and the peptides were treated at 5 and 50 μM, respectively. Mouse splenocytes from a group without LPS treatment (NON), a negative control group (NC) treated with LPS but no peptide, and groups treated with the peptides of the present invention at the above concentrations along with LPS were incubated as described above, and the cell culture medium was then collected. The secretion levels of TNFα, IL-1β, and IFNγ were determined using ELISA techniques with the TNF-alpha Quantikine ELISA Kit (R&D Systems, USA), IL-1 beta Quantikine ELISA Kit (R&D Systems, USA), and IFN-gamma Quantikine ELISA Kit (R&D Systems, USA).

[0081] As a result, it was confirmed that when an inflammatory response was induced by treatment with LPS, the secretion levels of TNFα, IL-1β, and IFNγ were significantly increased compared to the group without LPS treatment, whereas when treated with the peptide of the present invention, the secretion levels of TNFα, IL-1β, and IFNγ were reduced despite the presence of LPS treatment (see Figure 5).

[0082] <4-2> Confirmation of the amount of inflammatory cytokines secreted from cells induced by TNFα Mouse splenocytes were treated with the inflammatory cytokine TNFα to induce inflammation, and the secretion levels of the inflammatory cytokines IL-17, IL-1β, and IFNγ were measured. IL-17 is a pro-inflammatory cytokine, and IL-1β and IFNγ are as described above.

[0083] Mouse splenocytes were plated in a 24-well plate at 1 × 10 7The cells / well were seeded and cultured until the next day. After 4 hours in serum-free medium, the cells were treated with TNFα and the peptides of the present invention and cultured for an additional 24 hours. An inflammatory response was induced by treatment with 20 nM TNFα, and the peptides were treated at 5 and 50 μM, respectively. Mouse splenocytes from a group without TNFα treatment (NON), a negative control group (NC) treated with TNFα but no peptide, and groups treated with the peptides of the present invention at the above concentrations together with TNFα were incubated as described above, and the cell culture medium was then collected. IL-17, IL-1β, and IFNγ secretion levels were determined using ELISA techniques with the IL-17 Quantikine ELISA Kit (R&D Systems, USA), IL-1 beta Quantikine ELISA Kit (R&D Systems, USA), and IFN-gamma Quantikine ELISA Kit (R&D Systems, USA).

[0084] As a result, it was confirmed that when an inflammatory response was induced by treatment with TNFα, the secretion levels of IL-17, IL-1β, and IFNγ were significantly increased compared to the group not treated with TNFα. Treatment with 5 μM of the peptide of the present invention slightly increased the secretion level of IL-17, but decreased the secretion levels of IL-1β and IFNγ. When the amount of peptide was increased to 50 μM, the secretion levels of IL-17, IL-1β, and IFNγ all decreased, confirming the effect of the peptide of the present invention on the reduction of the secretion levels of inflammation-inducing markers depending on the concentration (see FIG. 6).

[0085] <4-3> Confirmation of the amount of inflammatory cytokines secreted from cells induced by LPS, TGFβ, and IL-23 Mouse splenocytes were treated with the inflammatory cytokines TGFβ and IL-23 to induce inflammation, and the secretion levels of IL-17, IL-1β, and IFNγ were measured. IL-17, IL-1β, and IFNγ are described above.

[0086] Mouse splenocytes were plated in a 24-well plate at 1 × 10 7 The cells / well were seeded and cultured until the next day. After changing to serum-free medium, 4 hours later, LPS, TGFβ, IL-23, and the peptides of the present invention were treated and cultured again for an additional 24 hours. Inflammatory responses were induced by treatment with 2 μg / ml LPS, 20 ng / ml TGFβ, and 20 ng / ml IL-23, and peptides were treated at 5 and 50 μM, respectively. Mouse splenocytes from a group (NON) in which no inflammatory responses were induced because they were not treated with LPS, TGFβ, or IL-23; a negative control group (NC) treated with LPS, TGFβ, and IL-23 but no peptide; and groups treated with the peptides of the present invention at the aforementioned concentrations together with LPS, TGFβ, and IL-23 were incubated as described above, and the cell culture medium was then collected. The secretion levels of IL-17, IL-1β, and IFNγ were determined using the collected cell culture medium via ELISA techniques using an IL-17 Quantikine ELISA Kit (R&D system, USA), an IL-1 beta Quantikine ELISA Kit (R&D system, USA), and an IFN-gamma Quantikine ELISA Kit (R&D system, USA).

[0087] As a result, it was confirmed that when an inflammatory response was induced by treatment with LPS, TGFβ, or IL-23, the secretion levels of IL-17, IL-1β, and IFNγ were significantly increased compared to a group not treated with LPS, TGFβ, or IL-23. Treatment with 5 μM of the peptide of the present invention slightly increased the secretion level of IL-17, but decreased the secretion levels of IL-1β and IFNγ. When the amount of peptide was increased to 50 μM, the secretion levels of IL-17, IL-1β, and IFNγ all decreased, confirming the effect of the peptide of the present invention on the concentration-dependent reduction in the secretion of inflammation-inducing markers (see FIG. 7).

[0088] Considering the results of the above experimental examples, it was confirmed that even in cells in which an inflammatory response was induced, treatment with the peptide of the present invention reduced the amount of proteins and gene expression involved in the inflammatory response, and reduced the expression and secretion of cytokines that promote the inflammatory response, thereby demonstrating an inhibitory effect on the inflammatory response. Furthermore, when a larger amount of the peptide of the present invention was treated, the above-mentioned effect was more pronounced, and therefore it can be seen that the inhibitory effect on the inflammatory response observed from the results of the above experimental examples is due to the peptide of the present invention.

[0089] Although the present invention has been described in detail above only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the technical concept of the present invention, and it is natural that such modifications and variations fall within the scope of the claims.

Claims

1. A peptide having anti-inflammatory activity, consisting of the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, which suppresses the expression of inflammatory cytokines.

3. The peptide according to claim 2, wherein the inflammatory cytokine is one or more selected from the group consisting of TNFα, IL-6, IL-17, IL-1β, and IFNγ.

4. The peptide according to claim 1, which inhibits the expression of Cox2.

5. The peptide according to claim 1, which inhibits the expression of iNos.

6. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising the peptide of claim 1 as an active ingredient.

7. The inflammatory diseases include rhinitis, bronchitis, periodontitis, pancreatitis, gastritis, gastric ulcer, inflammatory skin diseases, atopic dermatitis, encephilitis, sepsis, inflammatory bowel disease, chronic obstructive pulmonary disease, pulmonary shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathies, arthritis, inflammatory osteolysis, chronic inflammatory diseases caused by chronic viral or bacterial infections, colitis, inflammatory bowel disease, type 1 diabetes, rheumatoid arthritis, reactive arthritis, and the like.

7. The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 6, wherein the inflammatory disease is one or more selected from the group consisting of inflammatory bowel syndrome, Crohn's disease, Sjogren's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immune deficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis.

8. A functional health food for preventing or improving inflammatory diseases, comprising the peptide according to claim 1 as an active ingredient.

9. A cosmetic composition for preventing or ameliorating inflammatory diseases, comprising the peptide according to claim 1 as an active ingredient.

10. 10. The cosmetic composition for preventing or ameliorating an inflammatory disease according to claim 9, wherein the cosmetic composition has one dosage form selected from the group consisting of a solution, a suspension, an emulsion, a gel, a lotion, an essence, a cream, a powder, a soap, a shampoo, a conditioner, a pack mask, a surfactant-containing cleanser, a cleansing foam, a cleansing water, an oil, a liquid foundation, a cream foundation, and a spray.

Images