Physiologically active peptides and their uses

Abstract

The present invention relates to novel peptides having various physiological activities and their uses. The novel peptides according to the present invention can not only induce an increase in joint components in chondrocytes and increase chondrocyte differentiation and extracellular matrix production in mesenchymal stem cells, but also inhibit an increase in inflammatory cytokines and inflammatory proteins, and inhibit the differentiation and formation of osteoclasts, and therefore can be usefully used as an active ingredient in pharmaceutical compositions for regenerating cartilage or preventing or treating inflammatory diseases or osteoporosis, or in health functional foods for improving the above symptoms.

Description

[Technical Field]

[0001] The present invention relates to peptides having various physiological activities and uses thereof. [Background technology]

[0002] Typically, cartilage tissue that forms the joints of vertebrates lacks blood vessels, nerves, and lymphatic tissue, and therefore cannot be regenerated normally in vivo once damaged. Damage to such cartilage tissue in joints can result in severe pain and limitations on daily activities. If the damage becomes chronic, it can lead to fatal degenerative arthritis, hindering normal daily and professional activities. Therefore, the ability of damaged articular cartilage to recover and regenerate on its own is extremely limited. While various medical treatments have been attempted, it remains difficult to restore hyaline cartilage, the articular cartilage, to its natural state.

[0003] Clinically used surgical methods for cartilage regeneration include inducing the differentiation of stem cells into chondrocytes, osteochondral transplantation, in which autologous or allogeneic cartilage tissue is transplanted into the cartilage defect site, microfracture, in which damaged cartilage is thoroughly scraped away to expose the subchondral bone, and holes are drilled at regular intervals through which bone marrow cells leak out and regenerate into cartilage tissue, chondrocyte transplantation, in which chondrocytes are transplanted into the cartilage defect site to induce cartilage regeneration, and autologous chondrocyte transplantation to treat damaged articular cartilage.

[0004] However, this method requires separate surgeries, one for harvesting chondrocytes and the other for transplanting the ex vivo cultured chondrocytes back into the damaged area of ​​articular cartilage. Consequently, the two surgeries result in significant pain, aftereffects, and economic burden for the patient, and the surgical procedure is complicated. Furthermore, the harvested chondrocytes are mostly obtained from fully grown adults, and since the harvested cells do not proliferate or grow vigorously, it takes a considerable amount of time to obtain the number of cells required for transplantation during ex vivo cell culture. Furthermore, if the cells lose their differentiation ability and do not proliferate at all, treatment is not performed. Furthermore, ex vivo culture of chondrocytes can result in changes in the phenotype of the cells. Therefore, there is a need for the development of a technology that can more effectively regenerate cartilage tissue.

[0005] Meanwhile, inflammation is a type of defense reaction of living tissues that occurs when tissues or cells are damaged or injured, or when they are infected by an external source of infection (viruses, bacteria, fungi, allergens, etc.). It refers to a series of complex conditions caused by immune cells involved in various immune responses that gather around the damaged or infected area and the inflammatory factors they secrete.

[0006] Currently known therapeutic agents for inflammatory diseases include dexamethasone and cortisone, which contain adrenal corticosteroid components. However, although these are active therapeutic agents, they have the problem of being highly toxic and potentially inducing 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 may be problematic. As described above, the use of steroid-based drugs 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 non-steroidal drugs and can provide stable anti-inflammatory therapeutic effects without side effects.

[0007] At the same time, various compounds that inhibit bone resorption or promote bone formation have been disclosed as therapeutic agents for osteoporosis. However, since these conventional therapeutic agents often have toxicity and side effects, there is a need to develop new substances to replace them. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide peptides having diverse physiological activities.

[0009] Another object of the present invention is to provide a pharmaceutical composition for cartilage regeneration, or for preventing or treating inflammatory diseases or osteoporosis, which comprises the peptide as an active ingredient.

[0010] Another object of the present invention is to provide a health functional food containing the peptide as an active ingredient for cartilage regeneration, or for preventing or improving inflammatory diseases or osteoporosis.

[0011] Another object of the present invention is to provide a method for treating, preventing, or ameliorating osteoporosis, which comprises administering a therapeutically effective amount of the peptide to a subject.

[0012] Another object of the present invention is to provide uses of the peptide for cartilage regeneration, and for the treatment, prevention, or amelioration of inflammatory diseases or osteoporosis. [Means for solving the problem]

[0013] To achieve the above object, one aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0014] In one embodiment, the peptide of the present invention may have various physiological activities such as cartilage regeneration activity, anti-inflammatory activity, osteoporosis suppression activity, and the like.

[0015] In one embodiment, the peptide of the present invention can promote the differentiation of stem cells, such as umbilical cord blood-derived stem cells, peripheral blood-derived stem cells, bone marrow-derived stem cells, and mesenchymal stem cells, preferably mesenchymal stem cells, into chondrocytes.

[0016] In one embodiment, the peptide of the present invention can increase the synthesis of extracellular matrix (ECM) in chondrocytes and can also increase the expression of genes related to ECM production, such as collagen type II (COL2A1), COMP (Cartilage Oligomeric Matrix Protein), and PCP (Proteoglycan Core Protein), in chondrocytes.

[0017] In another embodiment, the peptide of the present invention may suppress the expression of pro-inflammatory cytokines, including, but not limited to, TNFα, IL-6, IL-17, IL-1β, and IFNγ.

[0018] In other embodiments, the peptides of the present invention may, for example, promote differentiation of macrophages into osteoclasts.

[0019] Another aspect of the present invention provides a pharmaceutical composition for cartilage regeneration, or for preventing or treating inflammatory diseases or osteoporosis, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0020] Another aspect of the present invention provides a health functional food for cartilage regeneration, or for preventing or ameliorating inflammatory diseases or osteoporosis, which contains a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0021] Another aspect of the present invention provides a method for treating, preventing, or ameliorating osteoporosis in a subject, comprising administering to the subject, e.g., an osteoporosis patient, a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0022] Another aspect of the present invention provides a method for treating, preventing, or ameliorating an inflammatory disease in a subject, comprising administering to the subject, e.g., a patient suffering from an inflammatory disease, a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0023] Another aspect of the present invention provides a method for regenerating cartilage in a subject, comprising administering to the subject, e.g., a patient in need of cartilage regeneration, a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0024] Another aspect of the present invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the treatment, prevention, or amelioration of osteoporosis or an inflammatory disease.

[0025] Another aspect of the present invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for cartilage regeneration. [Effects of the Invention]

[0026] The peptides provided by the present invention can induce an increase in joint components in chondrocytes, increase chondrogenic differentiation and extracellular matrix (ECM) production in mesenchymal stem cells, suppress the increase in inflammatory cytokines and inflammatory proteins, and inhibit the differentiation and formation of osteoclasts. Therefore, the peptides can be useful as active ingredients in pharmaceutical compositions for regenerating cartilage or preventing or treating inflammatory diseases or osteoporosis, or in health functional foods for ameliorating the symptoms of these diseases.

[0027] 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]

[0028] [Figure 1] 1 shows Alcian blue stained photographs showing the effect of the peptide of the present invention on ECM synthesis in chondrocytes, where "Non" indicates a group not treated with the peptide of the present invention. [Figure 2] 1 shows an electrophoretic photograph and a graph showing the effect of the peptide of the present invention on the expression of ECM-related genes in chondrocytes, where "Non" indicates a group not treated with the peptide of the present invention. [Figure 3a] 1 shows electrophoretic photographs and a graph showing the effect of the peptide of the present invention on the production of SOX5, SOX6, and SOX9 proteins, which are regulators of ECM synthesis, in chondrocytes, showing the results after 3 days of treatment with the peptide of the present invention. Non indicates a group not treated with the peptide of the present invention. [Figure 3b] 1 shows electrophoretic photographs and a graph showing the effect of the peptide of the present invention on the production of SOX5, SOX6, and SOX9 proteins, which are regulators of ECM synthesis, in chondrocytes, showing the results after 5 days of treatment with the peptide of the present invention. Non indicates a group not treated with the peptide of the present invention. [Figure 3c] 1 shows electrophoretic photographs and a graph showing the effect of the peptide of the present invention on the production of SOX5, SOX6, and SOX9 proteins, which are regulators of ECM synthesis, in chondrocytes, showing the results after 7 days of treatment with the peptide of the present invention. Non indicates a group not treated with the peptide of the present invention. [Figure 4a] 1 shows stained photographs showing the effect of the peptide of the present invention on the synthesis of ECM molecules in 3D cultured chondrocytes, which are toluidine blue stained photographs. Non indicates a group not treated with the peptide of the present invention. [Figure 4b] 1 shows Alcian blue stained photographs showing the effect of the peptide of the present invention on the synthesis of ECM molecules in 3D culture of chondrocytes. Non indicates a group not treated with the peptide of the present invention. [Figure 5] 1 shows alcian blue stained photographs showing the effect of the peptide of the present invention on the differentiation of mesenchymal stem cells (MSCs) into chondrocytes and ECM production. [Figure 6a]Electrophoresis photographs showing the effect of the peptide of the present invention on the expression of ECM-related genes in mesenchymal stem cells, showing the results after 3 days of treatment with the peptide of the present invention. Non indicates the group not treated with the peptide of the present invention. [Figure 6b] Electrophoresis photographs showing the effect of the peptide of the present invention on the expression of ECM-related genes in mesenchymal stem cells, showing the results 7 days after treatment with the peptide of the present invention. Non indicates a group not treated with the peptide of the present invention. [Figure 6c] Electrophoresis photographs showing the effect of the peptide of the present invention on the expression of ECM-related genes in mesenchymal stem cells, showing the results 14 days after treatment with the peptide of the present invention. Non indicates the group not treated with the peptide of the present invention. [Figure 7a] 1 shows electrophoresis photographs and a graph showing the effect of the peptide of the present invention on the production of SOX9 protein, a regulator of ECM synthesis, in mesenchymal stem cells, showing the results after 3 days of treatment with the peptide of the present invention. Non indicates a group not treated with the peptide of the present invention, and CM indicates a positive control group. [Figure 7b] 1 shows electrophoresis photographs and graphs showing the effect of the peptide of the present invention on the production of SOX9 protein, a regulator of ECM synthesis, in mesenchymal stem cells, showing the results after 7 days of treatment with the peptide of the present invention. Non indicates a group not treated with the peptide of the present invention, and CM indicates a positive control group. [Figure 7c] 1 shows electrophoresis photographs and graphs showing the effect of the peptide of the present invention on the production of SOX9 protein, a regulator of ECM synthesis, in mesenchymal stem cells, showing the results after 14 days of treatment with the peptide of the present invention. Non indicates a group not treated with the peptide of the present invention, and CM indicates a positive control group. [Figure 8] 1 shows an electrophoretic photograph and graph showing the effect of the peptide of the present invention on the expression of inflammatory cytokines in macrophages in which an inflammatory response has been induced, where CON indicates a group not treated with the peptide of the present invention. [Figure 9] 1 is an electrophoretic photograph showing the effect of the peptide of the present invention on the production of COX, an inflammatory protein, in macrophages in which an inflammatory response has been induced. [Figure 10a]1 shows stained photographs and a graph showing the effect of the peptide of the present invention on differentiation of macrophages into osteoclasts, and indicates that the peptide of the present invention inhibits differentiation into osteoclasts induced by RANKL. Non and CON indicate groups not treated with the peptide of the present invention. [Figure 10b] 1 shows staining photographs and a graph showing the effect of the peptide of the present invention on the differentiation of macrophages into osteoclasts, and the graph shows that the peptide of the present invention does not exhibit toxicity in mouse macrophages. Non and CON indicate groups not treated with the peptide of the present invention. [Figure 10c] 1 shows staining photographs and a graph showing the effect of the peptide of the present invention on the differentiation of macrophages into osteoclasts, and the graph shows that the peptide of the present invention inhibits the activity of TRAP, an osteoclast marker, in a density-dependent manner. Non and CON indicate groups not treated with the peptide of the present invention. [Figure 11] 1 shows immunofluorescence staining photographs showing the effect of the peptide of the present invention on the formation of an actin ring, which is essential for differentiation of macrophages into osteoclasts. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be described in detail below.

[0030] 1. Peptides of the present invention The present invention provides peptides having useful physiological activities, for example, cartilage regeneration activity, anti-inflammatory activity, or osteoporosis suppression activity.

[0031] The peptide refers to a polymer consisting of two or more amino acids linked by peptide bonds. If the size of the peptide itself is too large, it may not be able to be effectively delivered to target tissues or cells or may have a short half-life and be eliminated in the body in a short period of time. Therefore, the peptide of the present invention consists of 20 or less amino acids, for example, 15 or less, or 12 or less.

[0032] The peptide of the present invention may comprise or consist of the amino acid sequence of SEQ ID NO: 1. In one embodiment of the present invention, the peptide of the present invention may also include amino acid variants or fragments having a sequence different from that of SEQ ID NO: 1, which are obtained by deleting, inserting, substituting, or a combination thereof, amino acid residues constituting the amino acid sequence of SEQ ID NO: 1, without affecting useful physiological activities, such as cartilage regeneration activity, anti-inflammatory activity, or osteoporosis-suppressing activity. Amino acid replacements at the peptide level that do not overall alter the physiological activities of the peptide, such as cartilage regeneration activity, anti-inflammatory activity, or osteoporosis-suppressing activity, are known in the art. In some cases, modifications may be made by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Therefore, the present invention includes peptides comprising substantially the same amino acid sequence as or consisting of 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 75% or more, for example, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. The peptide of the present invention may further include a targeting sequence, a tag, a labeled residue, or an amino acid sequence specifically designed to increase half-life or peptide stability.

[0033] The peptides of the present invention can be obtained by various methods widely known in the art. In one embodiment of the present invention, the peptides of the present invention can be prepared in vitro using polynucleotide recombination and a protein expression system, or by chemical synthesis such as peptide synthesis, or by cell-free protein synthesis.

[0034] Furthermore, a protecting group may be attached to the N- or C-terminus of the peptide of the present invention to achieve better chemical stability, enhanced pharmacological properties (e.g., half-life, water 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" is used herein to mean not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protease enzymes, but also storage stability (e.g., room temperature storage stability).

[0035] In the present invention, the term "cartilage regeneration activity" is used to mean a series of in vivo or in vitro activities necessary for cartilage formation, without limitation. In one embodiment of the present invention, the cartilage regeneration activity may include the activity of promoting the synthesis of cartilage-constituting components, such as ECM, in chondrocytes. In another embodiment of the present invention, the cartilage regeneration activity may include the activity of increasing the expression of ECM regulatory factors in chondrocytes. In another embodiment of the present invention, the cartilage regeneration activity may include the activity of promoting the differentiation of stem cells, such as umbilical cord blood-derived stem cells, peripheral blood-derived stem cells, bone marrow-derived stem cells, and mesenchymal stem cells, preferably mesenchymal stem cells, into chondrocytes. In another embodiment of the present invention, the cartilage regeneration activity may include the activity of promoting the production of ECM and ECM regulatory factors or promoting the expression of genes related thereto in chondrocytes or stem cells, preferably mesenchymal stem cells.

[0036] To confirm the cartilage regeneration effect of the peptide of the present invention, in a preferred embodiment of the present invention, human chondrocytes were treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1 to confirm the formation of ECM. As a result, it was confirmed that treatment with the peptide of the present invention promoted ECM production in chondrocytes (see Figure 1).

[0037] In another embodiment of the present invention, human chondrocytes were treated with a peptide containing the amino acid sequence of SEQ ID NO: 1 to examine the level of expression of genes involved in ECM formation. As a result, it was confirmed that treatment with the peptide of the present invention promoted the expression of COL2A1, COMP, and PCP genes present in chondrocytes (see Figure 2).

[0038] In another embodiment of the present invention, human chondrocytes were treated with a peptide containing the amino acid sequence of SEQ ID NO: 1 to examine the level of expression of proteins that regulate ECM formation. As a result, it was confirmed that treatment with the peptide of the present invention increased the expression of various signaling proteins involved in ECM formation in chondrocytes (see Figures 3a to 3c).

[0039] In another embodiment of the present invention, the formation of ECM was confirmed by treating human chondrocytes in 3D culture with a peptide containing the amino acid sequence of SEQ ID NO: 1. As a result, it was confirmed that the production of signaling proteins involved in ECM formation in chondrocytes was increased by treatment with the peptide of the present invention (see Figures 4a and 4b).

[0040] In another embodiment of the present invention, we investigated whether mesenchymal stem cells could differentiate into chondrocytes and produce ECM. As a result, we confirmed that treatment with the peptide of the present invention promoted the differentiation of mesenchymal stem cells into chondrocytes and the production of ECM (see Figure 5).

[0041] In another embodiment of the present invention, mesenchymal stem cells were treated with a peptide containing the amino acid sequence of SEQ ID NO: 1 to examine the level of expression of genes involved in ECM production and ECM formation. As a result, it was confirmed that treatment with the peptide of the present invention promoted ECM production present in mesenchymal stem cells and the expression of genes and proteins that regulate ECM production (see Figures 6a to 6c and Figures 7a to 7c).

[0042] On the other hand, the peptide of the present invention may have physiological activities other than cartilage regeneration, such as anti-inflammatory activity.

[0043] In the present invention, "anti-inflammatory activity" is used to mean, without limitation, the activity of suppressing inflammation. Inflammation is a type of defense response of biological tissues to a certain stimulus, and refers to a pathological state of abscess formed when tissues or cells are damaged or infected by various infectious agents, such as bacteria, fungi, viruses, and allergens from the outside. The inflammatory response manifests as a complex physiological response, such as enzyme activation, secretion of inflammatory mediators, fluid infiltration, cell migration, and tissue destruction, which are expressed in association with inflammatory mediators and immune cells in local blood vessels and body fluids, as well as external symptoms such as erythema, edema, fever, and pain. The peptide of the present invention has the activity of suppressing inflammation, and is therefore effective in reducing and ameliorating a series of pathological conditions and symptoms.

[0044] Furthermore, the peptide of the present invention can suppress the expression of inflammatory cytokines or inflammatory proteins. When a wound is caused by an inflammatory response or an external infectious agent penetrates the wound site and enters the body, leukocytes responsible for the early stage of the immune response gather around the wound site or the infectious agent, and express and secrete inflammation-related cytokines and proteins, thereby inducing an inflammatory response. Therefore, by suppressing the expression of inflammatory cytokines or inflammatory proteins, the peptide can exhibit anti-inflammatory activity. Furthermore, the anti-inflammatory activity and inflammation-suppressing effect of the peptide of the present invention can be confirmed by checking the expression levels of the inflammatory cytokines and proteins.

[0045] 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 its excessive production can induce inflammatory diseases. IL-17 (interleukin 17) is also a pro-inflammatory cytokine, produced by Th17 cells and plays a role in inducing or mediating inflammatory responses. IFNγ (interferon γ) may be produced by T lymphocytes and macrophages, is secreted in response to externally invaded 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 by inhibiting the expression of such inflammatory cytokines and suppressing the secretion of the expressed cytokines.

[0046] Furthermore, the peptides of the present invention can suppress the expression of Cox2. Cox2 (cyclooxygenase 2) is an enzyme involved in stimulating the biosynthesis of prostaglandins, and its expression may 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 exhibit an anti-inflammatory effect by suppressing Cox2 expression.

[0047] To confirm the anti-inflammatory effect of the peptide of the present invention, in a preferred embodiment of the present invention, mouse macrophages were treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1 together with inflammatory cytokines, and the amount of Cox2 protein was measured. As a result, it was confirmed that although the inflammatory response of the cells was induced by the inflammatory cytokines, the amount of Cox2 protein was reduced by treatment with the peptide of the present invention (see Figure 8).

[0048] Furthermore, to confirm the effect of treatment with the peptide of the present invention in reducing the expression levels of inflammation-related genes, in a preferred embodiment of the present invention, mouse macrophages were treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1 together with inflammatory cytokines, and the mRNA levels of TNFα, IL-1β, and Cox2 genes were examined. As a result, it was confirmed that although the treated inflammatory cytokines induced an inflammatory response in the cells, the expression levels of the inflammatory cytokines and related enzyme genes were reduced by treatment with the peptide of the present invention (see Figure 9).

[0049] 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. Therefore, the peptide of the present invention can be usefully used as an active ingredient in a composition for preventing, treating, or ameliorating inflammatory diseases that are induced by inflammation or involve inflammatory responses.

[0050] At the same time, the peptide of the present invention may have physiological activities other than cartilage regeneration, such as osteoclast inhibitory activity.

[0051] In one embodiment of the present invention, mouse macrophages were treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1 together with RANKL (Receptor Activator of Nuclear factor Kappa-B ligand), which induces differentiation into osteoclasts, and the activity of proteins related to osteoclast differentiation was examined. As a result, it was confirmed that treatment with the peptide of the present invention reduced osteoclast differentiation and the expression of proteins related thereto, and also inhibited the formation of actin rings, which are essential for osteoclast differentiation (see Figures 10a to 10c and 11).

[0052] Increased osteoclast activity is closely related to osteoporosis, and therefore, the peptides of the present invention can be useful in preventing or treating osteoporosis by suppressing osteoclast activity.

[0053] 2. Pharmaceutical compositions containing the peptides of the present invention Another aspect of the present invention provides a pharmaceutical composition for cartilage regeneration, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0054] Yet 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.

[0055] Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0056] Since 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 of the present invention," the specific description will be referred to in the above section "1. Peptides of the present invention," and only the components specific to the pharmaceutical composition will be described below.

[0057] According to one embodiment of the present invention, the peptide of the present invention has the effect of promoting cartilage regeneration, and therefore, a pharmaceutical composition containing the peptide as an active ingredient can be used for cartilage regeneration.

[0058] According to another embodiment of the present invention, the peptide of the present invention has the effect of inhibiting the expression or secretion of inflammation-related cytokines or inflammation-related factors, and therefore, a pharmaceutical composition containing the peptide as an active ingredient can be used to prevent or treat inflammatory diseases.

[0059] According to another embodiment of the present invention, the peptide of the present invention has the effect of inhibiting the differentiation and activity of osteoclasts, and therefore, a pharmaceutical composition containing the peptide as an active ingredient can be used to prevent or treat osteoporosis.

[0060] In the present invention, the inflammatory disease may refer to a pathological condition in which inflammation induced by neutrophil chemotaxis occurs in leukocytes, and may include, without limitation, any disease caused by or accompanied by an inflammatory response. For example, the inflammatory disease may include rhinitis, bronchitis, periodontitis, pancreatic colitis, gastritis, gastric ulcer, inflammatory skin disease, atopic dermatitis, encephilitis, sepsis, inflammatory colitis, chronic obstructive pulmonary disease, pulmonary shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathies, 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 condition may be, but is not limited to, arthritis, osteoarthritis, psoriasis, scleroderma, osteoporosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, borreliosis, neuroborreliosis, tuberculosis, sarcoidosis, lupus, discoid lupus, lupus chilblains, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Crohn's disease, Sjogren's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue syndrome, myocardial infarction, amyotrophic lateral sclerosis, Parkinson's disease, or multiple sclerosis.

[0061] The pharmaceutical composition of the present invention may be used to promote cartilage regeneration, prevent the onset of inflammatory diseases by suppressing the expression or secretion of factors that induce inflammatory responses, prevent the onset of osteoporosis by suppressing osteoclast differentiation, or inhibit the progression of the disease by suppressing further inflammatory responses in damaged or injured cells in patients with the inflammatory diseases or the differentiation into osteoclasts in patients with osteoporosis, thereby treating the diseases.

[0062] 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 in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules, or gel (e.g., hydrogel), and may further contain a dispersant or stabilizer.

[0063] 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.

[0064] In addition, the pharmaceutically acceptable carrier may include, but is not limited to, ingredients commonly used in 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.

[0065] The pharmaceutical composition of the present invention can be administered orally or parenterally during clinical administration and may be used in the form of a common pharmaceutical formulation. That is, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during clinical administration. When formulated, it is 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, etc., and are prepared 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, and syrups, and may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. 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.

[0066] The pharmaceutical compositions of the present invention are 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 and sensitivity of the drug, the time and route of administration, the excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical compositions of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents for inflammatory diseases. They may be administered simultaneously with conventional therapeutic agents, separately, or sequentially, 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 provides maximum efficacy at the minimum dose without adverse effects, which can be easily determined by one skilled in the art.

[0067] 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, inactivation rate, and excretion rate of the active ingredient into the body, the type of disease, and concomitant medications, 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 at approximately 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, e.g., two to three times a day, at the discretion of a doctor or pharmacist.

[0068] 3. Health functional foods containing the peptide of the present invention Another aspect of the present invention provides a health functional food for cartilage regeneration, which contains a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0069] Yet 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.

[0070] Yet another aspect of the present invention provides a health functional food for preventing or ameliorating osteoporosis, which contains a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0071] Since 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 of the present invention," the specific explanation will be based on the above section "1. Peptides of the present invention," and only the unique composition of the health functional food will be described below.

[0072] Similar to the pharmaceutical composition, a health functional food containing the peptide of the present invention as an active ingredient can be useful for cartilage regeneration and the prevention or amelioration of inflammatory diseases or osteoporosis.

[0073] The health functional food may be used for the prevention or amelioration of a disease, either before or after the onset of the disease, simultaneously with or separately from a therapeutic drug.

[0074] In the health functional food of the present invention, the active ingredient may be added directly to the food or 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 appropriately determined 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, 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.

[0075] 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 added 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.

[0076] 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, thickening agents (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 proportions of these additives may also be appropriately selected by those skilled in the art.

[0077] The present invention will be described in more detail below with reference to examples.

[0078] However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0079] [Example 1] Preparation of peptides 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). The column used was an ACQUITY UPLC BEH300 C18 (2.1 mm x 100 mm, 1.7 μm, Waters Co., USA).

[0080] [Table 1]

[0081] [Experimental Example 1] Confirmation of promotion of ECM production in chondrocytes by peptide treatment To confirm the cartilage regeneration effect of treatment with the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1, human-derived chondrocytes were treated with the peptide of SEQ ID NO: 1, and then the increase in the formation of glycosaminoglycan, a component of ECM, was examined.

[0082] For this purpose, the human C28 / I2 chondrocyte cell line was cultured at 3 × 10 3 The cells were seeded into a 96-well plate at a density of 100 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. After replacing the medium with fresh medium, the cells were treated with the peptide of SEQ ID NO: 1 at concentrations of 30, 50, 100, and 150 μg / ml. The medium was changed every three days, and the peptide of SEQ ID NO: 1 was treated at these concentrations. On the seventh day, the medium was removed, and the cells were placed in 60 μl of 3.7% formalin in a 96-well plate for fixation for 1 minute. After removing the 3.7% formalin, 70 μl of Alcian blue staining solution (50 ml of 3% acetic acid + 0.5 g of 1% Asian Blue 8GX, pH 2.5) was added and incubated at 37°C for 24 hours. The staining solution was then removed, the cells were washed with triple-distilled water, dried, and then observed under a microscope.

[0083] As a result, it was confirmed that the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1 promoted ECM production in chondrocytes (see FIG. 1).

[0084] [Experimental Example 2] Confirmation of promotion of ECM gene expression in chondrocytes by peptide treatment After treating human-derived chondrocytes with a peptide having the amino acid sequence of SEQ ID NO: 1, we examined whether there was an increase in mRNA expression of collagen type II (COL2A1), cartilage oligomeric matrix protein (COMP), proteoglycan core protein (PCP), which are components of the ECM, as well as the GAPDH gene as a control.

[0085] For this purpose, the human C28 / I2 chondrocyte cell line was cultured at 8.9 × 10 4 The cells were seeded into 6-well plates at a density of 100 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. After replacing the medium with fresh medium, the cells were treated with the peptide of SEQ ID NO: 1 at concentrations of 50 and 100 μg / ml. The peptide of SEQ ID NO: 1 was treated at the different concentrations while changing the medium every three days. After removing the medium on the 9th day, the cells were harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit (Intron, Korea), and then PCR was performed for collagen type II, COMP, PCP, and GAPDH genes using PCR-free mix (Intron, Korea). Primers specific to collagen type II, COMP, PCP, and GAPDH are shown in Table 2 below:

[0086] [Table 2]

[0087] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention promoted the expression of collagen type II, COMP, and PCP genes related to ECM production in chondrocytes (see Figure 2).

[0088] [Experimental Example 3] Confirmation of ECM regulatory factor expression in chondrocytes by peptide treatment After treating human-derived chondrocytes with a peptide having the amino acid sequence of SEQ ID NO: 1, it was confirmed whether the expression of SOX ((sex determining region Y)-box)5, SOX6, and SOX9 proteins, which are ECM regulators, was increased.

[0089] For this purpose, the human C28 / I2 chondrocyte cell line was cultured at 8.9 × 10 4 The cells were seeded into 6-well plates at a density of 100 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. After replacing the medium with fresh medium, the cells were treated with the peptide of SEQ ID NO: 1 at concentrations of 30, 50, 100, and 150 μg / ml. The medium was changed every three days, and the peptide of SEQ ID NO: 1 was treated at these concentrations. After removing the medium on days 3, 7, and 9, the cells were harvested and cell lysates were prepared. Western blotting was performed using antibodies against the proteins. The antibodies used in the experiments were purchased from the following sources: Sox5 (Santacruz Biotechnology, USA), Sox6 (Santacruz Biotechnology, USA), and Sox9 (Millipore, USA).

[0090] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention increases the expression of SOX5, SOX6, and SOX9 proteins, which are ECM regulators, in chondrocytes (see FIGS. 3a to 3c).

[0091] [Experimental Example 4] Confirmation of promotion of ECM generation in 3D culture of chondrocytes by peptide treatment After treating 3D cultures of human-derived chondrocytes with a peptide having the amino acid sequence of SEQ ID NO: 1, it was confirmed whether or not the formation of glycosaminoglycan, a component of ECM, was increased.

[0092] For this purpose, 3 × 10 human C28 / I2 chondrocyte cell lines were used. 6 Cells were suspended in 1 ml of alginate solution (1.25% alginic acid, 20 mM HEPES, 150 mM NaCl, pH 7.4), and the cell-containing alginate solution was added dropwise to a polymerization solution (100 mM CaCl2, 10 mM HEPES, pH 7.4) to form beads. The beads were washed twice with DPBS and then distributed into conical tubes containing DMEM medium (5% FBS). The peptide of SEQ ID NO: 1 was treated at concentrations of 30, 50, and 100 μg / ml, with the medium being changed every three days. After removing the medium on the 14th day, the cells were fixed with 3.7% formaldehyde for 6 hours. Paraffin blocks were prepared by dehydration using a tissue processor (70%, 80%, 90%, 100% EtOH I, II, xylene I, II, sequentially for 30 minutes each) and embedding. The paraffin blocks were sectioned onto glass slides at a thickness of 0.4 μm and allowed to dry completely for at least 24 hours. The sections were hydrated (xylene I, II, 100% EtOH I, II, 90%, 80%, 70%, sequentially for 3 minutes each) and washed three times with running water for 10 seconds each. After staining with Alcian blue or toluidine blue for 1 hour, the sections were washed three times with running water for 10 seconds each. The sections were then immersed in 90%, 100% EtOH I, II for 10 seconds each, followed by immersion in xylene I and II for 3 minutes each, and then mounted. After drying for at least 24 hours, the sections were observed under a microscope.

[0093] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention promoted the production of glycosaminoglycan, a component of ECM, in 3D culture of chondrocytes (see Figures 4a and 4b).

[0094] [Experimental Example 5] Confirmation of promotion of chondrocyte differentiation and ECM production in mesenchymal stem cells by peptide treatment Adipose-derived mesenchymal stem cells were treated with a peptide having the amino acid sequence of SEQ ID NO: 1, and the effects on chondrogenic differentiation and ECM production of the stem cells were examined.

[0095] For this purpose, human AD-MSC (Adipose-Derived Mesenchymal Stem Cell) cells were cultured at 1.5 × 10 3 The cells were seeded into a 96-well plate at a density of 1000 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. After changing the medium to DMEM medium containing 5% FBS, the cells were treated with the peptide of SEQ ID NO: 1 at concentrations of 30, 50, 100, and 150 μg / ml. The medium was changed every three days, and the peptide of SEQ ID NO: 1 was treated at these concentrations. On the 21st day, the medium was removed, and 60 μl of 3.7% formalin was added to the 96-well plate for staining and fixation for 1 minute. After removing the 3.7% formalin, 70 μl of Alcian blue staining solution (50 ml of 3% acetic acid + 0.5 g of 1% Alcian Blue 8GX, pH 2.5) was added and incubated at 37°C for 24 hours. The staining solution was then removed, and the cells were washed with triple-distilled water, dried, and observed under a microscope.

[0096] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention promoted chondrogenic differentiation of adipose-derived mesenchymal stem cells and the production of glycosaminoglycans, which are components of ECM (see Figure 5).

[0097] [Experimental Example 6] Confirmation of promotion of ECM regulatory factor and ECM gene expression in mesenchymal stem cells by peptide treatment After treating adipose-derived mesenchymal stem cells with a peptide having the amino acid sequence of SEQ ID NO: 1, we confirmed whether there was an increase in the expression of COL2A1, COMP, collagen type 11A (COL 11A), ACP (Aggrecan core protein), PCP, and ACAN (aggrecan), which are related to the production of ECM, and the Sox9 gene, which is a regulator of ECM.

[0098] For this purpose, human AD-MSC cells were cultured at 1.5 × 10 3 The cells were seeded into a 96-well plate at a density of 100 cells / well and cultured for 24 hours in DMEM medium (cat. 11995-065, Gibco) containing 10% FBS. After changing the medium to DMEM medium containing 5% FBS, the cells were treated with the peptide of SEQ ID NO: 1 at 50 and 100 μg / ml concentrations. The medium was changed every three days, and the peptide of SEQ ID NO: 1 was treated at the respective concentrations. After removing the medium on days 3, 7, and 11, the cells were harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit (Intron, Korea), and PCR was performed using PCR-free mix (Intron, Korea) for the COL2A1, COMP, Sox9, COL11A, ACP, PCP, ACAN, and GAPDH genes. Primers specific to COL2A1, COMP, Sox9, COL11A, ACP, PCP, ACAN, and GAPDH are listed in Table 3 below.

[0099] [Table 3-1]

[0100] [Table 3-2]

[0101] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention promoted the expression of COL2A1, COMP, COL11A, ACP, PCP, and ACAN genes, which are associated with ECM production, and the Sox9 gene, which is an ECM regulator, in adipose-derived mesenchymal stem cells (see Figures 6a to 6c).

[0102] [Experimental Example 7] Confirmation of ECM regulatory factor expression in mesenchymal stem cells by peptide treatment Adipose-derived mesenchymal stem cells were treated with a peptide having the amino acid sequence of SEQ ID NO: 1, and then it was confirmed whether the expression of SOX9 protein, a regulator of ECM, increased.

[0103] For this purpose, human AD-MSC cells were cultured at 1.5 × 10 3 The cells were seeded into 96-well plates at a density of 100 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) containing 10% FBS for 24 hours. After changing the medium to DMEM medium containing 5% FBS, the cells were treated with the peptide of SEQ ID NO: 1 at concentrations of 50 and 100 μg / ml. The medium was changed every three days, and the peptide of SEQ ID NO: 1 was treated at the respective concentrations. A positive control (CM) solution containing dexamethasone (100 nM), ascorbic acid (50 μM), proline (40 μM), TGFβ1 (10 ng / ml), and 1X ITS was used. After removing the medium on days 3, 7, and 14, the cells were harvested, and cell lysates were prepared. Western blotting was performed using an antibody against the SOX9 protein (Millipore, USA).

[0104] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention increases the production of SOX9 protein, a regulator of ECM, in adipose-derived mesenchymal stem cells (see FIGS. 7a to 7c).

[0105] Taking the results of Experimental Examples 1 to 7 together, it can be seen that the peptide of the present invention having the base sequence of SEQ ID NO: 1 increases the activity of chondrocytes, promotes ECM synthesis, increases the expression of genes related to the production of the ECM and ECM regulatory factor genes, promotes the differentiation of mesenchymal stem cells into chondrocytes, and also increases the expression of genes related to the production of ECM and ECM regulatory factor genes in mesenchymal stem cells, thereby demonstrating excellent cartilage regeneration effects. Furthermore, when a larger amount of the peptide of the present invention is used, the above effects are more pronounced, and therefore it can be seen that the cartilage regeneration effects seen from the results of the above experiments are due to the peptide of the present invention.

[0106] [Experimental Example 8] Confirmation of inflammatory cytokine mRNA expression levels in cells induced by TNFα inflammation To confirm the anti-inflammatory effect of treatment with a peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention, in addition to the cartilage regeneration activity confirmed in Experimental Examples 1 to 7, a mouse RAW264.7 macrophage cell line was treated with TNFα to induce inflammation, and the expression levels of the 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 associated with 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 the TNFα, Cox2, and IL-1β genes.

[0107] For this purpose, the mouse RAW264.7 cell line was cultured at 2 × 10 5 The cells were seeded into 6-well plates at a density of 100 cells / well and cultured in α-MEM medium containing 10% FBS for 24 hours. The medium was replaced with serum-free α-MEM medium (1% penicillin) and then fasted for 6 hours. The cells were treated with 100 and 150 μg / ml of peptide of SEQ ID NO: 1 at concentrations of 100 and 150 μg / ml and then incubated for 1 hour. After 1 hour, the cells were treated with 20 nM TNF-α and incubated for 24 hours. The cells were then harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit (Intron, Korea), and PCR was performed for the TNFα, IL-1β, Cox2, and GAPDH genes using PCR Free Mix (Intron, Korea). Primers specific to the TNFα, IL-1β, Cox2, and GAPDH genes are listed in Table 4 below:

[0108] [Table 4]

[0109] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention inhibited the expression of inflammatory cytokine genes induced by treatment of mouse macrophages with inflammatory cytokines (see Figure 8).

[0110] [Experimental Example 9] Confirmation of inflammatory cytokine mRNA expression levels in cells induced by TNFα inflammation In addition to the results of Experimental Example 9, to further confirm the anti-inflammatory effect of treatment with the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention, the mouse RAW264.7 macrophage cell line was treated with TNFα to induce inflammation, and the expression level of COX2 protein, an inflammation induction marker, was determined.

[0111] For this purpose, the mouse RAW264.7 cell line was cultured at 2 × 10 5 After seeding into 6-well plates at a density of 100 cells / well, the cells were cultured in α-MEM medium containing 10% FBS for 24 hours. The medium was replaced with serum-free α-MEM medium (1% penicillin) and then fasted for 6 hours. The cells were treated with 100 and 150 μg / ml of SEQ ID NO: 1 peptide and incubated for 1 hour. After 1 hour, the cells were treated with 20 nM TNF-α and incubated for 24 hours. The cells were then harvested, and cell lysates were prepared. Western blotting was performed using an antibody against the COX2 protein (Cell Signaling Technology, USA).

[0112] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention inhibited the increase in inflammatory proteins induced by the treatment of inflammatory cytokines in mouse macrophages (see FIG. 9).

[0113] Considering the results of Experimental Examples 8 and 9, it can be seen that even in cells in which an inflammatory response has been induced, treatment with the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1 reduces the amount of proteins and gene expression associated with the inflammatory response, demonstrating an inhibitory effect on the inflammatory response. Furthermore, when a larger amount of the peptide of the present invention is treated, the above-mentioned effect is more pronounced, and therefore it can be seen that the inhibitory effect on the inflammatory response seen from the results of the above-mentioned Experimental Examples is due to the peptide of the present invention.

[0114] [Experimental Example 10] Confirmation of the inhibitory effect of RANKL-induced osteoclast differentiation In addition to the cartilage regeneration activity confirmed in Experimental Examples 1 to 7 and the anti-inflammatory effect confirmed in Experimental Examples 8 and 9, the inhibitory effect of osteoclast differentiation by treatment with a peptide having the amino acid sequence of Sequence No. 1 of the present invention was confirmed.

[0115] <10-1> Confirmation of the osteoclast differentiation inhibitory effect of the peptide of the present invention Mouse RAW264.7 cell line, 1.7 x 10 3 The cells were plated into a 96-well plate at a density of 1000 cells / well and cultured in DMEM medium for 24 hours. After 24 hours, the cells were treated with RANKL and SEQ ID NO: 1 peptide at concentrations of 10, 30, 100, and 150 μg / ml for 4 days to induce osteoclast differentiation. The medium was removed, the cells were fixed for 30 seconds, and washed twice with double-distilled water. Staining was carried out with 100 μl of TRAP staining solution (acid phosphatase kit, Sigma-Aldrich) at 37°C for 30 minutes. The staining solution was then removed, the cells were washed twice with double-distilled water, dried, and observed under a microscope.

[0116] As a result, it was confirmed that the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1 inhibited the differentiation of mouse macrophages into osteoclasts induced by RANKL (see FIG. 10a).

[0117] <10-2> Confirmation of cytotoxicity of the peptide of the present invention The effect of the peptide of the present invention on cell proliferation was confirmed using CCK-8 assay. CCK-8 reagent is reduced by cellular dehydrogenase to produce a colored product, and the difference in absorbance using this product allows the determination of cell proliferation.

[0118] For this purpose, the mouse RAW264.7 cell line was cultured at 1.7 × 10 3The cells were plated into a 96-well plate at a density of 1000 cells / well and cultured in DMEM medium for 24 hours. After 24 hours, the cells were treated with peptide of SEQ ID NO: 1 at concentrations of 10, 30, 30, 100, and 150 μg / ml and then incubated for 3 days. After removing the medium, 100 μl of CCK-8 solution (Dojindo, CCK-8 kit) was diluted 10-fold in the culture medium and treated. During incubation at 37°C, the absorbance at 450 nm was measured using a microplate reader at 30-minute intervals.

[0119] As a result, when the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention was treated, the absorbance was actually higher, confirming that the peptide of the present invention does not exhibit toxicity in mouse macrophages (see Figure 10b).

[0120] <10-3> Confirmation of the inhibitory effect of the peptide of the present invention on TRAP activity associated with osteoclast differentiation Mouse RAW264.7 cell line, 1.7 x 10 3 The cells were plated into 96-well plates at a density of 100 μg / well and cultured in DMEM medium for 24 hours. After 24 hours, they were treated with RANKL and the peptide of SEQ ID NO: 1 at concentrations of 10, 30, 100, and 150 μg / ml for 4 days to induce osteoclast differentiation. They were treated with 100 μl of TRAP activation solution (15 ml of TRAP buffer (0.1% sodium citrate + 50 μM sodium tartrate [pH 5.0]) + 4-nitrophenly phosphate disodium salt hexahydrate, Sigma, 1 tablet mix), incubated at 37°C for 1 hour, and then treated with 10 μl of 2 N NaOH. The absorbance at 405 nm was measured using a plate reader.

[0121] As a result, it was confirmed that the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1 inhibited the activity of TRAP, an osteoclast marker, in mouse macrophages in a concentration-dependent manner (see FIG. 10c).

[0122] [Experimental Example 11] Confirmation of RANKL-induced inhibition of actin ring formation in osteoclasts In addition to the results of Experimental Example 10, to further confirm the inhibitory effect of treatment with the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention on osteoclast differentiation, mouse RAW264.7 macrophage cell line was treated with RANKL to induce differentiation into osteoclasts, and it was confirmed whether the peptide of the present invention inhibits the formation of actin rings, which are essential for osteoclast differentiation.

[0123] For this purpose, the mouse RAW264.7 cell line was cultured at 5 × 10 3 The cells were seeded into a 96-well plate at a density of 100 cells / well and cultured in DMEM medium for 24 hours. After 24 hours, they were treated with RANKL and the peptide of SEQ ID NO: 1 at concentrations of 50 and 100 μg / ml, and then osteoclast differentiation was induced for 4 days. After removing the medium, the cells were washed three times with chilled PBS and fixed with 4% paraformaldehyde for 30 minutes. After washing three times with chilled PBS for 5 minutes, the cells were permeabilized with 0.3% Triton X-100 in PBS for 30 minutes. After washing three times with chilled PBS for 5 minutes, the cells were blocked with filtered 3% BSA in PBS for 1 hour with agitation. Primary antibody (Rhodamin (Red) [Invitrogen] diluted 1:100 in blocking medium) was added. TM After incubation in PBS for 2 hours with agitation, the wells were protected from light and washed three times with PBS for 15 minutes with agitation. After washing, DAPI mounting medium (VECTASHIELD® MOUNTING MEDIUM with DAPI) was added, and each well was covered with a coverslip and observed under a microscope.

[0124] As a result, it was confirmed that the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1 inhibited the formation of actin rings in mouse macrophages, which are essential for differentiation into osteoclasts (see FIG. 11).

[0125] Considering the results of Experimental Examples 10 and 11, it can be confirmed that the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1 can inhibit osteoclast differentiation and can therefore be used to prevent or treat osteoporosis. Furthermore, the effect of inhibiting osteoclast differentiation as seen from the results of the above Experimental Examples is due to the peptide of the present invention, since the effect is more pronounced when a larger amount of the peptide of the present invention is used.

[0126] [Production Example 1] Production of pharmaceutical composition <1-1> Manufacturing of powders 2g of the peptide of Example 1 1g lactose The ingredients were mixed and filled into an airtight bag to prepare a powder.

[0127] <1-2> Tablet manufacturing 100 mg of the peptide of Example 1 100mg corn starch Lactose 100mg Magnesium stearate 2mg The above ingredients were mixed and then compressed into tablets according to a conventional tablet manufacturing method.

[0128] <1-3> Manufacturing of capsules 100 mg of the peptide of Example 1 100mg corn starch Lactose 100mg Magnesium stearate 2mg After mixing the ingredients, the mixture was charged into a gelatin capsule according to a conventional capsule manufacturing method to prepare a capsule.

[0129] <1-4> Ring production 1g of the peptide of Example 1 Lactose 1.5g 1g glycerin 0.5g xylitol The above ingredients were mixed and then prepared in a conventional manner so that each ring weighed 4 g.

[0130] <1-5> Granule manufacturing 150 mg of the peptide of Example 1 Soybean extract 50mg Glucose 200mg Starch 600mg After mixing the ingredients, 100 mg of 30% ethanol was added and dried at 60°C to form granules, which were then filled into sachets.

[0131] [Production Example 2] Production of functional health food composition <2-1> Manufacture of flour foods 0.5 to 5.0 parts by weight of the peptide of Example 1 was added to wheat flour, and the mixture was used to produce bread, cakes, cookies, crackers, and noodles.

[0132] <2-2> Production of soups and gravies Health-promoting processed meat products, noodle soups and gravy were prepared by adding 0.1 to 5.0 parts by weight of the peptide of Example 1 to soups and gravy.

[0133] <2-3> Dairy product manufacturing 5 to 10 parts by weight of the peptide of Example 1 was added to milk, and various dairy products such as butter and ice cream were produced using the milk.

[0134] <2-4> Zen food production Brown rice, barley, glutinous rice, and adlay were gelatinized and dried using a known method, then roasted and ground into powder with a particle size of 60 mesh using a grinder. Black beans, black sesame seeds, and perilla seeds were also steamed and dried using a known method, then roasted and ground into powder with a particle size of 60 mesh using a grinder. The grains, nuts, and seeds prepared above and the peptide of Example 1 were blended in the following proportions to produce the product.

[0135] Grains (brown rice 30 parts by weight, adlay 15 parts by weight, barley 20 parts by weight), nuts and seeds (perilla 7 parts by weight, black beans 8 parts by weight, black sesame 7 parts by weight), peptide of Example 1 (3 parts by weight), reishi mushroom (0.5 parts by weight), rehmannia (0.5 parts by weight) <2-5> Manufacturing of health drinks The supplementary ingredients, such as liquid fructose (0.5%), oligosaccharides (2%), sugar (2%), salt (0.5%), and water (75%), were homogeneously mixed with 5 g of the peptide of Example 1, flash sterilized, and then packaged in small containers such as glass bottles and PET bottles.

[0136] Although the present invention has been described in detail above only with reference 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 to be understood that such modifications and variations are within the scope of the appended claims.

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1 , wherein the peptide has one or more physiological activities selected from the group consisting of cartilage regeneration activity, anti-inflammatory activity, and osteoporosis suppression activity.

3. The peptide of claim 1 , wherein the peptide promotes differentiation of stem cells into chondrocytes.

4. The peptide of claim 3, wherein the stem cells are one or more selected from the group consisting of umbilical cord blood-derived stem cells, peripheral blood-derived stem cells, bone marrow-derived stem cells, and mesenchymal stem cells.

5. The peptide of claim 1, wherein the peptide increases the synthesis of extracellular matrix (ECM) in chondrocytes.

6. The peptide of claim 1, wherein the peptide increases the expression of one or more genes selected from the group consisting of collagen type II (COL2A1), COMP (Cartilage Oligomeric Matrix Protein), and PCP (Proteoglycan Core Protein) in chondrocytes.

7. The peptide of claim 1 , wherein the peptide suppresses the expression of inflammatory cytokines.

8. The peptide according to claim 7, wherein the inflammatory cytokine is one or more selected from the group consisting of TNFα, IL-6, IL-17, IL-1β, and IFNγ.

9. The peptide of claim 1 , which inhibits differentiation of macrophages into osteoclasts.

10. A pharmaceutical composition for cartilage regeneration comprising the peptide according to any one of claims 1 to 9 as an active ingredient.

11. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising the peptide according to any one of claims 1 to 9 as an active ingredient.

12. A pharmaceutical composition for preventing or treating osteoporosis, comprising the peptide according to any one of claims 1 to 9 as an active ingredient.

13. A health functional food for cartilage regeneration, comprising the peptide according to any one of claims 1 to 9 as an active ingredient.

14. A functional health food for preventing or improving inflammatory diseases, comprising the peptide according to any one of claims 1 to 9 as an active ingredient.

15. A health functional food for preventing or improving osteoporosis, comprising the peptide according to any one of claims 1 to 9 as an active ingredient.

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