Peptides Having Physiological Activity 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 biological activities and their uses.

Background Art

[0002] Normally, since the cartilage tissue forming the joints of vertebrates does not have blood vessels, nerves, and lymphatic tissues, once damaged, it cannot be normally regenerated in vivo. When such joint cartilage tissue is damaged, it becomes restricted in daily activities along with severe pain, and when it becomes chronic, it induces fatal degenerative arthritis, hindering normal life and occupational activities. Therefore, damaged articular cartilage has very limited self-recovery and regeneration ability. Although various medical methods have been attempted, the current situation is still insufficient to restore hyaline cartilage, which is articular cartilage, to a natural state.

[0003] Clinically, surgical methods for cartilage regeneration include methods for inducing the differentiation of stem cells into chondrocytes, methods for transplanting autologous or allogeneic cartilage tissue to the cartilage defect site by osteochondral transplantation, methods for scraping and removing damaged cartilage by microfracture to expose the subchondral bone, making holes at regular intervals here, and attempting to regenerate the flowing-out bone marrow cells into cartilage tissue through this, methods for inducing cartilage regeneration by transplanting chondrocytes to the cartilage defect site by chondrocyte transplantation, methods for using autologous chondrocyte transplantation for the treatment of damaged articular cartilage, etc.

[0004] However, when using the above method, surgery is required both when harvesting chondrocytes and when transplanting the chondrocytes cultured in vitro back to the articular cartilage injury site. Ultimately, the two surgeries cause great pain, sequelae, and economic burden to the patient, and the surgical process is also complex. In addition, most of the harvested chondrocytes are obtained from fully grown adults, and the proliferation and growth of the harvested cells are not vigorous. Therefore, it takes a considerable period of time to obtain the number of cells required for transplantation during in vitro cell culture. If the cells lose their differentiation ability and do not proliferate at all, the treatment itself cannot be carried out. Since chondrocytes are cultured in vitro, there is a problem that the cell phenotype changes. Therefore, there is a real need to develop a technology that can more effectively regenerate cartilage tissue.

[0005] On the other hand, the inflammatory reaction is a kind of defense reaction of living tissues shown when tissues or cells are damaged or injured, or infected by external infection sources (such as viruses, bacteria, fungi, and allergy-inducing substances). It means a series of complex states caused by immune cells involved in various immune reactions that gather around the damaged or infected site and inflammatory factors secreted by these cells.

[0006] Currently known therapeutic agents for inflammatory diseases include dexamethasone and cortisone that use adrenal cortical hormone components. However, in these cases, although they have activity as therapeutic agents, there is a problem that they are highly toxic and may induce side effects such as edema. In addition, they do not act selectively on the cause of the induced inflammation, so there may be a problem that a severe level of immunosuppression phenomenon may occur. As reported above, there are side effects and problems associated with using drugs containing steroid components to treat inflammatory diseases. Therefore, it is an urgent situation to develop a therapeutic agent for inflammatory diseases that can exhibit the effect of stable inflammation suppression treatment without side effects by using drugs containing non-steroid components.

[0007] Meanwhile, while various types of compounds that suppress bone resorption or promote bone formation are currently disclosed as therapeutic agents for osteoporosis, such conventional therapeutic agents often have toxicity and side effects, and thus, the development of new substances to replace them is required.

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a peptide having various biological activities.

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

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

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

[0012] Another object of the present invention is to provide a use of the peptide for cartilage regeneration, or for the treatment, prevention, or improvement of inflammatory diseases or osteoporosis.

Means for Solving the Problems

[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 can have various biological activities such as cartilage regeneration activity, anti-inflammatory activity, and osteoporosis inhibitory activity.

[0015] In one embodiment, the peptide of the present invention can promote the differentiation of stem cells, for example, umbilical cord blood-derived stem cells, peripheral blood-derived stem cells, bone marrow-derived stem cells, 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. Further, the peptide of the present invention can increase the expression of genes related to the production of ECM such as collagen type II (COL2A1), aggrecan (ACAN), and proteoglycan core protein (PCP) in chondrocytes.

[0017] In other embodiments, the peptide of the present invention can suppress the expression of inflammatory cytokines. Examples of the inflammatory cytokines include, but are not limited to, TNFα, IL-1β, COX2, IL-6, IL-17, and IFNγ.

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

[0019] Another aspect of the present invention provides a pharmaceutical composition for cartilage regeneration, or for the prevention or treatment of inflammatory diseases or osteoporosis, comprising a peptide containing 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 the prevention or improvement of inflammatory diseases or osteoporosis, comprising a peptide containing 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 improving osteoporosis in a subject, comprising administering a therapeutically effective amount of a peptide containing the amino acid sequence of SEQ ID NO: 1 to the subject, for example, a patient with osteoporosis.

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

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

[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.

Advantages of the Invention

[0026] The peptide provided by the present invention can induce an increase in the components of joints in chondrocytes, increase the chondrogenic differentiation of mesenchymal stem cells and the production of extracellular matrix (ECM), suppress the increase of inflammatory cytokines and inflammatory proteins, and also suppress the differentiation and formation of osteoclasts. Therefore, the peptide can be usefully used as an active ingredient of a pharmaceutical composition for regenerating cartilage or preventing or treating an inflammatory disease or osteoporosis, or a health functional food for ameliorating the symptoms.

[0027] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those of ordinary skill in the art from the following description.

Brief Description of the Drawings

[0028]

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Mode for Carrying Out the Invention

[0029] Hereinafter, the present invention will be described in detail.

[0030] 1. Peptide of the Present Invention The present invention provides a peptide having useful physiological activities, such as cartilage regeneration activity, anti-inflammatory activity, or osteoporosis inhibitory activity.

[0031] The peptide means a polymer composed of two or more amino acids linked by peptide bonds. If the size of the peptide itself is too large, it cannot effectively flow into the target tissue or cells, or has the drawback of a short half-life and disappearing in the body in a short period. Therefore, the peptide of the present invention is composed of 20 or fewer, for example, 15 or fewer, or 12 or fewer amino acids.

[0032] The peptide of the present invention may include 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 has an amino acid residue that constitutes the amino acid sequence of SEQ ID NO: 1 deleted, inserted, substituted, or modified by a combination thereof within a range that does not affect useful physiological activities, such as cartilage regeneration activity, anti-inflammatory activity, or osteoporosis inhibitory activity, and may also include all mutants or fragments of amino acids having a sequence different from SEQ ID NO: 1. Amino acid exchanges at the peptide level that do not overall change the physiological activities of the peptide, such as cartilage regeneration activity, anti-inflammatory activity, or osteoporosis inhibitory activity, are known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Therefore, the present invention includes a peptide containing an amino acid sequence substantially identical to the peptide containing or consisting of the amino acid sequence of SEQ ID NO: 1, its mutants, or its active fragments. The substantially identical protein means an amino acid sequence having a sequence homology of 75% or more, for example, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more with the amino acid sequence of SEQ ID NO: 1. Further, the peptide of the present invention may further include a targeting sequence, a tag, a labeled residue, a half-life, or an amino acid sequence produced for a specific purpose of increasing peptide stability.

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

[0034] In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, water absorption, titer, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activities), and reduced antigenicity, a protecting group may be attached to the N-terminus or C-terminus of the peptide of the present invention. 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 polyethylene glycol (PEG), but any component that can modify the peptide, particularly enhance the stability of the peptide, may be included without limitation. The term "stability" is used to mean not only in vivo stability that protects the peptide of the present invention from the attack of proteolytic enzymes in the living body, but also storage stability (e.g., stability at room temperature).

[0035] In the present invention, "chondrogenic activity" is used to mean including without limitation a series of in vivo or in vitro activities necessary for cartilage formation. In one embodiment of the present invention, the chondrogenic activity may include an activity of promoting the synthesis of components constituting cartilage, e.g., ECM, in chondrocytes. In another embodiment of the present invention, the chondrogenic activity may include an activity of increasing the expression of ECM regulatory factors in chondrocytes. In another embodiment of the present invention, the chondrogenic activity may include an activity of promoting the differentiation of stem cells, e.g., stem cells derived from umbilical cord blood, peripheral blood, bone marrow, mesenchymal stem cells, preferably mesenchymal stem cells, into chondrocytes. In another embodiment of the present invention, the chondrogenic activity may include an 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] In order 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 the peptide containing the amino acid sequence of SEQ ID NO: 1 to confirm the formation of ECM. As a result, it was confirmed that the production of ECM by chondrocytes was promoted by the treatment with the peptide of the present invention (see Figure 2).

[0037] In another embodiment of the present invention, when human chondrocytes were treated with the peptide containing the amino acid sequence of SEQ ID NO: 1 to confirm the degree of expression of genes involved in ECM formation, it was confirmed that the expression of COL2A1, COMP, and PCP genes present in chondrocytes was promoted by the treatment with the peptide of the present invention (see Fig. 3).

[0038] In another embodiment of the present invention, when the formation of ECM by treatment with the peptide containing the amino acid sequence of SEQ ID NO: 1 was confirmed in a three-dimensional culture of human chondrocytes, it was confirmed that the production of signaling proteins involved in ECM formation in chondrocytes increased by the treatment with the peptide of the present invention (see Fig. 4).

[0039] In another embodiment of the present invention, when the differentiation of mesenchymal stem cells into chondrocytes and the ability to produce ECM were confirmed, it was confirmed that the treatment with the peptide of the present invention promoted the differentiation of mesenchymal stem cells into chondrocytes and the production of ECM (see Fig. 5).

[0040] In another embodiment of the present invention, when mesenchymal stem cells were treated with the peptide containing the amino acid sequence of SEQ ID NO: 1 to confirm the degree of expression of genes involved in ECM production and ECM formation, it was confirmed that the expression of genes and proteins that regulate ECM production and ECM production present in mesenchymal stem cells was promoted by the treatment with the peptide of the present invention (see Figs. 6a to 6c and Figs. 7a to 7c).

[0041] On the other hand, the peptide of the present invention can have physiological activities other than cartilage regeneration, for example, anti-inflammatory activity.

[0042] In the present invention, "anti-inflammatory activity" is used to mean an activity that suppresses inflammation without limitation, and the inflammation is a kind of defensive reaction of living tissue against a certain stimulus, and the pathological state of abscess formed when tissues and cells are damaged or infected by various infection sources such as bacteria, molds, viruses, and allergy-inducing substances derived from the outside. Further, the inflammatory reaction is a complex physiological reaction such as activation of enzymes, secretion of inflammatory mediators, body fluid infiltration, cell migration, and tissue destruction, in which local blood vessels and body fluids are associated with inflammatory mediators and immune cells, and external symptoms such as erythema, edema, fever, and pain are shown. Since the peptide of the present invention has an activity of suppressing inflammation, it has an effect of reducing and improving a series of pathological states and symptoms as described above.

[0043] In addition, the peptide of the present invention can suppress the expression of inflammatory cytokines or inflammatory proteins. When a wound occurs in an inflammatory reaction or an external infectant that has invaded the wound site enters the body, white blood cells responsible for the initial immune reaction gather around the wound site or the infectant, and express and secrete inflammation-related cytokines and proteins to induce an inflammatory reaction. Therefore, by suppressing the expression of inflammatory cytokines or inflammatory proteins, anti-inflammatory activity can be shown, and by confirming the expression level of the inflammatory cytokines and proteins, the anti-inflammatory activity and inflammation-suppressing effect of the peptide of the present invention can be confirmed.

[0044] The inflammatory cytokine may be one or more selected from the group consisting of TNFα, IL-6, IL-17, IL-1β, and IFNγ. The TNFα is an abbreviation of "Tumor Necrosis Factor α", which is a cytokine generated and secreted from macrophages and various cells activated during immune responses to bacterial infections and tumor diseases. It is known as a main mediator of inflammatory reactions and plays an important role in inflammatory diseases such as rheumatoid arthritis (RA), psoriatic arthritis, Crohn's disease, psoriasis, and ankylosing spondylitis (AS). The IL-6 (interleukin 6) is a cytokine produced by macrophages and various lymphocytes, etc., which plays a role in promoting inflammatory reactions and is known to be able to induce inflammatory diseases when overproduced. Similarly, the IL-17 (interleukin 17) corresponds to a cytokine that promotes inflammation and plays a role in inducing or mediating inflammatory reactions generated from Th17 cells. The IFNγ (interferon γ) may be generated by T lymphocytes and macrophages and is secreted in response to infections by externally invading viruses and bacteria, and is known to play a role in relation to autoimmune or autoinflammatory diseases. Therefore, the peptide of the present invention has the effect of suppressing inflammation by suppressing the expression of the inflammatory cytokines as described above and suppressing the secretion of the expressed cytokines.

[0045] In addition, the peptide of the present invention can suppress the expression of Cox2. The Cox2 (cyclooxygenase 2) is an enzyme that stimulates and participates in the biosynthesis process of prostaglandins, and may be regulated and expressed by NF-κB to regulate inflammatory reactions. Cox2 is a protein that is hardly expressed under normal conditions but is rapidly expressed by stimuli such as cytokines, inflammatory factors, and endotoxins. Therefore, by confirming the expression level of the Cox2 gene and the amount of the Cox2 protein, the anti-inflammatory activity of the peptide of the present invention can be confirmed, and the peptide of the present invention exhibits the effect of suppressing inflammation by suppressing the expression of Cox2.

[0046] To confirm the anti-inflammatory effect of the peptide of the present invention, in a preferred embodiment of the present invention, inflammatory cytokine was treated together with the peptide containing the amino acid sequence of SEQ ID NO: 1 in mouse macrophages, and the amount of Cox2 protein was confirmed. As a result, although the inflammatory cytokine induced an inflammatory reaction in the cells, it was confirmed that the amount of Cox2 protein decreased due to the treatment with the peptide of the present invention (see Figure 8).

[0047] In addition, to confirm the effect of reducing the expression level of inflammation-related genes by the treatment with the peptide of the present invention, in a preferred embodiment of the present invention, inflammatory cytokine was treated together with the peptide containing the amino acid sequence of SEQ ID NO: 1 in mouse macrophages, and the mRNA amounts of TNFα, IL-1β, and Cox2 genes were confirmed. As a result, although the inflammatory cytokine induced an inflammatory reaction in the cells, it was confirmed that the expression levels of the inflammatory cytokine and related enzyme genes as described above decreased due to the treatment with the peptide of the present invention (see Figure 9).

[0048] Therefore, it is clear that the peptide of the present invention has anti-inflammatory activity that can reduce and improve the inflammatory reaction by decreasing the expression and secretion of inflammatory cytokines such as TNFα, IL-6, IL-17, IL-1β, and IFNγ that can promote the inflammatory reaction and 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 of a composition for preventing, treating, and improving inflammatory diseases induced by or accompanied by an inflammatory reaction.

[0049] At the same time, the peptide of the present invention can have physiological activities other than cartilage regeneration, for example, osteoclast inhibitory activity.

[0050] In one embodiment of the present invention, mouse macrophages were treated with RANKL (Receptor Activator of Nuclear factor Kappa-B ligand) that induces differentiation into osteoclasts together with a peptide containing the amino acid sequence of SEQ ID NO: 1, and the differentiation into osteoclasts and the activity of proteins related to the differentiation into osteoclasts were confirmed. As a result, it was confirmed that the differentiation into osteoclasts and the expression of related proteins were decreased by the treatment with the peptide of the present invention, and the formation of the actin ring essential for the differentiation of osteoclasts was suppressed (see FIGS. 10a to 10c, FIG. 11, and FIG. 12).

[0051] An increase in osteoclast activity is closely related to osteoporosis. Therefore, the peptide of the present invention can be usefully used for the prevention or treatment of osteoporosis by suppressing osteoclast activity.

[0052] 2. Pharmaceutical composition containing the peptide of the present invention Another aspect of the present invention provides a pharmaceutical composition for cartilage regeneration containing, as an active ingredient, a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0053] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing, as an active ingredient, a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0054] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating osteoporosis containing, as an active ingredient, a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0055] Since the peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the item "1. Peptide of the present invention" above, for a specific description, the item "1. Peptide of the present invention" above is incorporated by reference, and hereinafter, only the configuration specific to the pharmaceutical composition will be described.

[0056] According to one embodiment of the present invention, since the peptide of the present invention has an effect of promoting cartilage regeneration, the pharmaceutical composition containing the peptide as an active ingredient can be used for the purpose of regenerating cartilage.

[0057] According to another embodiment of the present invention, since the peptide of the present invention has the effect of inhibiting the expression or secretion of inflammation-related cytokines and inflammation-related factors, the pharmaceutical composition containing the peptide as an active ingredient can be used for preventing or treating inflammatory diseases.

[0058] According to another embodiment of the present invention, since the peptide of the present invention has the effect of suppressing the differentiation and activity of osteoclasts, the pharmaceutical composition containing the peptide as an active ingredient can be used for preventing or treating osteoporosis.

[0059] In the present invention, the inflammatory disease may exhibit a pathological condition that induces inflammation by neutrophil chemotaxis in leukocytes, and may include any disease that develops due to an inflammatory reaction or involves an inflammatory reaction without limitation. For example, the inflammatory disease may be rhinitis, bronchitis, periodontitis, pancreatitis, gastritis, gastric ulcer, inflammatory skin disease, atopic dermatitis, encephalitis, sepsis, inflammatory bowel disease, chronic obstructive pulmonary disease, pulmonary septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic virus, or chronic inflammatory disease due to bacterial infection, colitis, inflammatory bowel disease, type 1 diabetes, rheumatoid arthritis, reactive arthritis, 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 erythematosus, discoid lupus erythematosus, 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, but is not limited thereto.

[0060] The pharmaceutical composition of the present invention may be used for the purpose of promoting cartilage regeneration, preventing the occurrence of inflammatory diseases by suppressing the expression and secretion of factors that induce inflammatory reactions, preventing the occurrence of osteoporosis by suppressing the differentiation of osteoclasts, inhibiting the exacerbation of the disease state by suppressing the further occurring inflammatory reactions in damaged or wounded cells of patients with the inflammatory disease or the differentiation into osteoclasts in patients with osteoporosis, and treating the disease.

[0061] On the other hand, the pharmaceutical composition containing the peptide of the present invention as an active ingredient can be formulated using a pharmaceutically acceptable carrier and / or excipient by a method that can be easily implemented by those having ordinary knowledge in the technical field to which the present invention pertains, and may be manufactured in the form of a unit volume or incorporated into a multi-dose container. At this time, 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, granule, tablet, capsule, or gel (e.g., hydrogel), and may further contain a dispersant or stabilizer.

[0062] Also, the peptide contained in the pharmaceutical composition may be carried by a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline, lipid, liposome, microspheres, or nanospherical particles. These may form a complex or be associated with the delivery means and may be 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.

[0063] In addition to the above, the pharmaceutically acceptable carrier may include, but is not limited to, 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, etc., which are commonly used in pharmaceutical formulations. Further, in addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be further included.

[0064] The pharmaceutical composition according to the present invention can be administered orally or parenterally during clinical administration and may be used in the form of general pharmaceutical formulations. That is, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration. When formulating, it is usually prepared using 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. Such solid formulations are prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with crude drug extracts or crude drug fermented products. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerol, gelatin, etc. may be used.

[0065] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The level of the effective volume may be determined according to factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route, and the excretion rate, the treatment period, elements including drugs used simultaneously, and other elements well known in the medical field. Other pharmaceutical compositions of the present invention may be administered as an individual therapeutic agent or in combination with a therapeutic agent for other inflammatory diseases, and may be administered simultaneously with, separately from, or sequentially to a conventional therapeutic agent, and may be administered in single or multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects considering all the above factors, and this can be easily determined by those skilled in the art.

[0066] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary according to the patient's age, gender, condition, weight, absorption degree of the active ingredient in the body, inactivation rate, excretion rate, disease type, and drugs used in combination, and may increase or decrease according to the administration route, severity of obesity, gender, weight, age, etc. As an example, the peptide of the present invention may be administered at about 0.0001 μg to 500 mg per kg of the patient's body weight per day, for example, 0.01 μg to 100 mg. Also, it may be administered in divided doses several times a day, for example, 2 to 3 times a day, at regular time intervals according to the judgment of a doctor or a pharmacist.

[0067] 3. Health functional food containing the peptide of the present invention Another aspect of the present invention provides a health functional food for cartilage regeneration containing, as an active ingredient, a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0068] Still another aspect of the present invention provides a health functional food for preventing or improving inflammatory diseases containing, as an active ingredient, a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0069] Still another aspect of the present invention provides a health functional food for preventing or improving osteoporosis containing, as an active ingredient, a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0070] The peptide containing the amino acid sequence of SEQ ID NO: 1 is identical to the peptide described in the item "1. The peptide of the present invention", so for the specific description, the item "1. The peptide of the present invention" is incorporated by reference, and hereinafter, only the specific composition of the health functional food will be described.

[0071] Similar to the pharmaceutical composition, the health functional food containing the peptide of the present invention as an active ingredient can be usefully used for cartilage regeneration, prevention or improvement of inflammatory diseases or osteoporosis.

[0072] The health functional food may be used before the onset stage of the disease or after the onset of the disease, simultaneously with or separately from the drug for treatment, for the prevention or improvement of the disease.

[0073] In the health functional food of the present invention, the active ingredient may be added to the food as it is or used together with other foods or food ingredients, and may be appropriately used by a normal method. The mixing amount of the active ingredient may be preferably determined according to its purpose of use (for prevention or improvement). Generally, when manufacturing food or beverage, the composition of the present invention may be added in an amount preferably of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health regulation, the amount may be below the above range.

[0074] In addition to containing the above-mentioned active ingredients, the health functional food of the present invention may contain other ingredients as essential ingredients without any special restrictions. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. Examples of the aforementioned natural carbohydrates may include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., which are ordinary sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrates may be appropriately determined by those skilled in the art according to their selection.

[0075] In addition to the above, the health functional food of the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, and heavy stabilizers (such as cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Such ingredients may be used independently or in combination, and the proportion of such additives may also be appropriately selected by those skilled in the art.

[0076] Hereinafter, the present invention will be described in detail with reference to examples.

[0077] However, the following examples are to specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0078] [Example 1] Production of Peptide Using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), a peptide having the amino acid sequence of SEQ ID NO: 1 described in Table 1 below was synthesized, and these synthesized peptides were purified by C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA). As a result of molecular weight analysis using a 3200 QTRAP LC-MS / MS system, it was confirmed that the peptide of the present invention corresponding to a molecular weight of 1250.5 Da was synthesized (FIGS. 1a and 1b).

[0079] [Table 1] [Experimental Example 1] Confirmation of promotion of ECM production by chondrocytes by peptide treatment To confirm the cartilage regeneration effect by treatment with the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1, after treating human-derived chondrocytes with the peptide of SEQ ID NO: 1, the increase in the formation of glycosaminoglycan, which is a component of ECM, was confirmed.

[0080] For this purpose, the human C28 / I2 chondrocyte cell line was dispensed into a 96-well plate at a density of 3×10 3 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. After replacing with fresh medium, the peptide of SEQ ID NO: 1 was treated at concentrations of 30, 50, 100, and 150 μg / ml. The peptide of SEQ ID NO: 1 was treated according to the above concentrations while changing the medium once every 3 days. After removing the medium on day 6, it was placed in a 96-well plate stained with 60 μl of 3.7% formalin and fixed 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. Then, the staining solution was removed, washed with triple-distilled water and dried, and observed under a microscope.

[0081] 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 ECM in chondrocytes (see Fig. 2).

[0082] [Experimental Example 2] Confirmation of promotion of ECM gene expression in chondrocytes by peptide treatment After treating human-derived chondrocytes with the peptide having the amino acid sequence of SEQ ID NO: 1, the mRNA expression of collagen type II (COL2A1, Collagen type II), aggrecan (ACAN), proteoglycan core protein (PCP, proteoglycan core protein), which are components of ECM, and the GAPDH gene as a control group was confirmed for increase.

[0083] For this purpose, human C28 / I2 chondrocyte cell line was dispensed into a 6-well plate at a density of 8.9×10 4 cells / well and cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. After replacing with fresh medium, the peptide of SEQ ID NO: 1 was treated at concentrations of 50 and 100 μg / ml. The peptide of SEQ ID NO: 1 was treated according to the above concentrations while changing the medium once every 3 days. After removing the medium on the 9th day, the cells were harvested and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit (Intron, Korea), PCR was performed on the collagen type II, ACAN, PCP, and GAPDH genes using a PCR-free mix (Intron, Korea). The primers specific for the collagen type II, ACAN, PCP, and GAPDH are as shown in Table 2 below:

[0084]

Table 2

[0085] [Experimental Example 3] Confirmation of promotion of ECM production in three-dimensional culture of chondrocytes by peptide treatment After treating the peptide having the amino acid sequence of SEQ ID NO: 1 in the three-dimensional culture of human-derived chondrocytes, the increase in the formation of glycosaminoglycan, which is a component of ECM, was confirmed.

[0086] For this purpose, 3×10 6 cells were suspended in 1 ml of an alginate solution (1.25% alginic acid, 20 mM HEPES, 150 mM NaCl, pH 7.4), and the alginate solution containing the cells was dropped one by one into a polymerization solution (100 mM CaCl2, 10 mM HEPES, pH 7.4) to form beads. After washing the formed beads twice with DPBS, the beads were dispensed into a conical tube filled with DMEM medium (5% FBS). The peptide of SEQ ID NO: 1 was treated at concentrations of 100 and 150 μg / ml, and the peptide of SEQ ID NO: 1 was treated separately at the above concentrations while changing the medium once every three days. After removing the medium on day 9, 3.7% formaldehyde was added and fixed for 6 hours. Dehydration was carried out using a tissue processor (in the order of 70%, 80%, 90%, 100% I, II of EtOH and 30 minutes each for xylene I, II), and embedding was performed to produce a paraffin block. After slicing the paraffin block to a thickness of 0.4 μm on a glass slide, it was completely dried for 24 hours or more. The sections were hydrated (3 minutes each in the order of xylene I, II, 100% I, II of EtOH, 90%, 80%, 70%), immersed in running water for 10 seconds and washed three times. After staining with Alcian blue or Toluidine blue for 1 hour, it was immersed in running water for 10 seconds and washed three times. After immersing in 90% and 100% I, II of EtOH for 10 seconds, it was immersed in xylene I, II for 3 minutes each, and then mounted. After drying for 24 hours or more, it was observed under a microscope.

[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 production of glycosaminoglycan, which is a component of ECM, in the three-dimensional culture of chondrocytes (see Fig. 4).

[0088] [Experimental Example 4] Confirmation of Chondrocyte Differentiation and Promotion of ECM Production of Mesenchymal Stem Cells by Peptide Treatment After treating adipose-derived mesenchymal stem cells with the peptide having the amino acid sequence of SEQ ID NO: 1, the effects on chondrogenic differentiation and ECM production of the stem cells were confirmed.

[0089] For this purpose, human AD-MSC (Adipose-Derived Mesenchymal Stem Cell) cells were dispensed into a 96-well plate at a density of 1.5×10 3 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. After alternating to DMEM medium containing 5% FBS, the peptide of SEQ ID NO: 1 was treated at concentrations of 30, 50, 100, and 150 μg / ml. The peptide of SEQ ID NO: 1 was treated according to the above concentrations while changing the medium once every three days. After removing the medium on day 15, 60 μl of 3.7% formalin was placed in the 96-well plate for staining and fixed for 1 minute. After removing 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 then incubated at 37°C for 24 hours. Thereafter, the staining solution was removed, washed with triple-distilled water and dried, and then observed under a microscope.

[0090] 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 glycosaminoglycan, which is a component of ECM (see Fig. 5).

[0091] [Experimental Example 5] Confirmation of Expression of ECM Regulatory Factors in Mesenchymal Stem Cells by Peptide Treatment After treating adipose-derived mesenchymal stem cells with the peptide having the amino acid sequence of SEQ ID NO: 1, it was confirmed whether the expression of SOX9 protein, which is a regulatory factor of ECM, increased.

[0092] For this purpose, human AD-MSC cells were at 1.5×10 3Dispensed into a 96-well plate at a cell / well density and cultured in DMEM medium (cat. 11995-065, Gibco) containing 10% FBS for 24 hours. After alternating to DMEM medium containing 5% FBS, the peptide of SEQ ID NO: 1 was treated at concentrations of 50 and 100 μg / ml. The peptide of SEQ ID NO: 1 was treated separately according to the above concentrations while changing the medium once every three days. At this time, as a positive control group (CM), a solution containing dexamethasone (100 nM) + ascorbic acid (50 μM) + proline (40 μM) + TGFβ1 (10 ng / ml) + 1X ITS was used. After removing the medium on days 3, 7, and 14, the cells were harvested, cell lysates were prepared, and Western blotting was performed using antibodies against the SOX9 and SOX5 proteins (Millipore, USA).

[0093] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention increased the production of the SOX9 protein, which is a regulator of ECM in adipose-derived mesenchymal stem cells (see FIGS. 6a to 6c).

[0094] [Experimental Example 6] Confirmation of promotion of ECM gene expression in mesenchymal stem cells by peptide treatment After treating adipose-derived mesenchymal stem cells with the peptide having the amino acid sequence of SEQ ID NO: 1, it was confirmed whether the expression of COMP (Cartilage Oligomeric matrix protein), collagen type 11A (COL 11A), PCP, and ACAN (aggrecan) genes related to the production of ECM increased or not.

[0095] For this purpose, human AD-MSC cells were 3Cells were dispensed into a 96-well plate at a density of cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) containing 10% FBS for 24 hours. After alternating to DMEM medium containing 5% FBS, the peptide of SEQ ID NO: 1 was treated at concentrations of 50 and 100 μg / ml. The peptide of SEQ ID NO: 1 was treated separately at the above concentrations while changing the medium once every three days. After removing the medium on days 3, 7, and 11, the cells were harvested and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit (Intron, Korea), PCR was performed on the COMP, COL 11A, PCP, ACAN, and GAPDH genes using a PCR-free mix (Intron, Korea). The primers specific to the COMP, COL 11A, PCP, ACAN, and GAPDH are as shown in Table 3 below:

[0096] [Table 3] 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 the COMP, COL11A, PCP, and ACAN genes related to ECM production in adipose-derived mesenchymal stem cells (see FIGS. 7a to 7c).

[0097] Taking the results of Experimental Examples 1 to 6 as described above into consideration, the peptide having the nucleotide sequence of SEQ ID NO: 1 of the present invention increased the activity of chondrocytes to promote ECM synthesis, increased the expression of genes related to the production of the ECM and ECM regulatory factor genes, promoted the differentiation of mesenchymal stem cells into chondrocytes, and also increased the expression of genes related to the production of the ECM and ECM regulatory factor genes in mesenchymal stem cells, and it can be confirmed that there is an excellent cartilage regeneration effect. In addition, when the peptide of the present invention is treated in a larger amount, the above effects appear more highly, so it can be seen from the results of the above experimental examples that the cartilage regeneration effect is the effect of the peptide of the present invention.

[0098] [Experimental Example 7] Confirmation of the expression level of inflammatory cytokine mRNA in cells induced by TNFα In addition to the cartilage regeneration activity confirmed in the above Experimental Examples 1 to 6, in order to confirm the anti-inflammatory effect by 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 levels of TNFα, IL-1β which are inflammatory cytokines, and the Cox2 gene which is an inflammation induction marker were confirmed. For the induction of inflammation, cytokine TNFα was treated. TNFα corresponds to a signal transduction protein related to the inflammatory reaction, IL-1β corresponds to a cytokine that promotes the inflammatory reaction, and Cox2 is an inflammation-related protein. Therefore, by measuring the expression levels of the TNFα, Cox2, and IL-1β genes, the degree of the inflammatory reaction can be confirmed.

[0099] For this purpose, the mouse RAW264.7 cell line was dispensed into a 6-well plate at a density of 2×10 5 cells / well, and then cultured in α-MEM medium containing 10% FBS for 24 hours. After alternating with serum-free α-MEM medium (1% penicillin), it was starved for 6 hours. After treating with the peptide of SEQ ID NO: 1 at concentrations of 100 and 150 μg / ml, it was incubated for 1 hour. After 1 hour, TNF-α was treated at 20 nM and incubated for 24 hours. Then, the cells were harvested and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit (Intron, Korea), PCR was performed on the TNFα, IL-1β, Cox2, and GAPDH genes using a PCR-free mix (Intron, Korea). The primers specific to the TNFα, IL-1β, Cox2, and GAPDH genes are as shown in Table 4 below:

[0100]

Table 4

[0101] [Experimental Example 8] Confirmation of the Expression Level of Inflammatory Cytokine mRNA in Cells Induced by TNFα In addition to the results of Experimental Example 7 above, in order to further confirm the anti-inflammatory effect by treatment with the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention, mouse RAW264.7 macrophage cell line was treated with TNFα to induce inflammation, and the expression level of COX2 protein, which is an inflammation induction marker, was confirmed.

[0102] For this purpose, mouse RAW264.7 cell line was dispensed into 6-well plates at a density of 2 × 10 5 cells / well, and then cultured in α-MEM medium containing 10% FBS for 24 hours. After alternating with serum-free α-MEM medium (1% penicillin), the cells were starved for 6 hours. After treating with the peptide of SEQ ID NO: 1 at concentrations of 100 and 150 μg / ml, the cells were incubated for 1 hour. After 1 hour, TNF-α was treated at 20 nM and incubated for 24 hours. Then, the cells were harvested, cell lysates were prepared, and Western blotting was performed using an antibody against the COX2 protein (Cell Signaling Technology, USA).

[0103] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention suppressed the increase in inflammatory proteins induced by the treatment of inflammatory cytokines in mouse macrophages (see Figure 9).

[0104] Taking the results of Experimental Example 7 and Experimental Example 8 as described above together, when the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention is treated in cells in which an inflammatory reaction is induced, the amount of proteins and the expression level of genes related to the inflammatory reaction decrease, and it can be confirmed that there is an inhibitory effect on the inflammatory reaction. In addition, when the peptide of the present invention is treated in a larger amount, the above effects are shown more highly. Therefore, it can be seen that the inhibitory effect on the inflammatory reaction seen from the results of the above experimental examples is the effect of the peptide of the present invention.

[0105] [Experimental Example 9] Confirmation of the Inhibitory Effect on Osteoclast Differentiation Induced by RANKL In addition to the cartilage regeneration activity confirmed in Experimental Examples 1 to 6 and the anti-inflammatory effect confirmed in Experimental Examples 7 and 8, the inhibitory effect of the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention on osteoclast differentiation was confirmed.

[0106] <9-1>Confirmation of the inhibitory effect of the peptide of the present invention on osteoclast differentiation The mouse RAW264.7 cell line was dispensed into a 96-well plate at a density of 1.7×10 3 cells / well and then cultured in DMEM medium for 24 hours. After 24 hours, the peptide of SEQ ID NO: 1 was treated at concentrations of 10, 30, 50, 100, and 150 μg / ml together with RANKL, and then osteoclast differentiation was induced for 4 days. After removing the medium, it was fixed for 30 seconds and washed twice with secondary distilled water. Staining was carried out at 37°C for 30 minutes with 100 μl of TRAP staining solution (acid phosphatase kit, Sigma aldrich). Then, the staining solution was removed, washed three times with secondary distilled water and dried, and then observed under a microscope.

[0107] 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 differentiation into osteoclasts induced by RANKL in mouse macrophages (see Fig. 10a).

[0108] <9-2>Confirmation of the cytotoxicity of the peptide of the present invention Using the CCK-8 assay method, the effect of the peptide of the present invention on cell growth was confirmed. The CCK-8 reagent is reduced by the dehydrogenase enzyme of cells to produce a colored product, and the difference in absorbance using this can be used to confirm whether cell growth is possible.

[0109] For this purpose, the mouse RAW264.7 cell line was adjusted to 1.7×10 3After dispensing into a 96-well plate at a cell / well density, it was cultured in DMEM medium for 24 hours. After 24 hours, after treating with the peptide of SEQ ID NO: 1 at concentrations of 10, 30, 50, 100, and 150 μg / ml, it was incubated for 3 days. After removing the medium, the CCK-8 solution (Dojindo, CCK-8 kit) was diluted 10-fold in the culture solution and 100 μl of it was added to each well. The absorbance at a wavelength of 450 nm was measured using a microplate reader at intervals of 30 minutes while incubating at 37°C.

[0110] As a result, when the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention was treated, it was shown that the absorbance was rather higher, and it was confirmed that the peptide of the present invention did not show toxicity in mouse macrophages (see Fig. 10b).

[0111] <9-3>Confirmation of the inhibitory effect of the peptide of the present invention on TRAP activity related to osteoclast differentiation The mouse RAW264.7 cell line was dispensed into a 96-well plate at a density of 1.7×10 3 cells / well and cultured in DMEM medium for 24 hours. After 24 hours, the peptide of SEQ ID NO: 1 was treated at concentrations of 10, 30, 50, 100, and 150 μg / ml together with RANKL, and then osteoclast differentiation was induced for 4 days. After treating with 100 μl of the TRAP activity solution (TRAP buffer (0.1 sodium citrate + 50 μM sodium tartrate [pH 5.0]) 15 ml + 4-Nitrophenly phosphate disodium salt hexahydrate, sigma, 1 tablet mix), it was incubated at 37°C for 1 hour, and then 10 μl of 2 N NaOH was added. The absorbance at a wavelength of 405 nm was measured using a plate reader.

[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 activity of TRAP, which is a marker of osteoclasts, in a concentration-dependent manner in mouse macrophages (see Fig. 10c).

[0113] [Experimental Example 10] Confirmation of the inhibitory effect on osteoclast differentiation induced by RANKL Mouse RAW264.7 cell line was dispensed into a 96-well plate at a density of 1.7×10 3 cells / well and then cultured in DMEM medium for 24 hours. After 24 hours, the peptide of SEQ ID NO: 1 was treated at concentrations of 50 and 100 together with RANKL, and then osteoclast differentiation was induced for 4 days. On the first day of induction, after removing the medium, the cells were harvested, cell lysates were prepared, and Western blotting was performed using an antibody against NFATc1 protein (Millipore, USA).

[0114] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention inhibits the production of NFATc1 protein, which is a main transcription factor in the process of differentiating from adipose-derived mesenchymal stem cells to osteoclasts (see Figure 11).

[0115] [Experimental Example 11] Confirmation of the inhibitory effect on actin ring formation of osteoclasts induced by RANKL In addition to the results of Experimental Example 9 and Experimental Example 10, in order to further confirm the inhibitory effect on osteoclast differentiation by treatment with the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention, 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 ring, which is essential for osteoclast differentiation.

[0116] For this purpose, mouse RAW264.7 cell line was adjusted to 5×10 3After dispensing into a 96-well plate at a cell / well density, it was cultured in DMEM medium for 24 hours. After 24 hours, after treating with the peptide of SEQ ID NO: 1 at concentrations of 50 and 100 μg / ml together with RANKL, osteoclast differentiation was induced for 4 days. After removing the medium, it was washed 3 times with cooled PBS and fixed with 4% paraformaldehyde for 30 minutes. After washing 3 times with cooled PBS for 5 minutes each, it was permeabilized with 0.3% Triton X-100 in PBS for 30 minutes. After washing 3 times with cooled PBS for 5 minutes each, it was blocked with filtered 3% BSA in PBS for 1 hour with stirring. It was incubated with a primary antibody (Rhodamin (Red) [Invitrogen TM ) diluted 1:100 in blocking medium for 2 hours with stirring. After blocking the light and washing 3 times with PBS for 15 minutes each with stirring, DAPI mounting medium (VECTASHIELD(R) MOUNTING MEDIUM with DAPI) was added, a coverslip was placed over each well, and then it was observed under a microscope.

[0117] As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention suppressed the formation of actin rings essential for differentiation into osteoclasts in mouse macrophages (see Fig. 12).

[0118] Taking the results of Experimental Example 9 and Experimental Example 11 as described above together, it can be confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention can suppress differentiation into osteoclasts and can thus be applied to the prevention or treatment of osteoporosis. Further, when the peptide of the present invention is treated in a larger amount, the above-described effects are shown more highly, so it can be seen that the osteoclast differentiation inhibitory effect seen from the results of the above experimental examples is the effect of the peptide of the present invention.

[0119] [Production Example 1] Production of Pharmaceutical Composition <1-1> Production of Powder 2 g of the peptide of Example 1 1 g of lactose The above components were mixed and filled into an airtight package to produce a powder.

[0120] <1-2>Manufacture of Tablets 100 mg of the peptide of Example 1 100 mg of corn starch 100 mg of lactose 2 mg of magnesium stearate After mixing the above components, tablets were manufactured by tableting according to the usual tablet manufacturing method.

[0121] <1-3>Manufacture of Capsules 100 mg of the peptide of Example 1 100 mg of corn starch 100 mg of lactose 2 mg of magnesium stearate After mixing the above components, capsules were manufactured by charging gelatin capsules according to the usual capsule manufacturing method.

[0122] <1-4>Manufacture of Rings 1 g of the peptide of Example 1 1.5 g of lactose 1 g of glycerin 0.5 g of xylitol After mixing the above components, they were manufactured to be 4 g per ring according to the usual method.

[0123] <1-5>Manufacture of Granules 150 mg of the peptide of Example 1 50 mg of soybean extract 200 mg of glucose 600 mg of starch After mixing the above components, 100 mg of 30% ethanol was added, dried at 60°C to form granules, and then filled into packages.

[0124] [Production Example 2] Manufacture of Health Functional Food Composition <2-1>Manufacture of Wheat Flour Foods 0.5 to 5.0 parts by weight of the peptide of Example 1 was added to wheat flour, and bread, cake, cookie, cracker and noodles were manufactured using this mixture.

[0125] <2-2> Manufacture of soups and gravies 0.1 to 5.0 parts by weight of the peptide of Example 1 were added to soups and gravies to produce meat processed products for health promotion, soups and gravies of noodles.

[0126] <2-3> Manufacture of dairy products 5 to 10 parts by weight of the peptide of Example 1 were added to milk, and various dairy products such as butter and ice cream were produced using the milk.

[0127] <2-4> Manufacture of Zen food After roasting the alpha - converted and dried brown rice, wheat, glutinous rice, and buckwheat by a known method, they were ground into a powder with a particle size of 60 mesh by a pulverizer. After roasting the black beans, black sesame seeds, and perilla seeds that had been steamed and dried by a known method, they were ground into a powder with a particle size of 60 mesh by a pulverizer. The grains, seeds, and the peptide of Example 1 produced above were blended and manufactured in the following ratio.

[0128] Grains (30 parts by weight of brown rice, 15 parts by weight of buckwheat, 20 parts by weight of wheat), seeds (7 parts by weight of perilla seeds, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds), peptide of Example 1 (3 parts by weight), Ganoderma lucidum (0.5 part by weight), Rehmannia glutinosa (0.5 part by weight) <2-5> Manufacture of health beverages Auxiliary materials such as liquid fructose (0.5%), oligosaccharides (2%), sugar (2%), salt (0.5%), and water (75%) and 5 g of the peptide of Example 1 were homogeneously blended and instantaneously sterilized, and then this was packaged in small packaging containers such as glass bottles and PET bottles for manufacture.

[0129] As described above, the present invention has been described in detail only for the described embodiments, but it is obvious to those of ordinary skill in the art that various modifications and corrections are possible within the scope of the technical idea of the present invention, and such modifications and corrections naturally belong to the appended claims.

Claims

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

1.

2. The peptide according to claim 1, having one or more biological activities selected from the group consisting of cartilage regeneration activity, anti-inflammatory activity, and osteoporosis inhibitory activity.

3. The peptide according to claim 1, which promotes the differentiation of stem cells into chondrocytes.

4. The peptide according to 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 according to claim 1, which increases the synthesis of extracellular matrix (ECM) in chondrocytes.

6. The peptide according to claim 1, which increases the expression of one or more genes selected from the group consisting of collagen type II (COL2A1), ACAN (aggrecan), and PCP (Proteoglycan Core Protein) in chondrocytes.

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

8. The peptide according to claim 7, wherein the inflammatory cytokines are one or more selected from the group consisting of TNFα, IL-1β, COX2, IL-6, IL-17, and IFNγ.

9. The peptide according to claim 1, which suppresses the 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, as an active ingredient, the peptide according to any one of claims 1 to 9.

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

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

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

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

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